WO2025259743A1 - Dual conjugate compounds for extrahepatic delivery - Google Patents

Dual conjugate compounds for extrahepatic delivery

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Publication number
WO2025259743A1
WO2025259743A1 PCT/US2025/033130 US2025033130W WO2025259743A1 WO 2025259743 A1 WO2025259743 A1 WO 2025259743A1 US 2025033130 W US2025033130 W US 2025033130W WO 2025259743 A1 WO2025259743 A1 WO 2025259743A1
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Prior art keywords
bond
dsrna agent
independently
group
aln
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PCT/US2025/033130
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French (fr)
Inventor
Jayaprakash K. NAIR
Joseph David PANARESE
Karyn SCHMIDT
Kevin Dooley
Haiqing Wang
Juan SALINAS
Mark Neil TOLENTINO
Ivan Zlatev
Punit Seth
Vasant R. Jadhav
Bryan INGOGLIA
Masaaki Nakata
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Alnylam Pharmaceuticals Inc
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Alnylam Pharmaceuticals Inc
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Publication of WO2025259743A1 publication Critical patent/WO2025259743A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • the ⁇ v ⁇ 6 integrin targeting ligand or the ⁇ v ⁇ 6 integrin targeting ligand together with a linker useful in the present disclosure comprises a structure selected from the table below: 6 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 7 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Z1 is a linker linking the the ⁇ v ⁇ 6 integrin targeting ligand to the remainder of the dsRNA agent and wherein * represents the bond to the remainder of the dsRNA agent. 8 ME1 ⁇ 53466565.v1 Atty.
  • the cardiac muscle disorder is selected from the group consisting of obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic cardiomyopathy (FHC); Heart failure with preserved ejection fraction (HFPEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina; myocardial infarction (MI); heart failure or heart failure with reduced ejection fraction 30 (HFREF); supraventricular tachycardia (SVT); hypertrophic cardiomyopathy (HCM); and PLN cardiomyopathy.
  • HOCM obstructive hypertrophic cardiomyopathy
  • FHC familial hypertrophic cardiomyopathy
  • HPF Heart failure with preserved ejection fraction
  • AFIB atrial fibrillation
  • VFIB ventricular fibrillation
  • angina myocardial infarction
  • MI myocardial infarction
  • HVF supraventricular tachycardia
  • HCM hypertrophic cardiomyopathy
  • PLN cardiomyopathy PLN cardiomyopathy
  • the therapeutic methods of the disclosure further include administering to 35 the subject an additional agent or a therapy suitable for treatment or prevention of an extrahepatic disorder.
  • 11 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG.1 is a bar graph showing AD-1812376 mediated SOD1 knockdown in quadriceps and gastrocnemius of a mouse model at Day 21 following a single IV dose of 2 mg/kg. PBS as used for the control group.
  • FIG.2 is a bar graph showing AD-1812376 mediated SOD1 knockdown in quadriceps, gastrocnemius and liver of non-human primates at Days 29 and 57 following a single IV dose of 1 mg/kg, 3 mg/kg or 9 mg/kg. PBS was used for the control group.
  • FIG.3 is a bar graph showing AD-2032892 mediated SOD1 knockdown in liver, quadriceps and gastrocnemius of a mouse model at Day 21 following administration of a single IV dose of 1 mg/kg, 10 3 mg/kg or 9 mg/kg. PBS was used for the control group.
  • FIG.4 is a bar graph showing AD-2032892 mediated NHP SOD1 knockdown in gastrocnemius, quadriceps and liver of non-human primates following administration of a single IV dose of 10 mg/kg. PBS was used for the control group.
  • FIG.5 is a bar graph showing NHP SOD1 knockdown mediated by AD-2432777, AD-2241090, 15 AD-2315832, AD-2315873 and AD-2315833 in biceps, gastrocnemius and quadriceps of non-human primates at Day 29 following administration of a single IV dose of 10 mg/kg. PBS was used for the control group.
  • FIG.6 is a bar graph showing NHP SOD1 knockdown mediated by AD-2432777 and AD- 2640041 in biceps, gastrocnemius and soleus of non-human primates at Day 28 following administration 20 of a single SC 10 mg/kg dose. PBS was used for the control group.
  • FIG.7 is a bar graph showing AD-2432777, AD-2902547, AD-2889354, AD-2889355, AD- 2889356, and AD-2889357 mediated SOD1 knockdown in quadriceps of a mouse model at Day 7 following administration of a single subcutaneous dose of 1 mg/kg. PBS was used for the control group.
  • FIG.8 is a bar graph showing AD-2432777, AD-2902547, AD-2889354, AD-2889355 and AD- 25 2889356, and AD-2889357 mediated SOD1 knockdown in heart of a mouse model at Day 7 following administration of a single subcutaneous dose of 1 mg/kg. PBS was used for the control group.
  • FIG.9 is a bar graph showing AD-2640041, AD-2700084, AD-2700085, AD-2700086, AD- 2700087, AD-2700088, AD-2700089, AD-2700090, AD-2700091, AD-2700092, AD-2700093, AD- 2700094, AD-2700095, AD-2700096, AD-2700097, and AD-2700099 mediated SOD1 knockdown in 30 quadricep of a mouse model at Day 7 following administration of a single IV dose of 0.3 mg/kg. PBS was used for the control group.
  • FIG.10 is a bar graph showing AD-2640041, AD-2700084, AD-2700085, AD-2700086, AD- 2700087, AD-2700088, AD-2700089, AD-2700090, AD-2700091, AD-2700092, AD-2700093, AD- 2700094, AD-2700095, AD-2700096, AD-2700097, and AD-2700099 mediated SOD1 knockdown in 35 gastrocnemius of a mouse model at Day 7 following administration of a single IV dose of 0.3 mg/kg. PBS was used for the control group. 12 ME1 ⁇ 53466565.v1 Atty.
  • FIG.11 is a bar graph showing AD-2640041, AD-2700084, AD-2700085, AD-2700086, AD- 2700087, AD-2700088, AD-2700089, AD-2700090, AD-2700091, AD-2700092, AD-2700093, AD- 2700094, AD-2700095, AD-2700096, AD-2700097, and AD-2700099 mediated SOD1 knockdown in heart of a mouse model at Day 7 following administration of a single IV dose of 0.3 mg/kg. PBS was 5 used for the control group.
  • FIG.12 is a bar graph showing AD-3214512 mediated HPRT1 knockdown in quadricep and heart of a mouse model at Day 7 following administration of a single subcutaneous dose of 1 mg/kg. PBS was used for the control group.
  • FIG.13 is a bar graph showing NHP DMPK knockdown mediaded by AD-3100656 in heart, 10 gastrocnemius, and quadriceps of non-human primates at Day 32 following a single subcutaneous dose of 3 mg/kg.
  • the control group was the average of animals treated with non-DMPK targeting siRNAs.
  • the DMPK mRNA % remaining is relative to two housekeeping genes (PPIB and ADD1).
  • the present disclosure is based, at least in part, on the surprising discovery that conjugating at least one alpha-v-beta-6 ( ⁇ v ⁇ 6) targeting ligand and at least one in vivo delivery enhancing moiety, e.g., a moiety comprising at least one C10-C26 hydrocarbon chain, to at least one strand of a dsRNA agent, e.g., the sense strand, provides surprisingly efficient in vivo delivery to extrahepatic tissue, i.e., muscle tissue, resulting in efficient entry and internalization of the dsRNA agent into extrahepatic tissue, e.g., muscle 20 tissue, e.g., skeletal muscle tissue and/or cardiac muscle tissue, and surprisingly good inhibition of target gene expression in extrahepatic tissue, e.g., muscle tissue, e.g., skeletal muscle tissue and/or cardiac muscle tissue.
  • a dsRNA agent e.g., the sense strand
  • dsRNA agents comprising at least one alpha-v-beta- 6 ( ⁇ v ⁇ 6) targeting ligand and at least one in vivo delivery enhancing moiety that mediate delivery to 25 extrahepatic tissue, e.g., muscle tissue, e.g., skeletal muscle tissue and/or cardiac muscle tissue, to inhibit the expression of a target gene as well as compositions, ustes, and methods for treating subjects that would benefit from inhibition and/or reduction of the expression of the target gene.
  • extrahepatic tissue e.g., muscle tissue, e.g., skeletal muscle tissue and/or cardiac muscle tissue
  • sense strand or antisense strand is understood as “sense strand or antisense strand or sense strand and antisense strand.”
  • the term “about” is used herein to mean within the typical ranges of tolerances in the art. For 10 example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.
  • the term “at least”, “no less than”, or “or more” prior to a number or series of numbers is 15 understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context.
  • the number of nucleotides in a nucleic acid molecule must be an integer.
  • “at least 19 nucleotides of a 21 nucleotide nucleic acid molecule” means that 19, 20, or 21 nucleotides have the indicated property.
  • nucleotide overhang As used herein, “no more than” or “or less” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the 25 numbers in the series or range. As used herein, ranges include both the upper and lower limit. As used herein, methods of detection can include determination that the amount of analyte present is below the level of detection of the method.
  • the indicated sequence takes precedence.
  • an alkyl group can be both a monovalent radical or divalent radical; in the latter case, it would be apparent to one skilled in the art that an additional hydrogen atom is removed from a 5 monovalent alkyl radical to provide a suitable divalent moiety.
  • oligonucleotides may be an RNA, a DNA, a single-stranded RNA, such as an antisense oligonucleotide (ASO), a double-stranded RNA, such as an siRNA, and oligonucleotide derivatives such as phosphorodiamidate morpholino oligomers (PMOs).
  • bicyclic aryls include, but are not limited to, azulenyl, naphthyl, 2,3- dihydroinden-1-yl, 2,3-dihydroinden-2-yl, 2,3-dihydroinden-3-yl, 2,3-dihydroinden-4-yl, 2,3- 15 ME1 ⁇ 53466565.v1 Atty.
  • the bicyclic aryl is (i) naphthyl or (ii) a phenyl ring fused to either a 5 or 20 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, or a 5 or 6 membered monocyclic heterocyclyl, wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • arylalkyl means an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined25 herein.
  • Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3- phenylpropyl, and 2-naphth-2-ylethyl.
  • azido means a -N 3 group.
  • carboxy means a -COOH group.
  • cyano and "nitrile” as used herein, mean a -CN group.
  • cycloalkyl as used herein, means a monocyclic or a bicyclic cycloalkyl ring system.
  • Monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 10 carbon atoms, where such groups are saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Bicyclic cycloalkyl ring systems are bridged monocyclic rings or fused bicyclic rings.
  • Bridged monocyclic rings contain a monocyclic cycloalkyl ring 35 where two non-adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form -(CH 2 ) w -, where w is 1, 2, or 3).
  • Representative examples of bridged bicyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, 16 ME1 ⁇ 53466565.v1 Atty.
  • Fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to a monocyclic cycloalkyl.
  • Representative examples of fused bicyclic ring systems include, but are not limited to, decaliyl.
  • Cycloalkyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • the fused bicyclic 5 cycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused to a 5 or 6 membered monocyclic cycloalkyl, wherein the fused bicyclic cycloalkyl is optionally substituted by one or two groups which are independently oxo or thia.
  • Cycloalkenyl refers to a monocyclic or a bicyclic cycloalkenyl ring system.
  • Monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such 10 groups are unsaturated (i.e., containing at least one annular carbon-carbon double bond), but not aromatic.
  • Examples of monocyclic ring systems include cyclopentenyl and cyclohexenyl.
  • Bicyclic cycloalkenyl rings are bridged monocyclic rings or a fused bicyclic rings.
  • Bridged monocyclic rings contain a monocyclic cycloalkenyl ring where two non-adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of 15 the form -(CH 2 ) w -, where w is 1, 2, or 3).
  • Representative examples of bicyclic cycloalkenyls include, but are not limited to, norbornenyl and bicyclo[2.2.2]oct-2-enyl.
  • Fused bicyclic cycloalkenyl ring systems contain a monocyclic cycloalkenyl ring fused to either a monocyclic cycloalkyl or a monocyclic cycloalkenyl. Cycloalkenyl groups are optionally substituted with one or two groups which are independently oxo or thia. 20
  • the term “monocyclic ring”, as used herein, comprises monocyclic aryl, monocyclic cycloalkyl, monocyclic cycloalkenyl and monocyclic heterocyclyl.
  • halo or "halogen” as used herein, means -CI, -Br, -I or -F.
  • H means hydrogen.
  • haloalkyl means at least one halogen, as defined herein, appended to 25 the parent molecular moiety through an alkyl group, as defined herein.
  • Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl.
  • heteroaryl as used herein, means a monocyclic heteroaryl or a bicyclic ring system containing at least one heteroaromatic ring (i.e., a monocyclic or bicyclic aromatic ring system containing 30 at least one heteroatom within the aromatic system).
  • the monocyclic heteroaryl can be a 5 or 6 membered ring.
  • the 5 membered ring consists of two double bonds and one, two, three or four nitrogen atoms and optionally one oxygen or sulfur atom.
  • the 6 membered ring consists of three double bonds and one, two, three or four nitrogen atoms.
  • the 5 or 6 membered heteroaryl is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heteroaryl.
  • monocyclic heteroaryl include, but are not limited to, furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, and triazinyl.
  • the bicyclic heteroaryl consists of a monocyclic heteroaryl fused to a phenyl, a monocyclic cycloalkyl, a monocyclic 17 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl.
  • the fused cycloalkyl or heterocyclyl portion of the bicyclic heteroaryl group is optionally substituted with one or two groups which are independently oxo or thia.
  • bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl, benzoxathiadiazolyl, 5 benzothiazolyl, cinnolinyl, 5,6-dihydroquinolin-2-yl, 5,6-dihydroquinolin-8-yl, 5,6-dihydroisoquinolin- 1-yl, furopyridinyl, indazolyl, indolyl, isoquinolinyl, naphthyridinyl, quinolinyl, purinyl, 5,6,7,8- tetrahydroquinolin-2-yl, 5,6,7,8-tetrahydroquinolin-3-yl, 5,6,7,8-tetrahydroquinolin-4-yl, 5,6,7,8- tetrahydroquinolin-5-yl, 5,6,7,8-tetrahydr
  • the fused bicyclic heteroaryl is a 5 or 6 membered monocyclic heteroaryl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted with one 15 or two groups which are independently oxo or thia.
  • heteroarylalkyl and "-alkylheteroaryl” as used herein, means a heteroaryl, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • Representative examples of heteroarylalkyl include, but are not limited to, fur-3-ylmethyl, 1H-imidazol- 2-ylmethylm 1H-imidazol-4-ylmethyl, 1-(pyridin-4-yl)ethyl, pyridin-3-ylmethyl, pyridin-4-ylmethyl, 20 pyrimidin-5-ylmethyl, 2-(pyrimidin-2-yl)propyl, thien-2-ylmethyl, and thien-3-ylmethyl.
  • heterocyclyl as used herein, means a monocyclic heterocycle or a bicyclic heterocycle.
  • the monocyclic heterocycle is a 3, 4, 5, 6, or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic.
  • the 3 or 4 membered ring contains 1 heteroatom selected from the group 25 consisting of O, N and S.
  • the 5 membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S.
  • the 6 or 7 membered ring contains zero, one, or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S.
  • monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxan-2-yl, 1,3-dioxolan-2-yl, 1,3-dithiolan-2-yl, 1,2- 30 dithiolan-3-yl, 1,2-dithiolan-4-yl, 1,3-dithian-2-yl, 1,2-dithian-3-yl, 1,2-dithian-4-yl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyranyl
  • the bicyclic heterocycle is a monocyclic heterocycle fused to either a monocyclic cycloalkyl, a monocyclic cycloalkenyl, or a monocyclic heterocycle.
  • Representative examples of bicyclic heterocyclyls include, but are not limited to, decahydroquinolinyl, decahydroisoquinolinyl, octahydro- 1H-indolyl, and octahydrobenzofuranyl.
  • Heterocyclyl groups are optionally substituted with one or two 18 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO groups which are independently oxo or thia.
  • the bicyclic heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring fused to a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, or a 5 or 6 membered monocyclic heterocyclyl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are independently oxo or thia.
  • hydroxy or “hydroxyl” as used herein means an -OH group.
  • thiol as used herein means an –SH group.
  • hydroxyl protecting group refers to a labile chemical moiety which protects a hydroxyl group against undesired reactions during synthetic procedure(s). After the synthetic procedure(s), the hydroxy protecting group may be selectively removed. Hydroxy protecting groups as 10 known in the art are described generally in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).
  • hydroxyl protecting groups include, but are not limited to, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4- bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl, isopropoxycarbonyl, diphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl) 15 ethoxycarbonyl, 2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, t-butyl, 2,2,2-trichloroethyl, 2-trimethylsilyl ethyl, l,l- dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl,
  • hydroxyl protecting groups can be selected from acetyl (Ac or — C(O)CH 3 ), benzoyl (Bz or — C(O)C 6 H 5 ), and trimethylsilyl (TMS or -Si(CH 3 ) 3 ).
  • nitro as used herein means a -NO 2 group.
  • saturated as used herein means the referenced chemical structure does not contain any multiple carbon-carbon bonds. For example, a saturated cycloalkyl group as defined herein includes cyclohexyl, cyclopropyl, and the like.
  • amine or “amino” encompasses compounds where a nitrogen atom is covalently 30 bonded to at least one carbon or heteroatom.
  • alkyl amino includes groups and compounds wherein the nitrogen is bound to at least one additional alkyl group.
  • dialkyl amino includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups.
  • unsaturated as used herein means the referenced chemical structure contains at least one multiple carbon-carbon bond (i.e., double or triple bond, or both), but is not aromatic.
  • an unsaturated cycloalkyl group as defined herein includes cyclohexenyl, cyclopentenyl, cyclohexadienyl, and the like.
  • the term “leaving group” as used herein means an atom or group (charged or uncharged) that becomes detached from an atom in what is considered to be the residual or main part of the substrate in a 19 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO specified reaction.
  • the specified reaction herein unless otherwise noted, is an S N 1 or an S N 2 reaction as is understood by one skilled in the art. In certain embodimentns, the specified reaction herein, is an S N 2 reaction.
  • substituted as used herein, whether preceded by the term“optionally” or not, means 5 that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
  • a“substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is 10 substituted, the substituent is either the same or different at each position.
  • Suitable substituents include, but are not limited to, halogen, hydroxy, thiol, nitro, alkoxy, azido, carboxy, cyano, amino, C 1-6 alkyl, C 2- 6 alkenyl, C 1-6 alkoxy, C 1-6 alkylamino.
  • support linking group means a divalent chemical moiety that covalent connects a surface-bound functional group of a solid support (e.g., an amino group of an amino-modified solid support) to another chemical moiety.
  • reactive pair means two functional groups known by one skilled in the art to be capable of reacting, alone or in the presence of other reagents, to form a covalent linkage between the two chemical entities which each contain one of member of the reactive pair, respectively; the latter, herein, is referred to as “linking group formed by a reactive pair”.
  • a reactive pair is a click pair, i.e., two functional groups capable of reacting in a click reaction to form a 20 covalent linkage.
  • Matael acceptor means an alpha, beta-unsaturated compound capable of reacting with a nucleophile at the beta-carbon of the electrophilic alkene of the alpha, beta -unsaturated compound.
  • alpha, beta-unsaturated compound include, but are not limited to, such as an alpha, beta-unsaturated aldehyde, ester, amide, sulfonyl, ketone, nitrile, or nitro.
  • Alpha, beta- 25 unsaturated refers to the carbon-carbon multiple bond that connects the carbon atom that is immediately adjacent to the referenced aldehyde, ester, amide, sulfonyl, ketone, nitrile, or nitro group, to its adjacent carbon atom.
  • Michael acceptor group include, but are not limited to, N-maleimide, acrylaldehyde, acrylonitrile, acrylic acid, acrylamide (e.g., N-isoproprylacrylamide), acrylate esters (e.g., methyl acrylate), vinyl sulfones, vinylsulfonates, and vinylsulfonamides.
  • nitrogen protecting group means those functional groups that are well known in the art and include those described in detail, for example, in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Suitable nitrogen protecting groups include but are not limited to, allyl (Alloc), acetyl (Ac), Adpoc (1-(1-Adamantyl)-1-methylethoxycarbonyl), Boc (tert-butyloxy 20 ME1 ⁇ 53466565.v1 Atty.
  • the term “remainder of the dsRNA agent”, refers to an oligonucleotide strand 15 (i.e. the sense strand or the antisense strand) that forms a dsRNA agent and any intervening linking group that connects the parent moiety (e.g., one or more ⁇ v ⁇ 6 integrin targeting ligand or one or more in vivo delivery enhancing moieties) to the oligonucleotide strand.
  • linker refers to a chemical moiety connecting at least one ⁇ v ⁇ 6 integrin targeting ligand or at least one in vivo 20 delivery enhancing moiety to at least one strand of the dsRNA agent of the disclosure.
  • target sequence or “target nucleic acid” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcription product.
  • the target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed 25 cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene.
  • the target sequence is within the protein coding region of the target gene.
  • the target sequence is within the 3’ UTR of the target gene.
  • the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state.
  • the target sequence may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length.
  • the target sequence can be about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21 ME1 ⁇ 53466565.v1 Atty.
  • the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In still other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in 5 length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.
  • strand comprising a sequence refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature. 10 “G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively.
  • ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, o r a surrogate replacement moiety (see, e.g., Table 1).
  • guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base 15 pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. It is understood that when a cDNA sequence is provided, the corresponding mRNA or RNAi agent would include a U in place of a T.
  • a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil.
  • nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the 20 disclosure by a nucleotide containing, for example, inosine.
  • adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the disclosure.
  • a T is a target gene sequence, or reverse complement thereof, would often be replaced by a U in an RNAi 25 agent of the disclosure.
  • RNAi agent refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • RNA interference is a process that directs the sequence-specific degradation of 30 mRNA. RNAi modulates, e.g., inhibits, the expression of a target gene in a cell, e.g., a cell within a subject, such as a mammalian subject.
  • RNAi agent and “dsRNA agent” may be used interchangeably herein.
  • an RNAi agent of the disclosure includes a single stranded RNAi that interacts with a target RNA sequence, e.g., a target mRNA sequence, to direct the cleavage of the target 35 RNA.
  • a target RNA sequence e.g., a target mRNA sequence
  • siRNAs double-stranded short interfering RNAs
  • Dicer Type III endonuclease
  • Dicer a ribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pair short 22 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO interfering RNAs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309).
  • RISC RNA-induced silencing complex
  • the disclosure relates to a single stranded RNA (ssRNA) (the antisense strand of a siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene.
  • ssRNA single stranded RNA
  • siRNA the antisense strand of a siRNA duplex
  • the term “siRNA” is also used herein to refer to an RNAi as described above.
  • the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA.
  • Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA.
  • the single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Patent No.8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of 15 which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.
  • an “RNAi agent” for use in the compositions and methods of the disclosure is a double stranded RNA and is referred to herein as a “double stranded RNAi agent,” 20 “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”.
  • dsRNA refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., a target mRNA sequence.
  • a double stranded RNA triggers the degradation of a target RNA, e.g., an mRNA, 25 through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.
  • a dsRNA molecule can include ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide, a modified nucleotide.
  • an “RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides.
  • modified nucleotide refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase.
  • modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases.
  • the modifications suitable for use in the agents of the disclosure include all types of modifications disclosed herein or known in the art.
  • RNAi agent any such 35 modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims. 23 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • inclusion of a deoxy-nucleotide – which is acknowledged as a naturally occurring form of nucleotide – if present within a RNAi agent can be considered to constitute a modified nucleotide.
  • the duplex region may be of any length that permits specific degradation of a desired target 5 RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 10 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20
  • the two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger 15 molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3’-end of one strand and the 5’-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.”
  • a hairpin loop can comprise at least one unpaired nucleotide.
  • the hairpin loop can comprise at at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides or nucleotides not directed to the 20 target site of the dsRNA.
  • the hairpin loop can be 10 or fewer nucleotides.
  • the hairpin loop can be 8 or fewer unpaired nucleotides.
  • the hairpin loop can be 4-10 unpaired nucleotides.
  • the hairpin loop can be 4-8 nucleotides.
  • the two strands of double-stranded oligomeric compound can be linked together.
  • the two strands can be linked to each other at both ends, or at one end only. By linking at one 25 end is meant that 5'-end of first strand is linked to the 3'-end of the second strand or 3'-end of first strand is linked to 5'-end of the second strand. When the two strands are linked to each other at both ends, 5'- end of first strand is linked to 3'-end of second strand and 3'-end of first strand is linked to 5'-end of second strand.
  • the two strands can be linked together by an oligonucleotide linker including, but not limited to, (N)n; wherein N is independently a modified or unmodified nucleotide and n is 3-23.
  • n is 3-10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10.
  • the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide.
  • N is a modified or unmodified nucleotide
  • R is a modified or unmodified purine nucleotide.
  • Some of the nucleotides in the linker can be involved in base-pair interactions with other nucleotides in the linker.
  • the two strands can also be linked together by a non-nucleosidic linker, e.g. a linker described herein.
  • Hairpin and dumbbell type oligomeric compounds will have a duplex region equal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs.
  • the duplex region can be equal to or 24 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO less than 200, 100, or 50, in length. In some embodiments, ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.
  • the hairpin oligomeric compounds can have a single strand overhang or terminal unpaired region, in some embodiments at the 3', and in some embodiments on the antisense side of the hairpin. In 5 some embodiments, the overhangs are 1-4, more generally 2-3 nucleotides in length.
  • the hairpin oligomeric compounds that can induce RNA interference are also referred to as "shRNA" herein. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected.
  • the connecting structure is referred to as a “linker.”
  • the RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex.
  • an RNAi may comprise one or more nucleotide overhangs.
  • an RNAi agent of the disclosure is a dsRNA, each strand of which is 24-30 nucleotides in length, that interacts with a target RNA sequence, e.g., a target mRNA sequence, to direct the cleavage of the target RNA.
  • a target RNA sequence e.g., a target mRNA sequence
  • long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev.15:485).
  • Dicer a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base20 pair short interfering RNAs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363).
  • the siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309).
  • RISC RNA-induced silencing complex
  • an RNAi agent of the disclosure is a dsRNA agent, each strand of which comprises 19-23 nucleotides that interacts with a target mRNA sequence to direct the cleavage of the target RNA.
  • a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 30 15:485).
  • Dicer a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3’ overhangs (Bernstein, et al., (2001) Nature 409:363).
  • the siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309).
  • RISC RNA-induced silencing complex
  • an RNAi agent of the disclosure is a dsRNA of 24-30 nucleotides that interacts with a target mRNA sequence to direct the cleavage of the target RNA. 25 ME1 ⁇ 53466565.v1 Atty.
  • nucleotide overhang refers to at least one unpaired nucleotide that protrudes from the duplex structure of a RNAi agent, e.g., a dsRNA. For example, when a 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa, there is a nucleotide overhang.
  • a dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can 5 comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more.
  • a nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.
  • the overhang(s) can be on the sense strand, the antisense strand or any combination thereof.
  • the nucleotide(s) of an overhang can be present on the 5'-end, 3'-end or both ends of either an antisense or sense strand of a dsRNA.
  • At least one strand comprises a 3’ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3’ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5’ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5’ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 15 nucleotides.
  • both the 3’ and the 5’ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.
  • the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., 0-3, 1-3, 2-4, 2- 5, 4-10, 5-10, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3’-end or the 5’-end.
  • the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 20 nucleotide, overhang at the 3’-end or the 5’-end.
  • one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
  • the overhang on the sense strand or the antisense strand, or both can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides in length.
  • an extended overhang is on the sense 25 strand of the duplex. In certain embodiments, an extended overhang is present on the 3’end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5’end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3’end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5’end of the antisense strand 30 of the duplex. In certain embodiments, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
  • the overhang includes a self-complementary portion such that the overhang is capable of forming a hairpin structure that is stable under physiological conditions.
  • One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended.
  • a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., 26 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO no nucleotide overhang at either end of the molecule. Most often such a molecule will be double stranded over its entire length.
  • the term “antisense strand” or "guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., a target mRNA 5 sequence.
  • region of complementarity refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., a target nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the 10 most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5’- or 3’-terminus of the RNAi agent.
  • RNA agent of the disclosure includes a nucleotide mismatch in the antisense strand.
  • the antisense strand of the double stranded RNA agent of the disclosure includes no more than 4 mismatches with the target mRNA, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the target mRNA.
  • the 20 antisense strand double stranded RNA agent of the disclosure includes no more than 4 mismatches with the sense strand, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the sense strand.
  • a double stranded RNA agent of the disclosure includes a nucleotide mismatch in the sense strand.
  • the sense strand of the double stranded RNA agent of the disclosure includes no more than 4 mismatches with the antisense strand, e.g., the sense strand includes 4, 3, 2, 1, or 25 0 mismatches with the antisense strand.
  • the nucleotide mismatch is, for example, within 5, 4, 3 nucleotides from the 3’-end of the iRNA.
  • the nucleotide mismatch is, for example, in the 3’-terminal nucleotide of the iRNA agent.
  • the mismatch(s) is not in the seed region.
  • an RNAi agent as described herein can contain one or more mismatches to the target 30 sequence.
  • a RNAi agent as described herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein contains no more than 2 mismatches. In one embodiment, an RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, an RNAi agent as described herein contains 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the 35 mismatch can optionally be restricted to be within the last 5 nucleotides from either the 5’- or 3’-end of the region of complementarity.
  • RNAi agent for a 23 nucleotide RNAi agent, the strand which is complementary to a region of a target gene, generally does not contain any mismatch within the central 13 nucleotides.
  • the methods described herein or methods known in the art can be used 27 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of a target gene. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of a target gene is important, especially if the particular region of complementarity i n a target gene is known to vary.
  • sense strand or “passenger strand” as used herein, refers to the strand of a RNAi agent that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.
  • substantially all of the nucleotides are modified are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.
  • cleavage region refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs.
  • the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at 15 the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.
  • the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a 20 duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person.
  • Such conditions can be, for example, “stringent conditions”, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 o C or 70 o C for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other 25 conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
  • Such sequences can be referred to as “fully complementary” with respect to each other herein.
  • first sequence is referred to as “substantially complementary” with respect to a second sequence herein
  • the two sequences can be fully complementary, or they can 35 form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression , in vitro or in vivo.
  • two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such 28 ME1 ⁇ 53466565.v1 Atty.
  • a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for 5 the purposes described herein.
  • “Complementary” sequences can also include, or be formed entirely from, non- Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled.
  • Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogsteen base pairing.
  • a polynucleotide that is “substantially complementary to at least part of” a 15 messenger RNA (mRNA) or target sequence refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest or target sequence (e.g., an mRNA encoding a target gene).
  • mRNA messenger RNA
  • target sequence e.g., an mRNA encoding a target gene.
  • a polynucleotide is complementary to at least a part of a target RNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding a target gene.
  • the antisense strand polynucleotides disclosed herein are fully complementary to the target gene sequence.
  • the antisense polynucleotides disclosed herein are substantially complementary to the target sequence and comprise a contiguous nucleotide sequence which is at least 80% complementary over its entire length to the equivalent region of the nucleotide sequence of the target sequence, such as about 85%, about 90%, about 91%, about 92%, 25 about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.
  • target genes include, for example, adrenoceptor beta 1 (ADRB1); calcium voltage- gated channel subunit alpha1 C (CACNA1C); calcium voltage-gated channel subunit alpha1 G (CACNA1G) (T type calcium cchannel); angiotensin II receptor type 1(AGTR1); Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium 30 Channel 1 (HCN1); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3 (HCN3); Potassium Voltage- Gated Channel Subfamily A Member 5 (KCNA5); Potassium Inwardly Rectifying Channel Subfamily J Member 3 (KCNJ3); Potassium Inwardly Rectifying Channel Subfamily J Member 4 (KCNJ4); phospholamban (PLN); calcium/calmodulin
  • Additional exemplary target genes also include, for example, myostatin (MSTN); Cholinergic Receptor Nicotinic Alpha 1 Subunit (CHRNA1); Cholinergic Receptor Nicotinic Beta 1 Subunit (CHRNB1); Cholinergic Receptor Nicotinic Delta Subunit (CHRND); Cholinergic Receptor Nicotinic 29 ME1 ⁇ 53466565.v1 Atty.
  • MSTN myostatin
  • CHRNA1 Cholinergic Receptor Nicotinic Alpha 1 Subunit
  • CHRNB1 Cholinergic Receptor Nicotinic Beta 1 Subunit
  • CHRND Cholinergic Receptor Nicotinic Delta Subunit
  • CHRNE Cholinergic Receptor Nicotinic Gamma Subunit
  • CHRNG Collagen Type XIII Alpha 1 Chain
  • LRP4 LDL Receptor Related Protein 4
  • MUSK Muscle Associated Receptor Tyrosine Kinase
  • RPSN Receptor Associated Protein Of The Synapse
  • SCN4A Sodium Voltage-Gated Channel Alpha Subunit 4
  • SCN4A Double Homeobox 4
  • DMPK 5 dystrophy myotonic protein kinase
  • GYS1 glycogen synthase 1
  • GYS1 glycogen synthase 1
  • GYS1 glycogen synthase 1
  • GYS1 glycogen synthase 1
  • GYS1 glycogen synthase 1
  • GYS1 glycogen synthase 1
  • GYS1 glycogen synthase 1
  • GYS1 glycogen synthase 1
  • GYS1 glycogen synthase 1
  • GAA glycogen synthas
  • ADRB1 calcium voltage-gated channel subunit alpha1 C
  • CACNA1C calcium voltage-gated channel subunit alpha1 C
  • CACNA1C is also known as calcium channel, voltage-dependent, L type, alpha 1C subunit; voltage-dependent L-type 15 calcium channel subunit alpha-1C; voltage-gated L-type calcium channel Cav1.2 alpha 1 subunit, splice variant 10; calcium channel, L type, alpha-1 polypeptide, isoform 1, cardiac muscle; calcium channel, cardic dihydropyridine-sensitive, alpha-1 subunit; voltage-dependent L-type Ca2+ channel alpha 1 subunit; voltage-gated calcium channel subunit alpha CaV1.2; DHPR, alpha-1 subunit; CACH2, CACN2, CACNL1A1, CCHL1A1, CaV1.2, LQT8, TS, or TS.
  • CACNA1G calcium voltage-gated channel subunit alpha1 G
  • CACNA1G is also known as calcium channel, voltage-dependent, T type, alpha 1G subunit; voltage-dependent T-type calcium channel subunit alpha-1G; voltage-gated calcium channel subunit alpha Cav3.1; NBR13 ; Cav3.1c; Ca(V)T.1; KIAA1123; SCA42ND; or SCA42.
  • angiotensin II receptor type 1 used interchangeably with the term “AGTR1,” refers to a receptor for the vasoconstricting peptide angiotensin II.
  • AGTR1 is also known as angiotensin receptor 1B, AT1, AT2R1, AGTR1A, AT2R1B, AGTR1B, HAT1R, AG2S, AT1B, AT2R1A, AT1AR, AT1BR, or AT1R.
  • SCN2A sodium Voltage-Gated Channel Alpha Subunit 2
  • SCN2A is also known as Nav1.2, HBSCII, SCN2A1, SCN2A2, HBSCI, EIEE11, BFIC3, BFIS3, BFNIS, DEE11, EA9, or HBA.
  • HBN1 Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1
  • HCN1 refers to a member of the hyperpolarization-activated cyclic 35 nucleotide-gated (HCN) channel family.
  • HCN1 is also known as potassium channel 1, BCNG-1, HAC-2, BCNG1, Potassium/Sodium Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 1; Brain Cyclic Nucleotide-Gated Channel 1; Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1; GEFSP10, EIEE24, or DEE24. 30 ME1 ⁇ 53466565.v1 Atty.
  • HCN4 Hydropolarization Activated Cyclic Nucleotide Gated Potassium Channel 4
  • HCN4 refers to a member of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family.
  • HCN4 is also known as Potassium/Sodium Hyperpolarization- Activated Cyclic Nucleotide-Gated Channel 4, Hyperpolarization Activated Cyclic Nucleotide-Gated 5 Potassium Channel 4, Hyperpolarization Activated Cyclic Nucleotide-Gated Cation Channel 4 or SSS2.
  • HCN3 hyperpolarization-activated Cyclic Nucleotide Gated Potassium Channel 3
  • HCN3 refers to a member of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family.
  • HCN3 is also known as Potassium/Sodium Hyperpolarization- Activated Cyclic Nucleotide-Gated Channel 3, Hyperpolarization Activated Cyclic Nucleotide-Gated 10 Potassium Channel 3, or KIAA1535.
  • KCNA5 is also known as HPCN1, HK2, Potassium Voltage-Gated Channel, Shaker-Related Subfamily, Member 5; Voltage-Gated Potassium Channel Subunit Kv1.5; Voltage-Gated Potassium 15 Channel HK2; Kv1.5; Insulinoma And Islet Potassium Channel; Cardiac Potassium Channel; Potassium Channel 1; ATFB7, HCK1 or PCN1.
  • KCNJ3 is also known as GIRK1, G Protein-Activated Inward Rectifier 20 Potassium Channel 1, KGA; Potassium Channel, Inwardly Rectifying Subfamily J Member 3; Inward Rectifier K(+) Channel Kir3.1; or Potassium Inwardly-Rectifying Channel Subfamily J Member 3 Splice Variant 1e.
  • KCNJ4 is also known as HIRK2, HRK1, IRK3, HIR, Kir2.3, inward rectifier potassium channel 4; Inward Rectifier K(+) Channel Kir2.3; Potassium Voltage-Gated Channel Subfamily J Member 4; Hippocampal Inward Rectifier Potassium Channel; or Hippocampal Inward Rectifier.
  • Phosphodiesterase 1 used interchangeably with the term “PDE1,” refers to a 30 member of the cyclic nucleotide phosphodiesterases families.
  • PDE1 is also known as Calcium/Calmodulin-Dependent 3',5'-Cyclic Nucleotide Phosphodiesterase 1; Calcium/Calmodulin- Stimulated Cyclic Nucleotide Phosphodiesterase; CAM-PDE 1, HSPDE1, HCAM1, or EC 3.1.4.
  • myostatin used interchangeably with the term “MSTN,” refers to a secreted ligand of the TGF-beta (transforming growth factor-beta) superfamily of proteins. Myostatin is also 35 known as GDF8, Growth/Differentiation Factor 8, or MSLHP.
  • CHRNA1 Cholinergic Receptor Nicotinic Alpha 1 Subunit
  • AChR muscle acetylcholine receptor
  • CHRNA1 is also known as Cholinergic Receptor, Nicotinic, Alpha Polypeptide 1; Acetylcholine Receptor, 31 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Nicotinic, Alpha 1 (Muscle); ACHRA; CHRNA; Muscle Nicotinic Acetylcholine Receptor; CMS1A, CMS1B, CMS2A, FCCMS, SCCMS, or ACHRD.
  • CHRNB1 Cholinergic Receptor Nicotinic Beta 1 Subunit
  • CHRNB1 is 5 associated with diseases associated such as Myasthenic Syndrome.
  • CHRNB1 is also known as Cholinergic Receptor, Nicotinic, Beta Polypeptide 1; Acetylcholine Receptor, Nicotinic, Beta 1 (Muscle); ACHRB; CHRNB; CMS1D, CMS2C, CMS2A, or SCCMS.
  • CHRND Cholinergic Receptor Nicotinic Delta Subunit
  • CHRND refers to a delta subunit of the muscle acetylcholine receptor (AChR).
  • CHRND is also 10 known as ACHRD, Cholinergic Receptor, Nicotinic, Delta Polypeptide; Acetylcholine Receptor, Nicotinic, Delta (Muscle); CMS2A; CMS3A, CMS3B, CMS3C, FCCMS, or SCCMS.
  • CHRNE Cholinergic Receptor Nicotinic Epsilon Subunit
  • CHRNE is also known as Cholinergic Receptor, Nicotinic, Epsilon; Acetylcholine Receptor, Nicotinic, Epsilon; ACHRE; CMS1D, CMS1E, 15 CMS2A, CMS4A, CMS4B, CMS4C, FCCMS, or SCCMS.
  • “Cholinergic Receptor Nicotinic Gamma Subunit,” used interchangeably with the term “CHRNG,” refers to a subunit of the acetylcholine receptor.
  • CHRNG is also known as Cholinergic Receptor, Nicotinic, Gamma; Acetylcholine Receptor, Nicotinic, Gamma; or ACHRG.
  • Collagen Type XIII Alpha 1 Chain used interchangeably with the term 20 “COL13A1,” refers to a synaptic extracellular-matrix protein involved in the formation and maintenance of the neuromuscular synapse. COL13A1 is also known as COLXIIIA1, Collagen Alpha-1(XIII) Chain, or CMS19.
  • LRP4 LDL Receptor Related Protein 4
  • LRP4 refers to a member of the low-density lipoprotein receptor-related protein family. LRP4 is also known as MEGF7, LRP-4, SOST2, CLSS, Low-Density Lipoprotein Receptor-Related Protein 4, Multiple 30 Epidermal Growth Factor-Like Domains 7, LRP10, KIAA0816, or CMS17.
  • MUSK Muscle Associated Receptor Tyrosine Kinase
  • NMJ neuromuscular junction
  • MUSK is also known as EC 2.7.10.1, FADS1, CMS9, FADS, Muscle, 35 Skeletal Receptor Tyrosine-Protein Kinase, or Muscle-Specific Kinase Receptor.
  • RAPSN Receptor Associated Protein Of The Synapse
  • RAPSN refers to a member of a family of proteins that are receptor associated proteins of the synapse.
  • RAPSN is also known as RNF205, 43 KDa Receptor-Associated Protein Of The Synapse, 32 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO RING Finger Protein 205, CMS1D, CMS1E, Acetylcholine Receptor-Associated 43 Kda Protein, RAPSYN, CMS11, CMS4C, FADS2, or FADS.
  • Sodium Voltage-Gated Channel Alpha Subunit 4 refers to a member of the voltage-gated sodium channel family. SCN4A is also 5 known as SkM1, Nav1.4, HYPP, Sodium Channel Protein Skeletal Muscle Subunit Alpha, Voltage- Gated Sodium Channel Subunit Alpha Nav1.4, HYKPP, Skeletal Muscle Voltage-Dependent Sodium Channel Type IV Alpha Subunit, CTC-264K15.6, Na(V)1.4, HOKPP2, CMS16, or NAC1A.
  • Double Homeobox 4 used interchangeably with the term “DUX4,” refers to a transcriptional activator of many genes.
  • DUX4 is also known as Double Homeobox Protein 10, Double 10 Homeobox Protein 4, Double Homeobox Protein 4/10, DUX4L, and DUX10.
  • phospholamban used interchangeably with the term “PLN,” refers to a crucial regulator of cardiac contractility. PLN is also known as CMD1P, PLB, Cardiac Phospholamban, or CMH.
  • calcium/calmodulin dependent protein kinase II delta used interchangeably 15 with the term “CAMK2D,” refers to a member of the serine/threonine protein kinase family and the Ca(2+)/calmodulin-dependent protein kinase subfamily.
  • CAMK2D is also known as Calcium/Calmodulin-Dependent Protein Kinase Type II Delta Chain, CaM Kinase II Delta Subunit, CaM Kinase II Subunit Delta, CAMKD, EC 2.7.11.17, or EC 2.7.11.
  • DMPK is also known as DM1 protein kinase, DM1PK, DM1, MT-PK, MDPK, DMK, myotonin-protein kinase, myotonic dystrophy associated protein kinase, dystrophia myotonica protein kinase, myotonin protein kinase A, thymopoietin homolog, or EC 2.7.11.1.
  • GYS1 glycogen synthase 1
  • GYS1 refers to an 25 enzyme that catalyzes the addition of glucose monomers to the growing glycogen molecule.
  • GYS1 is also known as muscle glycogen synthase, GSY, GYS, or EC 2.4.1.11.
  • “survival of motor neuron 1,” used interchangeably with the term “SMN1,” refers to one of a group of proteins called the SMN complex, which is important for the maintenance of specialized nerve cells called motor neurons.
  • SMN1 is also known as SMNT, TDRD16A, Gemin-1, 30 BCD541, GEMIN1, SMA1, SMA2, SMA3, SMA4, SMN, SMNT, tudoe domain containing 16A, complement of gems 1, or SMNC.
  • alpha-glucosidase used interchangeably with the term “GAA,” refers to an enzyme responsible for the degradation of glycogen to glucose in lysosomes. GAA is also known as lysosomal alpha-glucosidase, acid maltase, EC 3.2.1.20, glycogen storage disease type II, or LYAG. 35 Examples of target mRNA sequences, and variations thereof (e.g., variants provided in the SNP database) are readily available through publicly available databases, e.g., GenBank, UniProt, OMIM, UCSC Genome Browser, NCBI dbSNP, and the Macaca genome project website. 33 ME1 ⁇ 53466565.v1 Atty.
  • the double-stranded region of a dsRNA agent is equal to or at least, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.
  • the antisense strand of a dsRNA agent is equal to or at least 14, 15, 16, 5 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the sense strand of a dsRNA agent is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the sense and antisense strands of the dsRNA agent are each independently 15 to 30 nucleotides in length. 10 In one embodiment, the sense and antisense strands of the dsRNAagent are each independently 19 to 25 nucleotides in length. In one embodiment, the sense and antisense strands of the dsRNAagent are each independently 21 to 23 nucleotides in length.
  • the sense strand of the dsRNA agent is 21-nucleotides in length
  • the 15 antisense strand is 23-nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs having a 2-nucleotide long single stranded overhangs at the 3’-end.
  • an agent for use in the methods and compositions of the disclosure is a single-stranded antisense nucleic acid molecule that inhibits a target mRNA via an antisense inhibition mechanism.
  • the single-stranded antisense RNA molecule is complementary to a 20 sequence within the target mRNA.
  • the single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355.
  • the single-stranded antisense RNA molecule may be about 15 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence.
  • the single-stranded antisense RNA molecule may comprise a 25 sequence that is at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.
  • At least partial suppression of the expression of a target gene is assessed by a reduction of the amount of target mRNA which can be isolated from or detected in a first cell or group of cells in which a target gene is transcribed and which has or have been treated such that the expression 30 of a target gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).
  • the degree of inhibition may be expressed in terms of: (mRNA in control cells) - (mRNA in treated cells) ( mRNA in control cells) 100% 35
  • inhibition of expression is determined by the dual luciferase method wherein the RNAi agent is present at 10 nM.
  • contacting a cell with an RNAi agent includes contacting a cell by any possible means.
  • Contacting a cell with an RNAi agent includes contacting a cell 34 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO in vitro with the RNAi agent or contacting a cell in vivo with the RNAi agent.
  • the contacting may be done directly or indirectly.
  • the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.
  • an “alpha-v-beta-6 ( ⁇ v ⁇ 6) integrin targeting ligand”, as used herein, includes any moiety (e.g., peptides and small molecules) which bind an ⁇ v ⁇ 6 integrin and are able to mediate delivery of a dsRNA agent to which they are attached to skeletal muscle (e.g., a skeletal muscle cell or skeletal muscle tissue) and/or cardiac muscle (e.g., a cardiac muscle cell or cardiac muscle tissue).
  • the ⁇ v ⁇ 6 integrin targeting 15 ligands bind ⁇ v ⁇ 6 integrin or the ⁇ v ⁇ 6 integrin receptor on skeletal muscle/cardiac myocytes (cells).
  • contacting a cell with an RNAi agent includes “introducing” or “delivering the RNAi agent into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or 20 uptake of a RNAi agent can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing a RNAi agent into a cell may be in vitro or in vivo. For example, for in vivo introduction, a RNAi agent can be injected into a tissue site or administered systemically.
  • a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), or a non-primate (such as a a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse), or a bird that expresses the target gene, either endogenously or heterologously.
  • a primate such as a human, a non-human primate, e.g., a monkey, and a chimpanzee
  • a non-primate such as a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse
  • the subject is a human, such as a human being treated or assessed for a disease, disorder, or condition that would benefit 30 from reduction in target gene expression; a human at risk for a disease, disorder, or condition that would benefit from reduction in target gene expression; a human having a disease, disorder, or condition that would benefit from reduction in target gene expression; or human being treated for a disease, disorder, or condition that would benefit from reduction in target gene expression as described herein.
  • the subject is a female human.
  • the subject is a male human.
  • the subject is an adult subject.
  • the subject is a pediatric subject.
  • treating refers to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more signs or symptoms associated with target gene expression or target gene protein production, e.g., a target gene-associated disease, e.g., 35 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO a muscle disorder, e.g., a skeletal muscle disorder, and/or a cardiac muscle disorder, or symptoms associated with unwanted target gene expression; diminishing the extent of unwanted target activation or stabilization; amelioration or palliation of unwanted target activation or stabilization.
  • a target gene-associated disease e.g., 35 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • a muscle disorder e.g., a skeletal muscle disorder, and/or a cardiac muscle disorder, or symptoms associated with unwanted target gene expression
  • “Lower” in the context of the 10 level of a target gene in a subject is a decrease to a level accepted as within the range of normal for an individual without such disorder.
  • the expression of the target is normalized, i.e., decreased towards or to a level accepted as within the range of normal for an individual without such disorder, e.g., blood glucose level, blood uric acid level, blood lipid level, blood oxygen level, white blood cell count, kidney function, spleen function, liver function.
  • “lower” in a subject can 15 refer to lowering of gene expression or protein production in a cell in a subject does not require lowering of expression in all cells or tissues of a subject.
  • lowering in a subject can include lowering of gene expression or protein production in a subject.
  • the term “lower” can also be used in association with normalizing a symptom of a disease or condition, i.e. decreasing the difference between a level in a subject suffering from a target gene- 20 associated disease towards or to a level in a normal subject not suffering from a target gene-associated disease.
  • a disease is associated with an elevated value for a symptom
  • “normal” is considered to be the upper limit of normal.
  • a disease is associated with a decreased value for a symptom
  • “normal” is considered to be the lower limit of normal.
  • prevention when used in reference to a disease, disorder, or 25 condition thereof, that would benefit from a reduction in expression of a target gene or production of a target protein, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of a target gene-associated disease.
  • the failure to develop a disease, disorder, or condition, or the reduction in the development of a symptom associated with such a disease, disorder, or condition e.g., by at least about 10% on a clinically accepted 30 scale for that disease or disorder
  • the exhibition of delayed symptoms delayed e.g., by days, weeks, months or years
  • target gene-associated disease is a disease or disorder that would benefit from reduction in the expression or activity of the target gene.
  • target gene-associated disease is a disease or disorder that is caused by, or associated with expression or protein production of 35 the target gene.
  • target gene-associated disease includes a disease, disorder or condition that would benefit from a decrease in expression or protein activity of the target gene. Additional information regarding specific target genes and disease that would benefit from reduction in expression of the target gene are descrived below. 36 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, the target gene-associated disease is a muscle disorder.
  • Exemplary muscle disorders include Myostatin-related muscle hypertrophy, congenital myasthenic syndrome, facioscapulohumeral muscular dystrophy (FSHD), Spinal Muscular Atrophy (SMA), Myotonic Dystrophy Type 1 (DM1), Pompe disease, PLN cardiomyopathy, spasticity, 5 obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic cardiomyopathy (FHC); Heart failure with preserved ejection fraction (HFPEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina; myocardial infarction (MI); heart failure or heart failure with reduced ejection fraction (HFREF); supraventricular tachycardia (SVT); hypertrophic cardiomyopathy (HCM); and PLN cardiomyopathy.
  • FSHD facioscapulohumeral muscular dystrophy
  • SMA Spinal Muscular Atrophy
  • DM1 Myotonic Dystrophy Type 1
  • HOCM familial hypertrophic
  • the target gene-associated disease is a skeletal muscle disease or disorder.
  • the target gene-associated disease is a cardiac muscle disease or disorder.
  • Exemplary cardiac muscle disorders include obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic cardiomyopathy (FHC); Heart failure with preserved ejection fraction (HFPEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina; myocardial infarction (MI); heart failure 15 or heart failure with reduced ejection fraction (HFREF); supraventricular tachycardia (SVT); hypertrophic cardiomyopathy (HCM); and PLN cardiomyopathy.
  • HOCM obstructive hypertrophic cardiomyopathy
  • FHC familial hypertrophic cardiomyopathy
  • HPF Heart failure with preserved ejection fraction
  • AFIB atrial fibrillation
  • VFIB ventricular fibrillation
  • MI myocardial infarction
  • HVF supraventricular tachycardia
  • HCM hypertrophic cardiomyopathy
  • Heart failure (“HF”) or “congestive heart failure” (“CHF”) is a chronic condition in which the heart does not pump blood as well as it should. Heart failure can occur when the heart’s capacity to pump blood cannot keep up with the body’s need. Heart failure can occur if the heart cannot pump 20 (systolic) or fill (diastolic) adequately.
  • congestive refers to the resulting buildup of fluid in the ankles and feet, arms, lungs, and/or other organs.
  • HF-pEF heart failure with preserved left ventricular function
  • HF-pEF heart failure with preserved ejection fraction
  • a common cause of congestive heart failure is coronary artery disease. Risk factors for coronary artery disease can include high levels of cholesterol and/or triglyceride, high blood pressure, poor diet, a sedentary lifestyle, diabetes, smoking, being overweight or obese, and stress.
  • coronary artery disease several other conditions can damage the heart muscles, including inherited and genetic factors, some infections and autoimmune diseases and some treatments such as chemotherapy.
  • HCM Hemasarcoma
  • HOCM Hemasar cardiomyopathy
  • the thickened septum may cause a narrowing that can block or reduce the blood flow from the left ventricle to the aorta, which is a condition called “outflow tract obstruction.”
  • Both HCM and HOCM can be caused by heart muscle gene mutation, which may be inherited.
  • 37 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO “Familial hypertrophic cardiomyopathy” is an autosomal dominant disease characterized mainly by left ventricular hypertrophy. Thickening usually occurs in the interventricular septum.
  • Familial hypertrophic cardiomyopathy can cause abnormal heart rhythms (arrhythmias) that can be life threatening. People with familial hypertrophic cardiomyopathy have an increased risk of sudden death, even if they have no other symptoms of the condition. A small number of affected individuals develop potentially fatal heart failure, which may require heart transplantation. 10 Mutations in one of several genes can cause familial hypertrophic cardiomyopathy; the most commonly involved genes are MYH7, MYBPC3, TNNT2, and TNNI3.
  • AFIB cardiac fibrillation
  • VFIB cardiac fibrillation
  • Ventricular fibrillation is an emergency that requires immediate medical attention.
  • a “myocardial infarction” or “MI” occurs when the flow of blood to the heart is blocked.
  • the blockage can be a buildup of fat, cholesterol and other substances, which form a plaque in the arteries that feed the heart (coronary arteries).
  • “Supraventricular tachycardia” (“SVT”) is as an abnormally fast or erratic heartbeat that affects 25 the heart's atria. During an episode of SVT, the heart beats about 150 to 220 times per minute, but it can occasionally beat faster or slower.
  • HCM Hemophilic cardiomyopathy
  • Angina is a type of chest pain caused by reduced blood flow to the heart.
  • Angina is a symptom of coronary artery disease. 38 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Angina, also called angina pectoris, can be described as squeezing, pressure, heaviness, tightness or pain in the chest.
  • Phospholamban (PLN) cardiomyopathy refers to a specific subtype of hereditary cardiomyopathy caused by PLN p.(Arg14del), a pathogenic variant in the gene encoding PLN, which is a 5 protein with a central role in calcium homeostasis in cardiac tissue. This protein ensures proper contraction and relaxation of the human heart.
  • Carriers of this pathogenic variant have a high risk of developing dilated cardiomyopathy (DCM), arrhythmic cardiomyopathy (ACM), or both. They commonly have a high arrhythmic burden, with premature ventricular contractions (PVCs) and ventricular tachycardia, remarkable low-voltage electrocardiograms (ECGs), left ventricular dysfunction, 10 and a positive family history for sudden cardiac death.
  • PLN p.(Arg14del) cardiomyopathy has been found in several European countries, but also in the United States, Canada, and China. On a global scale it is a rare disease, but it is particularly common in The Netherlands, with the pathogenic variant being present in 12% of all ACM patients and 15% of all DCM patients.
  • Exemplary skeletal muscle disorders include Myostatin-related muscle hypertrophy, congenital 15 myasthenic syndrome, facioscapulohumeral muscular dystrophy (FSHD), Spinal Muscular Atrophy (SMA), Myotonic Dystrophy Type 1 (DM1), Pompe disease, PLN cardiomyopathy, and spasticity.
  • Myostatin-related muscle hypertrophy is a rare condition characterized by reduced body fat and increased muscle size. Affected individuals have up to twice the usual amount of muscle mass in their bodies. They also tend to have increased muscle strength.
  • Myostatin-related muscle hypertrophy is 20 caused by mutations in the MSTN gene. It follows an incomplete autosomal dominant pattern of inheritance.
  • CMS Congenital myasthenic syndromes
  • endplate myopathies e.g., CHRNA1, 25 CHRNB1, CHRBD, CHRNE, CHRNG, COL13A1, DOX7, LRP4, MUSK, RAPSN, or SCN4A.
  • CMS are clinically characterised by abnormal fatigability, or transient or permanent weakness of extra-ocular, facial, bulbar, truncal, respiratory, or limb muscles.
  • Onset of endplate myopathy is intrauterine, congenital, in infancy, or childhood, and rarely in adolescence.
  • FSHD Facioscapulohumeral muscular dystrophy
  • FSHD typically presents before age 20 years with weakness of the facial muscles and the stabilizers of the scapula or the dorsiflexors of the foot. There is extreme clinical variability. In some cases, congenital facial weakness may be present. In FSHD, the muscle weakness is 39 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO slowly progressive and approximately 20% of affected individuals eventually require a wheelchair. Life expectancy is not shortened. The incidence is approximately 4 individuals affected per 100,000 people.
  • Spinal muscular atrophy refers to a genetic disorder characterized by weakness and wasting (atrophy) in muscles used for movement (skeletal muscles).
  • spinal muscular atrophy There are many types of spinal muscular atrophy that are caused by changes in the same genes. The types differ in age of onset and severity of muscle weakness; however, there is overlap between the types. Other forms of spinal 10 muscular atrophy and related motor neuron diseases, such as spinal muscular atrophy with progressive myoclonic epilepsy, spinal muscular atrophy with lower extremity predominance, X-linked infantile spinal muscular atrophy, and spinal muscular atrophy with respiratory distress type 1 are caused by mutations in other genes.
  • SMN1 and SMN2 genes both provide instructions for making a protein called the survival motor neuron (SMN) protein. Normally, most functional SMN protein is produced from the SMN1 gene, with a small amount produced from the SMN2 gene. Several different versions of the SMN protein are produced from the SMN2 gene, but only one version is functional; the other versions are 20 smaller and quickly broken down.
  • the SMN protein is one of a group of proteins called the SMN complex, which is important for the maintenance of motor neurons.
  • Myotonic dystrophy refers to a part of a group of inherited disorders called muscular dystrophies. It is the most common form of muscular dystrophy that begins in adulthood. 25 Myotonic dystrophy is characterized by progressive muscle wasting and weakness. People with this disorder often have prolonged muscle contractions (myotonia) and are not able to relax certain muscles after use. Other signs and symptoms of myotonic dystrophy include clouding of the lens of the eye (cataracts) and abnormalities of the electrical signals that control the heartbeat (cardiac conduction defects).
  • myotonic dystrophy often develop during a person's twenties or thirties, although they can occur at any age. The severity of the condition varies widely among affected people, even among members of the same family. There are two major types of myotonic dystrophy: type 1 and type 2. Their signs and symptoms overlap, although type 2 tends to be milder than type 1. The muscle weakness associated with type 1 particularly affects muscles farthest from the center 35 of the body (distal muscles), such as those of the lower legs, hands, neck, and face.
  • Muscle weakness in type 2 primarily involves muscles close to the center of the body (proximal muscles), such as the those of the neck, shoulders, elbows, and hips.
  • the two types of myotonic dystrophy are caused by mutations in different genes. 40 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • Myotonic dystrophy type 1 is caused by mutations in the DMPK gene, while type 2 results from mutations in the CNBP gene.
  • the protein produced from the DMPK gene likely plays a role in communication within cells. It appears to be important for the correct functioning of cells in the heart, brain, and skeletal muscles (which are used for movement).
  • the protein produced from the CNBP gene is 5 found primarily in the heart and in skeletal muscles, where it helps regulate the function of other genes. Similar changes in the structure of the DMPK and CNBP genes cause myotonic dystrophy type 1 and type 2. In each case, a segment of DNA is abnormally repeated many times, forming an unstable region in the gene. The gene with the abnormal segment produces an unusually long messenger RNA, which is a molecular blueprint of the gene that guides the production of proteins. The unusually long messenger 10 RNA forms clumps inside the cell that interfere with the production of many other proteins. These changes prevent muscle cells and cells in other tissues from functioning normally, which leads to the signs and symptoms of myotonic dystrophy.
  • Pompe disease refers to an inherited disorder caused by the buildup of a complex 15 sugar called glycogen in the body's cells. The accumulation of glycogen in certain organs and tissues, especially muscles, impairs their ability to function normally. There are three types of Pompe disease, which differ in severity and the age at which they appear. These types are known as classic infantile- onset, non-classic infantile-onset, and late-onset. The classic form of infantile-onset Pompe disease begins within a few months of birth.
  • Pompe disease infants with this disorder typically experience muscle weakness 20 (myopathy), poor muscle tone (hypotonia), an enlarged liver (hepatomegaly), and heart defects. Affected infants may also fail to gain weight and grow at the expected rate (failure to thrive) and have breathing problems. If untreated, this form of Pompe disease leads to death from heart failure in the first year of life.
  • the non-classic form of infantile-onset Pompe disease usually appears by age 1. It is characterized by delayed motor skills (such as rolling over and sitting) and progressive muscle weakness.
  • the heart 25 may be abnormally large (cardiomegaly), but affected individuals usually do not experience heart failure. The muscle weakness in this disorder leads to serious breathing problems, and most children with non- classic infantile-onset Pompe disease live only into early childhood.
  • the late-onset type of Pompe disease may not become apparent until later in childhood, adolescence, or adulthood. Late-onset Pompe disease is usually milder than the infantile-onset forms of this disorder and is less likely to involve the 30 heart. Most individuals with late-onset Pompe disease experience progressive muscle weakness, especially in the legs and the trunk, including the muscles that control breathing. As the disorder progresses, breathing problems can lead to respiratory failure. Mutations in the GAA gene cause Pompe disease.
  • the GAA gene provides instructions for producing an enzyme called acid alpha-glucosidase (also known as acid maltase). This enzyme is active 35 in lysosomes, which are structures that serve as recycling centers within cells. The enzyme normally breaks down glycogen into a simpler sugar glucose, which is the main energy source for most cells.
  • Spasticity refers to a condition in which muscles stiffen or tighten, preventing normal fluid movement. The muscles remain contracted and resist being stretched, thus affecting 5 movement, speech and gait. Spasticity is generally caused by damage or disruption to the area of the brain and spinal cord that are responsible for controlling muscle and stretch reflexes.
  • Therapeutically effective amount is intended to include the amount of an RNAi agent that, when administered to a subject having a target gene-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease or one or more symptoms of disease).
  • the "therapeutically effective amount” may vary depending on the RNAi 15 agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.
  • “Prophylactically effective amount,” as used herein, is intended to include the amount of a RNAi agent that, when administered to a subject having a target gene-associated disorder, e.g., gout or diabetes, 20 is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease.
  • the “prophylactically effective amount” may vary depending on the RNAi agent, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of 25 the patient to be treated.
  • a “therapeutically-effective amount” or “prophylacticaly effective amount” also includes an amount of a RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment.
  • a RNAi agent employed in the methods of the present disclosure may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such 30 treatment.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable 35 benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent 42 ME1 ⁇ 53466565.v1 Atty.
  • materials which can serve as pharmaceutically-acceptable carriers 5 include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and 10 soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate
  • RNAi agents for pulmonary delivery are known in the art and will vary depending on the desired location for deposition of the agent, e.g., upper or lower respiratory system, and the type of device to be used for delivery, e.g., sprayer, nebulizer, dry powder inhaler.
  • “Pharmaceutically acceptable salts” of each of RNAi agents herein include, but are not limited to, a sodium salt, a calcium salt, a lithium salt, a potassium salt, an ammonium salt, a magnesium salt, an mixtures thereof.
  • RNAi agent when provided as a polycationic salt having one cation per free acid group of the optionally modified phosophodiester backbone and/or any other acidic modifications (e.g., 5’-terminal phosphonate groups).
  • an 25 oligonucleotide of “n” nucleotides in length contains n-1 optionally modified phosophodiesters, so that an oligonucleotide of 21 nt in length may be provided as a salt having up to 20 cations (e.g., 20 sodium cations).
  • an RNAi agentshaving a sense strand of 21 nt in length and an antisense strand of 23 nt in length may be provided as a salt having up to 42 cations (e.g., 42 sodium cations).
  • the RNAi agent may be provided as a salt having up to 44 cations (e.g., 44 sodium cations).
  • the term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject.
  • Tissue samples may include samples from tissues, 35 organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. 43 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO II.
  • dsRNA agent provides dual conjugated dsRNA agents for inhibiting expression of a target gene.
  • a dsRNA agent comprises an antisense strand and a sense strand; at 5 least one ⁇ v ⁇ 6 integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least one strand; and at least one in vivo delivery enhancing moiety conjugated to at least one strand.
  • the at least one in vivo delivery enhancing moiety may be conjugated to any internal position of the antisense strand or the sense strand. In some embodiments, the at least one in vivo delivery enhancing moiety may be conjugated to a position that is not an external position of the antisense strand 10 or the sense strand.
  • the at least one in vivo delivery enhancing moiety may be conjugated to an external positon of the antisense strand or the sense strand.
  • the term “in vivo delivery enhancing moiety” refers to a moiety which, when conjugated to a dsRNA agent, enhances delivery of the dsRNA agent to a target tissue, e.g., muscle, or a cell type, such as a muscle cell (e.g., a skeletal muscle cell or a cardiac muscle cell).
  • the in vivo delivery enhancing moiety has pharmacokinetic (PK) enhancing properties, such as increasing the residence time in the blood of the dsRNA agent, and/or increasing tissue PK, including increasing tissue exposure of the dsRNA agent.
  • PK pharmacokinetic
  • the in vivo delivery enhancing moiety can have a cooperative or synergistic effect with an ⁇ v ⁇ 6 compound in increasing tissue exposure when both elements are conjugated to the dsRNA agent.
  • the in vivo delivery enhancing moiety is lipophilic.
  • the in vivo enhancing moiety increases lipophilicity of the dsRNA agent and provides optimal hydrophobicity for the enhanced in vivo delivery of dsRNA to muscle tissue, e.g., skeletal muscle tissue or cardiac muscle tissue.
  • muscle tissue e.g., skeletal muscle tissue or cardiac muscle tissue.
  • One way to characterize lipophilicity is by the octanol-water partition coefficient, logKow, where Kow is the ratio of a chemical’s concentration in the octanol-phase to its concentration in the aqueous phase of a two-phase system at equilibrium.
  • the octanol-water partition coefficient is a laboratory- measured property of a substance.
  • a chemical substance is lipophilic in character when its logK ow exceeds 0.
  • the lipophilic moiety possesses a logK ow exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, 35 exceeding 5, or exceeding 10.
  • the logK ow of 6-amino hexanol is predicted to 44 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO be approximately 0.7.
  • the logK ow of cholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7.
  • the lipophilicity of a molecule can change with respect to the functional group it carries. For instance, adding a hydroxyl group or amine group to the end of a hydrocarbon chain, e.g., a C 22 5 hydrocarbon chain, can increase or decrease the partition coefficient (e.g., logK ow ) value of the hydrocarbon chain.
  • the hydrophobicity of the dsRNA agent conjugated to at least one in vivo delivery enhancing moiety can be measured by its protein binding characteristics.
  • the unbound fraction in the plasma protein binding assay of the dsRNA agent can be determined to positively correlate 10 to the relative hydrophobicity of the dsRNA agent, which can positively correlate to the silencing activity of the dsRNA agent.
  • the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein.
  • the hydrophobicity of the dsRNA agent measured by fraction of unbound dsRNA agent in the binding assay, exceeds 0.15, 15 exceeds 0.2, exceeds 0.25, exceeds 0.3, exceeds 0.35, exceeds 0.4, exceeds 0.45, or exceeds 0.5 for an enhanced in vivo delivery of dsRNA agent.
  • the at least one in vivo delivery enhancing agent may be attached to the dsRNA agent of the disclosure by any method known in the art, including via a functional grouping already present in the in vivo delivery enhancing moiety or introduced into the dsRNA agent, such as a hydroxy group (e.g., — 20 CO—CH2—OH).
  • the functional groups already present in the in vivo delivery enhancing moiety or introduced into the dsRNA agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. Conjugation of the dsRNA agent and the in vivo delivery enhancing moiety may occur, for example, through formation of an ether or a carboxylic or carbamoyl ester linkage between the hydroxy 25 and an alkyl group R—, an alkanoyl group RCO— or a substituted carbamoyl group RNHCO—.
  • the alkyl group R may be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chained or branched; and saturated or unsaturated).
  • Alkyl group R may be a butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl group, or the like.
  • more than one in vivo delivery enhancing moiety can be conjugated to 30 the dsRNA agent of the disclosure.
  • two or more in vivo delivery enhancing moieties are conjugated to the same strand of the dsRNA agent.
  • each strand of the dsRNA agent is conjugated to one or more in vivo delivery enhancing moieties.
  • two or more in vivo delivery enhancing moieties are conjugated to the same position (i.e., the same nucleobase, same sugar moiety, or same internucleosidic linkage) of the dsRNA agent.
  • the in vivo delivery enhancing moiety may comprise at least one C 10 -C 26 hydrocarbon chain, e.g., a C 10 hydrocarbon chain, a C 11 hydrocarbon chain, a C 12 hydrocarbon chain, a 5 C 13 hydrocarbon chain, a C 14 hydrocarbon chain, a C 15 hydrocarbon chain, a C 16 hydrocarbon chain, a C 17 hydrocarbon chain, a C 18 hydrocarbon chain, a C 19 hydrocarbon chain, a C 20 hydrocarbon chain, a C 21 hydrocarbon chain, a C 22 hydrocarbon chain, a C 23 hydrocarbon chain, a C 24 hydrocarbon chain, a C 25 hydrocarbon chain or a C 26 hydrocarbon chain.
  • a C 10 hydrocarbon chain e.g., a C 10 hydrocarbon chain, a C 11 hydrocarbon chain, a C 12 hydrocarbon chain, a 5 C 13 hydrocarbon chain, a C 14 hydrocarbon chain, a C 15 hydrocarbon chain, a C 16 hydrocarbon chain, a C 17 hydrocarbon chain, a C 18 hydrocarbon
  • the C 10 -C 26 hydrocarbon chain may be a straight hydrocarbon chain. In other embodiments, the C 10 -C 26 hydrocarbon chain may be a 10 branched hydrocarbon chain. In some embodiments, the C 10 -C 26 hydrocarbon chain may be a saturated hydrocarbon chain. In other embodiments, the C 10 -C 26 hydrocarbon chain may be an unsaturated hydrocarbon chain, e.g., comprising one or more double bonds and/or one or more triple bonds. In one embodiment, the in vivo delivery enhancing moiety may comprise at least one C 11 hydrocarbon chain. In one embodiment, the at least one in vivo delivery enhancing moiety may comprise 15 at least one C 17 hydrocarbon chain.
  • the in vivo delivery enhancing moiety may comprise at least one C 22 hydrocarbon chain.
  • the C 10 -C 26 hydrocarbon chain may be unsubstituted.
  • the C 10 -C 26 hydrocarbon chain may be substituted with at least one functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, 20 and alkyne.
  • the C 10 -C 26 hydrocarbon chain is substituted with a carboxylic acid group.
  • the C 10 -C 26 hydrocarbon chain is represented by –(CH 2 ) n -, wherein n is a number from 10 to 26, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26; and — is a bond connecting the C 10 -C 26 hydrocarbon chain to other portions of the in vivo delivery enhancing 25 moiety.
  • the C 10 -C 26 hydrocarbon chain is connected at both ends to the remainder of the in vivo delivery enhancing moiety.
  • the C 10 -C 26 hydrocarbon chain is represented by the following structure: – linker-(CH 2 ) n (CH 3 ), wherein n is a number from 10 to 25, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25; the linker is as described hereinbelow; and – is a bond connecting the C 10 -C 26 30 hydrocarbon chain to other portions of the in vivo delivery enhancing moiety.
  • the C 10 -C 26 hydrocarbon chain is connected at one end to other portions of the in vivo delivery enhancing moiety.
  • the C 10 -C 26 hydrocarbon chain is represented by the following structure: – linker-(CH 2 ) n -A, wherein n is a number from 10 to 26, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35 22, 23, 24, 25 or 26; the linker as described herein below and A is a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne; or A1 or A2 with a structure shown below: 46 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, A is carboxylic acid.
  • A is A1. In one embodiment, A is 5 A2.
  • Exemplary in vivo delivery enhancing moieties useful in the context of the present disclosure are described, e.g., in WO 2023/064530, the entire contents of which are incorporated herein by reference.
  • Lipophilic Moiety As noted above, the in vivo delivery enhancing moietycomprises a lipophilic moiety, such as at 10 least one C 10 -C 26 hydrocarbon chain. In some embodiments, the in vivo delivery enhancing moiety comprises a C 22 hydrocarbon chain, e.g., one or more C 22 hydrocarbon chains.
  • the C 22 hydrocarbon chain is a C 22 acid, e.g., a C 22 acid selected from the group consisting of docosanoic acid, 6-octyltetradecanoic acid, 10-hexylhexadecanoic acid, all-cis-7,10,13,16,19-docosapentaenoic acid, all-cis-4,7,10,13,16,19-docosahexaenoic acid, all-cis-13,16-docosadienoic acid, all-cis-7,10,13,16- 15 docosatetraenoic acid, all-cis-4,7,10,13,16-docosapentaenoic acid, and cis-13-docosenoic acid.
  • a C 22 acid selected from the group consisting of docosanoic acid, 6-octyltetradecanoic acid, 10-hexylhexadecanoic acid, all-c
  • the C22 hydrocarbon chain is a C22 alcohol, e.g. the C22 alcohol selected from the group consisting of 1-docosanol, 6-octyltetradecan-1-ol, 10-hexylhexadecan-1-ol, cis-13-docosen-1- ol, docosan-9-ol, docosan-2-ol, docosan-10-ol, docosan-11-ol, and cis-4,7,10,13,16,19-docosahexanol.
  • the C22 alcohol selected from the group consisting of 1-docosanol, 6-octyltetradecan-1-ol, 10-hexylhexadecan-1-ol, cis-13-docosen-1- ol, docosan-9-ol, docosan-2-ol, docosan-10-ol, docosan-11-ol, and cis-4,7,
  • the C 22 hydrocarbon chain is a C 22 amide, e.g., the C 22 amide selected from the group consisting of (E)-Docos-4-enamide, (E)-Docos-5-enamide, (Z)-Docos-9-enamide, (E)-Docos- 11-enamide,12-Docosenamide, (Z)-Docos-13-enamide, (Z)-N-Hydroxy-13-docoseneamide, (E)-Docos- 14-enamide, 6-cis-Docosenamide, 14-Docosenamide Docos-11-enamide, (4E,13E)-Docosa-4,13- 25 dienamide, and (5E,13E)-Docosa-5,13-dienamide.
  • the C 22 amide selected from the group consisting of (E)-Docos-4-enamide, (E)-Docos-5-enamide, (Z)-Docos-9-enamide, (E)-Do
  • the C 22 hydrocarbon chain includes, but are not limited to, 47 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO docosan-2-yl, docosan-3-yl, docosan-4-yl, docosan-5-yl, docosan-6-yl, docosan-7-yl, docosan-8-yl, docosan-9-yl, docosan-10-yl, docosan-11-yl, 2-(decyl)dodecan-1-yl, 2-(nonyl)tridecan-1-yl, 2- (octyl)tetradecan-1-yl, 2-(heptyl)pentadecan-1-yl, 2-(hexyl)hexadecan-1-yl, 2-(pentyl)heptadecan-1-yl, 2- (butyl)octadecan-1-yl
  • the at least one C10-C26 hydrocarbon chain comprised in the in vivo delivery enhancing moiety may be conjugated to the remainder of the dsRNA agent via a linker or via a 20 carrier or via an internucleotide phosphate linkage.
  • the at least one C10-C26 hydrocarbon chain is conjugated to the remainder of the dsRNA agent via a linker.
  • the linker may be a cleavable linker, i.e., the linker comprises a portion that can be cleavable chemically or enzymatically. For example, amide linkages can be enzymatically cleavable.
  • the linker comprises —(CH2)n-NH-C(O)- or –(CH2)n-NH-C(O)-(CH2)2-C(COOH)- 25 NH-C(O)-, wherein n is a number from 1 to 20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
  • the linker comprises –(CH)2-O-(CH2CH2)-(O)-(CH2CH2)-NH-C(O)-.
  • the in vivo delivery enhancing moiety comprises a naturally occurring lipophilic compound such as a steroid (e.g., cholesterol), Vitamin E, retinol, or retinoic acid.
  • the linker may comprise a group selected from the group consisting of an 30 ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, and a carbamate.
  • the linker may be selected from the group consisting of -(CH 2 )nNH-; -C(O)(CH 2 ) n NH-; - NR’’’’(CH2)nNH-, -C(O)-(CH 2 ) n -C(O)-; -C(O)-(CH 2 ) n -C(O)O-; -C(O)-O-; -C(O)-(CH 2 ) n -NH-C(O)-; - C(O)-(CH2) n -; -C(O)-NH-; -C(O)-; -(CH 2 ) n -C(O)-; -(CH 2 ) n -C(O)O-; -(CH 2 ) n -C(O)O-; -(CH 2 ) n -; and -(CH 2 ) n -NH-C(O)-; 35 where
  • n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19 or 20.
  • the ⁇ v ⁇ 6 integrin targeting ligand is linked to the remainder of the dsRNA agent as shown in the exemplary schematics below, wherein “2’-N6” refers to attachment of a lipophilic 5 moiety (e.g., C 22 ) at the 2’ position on the nucleotide at position 6 on the sense strand (counting from the 5’ end): 10
  • the in vivo delivery enhancing moiety that may be conjugated to the dsRNA agent of the disclosure is represented by the following structure: , 49 ME1 ⁇ 53466565.v1 Atty.
  • the in vivo delivery enhancing moiety may be attached to the remainder of the dsRNA agent at a 2’ position on an internal nucleotide. 50 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • the at least one in vivo delivery enhancing moiety may be conjugated to the dsRNA agent via a direct attachment to the ribosugar of the dsRNA agent, e.g., an antisense strand or a sense strand.
  • the at least one in vivo delivery enhancing moiety may be conjugated to the dsRNA agent via a linker or a carrier.
  • at least one in vivo delivery enhancing moiety may be conjugated to the iRNA agent via one or more linkers (tethers).
  • the at least one in vivo delivery enhancing moiety is conjugated to the dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide- thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole 10 from the azide-alkyne cycloaddition), or carbamate.
  • a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide- thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole 10 from the azide-alkyne cycloaddition), or carbamate.
  • the in vivo delivery enhancing moiety and the targeting moiety are independently present within: (a) an internally-modified nucleosides such as, 15 (b) a modified internucleotide linkage such as, -OP(Y)(X)O-, wherein Y is O or S (e.g., O), and (c) a 5’-terminal modification such as 20 P(Y’)(OH)-O-; or (ii) -P(Y)(OH)O-R L3 or -C(O)N(H)R L3 , wherein Y is O or S; and (iii) -R L3 , -C(O)R L3 , -C(O)N(H)R L3 , -S(O)2R L3 , -S(O)2N(H)R L3 or (d) a 3’-terminal modification such as 51 ME1 ⁇ 53466565.v1 Atty.
  • R 2 ’ or R 3 ’groups include, but are not limited to, hydrogen, halogen (e.g., 2’-fluoro), hydroxy, 2’-O-alkyl (e.g., 2’-OMethyl), 2’-O-methoxyalkyl (e.g., 2’-O-methoxymethyl, 2’-O- methoxyethyl, or 2’-O-2-methoxypropanyl) modification, 2’-O-allyl modification, 2’-C-allyl modification, 2'-O-N-methylacetamido (2'-O-NMA, i.e.
  • halogen e.g., 2’-fluoro
  • hydroxy hydroxy
  • 2’-O-alkyl e.g., 2’-OMethyl
  • 2’-O-methoxyalkyl e.g., 2’-O-methoxymethyl, 2’-O- methoxyethyl, or 2
  • R 2 ’ or R 3 ’ may be H, OH, F, OMe, O-methoxyalkyl, O-allyl, O-N-methylacetamido, O-dimethylaminoethoxyethyl, or O- aminopropyl.
  • R L1 , R L2 and R L3 are each a group containing a lipophilic moiety, such as a C 10-26 saturated or unsaturated hydrocarbon chain. In one embodiment, R L1 , R L2 and R L3 are each a group 20 containing a C 12-26 saturated or unsaturated hydrocarbon chain. In one embodiment, R L1 , R L2 and R L3 are each a group containing a C 12-24 saturated or unsaturated hydrocarbon chain. In one embodiment, R L1 , R L2 and R L3 are each a group containing a C 14-24 saturated or unsaturated hydrocarbon chain.
  • R L1 , R L2 and R L3 are each a group containing a C14-18 saturated or unsaturated hydrocarbon chain. In one embodiment, R L1 , R L2 and R L3 are each a group containing a C16 saturated or unsaturated 25 hydrocarbon chain. In one embodiment, R L1 , R L2 and R L3 are each a group containing a saturated or unsaturated C22-hydrocarbon chain. In other embodiments R L1 , R L2 and R L3 are each a group containing a lipophilic moiety, such as a lipophilic vitamin or steroid, including, but not limited to, Vitamin E, Vitamin A (retinol, retinoic acid), and cholesterol.
  • a lipophilic moiety such as a lipophilic vitamin or steroid, including, but not limited to, Vitamin E, Vitamin A (retinol, retinoic acid), and cholesterol.
  • R L3 when R L3 comprises a lipophilic moiety, then R L3 can be selected from the group consisting of: 52 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein integer m is 0-10 (e.g., 0; or 1-10 or 1-8; or 0-6; or 1; or 2; or 3; or 4; or 5; or 6; or 7; or 8); integer n is 1-21 (e.g., 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2; or 2 or 3 or 4 or 5 or 6); 5 W is C1-C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or t-butyl); R and R’ are each independently H or C1-C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl,
  • R L3 examples include, but are not limited to the following structures: 53 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 54 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Other examples of R L3 include, but are not limited to the following structures: Further examples of R L3 include, but are not limited to the following structures: 55 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO .
  • R L1 can be selected from the group consisting of -G 1 and - S(O)2G1.
  • Examples of R L1 include, but are not limited to, the following structures: Further examples of R L1 include, but are not limited to, the following structures: 5
  • R L2 is -C(O)R L3 , wherein R L3 is according to any of the preceding embodiments thereof.
  • R L2 can be selected from the group consisting of the following structures: 56 ME1 ⁇ 53466565.v1 Atty.
  • integer m is 0-8 (for instance, m is 0; or m is 1-8; or m is 0-6; or m is 1; or 2; or 3; or 4; or 5; or 6; or 7; or 8); integer n is 1-21 (for instance, 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2; or 2 or 3 or 4 or 5 or 6); 5 R and R’ are each independently H or an alkyl group such as a C1-C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl); G is G1 or a saturated or unsaturated C10-26 saturated or unsaturated hydrocarbon chain (e.g.,a C21 hydrocarbon chain,i.e., G together with the carbonyl to which it is attached may form a group with 22 carbons); for instance, G may be a linear or branched C21 al
  • R L2 is a nucleobase modified with a G or G1 group, wherein G and G 1 are 5 as defined above (e.g., a pyrimidine nucleobase modified at the 5’-position with a group comprising G or G1).
  • B1 include, wherein t is selected from 0 – 20 (e.g., 1-12, or 1-10, or 3-12, or 3-10).
  • in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: 10 wherein: B is an optionally modified nucleobase; 58 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO G 3 is a saturated or unsaturated C 1-20 hydrocarbon group (e.g., C 1-6 alkylene; C 2-6 alkylene; or hexylene); L K is a linking group such as -O-, -N(H)-, -S-, -S-S-, -C(O)O-, OC(O)-, -C(O)N(H)-, -N(H)C(O), -OC(O)N(H)-, -N(H)C(O)O-, -S(O) 2 -, -S(O) 2 O-, -S(O) 2 N(H)-, -P(O)
  • G2 when L K contains a carbonyl attached to G2 (e.g., (-N(H)C(O)- or -OC(O)-), 10 then G2 is a C21 hydrocarbon group. In another embodiment, when L K does not contain a carbonyl attached to G2, then G2 is a C22 hydrocarbon group.
  • R G is hydrogen. In another embodiment, R G is OH, In one embodiment, R G is COOH. In another embodiment, R G is CONH2. In one embodiment, R G is amino.
  • the monomers may also contain one or more 15 asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included.
  • substituent atoms fully described at the phosphorous atom, e.g., , where C’ is the 2’-carbon or 3’-carbon atom of a ribose ring, it is understood that the 20 oxygen having the broken bond is the 5'-oxygen of the subsequent nucleotide.
  • the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: ,wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 1 4 ,15, 16, 17, 18, 19, 20, or 21), such . 59 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 1 4 ,15, 16, 17, 18, 19, 20, or 21), such .
  • the in vivo delivery enhancing moiety is present within a modified 5 nucleoside of the formula: wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), and R G is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2, such as .
  • the in vivo delivery enhancing moiety is present within a modified n ucleoside of the formula: , wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 10 14 ,15, 16, 17, 18, 19, 20, or 21), and R G is hydrogen, hydroxy, amino, -COOH, or -C(O)NH 2 , such as .
  • the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , wherein n is an integer of 1-21, for instance, 1-12, 1- 10, 1-8, 1-6, 1-4, or 1-2, or 2 or 3 or 4 or 5 or 6); G is a C10-C22 hydrocarbon chain (e.g., a C16-C22 alkyl 15 chain, or a C16 alkyl chain, or a C22 alkyl chain)., optionally substituted with one or two groups selected from the group consisting of halogen, -OR G , -SR G , -N(R G )2, -C(O)OR G , -OC(O)R G , -C(O)N(R G )2, - N(R G )C(O)R G , -N(R G )C(O)OR G , -N(R G )SO2(R G ), or -SO2N(R G ), or
  • 60 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO n is 1. In one embodiment, n is 2-6. In one embodiment, n is 6. In one embodiment, G is a C 22 alkyl chain.
  • G is a C 16 alkyl chain
  • the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , wherein n is an integer of 1-21, for instance, 1-12, 1-10, 5 1-8, 1-6, 1-4, or 1-2, or 2 or 3 or 4 or 5 or 6); G is a C22 hydrocarbon chain, optionally substituted with one or two groups selected from the group consisting of halogen, -OR G , -SR G , -N(R G )2, -C(O)OR G , - OC(O)R G , -C(O)N(R G ) 2, -N(R G )C(O)R G , -N(R G )C(O)OR G , -N(R G )SO2(R G ), or -SO2N(R G )2, wherein each R G is independently hydrogen or C1-C6 alkyl; and nucle
  • n is 1. In one embodiment, n is 2-6. In one embodiment, n is 6. In one 10 embodiment, G is C10-C22 alkyl chain (e.g., a C14-C24 alkyl chain, C16-C22 alkyl chain, or a C16 alkyl chain, or a C22 alkyl chain). In one embodiment, the in vivo delivery enhancing moiety is present within a modified n ucleoside of the formula: one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: 15 .
  • the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 1 4 ,15, 16, 17, 18, 19, 20, or 21), such one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula , 61 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), such as .
  • the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula 62 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • the in vivo delivery enhancing moiety is present within a modified internucleotide linkage of the form, -OP(Y)(X)O-, wherein Y is O or S (e.g., O), and X is -N(H)(R L1 ), wherein R L1 is -G 1 or S(O) 2 -G 1 , each as defined above, wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic.
  • the in vivo delivery enhancing moiety is present within a modified internucleotide linkage of the form, -OP(O)(X)O-, wherein X is -N(H)(R L1 ), wherein R L1 is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) and the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic.
  • the in vivo delivery enhancing moiety is present within a a modified internucleotide linkage of the form, (such as ) , wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) and the 3’-O is from the preceding nucleoside and the 5’-O is from the subsequent nucleoside, and wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic.
  • the preceding nucleotide contains a 2’-fluoro modification.
  • the preceding nucleotide contains a 2’-O-methyl modification. In certain embodiments, the preceding nucleotide contains a 2’-H modification. In some embodiments, the in vivo delivery enhancing moiety is present within a 63 ME1 ⁇ 53466565.v1 Atty.
  • a modified internucleotide linkage of the form (such wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) and the 3’-O is from the preceding nucleoside and the 5’-O is from the subsequent nucleoside, and wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is 5 racemic.
  • the preceding nucleotide contains a 2’-fluoro modification.
  • the preceding nucleotide contains a 2’-O-methyl modification.
  • the preceding nucleotide contains a 2’-H modification.
  • the in vivo delivery enhancing moiety is present within a a modified internucleotide linkage of the form, -OP(Y)(X)O-, whereinY 10 wherein G1 is defined above, such a wherein n is selected from 7-23 (e.g.,11 – 21, or 1 1, 12, 13, 14 ,15, 16, 17, 18, 19, 20,
  • in vivo delivery enhancing moiety is conjugated to the 3’-end or 5’-end of one of the sense and antisense strands via a direct bond or through a carrier or linker.
  • in vivo delivery enhancing moiety is conjugated to the 3’-end of the sense or antisense 15 strand via a direct bond or through a carrier or linker. In some embodiments, in vivo delivery enhancing moiety is conjugated to the 5’-end of the sense or antisense strand via a direct bond or through a carrier or linker. In some embodiments, in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the sense and antisense strands (e.g., sense strand) and is of the formula , wherein X is O or 20 S (e.g., S); and R L3 is according to any of the preceding embodiments there.
  • R L3 can be , wherein n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23).
  • n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23).
  • n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23).
  • n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17,
  • RL3 can wherein m is selected from 1-6, and n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23).
  • m can be 2-5, or 1, or 2, or 3, or 4 or 5.
  • R L3 can be 20 selected from 1-6, and n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23).
  • m can be 2-5, or 1, or 2, or 3, or 4 or 5.
  • in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the sense and antisense strands (e.g., sense strand) and is of the formula: , or a salt thereof, 65 ME1 ⁇ 53466565.v1 Atty.
  • in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the sense and a ntisense strands and is of the formula (such as ), or a salt thereof, wherein q is selected from 0 – 18 (e.g., 1-11 or 1-8, or 3-11, or 3-8) and X is O or S (e.g., S).
  • a reactive pair e.g., a reaction between an azide and an alkyne or a cycloalkyne.
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula wherein m is selected from 1-10 (e.g., 3-6, or 3); R G is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2, and n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 5 19, or 20, or 21, or 22, or 23).
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and the 5’-terminal nucleotide is of the formula: 10 67 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- 5 terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is: .
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is , wherein 10 R L2 selected from: , 68 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 10 69 ME1 ⁇ 53466565.v1 Atty.
  • in vivo delivery enhancing moiety is bonded to the 3’-oxygen of the 3’- terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is: 5
  • in vivo delivery enhancing moiety is bonded to the 3’-oxygen of the 3’-terminal n ucleotide, and is of the formula -P(Y)(OH)-R3, wherein , wherein R L2 selected from: 10 70 ME1 ⁇ 53466565.v1 Atty.
  • in vivo delivery enhancing moiety is conjugated to the 3’-end or 5’-end of one of the sense and antisense strands via a carrier or linker, and the carrier or linker is an inverted abasic 5 nucleotide, such as an inverted abasic deoxyribonucleotide or an inverted abasic ribonucleotide, each connected to the remainder of the oligonucleotide via a phosphodiester (PO) or phosphorothioate (PS) linkage.
  • PO phosphodiester
  • PS phosphorothioate
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- 10 terminal nucleotide and is of the formula salt thereof, wherein each X is i ndependently O or S (e.g., each is S); Rligand is selected from the groups listed in Table R-1; and L is a divalent linking group (e.g., C1-20 alkyl or C1-10 alkyl-S-S-C1-10 alkyl).
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula 15 salt thereof, wherein each X is independently O or S (e.g., each is S) and R ligand is selected from a , wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21).
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula 71 ME1 ⁇ 53466565.v1 Atty.
  • each X is independently O or S (e.g., e ach is S) and Rligand is selected from a ,wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21).and L is a divalent linking group (e.g., C1-20 alkyl or C1-10 alkyl-S-S-C1-10 alkyl.
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula salt thereof, wherein each X is O or S (e.g., each is S) and R ligand is selected from a ,,wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21).
  • R L1 , R L2 , and R L3 are each a group containing at least one targeting moiety, such as an ⁇ v ⁇ 6 integrin targeting ligand described herein.
  • the targeting moiety can be selected from: 72 ME1 ⁇ 53466565.v1 Atty.
  • the broken bond is connected to a group of the formula **-L-ZZ-L’- , wherein ** represents the bond to the targeting ligand.
  • each R is independently C1-10alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl, isobutyl, butyl, 5 or hexyl); and R La is hydrogen, C1-10alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl, isobutyl, butyl, 5 or hexyl); and R La is hydrogen, C1-10alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl, isobutyl, butyl, 5 or hexyl); and R La is hydrogen, C1-10alkyl (e.g., methyl, ethyl, propyl
  • click adduct includes those adducts formed by a copper(I)-catalyzed azide-alkyne cycloaddition reaction, a strain-promoted azide-alkyne cycloaddition, a strain-promoted azide-trans-cycloalkene cycloaddition, and a thiol-maleimide Michael-addition reaction including, for 10 example, (i) an azide with a terminal alkyne or cycloalkyne (e.g.
  • L and L’ are independently one of: ( a) -L1-[G-L2]q-G-L3-* wherein q is 0 or an integer selected from 1-10; 15 (b) -L1-G-L2-G-L3-*; 74 ME1 ⁇ 53466565.v1 Atty.
  • L 1 is selected from one of the following groups: (a) a bond, C(O), C(S), C(NR N ), S(O) 2 , P(O)(OH), or P(S)(OH), wherein and R N is hydrogen or C 1-6 alkyl; (b) a bond, C(O), P(O)(OH), or P(S)(OH); 10 (c) a bond; (d) C(O); (e) P(O)(OH); or (f) P(S)(OH); each L 2 and L 3 is independently selected from one of the following groups: 15 (a) -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)O-, -OC(O)N(RN)-, - N(R N )C(O)O-, -N(R N )C(O)O-, -N(R N )C(O
  • each R’ is independently halogen, cyano, azido, nitro, -N(R b ) 2 , -O(R a ), -S(R 0 ), -C(O)OR 0 , - C(O)R 0 , -C(O)N(R 0 ) 2 , -C(NR 0 )OR 0 , -C(NR 0 )R 0 , -C(NR 0 )N(R 0 ) 2 , -C(S)OR 0 , -C(S)R 0 , -C(S)N(R 0 ) 2, -S(O) 2 R 0 , -S(O) 2 OR 0 , -S(O) 2 N(R 0 ) 2 , -N(R 0 )C(O)OR 0 , -N(R 0 0 )
  • q is 0, 1, 2, 3, 4, or 5. In another embodiment of L or L’, q is 0, 1, 10 2, 3, or 4. In another embodiment of L or L’, q is 0, 1, 2, or 3. In another embodiment of L or L’, q is 0, 1, or 2. In another embodiment of L or L’, q is 1, 2, 3, 4, or 5, In another embodiment of L or L’, q is 1, 2, 3, or 4. In another embodiment of L or L’, q is 1, 2, or 3. In another embodiment of L or L’, q is 1 or 2. In another embodiment of L or L’, q is 4. In another embodiment of L or L’, q is 3. In another embodiment of L or L’, q is 2.
  • L is one of: ( a) , wherein k is an integer from 1 to 10; L1 is bond, C(O), C(S), C(NRN), S(O)2, P(O)(OH), or P(S)(OH) (e.g., L 1 is a bond, C(O), P(O)(OH), or P(S)(OH)); and R N is hydrogen or C 1-6 alkyl; ( b) , wherein k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 20 3 to 10; or an integer from 4 to 10; or an integer from 5 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7; ( c) , wherein t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3); ( d) , wherein t is an integer from 0 to 10 (e.g., an integer from 0 to 10 (
  • s, s’, and s’’ are independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6); 5 independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); or and wherein in each of the preceding embodiments of L, * represents the bond to ZZ.
  • L’ is one of: (a)*-G-L 1 -, wherein L 1 is a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH); and G is C 1-10 alkyl, C 2- 10 alkenyl, C 2-10 alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; or L 1 is a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH); and G is C 1-10 alkyl; (b)*-G-[L 2 -G] q -L 1 -, wherein L 1 is a bond, CH 2 , C(O), S(O) 2 , P(O)(OH), or P(S)(OH); each L 2 is 15 independently -A-B-A-; each A is independently a bond, -O-, -S-, or -N(
  • each L 2 is independently C(O)(NR N ) , N(R N )C(O), OP(O)(OH)O, or OP(S)(OH)O wherein each R N is independently hydrogen or C 1-6 alkyl; and each G is independently C 1-10 alkyl, each of which is optionally substituted with 1 or 2 R groups; (g) -C 2-30 alkyl-*, such as -C 5-20 alkyl-* or -C 10-20 alkyl-*; 5 (h) -C 1-10 alkyl-*, optionally substituted with 1 or 2 R groups; (i) -C(O)-C 2-30 alkyl-*, such as -C(O)-C 5-20 alkyl-* or -C(O)-C 10-20 alkyl-*; wherein in each of (a) -
  • R L1 and R L3 that comprise a targeting ligand include, but are not limited to the following structures: Additional examples of R L1 and R L3 that comprise a targeting ligand include, but are not limited to the following structures: 78 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Examples of R L2 that comprise a targeting ligand include, but are not limited to the following structures: Additional examples of R L2 that comprise a targeting ligand include, but are not limited to the 5 following structure: 79 ME1 ⁇ 53466565.v1 Atty.
  • each R X is an integrin-receptor targeting ligand such as, 5 .
  • R L2 is .
  • targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, 10 and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is:
  • targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is: 80 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO .
  • R L1 , R L2 and R L3 can comprise a branched linking group ( ⁇ ) capable of 5 supporting multiple targeting ligands (e.g., at least 2; or 2-8; or 2-6; or 2-4; or 2; or 3).
  • the mutiple targeting moieties can be connected through an R L1 , R L2 , and R L3 of the form, (R X -L-ZZ-)z- ⁇ -T- , wherein each R X is a targeting moiety; z is at least 2; or 2-8; or 2-6; or 2-4; or 2; or 3, 10 T is -L’-T’-**, wherein ** is the bond to ⁇ , and T’ is O, S, N(H), C(O), S(O)2, C(O)N(H), N(H)C(O), OC(O), OC(O), -P(O)(OH)-, -P(S)(OH)-, -OP(O)(OH)-, -OP(S)(OH)-, -P(O)(OH)O-, - P(S)(OH)O-, -OP(O)(OH)O-, or -OP(S)(OH
  • T is selected from the following, wherein ** is the bond to ⁇ : ( a) -C(O)-X1-L5-X2-C(O)-**, wherein X1 and X2 are each independently C1-10alkyl; or C2- 10alkyl; or C4-10alkyl; or C6-10alkyl; or C2-8alkyl; or C2-6alkyl; or C2-4alkyl; ( b) -C(O)-C2-20alkyl-C(O)-**, such as -C(O)-C2-12alkyl-C(O)-**, (c) -C(O)-C6-20alkyl-C(O)-**, such as -C(O)-C6-12alkyl-C(O)-**, 20 (d) -C(O)-C10alkyl-C(O)-** and wherein each L 5 is a bond, ZZ, or - A 1 -B
  • T is selected from the following, ** is the bond to ⁇ : ( f) -N(H)C(O)-C2-20alkyl-C(O)-**, (g) - N(H)C(O)-C6-20alkyl-C(O)-**, such as - N(H)C(O)-C6-12alkyl-C(O)-**, 30 (h) - N(H)C(O)-C10alkyl-C(O)-**, (i) -C(O)-C2-20alkyl-C(O)N(H)-**, (j) -C(O)-C6-20alkyl-C(O)N(H)-**, 81 ME1 ⁇ 53466565.v1 Atty.
  • T is selected from the following, ** is the bond to ⁇ : ( a) -N(H)C(O)-X3-ZZ-X4-C(O)-**, (b) -C(O)-X3-ZZ-X4-C(O)N(H)-**, 10 (c) N(H)C(O)-X3-ZZ- X4-C(O)N(H)-**, wherein X 3 and X 4 are independently C2-12alkyl; or C4-10alkyl; or C6-10alkyl; or C4-8alkyl; and ZZ is as described above (such as -C(O)N(H)-, N(H)C(O)-, -OP(O)(OH)O-, -OP(S)(OH)O-, or a click adduct).
  • T is selected from the following (w) -L 6 -[G 5 -O]q5-G 5 -L 4 -**, wherein L 4 and L 6 are independently -A 1 -B 1 -A 1 -, wherein each A 1 15 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is hydrogen or C1-6alkyl; each B 1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G 5 is independently C1-10alkyl; (x) -C(O)-[CH2CH2-O]q5-G 5 -L 4 -**, L 4 is -A 1 -B 1 -A 1 -, wherein each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is hydrogen or C1-6al
  • L 4 is -A 1 -B 1 or -B 1 -A 1 -, wherein each A 1 is independently -O- or -N(H)-, and each B 1 is independently C(O), G 5 is C1-10alkyl (e.g., C2- 10alkyl or C2-6alkyl); and (z) -C(O)-[CH2CH2-O]q5- C2-10alkyl-C(O)N(H)-**; 25 wherein in each of the preceding ** is the bond to ⁇ , and q5, when present, is an integer selected from 1 to 20 (e.g., 1 to 10, or 2 to 10; or 2 – 8; or 1; or 2; or 3; or 4.)
  • Examples of branched linking group ( ⁇ ) include, but are not limited to, 82 ME1 ⁇ 53466565.v1 Atty.
  • branched R L2 examples include, but are not limited to, the following structures: 5 . I n some embodiments, targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is: 10 ( .g., ; . 85 ME1 ⁇ 53466565.v1 Atty.
  • targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is: .
  • targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, 5 and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is: .
  • targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is: wherein , r is an integer 10 selected from 1 - 10 (e.g., r is 7); and each R is: , wherein R X is a targeting ligand.
  • targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is: 15 ligand.
  • each R X is an integrin-receptor targeting ligand such as, 86 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO .
  • the dsRNA agent comprises a double-stranded region formed between the sense and antisense strands and optionally one or two single-stranded non-loop overhang, and wherein the one or more lipophilic moieties are conjugated to either the double-stranded region or the non-loop 5 overhang.
  • the dsRNA agent does not contain a loop (e.g., stem loop) region.
  • the dsRNA agent contains a loop (e.g., stem loop) region, and the one or more lipophilic moieties are not conjugated to the loop (e.g., stem loop) region.
  • the dsRNA agent comprises a sense strand of 10 to 53 nucleotides in length, in which the sense strand forms a duplex region with the antisense strand.
  • the sense 10 strand may be 10 to 49, 12 to 49, 12 to 45, 12 to 42, 12 to 40, 15 to 49, 15 to 45, 15 to 42, 15 to 40, 15 to 38, or 15 to 36 nucleotides in length.
  • the duplex region is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides in length. In some embodiments, the region of complementarity to the target sequence is at least 19 contiguous nucleotides in length.
  • the sense strand comprises at its 3′-end a stem-loop set forth as: S1-L-S2, 15 in which S1 is complementary to S2, and in which L forms a loop between S1 and S2.
  • the first 17 to 25 nucleotides counting from 5’ end of the sense strand forms a duplex region with the antisense strand, and the last 11 to 28 counting from 5’ end of the sense strand forms a 3′-end a stem-loop set forth as: S1-L-S2.
  • the length of the stem loop S1-L-S2 is 11 to 28, 13 to 26, or 15 to 24 20 nucleotides in length.
  • the stem loop S1-L-S2 is 16 nucleotides in length.
  • the stem loop S1-L-S2 comprises a sequence of GCAGCCGAAAGGCUGC (SEQ ID NO: 1).
  • L is at least 3, 4, or 5 nucleotides in length. In some embodiments, L comprises a sequence of GAAA. 25 In some embodiments, the sense strand is 36 nucleotides in length, the first 20 nucleotide counting from 5’ end of the sense strand forms a duplex region with the antisense strand, and the last 16 nucleotides forms a stem loop S 1 -L-S 2 . In one embodiment, the 16-nucleotide stem loop S 1 -L-S 2 has the sequence of GCAGCCGAAAGGCUGC (SEQ ID NO: 2), wherein L is GAAA.
  • the one or more lipophilic moieties are conjugated to a non-terminal 30 position of the sense strand. 87 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • the one or more lipophilic moieties are conjugated to one or more nucleotides of the stem loop S 1 -L-S 2 .
  • the one or more lipophilic moieties are conjugated to one or more nucleotides of the loop L.
  • S 1 and S 2 are complementary and contain 4-10 nucelotides, e.g., S 1 and S 2 5 each contain 6 complementary nucelotides.
  • S 1 and S 2 are complementary and contain 4-10 nucelotides and L is GAAA, e.g., S 1 and S 2 each contain 6 complementary nucelotides and L is GAAA.
  • the one or more lipophilic moieties containing one or more saturated or unsaturated C22 hydrocarbon chains are conjugated to one or more internal positions on at least one strand 10 of the dsRNA agent. Dual Conjugation
  • in vivo delivery enhancing moiety is connected in series with a targeting moiety, e.g., ⁇ v ⁇ 6 integrin targeting ligand, as described herein.
  • a sense or antisense strand 15 can contain a series modification at the 3’-end or 5’-end of the oligonucleotide, . such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises the t argeting moiety, e.g., ⁇ v ⁇ 6 integrin targeting ligand as described herein.
  • the series modification is of the form, wherein Q is selected 20 wherein RL2 is according to any preceding embodiment, wherein one of the broken bonds connects to a 5’-oxygen of a nucleoside or a 3’-oxygen of a nucleoside and the other connects to a 5’-terminal or 3’-terminal modification as described herein.
  • a sense or antisense strand can contain a series modification of the form, wherein RL2 is 25 according to any preceding embodiment, each Y is independently O or S; one of the broken bonds 88 ME1 ⁇ 53466565.v1 Atty.
  • a sense or antisense strand can contain a series modification of the form, wherein the broken bond connects to the 5’-oxygen of a 5’-terminal 5 nucleoside; each Y is independently O or S; one of R 51 and R 52 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety) and the other comprises a second ligand moiety (e.g., a targeting moiety, such as a ⁇ v ⁇ 6 integrin targeting ligand as described herein).
  • a lipophilic group e.g., an in vivo delivery enhancing moiety
  • a second ligand moiety e.g., a targeting moiety, such as a ⁇ v ⁇ 6 integrin targeting ligand as described herein.
  • a sense or antisense strand can contain a series modification of the form , wherein the broken bond connects to the 5’-oxygen of a 5’-terminal nucleoside; 10 each Y is independently O or S; one of R 51 and R 52 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety, such as any of R L or R L2 above) and the other comprises a second ligand moiety (e.g., a targeting moiety).
  • a lipophilic group e.g., an in vivo delivery enhancing moiety, such as any of R L or R L2 above
  • a second ligand moiety e.g., a targeting moiety
  • an oligonucleotide may have a series modification at the 5’-end of the formula: 15 is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); and each Y is independently O or S and R 510 and R 520 are 89 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein each Y is independently O or S.
  • a sense or antisense strand can contain a series modification of the form, wherein the broken bond connects to the 3’-oxygen of a 3’- 5 terminal nucleoside; each Y is independently O or S; one of R 31 and R 32 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety, such as any of R L2 above) and the other comprises a second ligand moiety (e.g., a targeting ligand).
  • a lipophilic group e.g., an in vivo delivery enhancing moiety, such as any of R L2 above
  • a second ligand moiety e.g., a targeting ligand
  • an oligonucleotide may have a series modification at the 3’-end of the formula: wherein n is selected from 7-2310 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); and each Y is independently O or S and R 310 and R 320 are wherein each Y is indepedently O or S. 90 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In another embodiment, can contain a single modification at the 5’-end, the 3’-end or at an i nternal position that contains both the in vivo delivery enhancing moiety and the targeting moiety.
  • a nd is of the formula -P(Y)(OH)-R3, wherein 5 wherein , wherein R L2 and R L3 comprises the in vivo delivery enhancing moiety (e.g., according to any in vivo delivery enhancing moiety embodiment of R L2 or R L3 above); R TG comprises the targeting moiety (e.g., R TG is according to Formula (X) (below), wherein R 5 is -L-ZZ-L’-, where L, ZZ, and L’ are defined for Formula (X) or an embodiment thereof; E is -C(O)N(H)-(CH 2 ) p -*, -N(H)C(O)-(CH 2 ) p -*, -C(O)O-(CH 2 ) p -*, -OC(O)-(CH 2 ) p -*, -
  • the single modification is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein wherein R D is as defined above.
  • R D is 10 15 92 ME1 ⁇ 53466565.v1 Atty.
  • RD is . In one embodiment of the 3’- or 5’-modification, RD is . In one embodiment of the 3’- or 5’-modification, RD is . In one embodiment of the 3’- or 5’-modification, RD i s ; for example, G1 can be C2-20alkyl.
  • n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 10 21, or 22, or 23);
  • R G is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2;
  • G 1 is C2-20alkyl.
  • n one embodiment of the 3’- or 5’-modification, wherein n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23);
  • R G is hydrogen, hydroxy, amino, -COOH, or - C(O)NH 2 ;
  • G 1 is C 2-20 alkyl, and -C(O)-R TG is 93 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO broken bond is the bond between R TG and the nitrogen.
  • a sense or antisense strand can contain two different ligand modifications, one at the 3'-end of the strand and the other at the 5'-end of the strand: such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises the t argeting moiety, e.g., ⁇ v ⁇ 6 integrin targeting ligand as described herein.
  • a sense or antisense strand can contain two different ligand modifications, one is an internal modified nuceloside or modified internucleotide linkage of the strand and the other at the 3’-end or 5'-end of the strand: such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises the 15 targeting moiety, e.g., ⁇ v ⁇ 6 integrin targeting ligand as described herein.
  • the modified nuceloside represented by L1 can be located at a position in the strand selected from positions 2 to n-1, where the strand contains n nucleotides (e.g., n-1 is 20 when n is 21).
  • segment (1) of the strand contains 5 nucleotides and segment (2) contains the remainder of the nucleotides within the strand.
  • “Position” herein when referring to a modified nucleotide, nucleoside, or 20 internucleotide linkage is counted from the 5’-end of the strand, for example, position 6 includes the 6th nucleotide from the 5’-end of the strand and the 6th internucleotide linkage counting from the 5’-end of the strand.
  • 94 ME1 ⁇ 53466565.v1 Atty.
  • L1 comprises the in vivo delivery enhancing moiety (e.g., C 10 -C 26 saturated or unsaturated hydrocarbon, such as a C 10 -C 26 alkyl group, or a C 14 -C 24 alkyl group, or a C 16 -C 22 alkyl group, or a C 16 alkyl group, or a C 22 alkyl group) and a sense or antisense strand can be represented by one of: 5 wherein B is an optionally modified nucleobase (e.g., A, C, G, U, or T); R L3 or R L1 comprises the in vivo delivery enhancing moiety, L2 comprises the targeting moiety.
  • B is an optionally modified nucleobase (e.g., A, C, G, U, or T)
  • R L3 or R L1 comprises the in vivo delivery enhancing moiety
  • L2 comprises the targeting moiety.
  • R L3 is selected from the group consisting of: , , , and wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); for example, in one embodiment, n is 13; and in another embodiment, n is 19. 10
  • R L1 is selected from the group consisting of: a , wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); for example, in one embodiment, n is 7; and in another embodiment, n is 10. 95 ME1 ⁇ 53466565.v1 Atty.
  • R L3 is selected from the group consisting of: another embodiment, R L1 is selected from the group consisting of: . 1 .
  • Linkers/Tethers 5 Linkers/Tethers are connected to the at least one in vivo delivery enhancing moiety at a “tethering attachment point (TAP).”
  • TAP tethering attachment point
  • Linkers/Tethers may include any C 1 -C 100 carbon-containing moiety, (e.g. C1-C75, C1-C50, C1-C20, C1-C10; C1, C2, C3, C4, C5, C6, C7, C8, C9, or C10), and may have at least one nitrogen atom.
  • the nitrogen atom forms part of a terminal amino or amido (NHC(O)-) group on the linker/tether, which may serve as a connection point for the lipophilic moiety.
  • linkers/tethers include TAP- nNH-; TAP-C(O)(CH2)nNH-; TAP-NR’’’’(CH2)nNH-, TAP-C(O)-(CH2)n-C(O)-; TAP-C(O)-(CH2)n-C(O)O-; TAP-C(O)-O-; TAP- C(O)-(CH2)n-NH-C(O)-; TAP-C(O)-(CH2)n-; TAP-C(O)-NH-; TAP-C(O)-; TAP-C(O)-; TAP-(CH2)n-C(O)-; TAP- (CH2)n-C(O)O-; TAP-(CH2)n-; or TAP-(
  • n is 5, 6, or 11.
  • the nitrogen may form part of a terminal oxyamino group, e.g., -ONH2, or hydrazino group, -NHNH2.
  • the linker/tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S.
  • Preferred tethered ligands may include, e.g., TAP-(CH2)nNH(LIGAND); TAP- C(O)(CH2)nNH(LIGAND); TAP-NR’’’’(CH2)nNH(LIGAND); TAP-(CH2)nONH(LIGAND); TAP-20 C(O)(CH2)nONH(LIGAND); TAP-NR’’’’(CH2)nONH(LIGAND); TAP-(CH2)nNHNH2(LIGAND), TAP- C(O)(CH2)nNHNH2(LIGAND); TAP-NR’’’’(CH2)nNHNH2(LIGAND); TAP-C(O)-(CH2)n- C(O)(LIGAND); TAP-C(O)-(CH2)n-C(O)O(LIGAND); TAP-C(O)-O(LIGAND); TAP-C(O)-(CH2)n-NH- C(O)(LIG
  • amino terminated linkers/tethers e.g., NH 2 , ONH 2 , NH 2 NH 2
  • the tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S.
  • the double bond can be cis or trans or E or Z.
  • the linker/tether may include an electrophilic moiety, preferably at the terminal position of the linker/tether.
  • electrophilic moieties include, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl ester.
  • Preferred linkers/tethers include TAP-(CH 2 ) n CHO; TAP- 5 C(O)(CH 2 ) n CHO; or TAP-NR’’’’(CH 2 ) n CHO, in which n is 1-6 and R’’’’ is C 1 -C 6 alkyl; or TAP- (CH 2 ) n C(O)ONHS; TAP-C(O)(CH 2 ) n C(O)ONHS; or TAP-NR’’’’(CH 2 ) n C(O)ONHS, in which n is 1-6 and R’’’’’ is C 1 -C 6 alkyl; TAP-(CH 2 ) n C(O)OC 6 F 5 ; TAP-C(O)(CH 2 ) n C(O) OC 6 F 5 ; or TAP-NR’’’’(CH 2 ) nC(O) OC 6 F 5 , in which n is 1-11 and R
  • Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether.
  • a nucleophilic group of a ligand e.g., a thiol or amino group
  • an electrophilic group on the tether it can be desirable for the monomer to include a phthalimido group (K) at t he terminal position of the l .
  • linker/tether e.g., alloc, monomethoxy trityl (MMT), trifluoroacetyl, Fmoc, or aryl sulfonyl (e.g., the aryl portion can be ortho-nitrophenyl or ortho, para-dinitrophenyl).
  • At least one of the linkers/tethers can be a redox cleavable linker, an acid cleavable linker, an esterase cleavable linker, a phosphatase cleavable linker, or a peptidase cleavable linker. 25 In one embodiment, at least one of the linkers/tethers can be a reductively cleavable linker (e.g., a disulfide group).
  • At least one of the linkers/tethers can be an acid cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group). In one embodiment, at least one of the linkers/tethers can be an esterase cleavable linker (e.g., an 30 ester group). In one embodiment, at least one of the linkers/tethers can be a phosphatase cleavable linker (e.g., a phosphate group). 97 ME1 ⁇ 53466565.v1 Atty.
  • At least one of the linkers/tethers can be a peptidase cleavable linker (e.g., a peptide bond).
  • Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher 5 levels or activities inside cells than in serum or blood.
  • degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade 10 an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
  • a cleavable linkage group such as a disulfide bond, can be susceptible to pH.
  • the pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 15 5.0.
  • Some tethers will have a linkage group that is cleaved at a preferred pH, thereby releasing the iRNA agent from a ligand (e.g., a targeting or cell-permeable ligand, such as cholesterol) inside the cell, or into the desired compartment of the cell.
  • a chemical junction e.g., a linking group that links a ligand to an iRNA agent can include a disulfide bond.
  • the ligand can be a targeting ligand or a second therapeutic agent that may complement the therapeutic effects of the iRNA agent.
  • a tether can include a linking group that is cleavable by a particular enzyme.
  • the type of linking 25 group incorporated into a tether can depend on the cell to be targeted by the iRNA agent. Tethers that contain peptide bonds can be conjugated to iRNA agents target to cell types rich in peptidases. In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or 30 when in contact with other non-target tissue, e.g., tissue the iRNA agent would be exposed to when administered to a subject.
  • tissue e.g., tissue the iRNA agent would be exposed to when administered to a subject.
  • the evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, 35 or in whole animals. It may be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals.
  • useful candidate compounds 98 ME1 ⁇ 53466565.v1 Atty.
  • cleavable linking groups are redox cleavable linking groups that are cleaved upon reduction or oxidation.
  • reductively cleavable linking group is a disulphide linking group (—S—S—).
  • a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular in vivo 10 delivery enhancing moiety
  • a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell.
  • DTT dithiothreitol
  • the candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions.
  • candidate compounds are cleaved by at most 10% in the blood.
  • useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions).
  • the rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.
  • Phosphate-Based Cleavable Linking Groups are cleaved by agents that degrade or hydrolyze the phosphate group.
  • An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells.
  • phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—25 , —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O— P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(O)(Rk)- O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—.
  • Preferred embodiments are —O— P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O— P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O— 30 P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(O)(H)—O—, —S—P(O)(H)—S— , —O—P(S)(H)—S—.
  • Acid Cleavable Linking Groups are linking groups that are cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of 99 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid.
  • acid cleavable linking groups include but are not limited to hydrazones, ketals, acetals, esters, and esters of amino acids.
  • Acid 5 cleavable groups can have the general formula —C ⁇ NN—, C(O)O, or —OC(O).
  • a preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl.
  • Ester-Based Linking Groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above. 15 7 .
  • Peptide-Based Cleaving Groups are cleaved by enzymes such as peptidases and proteases in cells.
  • Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not 20 include the amide group (—C(O)NH—).
  • the amide group can be formed between any alkylene, alkenylene or alkynelene.
  • a peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins.
  • the peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group.
  • Peptide cleavable linking groups have the general formula — 25 NHCHR 1 C(O)NHCHR 2 C(O)—, where R 1 and R 2 are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.
  • H. Biocleavable linkers/tethers The linkers can also include biocleavable linkers that are nucleotide and non-nucleotide linkers 30 or combinations thereof that connect two parts of a molecule.
  • the non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides, and derivatives thereof, aliphatic, alicyclic, hetercyclic, and combinations thereof.
  • At least one of the linkers is a bio-clevable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or 35 oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, and combinations thereof.
  • the bio-cleavable carbohydrate linker may have 1 to 10 saccharide units, which have at least one anomeric linkage capable of connecting two siRNA units.
  • bio-cleavable linkers include: 5 101 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 102 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . More discussion about the biocleavable linkers may be found in WO2018136620, entitled 5 “Endosomal Cleavable Linkers,” the entire contents of which are incorporated herein by reference. 8 .
  • the at least one in vivo delivery enhancing moiety is conjugated to the dsRNA agent via a carrier that replaces one or more nucleotide(s).
  • the carrier can be a cyclic group or an acyclic group.
  • the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin.
  • the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone.
  • the carrier replaces one or more nucleotide(s) in the internal position(s) of the dsRNA agent. In other embodiments, the carrier replaces the nucleotides at the terminal end of the sense strand or antisense strand. In one embodiment, the carrier replaces the terminal nucleotide on the 3’ end of the sense strand, thereby functioning as an end cap protecting the 3’ end of the sense strand. In one 20 embodiment, the carrier is a cyclic group having an amine, for instance, the carrier may be pyrrolidinyl, 103 ME1 ⁇ 53466565.v1 Atty.
  • pyrazolinyl pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.
  • a ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred 5 to herein as a ribose replacement modification subunit (RRMS).
  • the carrier can be a cyclic or acyclic moiety and include two “backbone attachment points” (e.g., hydroxyl groups) and a ligand (e.g., the lipophilic moiety).
  • the one or more C 22 hydrocarbon chains can be directly attached to the carrier or indirectly attached to the carrier by an intervening linker/tether, as described above.
  • the ligand-conjugated monomer subunit may be the 5’ or 3’ terminal subunit of the iRNA molecule, i.e., one of the two “W” groups may be a hydroxyl group, and the other “W” group may be a chain of two or more unmodified or modified ribonucleotides.
  • the ligand-conjugated monomer subunit may occupy an internal position, and both “W” groups may be one or more unmodified or modified ribonucleotides. More than one ligand-conjugated monomer subunit may be present in an 15 iRNA agent.
  • Cyclic sugar replacement-based monomers e.g., sugar replacement-based ligand-conjugated 20 monomers
  • the carriers may have the general formula (LCM-2) provided below (in that structure preferred backbone attachment points can be chosen from R 1 or R 2 ; R 3 or R 4 ; or R 9 and R 10 if Y is CR 9 R 10 (two positions are chosen to give two backbone attachment points, e.g., R 1 and R 4 , or R 4 and R 9 )).
  • Preferred tethering attachment points include R 7 ; R 5 or R 6 when X is CH 2 .
  • the carriers are described below as an entity, which can be 25 incorporated into a strand.
  • the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R 1 or R 2 ; R 3 or R 4 ; or R 9 or R 10 (when Y is CR 9 R 10 ), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone.
  • R 104 ME1 ⁇ 53466565.v1 Atty.
  • Docket No.121301-23820/ALN-523-WO groups can be -CH 2 -, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.
  • (LCM-2) 5 wherein: X is N(CO)R 7 , NR 7 or CH 2 ; Y is NR 8 , O, S, CR 9 R 10 ; Z is CR 11 R 12 or absent;
  • R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is, independently, H, OR a , or (CH 2 ) n OR b , provided 10 that at least two of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 are OR a and/or (CH 2 ) n OR b ;
  • Each of R 5 , R 6 , R 11 , and R 12 is, independently, a ligand, H, C 1 -C 6 alkyl optionally substituted with 1-3 R 13 , or C(O)NHR 7 ; or R 5 and R 11 together are C3-C8 cycloalkyl optionally substituted with R 14 ;
  • R 7 can be a lig
  • R b is P(O)(O-)H, P(OR 15 )N(R 16 )2 or L-R 17 ;
  • R c is H or C1-C6 alkyl;
  • R d is H or a ligand;
  • Each Ar is, independently, C6-C10 aryl optionally substituted with C1-C4 alkoxy; 30 n is 1-4; and q is 0-4.
  • Exemplary carriers include those in which, e.g., X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is absent; or X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is CR 11 R 12 ; or X is N(CO)R 7 or NR 7 , Y is NR 8 , and Z is CR 11 R 12 ; or X is N(CO)R 7 or NR 7 , Y is O, and Z is CR 11 R 12 ; or X is CH 2 ; Y is CR 9 R 10 ; Z is CR 11 R 12 , 105 ME1 ⁇ 53466565.v1 Atty.
  • the carrier may be based on the pyrroline ring system or the 4- hydroxyproline ring system, e.g., X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is absent (D). 5 .
  • OFG 1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the five-membered ring (-CH2OFG 1 in D).
  • OFG 2 is preferably attached directly to one of the carbons in the five-membered ring (-OFG 2 in D).
  • -CH2OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; or - CH2OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4.
  • CH2OFG 1 10 and OFG 2 may be geminally substituted to one of the above-referenced carbons.
  • -CH2OFG 1 may be attached to C-2 and OFG 2 may be attached to C-4.
  • the pyrroline- and 4-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon- carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon- carbon bonds
  • CH2OFG 1 and OFG 2 may be cis or trans with respect to one another 15 in any of the pairings delineated above Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH 2 OFG 1 and OFG 2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration 20 and the other center can have the S configuration and vice versa).
  • the tethering attachment point is preferably nitrogen.
  • Preferred examples of carrier D include the following: 106 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO .
  • the carrier may be based on the piperidine ring system (E), e.g., X is .
  • OFG 2 is preferably attached directly to one of the carbons in the six-membered ring (-OFG 2 in E).
  • -(CH2)nOFG 1 and OFG 2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4.
  • -(CH 2 ) n OFG 1 and 10 OFG 2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CH 2 ) n OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; -(CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-2; -(CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4; or -(CH 2 ) n OFG 1 may be attached to C-4 and OFG 2 may be attached to C- 3.
  • the piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein 15 bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • -(CH 2 ) n OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures.
  • the carriers may be based on the piperazine ring system (F), e.g., X is 25 N(CO)R7 or NR7, Y is NR8, and Z is CR11R12, or the morpholine ring system (G), e.g., X is N(CO)R7 or NR 7 , Y is O, and Z is CR 11 R 12 .
  • F piperazine ring system
  • X is 25 N(CO)R7 or NR7
  • Y is NR8
  • Z is CR11R12
  • G morpholine ring system
  • X is N(CO)R7 or NR 7
  • Y is O
  • Z is CR 11 R 12 .
  • OFG 1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the six-membered ring (-CH2OFG 1 in F or G).
  • OFG 2 is preferably attached directly to one of the carbons in the six-membered rings (-OFG 2 in F or G). 5 For both F and G, -CH2OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; or vice versa.
  • CH2OFG 1 and OFG 2 may be geminally substituted to one of the above- referenced carbons.
  • the piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • CH2OFG 1 and OFG 2 may be cis or trans with 10 respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures.
  • R can be, e.g., C1-C6 alkyl, preferably CH3.
  • the tethering attachment point is preferably nitrogen in both F and G.
  • OFG 2 is preferably attached directly to one of C-2, C-3, C-4, or C-5 (-OFG 2 in H).
  • -(CH2)nOFG 1 and OFG 2 may be disposed 25 in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5.
  • -(CH 2 ) n OFG 1 and OFG 2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CH2)nOFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; -(CH2)nOFG 1 may be attached to C-3 and OFG 2 may be attached to C- 108 ME1 ⁇ 53466565.v1 Atty.
  • the decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein 5 bond rotation is restricted about that particular linkage, e.g.
  • -(CH 2 ) n OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the 10 monomers are expressly included (e.g., the centers bearing CH2OFG 1 and OFG 2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa).
  • the substituents at C-1 and C-6 are trans with respect to one another.
  • the tethering attachment point is preferably C-6 or C-7.
  • Other carriers may include those based on 3-hydroxyproline (J). 15 .
  • -(CH2)nOFG 1 and OFG 2 may be cis or trans with respect to one another. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures.
  • the tethering attachment point is preferably nitrogen. Details about more representative cyclic, sugar replacement-based carriers can be found in U.S. Patent Nos.7,745,608 and 8,017,762, which are herein incorporated by reference in their entireties.
  • Acyclic sugar replacement-based monomers e.g., sugar replacement-based ligand-conjugated monomers, are also referred to herein as ribose replacement monomer subunit (RRMS) monomer compounds.
  • Preferred acyclic carriers can have formula LCM-3 or LCM-4: 109 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO .
  • each of x, y, and z can be, independently of one another, 0, 1, 2, or 3.
  • the tertiary carbon can have either the R or S configuration.
  • x is zero and y and z are each 1 in formula LCM-3 (e.g., based 5 on serinol), and y and z are each 1 in formula LCM-3.
  • formula LCM-3 or LCM-4 below can optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl. Details about more representative acyclic, sugar replacement-based carriers can be found in U.S. Patent Nos.7,745,608 and 8,017,762, which are herein incorporated by reference in their entireties.
  • the at least one in vivo delivery enhancing moiety may be conjugated to one or more internal 10 positions on at least one strand, e.g., a sense strand or an antisense strand.
  • Internal positions of a strand refers to the nucleotide on any position of the strand, except the terminal position from the 3’ end and 5’ end of the strand (e.g., excluding 2 positions: position 1 counting from the 3’ end and position 1 counting from the 5’ end).
  • the at least one in vivo delivery enhancing moiety may be conjugated to one 15 or more internal positions on at least one strand, which include all positions except the terminal two positions from each end of the strand (e.g., excluding 4 positions: positions 1 and 2 counting from the 3’ end and positions 1 and 2 counting from the 5’ end). In one embodiment, the at least one in vivo delivery enhancing moiety is conjugated to one or more internal positions on at least one strand, which include all positions except the terminal three positions from each end of the strand (e.g., excluding 6 positions: 20 positions 1, 2, and 3 counting from the 3’ end and positions 1, 2, and 3 counting from the 5’ end).
  • the at least one in vivo delivery enhancing moiety may be conjugated to one or more internal positions on at least one strand, except the cleavage site region of the sense strand, for instance, the at least one in vivo delivery enhancing moiety is not conjugated to positions 9-12 counting from the 5’-end of the sense strand, for example, the at least one in vivo delivery enhancing moiety is not 25 conjugated to positions 9-11 counting from the 5’-end of the sense strand.
  • the internal positions exclude positions 11-13 counting from the 3’-end of the sense strand.
  • the at least one in vivo delivery enhancing moiety may be conjugated to one or more internal positions on at least one strand, which exclude the cleavage site region of the antisense strand. For instance, the internal positions exclude positions 12-14 counting from the 5’-end of the 30 antisense strand. In one embodiment, the at least one in vivo delivery enhancing moiety may be conjugated to one or more internal positions on at least one strand, which exclude positions 11-13 on the sense strand, counting from the 3’-end, and positions 12-14 on the antisense strand, counting from the 5’-end. 110 ME1 ⁇ 53466565.v1 Atty.
  • the at least one in vivo delivery enhancing moiety may be conjugated to one or more of the following internal positions: positions 4-8 and 13-18 on the sense strand, and positions 6- 10 and 15-18 on the antisense strand, counting from the 5’end of each strand. In one embodiment, the at least one in vivo delivery enhancing moiety may be conjugated to one 5 or more of the following internal positions: positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5’end of each strand.
  • the at least one in vivo delivery enhancing moiety may be conjugated to position 6 on the sense strand, counting from the 5’end of each strand. I n some embodiments, the at least one in vivo delivery enhancing moiety may be conjugated to a 10 nucleobase, sugar moiety, or internucleosidic phosphate linkage of the dsRNA agent. In one embodiment, the at least one in vivo delivery enhancing moiety is conjugated to a sugar moiety of the dsRNA agent. In a further embodiment, the at least one in vivo delivery enhancing moiety is conjugated to the 2’ position of a ribose sugar of the dsRNA agent. 15 III.
  • a dsRNA agent comprises an antisense strand and a sense strand; at least one ⁇ v ⁇ 6 integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least one 20 strand; and at least one in vivo delivery enhancing moiety conjugated to at least one strand.
  • Integrins are cell surface receptors that, upon ligand binding, activate signal transduction pathways including signaling pathways involved in cytoskeleton organization and cell cycle regulation.
  • Integrins are also involved in cell attachment to the extracellular matrix and the integrin ligands comprise common extracellular matrix components, including fibronectin, collagen, laminin, fibrinogen, 25 thrombospondin, and glycoproteins (e.g., tenascin C, osteopontin, and nefronectin).
  • integrin heterodimers composed of an alpha and a beta subunit, e.g., ⁇ v ⁇ 6. It is the combination of the alpha and beta subunits which determines the ligand specificity and function of the integrin.
  • ⁇ v ⁇ 6 cytokine transforming growth factor-b1
  • LAP pro-peptide latency associated peptide
  • ⁇ v ⁇ 6 binds LAP, and through cytoskeletal force releases TGF- ⁇ 1.
  • TGF- ⁇ 1 regulates multiple processes including cell proliferation, differentiation, angiogenesis, epithelial-mesenchymal-transition (EMT) and immune suppression. These processes combine to heal wounds but when uncontrolled can 35 promote tissue pathologies.
  • the present disclosure provides dual conjugated dsRNA agents for inhibiting expression of a target gene that comprise an antisense strand and a sense strand; at least one ⁇ v ⁇ 6 integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least one strand; and at least one in vivo delivery enhancing moiety conjugated to at least one strand.
  • a target gene that comprise an antisense strand and a sense strand
  • at least one ⁇ v ⁇ 6 integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least one strand
  • at least one in vivo delivery enhancing moiety conjugated to at least one strand 5 A .
  • Alpha-v-Beta-6 ( ⁇ v ⁇ 6) Integrin Targeting Ligands Conjugated to dsRNA agents
  • the dsRNA agent of the present disclosure is conjugated to at least one ⁇ v ⁇ 6 integrin targeting ligand.
  • the at least one ⁇ v ⁇ 6 integrin targeting ligand may be conjugated to the dsRNA agent via a direct attachment to the dsRNA agent, e.g., a ribosugar of the dsRNA agent. 10
  • the at least one ⁇ v ⁇ 6 integrin targeting ligand may be conjugated to the dsRNA agent via a linker or a carrier as described herein.
  • Exemplary ⁇ v ⁇ 6 integrin targeting ligands that can conjugated to the dsRNA agents of the disclosure can be found, e.g., in U.S.
  • the ⁇ v ⁇ 6 integrin targeting ligand may be conjugated to the dsRNA agent via a linker comprising a compound of the structure L333 N-(aminocaproyl-DBCO)-4-hydroxyprolinol, wherein * represents the bond to the remainder of the 20 dsRNA agent.
  • the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: wherein 112 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 bond from R Lig to the nitrogen, # is the bond to the integrin ligand, and * represents the bond to the remainder of the dsRNA agent. 113 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: 5
  • the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: 10 114 ME1 ⁇ 53466565.v1 Atty.
  • the bond from R Lig to the nitrogen # is the bond to the integrin ligand, and * represents the bond to the remainder of the dsRNA agent.
  • the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: 10 wherein wherein 115 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 bond from R Lig to the nitrogen, # is the bond to the integrin ligand, and * represents the bond to the remainder of the dsRNA agent. 116 ME1 ⁇ 53466565.v1 Atty.
  • the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: to the re 5
  • the ⁇ v ⁇ 6 integrin targeting ligand or the the ⁇ v ⁇ 6 integrin targeting ligand to the dsRNA agent of the present disclosure comprises a structure selected from the table below: 117 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 118 ME1 ⁇ 53466565.v1 Atty.
  • the alpha-v-beta-6 ( ⁇ v ⁇ 6) integrin targeting ligand may be conjugated to the dsRNA agent of the disclosure, e.g., to at least one strand of the dsRNA agent of the disclosure, via a 5 carrier group.
  • the alpha-v-beta-6 ( ⁇ v ⁇ 6) integrin targeting ligand conjugated to the dsRNA agent of the disclosure is represented by a compound of the Formula (X): or a salt thereof, wherein: Y is O, N(H), S, or CH 2 ; (e.g., O or CH 2 ) 10 , wherein m is 0, 1, 2, 3, or 4; and each R 2 is independently R, or two R 2 groups on adjacent carbon atoms taken together with the atoms to which they are bound form a fused 4 – 8 membered ring (e.g,.
  • A is an 5-membered heteroaryl optionally substituted with 1 or 2 substituents independently selected from methyl, ethyl, fluoro, hydroxymethyl, 2-hydroxypropan-2-yl, trifluoromethyl, difluoromethyl, and fluoromethyl
  • Q is -COOR 1 or tetrazolyl (e.g., 1,2,3,4-tetrazol-5-yl), wherein R 1 is hydrogen or C 1-6 alkyl (e.g., 20 methyl);and R L is -N(R 3 )(R 4 ), -O(R 5 ), -S(R 5 ), or -R 5 , wherein R 3 and R 4 are either ( i) R3 is hydrogen or C1-6alkyl and R4 is R5; or (ii) R3 and R4 taken together with the nitrogen
  • Docket No.121301-23820/ALN-523-WO * is the bond to ZZ; q is 0 or an integer selected from 1 – 25; (e.g., 1-20, or 1-15); L 1 is a bond or -B-A-; each L 2 is independently -A-B-A-; 5 L 3 is a bond or -A-B-A-; each G is independently -D-E-F-, wherein D, E, and F are independently a bond, C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; 10 each A is independently a bond, -O-, -S-, or -N(R N )-; each B is independently a bond, CH2, C(O), C(S), C(NR N ), S(O), S
  • each R’ is independently halogen, cyano, azido, nitro, -N(R b ) 2 , -O(R a ), -S(R 0 ), - C(O)OR 0 , - C(O)R 0 , -C(O)N(R 0 ) 2 , -C(NR 0 )OR 0 , -C(NR 0 )R 0 , -C(NR 0 )N(R 0 ) 2 , -C(S)OR 0 , -C(S)R 0 , -C( S)N(R 0 ) 2 , -S(O) 2 R 0 , -S(O) 2 OR 0 , -S(O) 2 N(R 0 ) 2 , -N(R 0 )C(O)OR 0 , -N(R 0 ), -N(R 0 )OR 0
  • each -D-E-F- group at least one of D, E, and F is not a bond; and R L is not N-morpholinyl.
  • R 3 and R 4 do not form a morpholino ring.
  • B in each -A-B-A- group, B is only a bond when one of the A groups is not a bond.
  • each R group is independently selected from the group consisting of R’, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, heterocyclyl, aryl, heteroaryl, C3- 8cycloalkylC1-6alkyl, heterocyclylC1-6alkyl, aryl C1-6alkyl and heteroaryl1-6alkyl, each of which, other than R’, is optionally substituted with 1, 2, or 3 R’ groups, wherein each R’ is independently halogen, cyano, azido, nitro, -N(R b )2, -O(R a ), -S(R 0 ), -C(O)OR 0 , -C(O)R 0 , -C(O)N(R 0 )2, -N(R 0 )C(O)R 0 , -OC(O)OR 0 , or
  • each R group is independently selected from the group consisting of R’, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, C2-6alkynyl, C3-6cycloalkyl, 5-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl, and benzyl, each of which, other than R’, is optionally substituted with 1, 2, or 325 R’ groups, wherein each R’ is independently halogen, cyano, azido, nitro, -N(R b )2, -O(R a ), -S(R 0 ), - C(O)OR 0 , -C(O)R 0 , -C(O)N(R 0 )2, -N(R 0 )C(O)R 0 , -OC(O)OR 0 , or -OC(O)R 0 , wherein each R 0 is independently hydrogen or C1-6alkyl
  • each R group is independently selected from the group consisting of R’, 30 C 1-6 alkyl, C 3-6 cycloalkyl, 5-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl, and benzyl, each of which, other than R’, is optionally substituted with 1, 2, or 3 R’ groups, wherein each R’ is independently halogen, cyano, azido, nitro, -N(R b ) 2 , -O(R a ), -S(R 0 ), -C(O)OR 0 , - C(O)R 0 , -C(O)N(R 0 ) 2 , -N(R 0 )C(O)R 0 , -OC(O)OR 0 , or -OC(O)R 0 , wherein each R 0 is independently hydrogen or C 1-6 alkyl; each R a is independently hydrogen or C 1-6 alkyl; and
  • Formula (X) is selected from an optionally substituted N- or a C-linked pyrazole, an optionally substituted N- or a C-linked triazole, and an optionally substituted N- or C-linked imidazole, wherein the optionally substituted N- or a C-linked pyrazole, the optionally substituted N- or a 10 C-linked triazole, and the optionally substituted N- or C-linked imidazole is optionally substituted by 1 or 2 substituents independently selected from methyl, ethyl, fluoro, hydroxymethyl, 2-hydroxypropan-2-yl, trifluoromethyl, difluoromethyl,
  • R 1 is hydrogen, methyl, or ethyl
  • R 2 is hydrogen or fluoro
  • R3 is hydrogen, methyl, or ethyl.
  • A is .
  • A is selected from: 123 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO R Y
  • Formula (X) In some embodiments of any one of Formula (X), in R Y , each R 2 is independently R as defined in Formula (X). 5 In some embodiments,R In some embodiments,R .
  • R Y in R Y , two R 2 groups on adjacent carbon atoms taken together with the atoms to which they are bound form a fused 4 – 8 membered ring that is optionally substituted with 1, 2, 3 or 4 groups independently selected from group consisting of R and a nitrogen protecting group, wherein R is 10 as defined in Formula (IV).
  • R Y is NH wherein p is 0, 1, 2, 3 or 4; and each R 21 is independently selected from group consisting of R and a nitrogen protecting group, wherein R is as defined in Formula (IV).
  • R Y is , wherein p is 0, 1, 2, 3 or 4; and each R 21 is 15 independently selected from the group consisting of R and a nitrogen protecting group, wherein R is as defined in Formula (IV).
  • R Y is , wherein p is 0, 1, 2, 3 or 4; and each R 21 is 15 independently selected from the group consisting of R and a nitrogen protecting group, wherein R is as defined in Formula (IV).
  • R P is a nitrogen protecting group. In some embodiments, 5 group.
  • R P is 10 , wherein r is 0, 1, 2, or 3; each R P2 is independently halogen, nitro, cyano, C1- 4alkoxy, C 1-4 alkyl, C 1-4 haloalkyl; and each R P3 is independently hydrogen, methyl, or ethyl.
  • R P is methoxyacetyl (mac), phenoxyacetyl (pac), 2- chlorophenoxyacetyl, 3-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2,4-dichlorophenoxyacetyl, 2- 125 ME1 ⁇ 53466565.v1 Atty.
  • R P is methoxyacetyl (mac), phenoxyacetyl (pac), 2- chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2-methylphenoxyacetyl, 4-methylphenoxyacetyl, or 4- isopropylphenoxyacetyl. 10
  • R P is methoxyacetyl (mac).
  • R P is phenoxyacetyl (pac).
  • R 1 is C1-6alkyl , wherein r is 0, 1, 2, or 3; each R P2 is independently halogen, nitro, cyano, C 1-4 alkoxy, C 1-4 alkyl, C 1-4 haloalkyl; and each R P3 is independently hydrogen, methyl, or ethyl.
  • R 1 is C 1-6 alkyl and R P is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 3-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2,4- dichlorophenoxyacetyl, 2-methylphenoxyacetyl, 3-methylphenoxyacetyl, 4-methylphenoxyacetyl, 4- chloro-2-methylphenoxyacetyl, 2-nitrophenoxyacetyl, 3-nitrophenoxyacetyl, 4-nitrophenoxyacetyl, 2- isopropylphenoxyacetyl, 3-isopropylphenoxyacetyl, 4-isopropylphenoxyacetyl, 2-(t-butyl)phenoxyacetyl,20 3-(t-butyl)phenoxyacetyl, 4-(t-butyl)phenoxyacetyl, 2-fluorophenoxyacetyl,
  • R 1 when present, R 1 is C1-6alkyl and R P is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2-methylphenoxyacetyl, 4- 25 methylphenoxyacetyl, or 4-isopropylphenoxyacetyl.
  • R 1 when present, R 1 is C1-6alkyl and R P is methoxyacetyl (mac). In some embodiments, when present, R 1 is C1-6alkyl and R P is phenoxyacetyl (pac).
  • R 1 is methyl 1, 2, or 3; each R P2 is independently halogen, nitro, cyano, C 1-4 alkoxy, C 1-4 alkyl, C 1-4 haloalkyl; and each 30 R P3 is independently hydrogen, methyl, or ethyl.
  • R 1 is methyl 1, 2, or 3; each R P2 is independently halogen, nitro, cyano, C 1-4 alkoxy, C 1-4 alkyl, C 1-4 haloalkyl; and each 30 R P3 is independently hydrogen, methyl, or ethyl.
  • R 1 is methyl and R P is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 3-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2,4- dichlorophenoxyacetyl, 2-methylphenoxyacetyl, 3-methylphenoxyacetyl, 4-methylphenoxyacetyl, 4- chloro-2-methylphenoxyacetyl, 2-nitrophenoxyacetyl, 3-nitrophenoxyacetyl, 4-nitrophenoxyacetyl, 2- 5 isopropylphenoxyacetyl, 3-isopropylphenoxyacetyl, 4-isopropylphenoxyacetyl, 2-(t-butyl)phenoxyacetyl, 3-(t-butyl)phenoxyacetyl, 4-(t-butyl)phenoxyacetyl, 2-fluor
  • R 1 when present, R 1 is methyl and R P is methoxyacetyl (mac), phenoxyacetyl 10 (pac), 2-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2-methylphenoxyacetyl, 4-methylphenoxyacetyl, or 4-isopropylphenoxyacetyl.
  • R 1 when present, R 1 is methyl and R P is methoxyacetyl (mac). In some embodiments, when present, R 1 is methyl and R P is phenoxyacetyl (pac).
  • R 1 is hydrogen wherein r is 15 0, 1, 2, or 3; each R P2 is independently halogen, nitro, cyano, C 1-4 alkoxy, C 1-4 alkyl, C 1-4 haloalkyl; and each R P3 is independently hydrogen, methyl, or ethyl.
  • R 1 is hydrogen and R P is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 3-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2,4- dichlorophenoxyacetyl, 2-methylphenoxyacetyl, 3-methylphenoxyacetyl, 4-methylphenoxyacetyl, 4-20 chloro-2-methylphenoxyacetyl, 2-nitrophenoxyacetyl, 3-nitrophenoxyacetyl, 4-nitrophenoxyacetyl, 2- isopropylphenoxyacetyl, 3-isopropylphenoxyacetyl, 4-isopropylphenoxyacetyl, 2-(t-butyl)phenoxyacetyl, 3-(t-butyl)phenoxyacetyl, 4-(t-butyl)phenoxyacetyl, 2-fluorophenoxyacetyl, 3-fluorophenoxyacetyl, 2-fluorophenoxy
  • R 1 when present, R 1 is hydrogen and R P is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2-methylphenoxyacetyl, 4- methylphenoxyacetyl, or 4-isopropylphenoxyacetyl.
  • R 1 when present, R 1 is hydrogen and R P is methoxyacetyl (mac).
  • R 1 when present, R 1 is hydrogen and R P is phenoxyacetyl (pac).
  • R T is R T1 . 127 ME1 ⁇ 53466565.v1 Atty.
  • R T is -G 0 -OR T1 , wherein R T1 is as defined for Formula (X) and G 0 is selected from: ( a) G0 is absent or -D0-E0-F0-, wherein D0, E0, and F0 are independently a bond, C1-10alkyl, C2- 10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of 5 which is optionally substituted with 1, 2, 3, or 4 R groups; ( b) G0 is -D0-E0-F0-, wherein D 0 and F 0 are independently a bond or C 1-10 alkyl optionally substituted with 1, 2, 3, or 4 R groups; and E 0 is C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is
  • G0 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups (e.g., 1 or 2 R groups);
  • G0 is C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups;
  • 5 G0 is C1-10alkyl optionally substituted with 1 or 2 R groups;
  • G0 is C1-10alkyl, optionally substituted with -O(Ra), wherein Ra is independently hydrogen or C1- 6alkyl;
  • j G0 is C1-10alkyl, and 10
  • G0 is absent (a bond); wherein * represents the bond to L’, the broken bond represents the bond to OR T1 , and R is -C 1-6 alkyl- OR a or -OR a , wherein R a is
  • Formula (X) 15 In some embodiments of Formula (X), -L’- is *-G-L1-, wherein * is the bond to ZZ; and (a) L1 is a bond or -B-A-, wherein A is a bond, -O-, -S-, or -N(R N )-; B is a bond, CH 2 , C(O), C(S), C(NR N ), S(O), S(O) 2 , P(O)(OH), P(S)(OH), or P(S)(SH); each R N is independently hydrogen or C 1-6 alkyl; and 20 G is -D-E-F-, wherein D, E, and F are independently a bond, C 1-10 alkyl, C 2-10 alkenyl, C 2- 10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1,
  • G is C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; or ( e) L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and G is C1-10alkyl.
  • -L’- is *-G-[L2-G]q-L1-, wherein * is the bond to ZZ; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer selected from 1 to 8, an integer selected from 1 to 5, or an integer selected from 1 to 3); and ( a) L1 is a bond or -B-A-; each L2 is independently -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-; each B is independently a bond, CH2, C(O), C(S), C(NR N ), 10 S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each R N is independently hydrogen or C1-6alkyl; each G is independently -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkeny
  • each L1 is a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); 20 each L 2 is independently -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-, wherein R N is hydrogen or C1-6alkyl each B is independently a bond, CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G is independently C1-10alkyl, optionally substituted with 1, 2, 3, or 4 R groups; ( d) L1 is a bond, CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each L2 is independently - 25 A-B-A-; each A is independently a bond, -O-, -S-, or -N(R
  • -L’- is -[G-L2]q-G-L3-*, wherein * is the bond to ZZ; and 130 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO ( i) q is 0, 1, 2, 3, 4, or 5; each L2 is independently -A-B-A-; L3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-; each B is independently a bond, CH 2 , C(O), C(S), C(NR N ), S(O), S(O) 2 , P(O)(OH), P(S)(OH), or P(S)(SH); each R N is independently hydrogen or C 1-6 alkyl; each G is independently C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, each 5 of which
  • -L’- is *-L 3 -G-L 1 -, wherein * is the bond to ZZ; and ( a) L1 and L3 are independently -A-B-A-; each G is independently C1-10alkyl, C2-10alkenyl, C2- 10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is30 optionally substituted with 1, 2, or 3 R groups; each A is independently a bond, -O-, -S-, or - N(R N )-, wherein R N is hydrogen or C 1-6 alkyl; and each B is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH); ( b) L3 is -C(O)O- or C(O)N(RN)-, wherein RN is hydrogen or C1-6alkyl; L1 is -
  • L3 is -C(O)O- or C(O)N(RN)-, wherein RN is hydrogen or C1-6alkyl; L1 is -OP(O)(OH)O- or - OP(S)(OH)O-; and each G is independently C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with one R group;
  • L3 is -C(O)O- or C(O)N(RN)-, wherein RN is hydrogen or C1-6alkyl;
  • L1 is -OP(O)(OH)O- or - 5 OP(S)(OH)O-; and each G is independently C 3-10 cycloalkyl or 3-10 membered heterocyclyl; or
  • e) L3 is -C(O)O- or C(O)N(RN)-, wherein RN
  • -L’- is *-G-, wherein * is the bond to ZZ; and G is C1-10alkyl is optionally substituted with 1 or 2 R groups.
  • -L’- is --L 1 -[G-L 2 ]q-G-L 3 -*, wherein * is the bond to ZZ.
  • -L’- is -L 1 -G-L 3 -*, wherein * is the bond to ZZ.
  • -L’- is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein * is the bond to ZZ; q is 0, 1, 2, 3, 4, 15 or 5; L 1 is a bond or -B-A-; each L 2 is independently -A-B-A-; L 3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-; each B is independently a bond, CH2, C(O), C(S), C(NR N ), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each R N is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • -L’- is -L 1 -[G-L 2 ]q-G-*, wherein * is the bond to ZZ; q is 0, 1, 2, or 3; L 1 is a bond or -B-A-; each L 2 is independently a bond, C(O)O, OC(O), C(O)(NR N ), N(R N )C(O), SO2N(R N ), N(R N )SO 2 , OP(O)(OH), OP(S)(OH), P(O)(OH)O, P(S)(OH)O, OP(O)(OH)O, or OP(S)(OH)O, wherein each R N is independently hydrogen or C 1-6 alkyl; and each G is independently C 1-10 alkyl, C 2-10 alkenyl, C 2- 10 alkynyl, each of which is optionally substituted with 1 or 2 R groups.
  • -L’- is -L 1 -[G-L 2 ] q -G-*, wherein * is the bond to ZZ; q is 0, 1, 2, or 3; L 1 is a bond or -B-A-; each L 2 is independently a bond, C(O)O, OC(O), C(O)(NR N ), N(R N )C(O), OP(O)(OH)O, or OP(S)(OH)O, wherein each R N is independently hydrogen or C 1-6 alkyl; each G is independently C 1-10 alkyl or C 2-10 alkenyl, each of which is optionally substituted with 1 or 2 R groups.
  • -L’- is -[G-L 2 ] q -G-*, wherein * is the bond to ZZ; and 30 (a) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each L2 is independently C(O)O or OC(O); each G is independently C 1-10 alkyl, each of which is optionally substituted with 1 or 2 R groups; ( b) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each L2 is independently C(O)(NR N ) or N(R N )C(O), wherein each R N is independently hydrogen or C 1-6 alkyl; and 132 ME1 ⁇ 53466565.v1 Atty.
  • each G is independently C 1-10 alkyl, each of which is optionally substituted with 1 or 2 R groups;
  • ( c) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2);
  • each L2 is independently OP(O)(OH)O, or OP(S)(OH)O (e.g., each is OP(O)(OH)O); and each G is independently 5 C 1-10 alkyl, each of which is optionally substituted with 1 or 2 R groups;
  • ( d) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2);
  • each L2 is a bond; and each G is independently C 1-10 alkyl, each of which is optionally substituted with 1 or 2 R groups.
  • -L’- is -C2-30alkyl-*, wherein * is the bond to ZZ, such as -C5-20alkyl-* or -10 C10-20alkyl-*.
  • -L’- is -C(O)-C2-30alkyl-*, wherein * is the bond to ZZ, such as -C(O)- C5-20alkyl-* or -C(O)-C10-20alkyl-*.
  • L’-R T Embodiments wherein * is the 15 bond to ZZ; L 1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and R T1 is as defined for Formula ( X). In some embodiments, - wherein * is the bond to ZZ; L 1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and R T1 is as defined for Formula (X).
  • - wherein * is the 20 bond to ZZ; L 1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); R is -OH or -C1-6alkyl-OH; and R T1 is as defined for Formula (X). 133 ME1 ⁇ 53466565.v1 Atty.
  • L 1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH);
  • R is -OH or -C 1-6 alkyl-OH; and
  • R T1 is as defined for Formula (X).
  • -L’-R T is 5 wherein* is the bond to ZZ; L 1 is a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH); R is -OH or -C 1- 6 alkyl-OH; and R T1 is as defined for Form
  • -L’-R T is , wherein * is the bond to ZZ; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 10 1 to 5, or an integer from 1 to 3); each L 2 is ( i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)
  • each L 2 is 10 (i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, - OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl; or ( ii) independently selected from the group consisting of -C(O)O
  • -L’-RT is wherein * is the bond to ZZ; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, or an integer from 1 to 3); each L 2 is 5 (i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, - OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(R N )-,
  • -L’-R T is wherein * is the bond to ZZ; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1-8; or 1-5; or 1-3); 25 each L 2 is ( i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, - OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl; or 136 ME1 ⁇ 53466565.v1 Atty.
  • each L 2 is 20 (i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, - OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl; or ( ii) independently selected from the group consisting of -C(O)O-, -OC(O)
  • -L’-R T is 10 wherein* is the bond to ZZ; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, or an integer from 1 to 3); each L 2 is ( i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - 15 N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, - OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl; or ( ii) independently selected from the group consisting of -
  • each L 2 is 5 (i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, - OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl; or ( ii) independently selected from the group consisting of -
  • -L’-R T is 25 wherein* is the bond to ZZ; L 1 is a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH); R is -O(R a ) or -C 1- 6 alkyl-O(R a ), wherein R a is hydrogen; and R T1 is as defined for Formula (X). 139 ME1 ⁇ 53466565.v1 Atty.
  • R L is -N(R 3 )(R 4 ), -O(R 5 ), -S(R 5 ), or -R 5 , wherein R 5 is according to one Formulae (x-a) through (x-ab): 140 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (x-ab) wherein R P3 is hydrogen and L, ZZ, L’ and R T1 are as defined for Formula (X) or any embodiment herein.
  • R L is according to one of Formulae (xi-a) through (xi-m): 141 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (xi-m) wherein R P3 is hydrogen, and L, ZZ, L’ and R T1 are as defined for Formula (X) or any embodiment herein.
  • R L is according to one of Formulae (xi-n) through (xi-z): 142 ME1 ⁇ 53466565.v1 Atty.
  • R P3 is hydrogen
  • L, ZZ, L’ and R T1 are as defined for Formula (X) or any embodiment herein.
  • R L is -N(R 3 )(R 4 ), wherein R 3 is hydrogen or C1-6alkyl and R 4 is R 5 .
  • R L is -N(R 3 )(R 4 ), wherein R 3 is hydrogen and R 4 is R 5 .
  • R L is -N(R 3 )(R 4 ), wherein R 3 is C1-3alkyl and R 4 is R 5 .
  • R L is -N(R 3 )(R 4 ), wherein R 3 is methyl and R 4 is R 5 .
  • R L is --N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R 5 .
  • R L is --N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen 10 atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R 5 , provided that R 3 and R 4 taken together with the nitrogen atom to which they are attached do not form a morpholino group.
  • R L is --N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen atom to which they are attached form a group that is piperidinyl, piperazinyl, pyrrolidinyl, 15 imidazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, azetidinyl, pyrrolinyl, imidazolinyl, or pyrazolinyl, each substituted with R 5 .
  • 143 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, .
  • R L is -N(R 3 )(R 4 ). In one embodiment the Formula (X), or a salt thereof, R L is -N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl 5 group that is substituted with R 5 . In some embodiments, R L is -O(R 5 ). In some embodiments, R L is -R 5 .
  • RL is , wherein * is a bond to a ZZ group; s is 1 – 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an 10 integer from 4 to 6). I n some embodiments, RL is , wherein * is a bond to a ZZ group; t is 0 to 10 (e.g., 1-5; or 1-3; or 1; or 2; or 3).
  • L Embodiments, Formula (X) 15 In some embodiments of any one of Formula (X) and any embodiments thereof, L is -L 1 -[G-L 2 ]q- G-L 3 -*, wherein * is the bond to ZZ.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 0, 1, 2, 3, 4, or 5.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 0, 1, 2, 3, or 4
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 0, 1, 2, or 3.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 0, 1, or 2.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 1, 2, 3, 4, or 5.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 1, 2, 3, or 4.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 1, 2, or 3.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 1 or 2.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 4. In another embodiment , L is - L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 3. In another embodiment, L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 2. In another embodiment, L is -L 1 -G-L 2 -G-L 3 -*. In another embodiment, L is -L 1 -G-L 3 -*. In another embodiment, L is -G-L 3 -*. In another embodiment, L is -L 1 -G-*. In another embodiment, L is -L 1 -G-*.
  • L is - G-*. 30 In another embodiment, wherein L is -[G-L 2 ] q -G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q i s 3; or q is 4; or q is 5).
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R N )-, -N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, - 144 ME1 ⁇ 53466565.v1 Atty.
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R N )-, -N(R N )C(O)-, -OC(O)N(R N )-, -N(R N )C(O)O-,- 5 N(R N )C(O)N(R N )-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)N(R N )-, -N(R N )C(O)-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -O-, and - N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • each instance of A-B-A- is independently selected from the group 10 consisting of -C(O)O-, -OC(O)-, -C(O)N(R N )-, -N(R N )C(O)-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl.
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)N(R N )-, -N(R N )C(O)-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl.
  • D and F are each independently a bond, C1-10alkyl, C2-10alkenyl, or C2- 10alkynyl, each optionally substituted with 1, 2, 3, or 4 R groups; and E is C1-10alkyl, C2-10alkenyl, C2- 10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • D and F are each independently a bond or C1-10alkyl optionally 20 substituted with 1, 2, 3, or 4 R groups; and E is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3- 10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted 25 with 1, 2, 3, or 4 R groups.
  • each G is independently C1-10alkyl, optionally substituted with 1, 2, or 3 R groups. In some embodiments , each G is independently C1-10alkyl, optionally substituted with 1 or 2 R groups. In some embodiments, each G is independently C 1-10 alkyl, optionally substituted with one R group . In some embodiments, each G is independently C 1-10 alkyl.
  • L is -L 1 -G-L 3 -*, wherein * is the bond to ZZ ;
  • G is -D-E-F-, wherein D, E, and F are independently a bond, C 1-10 alkyl, C 2-10 alkenyl, C 2- 10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups;
  • L 1 is -B-A-; 35 L 3 is a bond or -A-B-A-; 145 ME1 ⁇ 53466565.v1 Atty.
  • each A is independently a bond, -O-, -S-, or -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • each B is independently a bond, CH 2 , C(O), S(O) 2 , P(O)(OH), or P(S)(OH); and
  • L is -L 1 -G-L 3 -*, wherein * is the bond to ZZ;
  • 5 G is C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups;
  • L 1 is -B-;
  • L 3 is a bond or -A-B-A-;
  • each A is independently a bond, -O-, -S-, or -N(R N )
  • L is -L 1 -G-*, wherein * is the bond to ZZ;
  • 20 G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1 or 2 R groups;
  • L 1 is -B-A-, wherein A is a bond, -O-, -S-, or -N(R N )-, each R N is independently hydrogen or C1-6alkyl; and B is a bond, C(O), S(O)2, P(O)(OH), or P(S)(OH).
  • L is -L 1 -G-*, wherein * is the bond to ZZ; G is C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups; L 1 is bond, C(O), S(O)2, P(O)(OH), or P(S)(OH); and R N is hydrogen or C 1-6 alkyl.
  • L is -L 1 -G-*, wherein * is the bond to ZZ , G is C 1-10 alkyl or C 2-10 alkenyl, 30 and L 1 is -B-A-, wherein, A is a bond, -O-, -S-, or -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl; and B is a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH).
  • L is -L 1 -G-*, wherein * is the bond to ZZ , G is C 1-10 alkyl or C 2-10 alkenyl, L 1 is -O-, -S-, or -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • L is -L 1 -G-*, wherein * is the bond to ZZ , G is C 2-10 alkyl, L 1 is -O-, -S-, 35 or -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • 146 ME1 ⁇ 53466565.v1 Atty.
  • L is -L 1 -G-*, wherein * is the bond to ZZ , G is C 2-10 alkyl, L 1 is -O- (e.g., G is C 3- 10 alkyl or C 4-10 alkyl or C 5-10 alkyl or C 6-10 alkyl or C 2-8 alkyl or C 4-8 alkyl or C 4-8 alkyl or C 4 alkyl or C 5 alkyl o r C6alkyl).
  • L is wherein * is the bond to ZZ; k is an integer from 1 to 10; L 1 is bond, C(O), C(S), C(NR N ), S(O) 2 , P(O)(OH), or P(S)(OH); and R N is hydrogen or C 1- 5 6 alkyl.
  • L is wherein * is the bond to ZZ; k is an integer from 1 to 10; L 1 is bond, C(O), P(O)(OH), or P(S)(OH).
  • L is , wherein * is the bond to ZZ; k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 3 to 10; or an integer from 4 to 10; or an integer from 5 10 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7.
  • L is L wherein * is the bond to ZZ; L 1 is bond; and k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 3 to 10; or an integer from 4 to 10; or an integer from 5 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7.
  • each G is indepdently C 1-10 alkyl and L 2 is O, S, or N(H).
  • L is -[G-L 2 ] q -G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q 15 is 3; or q is 4; or q is 5)
  • each G is indepdently C 1-10 alkyl and L 2 is O, S, or N(H).
  • L is -[G-L 2 ] q -G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q is 3; or q is 4; or q is 5)
  • each G is indepdently C 1-10 alkyl and L 2 is O.
  • L is -[CH 2 CH 2 O] q -G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q is 3; or q is 4; or q is 5), G is indepdently C 1-10 alkyl.
  • L is , wherein * is the bond to ZZ; t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3). I n some embodiments, L is , wherein * is the bond to ZZ.
  • L is , wherein * is the bond to ZZ; t is 1 to 10 (e.g., 1-5; or 1-3; or 1; or 2; or 3); and z is 1-10 (e.g., 1-6; or 1-4; or 1; or 2; or 3 or 4 or 5 or 6). 25 In some embodiments, L is , wherein * is the bond to ZZ, and t is an integer from 1 to 5 (e.g., 1; or 2; or 3). 147 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO I n some embodiments, L is , wherein * is the bond to ZZ.
  • L is wherein * is the bond to ZZ, t is an integer from 0 to 10 (e.g., an integer from 1 to 5 or 1; or 2; or 3); a is an integer from 1 to 3; and s and s’ are each independently an integer from 1 to 24 (e.g., an integer from 1 to 16; an integer from 1 to 10; an integer from 3 to 10; an 5 integer from 3 to 7; or an integer from 4 to 6).
  • * is the bond to ZZ; a is 1, 2 or 3; and each s, s’, and s” independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6).
  • L is wherein * is the bond to ZZ; and s, s’, 10 and s’’ are independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6).
  • L is , wherein * is the bond to ZZ and each s, s’, and s” independently is an integer from 1 to 24(e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6) 15
  • L is wherein * is the bond to ZZ; s and k are independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 10 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6).
  • L is wherein * is the bond to ZZ and w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6).
  • w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6).
  • L is C 1-20 alkyl. In some embodiments, L is C 2-20 alkyl. In some embodiments, L is C 6-20 alkyl. In some embodiments, L is C 8-12 alkyl. In some embodiments, L is C 10 alkyl.
  • L is -G-L 2 -G-*, wherein L 2 is -A-B-A-; each A is independently a bond, 5 -O-, -S-, or -N(R N )-; each B is independently a bond, CH 2 , C(O), C(S), C(NR N ), S(O), S(O) 2 , P(O)(OH), P(S)(OH), or P(S)(SH); each R N is independently hydrogen or C 1-6 alkyl, or two R N within an -A-B-A- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; and each G is independently C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • L is -G 10 -L 2 -G 20 -*, wherein L 2 is -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-; each B is independently a bond, CH2, C(O), C(S), C(NR N ), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each R N is independently hydrogen or C1-6alkyl, or two R N within an - A-B-A- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; G 10 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, 15 aryl, or heteroaryl; and G 20 is C1-10alkyl, C2-10alkenyl, C2-10alkyl
  • L is -G 10 -L 2 -G 20 -*, wherein L 2 is a bond, -O-, -S-, or -N(R N )-; R N is independently hydrogen or C1-6alkyl, G 10 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl; and G 20 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 20 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • L is -G 10 -L 2 -G 20 -*, wherein L 2 is a bond, -O-, -S-, or -N(R N )-; R N is independently hydrogen or C1-6alkyl, G 10 is aryl or heteroaryl; and G 20 is C1-10alkyl or C2-10alkenyl.
  • L is -G 10 -L 2 -G 20 -*, wherein L 2 is a bond, -O-, G 10 is aryl (e.g., phenyl);25 and G 20 is C1-10alkyl.
  • RP3 when present, is hydrogen.
  • the compound of Formula (X) is according to one of Formulae (X-b) through (X-e) and (X-x) through (X-ab): 149 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (X-x) 150 ME1 ⁇ 53466565.v1 Atty.
  • R P is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group), wheren R and the remaining variables are as defined in Formula (X).
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • R 1 is hydrogen and R P is hydrogen.
  • R 1 is hydrogen and a nitrogen protecting group.
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and a nitrogen protecting group.
  • 10 -L-ZZ-L’-R T Embodiments In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-R T is 151 ME1 ⁇ 53466565.v1 Atty.
  • -L-ZZ-L’-R T is (x-b).
  • -L-ZZ-L’-R T is (x-c).
  • -L-ZZ-L’-R T is In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-R T is In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-R T is In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) 10 through (X-ab), -L-ZZ-L’-R T is (x-f).
  • -L-ZZ-L’-R T is 152 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (x-g).
  • -L-ZZ-L’-R T is (x-h).
  • -L-ZZ-L’-R T is (x-i). 5
  • -L-ZZ-L’-R T is (x-j).
  • -L-ZZ-L’-R T is (x-k).
  • -L-ZZ-L’-R T is 153 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • -L-ZZ-L’-R T is (x-m).
  • -L-ZZ-L’-R T is (x-n). 5 In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-R T is (x-o). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-R T is (x-p).
  • -L-ZZ-L’-R T is 154 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (x-q).
  • -L-ZZ-L’-R T is (x-r)
  • -L-ZZ-L’-R T is (x-s).
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to the 3’-end of the oligonucleotide, the 5’-end of the oligonucleotide, or an internal 2’- or 3’ position on a internal nucleotide (i.e., a nucleotide that is not the 5’-terminal or 3’-terminal nucleoside).
  • L L is a bond.
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to 15 the 3’-end of the oligonucleotide, such as one of : directly to the 3’-carbon of the 3’-terminal nucleoside; directly to the 3’-O of the 3’-terminal nucleoside; directly to the 4’-carbon of the 3’-terminal nucleoside; directly to the 2’-carbon of the 3’-terminal nucleoside; or 20 directly to the 2’-O of the 3’-terminal nucleoside.
  • L L is a bond.
  • R T1 is -L L -oligonucleotide, wherein L L L is a divalent linker that connects to 15 the 3’-end of the oligonucleotide, such as one of : directly to the 3’-carbon of the 3’-terminal nucleoside; directly to the 3’
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to the 5'-end of the oligonucleotide, such as one of: directly to the 5’-carbon of the 5’-terminal nucleoside; directly to the 5’-O of the 5’-terminal nucleoside; 5 directly to the 4’-carbon of the 5’-terminal nucleoside; directly to the 2’-carbon of the 5’-terminal nucleoside; or directly to the 2’-O of the 5’-terminal nucleoside.
  • L L is a divalent linker that connects to the 5'-end of the oligonucleotide, such as one of: directly to the 5’-carbon of the 5’-terminal nucleoside; directly to the 5’-O of the 5’-terminal nucleoside; 5 directly to the 4’-carbon of the 5’-terminal nucleoside; directly to the 2’-carbon of the
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to an internal 2’- or 3’ position on an internal nucleotide. 10 In some embodiments, R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to an internal 2’- position on a internal nucleotide.
  • R T is R T1 , wherein R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to an internucleotide linkage (i.e., to an oxygen atom in a phosphodiester linkage to form a phosphotriester; or to a nitrogen when the internucleotide linkage is a phosphoroamidate).
  • L L is a divalent linker that connects to an internucleotide linkage (i.e., to an oxygen atom in a phosphodiester linkage to form a phosphotriester; or to a nitrogen when the internucleotide linkage is a phosphoroamidate).
  • L L when L L connects to a carbon atom on a nucleoside, then L L is -B 3 -A 3 -, wherein B 3 is -P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH)-; and A 3 is -O-, -S-, or -N(H)-.
  • L L when L L connects to a carbon atom on a nucleoside, then L L is -B 3 -A 3 -, wherein B 3 is -P(O)(OH)- or-P(S)(OH)-; and A 3 is -O-.
  • L L when L L connects to a oxygen atom on a nucleoside, then L L is - P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH). In another embodiment, when L L connects to a oxygen atom on a nucleoside, then L L is -P(O)(OH)- or -P(S)(OH)-. In another embodiment, when L L connects to a oxygen atom on a nucleoside, then L L is -P(O)(OH)-. In another embodiment, when L L connects to a oxygen atom on a nucleoside, then L L is -P(S)(OH)-.
  • LL when LL connects to an oxygen atom on a nucleoside, then LL is -B3-A3- L L1 -A 3 -B 3 -, wherein B 3 is a bond, -C(O)-, C(S)-, C(NH), S(O), S(O)2, -P(O)(OH)-, -P(S)(OH)-, or - P(S)(SH); A 3 is a bond, -O-, -S-, or -N(H)- ; and L L1 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 1 0 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl.
  • R T is R T1 wherein R T1 is is -L L -oligonucleotide, when L L connects to a oxygen atom on a nucleoside, and L L is a bond, the nucleoside is of Formula (X-f), 156 ME1 ⁇ 53466565.v1 Atty.
  • B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil);
  • L is according any of the preceding embodiments; and * represent the bond to ZZ.
  • -L’- is -L 1 -G-L 3 -*, wherein * is the bond to ZZ.
  • -L’- is -C 2-30 alkyl-*, wherein the alkyl is optionally substituted with one or two R groups, and * is the bond to ZZ.
  • -L’- is -C 2-30 alkyl-*, wherein the alkyl is optionally substituted with one or two groups selected from the group consisting of halogen, hydroxy, C 1- 6 alkoxy, amino, C l-6 alkylamino, di(C 1-6 alkylamino), cyano, carboxy , and * is the bond to ZZ.
  • -L’- is -C 2-30 alkyl-*, wherein the alkyl is optionally substituted with one group selected from 10 the group consisting of halogen, hydroxy, C 1-6 alkoxy, amino, C l-6 alkylamino, di(C 1-6 alkylamino), cyano, carboxy , and * is the bond to ZZ.
  • -L’- is -C 2-30 alkyl-*, wherein the alkyl is optionally substituted with one group selected from the group consisting of hydroxy, amino, and carboxy , and * is the bond to ZZ.
  • -L’- is -C 2-30 alkyl-*, wherein the alkyl is optionally substituted with hydroxy, and * is the bond to ZZ.
  • -L’- is -C 2-30 alkyl-*, wherein * is the bond to ZZ.
  • - L’- is -C 2-16 alkyl-*, wherein * is the bond to ZZ.
  • -L’- is -C 4-12 alkyl-*, wherein * is the bond to ZZ.
  • -L’- is -C 4-10 alkyl-*, wherein * is the bond to ZZ.
  • -L’- is -C 5-10 alkyl-*, wherein * is the bond to ZZ. In some embodiments, -L’- is -C 6 alkyl-*, wherein * is the bond to ZZ. In some embodiments, -L’- is -C 8 alkyl-*, wherein * is the bond to ZZ. In 20 some embodiments, -L’- is -C10alkyl-*, wherein * is the bond to ZZ.
  • -L’- is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein * is the bond to ZZ; q is 0, 1, 2, 3, 4, or 5; L 1 is a bond or -B-A-; each L 2 is independently -A-B-A-; 25 L 3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-; each B is independently a bond, CH2, C(O), C(S), C(NR N ), S(O)2, P(O)(OH), or P(S)(OH); each R N is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally 30 substituted with 1, 2, 3, or 4 R groups.
  • -L’- is -L 1 -[G-L 2 ] q -G-*, wherein * is the bond to ZZ, q is 0, 1, 2, or 3;
  • -L’- is -[G-L 2 ]q-G-*, wherein * is the bond to ZZ, q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each G is independently C1-10alkyl, each of which is optionally 15 substituted with 1 or 2 R groups, and one of: ( a) each L2 is independently C(O)O or OC(O); (b) each L2 is independently C(O)(NRN) or N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl ( c) each L2 is independently OP(O)(OH)O, or OP(S)(OH)O (e.g., each is OP(O)(OH)O); 20 or ( d) each L2 is a bond.
  • the compound of Formula (X) is according to one of Formulae (X-g) through (X-q): 158 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (X-q) wherein B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); each m is independently integer selected from 1-20 (e.g., 2-12, or 2-10; or 2-6; or 2; or 3; or 4; or 5; or 6).
  • B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); each n is independently 0
  • the compound of Formula (X) is according to one of Formulae (X-r) through (X-w): 159 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 L and ZZ are as defined in Formula (X) or in any embodiment preceding or below; 160 ME1 ⁇ 53466565.v1 Atty.
  • Docket No.121301-23820/ALN-523-WO B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); each m is independently integer selected from 1-20 (e.g., 2-12, or 2-10; or 2-6; or 2; or 3; or 4; or 5; or 6); 5 each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); R P is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group); and R 1 is hydrogen or C 1-6 alkyl (e.g., methyl or t-butyl).
  • nucleobase e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil
  • each m is independently integer selected from 1-20
  • R 1 is hydrogen. In another embodiment of Formulae (X-r) through (X-w), R 1 is C1-6alkyl (e.g., methyl or t-butyl). In another embodiment of 10 Formulae (X-r) through (X-w), R 1 is hydrogen and R P is hydrogen. In another embodiment of Formulae (X-r) through (X-w), R 1 is hydrogen and a nitrogen protecting group. In another embodiment of Formulae (X-r) through (X-w), R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl) and a nitrogen protecting group.
  • two adjacent nucleosides in the oligonucleotide have one of the formula 15 according any of the preceding embodiments; and B is an optionally modified nucleobase.
  • each nucleoside of the two adjacent nucleosides is independently according to any one of Formula (X-f) 20 through (X-q).
  • each nucleoside is according to the same Formula. 161 ME1 ⁇ 53466565.v1 Atty.
  • three adjacent nucleosides in the oligonucleotide have the formula , 5 wherein each Y is independently O or S (or O; or S; or O then S or S then O, 5’ followed by 3’); represents the remainder for the oligonucleotide, L’ is according any of the preceding e mbodiments;and B is an optionally modified nucleobase; e.g., each Y is O .
  • each nucleoside of the three adjacent nucleosides is independently according to any one of Formula (X-f) through (X-q). In certain embodiments, each nucleoside is according to the same Formula.
  • each nucleoside in the oligonucleotide has the formula , , 162 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein each Y is independently O or S; represents the remainder for the oligonucleotide, L’ is according any of the preceding embodiments;and B is an optionally modified nucleobase; e.g., each 5 Y is O.
  • each nucleoside of the four adjacent nucleosides is independently according to any one of Formula (X-f) through (X-q). In certain embodiments, each nucleoside is according to the same Formula.
  • each nucleoside is according to the same Formula (X-u) or (X-w).
  • R T is R T1
  • R T1 is -L L -oligonucleotide
  • the internucleotide linkage 10 can be of the formula, including the 3’ and 5’ oxygen atoms of the preceding and following nucelosides, respectively, (X-pa) (X-pb) (X-pc) wherein L’ can be, for example a bond, -S(O)2- or, in for Formula (X-pc), a 5 -8 membered heterocyclyl ring optionally substituted with 1 or 2 R groups, as defined herein ; and * represent the bond to ZZ.
  • the preceding includes, 15 wherein * represent the bond to ZZ; and R N5 is hydrogen or C 1-10 alkyl. 163 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • the preceding includes, (X-pg) (X-ph) (X-pi) wherein * represent the bond to ZZ; m is an integer selected from 1 – 20 (e.g., 1-10, or 2-20, or 2-10, or 4-10, or 4-8; or 6-12; or 5; or 6; or 7; or 8; or 9; or 10), and R N5 is hydrogen or C 1-10 alkyl.
  • the compound of Formula (X-pd) is 5 wherein Y’ is O or S, and R Y , Y, R 1 , L, L’, and ZZ are as defined for Formula (X).
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • the compound is 10 164 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S, and R P , R 1 , L, L’, and ZZ are as defined for Formula (X).
  • the compound is 5 165 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein each m is an integer selected from 1 – 20 (e.g., 2-20, 2-10, 1-10, 2-16, 4-16, 4-8, or 6- 5 12), each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); Y’ is O or S, and R P , R 1 , L, L’, and ZZ are as defined for Formula (X).
  • the compound of Formula (X-pe) is wherein Y’ is O or S, and R Y , Y, R 1 , L, L’, and ZZ are as defined for Formula (X).
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • 166 ⁇ 53466565.v1 Atty.
  • Y’ is O and R 1 is hydrogen. In one embodiment, Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 10 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is S
  • R 1 is hydrogen
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound is 20 167 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S, and R P , R 1 , L, L’, and ZZ are as defined for Formula (X).
  • 5 Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen. In another embodiment, R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen. In another embodiment, R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 168 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • Y’ is O and R 1 is hydrogen. In one embodiment, Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen . In one embodiment, Y’ is O and R P is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is O
  • R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 10 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound is 20 169 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein each m is an integer selected from 1 – 20 (e.g., 2-20, 2-10, 1-10, 2-16, 4-16, 4-8, or 6-12), Y’ is 5 O or S, and R P , R 1 , L, L’, and ZZ are as defined for Formula (X).
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., 10 t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 170 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • Y’ is O and R 1 is hydrogen. In one embodiment, Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen .
  • R P is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 10 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at the 5’-end of the 20 oligonucleotide, in one embodiment, R T1 can be represented as Formula (X-5’) wherein L L is -P(Y)(OH)-, wherein Y is O or S (e.g., S); and * represents the bond to remainder of the compound of Formula (X).
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at the 25 5’-end of the oligonucleotide, in one embodiment, R T1 can be represented as Formula (X-5’o) or Formula (X-5’s), wherein * represents the bond to remainder of the compound of Formula (X). 171 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In an embodiment of Formula (X-5’), (X-5’o), and Formula (X-5’s), -L-ZZ-L’-R T represents any one of Formulae (x-a) through (x-s) .
  • -L-ZZ-L’-R T represents Formula In other embodiments-L-ZZ-L’-R T represents one of the following formulae: 5 In other embodiments of Formula (X-5’), (X-5’o), and Formula (X-5’s), L-ZZ-L’-R T represents, (x-m) In other embodiments of Formula (X-5’), (X-5’o), and Formula (X-5’s), L-ZZ-L’-R T represents, (x-s).
  • the compound of Formula (X) is 172 ME1 ⁇ 53466565.v1 Atty.
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at the 3’-end of the 5 oligonucleotide, in one embodiment, R T1 can be represented as Formula (X-3’) wherein L L is -P(Y)(OH)-, wherein Y is O or S (e.g., S); and * represents the bond to remainder of the compound of Formula (X).
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at the 10 3’-end of the oligonucleotide, in one embodiment, R T1 can be represented as Formula (X-3’o) or Formula (X-3’s), wherein * represents the bond to remainder of the compound of Formula (X).
  • -L-ZZ-L’-R T represents any 15 one of Formulae (x-a) through (x-ab) .
  • -L-ZZ-L’-R T represents Formula (x-e), .
  • -L-ZZ-L’-R T represents one of the following formulae: 173 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (x-a) (x-b) (x-c) (x-aa) (x-ab)
  • L-ZZ-L’-R T represents In other embodiments of Formula (X-3’), (X-3’o), and Formula (X-3’s)
  • L-ZZ-L’-R T represents 174 ME1 ⁇ 53466565.v1 Atty.
  • L-ZZ-L’-R T represents (x-p).
  • L-ZZ-L’-R T represents In other embodiments of Formula (X-3’), (X-3’o), and Formula (X-3’s), L-ZZ-L’-R T represents (x-r). 5
  • R L is 175 ME1 ⁇ 53466565.v1 Atty.
  • the compound of Formula (X) is 5 wherein Y' is O or S, and R Y , R 1 , Y, L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • the compound of Formula (X) is 176 ME1 ⁇ 53466565.v1 Atty.
  • Y’ is O or S
  • R Y , Y, R 1 , L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof 5
  • the compound of Formula (X) is , wherein Y’ is O or S, and R P , R 1 , L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S. 177 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen . In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 10 (pac)). In one embodiment, Y’ is S and R P is hydrogen. In one embodiment, Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen 15 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen 15 protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxy
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is 20 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 178 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein Y’ is O or S, and R P , R 1 , L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- 5 butyl).
  • -COOR 1 is replaced with a carboxylic acid mimic, such as a tetrazolyl group (e.g., 1,2,3,4-tetrazol-5-yl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C 1-6 alkyl 10 (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen.
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is S and R P is a 15 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a 15 nitrogen protecting group
  • Y’ is O
  • R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t- butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 25 (pac)).
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 179 ME1 ⁇ 53466565.v1 Atty.
  • the compound of Formula (X) is wherein Y’ is O or S, and R P , R 1 , L’, and L are as defined for Formula (X) or any embodiment thereof. 5
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen. In one embodiment, Y’ is O and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen . In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 15 (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl 15
  • Y’ is S and R P is hydrogen .
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen 20 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t- butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is 5 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 10 , wherein each m is independently an integer selected from 1-10; n is 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • each m is independently an integer selected from 2-10; or 2-8, or 2-6. 15
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 181 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (pac)).
  • Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 5 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- 10 butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 15 wherein Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- 20 butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen . In one embodiment, Y’ is O and R P is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen. In one embodiment, Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 10 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 20 183 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S, and R P , R 1 , L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). 10
  • Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is O, R 1 is C1-6alkyl (e.g.,20 methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 184 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S, and R P , R 1 , L’, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen. In another embodiment, R 1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, R P is hydrogen. In another embodiment, R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R 1 is hydrogen. In one embodiment, Y’ is O and R 1 is C1-6alkyl 10 (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen. In one embodiment, Y’ is S and R P is a 15 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one 20 embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), 185 ME1 ⁇ 53466565.v1 Atty.
  • Boc t-butoxycarbonyl
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t- butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 5 (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 5 (pac)).
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 10 wherein each m is independently an integer selected from 1-10; n is 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • each m is independently an integer selected from 2-10; or 2-8, or 2-6.
  • Y’ is O.
  • Y’ is S. 15
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C 1-6 alkyl 20 (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 186 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (pac)).
  • Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen 5 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t- butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is 10 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 15 wherein Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen . In one embodiment, Y’ is O and R P is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen. In one embodiment, Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 10 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 20 , wherein Y’ is O or S, and R P , R 1 , L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is O
  • R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 10 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 20 wherein Y’ is O or S, and R P , R 1 , L’, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • 189 ME1 ⁇ 53466565.v1 Atty.
  • Y’ is O and R 1 is hydrogen. In one embodiment, Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 10 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is S
  • R 1 is hydrogen
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 20 wherein each m is independently an integer selected from 1-10; Y’ is O or S, and R P and R 1 , are as defined for Formula (X) or any embodiment thereof.
  • each m is independently an integer selected from 2-10; or 2-8, or 2-6.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen. In another embodiment, R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen. In another embodiment, R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 190 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • Y’ is O and R 1 is hydrogen. In one embodiment, Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen . In one embodiment, Y’ is O and R P is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is O
  • R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 10 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 20 wherein Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 191 ME1 ⁇ 53466565.v1 Atty.
  • Y’ is O and R 1 is hydrogen. In one embodiment, Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 10 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is S
  • R 1 is hydrogen
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 20 , wherein Y’ is O or S, and R P , R 1 , L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl). 25
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen .
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 5 (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), 10 benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen protecting group e.g., t-butoxycarbonyl (Boc), 10 benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting 15 group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is wherein Y’ is O or S, and R P , R 1 , L’, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S. 20
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1-6alkyl 25 (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen. In one embodiment, Y’ is S and R P is a 193 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen 5 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen 5 protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxy
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t- butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is 10 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 15 wherein each m is independently an integer selected from 1-10; Y’ is O or S, and R P and R 1 , are as defined for Formula (X) or any embodiment thereof.
  • each m is independently an integer selected from 2-10; or 2-8, or 2-6.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen. In another embodiment, R 1 is C 1-6 alkyl (e.g., methyl or t- 20 butyl). In one embodiment, R P is hydrogen. In another embodiment, R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R 1 is hydrogen. In one embodiment, Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen.
  • Y’ is S 25 and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen.
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 194 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (pac)).
  • Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is O
  • R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 5 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- 10 butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 15 wherein Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). 25 In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen.
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), 5 benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t- butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen 10 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is , wherein Y’ is O or S, and R P , R 1 , L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S. 15
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C 1-6 alkyl 20 (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen. In one embodiment, Y’ is S and R P is a nitrogen protecting 25 group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), 196 ME1 ⁇ 53466565.v1 Atty.
  • Boc t-butoxycarbonyl
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen. In one embodiment, Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t- butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 5 (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 5 (pac)).
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is , wherein Y’ is O or S, and R P , R 1 , L’, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one 20 embodiment, Y’ is S and R P is hydrogen.
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t- butyl) and R P is hydrogen.
  • Y’ is O
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 197 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (pac)).
  • Y’ is S
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is , 5 wherein each m is independently an integer selected from 1-10; Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • each m is independently an integer selected from 2-10; or 2-8, or 2-6.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen. In another embodiment, R 1 is C 1-6 alkyl (e.g., methyl or t- 10 butyl). In one embodiment, R P is hydrogen. In another embodiment, R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R 1 is hydrogen. In one embodiment, Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen.
  • Y’ is S 15 and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen. In one embodiment, Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is O, R 1 is C1-6alkyl (e.g.,25 methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 198 ME1 ⁇ 53466565.v1 Atty.
  • Y’ is S
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is , 5 wherein Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group 10 (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen 15 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen. In one embodiment, Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen 20 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen 20 protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxy
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is 25 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • 199 ME1 ⁇ 53466565.v1 Atty.
  • Formula (X) In embodiments of Formula (X), Formula (X-a) through (X-w), including embodiments of Formulae (x-a) through (x-s) and (xi-a) through (xi-z) ZZ is a linking group formed by a reactive pair.
  • ZZ comprises a group selected from the group consisting of roup or wherein R La is hydrogen, C1-10alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl, isobutyl, butyl, or hexyl), C 3-8 cycloalkyl, 3-8 membered heterocyclyl, aryl (e.g., phenyl), or heteroaryl (e.g., 2-pyridyl).
  • ZZ comprises Group(1).
  • ZZ comprises Group(2).
  • ZZ comprises Group(3).
  • ZZ comprises Group(4).
  • ZZ comprises Group(5). In some embodiments, ZZ comprises Group(6). In some embodiments, ZZ comprises Group(7). In some embodiments, ZZ comprises Group(8). In some embodiments, ZZ comprises Group(9). In some embodiments, ZZ comprises Group(10). In some embodiments, ZZ comprises Group(11). In some embodiments, ZZ comprises Group(12). 5 In some embodiments, wherein ZZ is -A’-B’-A’-.
  • ZZ is -A’-B’-A’-, wherein each A’ is independently a bond, -O-, -S-, or -N(R N3 )-, wherein R N3 is independently hydrogen or C 1-6 alkyl and each B’ is independently CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). 10
  • ZZ is CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH).
  • ZZ is C(O), C(S), or S(O)2.
  • ZZ is P(O)(OH), or P(S)(OH).
  • ZZ is –C(O)-.
  • ZZ is -A’-B’- or -B’-A’- wherein 15 each A’ is independently -O-, -S-, or -N(R N3 )-; each B’ is independently CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and each R N3 is independently hydrogen or C1-6alkyl.
  • ZZ is -A’-B’- or -B’-A’- wherein each A’ is independently -O- or -N(R N3 )-, wherein R N3 is independently hydrogen or C1-6alkyl.
  • each B’ is independently CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); and each R N3 is independently hydrogen or C1-6alkyl.
  • R N3 is independently hydrogen or C1-6alkyl.
  • ZZ is -CH2O- or -OCH2-.
  • ZZ is -S-S-.
  • ZZ is -C(O)N(R N3 )-, -N(R N3 )C(O)-, -C(O)O-, -OC(O)-, -OC(O)N(R N3 )-, - N(R N3 )C(O)O-, -N(R N3 )C(O)N(R N3 )-, -S(O) 2 N(R N3 )-, or -N(R N3 )S(O) 2 -, wherein R N3 is independently hydrogen or C 1-6 alkyl.
  • ZZ is -C(O)N(R N3 )- or -N(R N3 )C(O)-, wherein R N3 is independently 35 hydrogen or C 1-6 alkyl. In some embodiments, ZZ is -C(O)O- or -OC(O)-. 201 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, ZZ is -OC(O)N(R N3 )-, -N(R N3 )C(O)O-, or -N(R N3 )C(O)N(R N3 )-, wherein R N3 is independently hydrogen or C 1-6 alkyl.
  • ZZ is -N(R N3 )C(O)N(R N3 )-, wherein R N3 is independently hydrogen or C 1-6 alkyl. 5 In some embodiments, ZZ is -OC(O)N(R N3 )- or -N(R N3 )C(O)O-, wherein R N3 is independently hydrogen or C 1-6 alkyl. In some embodiments, ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -OP(O)(OH)-, -OP(S)(OH)-, - P(O)(OH)O-, or -P(S)(OH)O-.
  • ZZ is -OP(O)(OH)O- or -OP(S)(OH)O-. 10 In some embodiments, ZZ is -OP(S)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)-, -OP(S)(OH)-, -P(O)(OH)O-, or -P(S)(OH)O-. In some embodiments, ZZ is -OP(S)(OH)- or -P(S)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)- or -P(O)(OH)O-.
  • the compound of Formula (X) is selected from the group consisting of: (X-a) (X-b) 202 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (X-c) (X-d) In another embodiment, the compound of Formula (X) is selected from the group consisting of: 203 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 204 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 205 ME1 ⁇ 53466565.v1 Atty.
  • the compound of Formula (X) is selected from the group consisting of: 207 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein * represents the bond to the remainder of the dsRNA agent.
  • the compound of Formula (X) is selected from the group consisting of: 208 ME1 ⁇ 53466565.v1 Atty.
  • the alpha-v-beta-6 ( ⁇ v ⁇ 6) integrin targeting ligand may be conjugated to the dsRNA agent of the disclosure, e.g., to at least one strand of the dsRNA agent of the disclosure, via a branched carrier group, e.g., a b.
  • R T1 is -L L -oligonucleotide, wherein L L is an oligonucleotide linking group connecting the ⁇ v ⁇ 6 integrin targeting ligand to the oligonucleotide comprised in the dsRNA agent; and each ⁇ is a compound of the Formula 5 wherein: Y is O, N(H), S, or CH2; R 1 is hydrogen, C1-6alkyl (e.g., methyl), or a carboxylic acid mimic.; R Y is , wherein m is 0, 1, 2, 3, or 4; and 10 each R 2 is independently R, or two R 2 groups on adjacent carbon atoms taken together with the atoms to which they are bound form a fused 4 – 8 membered ring that is optionally substituted by 1, 2, 3 or 4 groups independently selected from group consisting of R and a nitrogen protecting group; A is an 5-membered heteroaryl optionally substituted with 1 or 2 substitu
  • Embodiment for the variables of Formula (XII), and (XV) that are the same as Formula (X) are a s described above for Formula (X).
  • embodiments for the variable ZZ are each as described above for Formula (X); embodiments for the variable R T and L are each as described above for Formula (X).
  • T is a bond or **-L 6 -G 1 -[L 5 -G 1 ] q1 -L 4 -, wherein ** is the bond to R T ; 25 q1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each L 4 , L 5 , and L 6 are independently a bond, -A 1 -B 1 -A 1 - or ZZ 1 ; 212 ME1 ⁇ 53466565.v1 Atty.
  • Docket No.121301-23820/ALN-523-WO ZZ 1 is a group formed by reaction of a reactive pair (e.g., a reaction between an azide and an alkyne or a cycloalkyne); each G 1 is independently -D 1 -E 1 -F 1 -, wherein D 1 , E 1 , and F 1 are independently a bond, C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of 5 which is optionally substituted with 1, 2, or 3 R groups; each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-; each B 1 is independently a bond, C(O), C(S), C(NR N1 ), S(O), S(O) 2 , P(O)(OH), P(S)(OH), or P(
  • T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ;
  • L 4 and L 6 are independently -A 1 -B 1 -A 1 -;
  • each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond);
  • each G 1 is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered 15 heterocyclyl, aryl, or heteroaryl (e.g., C1-10alkyl or C2-10alkenyl);
  • each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C1-6alkyl; and each B 1 is
  • T is **-L 6 -G 1 -[L 5 -G 1 ]q1-L 4 -, wherein ** is the bond to R T ; q1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each L 4 , L 5 , and L 6 are independently a bond or -A 1 -B 1 -A 1 -, or ZZ 1 ; ZZ 1 is a group formed by reaction of an azide and alkyne or cycloalkyne; 25 each G 1 is independently C1-10alkyl, each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-; each B 1 is independently a bond, C(O), C(S), C(NR N1 ), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); and each R N1 is independently hydrogen or C 1-6 alkyl.
  • one L 5 is ZZ 1 .
  • ZZ 1 comprises: 213 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO .
  • T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ;
  • L 4 and L 6 are independently -A 1 -B 1 - or -B 1 -A 1 -;
  • 5 each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond);
  • each G 1 is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl (e.g., C
  • T is **-B 1 -G 1 -L 5 -G 1 -B 1 -, wherein ** is the bond to R T ; each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond); each G 1 is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered 15 heterocyclyl, aryl, or heteroaryl (e.g., C1-10alkyl or C2-10alkenyl); each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C1-6alkyl; and each B 1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or
  • T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ; 20 L 4 and L 6 are independently -A 1 -B 1 -A 1 -; each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond); each G 1 is independently C 1-10 alkyl or C 2-10 alkenyl; each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C 1-6 alkyl; and 25 each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH).
  • T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ;
  • L 4 and L 6 are independently -B 1 -A 1 - or -A 1 -B 1 -; each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond);
  • each G 1 is independently C 1-10 alkyl or C 2-10 alkenyl;
  • each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C 1-6 alkyl; and each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH).
  • T is **-B 1 -G 1 -L 5 -G 1 -B 1 -, wherein ** is the bond to R T ; 214 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond); each G 1 is independently C 1-10 alkyl or C 2-10 alkenyl; each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C 1-6 alkyl; and 5 each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH).
  • each L 5 is a bond or - A 1 -B 1
  • T is **-B 1 -C 1-10 alkyl-L 5 -C 1-10 alkyl-B 1 -, wherein ** is the bond to R T ; each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond); each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or 10 C1-6alkyl; and each B 1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH).
  • T is -L 4 -G 1 -L 6 -**, wherein ** is the bond to R T ;
  • L 4 and L 6 are independently -A 1 -B 1 -A 1 -;
  • each G 1 is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered 15 heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups;
  • each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is hydrogen or C1-6alkyl;
  • each B 1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH).
  • T is -L 4 -G 1 -L 6 -**, wherein ** is the bond to R T ;
  • L 4 is -C(O)O- or C(O)N(R N1 )-, wherein R N1 is hydrogen or C1-6alkyl;
  • L 6 is -OP(O)(OH)O- or -OP(S)(OH)O-; and each G 1 is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups.
  • T is one of: 25 (d) **-L 6 -G 1 -ZZ 1 -L 5 -G 1 -L 4 -, (e) **-L 6 -G 1 -L 5 -G 1 -ZZ 1 -G 1 -L 4 -; or (f) **-L 6 -G 1 -ZZ 1 -G 1 -L 4 -, 30 wherein the sum of q2 and q3 is less than q1.
  • q2 and q3 are independently 0, 1, 2, 3 or 4.
  • each G 1 is independently C 1-20 alkyl (e.g., C 2- 15 alkyl; or C 2-12 alkyl; or C 2-10 alkyl; or C 2-8 alkyl; or C 2-6 alkyl).
  • each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, - OC(O)O-, -OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O,35 -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • each L 5 is independently - 215 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, each L 5 is independently - C(O)N(H)- or -N(H)C(O)-.
  • each G 1 is independently C 1-20 alkyl (e.g., C 2-15 alkyl; or C 2-12 alkyl; or C 2- 10 alkyl); and each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - 5 OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(
  • each G 1 is independently C 1-20 alkyl (e.g., C 2-15 alkyl; or C 2-12 alkyl; or C 2- 10 alkyl); and each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-.
  • L 6 is a bond.
  • L 6 is -C(O)-, -S(O)2-, -P(O)(OH)-,10 or -P(S)(OH)-.
  • L 6 is -P(O)(OH)- or -P(S)(OH)-.
  • L 6 is - P(O)(OH)-. In certain embodiments, L 6 is -P(S)(OH)-. In certain embodiments, L 6 is -C(O)-. In certain embodiments, L 4 is -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • L 4 is -P(O)(OH)O-, -OP(S)(OH)-,15 -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • L 4 is -C(O)O-, - OC(O)-, -C(O)N(H)-, or -N(H)C(O)-.
  • T is one of: 20 (d) **-L 6 -G 1 -ZZ 1 -L 5 -G 1 -L 4 -, (e) **-L 6 -G 1 -L 5 -G 1 -ZZ 1 -G 1 -L 4 -; or (f) **-L 6 -G 1 -ZZ 1 -G 1 -L 4 -, wherein the sum of q2 and q3 is less than q1; and ZZ 1 is selected from the group consisting of: 216 ME1 ⁇ 53466565.v1 Atty.
  • R La is hydrogen, C 1-10 alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl, isobutyl, butyl, or hexyl), C 3-8 cycloalkyl, 3-8 membered heterocyclyl, aryl (e.g., phenyl), or heteroaryl (e.g., 2-pyridyl); and the sum of q2 and q3 is less than q1 In certain embodiments, q2 and q3 are independently 0, 1, 2, 3 or 4.
  • each G 1 is independently C 1-20 alkyl (e.g., C 2-15 alkyl; or C 2-12 alkyl; or C 2-10 alkyl; or C 2-8 alkyl; or C 2- 6 alkyl).
  • each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, - OC(O)O-, -OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • each L 5 is independently -10 C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, each L 5 is independently - C(O)N(H)- or -N(H)C(O)-.
  • each G 1 is independently C1-20 alkyl (e.g., C2-15alkyl; or C2-12alkyl; or C2- 10alkyl); and each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - 15 OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(
  • each G 1 is independently C1-20 alkyl (e.g., C2-15alkyl; or C2-12alkyl; or C2- 10alkyl); and each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-.
  • L 6 is a bond.
  • L 6 is -C(O)-, -S(O)2-, -P(O)(OH)-, or -P(S)(OH)-.
  • L 6 is -P(O)(OH)- or -P(S)(OH)-.
  • L 6 is - 20 P(O)(OH)-. In certain embodiments, L 6 is -P(S)(OH)-. In certain embodiments, L 6 is -C(O)-. In certain embodiments, L 4 is -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • L 4 is -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • L 4 is -C(O)O-, - 25 OC(O)-, -C(O)N(H)-, or -N(H)C(O)-.
  • T is one of: 30 (e) **-L 6 -C 2-12 alkyl-L 5 -C 2-12 alkyl-ZZ 1 -C 2-12 alkyl-L 4 -; or (f) **-L 6 -C 2-12 alkyl-ZZ 1 -C 2-12 alkyl-L 4 -, 217 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein the sum of q2 and q3 is less than q1 In certain embodiments, q2 and q3 are independently 0, 1, 2, 3 or 4.
  • each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, - N(H)C(O)-, -OC(O)O-, -OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, - OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • each L 5 is 5 independently -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, each L 5 is independently -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, L 6 is a bond. In certain embodiments, L 6 is -C(O)-, -S(O) 2 -, -P(O)(OH)-, or -P(S)(OH)-. In certain embodiments, L 6 is -P(O)(OH)- or -P(S)(OH)-.
  • L 6 is - P(O)(OH)-. In certain embodiments, L 6 is -P(S)(OH)-. In certain embodiments, L 6 is -C(O)-. 10 In certain embodiments, L 4 is -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • L 4 is -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • L 4 is -C(O)O-, - OC(O)-, -C(O)N(H)-, or -N(H)C(O)-.
  • T is selected from the following, wherein ** is the bond to R T : ( q) **-C(O)-C1-10alkyl-L5-C1-10alkylC(O)-, (r) **-C(O)-C2-10alkyl-L5-C2-10alkyl-C(O)-, (s) **-C(O)-C4-10alkyl-L5-C4-10alkyl-C(O)-, (t) **-C(O)-C6-10alkyl-L5-C6-10alkyl-C(O)-, 20 ( x) **-C(O)-C2-20alkyl-C(O)-, (y) **-C(O)-C2-12alkyl-C(O)-, 25 (z) **-C(O)-C6-20alkyl-C(O)-, (aa)**-C(O)-C6-12alkyl-C(O)-, ( b
  • T is selected from the following, wherein ** is the bond to R T : ( dd) **-N(H)C(O)-C2-20alkyl-C(O)-, (ee)**- N(H)C(O)-C 6-20 alkyl-C(O)-, 35 (ff) **- N(H)C(O)-C6-12alkyl-C(O)-, (gg) **- N(H)C(O)-C10alkyl-C(O)-, 218 ME1 ⁇ 53466565.v1 Atty.
  • T is -L 4 -G 1 -L 6 -**, wherein ** is the bond to R T ;
  • L 4 is -C(O)O- or C(O)N(R N1 )-, wherein R N1 is hydrogen or C1-6alkyl;
  • L 6 is -OP(O)(OH)O- or -OP(S)(OH)O-; and each G 1 is independently C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with one R group.
  • T is -L 4 -G 1 -L 6 -**, wherein ** is the bond to R T ;
  • L 4 is -C(O)O- or C(O)N(R N1 )-, wherein R N1 is hydrogen or C1-6alkyl;
  • L 6 is -OP(O)(OH)O- or -OP(S)(OH)O-; and each G 1 is independently C3-10cycloalkyl or 3-10 membered heterocyclyl.
  • T is -L 4 -G 1 -L 6 -**, wherein ** is the bond to R T ; 20 L 4 is -C(O)O- or C(O)N(R N1 )-, wherein R N1 is hydrogen or C1-6alkyl; L 6 is -OP(O)(OH)O- or -OP(S)(OH)O-; and each G 1 is independently C3-10cycloalkyl or 3-10 membered heterocyclyl.
  • ** is the bond to RT; and X is O or S (e.g., S).
  • T is **-L 6 -[G 5 -O] q5 -G 5 -L 4 -, wherein ** is the bond to R T ;
  • q5 is an integer selected from 1 to 20;
  • L 4 and L 6 are independently -A 1 -B 1 -A 1 -, wherein each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is hydrogen 30 or C1-6alkyl;
  • each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH);
  • T is **-C(O)-[CH2CH2-O]q5-G 5 -L 4 -, wherein ** is the bond to R T ; 219 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO q5 is an integer selected from 1 to 20; L 4 is -A 1 -B 1 -A 1 -, wherein each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is hydrogen or C 1-6 alkyl; 5 each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH); G 5 is C 1-10 alkyl.
  • T is **-C(O)-[CH 2 CH 2 -O] q5 -G 5 -L 4 -, wherein ** is the bond to R T ;
  • q5 is an integer selected from 1 to 20;
  • L 4 is -A 1 -B 1 or -B 1 -A 1 -, wherein 10 each A 1 is independently -O- or -N(H)-, each B 1 is independently C(O),
  • G 5 is C1-10alkyl (e.g., C2-10alkyl or C2-6alkyl).
  • T is **-C(O)-[CH2CH2-O]q5- C2-10alkyl-C(O)N(H)-, wherein ** is the bond to R T , wherein q5 is an integer selected from 1 to 20 (e.g., 1 to 10, or 2 to 10; or 2 – 8; or 1; or 2; or 15 3; or 4.)
  • Formula (XV) is according to one of Formulae (XVa) through (XVc), respectively: (XVc).
  • ⁇ is #–[G 2 -L 7 ] q2 -* or #–G 3 -([L 7 -G 4 ] q3 -*) y , wherein # is the bond to T; y is 1, 2, 3, 4, or 5; 25 q2 is 1, 2, 3, 4, 5, 6, 7, or 8; q3 is 0, 1, 2, 3, 4, 5, 6, 7, or 8; each G 2 , G 3 , and G 4 is independently -D 2 -E 2 -F 2 -, wherein 220 ME1 ⁇ 53466565.v1 Atty.
  • each G 2 and G 4 optionally contains at least one bond to a ZZ (e.g., one bond to a 5 ZZ); or G 3 is N and y is 2; each L 7 is independently -A 2 -B 2 -A 2 -; wherein each A 2 is independently a bond, -O-, -S-, or -N(R N2 )-; each B 2 is independently a bond, C(O), C(S), C(NR N2 ), S(O), S(O)2, P(O)(OH), 10 P(S)(OH), or
  • ⁇ is #–[G2-L7]q2-* wherein # is the bond to T; q2 is 1, 2, 3, 4, 5, 6, 7, or 8; 25 q3 is 0, 1, 2, 3, 4, 5, 6, 7, or 8; each G 2 , G 3 , and G 4 is independently -D 2 -E 2 -F 2 -, wherein D 2 , E 2 , and F 2 are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 1 0 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 R B groups, and 30 wherein each G 2 optionally contains at least one bond to ZZ; each L 7 is independently -A 2 -B 2 -A 2 -; each A 2 is independently a bond, -O-, -S-, or -N(R N2 )-; each B 2 is independently
  • each R N2 is independently hydrogen, C 1-6 alkyl, a bond to a ZZ, or two R N2 within an -A 2 -B 2 -A 2 - group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; each R B is independently halogen, cyano, azido, nitro, -N(R 10 ) 2 , -O(R 10 ), -S(R 10 ), - 5 C(O)OR 10 , -C(O)R 10 , -C(O)N(R 10 ) 2 , -C(NR 10 )OR 10 , -C(NR 10 )R 10 , -C(NR 10 )N(R 10 ) 2 , - C(S)OR 10 , - C(S)R 10 , -C(S)N(R 10 ) 2 , -S(O) 2 R
  • - ⁇ - is #–[G 2 -L 7 ]q2-*, where # is the bond to T and * is a bond to a ZZ group.
  • # is the bond to T
  • each G 2 is independently C1-10alkyl, C2-10alkenyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 R B groups
  • each L 7 is independently -A 2 -B 2 -A 2 -, wherein each A 2 is independently a bond, -O-, -S-, or - N(R N2 )-; each B 2 is independently a bond, C(O), S(O)2, P(O)(OH), or P(S)(OH).
  • each G 2 is independently C 1-10 alkyl, each optionally substituted with 1 or 2 R B groups. In one embodiment, each G 2 is independently C 1-10 alkyl. In one embodiment, each L 7 is independently -A 2 -B 2 -A 2 -, wherein each A 2 is independently a bond, -O-, -S-, or -N(R N2 )-; and each B 2 is independently a bond, C(O) or S(O) 2, provided that at least one A 2 is not a bond.
  • each L 7 is independently -A 2 -B 2 - or - B 2 -A 2 -, wherein each A 2 is independently, -O-, -S-, or -N(R N2 )-; and each B 2 is independently a bond, C(O) or S(O) 2.
  • such embodiments include each of the following, 222 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein # is the bond to T and each * is a bond to a ZZ group. ; and each G 2 is independently C 1-10 alkyl.
  • such embodiments include each of the following, wherein # is the bond to T and each * is a bond to a ZZ; wherein # is the bond to T and each * is a bond to a ZZ group. 5 In one embodiment, -#–G 3 -([L 7 -G 4 ] q3 -*) y ,where # is the bond to T and * is a bond to a ZZ group.
  • such embodiments include each of the following, 223 ME1 ⁇ 53466565.v1 Atty.
  • each L 7 is selected from the group consisting of -O-, -S-, -N(H)-, -C(O)O-, -OC(O)-, -C(O)N(H)- , -OC(O)O-, -N(H)C(O)O-, -OC(O)N(H)-, -OP(O)(OH)O-, or -OP(S)(OH)O-; and each G 4 is independently -D 2 -E 2 -F 2 -, wherein 5 each D 2 and F 2 are independently a bond or C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted
  • each L 7 is selected from the group consisting of -O-, -S-, -N(H)-, -N(H)C(O)-, -C(O)N(H)-, - OP(O)(OH)O-, and -OP(S)(OH)O-; and each G 4 is independently -D 2 -E 2 -F 2 -, wherein 15 each D 2 and F 2 are independently a bond or C1-10alkyl; each E 2 is independently C1-10alkyl, C2-10alkenyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 R B groups.
  • each L 7 is selected from the group consisting of -O-, -S-, -N(H)-, -N(H)C(O)-,-C(O)N(H)-, - OP(O)(OH)O-, and -OP(S)(OH)O-; and each G 4 is independently C 1-10 alkyl, C 2-10 alkenyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 R B .
  • each L 7 is selected from the group consisting of -O-, -S-, -N(H)-, -N(H)C(O)-, -C(O)N(H)-, - OP(O)(OH)O-, and -OP(S)(OH)O-; and each G 4 is independently C 1-10 alkyl which is optionally substituted with 1 or, 2 5 R B groups.
  • each L 7 is selected from the group consisting of -O-, -S-, -N(H)-, --N(H)C(O)-, C(O)N(H)-, - 30 OP(O)(OH)O-, and -OP(S)(OH)O-; and each G 4 is independently C 1-10 alkyl.
  • each L 7 is selected from the group consisting of --N(H)C(O)- and C(O)N(H)-; 224 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO and each G 4 is independently C 1-10 alkyl.
  • such embodiments include each of the following, wherein each * is a bond to a ZZ; wherein # is the bond to T and each * is a bond to a ZZ group.
  • ZZ is a linking group formed by a reactive pair.
  • ZZ comprises a group selected from the group consisting of 225 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • ZZ comprises Group(1).
  • ZZ comprises Group(2).
  • ZZ comprises Group(3).
  • ZZ comprises Group(4).
  • ZZ comprises Group(5).
  • ZZ comprises Group(6).
  • ZZ comprises Group(7).
  • ZZ comprises Group(8). In some 5 embodiments, ZZ comprises Group(9). In some embodiments, ZZ comprises Group(10). In some embodiments, ZZ comprises Group(11). In some embodiments, ZZ comprises Group(12). In some embodiments, wherein ZZ is -A’-B’-A’-. In some embodiments, ZZ is -A’-B’-A’-, wherein each A’ is independently a bond, -O-, -S-, or -N(R N3 )-, wherein R N3 is independently 10 hydrogen or C1-6alkyl and each B’ is independently CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH).
  • ZZ is CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, ZZ is C(O), C(S), or S(O)2. In some embodiments, ZZ is P(O)(OH), or P(S)(OH). 15 In some embodiments, ZZ is –C(O)-.
  • ZZ is -A’-B’- or -B’-A’- wherein each A’ is independently -O-, -S-, or -N(R N3 )-; each B’ is independently CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and 226 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each R N3 is independently hydrogen or C 1-6 alkyl.
  • ZZ is -A’-B’- or -B’-A’- wherein each A’ is independently -O- or -N(R N3 )-, wherein R N3 is independently hydrogen or C 1-6 alkyl.
  • each B’ is independently CH 2 , C(O), S(O) 2 , P(O)(OH), or P(S)(OH); and 5 each R N3 is independently hydrogen or C 1-6 alkyl.
  • R N3 is independently hydrogen or 10 C1-6alkyl.
  • ZZ is -CH2O- or -OCH2-.
  • ZZ is -S-S-.
  • ZZ is -C(O)N(R N3 )-, -N(R N3 )C(O)-, -C(O)O-, -OC(O)-, -OC(O)N(R N3 )-, - N(R N3 )C(O)O-, -N(R N3 )C(O)N(R N3 )-, -S(O)2N(R N3 )-, or -N(R N3 )S(O)2-, wherein R N3 is independently hydrogen or C1-6alkyl.
  • ZZ is -C(O)N(R N3 )- or -N(R N3 )C(O)-, wherein R N3 is independently hydrogen or C1-6alkyl. 20 In some embodiments, ZZ is -C(O)O- or -OC(O)-. In some embodiments, ZZ is -OC(O)N(R N3 )-, -N(R N3 )C(O)O-, or -N(R N3 )C(O)N(R N3 )-, wherein R N3 is independently hydrogen or C1-6alkyl.
  • ZZ is -N(R N3 )C(O)N(R N3 )-, wherein R N3 is independently hydrogen or C1-6alkyl. 25 In some embodiments, ZZ is -OC(O)N(R N3 )- or -N(R N3 )C(O)O-, wherein R N3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -OP(O)(OH)-, -OP(S)(OH)-, - P(O)(OH)O-, or -P(S)(OH)O-.
  • ZZ is -OP(O)(OH)O- or -OP(S)(OH)O-. 30 In some embodiments, ZZ is -OP(S)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)-, -OP(S)(OH)-, -P(O)(OH)O-, or -P(S)(OH)O-. In some embodiments, ZZ is -OP(S)(OH)- or -P(S)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)- or -P(O)(OH)O-.
  • R T Embodiments, Formulae ( 5 In some embodiments of Formula (XV), R T is R T1 and is as defined for Formula (X).
  • R T is -G 0 -OR T1 , wherein R T1 is as defined for Formula (X) and G 0 is selected from: ( c) G0 is -D0-E0-F0-, wherein D 0 and F 0 are independently a bond or C1-10alkyl optionally substituted with 1, 2, 3, or 4 10 R groups; and E 0 is C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; ( d) G0 is -D0-E0-F0-, wherein D 0 and F 0 are independently a bond or C1-10alkyl optionally substituted with 1, 2, 3, or 4 15 R groups; and E 0 is 3-10 membered heterocyclyl optionally substituted with 1, 2, 3, or 4 R groups; and ( e) G0 is 3-10 membered heterocyclyl optionally substituted with 1 or 2 R groups; examples ( c) G0 is -D
  • G0 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 5 3, or 4 R groups (e.g., 1 or 2 R groups);
  • G0 is C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups;
  • G0 is C1-10alkyl optionally substituted with 1 or 2 R groups;
  • G0 is C1-10alkyl, optionally substituted with -O(Ra), wherein Ra is independently hydrogen, 10 C 1-6 alkyl, or a hydroxyl protecting group; e.g., , ( l) G0 is C1-10alkyl, and ( m) G0 is absent (a bond); wherein * represents the bond to T, the broken bond represents the bond to OR T1 , and R is -C1-6alkyl-OR
  • R Y is defined in the structure of Formula (XII) according to any one of the R Y Embodiments of Formula (IV). 20
  • Formula (XII) has the structure described above for any embodiment of Formula (IV), where the variable Z is replaced by a bond to a ZZ group.
  • Formula (XII) has the structure of any one of Formula (XII-a) through (XII-h) and (XII-t): 229 ME1 ⁇ 53466565.v1 Atty.
  • L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein * is the bond to a ZZ group. 5 In another embodiment, wherein L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 0, 1, 2, 3, 4, or 5.
  • L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 0, 1, 2, 3, or 4
  • L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 0, 1, 2, or 3.
  • L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 0, 1, or 2.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 1, 2, 3, 4, or 5.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 1, 2, 3, or 4.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 1, 2, or 3.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 1 or 2.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 4.
  • L is - L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 3.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 2. 15
  • L is -L 1 -G-L 2 -G-L 3 -*.
  • nts L is -L 1 -G-L 3 -*.
  • L is -G-L 3 -*.
  • L is -L 1 -G-*.
  • L is - G-*.
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R N )-, -N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl.
  • each instance of A-B-A- is independently selected from the group25 consisting of -C(O)O-, -OC(O)-, -C(O)N(R N )-, -N(R N )C(O)-, -OC(O)N(R N )-, -N(R N )C(O)O-,- N(R N )C(O)N(R N )-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl.
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)N(R N )-, -N(R N )C(O)-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -O-, and - N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R N )-, -N(R N )C(O)-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)N(R N )-, -N(R N )C(O)-, -O-, and -N(R N )-, wherein each R N is independently hydrogen 35 or C 1-6 alkyl.
  • 231 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO D and F are each independently a bond, C 1-10 alkyl, C 2-10 alkenyl, or C 2-10 alkynyl, each optionally substituted with 1, 2, 3, or 4 R groups; and E is C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • D and F are each independently a bond or C 1-10 alkyl optionally substituted with 1, 2, 3, or 4 R groups; and E is C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • each G is independently C1-10alkyl, optionally substituted with 1, 2, or 3 R groups. 15 In some embodiments, each G is independently C1-10alkyl, optionally substituted with 1 or 2 R groups. In some embodiments, each G is independently C1-10alkyl, optionally substituted with one R group . In some embodiments, each G is independently C1-10alkyl.
  • L is -L 1 -G-L 3 -*, wherein * is the bond to a ZZ group ;
  • 20 G is -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2- 10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups;
  • L 1 is -B-A-;
  • L 3 is a bond or -A-B-A-;
  • 25 each A is independently a bond, -O-, -S-, or -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl.
  • each B is independently a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); and
  • L is -L 1 -G-L 3 -*, wherein * is the bond to a ZZ group;
  • G is C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or 30 heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups;
  • L 1 is -B-;
  • L 3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl; and 35 each B is independently a bond, CH 2 , C(O), S(O) 2 , P(O)(OH), or P(S)(OH;
  • Docket No.121301-23820/ALN-523-WO G is C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups;
  • L 1 is -B-A-, wherein, A is a bond, -O-, -S-, or -N(R N )-, wherein each R N is independently hydrogen or C 1- 5 6 alkyl; and B is a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH).
  • L is -L 1 -G-*, wherein * is the bond to a ZZ group; G is C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, each of which is optionally substituted with 1 or 2 R groups; 10 L 1 is -B-A-, wherein A is a bond, -O-, -S-, or -N(R N )-, each R N is independently hydrogen or C1-6alkyl; and B is a bond, C(O), S(O)2, P(O)(OH), or P(S)(OH).
  • L is -L 1 -G-*, wherein * is the bond to a ZZ group; G is C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups; 15 L 1 is bond, C(O), S(O)2, P(O)(OH), or P(S)(OH); and R N is hydrogen or C1-6alkyl.
  • L is wherein * is the bond to a ZZ group; k is an integer from 1 to 10; L 1 is bond, C(O), C(S), C(NR N ), S(O) 2 , P(O)(OH), or P(S)(OH); and R N is hydrogen or C 1- 6 alkyl.
  • L is wherein * is the bond to a ZZ group; k is an integer from 1 to 10; L 1 is bond, C(O), P(O)(OH), or P(S)(OH). In some embodiments, L is , wherein * is the bond to a ZZ group; k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 3 to 10; or an integer from 4 to 10; or an integer from 5 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7.
  • L is , wherein * is the bond to a ZZ group; t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3). In some embodiments, 233 ME1 ⁇ 53466565.v1 Atty.
  • t is an integer from 0 to 10 (e.g., an integer from 1 to 5 or 1; or 2; or 3); a is an integer from 1 to 3; and s and s’ are each independently an integer from 1 to 24 (e.g., an integer from 1 to 16; an integer from 1 to 10; an integer from 3 to 10; an integer from 3 to 7; or an integer from 4 to 6).
  • * is the bond to a ZZ group; a is 1, 2 or 3; and each s, s’, and s” independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6).
  • L is wherein * is the bond to a ZZ group; and s, s’, and s’’ are independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer10 from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6).
  • L is , wherein * is the bond to a ZZ group; and each s, s’, and s” independently is an integer from 1 to 24(e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6).
  • L is wherein * is the bond to ZZ; s and k 15 are independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 10 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6).
  • L is wherein * is the bond to ZZ and 20 w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6).
  • L is C 1-20 alkyl. In some embodiments, L is C 2-20 alkyl. In some embodiments, L is C 6-20 alkyl. In some embodiments, L is C 8-12 alkyl. In some embodiments, L is C 10 alkyl. 234 ME1 ⁇ 53466565.v1 Atty.
  • L is -L 1 -G-*, wherein * is the bond to ZZ , G is C 1-10 alkyl or C 2-10 alkenyl, and L 1 is -B-A-, wherein, A is a bond, -O-, -S-, or -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl; and B is a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH).
  • L is -L 1 -G-*, wherein * is the bond to ZZ , G is C 1-10 alkyl or C 2-10 alkenyl, 5 L 1 is -O-, -S-, or -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • L is -L 1 -G-*, wherein * is the bond to ZZ , G is C 2-10 alkyl, L 1 is -O-, -S-, or -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • L is -L 1 -G-*, wherein * is the bond to ZZ , G is C 2-10 alkyl, L 1 is -O- (e.g., G is C 3- 10alkyl or C4-10alkyl or C5-10alkyl or C6-10alkyl or C2-8alkyl or C4-8alkyl or C4-8alkyl or C4alkyl or C5alkyl 10 or C6alkyl).
  • L is L wherein * is the bond to ZZ; L 1 is bond; and k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 3 to 10; or an integer from 4 to 10; or an integer from 5 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7.
  • L is -[G-L 2 ]q-G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q 15 is 3; or q is 4; or q is 5)
  • each G is indepdently C1-10alkyl and L 2 is O, S, or N(H).
  • each G is indepdently C1-10alkyl and L 2 is O.
  • L is -[CH2CH2O]q-G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q is 3; or q is 4; or q is 5)
  • G is indepdently C1-10alkyl.
  • L is , wherein * is the bond to ZZ; t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3). I n some embodiments, L is , wherein * is the bond to ZZ. In some embodiments, L is , wherein * is the bond to ZZ; t is 1 to 10 (e.g., 1-5; or 1-3; or 1; or 2; or 3); and z is 1-10 (e.g., 1-6; or 1-4; or 1; or 2; or 3 or 4 or 5 or 6).
  • L is , wherein * is the bond to ZZ, and t is an integer from 1 to 5 (e.g., 1; or 2; or 3). I n some embodiments, L is , wherein * is the bond to ZZ.
  • L is -G-L 2 -G-*, wherein L 2 is -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-; each B is independently a bond, CH2, C(O), C(S), C(NR N ), S(O), S(O)2, P(O)(OH), 30 P(S)(OH), or P(S)(SH); each R N is independently hydrogen or C1-6alkyl, or two R N within an -A-B-A- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; 235 ME1 ⁇ 53466565.v1 Atty.
  • each G is independently C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • L is -G 10 -L 2 -G 20 -*, wherein L 2 is -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-; each B is independently a bond, CH 2 , C(O), C(S), C(NR N ), S(O), S(O) 2 , 5 P(O)(OH), P(S)(OH), or P(S)(SH); each R N is independently hydrogen or C 1-6 alkyl, or two R N within an - A-B-A- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; G 10 is C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl; and G 20 is C 1-10 alkyl, C 2-10 alkenyl,
  • L is -G 10 -L 2 -G 20 -*, wherein L 2 is a bond, -O-, -S-, or -N(R N )-; R N is independently hydrogen or C1-6alkyl, G 10 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl; and G 20 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • L is -G 10 -L 2 -G 20 -*, wherein L 2 is a bond, -O-, -S-, or -N(R N )-; R N is independently hydrogen or C1-6alkyl, G 10 is aryl or heteroaryl; and G 20 is C1-10alkyl or C2-10alkenyl.
  • L is -G 10 -L 2 -G 20 -*, wherein L 2 is a bond, -O-, G 10 is aryl (e.g., phenyl); and G 20 is C1-10alkyl.
  • R L is -N(R 3 )(R 4 ), wherein R 3 is hydrogen or C1-6alkyl and R 4 is R 5 .
  • R L is -N(R 3 )(R 4 ), wherein R 3 is hydrogen and R 4 is R 5 .
  • R L is -N(R 3 )(R 4 ), wherein R 3 is C1-3alkyl and R 4 is R 5 .
  • R L is -N(R 3 )(R 4 ), wherein R 3 is methyl and R 4 is R 5 .
  • R L is --N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R 5 .
  • R L is --N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen 30 atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R 5 , provided that R 3 and R 4 taken together with the nitrogen atom to which they are attached do not form a morpholino group.
  • R L is --N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen atom to which they are attached form a group that is piperidinyl, piperazinyl, pyrrolidinyl, 35 imidazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, azetidinyl, pyrrolinyl, imidazolinyl, or pyrazolinyl, each substituted with R 5 . 236 ME1 ⁇ 53466565.v1 Atty.
  • RL is .
  • RL is , group; t is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); a is an integer from 1 to 3 and s and s’ are each independently an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 5 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6).
  • * is a bond to a ZZ group; a is an integer from 1 to 3; and s, s’, and s’’ are each independently an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6).
  • 10 is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6.
  • R L is 15 and s’’ are independently an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and t is an integer from 1 to 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, an integer from 1 to 3, or 1, or 2, or 3).
  • R L is 237 ME1 ⁇ 53466565.v1 Atty.
  • R L is -O(R 5 ).
  • * is a bond to a ZZ group
  • s is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6)
  • t is an integer from 0 to 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, an integer from 1 to 3, or 1, or 2, or 3).
  • R L is -R 5 .
  • RL is , wherein * is a bond to a ZZ group; s is 1 – 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6). I n some embodiments, RL is , wherein * is a bond to a ZZ group; t is 0 to 10 (e.g., 1-5; or 1-3; or 1; or 2; or 3).
  • R L is , , , 238 ME1 ⁇ 53466565.v1 Atty.
  • the compound of Formula (XII) is according to one of Formulae (XII-i) through (XII-l) and (XII-s): (XII-s) 5 wherein * is a bond to a ZZ group, p is 0, 1, 2, or 3 (e.g., 0); each R 21 is independently selected from group consisting of R and a nitrogen protecting group, and L is defined according to Formula (XII), or according to any of the preceding embodiments of L. 240 ME1 ⁇ 53466565.v1 Atty.
  • R 1 is hydrogen. In another embodiment of Formulae (XII-i) through (XII-l) and (XII-s), R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • the compound of Formula (XII) is according to one of Formulae (XII-m) through (XII-q) and (XII-t): 5 or a salt thereof, wherein * is a bond to a ZZ group, p is 0, 1 or 2; each R 21 is independently selected from group consisting of R; R P is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group), and L is defined according to Formula (XII), or according to any of the preceding embodiments 10 of L, wheren R and the remaining variables are as defined in Formula (XII). 241 ME1 ⁇ 53466565.v1 Atty.
  • the compound of Formula (XII) is according to one of Formulae (XII-u) through (XII-z): or a salt thereof, wherein * is a bond to a ZZ group, p is 0, 1 or 2; each R 21 is independently selected from group consisting of R; 5 R P is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group), and L is defined according to Formula (XII), or according to any of the preceding embodiments of L, wheren R and the remaining variables are as defined in Formula (XII).
  • R 1 is hydrogen. In another embodiment of Formulae (XII-m) through (XII-q) and (XII-t) through (XII-z), R 1 is 10 C 1-6 alkyl (e.g., methyl or t-butyl).
  • R N3 is independently hydrogen or C 1-6 alkyl; ⁇ is selected from, wherein # is the bond to T and each * is a bond to a ZZ group; and 5 , wherein ** represents the bond to T, and the remaining variables are as defined in Formula (XV), and any embodiment thereof.
  • ⁇ is selected from Formula (XII) and any one of Formula (XII-a) through (XII-q) and (XII-t) through (XII-z); 10 ZZ is - C(O)N(R N3 )-, -N(R N3 )C(O)-, -C(O)O-, -OC(O)-, wherein R N3 is independently hydrogen or C 1- 6alkyl; ⁇ is selected from, 243 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein # is the bond to T and each * is a bond to a ZZ group.
  • each G 2 is independently C1-10alkyl; and wherein ** represents the bond to T, and the remaining variables are as defined in Formula (XV), and any embodiment thereof.
  • R N3 is independently hydrogen or C 1-6 alkyl; ⁇ is selected from, wherein # is the bond to T and each * is a bond to a ZZ group, G 3 is C1-10alkyl; 15 each G 4 is independently C1-10alkyl; and 244 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein ** represents the bond to T, and the remaining variables are as defined in Formula (XV), and any embodiment thereof.
  • ⁇ is selected from Formula (XII) and any one of Formula (XII-a) through (XII-q) and (XII-t) through 5 (XII-z);
  • ZZ is -C(O)-, -C(O)N(R N3 )-, -N(R N3 )C(O)-, -C(O)O-, -OC(O)-, wherein R N3 is independently hydrogen or C1-6alkyl;
  • ⁇ is selected from, , , wherein # is the bond to T and each * is a bond to a ZZ group; and 10 and the remaining variables are as defined in Formula (XV), and any embodiment thereof.
  • ⁇ is selected from Formula (XII) and any one of Formula (XII-i) through (XII-q) and (XII-t) through (XII-z); 245 ME1 ⁇ 53466565.v1 Atty.
  • 15 ⁇ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) through (XII-z); ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -C(O)N(R N3 )-, -N(R N3 )C(O)-, -C(O)O-, -OC(O)-, wherein R N3 is independently hydrogen or C1-6alkyl; ⁇ is 246 ME1 ⁇ 53466565.v1 Atty.
  • ⁇ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) through (XII-z); ZZ is -C(O)N(H)- or -N(H)C(O)-; 15 T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ; L 4 is -A 1 -B 1 -; L 6 is -B 1 -; 247 ME1 ⁇ 53466565.v1 Atty.
  • R P3 is hydrogen
  • 10 ⁇ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) through (XII-z)
  • ZZ is -C(O)N(H)- or -N(H)C(O)-
  • T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ;
  • L 4 is -A 1 -B 1 -;
  • L 6 is -B 1 -;
  • 15 L 5 is a bond, -B 1 -A 1 -, or -A 1 -B 1 -;
  • each G 1 is independently C 1-10 alkyl;
  • each A 1 is independently a bond, -O- or -N(H);
  • each B 1 is C(O); and the remaining variables are as defined in Formula (XV), and any embodiment thereof.
  • ⁇ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) through (XII-z);
  • ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -C(O)N(R N3 )-, -N(R N3 )C(O)-, -C(O)O-, -OC(O)-, wherein R N3 is independently hydrogen or C1-6alkyl; 248 ME1 ⁇ 53466565.v1 Atty.
  • T is ***-L 6 -[G 5 -O]q5-G 5 -L 4 -, wherein q5 is an integer selected from 1 to 20; L 4 and L 6 are independently -A 1 -B 1 -A 1 -, wherein 5 each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is hydrogen or C 1- 6 alkyl; and each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH); each G 5 is independently C 1-10 alkyl; and wherein ** represents the bond to T, 10 and the remaining variables are as defined in Formula (XV), and any embodiment thereof.
  • the compound of formula (XV) is of the structure, wherein 15 ⁇ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) t hrough (XII-z), wherein L is wherein * is the bond to ZZ; s and k are independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 10 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer 20 from 4 to 6); ; 249 ME1 ⁇ 53466565.v1 Atty.
  • Docket No.121301-23820/ALN-523-WO q5 is an integer selected from 1 to 20;
  • L 4 and L 6 are independently -A 1 -B 1 -A 1 -, wherein each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is hydrogen or C 1-6 alkyl; and each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), 5 or P(S)(OH); each G 5 is independently C 1-10 alkyl; and the remaining variables are as defined in Formula (XV), and any embodiment thereof.
  • ⁇ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) t hrough (XII-z), wherein L is wherein * is the bond to ZZ and w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6; 15 ZZ is -C(O)N(H)-, -N(H)C(O)-; T is **-C(O)-[CH2CH2-O]q5- C2-10alkyl-C(O)N(H)-, wherein ** is the bond to the pyrrolidine ring and q5 is an integer selected from 1 to 20 (e.g., 1 to 10, or 2 to 10; or 2 – 8; or 1; or 2; or 3; or 4); and
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to the 3’-end of the oligonucleotide, the 5’-end of the oligonucleotide, or an internal 2’- or 3’ position on a internal nucleotide (i.e., a nucleotide that is not the 5’-terminal or 3’-terminal nucleoside).
  • L L is a divalent linker that connects to the 3’-end of the oligonucleotide, the 5’-end of the oligonucleotide, or an internal 2’- or 3’ position on a internal nucleotide (i.e., a nucleotide that is not the 5’-terminal or 3’-terminal nucleoside).
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to the 3’-end of the oligonucleotide, such as one of : directly to the 3’-carbon of the 3’-terminal nucleoside; directly to the 3’-O of the 3’-terminal nucleoside; directly to the 4’-carbon of the 3’-terminal nucleoside; 250 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO directly to the 2’-carbon of the 3’-terminal nucleoside; or directly to the 2’-O of the 3’-terminal nucleoside.
  • L L is a divalent linker that connects to the 3’-end of the oligonucleotide, such as one of : directly to the 3’-carbon of the 3’-terminal nucleoside; directly to the 3’-O of the 3’-terminal nu
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to the 5'-end of the oligonucleotide, such as one of: 5 directly to the 5’-carbon of the 5’-terminal nucleoside; directly to the 5’-O of the 5’-terminal nucleoside; directly to the 4’-carbon of the 5’-terminal nucleoside; directly to the 2’-carbon of the 5’-terminal nucleoside; or directly to the 2’-O of the 5’-terminal nucleoside.
  • L L is a divalent linker that connects to the 5'-end of the oligonucleotide, such as one of: 5 directly to the 5’-carbon of the 5’-terminal nucleoside; directly to the 5’-O of the 5’-terminal nucleoside; directly to the 4’-carbon of the 5’-terminal nucleoside; directly to the 2’-carbon of the
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to an internal 2’- or 3’ position on an internal nucleotide. In some embodiments, R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to an internal 2’- position on a internal nucleotide.
  • L L when L L connects to a 15 carbon aton on a nucleoside, then L L is -B 3 -A 3 -, wherein B 3 is -P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH)-; and A 3 is -O-, -S-, or -N(H)- .
  • L L when L L connects to a carbon atom on a nucleoside, then L L is -B 3 -A 3 -, wherein B 3 is -P(O)(OH)- or-P(S)(OH)-; and A 3 is -O-.
  • L L when L L connects to a oxygen atom on a nucleoside, then L L is - 20 P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH). In another embodiment, when L L connects to a oxygen atom on a nucleoside, then L L is - P(O)(OH)- or -P(S)(OH)-. In another embodiment, when L L connects to a oxygen atom on a nucleoside, then L L is - P(O)(OH)-. 25 In another embodiment, when L L connects to a oxygen atom on a nucleoside, then L L is - P(S)(OH)-.
  • L L when L L connects to a oxygen atom on a nucleoside, then L L is -B3-A3-LL1-A3-B3-, wherein B 3 is a bond, -C(O)-, C(S)-, C(NH), S(O), S(O) 2 , -P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH); A 3 is a bond, - 30 O-, -S-, or -N(H)- ; and L L1 is C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl.
  • LL when LL connects to a oxygen atom on a nucleoside, then LL is -B3-LL1- B 3 -, wherein B 3 is -C(O)-; and L L1 is C 1-10 alkyl.
  • R T is R T1
  • R T1 is -L L -oligonucleotide
  • the nucleoside is of Formula (XV-f), 251 ME1 ⁇ 53466565.v1 Atty.
  • B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); T is according any of the preceding embodiments; and * represent the bond to ZZ.
  • -T- is -L 1 -G-L 3 -*, wherein * is the bond to ZZ. 5
  • -T- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one or two R groups, and * is the bond to ZZ.
  • -T- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one or two groups selected from the group consisting of halogen, hydroxy, C1- 6alkoxy, amino, Cl-6alkylamino, di(C1-6alkylamino), cyano, carboxy , and * is the bond to ZZ.
  • -T- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one group selected from 10 the group consisting of halogen, hydroxy, C1-6alkoxy, amino, Cl-6alkylamino, di(C1-6alkylamino), cyano, carboxy , and * is the bond to ZZ.
  • -T- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one group selected from the group consisting of hydroxy, amino, and carboxy , and * is the bond to ZZ.
  • -T- is -C 2-30 alkyl-*, wherein the alkyl is optionally substituted with hydroxy, and * is the bond to ZZ.
  • -T- is -C2-30alkyl-*, wherein * is the bond to ZZ.
  • - T- is -C 2-16 alkyl-*, wherein * is the bond to ZZ.
  • -T- is -C 4-12 alkyl-*, wherein * is the bond to ZZ. In some embodiments, -T- is -C 4-10 alkyl-*, wherein * is the bond to ZZ. In some embodiments, -T- is -C 5-10 alkyl-*, wherein * is the bond to ZZ. In some embodiments, -T- is -C 6 alkyl-*, wherein * is the bond to ZZ. In some embodiments, -T- is -C 8 alkyl-*, wherein * is the bond to ZZ. In some embodiments, 20 -T- is -C 10 alkyl-*, wherein * is the bond to ZZ.
  • -T- is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein * is the bond to ZZ; q is 0, 1, 2, 3, 4, or 5; L 1 is a bond or -B-A-; 25 each L 2 is independently -A-B-A-; L 3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-; each B is independently a bond, CH 2 , C(O), C(S), C(NR N ), S(O) 2 , P(O)(OH), or P(S)(OH); each R N is independently hydrogen or C 1-6 alkyl; and 30 each G is independently C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • -T- is -L 1 -[G-L 2 ] q -G-*, wherein * is the bond to ZZ, q is 0, 1, 2, or 3;
  • -T- is -[G-L 2 ]q-G-*, wherein * is the bond to ZZ, q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each G is independently C1-10alkyl, each of which is optionally 15 substituted with 1 or 2 R groups.and ( c) each L2 is independently C(O)O or OC(O); (d) each L2 is independently C(O)(NRN) or N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl ( e) each L2 is independently OP(O)(OH)O, or OP(S)(OH)O (e.g., each is OP(O)(OH)O); 20 or ( f) each L2 is a bond.
  • the compound of Formula (XV) is according to one of Formulae (XV-g) through (XV-q): 253 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); 5 each m is independently integer selected from 1-20 (e.g., 2-12, or 2-10; or 2-6; or 2; or 3; or 4; or 5; or 6).z 254 ME1 ⁇ 53466565.v1 Atty.
  • B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine
  • x is an integer selected from 2 to 8, L and ZZ are as defined in Formula (XV) or in any embodiment preceding or below;
  • 5 B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); each m is independently integer selected from 1-20 (e.g., 2-12, or 2-10; or 2-6; or 2; or 3; or 4; or 5; or 6); 10 each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); R P is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group); and 255 ME1 ⁇ 53466565.v1 Atty.
  • R 1 is hydrogen or C 1-6 alkyl (e.g., methyl or t-butyl).
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • R 1 is hydrogen and R P is hydrogen.
  • R 1 is hydrogen and a nitrogen protecting group.
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and a nitrogen protecting group.
  • R T is R T1 , wherein R T1 is -L L -oligonucleotide
  • the internucleotide linkage can be of the formula, including the 3’ and 5’ oxygen atoms of the preceding and following nucelosides, respectively, (XV-pa) (XV-pb) pc) wherein T can be, for example a bond, -S(O) 2 - or, in for Formula ( membered heterocyclyl ring optionally substituted with 1 or 2 R groups, as defined herein ; and * represent the bond to ⁇ .
  • the preceding includes, wherein * represent the bond to ⁇ ; and R N5 is hydrogen or C1-10 alkyl.
  • the preceding includes, (XV-pg) (XV-ph) (XV-pi) wherein * represent the bond to ⁇ ; m is an integer selected from 1 – 20 (e.g., 1-10, or 2-20, or 2-10, or 4- 10, or 4-8; or 6-12; or 5; or 6; or 7; or 8; or 9; or 10), and R N5 is hydrogen or C1-10 alkyl.
  • the compound of Formula (XV-pd) is 256 ME1 ⁇ 53466565.v1 Atty.
  • Docket No.121301-23820/ALN-523-WO 5 x is an integer selected from 2 to 8, m is an integer selected from 1 – 10; n is an integer selected from 1-6; and R Y , Y, R 1 , R P , L, T, ⁇ , and ZZ are as defined for Formula (XV).
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • the compound is 257 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Y ' 5 258 ME1 ⁇ 53466565.v1 Atty.
  • each m is an integer selected from 1 – 20 (e.g., 2-20, 2-10, 1-10, 2-16, 4-16, 4-8, or 6-12), n is an integer selected from 1-6; x is an integer selected from 2 to 8, Y’ is O or S, and R 1 , ⁇ , R P and ZZ are as defined for Formula (XV).
  • the compound of Formula (XV-pe) is 5 , 259 ME1 ⁇ 53466565.v1 Atty.
  • Y’ is O. In another embodiment, Y’ is S. In one embodiment, R 1 is hydrogen. In another embodiment, R 1 is C 1-6 alkyl (e.g., methyl or t- 5 butyl). In one embodiment, R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S 10 and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 15 (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), 20 benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen 25 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound is 260 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 261 ME1 ⁇ 53466565.v1 Atty.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 15 (pac)).
  • Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen 20 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is 25 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 262 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (pac)).
  • a nitrogen protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 262 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (pac)).
  • Y’ is S
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • L L connects to a oxygen atom on a nucleoside
  • L L is-a bond.
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at the 5’-end of the 5 oligonucleotide, in one embodiment, R T1 can be represented as Formula (XV-5’) wherein L L is -P(Y)(OH)-, wherein Y is O or S (e.g., S); and * represents the bond to remainder of the compound of Formula (XV).
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at 10 the 5’-end of the oligonucleotide, in one embodiment, R T1 can be represented as Formula (XV-5’o) or Formula (XV-5’s), wherein * represents the bond to remainder of the compound of Formula (XV).
  • a compound of Formula (XV) can be represented by, 15 , , 263 ME1 ⁇ 53466565.v1 Atty.
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at the 3’-end of the oligonucleotide, in one embodiment, R T1 can be represented as Formula (XV-3’) wherein L L is -P(Y)(OH)-, wherein Y is O or S (e.g., S); and * represents the bond to remainder of the compound of Formula (XV).
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at the 3’-end of the oligonucleotide, in one embodiment, R T1 can be represented as Formula (XV-3’o) or Formula (XV-3’s), wherein * represents the bond to remainder of the compound of Formula (XV). 15 In another embodiment, a compound of Formula (XV) can be represented by, , 264 ME1 ⁇ 53466565.v1 Atty.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • the compound of Formula (XV) is 10 wherein Y’ is O or S; each ZZ is N(H)C(O) or C(O)N(H); 265 ME1 ⁇ 53466565.v1 Atty.
  • each m is an integer selected from 1 – 10.
  • each wherein m is an integer selected from 1 – 10; 266 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO
  • each wherein m is an integer selected from 1 – 10; I n another embodiment of Formula (XV-x), each wherein m is 5 an integer selected from 1 – 10.
  • each wherein m is an integer selected from 1 – 10. wherein m is an integer selected from 1 – 10; and n is an integer selsected from 1 – 6.
  • L 4 and L 6 are independently -B 1 -A 1 - or -A 1 -B 1 -;
  • L 5 is a bond, -B 1 -A 1 - or -A 1 -B 1 -;
  • each G 1 is independently C 1-10 alkyl;
  • each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is 10 independently hydrogen or C 1-6 alkyl; and each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH).
  • L 4 is -B 1 -A 1 - or -A 1 -B 1 -;
  • L 6 is B 1 ;
  • L 5 is a bond, -B 1 -A 1 - or -A 1 -B 1 -;
  • each G 1 is independently C1- 10alkyl;
  • each A 1 is independently -O- or -N(H)-, and
  • each B 1 is independently C(O), S(O)2,15 P(O)(OH), or P(S)(OH).
  • -L 4 -G 1 -L 5 -G 1 -L 4 - is -B 1 -C1-10alkyl-L 5 -C1-10alkyl-B 1 -A 1 -;
  • L 5 is a bond, -B 1 -A 1 - or -A 1 -B 1 -;
  • each A 1 is 268 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO independently -O- or -N(H)-, and each B 1 is independently C(O), S(O) 2 , P(O)(OH), or P(S)(OH).
  • -L 4 -G 1 -L 5 -G 1 -L 4 - is -C(O)-C1-10alkyl-L 5 -C1-10alkyl-C(O)N(H)-, wherein L 5 is a bond, -B 1 -A 1 - or -A 1 -B 1 -, 5 wherein A 1 is -O- or -N(H)-, and B 1 is C(O), S(O)2, P(O)(OH), or P(S)(OH).
  • -L 4 -G 1 -L 5 -G 1 -L 4 - is -C(O)-C 2-20 alkyl-C(O)N(H)- (e.g., -C(O)-C 6-12 alkyl-C(O)N(H)-, or -C(O)-C 8-12 alkyl- C(O)N(H)-, or -C(O)-C10alkyl-C(O)N(H)-).
  • the compound of Formula (XV) is 10 is an integer selected from 1 – 10; 15 n in an integer selected from 1 – 6; L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond); each G 1 is independently C 1-10 alkyl; each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C 1-6 alkyl; and 20 each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH).
  • each ⁇ is 269 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein m is an integer selected from 1 – 10; R 1 is hydrogen or C1-6alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • each ⁇ is 5 , wherein m is an integer selected from 1 – 10; R 1 is hydrogen or C 1-6 alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • R 1 is hydrogen or C1-6alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • each ⁇ is 5 hydrogen).
  • the compound of Formula (XV) is 272 ME1 ⁇ 53466565.v1 Atty.
  • each ZZ is N(H)C(O) or C(O)N(H); each 5 is an integer selected from 1 – 10; n is an integer selected from 1-6; L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond); each G 1 is independently C 1-10 alkyl; each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or 10 C 1-6 alkyl; and each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH).
  • each ⁇ is , wherein m is an integer selected from 1 – 10; R 1 is hydrogen or C1-6alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., 15 hydrogen).
  • each ⁇ is 273 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein m is an integer selected from 1 – 10; R 1 is hydrogen or C 1-6 alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • each wherein m is an integer selected from 1 – 10; R 1 is hydrogen or C 1-6 alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen). 10 nitrogen protecting group (e.g., hydrogen)..
  • each ⁇ is 274 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein m is an integer selected from 1 – 10; R 1 is hydrogen or C1-6alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • R 1 is hydrogen or 10 C 1-6 alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • Formula (XV-x2) each , wherein R 1 is hydrogen or C 1-6 alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • R 1 is hydrogen or C1-6alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • Y’ is O. In another embodiment, Y’ is S.
  • R 1 is hydrogen. In another embodiment, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment of Formula (XV-x), (XV-x1), and (XV-x2), and each of the preceding embodiments thereof, R P is hydrogen. In another embodiment, R P is a nitrogen protecting group (e.g., t- 15 butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc butoxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O and R 1 is hydrogen. In one embodiment, Y’ is O and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). 20 In one embodiment of Formula (XV-x), (XV-x1), and (XV-x2), and each of the preceding embodiments thereof, Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is S and R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is 5 hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t- 10 butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound in other embodiments of Formula (XV), is of the structure: 15 277 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In other embodiments of Formula (XV), the compound is of the structure: 5 In other embodiments of Formula (XV), the compound is of the structure: 278 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . 5 In some embodiments of Formula (XV), the compound is of the structure: , 279 ME1 ⁇ 53466565.v1 Atty.
  • the compound of Formula (XV) is 5 wherein * represents the bond to the remainder of the dsRNA agent. 280 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In another embodiment, the compound of Formula (XV) is, 5 wherein * represents the bond to the remainder of the dsRNA agent. In another embodiment, the compound of Formula (XV) is, 10 wherein * represents the bond to the remainder of the dsRNA agent. 281 ME1 ⁇ 53466565.v1 Atty.
  • the compound of Formula (XV) is, 5 wherein * represents the bond to the remainder of the dsRNA agent.
  • the compound of Formula (XV) is, wherein B is an optionally modified nucleobase (e.g., cytosine, uracil, 5-methylcytosine, 5- methyluridine); and 282 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein * represents the bond to the remainder of the dsRNA agent. 5 III.
  • iRNA agents that inhibit the expression of a target gene in extrahepatic tissue, e.g., muscle tissue, e.g., skeletal muscle tissue and/or cardiac muscle tissue.
  • the iRNA agent includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a target gene in a skeletal and/or cardiac muscle cell or tissue, such as a cell or tissue within 10 a subject, e.g., a mammal, such as a human having a muscle disorder or disease, e.g., a skeletal and/or cardiac disorder or disease.
  • dsRNA double stranded ribonucleic acid
  • Any target gene can be inhibited by the iRNA agents provided herein.
  • the target gene is any gene involved in a muscle disorder or disease, e.g., a skeletal and/or cardiac muscle disorder or disease.
  • skeletal and/or cardiac muscle target genes include any genes involved 15 in a skeletal muscle and/or a cardiac muscle disorder or disease, such as, for example, adrenoceptor beta 1 (ADRB1); calcium voltage-gated channel subunit alpha1 C (CACNA1C); calcium voltage-gated channel subunit alpha1 G (CACNA1G) (T type calcium cchannel); angiotensin II receptor type 1(AGTR1); Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1 (HCN1); Hyperpolarization Activated Cyclic Nucleotide 20 Gated Potassium Channel 4 (HCN4); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3 (HCN3); Potassium Vol
  • the iRNA agents provided herein comprise a sense strand and an antisense strand and at least one of the strands is modified for targeting delivery to extrahepatic tissue, e.g., muscle tissue, e.g., skeletal muscle tissue and/or cardiac muscle tissue.
  • the iRNA agents are modified by conjugation to at least one alpha-v-beta-6 ( ⁇ v ⁇ 6) integrin ligand and conjugation to at least one in vivo 10 delivery enhancing moiety.
  • integrin ligand is meant any ligand that binds to an integrin or an integrin receptor. Integrin ligands comprise binding sequences that are recognized and bound by integrins or ntegrin receptors.
  • ⁇ v ⁇ 6 integrin targeting ligands include peptides and small molecules, which bind ⁇ v ⁇ 6 integrins or ⁇ v ⁇ 6 integrin receptors ( ⁇ v ⁇ 6 integrin targeting ligands) have been designed and synthesized herein and in, e.g., U.S. 15 Patent 6,410,526, the entire contents of which are incorporated herein by reference.
  • An exemplary linker to conjugate a ⁇ v ⁇ 6 integrin targeting ligand to the dsRNA agent is: 284 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 , 285 ME1 ⁇ 53466565.v1 Atty.
  • At least one of the strands of the iRNA agent is conjugated to at least one ⁇ v ⁇ 6 integrin targeting ligand and at least one in vivo delivery enhancing moiety.
  • the iRNA agent may be conjugated to one, two, three, four, or more ⁇ v ⁇ 6 integrin targeting ligands and one, 5 two, three, four or more in vivo delivery enhancing moieties.
  • the ⁇ v ⁇ 6 integrin targeting ligand is conjugated to the sense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand may be conjugated to an extrernal position of sense strand, e.g., the 3’-end of the sense strand, to the 5’-end of the sense strand, or to both the 5-end and the 3’-end of the sense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand may be conjugated to an internal 10 position of the sense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand is conjugated to the antisense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand may be conjugated to an external position of the antisense strand, e.g., the 3’-end of the antisense strand, to the 5’-end of the antisense strand, or to both the 5-end and the 3’- end of the antisense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand may be conjugated 15 to an internal position of the antisense strand.
  • the in vivo delivery enhancing moiety is conjugated to the sense strand.
  • the in vivo delivery enhancing moiety may be conjugated to an extrernal position of sense strand, e.g., the 3’-end of the sense strand, to the 5’-end of the sense strand, or to both the 5-end and the 3’-end of the sense strand.
  • the in vivo delivery enhancing moiety may be conjugated to an 20 internal position of the sense strand.
  • the in vivo delivery enhancing moiety is not conjugated to an external position of the sense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand is conjugated to the antisense strand.
  • the in vivo delivery enhancing moiety may be conjugated to an external position of the antisense strand, e.g., the 3’-end of the antisense strand, to the 5’-end of the antisense strand, or to both the 5-end and the 25 3’-end of the antisense strand.
  • the in vivo delivery enhancing moiety may be conjugated to an internal position of the antisense strand.
  • the in vivo delivery enhancing moiety is not conjugated to an external position of the antisense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand and the in vivo delivery enhancing moiety are both conjugated to the sense strand of the dsRNA agent.
  • the ⁇ v ⁇ 6 integrin 30 targeting ligand is conjugated to an external position of the sense strand, e.g., the 3’-end of the sense strand or the 5’-end of the sense strand, and the in vivo delivery enhancing moiety is conjugated to an internal position of the sense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand and the in vivo delivery enhancing moiety are both conjugated to the antisense strand of the dsRNA agent.
  • the ⁇ v ⁇ 6 integrin targeting ligand is conjugated to a sense strand
  • the 5 in vivo delivery enhancing moiety is conjugated to an antisense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand is conjugated to an antisense strand
  • the in vivo delivery enhancing moiety is conjugated to a sense strand.
  • the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a target gene.
  • the region of 10 complementarity is about 15-30 nucleotides or less in length.
  • the RNAi agent Upon contact with a cell expressing the target gene, the RNAi agent inhibits the expression of the target gene (e.g., a human gene, a primate gene, a non-primate gene) by at least 30% as compared to a similar cell not contacted with the RNAi agent or an RNAi agent not complementary to the target gene.
  • Expression of the gene may be assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as 15 by immunofluorescence analysis, using, for example, western blotting or flowcytometric techniques.
  • a dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used.
  • One strand of a dsRNA includes a region of complementarity that is substantially complementary, or fully complementary, to a target sequence.
  • the target sequence can be derived from the sequence of an mRNA 20 formed during the expression of a target gene.
  • the other strand includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.
  • the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.
  • the duplex structure is 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.
  • the duplex structure is 30 18 to 25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24 or 24-25 base pairs in length, for example, 19-21 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.
  • the region of complementarity to the target sequence is 15 to 30 nucleotides in length, 35 e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, for 287 ME1 ⁇ 53466565.v1 Atty.
  • the duplex structure is 19 to 30 base pairs in length.
  • the region of complementarity to the target sequence is 19 to 30 nucleotides in length.
  • the dsRNA is 15 to 23 nucleotides in length, 19 to 23 nucleotides in length, or 25 to 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme.
  • dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer.
  • the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an 10 mRNA molecule.
  • a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).
  • the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 15 to 36 base pairs, e.g., 15-36, 15-35, 15-34, 15-33, 15-32, 15- 15 31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 20-24,20-23, 20
  • an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA.
  • a miRNA is a dsRNA.
  • a dsRNA is not a naturally occurring miRNA.
  • an RNAi agent useful to target expression or a target gene is not generated in the target cell by cleavage of a larger dsRNA.
  • a dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides.
  • a nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.
  • the overhang(s) can be on the sense strand, the antisense strand or any combination thereof.
  • the nucleotide(s) of an overhang can be present on the 5'-end, 3'-end or both ends of either an antisense or sense strand of a 30 dsRNA.
  • a dsRNA can be synthesized by standard methods known in the art.
  • Double stranded RNAi compounds of the disclosure may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the dsRNA compound can be prepared using solution-phase or solid-phase 35 organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Similarly, single-stranded oligonucleotides of the disclosure can be prepared using solution-phase or solid-phase organic synthesis or both. 288 ME1 ⁇ 53466565.v1 Atty.
  • a dsRNA of the disclosure includes at least two nucleotide sequences, a sense sequence and an antisense sequence.
  • one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of a target gene.
  • a dsRNA will include 5 two oligonucleotides, where one oligonucleotide is described as the sense strand (passenger strand), and the second oligonucleotide is described as the corresponding antisense strand (guide strand).
  • the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide. 10 It will be understood that, although the sequences provided herein may be described as modified or conjugated sequences, the RNA of the RNAi agent of the disclosure e.g., a dsRNA of the disclosure, may comprise any one of the sequences set forth herein that is un-modified, un-conjugated, or modified or conjugated differently than described therein.
  • dsRNAs having a duplex structure of about 20 to 23 base 15 pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888).
  • RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).
  • dsRNAs described herein can include at least one strand of a 20 length of minimally 21 nucleotides.
  • dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein, and differing in their ability to inhibit the expression of a target gene by not more than 10, 15, 20, 25, 30, 35, 40, 45 or 50 % inhibition from a dsRNA 25 comprising the full sequence using the in vitro assay with, e.g., skeletal and/or cardiac muscle cells and a 10 nM concentration of the RNA agent and the PCR assay as provided in the examples herein, are contemplated to be within the scope of the present disclosure.
  • RNA agents described herein identify a site(s) in a target gene mRNA transcript that is susceptible to RISC-mediated cleavage.
  • the present disclosure further features RNAi 30 agents that target within this site(s).
  • an RNAi agent is said to “target within” a particular site of an mRNA transcript if the RNAi agent promotes cleavage of the mRNA transcript anywhere within that particular site.
  • Such an RNAi agent will generally include at least about 15 contiguous nucleotides, preferably at least 19 nucleotides, from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a target 35 gene.
  • the RNA of an RNAi agent of the disclosure is chemically modified to enhance stability or other beneficial characteristics.
  • substantially all of the nucleotides of an RNAi agent of the disclosure are modified.
  • all of the nucleotides of an RNAi agent of the disclosure are modified.
  • RNAi agents of the disclosure in which “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or unmodified nucleotides. In still other embodiments of the disclosure, RNAi agents of the disclosure can include not more than 5, 4, 3, 2 or 1 modified nucleotides.
  • the nucleic acids featured in the disclosure can be synthesized or modified by methods well 10 established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference.
  • Modifications include, for example, end modifications, e.g., 5’-end modifications (phosphorylation, conjugation, inverted linkages) or 3’-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, 15 destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2’-position or 4’-position) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5’-end modifications (phosphorylation, conjugation, inverted linkages) or 3’-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.
  • base modifications e.g., replacement with stabilizing bases, 15 destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucle
  • RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or no natural internucleoside 20 linkages.
  • RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone.
  • modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • a modified RNAi agent will have a phosphorus atom in its internucleoside backbone.
  • RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' 30 linkages, 2'-5'-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • the dsRNA agents of the disclosure are in a free acid form. In other embodiments of the disclosure, the dsRNA agents of the disclosure are in a salt form. In one embodiment, the dsRNA agents of the disclosure are in a sodium salt form. In certain 35 embodiments, when the dsRNA agents of the disclosure are in the sodium salt form, sodium ions are present in the agent as counterions for substantially all of the phosphodiester and/or phosphorothioate groups present in the agent.
  • Agents in which substantially all of the phosphodiester and/or phosphorothioate linkages have a sodium counterion include not more than 5, 4, 3, 2, or 1 phosphodiester 290 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO and/or phosphorothioate linkages without a sodium counterion.
  • sodium ions are present in the agent as counterions for all of the phosphodiester and/or phosphorothioate groups present in the agent.
  • Representative U.S. patents that teach the preparation of the above phosphorus-containing 5 linkages include, but are not limited to, U.S.
  • Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic 15 internucleoside linkages.
  • patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.
  • RNA mimetics are contemplated for use in RNAi agents, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with alternate groups.
  • the nucleobase units are maintained for hybridization with an appropriate nucleic acid target compound.
  • a RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA 30 compounds the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos.5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. 35 Additional PNA compounds suitable for use in the RNAi agents of the disclosure are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
  • RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones and in particular --CH 2 --NH--CH 2 -, --CH 2 --N(CH 3 )-- 291 ME1 ⁇ 53466565.v1 Atty.
  • RNAs featured herein have morpholino backbone structures of the above-referenced US5,034,506.
  • the 5 native phosphodiester backbone can be represented as -O-P(O)(OH)-OCH 2 -.
  • Modified RNAs can also contain one or more substituted sugar moieties.
  • the RNAi agents, e.g., dsRNAs, featured herein can include one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O- , S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • Exemplary suitable 10 modifications include O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10.
  • dsRNAs include one of the following at the 2' position: C 1 to C 10 alkyl, substituted alkyl, alkaryl, aralkyl, O- alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA 15 cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an RNAi agent, or a group for improving the pharmacodynamic properties of an RNAi agent, and other substituents having similar properties.
  • the modification includes a 2'-methoxyethoxy (2'-O--CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group.
  • Another exemplary modification is 2'- 20 dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O- dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH 2 --O--CH 2 --N(CH 3 ) 2 .
  • modifications include: 5’-Me-2’-F nucleotides, 5’-Me-2’-OMe nucleotides, 5’-Me-2’-deoxynucleotides, (both R and S isomers in these three families); 2’-alkoxyalkyl; and 2’-NMA (N-methylacetamide).
  • 25 Other modifications include 2'-methoxy (2'-OCH 3 ), 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ), 2’- O-hexadecyl, and 2'-fluoro (2'-F).
  • RNAi agents can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • Representative U.S. patents that teach the 30 preparation of such modified sugar structures include, but are not limited to, U.S. Pat.
  • RNAi agent of the disclosure can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobase often referred to in the art simply as “base” nucleobases
  • “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5- 292 ME1 ⁇ 53466565.v1 Atty.
  • methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8- 5 thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5- bromo, 5-trifluoromethyl and other 5-substit
  • nucleobases include those disclosed in U.S. Pat. No.3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. 10 ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993.
  • modified nucleobases are particularly useful for increasing the binding 15 affinity of the oligomeric compounds featured in the disclosure.
  • These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C (Sanghvi, Y. S., Crooke, S. T.
  • RNAi agent of the disclosure can also be modified to include one or more bicyclic sugar moieties.
  • a “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms.
  • a “bicyclic 30 nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system.
  • the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring.
  • an agent of the disclosure may include one or more locked nucleic acids (LNA).
  • a locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' 35 and 4' carbons.
  • an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo structural conformation.
  • the addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR.
  • bicyclic nucleosides for use in the polynucleotides of the disclosure include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms.
  • the antisense polynucleotide agents of the disclosure include one or more 5 bicyclic nucleosides comprising a 4′ to 2′ bridge.
  • 4′ to 2′ bridged bicyclic nucleosides include but are not limited to 4′-(CH 2 )—O-2′ (LNA); 4′-(CH 2 )—S-2′; 4′-(CH 2 ) 2 —O-2′ (ENA); 4′- CH(CH 3 )—O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH 2 OCH 3 )—O-2′ (and analogs thereof; see, e.g., U.S. Pat.
  • RNAi agent of the disclosure can also be modified to include one or more constrained ethyl nucleotides.
  • a "constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising 25 a bicyclic sugar moiety comprising a 4'-CH(CH 3 )-O-2' bridge.
  • a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”
  • An RNAi agent of the disclosure may also include one or more “conformationally restricted nucleotides” (“CRN”).
  • CRN are nucleotide analogs with a linker connecting the C2’and C4’ carbons of ribose or the C3 and -C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and 30 increase the hybridization affinity to mRNA.
  • the linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.
  • an RNAi agent of the disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides.
  • UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked "sugar” residue.
  • UNA also encompasses monomer with bonds between C1'-C4' have been removed (i.e. the covalent carbon-oxygen- 294 ME1 ⁇ 53466565.v1 Atty.
  • RNAi agent of the disclosure may also include one or more “cyclohexene nucleic acids” or (“CeNA”).
  • CeNA are nucleotide analogs with a replacement of the furanose moiety of DNA by a 10 cyclohexene ring. Incorporation of cylcohexenyl nucleosides in a DNA chain increases the stability of a DNA/RNA hybrid.
  • CeNA is stable against degradation in serum and a CeNA/RNA hybrid is able to activate E. Coli RNase H, resulting in cleavage of the RNA strand. (see Wang et al., Am. Chem. Soc. 2000, 122, 36, 8595–8602, hereby incorporated by reference).
  • RNA molecules can include N-15 (acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N- (acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4- hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"- phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in WO 2011/005861.
  • RNA molecules include inverted 20 nucleotides, including inverted abasic nucleotides.
  • each end of a nucleotide comprises an inverted abasic nucleotide, e.g., a 3’->3’ linked nucleotide at the 3’-end of the oligonucleotide and a 5'->5' linked nucleotide at the 5’-end of the oligonucleotide.
  • the inverted abasic nucleotide can be linked via a phosphodiester or phosphothioate internucleotide linkage.
  • RNAi agent of the disclosure include a 5’ phosphate or 25 5’ phosphate mimic, e.g., a 5’-terminal phosphate or phosphate mimic on the antisense strand of an RNAi agent. Suitable phosphate mimics are disclosed in, for example US 2012/0157511, the entire contents of which are incorporated herein by reference. A.
  • the double-stranded RNAi agents of the disclosure include agents with chemical modifications as disclosed, for example, in WO 2013/075035, the entire contents of which are incorporated herein by reference. As shown herein and in WO 2013/075035, a superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand or antisense strand of an RNAi agent, particularly at or near the cleavage 35 site. In some embodiments, the sense strand and antisense strand of the RNAi agent may otherwise be completely modified.
  • the introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand.
  • the RNAi agent may be optionally modified with a (S)-glycol nucleic acid 295 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (GNA) modification, for instance on one or more residues of the antisense strand.
  • the resulting RNAi agents present superior gene silencing activity.
  • the disclosure provides double stranded RNAi agents capable of inhibiting the expression of a target gene in vivo.
  • the RNAi agent comprises a sense strand and an antisense strand. 5
  • Each strand of the RNAi agent may be 15-30 nucleotides in length.
  • each strand may be 16- 30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. In certain embodiments, each strand is 19-23 nucleotides in length.
  • RNAi agent a duplex double stranded RNA
  • the duplex region of an RNAi agent may be 15-30 nucleotide pairs in length.
  • the duplex region can be 16-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17 - 23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in 15 length, 19- 21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length.
  • the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.
  • the duplex region is 19-21 nucleotide pairs in length.
  • the RNAi agent may contain one or more overhang regions or capping 20 groups at the 3’-end, 5’-end, or both ends of one or both strands.
  • the overhang can be 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1- 2 nucleotides in length.
  • the nucleotide overhang region is 2 nucleotides in length.
  • the overhangs can be the result of one strand being longer than the other, or the result of two strands of 25 the same length being staggered.
  • the overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence.
  • the first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
  • the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2’-sugar modified, 30 such as, 2-F, 2’-O-methyl, thymidine (T), and any combinations thereof.
  • TT can be an overhang sequence for either end on either strand.
  • the overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence.
  • the 5’- or 3’- overhangs at the sense strand, antisense strand or both strands of the RNAi agent 35 may be phosphorylated.
  • the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different.
  • the overhang is present at the 3’-end of the sense strand, antisense strand, or both 296 ME1 ⁇ 53466565.v1 Atty.
  • this 3’-overhang is present in the antisense strand. In one embodiment, this 3’-overhang is present in the sense strand.
  • the dsRNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability.
  • the single-stranded overhang 5 may be located at the 3'-terminal end of the sense strand or, alternatively, at the 3'-terminal end of the antisense strand.
  • the RNAi may also have a blunt end, located at the 5’-end of the antisense strand (i.e., the 3’-end of the sense strand) or vice versa.
  • the antisense strand of the RNAi has a nucleotide overhang at the 3’-end, and the 5’-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5’-end of the antisense strand and 3’-end overhang of the antisense strand favor the guide 10 strand loading into RISC process.
  • the RNAi agent is a double blunt-ended of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5’end.
  • the antisense strand contains at least one motif of three 2’-O- methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5’end.
  • the RNAi agent is a double blunt-ended of 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5’end.
  • the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5’end.
  • the RNAi agent is a double blunt-ended of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5’end.
  • the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5’end.
  • the RNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5’end; the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5’end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucleotide 30 overhang.
  • the 2 nucleotide overhang is at the 3’-end of the antisense strand.
  • the RNAi agent additionally has 35 two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5’-end of the sense strand and at the 5’-end of the antisense strand.
  • every nucleotide in the sense strand and the antisense strand of the RNAi agent including the nucleotides that are part of the 297 ME1 ⁇ 53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO motifs are modified nucleotides.
  • each residue is independently modified with a 2’-O- methyl or 2’-fluoro, e.g., in an alternating motif.
  • the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5' terminal nucleotide (position 1) 5 positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3' terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1- 23 of sense strand to form a duplex; wherein at least the 3 ' terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3' terminal nucleotides are unpaired with sense strand, thereby forming a 3' single stranded overhang of 10 1-6 nucleotides; wherein the 5' terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming
  • the antisense strand contains at least one motif of three 2’-O-methyl modifications on 20 three consecutive nucleotides at or near the cleavage site.
  • the dsRNAi agent comprises sense and antisense strands, wherein the dsRNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2’-O- methyl modifications on three consecutive nucleotides at position 11, 12, and 13 from the 5’ end; 25 wherein the 3’ end of the first strand and the 5’ end of the second strand form a blunt end and the second strand is 1-4 nucleotides longer at its 3’ end than the first strand, wherein the duplex region which is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotide of the second strand length to reduce target gene expression when
  • the RNAi agent further comprises a ligand.
  • the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.
  • the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand. 298 ME1 ⁇ 53466565.v1 Atty.
  • the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5’-end.
  • the motifs of three identical modifications may occur at the 9, 10, and 11 positions; 10, 11, and 12 positions; 11, 12, and 13 positions; 12, 13, and 14 positions; or 13, 14, and 15 positions of the antisense strand, the count starting 5 from the 1 st nucleotide from the 5’-end of the antisense strand, or, the count starting from the 1 st paired nucleotide within the duplex region from the 5’- end of the antisense strand.
  • the cleavage site in the antisense strand may also change according to the length of the duplex region of the RNAi from the 5’- end.
  • the sense strand of the RNAi agent may contain at least one motif of three identical 10 modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand.
  • the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide 15 overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand.
  • at least two nucleotides may overlap, or all three nucleotides may overlap.
  • the sense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides.
  • the first motif may occur at or near the 20 cleavage site of the strand and the other motifs may be a wing modification.
  • the term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adjacent to the first motif or is separated by at least one or more nucleotides.
  • the motifs are immediately adjacent to each other, then the chemistry of the motifs are distinct from each other and when the motifs are 25 separated by one or more nucleotide than the chemistries can be the same or different.

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Abstract

The present disclosure provides double stranded ribonucleic acid (dsRNA) agents for inhibiting expression of a target gene, comprising an antisense strand which is complementary to the target gene; a sense strand which is complementary to the antisense strand and forms a double stranded region with the antisense strand; at least one alpha-v-beta-6 (αvβ6) integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least one strand; and at least one in vivo delivery enhancing moiety conjugated to one or more internal positions on at least one strand. The present disclosure also provides compositions comprising such dsRNA agents, and methods of use thereof for treating a subject having a disorder that would benefit from reduction in expression of the target gene.

Description

Atty. Docket No.121301-23820/ALN-523-WO DUAL CONJUGATE COMPOUNDS FOR EXTRAHEPATIC DELIVERY RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No.63/659,097, filed on 5 June 12, 2024. The entire contents of the foregoing application are hereby incorporated herein by reference. SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in .XML 10 format and is hereby incorporated by reference in its entirety. Said .XML copy, created on June 5, 2025, is named “ALN-523_ST.xml” and is 608,848 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. BACKGROUND OF THE DISCLOSURE 15 Efficient delivery of an RNAi agent to cells in vivo requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins. RNAi-based therapeutics show promising clinical data for treatment of liver-associated disorders. However, RNAi delivery into extra-hepatic tissues remains an obstacle, limiting the use of RNAi-based therapies. One of the limiting factors is the ability to deliver intact RNAi efficiently to extra-hepatic tissues, 20 such as muscle tissues, e.g., skeletal muscle tissues and/or cardiac muscle tissues. Previous work has used delivery reagents such as liposomes, cationic lipids, and nanoparticles forming complexes to aid the intracellular internalization of RNAi agents into extra-hepatic cells. However, only limited success in delivering RNAi agents to extra-hepatic tissues, like muscle tissue, after systemic administration has been reported. For example, although cholesterol-conjugated RNAi 25 agents are delivered to muscles after intravenous injection, a high dose (50 mg/kg) is required to achieve sustainable gene silencing. In addition, cholesterol conjugates are highly toxic at high concentrations, limiting their potential for clinical applications. Thus, systemic delivery of oligonucleotides to muscle tissue remains a challenge and, accordingly, there is a continuing need for new and improved compositions and methods for delivering 30 RNAi agents in vivo. SUMMARY OF THE DISCLOSURE The present disclosure is based, at least in part, on the surprising discovery that conjugating at least one alpha-v-beta-6 (αvβ6) integrin targeting ligand and at least one in vivo delivery enhancing 35 moiety, e.g., a moiety comprising at least one C10-C26 hydrocarbon chain conjugated to at least one strand of a dsRNA agent, e.g., the sense strand, provides surprisingly efficient in vivo delivery to extrahepatic tissue, i.e., muscle tissue, resulting in efficient entry and internalization of the dsRNA agent into muscle tissue ( e.g., skeletal muscle tissue and/or cardiac muscle tissue), and surpringly good 1 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO inhibition of target gene expression in muscle tissue (e.g., skeletal muscle tissue and/or cardiac muscle tissue). Accordingly, in one aspect, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a target gene, comprising: an antisense strand which is 5 complementary to the target gene; a sense strand which is complementary to the antisense strand and forms a double stranded region with the antisense strand; at least one αvβ6 integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least one strand; and at least one in vivo delivery enhancing moiety conjugated to one or more internal positions on at least one strand. The dsRNA agent of claim 1, wherein the in vivo delivery enhancing moiety comprises at least 10 one C10-C26 hydrocarbon chain. In some embodiments, the in vivo delivery enhancing moiety comprises at least one C22 hydrocarbon chain, at least one C17 hydrocarbon chain or at least one C11 hydrocarbon chain. In some embodiments, the at least one C10-C26 hydrocarbon chain is unsubstituted or substituted with at least one functional group selected from the group consisting of hydroxyl, amine, 15 carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne, e.g., a carboxylic acid group. In some embodiments, the at least one C10-C26 hydrocarbon chain is covalently attached to the following moiety: wherein is a bond connecting the moiety to the at least one C10-C26 hydrocarbon. 20 In some embodiments, the in vivo delivery enhancing moiety is attached to the dsRNA agent via a linker or via a carrier or via an internucleotide phosphate linkage. In some embodiments, the in vivo delivery enhancing moiety is attached to the dsRNA agent via a linker, e.g., a linker comprising an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction,25 or carbamate. In some embodiments, the linker is selected from the group consisting of -(CH2)nNH-; - C(O)(CH2)nNH-; -NR’’’’(CH2)nNH-, -C(O)-(CH2)n-C(O)-; -C(O)-(CH2)n-C(O)O-; -C(O)-O-; -C(O)- (CH2)n-NH-C(O)-; -C(O)-(CH2)n-; -C(O)-NH-; -C(O)-; -(CH2)n-C(O)-; -(CH2)n-C(O)O-; -(CH2)n-; and - (CH2)n-NH-C(O)-; wherein n is a number from 1 to 20; and R’’’’ is C1-C6 alkyl. In some embodiments, the linker comprises –(CH2)n-NH-C(O)- or –(CH2)n-NH-C(O)-(CH2)2-C(COOH)-NH-C(O)-, wherein n is30 a number from 1 to 20. In some embodiments, the linker comprises –(CH)2-O-(CH2CH2)-(O)-(CH2CH2)- NH-C(O)-. In some embodiments, the in vivo delivery enhancing moiety comprises the following structure: 2 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , , 3 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein the broken bond represents the bond to the remainder of the dsRNA agent. In one embodiment, the αvβ6 integrin targeting ligand comprises the structure: 5 the broken bond represents the bond to the remainder of the dsRNA agent. In one embodiment, the αvβ6 integrin targeting ligand comprises the structure: wherein RLig is 4 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO broken bond represents the bond to the remainder of the dsRNA agent. In one embodiment, the αvβ6 integrin targeting ligand comprises the structure: 5 In one embodiment, the αvβ6 integrin targeting ligand comprises the structure: 10 5 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO the broken bond represents the bond to the remainder of the dsRNA agent. In one embodiment, the αvβ6 integrin targeting ligand comprises the structure: 5 broken bond represents the bond to the remainder of the dsRNA agent. In some embodiments, the αvβ6 integrin targeting ligand or the αvβ6 integrin targeting ligand together with a linker useful in the present disclosure comprises a structure selected from the table below: 6 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 7 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Z1 is a linker linking the the αvβ6 integrin targeting ligand to the remainder of the dsRNA agent and wherein * represents the bond to the remainder of the dsRNA agent. 8 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, the at least one αvβ6 integrin targeting ligand is conjugated to the sense strand, e.g., to the 3’-end of the sense strand or the 5’-end of the sense strand. In some embodiments, the at least one αvβ6 integrin targeting ligand is conjugated to both the 5’-end and the 3’-end of the sense 5 strand. In some embodiments, the at least one αvβ6 integrin targeting ligand is conjugated to an internal position of the sense strand. In some embodiments, the at least one αvβ6 integrin targeting ligand is conjugated to the antisense strand, e.g., to the 3’ end antisense strand or the 5’ end of the antisense strand. In some embodiments, the at least one αvβ6 integrin targeting ligand is conjugated to both the 5’-end and the 3’- 10 end of the antisense strand. In some embodiments, the at least one αvβ6 integrin targeting ligand is conjugated to an internal position of the antisense strand. In some embodiments, the at least one in vivo delivery enhancing moiety is conjugated to an internal position of the sense strand. In some embodiments, the at least one in vivo delivery enhancing moiety is conjugated to an internal position or an external position of the sense strand. In some 15 embodiments, the at least one in vivo delivery enhancing moiety is conjugated to an external position of the sense strand. In other embodiments, the at least one in vivo delivery enhancing moiety is not conjugated to an internal position of the sense strand. In some embodiments, the at least one in vivo delivery enhancing moiety is conjugated to one or more of the following internal positions: positions 4-8 and 13-18 on the sense strand, counting from the 20 5’ end. In some embodiments, the at least one in vivo delivery enhancing moiety is conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, 16 and 17 on the sense strand, counting from the 5’-end. In one embodiment, the at least one in vivo delivery enhancing moiety is conjugated to position 6 on the sense strand, counting from the 5’-end. In some embodiments, the at least one in vivo delivery enhancing moiety is conjugated to an 25 internal position of the antisense strand. In some embodiments, the at least one in vivo delivery enhancing moiety is conjugated to an internal position or an external position of the antisense strand. In some embodiments, the at least one in vivo delivery enhancing moiety is conjugated to an external position of the antisense strand. In other embodiments, the at least one in vivo delivery enhancing moiety is not conjugated to an internal position of the antisense strand. 30 In some embodiments, the at least one in vivo delivery enhancing moiety is conjugated to one or more of the following internal positions: positions 6-10 and 15-18 on the antisense strand, counting from the 5’-end. In some embodiments, the at least one in vivo delivery enhancing moiety is conjugated to one or more of the following internal positions: positions 15, 16 and 17 on the antisense strand, counting from the 5’-end. 35 In some embodiments, the at least one αvβ6 integrin targeting ligand and the at least one in vivo delivery enhancing moiety are both conjugated to the sense strand. In some embodiments, 9 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO the at least one αvβ6 integrin targeting ligand and the at least one in vivo delivery enhancing moiety are both conjugated to the antisense strand. In some embodiments, the at least one αvβ6 integrin targeting ligand is conjugated to the sense strand and the at least one in vivo delivery enhancing moiety is conjugated to the antisense strand. In 5 some embodiments, the at least one αvβ6 integrin targeting ligand is conjugated to the antisense strand and the at least one in vivo delivery enhancing moiety is conjugated to the sense strand. In some embodiments, the sense strand and the antisense strand are each independently 15-30 nucleotides in length; 19 to 25 nucleotides in length; or 21 to 23 nucleotides in length. In some embodiments, the target gene is selected from the group consisting of myostatin 10 (MSTN); Cholinergic Receptor Nicotinic Alpha 1 Subunit (CHRNA1); Cholinergic Receptor Nicotinic Beta 1 Subunit (CHRNB1); Cholinergic Receptor Nicotinic Delta Subunit (CHRND); Cholinergic Receptor Nicotinic Epsilon Subunit (CHRNE); Cholinergic Receptor Nicotinic Gamma Subunit (CHRNG); Collagen Type XIII Alpha 1 Chain (COL13A1); Docking Protein 7 (DOK7); LDL Receptor Related Protein 4 (LRP4); Muscle Associated Receptor Tyrosine Kinase (MUSK); Receptor Associated 15 Protein Of The Synapse (RAPSN); Sodium Voltage-Gated Channel Alpha Subunit 4 (SCN4A); and Double Homeobox 4 (DUX4), dystrophy myotonic protein kinase (DMPK), glycogen synthase 1 (GYS1), survival of motor neuron 1 (SMN1), alpha-glucosidase (GAA); adrenoceptor beta 1 (ADRB1); calcium voltage-gated channel subunit alpha1 C (CACNA1C); calcium voltage-gated channel subunit alpha1 G (CACNA1G) (T type calcium cchannel); angiotensin II receptor type 1(AGTR1); Sodium 20 Voltage-Gated Channel Alpha Subunit 2 (SCN2A); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1 (HCN1); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3 (HCN3); Potassium Voltage-Gated Channel Subfamily A Member 5 (KCNA5); Potassium Inwardly Rectifying Channel Subfamily J Member 3 (KCNJ3); Potassium Inwardly Rectifying Channel Subfamily J Member 25 4 (KCNJ4); phospholamban (PLN); calcium/calmodulin dependent protein kinase II delta (CAMK2D); or Phosphodiesterase 1 (PDE1). In one aspect, the present disclosure provides cells containing any of the dsRNA agents of the disclosure. In another aspect, the present disclosure provides a pharmaceutical composition for inhibiting 30 expression of the target gene, comprising any of the dsRNA agents of the disclosure. In one embodiment, the dsRNA agent is present in a buffer solution, e.g., a buffer solution comprising acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). In other embodiments, the dsRNA agent is present in an unbuffered solution, e.g., in water or normal saline. 35 In one aspect, the present disclosure provides a method of inhibiting expression of a target gene in a skeletal muscle cell and/or a cardiac muscle cell. The method includes contacting the cell with any of the dsRNA agents of the disclosure or any of the pharmaceutical compositions of the disclosure, e.g., 10 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO and maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the target gene in the skeletal muscle cell and/or cardiac muscle cell, thereby inhibiting expression of the target gene in the skeletal muscle cell and/or cardiac muscle cell. In one embodiment, the cell is within a subject, e.g., human subject. 5 In certain embodiments, contacting the cell with the dsRNA agent or pharmaceutical composition inhibits the expression of the target gene by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In one embodiment, inhibiting expression of the target gene decreases the target gene protein level in serum of the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. 10 In one aspect, the present disclosure provides a method of treating a subject having a muscle disorder, e.g., a skeletal muscle disorder and/or a cardiac muscle disorder. The method includes administering to the subject a therapeutically effective amount of any of the dsRNA agents of the disclosure or any of the pharmaceutical compositions of the disclosure, thereby treating the subject. In one embodiment, the muscle disorder is selected from the group consisting of Myostatin- 15 related muscle hypertrophy, congenital myasthenic syndrome, facioscapulohumeral muscular dystrophy (FSHD), Spinal Muscular Atrophy (SMA), Myotonic Dystrophy Type 1 (DM1), Pompe disease, PLN cardiomyopathy, spasticity, obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic cardiomyopathy (FHC); Heart failure with preserved ejection fraction (HFPEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina; myocardial infarction (MI); heart failure or heart failure 20 with reduced ejection fraction (HFREF); supraventricular tachycardia (SVT); hypertrophic cardiomyopathy (HCM); and PLN cardiomyopathy. In one embodiment, the skeletal muscle disorder is selected from the group consisting of Myostatin-related muscle hypertrophy, congenital myasthenic syndrome, facioscapulohumeral muscular dystrophy (FSHD), Spinal Muscular Atrophy (SMA), Myotonic Dystrophy Type 1 (DM1), Pompe 25 disease, PLN cardiomyopathy, and spasticity. In one embodiment, the cardiac muscle disorder is selected from the group consisting of obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic cardiomyopathy (FHC); Heart failure with preserved ejection fraction (HFPEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina; myocardial infarction (MI); heart failure or heart failure with reduced ejection fraction 30 (HFREF); supraventricular tachycardia (SVT); hypertrophic cardiomyopathy (HCM); and PLN cardiomyopathy. The dsRNA agent or pharmaceutical composition may be administered to the subject subcutaneously, intramusclularly, intravenously, or via inhalation. In one embodiment, the therapeutic methods of the disclosure further include administering to 35 the subject an additional agent or a therapy suitable for treatment or prevention of an extrahepatic disorder. 11 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 is a bar graph showing AD-1812376 mediated SOD1 knockdown in quadriceps and gastrocnemius of a mouse model at Day 21 following a single IV dose of 2 mg/kg. PBS as used for the control group. 5 FIG.2: is a bar graph showing AD-1812376 mediated SOD1 knockdown in quadriceps, gastrocnemius and liver of non-human primates at Days 29 and 57 following a single IV dose of 1 mg/kg, 3 mg/kg or 9 mg/kg. PBS was used for the control group. FIG.3: is a bar graph showing AD-2032892 mediated SOD1 knockdown in liver, quadriceps and gastrocnemius of a mouse model at Day 21 following administration of a single IV dose of 1 mg/kg, 10 3 mg/kg or 9 mg/kg. PBS was used for the control group. FIG.4: is a bar graph showing AD-2032892 mediated NHP SOD1 knockdown in gastrocnemius, quadriceps and liver of non-human primates following administration of a single IV dose of 10 mg/kg. PBS was used for the control group. FIG.5: is a bar graph showing NHP SOD1 knockdown mediated by AD-2432777, AD-2241090, 15 AD-2315832, AD-2315873 and AD-2315833 in biceps, gastrocnemius and quadriceps of non-human primates at Day 29 following administration of a single IV dose of 10 mg/kg. PBS was used for the control group. FIG.6: is a bar graph showing NHP SOD1 knockdown mediated by AD-2432777 and AD- 2640041 in biceps, gastrocnemius and soleus of non-human primates at Day 28 following administration 20 of a single SC 10 mg/kg dose. PBS was used for the control group. FIG.7: is a bar graph showing AD-2432777, AD-2902547, AD-2889354, AD-2889355, AD- 2889356, and AD-2889357 mediated SOD1 knockdown in quadriceps of a mouse model at Day 7 following administration of a single subcutaneous dose of 1 mg/kg. PBS was used for the control group. FIG.8: is a bar graph showing AD-2432777, AD-2902547, AD-2889354, AD-2889355 and AD- 25 2889356, and AD-2889357 mediated SOD1 knockdown in heart of a mouse model at Day 7 following administration of a single subcutaneous dose of 1 mg/kg. PBS was used for the control group. FIG.9: is a bar graph showing AD-2640041, AD-2700084, AD-2700085, AD-2700086, AD- 2700087, AD-2700088, AD-2700089, AD-2700090, AD-2700091, AD-2700092, AD-2700093, AD- 2700094, AD-2700095, AD-2700096, AD-2700097, and AD-2700099 mediated SOD1 knockdown in 30 quadricep of a mouse model at Day 7 following administration of a single IV dose of 0.3 mg/kg. PBS was used for the control group. FIG.10: is a bar graph showing AD-2640041, AD-2700084, AD-2700085, AD-2700086, AD- 2700087, AD-2700088, AD-2700089, AD-2700090, AD-2700091, AD-2700092, AD-2700093, AD- 2700094, AD-2700095, AD-2700096, AD-2700097, and AD-2700099 mediated SOD1 knockdown in 35 gastrocnemius of a mouse model at Day 7 following administration of a single IV dose of 0.3 mg/kg. PBS was used for the control group. 12 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO FIG.11: is a bar graph showing AD-2640041, AD-2700084, AD-2700085, AD-2700086, AD- 2700087, AD-2700088, AD-2700089, AD-2700090, AD-2700091, AD-2700092, AD-2700093, AD- 2700094, AD-2700095, AD-2700096, AD-2700097, and AD-2700099 mediated SOD1 knockdown in heart of a mouse model at Day 7 following administration of a single IV dose of 0.3 mg/kg. PBS was 5 used for the control group. FIG.12: is a bar graph showing AD-3214512 mediated HPRT1 knockdown in quadricep and heart of a mouse model at Day 7 following administration of a single subcutaneous dose of 1 mg/kg. PBS was used for the control group. FIG.13 is a bar graph showing NHP DMPK knockdown mediaded by AD-3100656 in heart, 10 gastrocnemius, and quadriceps of non-human primates at Day 32 following a single subcutaneous dose of 3 mg/kg. The control group was the average of animals treated with non-DMPK targeting siRNAs. The DMPK mRNA % remaining is relative to two housekeeping genes (PPIB and ADD1). DETAILED DESCRIPTION 15 The present disclosure is based, at least in part, on the surprising discovery that conjugating at least one alpha-v-beta-6 (αvβ6) targeting ligand and at least one in vivo delivery enhancing moiety, e.g., a moiety comprising at least one C10-C26 hydrocarbon chain, to at least one strand of a dsRNA agent, e.g., the sense strand, provides surprisingly efficient in vivo delivery to extrahepatic tissue, i.e., muscle tissue, resulting in efficient entry and internalization of the dsRNA agent into extrahepatic tissue, e.g., muscle 20 tissue, e.g., skeletal muscle tissue and/or cardiac muscle tissue, and surprisingly good inhibition of target gene expression in extrahepatic tissue, e.g., muscle tissue, e.g., skeletal muscle tissue and/or cardiac muscle tissue. The following detailed description discloses dsRNA agents comprising at least one alpha-v-beta- 6 (αvβ6) targeting ligand and at least one in vivo delivery enhancing moiety that mediate delivery to 25 extrahepatic tissue, e.g., muscle tissue, e.g., skeletal muscle tissue and/or cardiac muscle tissue, to inhibit the expression of a target gene as well as compositions, ustes, and methods for treating subjects that would benefit from inhibition and/or reduction of the expression of the target gene. I. Definitions 30 In order that the present disclosure may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this disclosure. In order that the present disclosure may be more readily understood, certain terms are first 35 defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this disclosure. 13 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements. The term "including" is used herein to mean, and is used interchangeably with, the phrase 5 "including but not limited to". The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise. For example, “sense strand or antisense strand” is understood as “sense strand or antisense strand or sense strand and antisense strand.” The term “about” is used herein to mean within the typical ranges of tolerances in the art. For 10 example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range. The term “at least”, “no less than”, or “or more” prior to a number or series of numbers is 15 understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 19 nucleotides of a 21 nucleotide nucleic acid molecule” means that 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the 20 numbers in the series or range. As used herein, “no more than” or “or less” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the 25 numbers in the series or range. As used herein, ranges include both the upper and lower limit. As used herein, methods of detection can include determination that the amount of analyte present is below the level of detection of the method. In the event of a conflict between an indicated target site and the nucleotide sequence for a sense or antisense strand, the indicated sequence takes precedence. 30 In the event of a conflict between a sequence and its indicated site on a transcript or other sequence, the nucleotide sequence recited in the specification takes precedence. Terms used herein may be preceded and/or followed by a single dash, or a double dash, "=", to indicate the bond order of the bond between the named substituent and its parent moiety; a single dash indicates a single bond and a double dash indicates a double bond or a pair of single bonds in the case of 35 a spiro-substituent. In the absence of a single or double dash it is understood that a single bond is formed between the substituent and its parent moiety; further, substituents are intended to be read "left to right" unless a dash indicates otherwise. For example, C1-6alkoxycarbonyloxy and -OC(O)C1-66alkyl indicate the same functionality; similarly arylalkyl, arylalkyl-, and -alkylaryl indicate the same functionality. 14 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Further, certain terms herein may be used as both monovalent and divalent linking radicals as would be familiar to those skilled in the art, and by their presentation linking between two other moieties. For example, an alkyl group can be both a monovalent radical or divalent radical; in the latter case, it would be apparent to one skilled in the art that an additional hydrogen atom is removed from a 5 monovalent alkyl radical to provide a suitable divalent moiety. Throughout the disclosure, is used to represent an oligonucleotide; such oligonucleotides may be an RNA, a DNA, a single-stranded RNA, such as an antisense oligonucleotide (ASO), a double-stranded RNA, such as an siRNA, and oligonucleotide derivatives such as phosphorodiamidate morpholino oligomers (PMOs). 10 The term “conjugated”, as used herein in reference, e.g., to a moiety conjugated to a dsRNA agent of the disclosure, e.g., at least one αvβ6 integrin targeting ligand conjugated to at least one strand of a dsRNA agent of the disclosure, or at least one in vivo delivery enhancing moiety conjugated to at least one strand of a dsRNA agent of the disclosure, means that the moiety is covalently attached to a dsRNA agent directly or via a linker or via a carrier or via an internucleotide phosphate linkage. 15 The term "alkenyl" as used herein, means a straight or branched chain hydrocarbon containing from 2 to 10 carbons, unless otherwise specified, and containing at least one carbon-carbon double bond. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2- propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl, and 3,7- dimethylocta-2,6-dienyl. 20 The term “alkynyl” as used herein means a straight or branched hydrocarbon chain containing from 2 to 10 carbons, unless otherwise specified, and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited to, 1-butynyl, 2-butynyl, 1-propynyl and the like. The term "alkoxy" as used herein, means an alkyl group, as defined herein, appended to the 25 parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, 2-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, and n-hexyloxy. The term "alkyl" as used herein, means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms, unless otherwise specified. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec -butyl, iso-butyl, tert-butyl, n-pentyl, 30 isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. When an "alkyl" group is a divalent linking group between two other moieties, then it may also be a straight or branched chain; examples include, but are not limited to -CH2-, - CH2CH2-, -CH2CH2CHC(CH3)-, -CH2CH(CH2CH3)CH2-. The term "aryl," as used herein, means a phenyl (i.e., monocyclic aryl); naphthyl or azulenyl; a 35 bicyclic ring system containing a phenyl fused to a cycloalkyl, cycloalkenyl, or heterocyclyl ring. Representative examples of the bicyclic aryls include, but are not limited to, azulenyl, naphthyl, 2,3- dihydroinden-1-yl, 2,3-dihydroinden-2-yl, 2,3-dihydroinden-3-yl, 2,3-dihydroinden-4-yl, 2,3- 15 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO dihydroinden-5-yl, 2,3-dihydroindol-1-yl, indolin-2-yl, indolin-3-yl, indolin-4-yl, indolin-5-yl, indolin-6- yl, indolin-7-yl, inden-1-yl, inden-2-yl, inden-3-yl, inden-4-yl, dihydronaphthalen-2-yl, dihydronaphthalen-3-yl, dihydronaphthalen-4-yl, dihydronaphthalen-1-yl, 5,6,7,8-tetrahydronaphthalen- 1-yl, 5,6,7,8-tetrahydronaphthalen-2-yl, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, 2,3- 5 dihydrobenzofuran-4-yl, 2,3-dihydrobenzofuran-5-yl, 2,3-dihydrobenzofuran-6-yl, 2,3- dihydrobenzofuran-7-yl, 2,3-dihydrobenzothien-2-yl, 2,3-dihydrobenzothien-3-yl, 2,3- dihydrobenzothien-4-yl, 2,3-dihydrobenzothien-5-yl, 2,3-dihydrobenzothien-6-yl, 2,3- dihydrobenzothien-7-yl, 2,3-dihydrobenzothien-8-yl, benzo[d][1,3]dioxol-4-yl, benzo[d][1,3]dioxol-5-yl, 2H-chromen-2-on-3-yl, 2H-chromen-2-on-4-yl, 2H-chromen-2-on-5-yl, 2H-chromen-2-on-6-yl, 2H- 10 chromen-2-on-7-yl, 2H-chromen-2-on-8-yl, isoindoline-1,3-dion-2-yl,isoindoline-1,3-dion-4-yl, isoindoline-1,3-dion-5-yl, inden-1-on-2-yl, inden-1-on-3-yl, inden-1-on-4-yl, inden-1-on-5-yl, inden-1- on-6-yl, inden-1-on-7-yl, 2,3-dihydrobenzo[b][1,4]dioxan-2-yl, 2,3-dihydrobenzo[b][1,4]dioxan-5-yl, 2,3-dihydrobenzo[b][1,4]dioxan-6-yl, 2H-benzo[b][1,4]oxazin-3(4H)-on-5-yl, 2H-benzo[b][1,4]oxazin- 3(4H)-on-6-yl, 2H-benzo[b][1,4]oxazin-3(4H)-on-7-yl, 2H-benzo[b][1,4]oxazin3(4H)-on-8-yl, 15 quinazolin-4(3H)-on-5-yl, quinazolin-4(3H)-on-6-yl, quinazolin-4(3H)-on-7-yl, quinazolin-4(3H)-on-8- yl, quinoxalin-2(1H)-on-5-yl, quinoxalin-2(1H)-on-6-yl, quinoxalin-2(1H)-on-7-yl, quinoxalin-2(1H)-on- 8-yl, benzo[d]thiazol-2(3H)-on-3-yl, benzo[d]thiazol-2(3H)-on-4-yl, benzo[d]thiazol-2(3H)-on-5-yl, benzo[d]thiazol-2(3H)-on-6-yl, and, benzo[d]thiazol-2(3H)-on-7-yl. In certain embodiments, the bicyclic aryl is (i) naphthyl or (ii) a phenyl ring fused to either a 5 or 20 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, or a 5 or 6 membered monocyclic heterocyclyl, wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia. The term "arylalkyl," “aralkyl”, "-alkylaryl," and "arylalkyl-" as used herein, means an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined25 herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3- phenylpropyl, and 2-naphth-2-ylethyl. The term “azido” means a -N3 group. The term “carboxy” means a -COOH group. The terms "cyano" and "nitrile" as used herein, mean a -CN group. 30 The term "cycloalkyl" as used herein, means a monocyclic or a bicyclic cycloalkyl ring system. Monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 10 carbon atoms, where such groups are saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyl ring systems are bridged monocyclic rings or fused bicyclic rings. Bridged monocyclic rings contain a monocyclic cycloalkyl ring 35 where two non-adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form -(CH2)w-, where w is 1, 2, or 3). Representative examples of bridged bicyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, 16 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane. Fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to a monocyclic cycloalkyl. Representative examples of fused bicyclic ring systems include, but are not limited to, decaliyl. Cycloalkyl groups are optionally substituted with one or two groups which are independently oxo or thia. In certain embodiments, the fused bicyclic 5 cycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused to a 5 or 6 membered monocyclic cycloalkyl, wherein the fused bicyclic cycloalkyl is optionally substituted by one or two groups which are independently oxo or thia. "Cycloalkenyl" as used herein refers to a monocyclic or a bicyclic cycloalkenyl ring system. Monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such 10 groups are unsaturated (i.e., containing at least one annular carbon-carbon double bond), but not aromatic. Examples of monocyclic ring systems include cyclopentenyl and cyclohexenyl. Bicyclic cycloalkenyl rings are bridged monocyclic rings or a fused bicyclic rings. Bridged monocyclic rings contain a monocyclic cycloalkenyl ring where two non-adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of 15 the form -(CH2)w-, where w is 1, 2, or 3). Representative examples of bicyclic cycloalkenyls include, but are not limited to, norbornenyl and bicyclo[2.2.2]oct-2-enyl. Fused bicyclic cycloalkenyl ring systems contain a monocyclic cycloalkenyl ring fused to either a monocyclic cycloalkyl or a monocyclic cycloalkenyl. Cycloalkenyl groups are optionally substituted with one or two groups which are independently oxo or thia. 20 The term “monocyclic ring”, as used herein, comprises monocyclic aryl, monocyclic cycloalkyl, monocyclic cycloalkenyl and monocyclic heterocyclyl. The term "halo" or "halogen" as used herein, means -CI, -Br, -I or -F. The term “H” means hydrogen. The term "haloalkyl" as used herein, means at least one halogen, as defined herein, appended to 25 the parent molecular moiety through an alkyl group, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl. The term "heteroaryl," as used herein, means a monocyclic heteroaryl or a bicyclic ring system containing at least one heteroaromatic ring (i.e., a monocyclic or bicyclic aromatic ring system containing 30 at least one heteroatom within the aromatic system). The monocyclic heteroaryl can be a 5 or 6 membered ring. The 5 membered ring consists of two double bonds and one, two, three or four nitrogen atoms and optionally one oxygen or sulfur atom. The 6 membered ring consists of three double bonds and one, two, three or four nitrogen atoms. The 5 or 6 membered heteroaryl is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heteroaryl. 35 Representative examples of monocyclic heteroaryl include, but are not limited to, furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, and triazinyl. The bicyclic heteroaryl consists of a monocyclic heteroaryl fused to a phenyl, a monocyclic cycloalkyl, a monocyclic 17 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. The fused cycloalkyl or heterocyclyl portion of the bicyclic heteroaryl group is optionally substituted with one or two groups which are independently oxo or thia. Representative examples of bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl, benzoxathiadiazolyl, 5 benzothiazolyl, cinnolinyl, 5,6-dihydroquinolin-2-yl, 5,6-dihydroquinolin-8-yl, 5,6-dihydroisoquinolin- 1-yl, furopyridinyl, indazolyl, indolyl, isoquinolinyl, naphthyridinyl, quinolinyl, purinyl, 5,6,7,8- tetrahydroquinolin-2-yl, 5,6,7,8-tetrahydroquinolin-3-yl, 5,6,7,8-tetrahydroquinolin-4-yl, 5,6,7,8- tetrahydroquinolin-5-yl, 5,6,7,8-tetrahydroquinolin-6-yl, 5,6,7,8-tetrahydroquinolin-7-yl, 5,6,7,8- tetrahydroquinolin-8-yl, 5,6,7,8-tetrahydroisoquinolin-1-yl, thienopyridinyl, 4,5,6,7- 10 tetrahydrobenzo[c][1,2,5]oxadiazol-4-yl, and 6,7-dihydrobenzo[c][1,2,5]oxadiazol-4(5H)-onyl. In certain embodiments, the fused bicyclic heteroaryl is a 5 or 6 membered monocyclic heteroaryl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted with one 15 or two groups which are independently oxo or thia. The term "heteroarylalkyl" and "-alkylheteroaryl" as used herein, means a heteroaryl, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of heteroarylalkyl include, but are not limited to, fur-3-ylmethyl, 1H-imidazol- 2-ylmethylm 1H-imidazol-4-ylmethyl, 1-(pyridin-4-yl)ethyl, pyridin-3-ylmethyl, pyridin-4-ylmethyl, 20 pyrimidin-5-ylmethyl, 2-(pyrimidin-2-yl)propyl, thien-2-ylmethyl, and thien-3-ylmethyl. The term "heterocyclyl" as used herein, means a monocyclic heterocycle or a bicyclic heterocycle. The monocyclic heterocycle is a 3, 4, 5, 6, or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic. The 3 or 4 membered ring contains 1 heteroatom selected from the group 25 consisting of O, N and S. The 5 membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. The 6 or 7 membered ring contains zero, one, or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxan-2-yl, 1,3-dioxolan-2-yl, 1,3-dithiolan-2-yl, 1,2- 30 dithiolan-3-yl, 1,2-dithiolan-4-yl, 1,3-dithian-2-yl, 1,2-dithian-3-yl, 1,2-dithian-4-yl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and 35 trithianyl. The bicyclic heterocycle is a monocyclic heterocycle fused to either a monocyclic cycloalkyl, a monocyclic cycloalkenyl, or a monocyclic heterocycle. Representative examples of bicyclic heterocyclyls include, but are not limited to, decahydroquinolinyl, decahydroisoquinolinyl, octahydro- 1H-indolyl, and octahydrobenzofuranyl. Heterocyclyl groups are optionally substituted with one or two 18 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO groups which are independently oxo or thia. In certain embodiments, the bicyclic heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring fused to a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, or a 5 or 6 membered monocyclic heterocyclyl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are independently oxo or thia. 5 The term "hydroxy" or “hydroxyl” as used herein means an -OH group. The term “thiol” as used herein means an –SH group. The term "hydroxyl protecting group," as used herein, refers to a labile chemical moiety which protects a hydroxyl group against undesired reactions during synthetic procedure(s). After the synthetic procedure(s), the hydroxy protecting group may be selectively removed. Hydroxy protecting groups as 10 known in the art are described generally in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley &amp; Sons, New York (1999). Examples of hydroxyl protecting groups include, but are not limited to, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4- bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl, isopropoxycarbonyl, diphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl) 15 ethoxycarbonyl, 2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, t-butyl, 2,2,2-trichloroethyl, 2-trimethylsilyl ethyl, l,l- dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl, para-methoxybenzyldiphenylmethyl, triphenylmethyl (trityl), tetrahydrofuryl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2- trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, para-toluenesulfonyl, tert- 20 butyldimethylsilyl (TBDMS), trimethylsilyl, triethylsilyl, and triisopropylsilyl. In certain embodiments, hydroxyl protecting groups can be selected from acetyl (Ac or — C(O)CH3), benzoyl (Bz or — C(O)C6H5), and trimethylsilyl (TMS or -Si(CH3)3). The term "nitro" as used herein means a -NO2 group. The term "oxo" as used herein means a =O group. 25 The term "saturated" as used herein means the referenced chemical structure does not contain any multiple carbon-carbon bonds. For example, a saturated cycloalkyl group as defined herein includes cyclohexyl, cyclopropyl, and the like. The term "thia" as used herein means a =S group. The term “amine” or “amino” encompasses compounds where a nitrogen atom is covalently 30 bonded to at least one carbon or heteroatom. The term “alkyl amino” includes groups and compounds wherein the nitrogen is bound to at least one additional alkyl group. The term “dialkyl amino” includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups. The term "unsaturated" as used herein means the referenced chemical structure contains at least one multiple carbon-carbon bond (i.e., double or triple bond, or both), but is not aromatic. For example, 35 an unsaturated cycloalkyl group as defined herein includes cyclohexenyl, cyclopentenyl, cyclohexadienyl, and the like. The term “leaving group” as used herein means an atom or group (charged or uncharged) that becomes detached from an atom in what is considered to be the residual or main part of the substrate in a 19 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO specified reaction. For example, the specified reaction herein, unless otherwise noted, is an SN1 or an SN2 reaction as is understood by one skilled in the art. In certain embodimentns, the specified reaction herein, is an SN2 reaction. The term“substituted” as used herein, whether preceded by the term“optionally” or not, means 5 that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a“substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is 10 substituted, the substituent is either the same or different at each position. Suitable substituents include, but are not limited to, halogen, hydroxy, thiol, nitro, alkoxy, azido, carboxy, cyano, amino, C1-6alkyl, C2- 6alkenyl, C1-6alkoxy, C1-6alkylamino.The term “support linking group as used herein means a divalent chemical moiety that covalent connects a surface-bound functional group of a solid support (e.g., an amino group of an amino-modified solid support) to another chemical moiety. 15 The term “reactive pair” as used herein means two functional groups known by one skilled in the art to be capable of reacting, alone or in the presence of other reagents, to form a covalent linkage between the two chemical entities which each contain one of member of the reactive pair, respectively; the latter, herein, is referred to as “linking group formed by a reactive pair”. In some embodiments, a reactive pair is a click pair, i.e., two functional groups capable of reacting in a click reaction to form a 20 covalent linkage. The term “Michael acceptor” as used herein means an alpha, beta-unsaturated compound capable of reacting with a nucleophile at the beta-carbon of the electrophilic alkene of the alpha, beta -unsaturated compound. Examples of alpha, beta-unsaturated compound include, but are not limited to, such as an alpha, beta-unsaturated aldehyde, ester, amide, sulfonyl, ketone, nitrile, or nitro. “Alpha, beta- 25 unsaturated” refers to the carbon-carbon multiple bond that connects the carbon atom that is immediately adjacent to the referenced aldehyde, ester, amide, sulfonyl, ketone, nitrile, or nitro group, to its adjacent carbon atom. Examples of Michael acceptor group include, but are not limited to, N-maleimide, acrylaldehyde, acrylonitrile, acrylic acid, acrylamide (e.g., N-isoproprylacrylamide), acrylate esters (e.g., methyl acrylate), vinyl sulfones, vinylsulfonates, and vinylsulfonamides. 30 The term “nitrogen protecting group” as used herein means those functional groups that are well known in the art and include those described in detail, for example, in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Suitable nitrogen protecting groups include but are not limited to, allyl (Alloc), acetyl (Ac), Adpoc (1-(1-Adamantyl)-1-methylethoxycarbonyl), Boc (tert-butyloxy 20 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO carbonyl), Dde Dnp (2,4-dinitrophenyl), Mmt (4- methoxytrityl), Mtt (4-methyltrityl), Teoc (2-trimethylsilylethoxycarbonyl, Tfa (trifluoroacetyl), optionally substituted trityl (e.g., trityl (Trt), 2-chlorotrityl (Clt), 4-methoxytrityl (Mmt), 4-methyltrityl (Mtt), 4,4’-dimethoxytrityl (DMT)), and 4,4’,4’’-trimethoxytrityl), optionally substituted 5 benzyloxycarbonyl (e.g., benzyloxycarbonyl (Z) or 2-chlorobenzyloxycarbonyl (2ClZ)), fluorenylmethoxycarbonyl (Fmoc), methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 3-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2,4-dichlorophenoxyacetyl, 2-methylphenoxyacetyl, 3- methylphenoxyacetyl, 4-methylphenoxyacetyl, 4-chloro-2-methylphenoxyacetyl, 2-nitrophenoxyacetyl, 3-nitrophenoxyacetyl, 4-nitrophenoxyacetyl, 2-isopropylphenoxyacetyl, 3-isopropylphenoxyacetyl, 4- 10 isopropylphenoxyacetyl, 2-(t-butyl)phenoxyacetyl, 3-(t-butyl)phenoxyacetyl, 4-(t-butyl)phenoxyacetyl, 2-fluorophenoxyacetyl, 3-fluorophenoxyacetyl, 4-fluorophenoxyacetyl, 2,4-difluorophenoxyacetyl, 4- (trifluoromethoxy)phenoxyacetyl, 2-phenoxypropanoyl, 2-(4-chloro-2-methylphenoxy)propanoyl, and 2- (4-chlorophenoxy)propanoyl. As used herein, the term “remainder of the dsRNA agent”, refers to an oligonucleotide strand 15 (i.e. the sense strand or the antisense strand) that forms a dsRNA agent and any intervening linking group that connects the parent moiety (e.g., one or more αvβ6 integrin targeting ligand or one or more in vivo delivery enhancing moieties) to the oligonucleotide strand.. The terms “linker”, “linking group”, “carrier” and “carrier group”, in some embodiments, each refer to a chemical moiety connecting at least one αvβ6 integrin targeting ligand or at least one in vivo 20 delivery enhancing moiety to at least one strand of the dsRNA agent of the disclosure. As used herein, “target sequence” or “target nucleic acid” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodment, the target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed 25 cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene. In one embodiment, the target sequence is within the protein coding region of the target gene. In another embodiment, the target sequence is within the 3’ UTR of the target gene. The target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state. 30 The target sequence may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In some embodiments, the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In still other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in 5 length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure. As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature. 10 “G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 1). The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base 15 pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. It is understood that when a cDNA sequence is provided, the corresponding mRNA or RNAi agent would include a U in place of a T. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the 20 disclosure by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the disclosure. Further, one of skill in the art that a T is a target gene sequence, or reverse complement thereof, would often be replaced by a U in an RNAi 25 agent of the disclosure. The terms “iRNA”, “RNAi agent,” “iRNA agent,” “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs the sequence-specific degradation of 30 mRNA. RNAi modulates, e.g., inhibits, the expression of a target gene in a cell, e.g., a cell within a subject, such as a mammalian subject. The terms “RNAi agent” and “dsRNA agent” may be used interchangeably herein. In one embodiment, an RNAi agent of the disclosure includes a single stranded RNAi that interacts with a target RNA sequence, e.g., a target mRNA sequence, to direct the cleavage of the target 35 RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into double-stranded short interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev.15:485). Dicer, a ribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pair short 22 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO interfering RNAs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or 5 more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev.15:188). Thus, in one aspect the disclosure relates to a single stranded RNA (ssRNA) (the antisense strand of a siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above. 10 In another embodiment, the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Patent No.8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of 15 which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894. In another embodiment, an “RNAi agent” for use in the compositions and methods of the disclosure is a double stranded RNA and is referred to herein as a “double stranded RNAi agent,” 20 “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA” refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., a target mRNA sequence. In some embodiments of the disclosure, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, 25 through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi. In general, a dsRNA molecule can include ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide, a modified nucleotide. In addition, as used in this specification, an “RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides. 30 As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the disclosure include all types of modifications disclosed herein or known in the art. Any such 35 modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims. 23 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In certain embodiments of the instant disclosure, inclusion of a deoxy-nucleotide – which is acknowledged as a naturally occurring form of nucleotide – if present within a RNAi agent can be considered to constitute a modified nucleotide. The duplex region may be of any length that permits specific degradation of a desired target 5 RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 10 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger 15 molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3’-end of one strand and the 5’-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides or nucleotides not directed to the 20 target site of the dsRNA. In some embodiments, the hairpin loop can be 10 or fewer nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides. In certain embodiment, the two strands of double-stranded oligomeric compound can be linked together. The two strands can be linked to each other at both ends, or at one end only. By linking at one 25 end is meant that 5'-end of first strand is linked to the 3'-end of the second strand or 3'-end of first strand is linked to 5'-end of the second strand. When the two strands are linked to each other at both ends, 5'- end of first strand is linked to 3'-end of second strand and 3'-end of first strand is linked to 5'-end of second strand. The two strands can be linked together by an oligonucleotide linker including, but not limited to, (N)n; wherein N is independently a modified or unmodified nucleotide and n is 3-23. In some 30 embodiemtns, n is 3-10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker can be involved in base-pair interactions with other nucleotides in the linker. The two strands can also be linked together by a non-nucleosidic linker, e.g. a linker described herein. It will be 35 appreciated by one of skill in the art that any oligonucleotide chemical modifications or variations describe herein can be used in the oligonucleotide linker. Hairpin and dumbbell type oligomeric compounds will have a duplex region equal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be equal to or 24 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO less than 200, 100, or 50, in length. In some embodiments, ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin oligomeric compounds can have a single strand overhang or terminal unpaired region, in some embodiments at the 3', and in some embodiments on the antisense side of the hairpin. In 5 some embodiments, the overhangs are 1-4, more generally 2-3 nucleotides in length. The hairpin oligomeric compounds that can induce RNA interference are also referred to as "shRNA" herein. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3’-end of 10 one strand and the 5’-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs. 15 In one embodiment, an RNAi agent of the disclosure is a dsRNA, each strand of which is 24-30 nucleotides in length, that interacts with a target RNA sequence, e.g., a target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev.15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base20 pair short interfering RNAs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) 25 Genes Dev.15:188). In one embodiment, an RNAi agent of the disclosure is a dsRNA agent, each strand of which comprises 19-23 nucleotides that interacts with a target mRNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 30 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3’ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one 35 or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev.15:188). In one embodiment, an RNAi agent of the disclosure is a dsRNA of 24-30 nucleotides that interacts with a target mRNA sequence to direct the cleavage of the target RNA. 25 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of a RNAi agent, e.g., a dsRNA. For example, when a 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can 5 comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5'-end, 3'-end or both ends of either an antisense or sense strand of a dsRNA. 10 In one embodiment of the dsRNA, at least one strand comprises a 3’ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3’ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5’ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5’ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 15 nucleotides. In still other embodiments, both the 3’ and the 5’ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide. In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., 0-3, 1-3, 2-4, 2- 5, 4-10, 5-10, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3’-end or the 5’-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 20 nucleotide, overhang at the 3’-end or the 5’-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense 25 strand of the duplex. In certain embodiments, an extended overhang is present on the 3’end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5’end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3’end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5’end of the antisense strand 30 of the duplex. In certain embodiments, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. In certain embodiments, the overhang includes a self-complementary portion such that the overhang is capable of forming a hairpin structure that is stable under physiological conditions. The terms “blunt” or “blunt ended”, as used herein in reference to a dsRNA, mean that there are 35 no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., 26 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO no nucleotide overhang at either end of the molecule. Most often such a molecule will be double stranded over its entire length. The term “antisense strand” or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., a target mRNA 5 sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., a target nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the 10 most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5’- or 3’-terminus of the RNAi agent. As used herein, the term “internal position” refers to any position of the antisense strand or the sense strand that is not a terminal position, i.e., a 3’ terminal position or a 5’ terminal position. As used herein, the term “external position” refers to a terminal position of the antisense strand 15 or the sense strand, i.e., a 3’ terminal position or a 5’ terminal position. In some embodiments, a double stranded RNA agent of the disclosure includes a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of the double stranded RNA agent of the disclosure includes no more than 4 mismatches with the target mRNA, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the 20 antisense strand double stranded RNA agent of the disclosure includes no more than 4 mismatches with the sense strand, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, a double stranded RNA agent of the disclosure includes a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of the double stranded RNA agent of the disclosure includes no more than 4 mismatches with the antisense strand, e.g., the sense strand includes 4, 3, 2, 1, or 25 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, 3 nucleotides from the 3’-end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3’-terminal nucleotide of the iRNA agent. In some embodiments, the mismatch(s) is not in the seed region. Thus, an RNAi agent as described herein can contain one or more mismatches to the target 30 sequence. In one embodiment, a RNAi agent as described herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein contains no more than 2 mismatches. In one embodiment, an RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, an RNAi agent as described herein contains 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the 35 mismatch can optionally be restricted to be within the last 5 nucleotides from either the 5’- or 3’-end of the region of complementarity. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand which is complementary to a region of a target gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used 27 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of a target gene. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of a target gene is important, especially if the particular region of complementarity in a target gene is known to vary. 5 The term “sense strand” or "passenger strand" as used herein, refers to the strand of a RNAi agent that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein. As used herein, “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides. 10 As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at 15 the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13. As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a 20 duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can be, for example, “stringent conditions”, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 oC or 70 oC for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other 25 conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides. Complementary sequences within an RNAi agent, e.g., within a dsRNA as described herein, 30 include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can 35 form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression , in vitro or in vivo. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such 28 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for 5 the purposes described herein. “Complementary” sequences, as used herein, can also include, or be formed entirely from, non- Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogsteen base pairing. 10 The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between two oligonucleotides or polynucleotides, such as the antisense strand of a RNAi agent and a target sequence, as will be understood from the context of their use. As used herein, a polynucleotide that is “substantially complementary to at least part of” a 15 messenger RNA (mRNA) or target sequence refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest or target sequence (e.g., an mRNA encoding a target gene). For example, a polynucleotide is complementary to at least a part of a target RNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding a target gene. 20 Accordingly, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target gene sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target sequence and comprise a contiguous nucleotide sequence which is at least 80% complementary over its entire length to the equivalent region of the nucleotide sequence of the target sequence, such as about 85%, about 90%, about 91%, about 92%, 25 about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary. Exemplary target genes include, for example, adrenoceptor beta 1 (ADRB1); calcium voltage- gated channel subunit alpha1 C (CACNA1C); calcium voltage-gated channel subunit alpha1 G (CACNA1G) (T type calcium cchannel); angiotensin II receptor type 1(AGTR1); Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium 30 Channel 1 (HCN1); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3 (HCN3); Potassium Voltage- Gated Channel Subfamily A Member 5 (KCNA5); Potassium Inwardly Rectifying Channel Subfamily J Member 3 (KCNJ3); Potassium Inwardly Rectifying Channel Subfamily J Member 4 (KCNJ4); phospholamban (PLN); calcium/calmodulin dependent protein kinase II delta (CAMK2D); or 35 Phosphodiesterase 1 (PDE1). Additional exemplary target genes also include, for example, myostatin (MSTN); Cholinergic Receptor Nicotinic Alpha 1 Subunit (CHRNA1); Cholinergic Receptor Nicotinic Beta 1 Subunit (CHRNB1); Cholinergic Receptor Nicotinic Delta Subunit (CHRND); Cholinergic Receptor Nicotinic 29 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Epsilon Subunit (CHRNE); Cholinergic Receptor Nicotinic Gamma Subunit (CHRNG); Collagen Type XIII Alpha 1 Chain (COL13A1); Docking Protein 7 (DOK7); LDL Receptor Related Protein 4 (LRP4); Muscle Associated Receptor Tyrosine Kinase (MUSK); Receptor Associated Protein Of The Synapse (RAPSN); Sodium Voltage-Gated Channel Alpha Subunit 4 (SCN4A); Double Homeobox 4 (DUX4), 5 dystrophy myotonic protein kinase (DMPK), glycogen synthase 1 (GYS1), survival of motor neuron 1 (SMN1), and alpha-glucosidase (GAA). As used herein, “adrenoceptor beta 1,” used interchangeably with the term “ADRB1,” refers to a member of the adrenergic receptor family. ADRB1 is also known as ADRB1R, beta-1 adrenergic receptor, B1AR, BETA1AR, FNSS2, or RHR. 10 In one embodiment, the target gene is calcium voltage-gated channel subunit alpha1 C (CACNA1C). As used herein, “calcium voltage-gated channel subunit alpha1 C,” used interchangeably with the term “CACNA1C,” refers to an alpha-1 subunit of a voltage-dependent calcium channel. CACNA1C is also known as calcium channel, voltage-dependent, L type, alpha 1C subunit; voltage-dependent L-type 15 calcium channel subunit alpha-1C; voltage-gated L-type calcium channel Cav1.2 alpha 1 subunit, splice variant 10; calcium channel, L type, alpha-1 polypeptide, isoform 1, cardiac muscle; calcium channel, cardic dihydropyridine-sensitive, alpha-1 subunit; voltage-dependent L-type Ca2+ channel alpha 1 subunit; voltage-gated calcium channel subunit alpha CaV1.2; DHPR, alpha-1 subunit; CACH2, CACN2, CACNL1A1, CCHL1A1, CaV1.2, LQT8, TS, or TS. LQT8 20 As used herein, “calcium voltage-gated channel subunit alpha1 G,” used interchangeably with the term “CACNA1G,” refers to a T-type, low-voltage activated calcium channel. CACNA1G is also known as calcium channel, voltage-dependent, T type, alpha 1G subunit; voltage-dependent T-type calcium channel subunit alpha-1G; voltage-gated calcium channel subunit alpha Cav3.1; NBR13 ; Cav3.1c; Ca(V)T.1; KIAA1123; SCA42ND; or SCA42. 25 As used herein, “angiotensin II receptor type 1,” used interchangeably with the term “AGTR1,” refers to a receptor for the vasoconstricting peptide angiotensin II. AGTR1 is also known as angiotensin receptor 1B, AT1, AT2R1, AGTR1A, AT2R1B, AGTR1B, HAT1R, AG2S, AT1B, AT2R1A, AT1AR, AT1BR, or AT1R. As used herein, “Sodium Voltage-Gated Channel Alpha Subunit 2,” used interchangeably with 30 the term “SCN2A,” refers to a member of the voltage-gated sodium channel family. SCN2A is also known as Nav1.2, HBSCII, SCN2A1, SCN2A2, HBSCI, EIEE11, BFIC3, BFIS3, BFNIS, DEE11, EA9, or HBA. As used herein, “Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1,” used interchangeably with the term “HCN1,” refers to a member of the hyperpolarization-activated cyclic 35 nucleotide-gated (HCN) channel family. HCN1 is also known as potassium channel 1, BCNG-1, HAC-2, BCNG1, Potassium/Sodium Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 1; Brain Cyclic Nucleotide-Gated Channel 1; Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1; GEFSP10, EIEE24, or DEE24. 30 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO As used herein, “Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4,” used interchangeably with the term “HCN4,” refers to a member of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family. HCN4 is also known as Potassium/Sodium Hyperpolarization- Activated Cyclic Nucleotide-Gated Channel 4, Hyperpolarization Activated Cyclic Nucleotide-Gated 5 Potassium Channel 4, Hyperpolarization Activated Cyclic Nucleotide-Gated Cation Channel 4 or SSS2. As used herein, “Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3,” used interchangeably with the term “HCN3,” refers to a member of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family. HCN3 is also known as Potassium/Sodium Hyperpolarization- Activated Cyclic Nucleotide-Gated Channel 3, Hyperpolarization Activated Cyclic Nucleotide-Gated 10 Potassium Channel 3, or KIAA1535. As used herein, “Potassium Voltage-Gated Channel Subfamily A Member 5,” used interchangeably with the term “KCNA5,” refers to a member of the voltage-gated potassium channel family. KCNA5 is also known as HPCN1, HK2, Potassium Voltage-Gated Channel, Shaker-Related Subfamily, Member 5; Voltage-Gated Potassium Channel Subunit Kv1.5; Voltage-Gated Potassium 15 Channel HK2; Kv1.5; Insulinoma And Islet Potassium Channel; Cardiac Potassium Channel; Potassium Channel 1; ATFB7, HCK1 or PCN1. As used herein, “Potassium Inwardly Rectifying Channel Subfamily J Member 3,” used interchangeably with the term “KCNJ3,” refers to an integral membrane protein and an inward-rectifier type potassium channel. KCNJ3 is also known as GIRK1, G Protein-Activated Inward Rectifier 20 Potassium Channel 1, KGA; Potassium Channel, Inwardly Rectifying Subfamily J Member 3; Inward Rectifier K(+) Channel Kir3.1; or Potassium Inwardly-Rectifying Channel Subfamily J Member 3 Splice Variant 1e. As used herein, “Potassium Inwardly Rectifying Channel Subfamily J Member 4,” used interchangeably with the term “KCNJ4,” refers to an integral membrane protein and inward-rectifier type 25 potassium channel. KCNJ4 is also known as HIRK2, HRK1, IRK3, HIR, Kir2.3, inward rectifier potassium channel 4; Inward Rectifier K(+) Channel Kir2.3; Potassium Voltage-Gated Channel Subfamily J Member 4; Hippocampal Inward Rectifier Potassium Channel; or Hippocampal Inward Rectifier. As used herein, “Phosphodiesterase 1,” used interchangeably with the term “PDE1,” refers to a 30 member of the cyclic nucleotide phosphodiesterases families. PDE1 is also known as Calcium/Calmodulin-Dependent 3',5'-Cyclic Nucleotide Phosphodiesterase 1; Calcium/Calmodulin- Stimulated Cyclic Nucleotide Phosphodiesterase; CAM-PDE 1, HSPDE1, HCAM1, or EC 3.1.4. As used herein, “myostatin,” used interchangeably with the term “MSTN,” refers to a secreted ligand of the TGF-beta (transforming growth factor-beta) superfamily of proteins. Myostatin is also 35 known as GDF8, Growth/Differentiation Factor 8, or MSLHP. As used herein, “Cholinergic Receptor Nicotinic Alpha 1 Subunit,” used interchangeably with the term “CHRNA1,” refers to an alpha subunit of the muscle acetylcholine receptor (AChR). CHRNA1 is also known as Cholinergic Receptor, Nicotinic, Alpha Polypeptide 1; Acetylcholine Receptor, 31 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Nicotinic, Alpha 1 (Muscle); ACHRA; CHRNA; Muscle Nicotinic Acetylcholine Receptor; CMS1A, CMS1B, CMS2A, FCCMS, SCCMS, or ACHRD. As used herein, “Cholinergic Receptor Nicotinic Beta 1 Subunit,” used interchangeably with the term “CHRNB1,” refers to a beta subunit of the muscle acetylcholine receptor (AChR). CHRNB1 is 5 associated with diseases associated such as Myasthenic Syndrome. CHRNB1 is also known as Cholinergic Receptor, Nicotinic, Beta Polypeptide 1; Acetylcholine Receptor, Nicotinic, Beta 1 (Muscle); ACHRB; CHRNB; CMS1D, CMS2C, CMS2A, or SCCMS. As used herein, “Cholinergic Receptor Nicotinic Delta Subunit,” used interchangeably with the term “CHRND,” refers to a delta subunit of the muscle acetylcholine receptor (AChR). CHRND is also 10 known as ACHRD, Cholinergic Receptor, Nicotinic, Delta Polypeptide; Acetylcholine Receptor, Nicotinic, Delta (Muscle); CMS2A; CMS3A, CMS3B, CMS3C, FCCMS, or SCCMS. As used herein, “Cholinergic Receptor Nicotinic Epsilon Subunit,” used interchangeably with the term “CHRNE,” refers to a subunit of the acetylcholine receptor. CHRNE is also known as Cholinergic Receptor, Nicotinic, Epsilon; Acetylcholine Receptor, Nicotinic, Epsilon; ACHRE; CMS1D, CMS1E, 15 CMS2A, CMS4A, CMS4B, CMS4C, FCCMS, or SCCMS. As used herein, “Cholinergic Receptor Nicotinic Gamma Subunit,” used interchangeably with the term “CHRNG,” refers to a subunit of the acetylcholine receptor. CHRNG is also known as Cholinergic Receptor, Nicotinic, Gamma; Acetylcholine Receptor, Nicotinic, Gamma; or ACHRG. As used herein, “Collagen Type XIII Alpha 1 Chain,” used interchangeably with the term 20 “COL13A1,” refers to a synaptic extracellular-matrix protein involved in the formation and maintenance of the neuromuscular synapse. COL13A1 is also known as COLXIIIA1, Collagen Alpha-1(XIII) Chain, or CMS19. As used herein, “Docking Protein 7,” used interchangeably with the term “DOK7,” refers to a protein that is essential for neuromuscular synaptogenesis. DOK7 is also known as C4orf25, Downstream 25 Of Tyrosine Kinase 7, FLJ33718, FLJ39137, Chromosome 4 Open Reading Frame 25, CMS10, CMS1B, or FADS3. As used herein, “LDL Receptor Related Protein 4,” used interchangeably with the term “LRP4,” refers to a member of the low-density lipoprotein receptor-related protein family. LRP4 is also known as MEGF7, LRP-4, SOST2, CLSS, Low-Density Lipoprotein Receptor-Related Protein 4, Multiple 30 Epidermal Growth Factor-Like Domains 7, LRP10, KIAA0816, or CMS17. As used herein, “Muscle Associated Receptor Tyrosine Kinase,” used interchangeably with the term “MUSK,” refers to a muscle-specific tyrosine kinase receptor, which plays a central role in the formation and the maintenance of the neuromuscular junction (NMJ), the synapse between the motor neuron and the skeletal muscle. MUSK is also known as EC 2.7.10.1, FADS1, CMS9, FADS, Muscle, 35 Skeletal Receptor Tyrosine-Protein Kinase, or Muscle-Specific Kinase Receptor. As used herein, “Receptor Associated Protein Of The Synapse,” used interchangeably with the term “RAPSN,” refers to a member of a family of proteins that are receptor associated proteins of the synapse. RAPSN is also known as RNF205, 43 KDa Receptor-Associated Protein Of The Synapse, 32 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO RING Finger Protein 205, CMS1D, CMS1E, Acetylcholine Receptor-Associated 43 Kda Protein, RAPSYN, CMS11, CMS4C, FADS2, or FADS. As used herein, “Sodium Voltage-Gated Channel Alpha Subunit 4,” used interchangeably with the term “SCN4A,” refers to a member of the voltage-gated sodium channel family. SCN4A is also 5 known as SkM1, Nav1.4, HYPP, Sodium Channel Protein Skeletal Muscle Subunit Alpha, Voltage- Gated Sodium Channel Subunit Alpha Nav1.4, HYKPP, Skeletal Muscle Voltage-Dependent Sodium Channel Type IV Alpha Subunit, CTC-264K15.6, Na(V)1.4, HOKPP2, CMS16, or NAC1A. As used herein, “Double Homeobox 4,” used interchangeably with the term “DUX4,” refers to a transcriptional activator of many genes. DUX4 is also known as Double Homeobox Protein 10, Double 10 Homeobox Protein 4, Double Homeobox Protein 4/10, DUX4L, and DUX10. As used herein, “phospholamban,” used interchangeably with the term “PLN,” refers to a crucial regulator of cardiac contractility. PLN is also known as CMD1P, PLB, Cardiac Phospholamban, or CMH. As used herein, “calcium/calmodulin dependent protein kinase II delta,” used interchangeably 15 with the term “CAMK2D,” refers to a member of the serine/threonine protein kinase family and the Ca(2+)/calmodulin-dependent protein kinase subfamily. CAMK2D is also known as Calcium/Calmodulin-Dependent Protein Kinase Type II Delta Chain, CaM Kinase II Delta Subunit, CaM Kinase II Subunit Delta, CAMKD, EC 2.7.11.17, or EC 2.7.11. As used herein, “dystrophy myotonic protein kinase,” used interchangeably with the term 20 “DMPK,” refers to a non-receptor serine/threonine protein kinase which is necessary for the maintenance of skeletal muscle structure and function. DMPK is also known as DM1 protein kinase, DM1PK, DM1, MT-PK, MDPK, DMK, myotonin-protein kinase, myotonic dystrophy associated protein kinase, dystrophia myotonica protein kinase, myotonin protein kinase A, thymopoietin homolog, or EC 2.7.11.1. As used herein, “glycogen synthase 1,” used interchangeably with the term “GYS1,” refers to an 25 enzyme that catalyzes the addition of glucose monomers to the growing glycogen molecule. GYS1 is also known as muscle glycogen synthase, GSY, GYS, or EC 2.4.1.11. As used herein, “survival of motor neuron 1,” used interchangeably with the term “SMN1,” refers to one of a group of proteins called the SMN complex, which is important for the maintenance of specialized nerve cells called motor neurons. SMN1 is also known as SMNT, TDRD16A, Gemin-1, 30 BCD541, GEMIN1, SMA1, SMA2, SMA3, SMA4, SMN, SMNT, tudoe domain containing 16A, complement of gems 1, or SMNC. As used herein, “alpha-glucosidase,” used interchangeably with the term “GAA,” refers to an enzyme responsible for the degradation of glycogen to glucose in lysosomes. GAA is also known as lysosomal alpha-glucosidase, acid maltase, EC 3.2.1.20, glycogen storage disease type II, or LYAG. 35 Examples of target mRNA sequences, and variations thereof (e.g., variants provided in the SNP database) are readily available through publicly available databases, e.g., GenBank, UniProt, OMIM, UCSC Genome Browser, NCBI dbSNP, and the Macaca genome project website. 33 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, the double-stranded region of a dsRNA agent is equal to or at least, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length. In some embodiments, the antisense strand of a dsRNA agent is equal to or at least 14, 15, 16, 5 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the sense strand of a dsRNA agent is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In one embodiment, the sense and antisense strands of the dsRNA agent are each independently 15 to 30 nucleotides in length. 10 In one embodiment, the sense and antisense strands of the dsRNAagent are each independently 19 to 25 nucleotides in length. In one embodiment, the sense and antisense strands of the dsRNAagent are each independently 21 to 23 nucleotides in length. In one embodiment, the sense strand of the dsRNA agent is 21-nucleotides in length, and the 15 antisense strand is 23-nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs having a 2-nucleotide long single stranded overhangs at the 3’-end. In one aspect of the disclosure, an agent for use in the methods and compositions of the disclosure is a single-stranded antisense nucleic acid molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a 20 sequence within the target mRNA. The single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense RNA molecule may be about 15 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense RNA molecule may comprise a 25 sequence that is at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein. In one embodiment, at least partial suppression of the expression of a target gene, is assessed by a reduction of the amount of target mRNA which can be isolated from or detected in a first cell or group of cells in which a target gene is transcribed and which has or have been treated such that the expression 30 of a target gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition may be expressed in terms of: (mRNA in control cells) - (mRNA in treated cells) (mRNA in control cells) 100% 35 In one embodiment, inhibition of expression is determined by the dual luciferase method wherein the RNAi agent is present at 10 nM. The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell 34 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO in vitro with the RNAi agent or contacting a cell in vivo with the RNAi agent. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell. 5 Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, or to the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. In some embodiments, the RNAi agent may contain or be coupled to a ligand, e.g., one or 10 more alpha-v-beta-6 (αvβ6) integrin targeting ligand. An “alpha-v-beta-6 (αvβ6) integrin targeting ligand”, as used herein, includes any moiety (e.g., peptides and small molecules) which bind an αvβ6 integrin and are able to mediate delivery of a dsRNA agent to which they are attached to skeletal muscle (e.g., a skeletal muscle cell or skeletal muscle tissue) and/or cardiac muscle (e.g., a cardiac muscle cell or cardiac muscle tissue). The αvβ6 integrin targeting 15 ligands bind αvβ6 integrin or the αvβ6 integrin receptor on skeletal muscle/cardiac myocytes (cells). Exemplary αvβ6 integrin targeting ligands are described in Section II below. Exemplary αvβ6 integrin targeting ligands are also described in WO2024/086633, incorporated by reference herein. In one embodiment, contacting a cell with an RNAi agent includes “introducing” or “delivering the RNAi agent into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or 20 uptake of a RNAi agent can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing a RNAi agent into a cell may be in vitro or in vivo. For example, for in vivo introduction, a RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or are known in the art. 25 As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), or a non-primate (such as a a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse), or a bird that expresses the target gene, either endogenously or heterologously. In one embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder, or condition that would benefit 30 from reduction in target gene expression; a human at risk for a disease, disorder, or condition that would benefit from reduction in target gene expression; a human having a disease, disorder, or condition that would benefit from reduction in target gene expression; or human being treated for a disease, disorder, or condition that would benefit from reduction in target gene expression as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one 35 embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject. As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more signs or symptoms associated with target gene expression or target gene protein production, e.g., a target gene-associated disease, e.g., 35 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO a muscle disorder, e.g., a skeletal muscle disorder, and/or a cardiac muscle disorder, or symptoms associated with unwanted target gene expression; diminishing the extent of unwanted target activation or stabilization; amelioration or palliation of unwanted target activation or stabilization. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment. 5 The term “lower” in the context of the level of a target gene in a subject or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, a decrease is at least 20%. In certain embodiments, the decrease is at least 50% in a disease marker, e.g., protein or gene expression level. “Lower” in the context of the 10 level of a target gene in a subject is a decrease to a level accepted as within the range of normal for an individual without such disorder. In certain embodiments, the expression of the target is normalized, i.e., decreased towards or to a level accepted as within the range of normal for an individual without such disorder, e.g., blood glucose level, blood uric acid level, blood lipid level, blood oxygen level, white blood cell count, kidney function, spleen function, liver function. As used here, “lower” in a subject can 15 refer to lowering of gene expression or protein production in a cell in a subject does not require lowering of expression in all cells or tissues of a subject. For example, as used herein, lowering in a subject can include lowering of gene expression or protein production in a subject. The term “lower” can also be used in association with normalizing a symptom of a disease or condition, i.e. decreasing the difference between a level in a subject suffering from a target gene- 20 associated disease towards or to a level in a normal subject not suffering from a target gene-associated disease. As used herein, if a disease is associated with an elevated value for a symptom, “normal” is considered to be the upper limit of normal. If a disease is associated with a decreased value for a symptom, “normal” is considered to be the lower limit of normal. As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder, or 25 condition thereof, that would benefit from a reduction in expression of a target gene or production of a target protein, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of a target gene-associated disease. The failure to develop a disease, disorder, or condition, or the reduction in the development of a symptom associated with such a disease, disorder, or condition (e.g., by at least about 10% on a clinically accepted 30 scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention. As used herein, the term “target gene-associated disease,” is a disease or disorder that would benefit from reduction in the expression or activity of the target gene. The term “target gene-associated disease,” is a disease or disorder that is caused by, or associated with expression or protein production of 35 the target gene. The term "target gene-associated disease” includes a disease, disorder or condition that would benefit from a decrease in expression or protein activity of the target gene. Additional information regarding specific target genes and disease that would benefit from reduction in expression of the target gene are descrived below. 36 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, the target gene-associated disease is a muscle disorder. Exemplary muscle disorders include Myostatin-related muscle hypertrophy, congenital myasthenic syndrome, facioscapulohumeral muscular dystrophy (FSHD), Spinal Muscular Atrophy (SMA), Myotonic Dystrophy Type 1 (DM1), Pompe disease, PLN cardiomyopathy, spasticity, 5 obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic cardiomyopathy (FHC); Heart failure with preserved ejection fraction (HFPEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina; myocardial infarction (MI); heart failure or heart failure with reduced ejection fraction (HFREF); supraventricular tachycardia (SVT); hypertrophic cardiomyopathy (HCM); and PLN cardiomyopathy. 10 In one embodiment, the target gene-associated disease is a skeletal muscle disease or disorder. In one embodiment, the target gene-associated disease is a cardiac muscle disease or disorder. Exemplary cardiac muscle disorders include obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic cardiomyopathy (FHC); Heart failure with preserved ejection fraction (HFPEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina; myocardial infarction (MI); heart failure 15 or heart failure with reduced ejection fraction (HFREF); supraventricular tachycardia (SVT); hypertrophic cardiomyopathy (HCM); and PLN cardiomyopathy. “Heart failure” (“HF”) or “congestive heart failure” (“CHF”) is a chronic condition in which the heart does not pump blood as well as it should. Heart failure can occur when the heart’s capacity to pump blood cannot keep up with the body’s need. Heart failure can occur if the heart cannot pump 20 (systolic) or fill (diastolic) adequately. The term “congestive” refers to the resulting buildup of fluid in the ankles and feet, arms, lungs, and/or other organs. One type of heart failure is “heart failure with preserved left ventricular function” (“HF-pEF”) also known as “heart failure with preserved ejection fraction” (“HF-pEF”) is a condition in which the heart contracts and pumps normally, but the ventricles are thicker and stiffer than normal.. 25 A common cause of congestive heart failure is coronary artery disease. Risk factors for coronary artery disease can include high levels of cholesterol and/or triglyceride, high blood pressure, poor diet, a sedentary lifestyle, diabetes, smoking, being overweight or obese, and stress. In addition to coronary artery disease, several other conditions can damage the heart muscles, including inherited and genetic factors, some infections and autoimmune diseases and some treatments such as chemotherapy. 30 Symptoms of CHF can include shortness of breath, fatigue, swollen legs, and rapid heartbeat. “Hypertrophic cardiomyopathy” (“HCM”) refers to impaired heart function associated with abnormally thick heart muscle in the absence of other heart disease; e.g., valvular heart disease. “Hypertrophic obstructive cardiomyopathy” (“HOCM”) is a subtype of HCM, where the wall (septum) between the two bottom chambers of the heart thickens. The walls of the pumping chamber can also 35 become stiff. The thickened septum may cause a narrowing that can block or reduce the blood flow from the left ventricle to the aorta, which is a condition called “outflow tract obstruction.” Both HCM and HOCM can be caused by heart muscle gene mutation, which may be inherited. 37 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO “Familial hypertrophic cardiomyopathy” is an autosomal dominant disease characterized mainly by left ventricular hypertrophy. Thickening usually occurs in the interventricular septum. In some, thickening of the interventricular septum impedes the flow of oxygen-rich blood from the heart, which may lead to an abnormal heart sound during a heartbeat (heart murmur) and other signs and symptoms of 5 the condition. Familial hypertrophic cardiomyopathy can cause abnormal heart rhythms (arrhythmias) that can be life threatening. People with familial hypertrophic cardiomyopathy have an increased risk of sudden death, even if they have no other symptoms of the condition. A small number of affected individuals develop potentially fatal heart failure, which may require heart transplantation. 10 Mutations in one of several genes can cause familial hypertrophic cardiomyopathy; the most commonly involved genes are MYH7, MYBPC3, TNNT2, and TNNI3. Other genes, including some that have not been identified, may also be involved in this condition. “Atrial fibrillation” (“AFIB”) is when the atria beat chaotically and irregularly - out of coordination with the ventricles. The result can be a fast and irregular heart rhythm. The heart rate in 15 atrial fibrillation can range from 100 to 175 beats a minute. The normal range for a heart rate is 60 to 100 beats a minute. “Ventricular fibrillation” (“VFIB”) is a type of abnormal heart rhythm (arrhythmia). During ventricular fibrillation, disorganized heart signals cause the ventricles to twitch (quiver) uselessly. As a result, the heart does not pump blood to the rest of the body. 20 Ventricular fibrillation is an emergency that requires immediate medical attention. A “myocardial infarction” or “MI” occurs when the flow of blood to the heart is blocked. The blockage can be a buildup of fat, cholesterol and other substances, which form a plaque in the arteries that feed the heart (coronary arteries). “Supraventricular tachycardia” (“SVT”) is as an abnormally fast or erratic heartbeat that affects 25 the heart's atria. During an episode of SVT, the heart beats about 150 to 220 times per minute, but it can occasionally beat faster or slower. Things that may cause an SVT episode include age, coronary artery disease, previous heart surgery, heart disease, heart failure, other heart problems, such as Wolff-Parkinson-White syndrome, chronic lung disease, consuming too much caffeine, drinking too much alcohol, drug use, particularly 30 stimulants such as cocaine and methamphetamines, pregnancy, smoking, thyroid disease, tobacco, sleep apnea, diabetes, and certain medications, including asthma medications and over-the-counter cold and allergy drugs. “Hypertrophic cardiomyopathy” (“HCM”) is a disease in which the heart muscle becomes abnormally thick (hypertrophied). The thickened heart muscle can make it harder for the heart to pump 35 blood. “Angina” is a type of chest pain caused by reduced blood flow to the heart. Angina is a symptom of coronary artery disease. 38 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Angina, also called angina pectoris, can be described as squeezing, pressure, heaviness, tightness or pain in the chest. Phospholamban (PLN) cardiomyopathy, as used herein, refers to a specific subtype of hereditary cardiomyopathy caused by PLN p.(Arg14del), a pathogenic variant in the gene encoding PLN, which is a 5 protein with a central role in calcium homeostasis in cardiac tissue. This protein ensures proper contraction and relaxation of the human heart. Carriers of this pathogenic variant have a high risk of developing dilated cardiomyopathy (DCM), arrhythmic cardiomyopathy (ACM), or both. They commonly have a high arrhythmic burden, with premature ventricular contractions (PVCs) and ventricular tachycardia, remarkable low-voltage electrocardiograms (ECGs), left ventricular dysfunction, 10 and a positive family history for sudden cardiac death. PLN p.(Arg14del) cardiomyopathy has been found in several European countries, but also in the United States, Canada, and China. On a global scale it is a rare disease, but it is particularly common in The Netherlands, with the pathogenic variant being present in 12% of all ACM patients and 15% of all DCM patients. Exemplary skeletal muscle disorders include Myostatin-related muscle hypertrophy, congenital 15 myasthenic syndrome, facioscapulohumeral muscular dystrophy (FSHD), Spinal Muscular Atrophy (SMA), Myotonic Dystrophy Type 1 (DM1), Pompe disease, PLN cardiomyopathy, and spasticity. Myostatin-related muscle hypertrophy is a rare condition characterized by reduced body fat and increased muscle size. Affected individuals have up to twice the usual amount of muscle mass in their bodies. They also tend to have increased muscle strength. Myostatin-related muscle hypertrophy is 20 caused by mutations in the MSTN gene. It follows an incomplete autosomal dominant pattern of inheritance. Congenital myasthenic syndromes (CMS) are a heterogeneous group of early-onset genetic neuromuscular transmission disorders due to mutations in proteins involved in the organisation, maintenance, function, or modification of the motor endplate (endplate myopathies), e.g., CHRNA1, 25 CHRNB1, CHRBD, CHRNE, CHRNG, COL13A1, DOX7, LRP4, MUSK, RAPSN, or SCN4A. CMS are clinically characterised by abnormal fatigability, or transient or permanent weakness of extra-ocular, facial, bulbar, truncal, respiratory, or limb muscles. Onset of endplate myopathy is intrauterine, congenital, in infancy, or childhood, and rarely in adolescence. Severity ranges from mild, phasic weakness, to disabling, permanent muscle weakness, respiratory insufficiency, and early death. All 30 subtypes of CMS share the clinical features of fatigability and muscle weakness, but age of onset, presenting symptoms, and response to treatment vary depending on the molecular mechanism that results from the underlying genetic defect. The term CMS is misleading since not all CMS are congenital. See, Finsterer (2019) Orphanet J Rare Dis.14: 57 for a review. Facioscapulohumeral muscular dystrophy (FSHD) type 1 is an autosomal dominant condition 35 caused by mutations in DUX4. FSHD typically presents before age 20 years with weakness of the facial muscles and the stabilizers of the scapula or the dorsiflexors of the foot. There is extreme clinical variability. In some cases, congenital facial weakness may be present. In FSHD, the muscle weakness is 39 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO slowly progressive and approximately 20% of affected individuals eventually require a wheelchair. Life expectancy is not shortened. The incidence is approximately 4 individuals affected per 100,000 people. Spinal muscular atrophy, as used herein, refers to a genetic disorder characterized by weakness and wasting (atrophy) in muscles used for movement (skeletal muscles). It is caused by a loss of 5 specialized nerve cells, called motor neurons that control muscle movement. The weakness can be more severe in the muscles that are close to the center of the body (proximal) compared to muscles away from the body's center (distal). The muscle weakness usually worsens with age. There are many types of spinal muscular atrophy that are caused by changes in the same genes. The types differ in age of onset and severity of muscle weakness; however, there is overlap between the types. Other forms of spinal 10 muscular atrophy and related motor neuron diseases, such as spinal muscular atrophy with progressive myoclonic epilepsy, spinal muscular atrophy with lower extremity predominance, X-linked infantile spinal muscular atrophy, and spinal muscular atrophy with respiratory distress type 1 are caused by mutations in other genes. Mutations in the SMN1 gene cause all types of spinal muscular atrophy described above. The 15 number of copies of the SMN2 gene modifies the severity of the condition and helps determine which type develops. The SMN1 and SMN2 genes both provide instructions for making a protein called the survival motor neuron (SMN) protein. Normally, most functional SMN protein is produced from the SMN1 gene, with a small amount produced from the SMN2 gene. Several different versions of the SMN protein are produced from the SMN2 gene, but only one version is functional; the other versions are 20 smaller and quickly broken down. The SMN protein is one of a group of proteins called the SMN complex, which is important for the maintenance of motor neurons. Motor neurons transmit signals from the brain and spinal cord that tell skeletal muscles to tense (contract), which allows the body to move. Myotonic dystrophy, as used herein, refers to a part of a group of inherited disorders called muscular dystrophies. It is the most common form of muscular dystrophy that begins in adulthood. 25 Myotonic dystrophy is characterized by progressive muscle wasting and weakness. People with this disorder often have prolonged muscle contractions (myotonia) and are not able to relax certain muscles after use. Other signs and symptoms of myotonic dystrophy include clouding of the lens of the eye (cataracts) and abnormalities of the electrical signals that control the heartbeat (cardiac conduction defects). Some affected individuals develop a condition called diabetes mellitus, in which blood sugar 30 levels can become dangerously high. The features of myotonic dystrophy often develop during a person's twenties or thirties, although they can occur at any age. The severity of the condition varies widely among affected people, even among members of the same family. There are two major types of myotonic dystrophy: type 1 and type 2. Their signs and symptoms overlap, although type 2 tends to be milder than type 1. The muscle weakness associated with type 1 particularly affects muscles farthest from the center 35 of the body (distal muscles), such as those of the lower legs, hands, neck, and face. Muscle weakness in type 2 primarily involves muscles close to the center of the body (proximal muscles), such as the those of the neck, shoulders, elbows, and hips. The two types of myotonic dystrophy are caused by mutations in different genes. 40 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Myotonic dystrophy type 1 is caused by mutations in the DMPK gene, while type 2 results from mutations in the CNBP gene. The protein produced from the DMPK gene likely plays a role in communication within cells. It appears to be important for the correct functioning of cells in the heart, brain, and skeletal muscles (which are used for movement). The protein produced from the CNBP gene is 5 found primarily in the heart and in skeletal muscles, where it helps regulate the function of other genes. Similar changes in the structure of the DMPK and CNBP genes cause myotonic dystrophy type 1 and type 2. In each case, a segment of DNA is abnormally repeated many times, forming an unstable region in the gene. The gene with the abnormal segment produces an unusually long messenger RNA, which is a molecular blueprint of the gene that guides the production of proteins. The unusually long messenger 10 RNA forms clumps inside the cell that interfere with the production of many other proteins. These changes prevent muscle cells and cells in other tissues from functioning normally, which leads to the signs and symptoms of myotonic dystrophy. If these changes affect the DMPK gene, the result is myotonic dystrophy type 1, if the CNBP gene is affected, the result is myotonic dystrophy type 2. Pompe disease, as used herein, refers to an inherited disorder caused by the buildup of a complex 15 sugar called glycogen in the body's cells. The accumulation of glycogen in certain organs and tissues, especially muscles, impairs their ability to function normally. There are three types of Pompe disease, which differ in severity and the age at which they appear. These types are known as classic infantile- onset, non-classic infantile-onset, and late-onset. The classic form of infantile-onset Pompe disease begins within a few months of birth. Infants with this disorder typically experience muscle weakness 20 (myopathy), poor muscle tone (hypotonia), an enlarged liver (hepatomegaly), and heart defects. Affected infants may also fail to gain weight and grow at the expected rate (failure to thrive) and have breathing problems. If untreated, this form of Pompe disease leads to death from heart failure in the first year of life. The non-classic form of infantile-onset Pompe disease usually appears by age 1. It is characterized by delayed motor skills (such as rolling over and sitting) and progressive muscle weakness. The heart 25 may be abnormally large (cardiomegaly), but affected individuals usually do not experience heart failure. The muscle weakness in this disorder leads to serious breathing problems, and most children with non- classic infantile-onset Pompe disease live only into early childhood. The late-onset type of Pompe disease may not become apparent until later in childhood, adolescence, or adulthood. Late-onset Pompe disease is usually milder than the infantile-onset forms of this disorder and is less likely to involve the 30 heart. Most individuals with late-onset Pompe disease experience progressive muscle weakness, especially in the legs and the trunk, including the muscles that control breathing. As the disorder progresses, breathing problems can lead to respiratory failure. Mutations in the GAA gene cause Pompe disease. The GAA gene provides instructions for producing an enzyme called acid alpha-glucosidase (also known as acid maltase). This enzyme is active 35 in lysosomes, which are structures that serve as recycling centers within cells. The enzyme normally breaks down glycogen into a simpler sugar glucose, which is the main energy source for most cells. Mutations in the GAA gene prevent acid alpha-glucosidase from breaking down glycogen effectively, which allows this sugar to build up to toxic levels in lysosomes. This buildup damages organs and tissues 41 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO throughout the body, particularly the muscles, leading to the progressive signs and symptoms of Pompe disease. Spasticity, as used herein, refers to a condition in which muscles stiffen or tighten, preventing normal fluid movement. The muscles remain contracted and resist being stretched, thus affecting 5 movement, speech and gait. Spasticity is generally caused by damage or disruption to the area of the brain and spinal cord that are responsible for controlling muscle and stretch reflexes. These disruptions can be due to an imbalance in the inhibitory and excitatory signals sent to the muscles, causing them to lock in place. Spasticity can be harmful to growing children as it can affect muscles and joints. People with brain injury, spinal cord injury, cerebral palsy or multiple sclerosis can have varying degrees of 10 spasticity. "Therapeutically effective amount," as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a target gene-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease or one or more symptoms of disease). The "therapeutically effective amount" may vary depending on the RNAi 15 agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated. “Prophylactically effective amount,” as used herein, is intended to include the amount of a RNAi agent that, when administered to a subject having a target gene-associated disorder, e.g., gout or diabetes, 20 is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of 25 the patient to be treated. A "therapeutically-effective amount" or “prophylacticaly effective amount” also includes an amount of a RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. A RNAi agent employed in the methods of the present disclosure may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such 30 treatment. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable 35 benefit/risk ratio. The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent 42 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers 5 include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and 10 soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum 15 component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations. Pharmaceutically acceptable carriers for pulmonary delivery are known in the art and will vary depending on the desired location for deposition of the agent, e.g., upper or lower respiratory system, and the type of device to be used for delivery, e.g., sprayer, nebulizer, dry powder inhaler. 20 “Pharmaceutically acceptable salts” of each of RNAi agents herein include, but are not limited to, a sodium salt, a calcium salt, a lithium salt, a potassium salt, an ammonium salt, a magnesium salt, an mixtures thereof. One skilled in the art will appreciate that the RNAi agent, when provided as a polycationic salt having one cation per free acid group of the optionally modified phosophodiester backbone and/or any other acidic modifications (e.g., 5’-terminal phosphonate groups). For example, an 25 oligonucleotide of “n” nucleotides in length contains n-1 optionally modified phosophodiesters, so that an oligonucleotide of 21 nt in length may be provided as a salt having up to 20 cations (e.g., 20 sodium cations). Similarly, an RNAi agentshaving a sense strand of 21 nt in length and an antisense strand of 23 nt in length may be provided as a salt having up to 42 cations (e.g., 42 sodium cations). In the preceding example, where the RNAi agent also includes a 5’-terminal phosphate or a 5’-terminal vinylphosphonate 30 group, the RNAi agent may be provided as a salt having up to 44 cations (e.g., 44 sodium cations). The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, bronchial fluids, sputum, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, sputum, and the like. Tissue samples may include samples from tissues, 35 organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. 43 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO II. In Vivo Delivery Enhancing Moiety Conjugated to dsRNA agent The present disclosure provides dual conjugated dsRNA agents for inhibiting expression of a target gene. In some embodiments, a dsRNA agent comprises an antisense strand and a sense strand; at 5 least one αvβ6 integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least one strand; and at least one in vivo delivery enhancing moiety conjugated to at least one strand. The at least one in vivo delivery enhancing moiety may be conjugated to any internal position of the antisense strand or the sense strand. In some embodiments, the at least one in vivo delivery enhancing moiety may be conjugated to a position that is not an external position of the antisense strand 10 or the sense strand. In other embodiments, the at least one in vivo delivery enhancing moiety may be conjugated to an external positon of the antisense strand or the sense strand. As used herein, the term “in vivo delivery enhancing moiety” refers to a moiety which, when conjugated to a dsRNA agent, enhances delivery of the dsRNA agent to a target tissue, e.g., muscle, or a cell type, such as a muscle cell (e.g., a skeletal muscle cell or a cardiac muscle cell). In some 15 embodiments, the in vivo delivery enhancing moiety has pharmacokinetic (PK) enhancing properties, such as increasing the residence time in the blood of the dsRNA agent, and/or increasing tissue PK, including increasing tissue exposure of the dsRNA agent. In some embodiments, the in vivo delivery enhancing moiety can have a cooperative or synergistic effect with an αvβ6 compound in increasing tissue exposure when both elements are conjugated to the dsRNA agent. 20 In some embodiments, the in vivo delivery enhancing moiety is lipophilic. Thus, when conjugated to one or more internal position(s) of the dsRNA agent of the disclosure, the in vivo enhancing moiety increases lipophilicity of the dsRNA agent and provides optimal hydrophobicity for the enhanced in vivo delivery of dsRNA to muscle tissue, e.g., skeletal muscle tissue or cardiac muscle tissue. 25 One way to characterize lipophilicity is by the octanol-water partition coefficient, logKow, where Kow is the ratio of a chemical’s concentration in the octanol-phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory- measured property of a substance. However, it may also be predicted by using coefficients attributed to the structural components of a chemical which are calculated using first-principle or empirical methods 30 (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci.41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of the tendency of the substance to prefer a non-aqueous or oily milieu rather than water (i.e. its hydrophilic/lipophilic balance). In principle, a chemical substance is lipophilic in character when its logKow exceeds 0. Typically, the lipophilic moiety possesses a logKow exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, 35 exceeding 5, or exceeding 10. For instance, the logKow of 6-amino hexanol, for instance, is predicted to 44 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO be approximately 0.7. Using the same method, the logKow of cholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7. The lipophilicity of a molecule can change with respect to the functional group it carries. For instance, adding a hydroxyl group or amine group to the end of a hydrocarbon chain, e.g., a C22 5 hydrocarbon chain, can increase or decrease the partition coefficient (e.g., logKow) value of the hydrocarbon chain. Alternatively, the hydrophobicity of the dsRNA agent conjugated to at least one in vivo delivery enhancing moiety can be measured by its protein binding characteristics. For instance, the unbound fraction in the plasma protein binding assay of the dsRNA agent can be determined to positively correlate 10 to the relative hydrophobicity of the dsRNA agent, which can positively correlate to the silencing activity of the dsRNA agent. In one embodiment, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. In some embodiments, the hydrophobicity of the dsRNA agent, measured by fraction of unbound dsRNA agent in the binding assay, exceeds 0.15, 15 exceeds 0.2, exceeds 0.25, exceeds 0.3, exceeds 0.35, exceeds 0.4, exceeds 0.45, or exceeds 0.5 for an enhanced in vivo delivery of dsRNA agent. The at least one in vivo delivery enhancing agent may be attached to the dsRNA agent of the disclosure by any method known in the art, including via a functional grouping already present in the in vivo delivery enhancing moiety or introduced into the dsRNA agent, such as a hydroxy group (e.g., — 20 CO—CH2—OH). The functional groups already present in the in vivo delivery enhancing moiety or introduced into the dsRNA agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. Conjugation of the dsRNA agent and the in vivo delivery enhancing moiety may occur, for example, through formation of an ether or a carboxylic or carbamoyl ester linkage between the hydroxy 25 and an alkyl group R—, an alkanoyl group RCO— or a substituted carbamoyl group RNHCO—. The alkyl group R may be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chained or branched; and saturated or unsaturated). Alkyl group R may be a butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl group, or the like. In certain embodiments, more than one in vivo delivery enhancing moiety can be conjugated to 30 the dsRNA agent of the disclosure. In one embodiment, two or more in vivo delivery enhancing moieties are conjugated to the same strand of the dsRNA agent. In one embodiment, each strand of the dsRNA agent is conjugated to one or more in vivo delivery enhancing moieties. In one embodiment, two or more in vivo delivery enhancing moieties are conjugated to the same position (i.e., the same nucleobase, same sugar moiety, or same internucleosidic linkage) of the dsRNA agent. This can be achieved by, e.g., 35 conjugating the two or more in vivo delivery enhancing moieties via a carrier, and/or conjugating the two or more in vivo delivery enhancing moieties via a branched linker, and/or conjugating the two or more in 45 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO vivo delivery enhancing moieties via one or more linkers, with one or more linkers linking the in vivo delivery enhancing moieties consecutively. In some embodiments, the in vivo delivery enhancing moiety may comprise at least one C10-C26 hydrocarbon chain, e.g., a C10 hydrocarbon chain, a C11 hydrocarbon chain, a C12 hydrocarbon chain, a 5 C13 hydrocarbon chain, a C14 hydrocarbon chain, a C15 hydrocarbon chain, a C16 hydrocarbon chain, a C17 hydrocarbon chain, a C18 hydrocarbon chain, a C19 hydrocarbon chain, a C20 hydrocarbon chain, a C21 hydrocarbon chain, a C22 hydrocarbon chain, a C23 hydrocarbon chain, a C24 hydrocarbon chain, a C25 hydrocarbon chain or a C26 hydrocarbon chain. In some embodiments, the C10-C26 hydrocarbon chain may be a straight hydrocarbon chain. In other embodiments, the C10-C26 hydrocarbon chain may be a 10 branched hydrocarbon chain. In some embodiments, the C10-C26 hydrocarbon chain may be a saturated hydrocarbon chain. In other embodiments, the C10-C26 hydrocarbon chain may be an unsaturated hydrocarbon chain, e.g., comprising one or more double bonds and/or one or more triple bonds. In one embodiment, the in vivo delivery enhancing moiety may comprise at least one C11 hydrocarbon chain. In one embodiment, the at least one in vivo delivery enhancing moiety may comprise 15 at least one C17 hydrocarbon chain. In one embodiment, the in vivo delivery enhancing moiety may comprise at least one C22 hydrocarbon chain. In some embodiments, the C10-C26 hydrocarbon chain may be unsubstituted. In other embodiments, the C10-C26 hydrocarbon chain may be substituted with at least one functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, 20 and alkyne. In one embodiment, the C10-C26 hydrocarbon chain is substituted with a carboxylic acid group. In some embodiments, the C10-C26 hydrocarbon chain is represented by –(CH2)n-, wherein n is a number from 10 to 26, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26; and – is a bond connecting the C10-C26 hydrocarbon chain to other portions of the in vivo delivery enhancing 25 moiety. In this embodiment, the C10-C26 hydrocarbon chain is connected at both ends to the remainder of the in vivo delivery enhancing moiety. In some embodiments, the C10-C26 hydrocarbon chain is represented by the following structure: – linker-(CH2)n(CH3), wherein n is a number from 10 to 25, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25; the linker is as described hereinbelow; and – is a bond connecting the C10-C26 30 hydrocarbon chain to other portions of the in vivo delivery enhancing moiety. In this embodiment, the C10-C26 hydrocarbon chain is connected at one end to other portions of the in vivo delivery enhancing moiety. In some embodiments, the C10-C26 hydrocarbon chain is represented by the following structure: – linker-(CH2)n-A, wherein n is a number from 10 to 26, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35 22, 23, 24, 25 or 26; the linker as described herein below and A is a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne; or A1 or A2 with a structure shown below: 46 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, A is carboxylic acid. In one embodiment, A is A1. In one embodiment, A is 5 A2. Exemplary in vivo delivery enhancing moieties useful in the context of the present disclosure are described, e.g., in WO 2023/064530, the entire contents of which are incorporated herein by reference. Lipophilic Moiety As noted above, the in vivo delivery enhancing moietycomprises a lipophilic moiety, such as at 10 least one C10-C26 hydrocarbon chain. In some embodiments, the in vivo delivery enhancing moiety comprises a C22 hydrocarbon chain, e.g., one or more C22 hydrocarbon chains. In some embodiments, the C22 hydrocarbon chain is a C22 acid, e.g., a C22 acid selected from the group consisting of docosanoic acid, 6-octyltetradecanoic acid, 10-hexylhexadecanoic acid, all-cis-7,10,13,16,19-docosapentaenoic acid, all-cis-4,7,10,13,16,19-docosahexaenoic acid, all-cis-13,16-docosadienoic acid, all-cis-7,10,13,16- 15 docosatetraenoic acid, all-cis-4,7,10,13,16-docosapentaenoic acid, and cis-13-docosenoic acid. In one embodiment, the C22 hydrocarbon chain is a C22 alcohol, e.g. the C22 alcohol selected from the group consisting of 1-docosanol, 6-octyltetradecan-1-ol, 10-hexylhexadecan-1-ol, cis-13-docosen-1- ol, docosan-9-ol, docosan-2-ol, docosan-10-ol, docosan-11-ol, and cis-4,7,10,13,16,19-docosahexanol. 20 In one embodiment, the C22 hydrocarbon chain is a C22 amide, e.g., the C22 amide selected from the group consisting of (E)-Docos-4-enamide, (E)-Docos-5-enamide, (Z)-Docos-9-enamide, (E)-Docos- 11-enamide,12-Docosenamide, (Z)-Docos-13-enamide, (Z)-N-Hydroxy-13-docoseneamide, (E)-Docos- 14-enamide, 6-cis-Docosenamide, 14-Docosenamide Docos-11-enamide, (4E,13E)-Docosa-4,13- 25 dienamide, and (5E,13E)-Docosa-5,13-dienamide. In one embodiment, the C22 hydrocarbon chain includes, but are not limited to, 47 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO docosan-2-yl, docosan-3-yl, docosan-4-yl, docosan-5-yl, docosan-6-yl, docosan-7-yl, docosan-8-yl, docosan-9-yl, docosan-10-yl, docosan-11-yl, 2-(decyl)dodecan-1-yl, 2-(nonyl)tridecan-1-yl, 2- (octyl)tetradecan-1-yl, 2-(heptyl)pentadecan-1-yl, 2-(hexyl)hexadecan-1-yl, 2-(pentyl)heptadecan-1-yl, 2- (butyl)octadecan-1-yl, 2-(propyl)nonadecan-1-yl, 2-(ethyl)eicosan-1-yl, 2-(methyl)henicosan-1-yl, 3- 5 (nonyl)tridecan-1-yl, 3-(octyl)tetradecan-1-yl, 3-(heptyl)pentadecan-1-yl, 3-(hexyl)hexadecan-1-yl, 3- (pentyl)heptadecan-1-yl, 3-(butyl)octadecan-1-yl, 3-(propyl)nonadecan-1-yl, 3-(ethyl)eicosan-1-yl, 3- (methyl)henicosan-1-yl, 4-(octyl)tetradecan-1-yl, 4-(heptyl)pentadecan-1-yl, 4-(hexyl)hexadecan-1-yl, 4- (pentyl)heptadecan-1-yl, 4-(butyl)octadecan-1-yl, 4-(propyl)nonadecan-1-yl, 4-(ethyl)eicosan-1-yl, 4- (methyl)henicosan-1-yl, 5-(heptyl)pentadecan-1-yl, 5-(hexyl)hexadecan-1-yl, 5-(pentyl)heptadecan-1-yl,10 5-(butyl)octadecan-1-yl, 5-(propyl)nonadecan-1-yl, 5-(ethyl)eicosan-1-yl, 5-(methyl)henicosan-1-yl, 6- (hexyl)hexadecan-1-yl, 6-(pentyl)heptadecan-1-yl, 6-(butyl)octadecan-1-yl, 6-(propyl)nonadecan-1-yl, 6- (ethyl)eicosan-1-yl, 6-(methyl)henicosan-1-yl, 7-(pentyl)heptadecan-1-yl, 7-(butyl)octadecan-1-yl, 7- (propyl)nonadecan-1-yl, 7-(ethyl)eicosan-1-yl, 7-(methyl)henicosan-1-yl, 8-(butyl)octadecan-1-yl, 8- (propyl)nonadecan-1-yl, 8-(ethyl)eicosan-1-yl, 8-(methyl)henicosan-1-yl, 9-(propyl)nonadecan-1-yl, 9- 15 (ethyl)eicosan-1-yl, 9-(methyl)henicosan-1-yl, 10-(ethyl)eicosan-1-yl, 10-(methyl)henicosan-1-yl, and 11-(methyl)henicosan-1-yl, which is substituted at the 2’-oxygen of a nucleoside of an oligonucleotide herein. In some embodiments, the at least one C10-C26 hydrocarbon chain comprised in the in vivo delivery enhancing moiety may be conjugated to the remainder of the dsRNA agent via a linker or via a 20 carrier or via an internucleotide phosphate linkage. In one embodiments, the at least one C10-C26 hydrocarbon chain is conjugated to the remainder of the dsRNA agent via a linker. In some embodiments, the linker may be a cleavable linker, i.e., the linker comprises a portion that can be cleavable chemically or enzymatically. For example, amide linkages can be enzymatically cleavable. In some embodiments, the linker comprises –(CH2)n-NH-C(O)- or –(CH2)n-NH-C(O)-(CH2)2-C(COOH)- 25 NH-C(O)-, wherein n is a number from 1 to 20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In one embodiment, the linker comprises –(CH)2-O-(CH2CH2)-(O)-(CH2CH2)-NH-C(O)-. In other embodiments, the in vivo delivery enhancing moiety comprises a naturally occurring lipophilic compound such as a steroid (e.g., cholesterol), Vitamin E, retinol, or retinoic acid. In some embodiments, the linker may comprise a group selected from the group consisting of an 30 ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, and a carbamate. In some embodiments, the linker may be selected from the group consisting of -(CH2)nNH-; -C(O)(CH2)nNH-; - NR’’’’(CH2)nNH-, -C(O)-(CH2)n-C(O)-; -C(O)-(CH2)n-C(O)O-; -C(O)-O-; -C(O)-(CH2)n-NH-C(O)-; - C(O)-(CH2)n-; -C(O)-NH-; -C(O)-; -(CH2)n-C(O)-; -(CH2)n-C(O)O-; -(CH2)n-; and -(CH2)n-NH-C(O)-; 35 wherein n is a number from 1 to 20; and R’’’’ is C1-C6 alkyl, e.g., C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, 48 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO C5 alkyl or C6 alkyl. In some embodiments, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19 or 20. In some embodiments, the αvβ6 integrin targeting ligand is linked to the remainder of the dsRNA agent as shown in the exemplary schematics below, wherein “2’-N6” refers to attachment of a lipophilic 5 moiety (e.g., C22) at the 2’ position on the nucleotide at position 6 on the sense strand (counting from the 5’ end): 10 In some embodiments, the in vivo delivery enhancing moiety that may be conjugated to the dsRNA agent of the disclosure is represented by the following structure: , 49 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein * represents the bond to the remainder of the dsRNA agent (e.g., to a 2’-oxygen of a nucleoside). The in vivo delivery enhancing moiety may be attached to the remainder of the dsRNA agent at a 2’ position on an internal nucleotide. 50 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO The at least one in vivo delivery enhancing moiety may be conjugated to the dsRNA agent via a direct attachment to the ribosugar of the dsRNA agent, e.g., an antisense strand or a sense strand. Alternatively, the at least one in vivo delivery enhancing moiety may be conjugated to the dsRNA agent via a linker or a carrier. 5 In certain embodiments, at least one in vivo delivery enhancing moiety may be conjugated to the iRNA agent via one or more linkers (tethers). In one embodiment, the at least one in vivo delivery enhancing moiety is conjugated to the dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide- thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole 10 from the azide-alkyne cycloaddition), or carbamate. In some embodiments, the in vivo delivery enhancing moiety and the targeting moiety are independently present within: (a) an internally-modified nucleosides such as, 15 (b) a modified internucleotide linkage such as, -OP(Y)(X)O-, wherein Y is O or S (e.g., O), and (c) a 5’-terminal modification such as 20 P(Y’)(OH)-O-; or (ii) -P(Y)(OH)O-RL3 or -C(O)N(H)RL3, wherein Y is O or S; and (iii) -RL3, -C(O)RL3, -C(O)N(H)RL3, -S(O)2RL3, -S(O)2N(H)RL3 or (d) a 3’-terminal modification such as 51 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (i) -P(Y)(OH)-R3, wherein ; or (ii) -RL3, -C(O)RL3, -C(O)N(H)RL3, -S(O)2RL3, -S(O)2N(H)RL3; wherein: 5 B is an optionally modified nucleobase; B1 is a nucleobase modified with a lipophilic moiety or a targeting moiety (e.g., a pyrimidine nucleobase modified at the 5-position); RL3, RL1, and RL2 are each a group containing a lipophilic moiety or a targeting moiety; R2’ or R3’ may be any functional group that is an acceptable 2’-modification for a ribose sugar. 10 Examples of suitable R2’ or R3’groups include, but are not limited to, hydrogen, halogen (e.g., 2’-fluoro), hydroxy, 2’-O-alkyl (e.g., 2’-OMethyl), 2’-O-methoxyalkyl (e.g., 2’-O-methoxymethyl, 2’-O- methoxyethyl, or 2’-O-2-methoxypropanyl) modification, 2’-O-allyl modification, 2’-C-allyl modification, 2'-O-N-methylacetamido (2'-O-NMA, i.e. -OCH2C(O)N(H)Me) modification, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) modification, 2'- 15 O-aminopropyl (2'-O-AP) modification, or 2'-ara-F modification. For instance, R2’ or R3’ may be H, OH, F, OMe, O-methoxyalkyl, O-allyl, O-N-methylacetamido, O-dimethylaminoethoxyethyl, or O- aminopropyl. In one embodiment, RL1, RL2 and RL3 are each a group containing a lipophilic moiety, such as a C10-26 saturated or unsaturated hydrocarbon chain. In one embodiment, RL1, RL2 and RL3 are each a group 20 containing a C12-26 saturated or unsaturated hydrocarbon chain. In one embodiment, RL1, RL2 and RL3 are each a group containing a C12-24 saturated or unsaturated hydrocarbon chain. In one embodiment, RL1, RL2 and RL3 are each a group containing a C14-24 saturated or unsaturated hydrocarbon chain. In one embodiment, RL1, RL2 and RL3 are each a group containing a C14-18 saturated or unsaturated hydrocarbon chain. In one embodiment, RL1, RL2 and RL3 are each a group containing a C16 saturated or unsaturated 25 hydrocarbon chain. In one embodiment, RL1, RL2 and RL3 are each a group containing a saturated or unsaturated C22-hydrocarbon chain. In other embodiments RL1, RL2 and RL3 are each a group containing a lipophilic moiety, such as a lipophilic vitamin or steroid, including, but not limited to, Vitamin E, Vitamin A (retinol, retinoic acid), and cholesterol. 30 In one embodiment, when RL3 comprises a lipophilic moiety, then RL3 can be selected from the group consisting of: 52 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein integer m is 0-10 (e.g., 0; or 1-10 or 1-8; or 0-6; or 1; or 2; or 3; or 4; or 5; or 6; or 7; or 8); integer n is 1-21 (e.g., 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2; or 2 or 3 or 4 or 5 or 6); 5 W is C1-C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or t-butyl); R and R’ are each independently H or C1-C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, or t- butyl); G is G1 or a saturated or unsaturated C10-26 saturated or unsaturated hydrocarbon chain (e.g.,a C21 hydrocarbon chain e., G together with the carbonyl to which it is attached may form a group with 22 10 carbons) (for instance, G may be a linear or branched C21 alkyl group), wherein G is optionally substituted with one or two groups selected from the group consisting of halogen, -ORG, -SRG, -N(RG)2, - C(O)ORG, -OC(O)RG, -C(O)N(RG) 2, -N(RG)C(O)RG, -N(RG)C(O)ORG, -N(RG)SO2(RG), or -SO2N(RG)2, wherein each RG is independently hydrogen or C1-C6 alkyl (for instance, G is optionally substituted with a -ORG, -C(O)ORG, or -N(RG)C(O)RG); and 15 G1 is a saturated or unsaturated C10-26 saturated or unsaturated hydrocarbon chain (e.g., a C22 hydrocarbon chain,for instance, G1 may be a linear or branched C22 alkyl group), wherein G1 is optionally substituted with one or two groups selected from the group consisting of halogen, -ORG1, -SRG1, - N(RG1)2, -C(O)ORG1, -OC(O)RG1, -C(O)N(RG1)2, -N(RG1)C(O)RG1, -N(RG1)C(O)ORG1, -N(RG1)SO2(RG1), or -SO2N(RG1)2, wherein each RG1 is independently hydrogen or C1-C6 alkyl (for instance, G1 is optionally 20 substituted with a -ORG1, -C(O)ORG1, or -N(RG1)C(O)RG1). Examples of RL3 include, but are not limited to the following structures: 53 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 54 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Other examples of RL3 include, but are not limited to the following structures: Further examples of RL3 include, but are not limited to the following structures: 55 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . In another embodiment, RL1 can be selected from the group consisting of -G1 and - S(O)2G1.Examples of RL1 include, but are not limited to, the following structures: Further examples of RL1 include, but are not limited to, the following structures: 5 In another embodiment, RL2 is -C(O)RL3, wherein RL3 is according to any of the preceding embodiments thereof. For example, RL2 can be selected from the group consisting of the following structures: 56 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO integer m is 0-8 (for instance, m is 0; or m is 1-8; or m is 0-6; or m is 1; or 2; or 3; or 4; or 5; or 6; or 7; or 8); integer n is 1-21 (for instance, 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2; or 2 or 3 or 4 or 5 or 6); 5 R and R’ are each independently H or an alkyl group such as a C1-C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl); G is G1 or a saturated or unsaturated C10-26 saturated or unsaturated hydrocarbon chain (e.g.,a C21 hydrocarbon chain,i.e., G together with the carbonyl to which it is attached may form a group with 22 carbons); for instance, G may be a linear or branched C21 alkyl group), wherein G is optionally 10 substituted with one or two groups selected from the group consisting of halogen, -ORG, -SRG, -N(RG)2, - C(O)ORG, -OC(O)RG, -C(O)N(RG) 2, -N(RG)C(O)RG, -N(RG)C(O)ORG, -N(RG)SO2(RG), or -SO2N(RG)2, wherein each RG is independently hydrogen or C1-C6 alkyl (for instance, G is optionally substituted with a -ORG, -C(O)ORG, or -N(RG)C(O)RG); and G1 is a saturated or unsaturated C10-26 saturated or unsaturated hydrocarbon chain (e.g.,a C22 15 hydrocarbon chain,(for instance, G1 may be a linear or branched C22 alkyl group), wherein G1 is optionally substituted with one or two groups selected from the group consisting of halogen, -ORG1, - SRG1, -N(RG1)2, -C(O)ORG1, -OC(O)RG1, -C(O)N(RG1)2, -N(RG1)C(O)RG1, -N(RG1)C(O)ORG1, - N(RG1)SO2(RG1), or -SO2N(RG1)2, wherein each RG1 is independently hydrogen or C1-C6 alkyl (for instance, G1 is optionally substituted with a -ORG1, -C(O)ORG1, or -N(RG1)C(O)RG1). 20 Additional examples of RL2 include, but are not limited to the following structures: 57 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Further examples of RL2 include, but are not limited to the following structures: In some embodiments, B1 is a nucleobase modified with a G or G1 group, wherein G and G1 are 5 as defined above (e.g., a pyrimidine nucleobase modified at the 5’-position with a group comprising G or G1). Examples of B1 include, , wherein t is selected from 0 – 20 (e.g., 1-12, or 1-10, or 3-12, or 3-10). In some embodiments, in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: 10 wherein: B is an optionally modified nucleobase; 58 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO G3 is a saturated or unsaturated C1-20 hydrocarbon group (e.g., C1-6 alkylene; C2-6 alkylene; or hexylene); LK is a linking group such as -O-, -N(H)-, -S-, -S-S-, -C(O)O-, OC(O)-, -C(O)N(H)-, -N(H)C(O), -OC(O)N(H)-, -N(H)C(O)O-, -S(O)2-, -S(O)2O-, -S(O)2N(H)-, -P(O)(OH)O-, -OP(O)(OH)-, - 5 P(S)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O-, -OP(S)(OH)O-, G2 is a saturated or unsaturated C10-26 hydrocarbon group (e.g., a C14-C24 hydrocarbon group, a C16-C22 hydrocarbon group, or a C21-C22 hydrocarbon group; and RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2. In one embodiment, when LK contains a carbonyl attached to G2 (e.g., (-N(H)C(O)- or -OC(O)-), 10 then G2 is a C21 hydrocarbon group. In another embodiment, when LK does not contain a carbonyl attached to G2, then G2 is a C22 hydrocarbon group. In one embodiment, RG is hydrogen. In another embodiment, RG is OH, In one embodiment, RG is COOH. In another embodiment, RG is CONH2. In one embodiment, RG is amino. In the above structures for the lipophilic monomers, the monomers may also contain one or more 15 asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. Further, in the preceding and throughout the present application, where a modified internucleotide linkage is shown with substituent atoms fully described at the phosphorous atom, e.g., , where C’ is the 2’-carbon or 3’-carbon atom of a ribose ring, it is understood that the 20 oxygen having the broken bond is the 5'-oxygen of the subsequent nucleotide. In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: ,wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), such . 59 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , , wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), such . In one embodiment, the in vivo delivery enhancing moiety is present within a modified 5 nucleoside of the formula: wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), and RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2, such as . In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13,10 14 ,15, 16, 17, 18, 19, 20, or 21), and RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2, such as . In some embodiments, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , wherein n is an integer of 1-21, for instance, 1-12, 1- 10, 1-8, 1-6, 1-4, or 1-2, or 2 or 3 or 4 or 5 or 6); G is a C10-C22 hydrocarbon chain (e.g., a C16-C22 alkyl 15 chain, or a C16 alkyl chain, or a C22 alkyl chain)., optionally substituted with one or two groups selected from the group consisting of halogen, -ORG, -SRG, -N(RG)2, -C(O)ORG, -OC(O)RG, -C(O)N(RG)2, - N(RG)C(O)RG, -N(RG)C(O)ORG, -N(RG)SO2(RG), or -SO2N(RG)2, wherein each RG is independently hydrogen or C1-C6 alkyl; and nucleobase B is a modified or unmodified nucleobase. In one embodiment, 60 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO n is 1. In one embodiment, n is 2-6. In one embodiment, n is 6. In one embodiment, G is a C22 alkyl chain. In one embodiment, G is a C16 alkyl chain In some embodiments, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , wherein n is an integer of 1-21, for instance, 1-12, 1-10, 5 1-8, 1-6, 1-4, or 1-2, or 2 or 3 or 4 or 5 or 6); G is a C22 hydrocarbon chain, optionally substituted with one or two groups selected from the group consisting of halogen, -ORG, -SRG, -N(RG)2, -C(O)ORG, - OC(O)RG, -C(O)N(RG) 2, -N(RG)C(O)RG, -N(RG)C(O)ORG, -N(RG)SO2(RG), or -SO2N(RG)2, wherein each RG is independently hydrogen or C1-C6 alkyl; and nucleobase B is a modified or unmodified nucleobase. In one embodiment, n is 1. In one embodiment, n is 2-6. In one embodiment, n is 6. In one 10 embodiment, G is C10-C22 alkyl chain (e.g., a C14-C24 alkyl chain, C16-C22 alkyl chain, or a C16 alkyl chain, or a C22 alkyl chain). In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: 15 . In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), such one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula , 61 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), such as . In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula 62 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, the in vivo delivery enhancing moiety is present within a modified internucleotide linkage of the form, -OP(Y)(X)O-, wherein Y is O or S (e.g., O), and X is -N(H)(RL1), wherein RL1 is -G1 or S(O)2-G1, each as defined above, wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic. 5 In some embodiments, the in vivo delivery enhancing moiety is present within a modified internucleotide linkage of the form, -OP(O)(X)O-, wherein X is -N(H)(RL1), wherein RL1 is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) and the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic. 10 In some embodiments, the in vivo delivery enhancing moiety is present within a a modified internucleotide linkage of the form, (such as ) , wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) and the 3’-O is from the preceding nucleoside and the 5’-O is from the subsequent nucleoside, and wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic. In certain 15 embodiments, the preceding nucleotide contains a 2’-fluoro modification. In certain embodiments, the preceding nucleotide contains a 2’-O-methyl modification. In certain embodiments, the preceding nucleotide contains a 2’-H modification. In some embodiments, the in vivo delivery enhancing moiety is present within a 63 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO a modified internucleotide linkage of the form , (such wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) and the 3’-O is from the preceding nucleoside and the 5’-O is from the subsequent nucleoside, and wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is 5 racemic. In certain embodiments, the preceding nucleotide contains a 2’-fluoro modification. In certain embodiments, the preceding nucleotide contains a 2’-O-methyl modification. In certain embodiments, the preceding nucleotide contains a 2’-H modification. In some embodiments, the in vivo delivery enhancing moiety is present within a a modified internucleotide linkage of the form, -OP(Y)(X)O-, whereinY 10 wherein G1 is defined above, such a wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, In some embodiments, in vivo delivery enhancing moiety is conjugated to the 3’-end or 5’-end of one of the sense and antisense strands via a direct bond or through a carrier or linker. In some embodiments, in vivo delivery enhancing moiety is conjugated to the 3’-end of the sense or antisense 15 strand via a direct bond or through a carrier or linker. In some embodiments, in vivo delivery enhancing moiety is conjugated to the 5’-end of the sense or antisense strand via a direct bond or through a carrier or linker. In some embodiments, in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the sense and antisense strands (e.g., sense strand) and is of the formula , wherein X is O or 20 S (e.g., S); and RL3 is according to any of the preceding embodiments there. For example, RL3 can be , wherein n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23). In another 64 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO example, RL3 can wherein m is selected from 1-6, and n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23). For example, m can be 2-5, or 1, or 2, or 3, or 4 or 5. 5 In some embodiments, in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the sense and antisense strands (e.g., sense strand) and is of the formula -RL3, wherein RL3 is according to 10 15 to the alpha-amino acid carbon, Ph is phenyl, Y is =O or =S, p is selected from 1 – 6; and ZZ1 is a group formed by reaction of a reactive pairFor example, m can be 2-5, or 1, or 2, or 3, or 4 or 5. In another example, RL3 can be 20 selected from 1-6, and n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23). For example, m can be 2-5, or 1, or 2, or 3, or 4 or 5. In some embodiments, in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the sense and antisense strands (e.g., sense strand) and is of the formula: , or a salt thereof, 65 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein X is O or S (e.g., S); L is a divalent linking group (e.g., C1-20 alkyl or C1-10 alkyl-S-S-C1-10 alkyl). In one embodiment, in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the sense and antisense strands and is of the formula (such as ), or a salt thereof, wherein q is selected from 0 – 18 (e.g., 1-11 or 1-8, or 3-11, or 3-8) and X is O or S (e.g., S). 5 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21); and a, b, and c are independently selected from 1- 22, provided that the sum of a + b + c is selected from 2 to 22. In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- 10 terminal nucleotide, and is of the formula 15 20 to the alpha-amino acid carbon, Ph is phenyl, Y is =O or =S, p is selected from 1 – 6; RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2; and ZZ1 is a group formed by reaction of a reactive pair (e.g., a reaction between an azide and an alkyne or a cycloalkyne). 66 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula wherein m is selected from 1-10 (e.g., 3-6, or 3); RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2, and n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 5 19, or 20, or 21, or 22, or 23). For example, m can be 3-6 and RG is hydrogen; or m can be 3-6 and RG is COOH. In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and the 5’-terminal nucleotide is of the formula: 10 67 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- 5 terminal nucleotide, and is of the formula -P(Y)(OH)-R5, wherein Y is O or S and R5 is: . In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula -P(Y)(OH)-R5, wherein Y is O or S and R5 is , wherein 10 RL2 selected from: , 68 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 10 69 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, in vivo delivery enhancing moiety is bonded to the 3’-oxygen of the 3’- terminal nucleotide, and is of the formula -P(Y)(OH)-R3, wherein Y is O or S and R3 is: 5 In one embodiment, in vivo delivery enhancing moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R3, wherein , wherein RL2 selected from: 10 70 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, in vivo delivery enhancing moiety is conjugated to the 3’-end or 5’-end of one of the sense and antisense strands via a carrier or linker, and the carrier or linker is an inverted abasic 5 nucleotide, such as an inverted abasic deoxyribonucleotide or an inverted abasic ribonucleotide, each connected to the remainder of the oligonucleotide via a phosphodiester (PO) or phosphorothioate (PS) linkage. Examples include, but are not limited to, , wherein Q2 is a bond, C(O), S(O)2, or -P(Y’)(OH)-O-, Y and Y’ are independently O or S; and RL3 is as defined above. In some embodiments, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- 10 terminal nucleotide and is of the formula salt thereof, wherein each X is independently O or S (e.g., each is S); Rligand is selected from the groups listed in Table R-1; and L is a divalent linking group (e.g., C1-20 alkyl or C1-10 alkyl-S-S-C1-10 alkyl). For example, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula 15 salt thereof, wherein each X is independently O or S (e.g., each is S) and Rligand is selected from a , wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21). In some embodiments, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula 71 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO salt thereof, wherein each X is independently O or S (e.g., each is S) and Rligand is selected from a ,wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21).and L is a divalent linking group (e.g., C1-20 alkyl or C1-10 alkyl-S-S-C1-10 alkyl. 5 In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula salt thereof, wherein each X is O or S (e.g., each is S) and Rligand is selected from a ,,wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21). 10 In another embodiment, RL1, RL2, and RL3 are each a group containing at least one targeting moiety, such as an αvβ6 integrin targeting ligand described herein. In one embodiment, the targeting moiety can be selected from: 72 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, the broken bond is connected to a group of the formula **-L-ZZ-L’- , wherein ** represents the bond to the targeting ligand. ZZ is a bridging group that may be formed, for example, by reaction of two functional groups, and can be selected from the group consisting of, -C(H)=N-, -C(H)=N-N(H)-, -C(H)=N-N(H)C(O)-, -5 C(H)=N-N(R)-, -C(H)=N-N(R)C(O)-, -C(H)=N-O-, -C(O)N(H)-, -C(O)N(H)-N(H)-, -C(O)N(H)-N(R)-, - C(O)N(R)-, -C(O)O-, -C(O)S-, -C(O)N(H)-, -C(O)N(H)-N(H)-, -C(O)N(H)-N(R)-, -C(O)N(R)-, - C(S)N(H)-, -C(S)N(R)-, -C(S)O-, -C(S)S-, -C(S)N(H)-, -C(S)N(R)-, -N(H)C(O)N(H)-, -N(H)C(O)N(R)-, 73 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO -N(H)C(O)O-, -N(H)C(O)S-, -N(H)C(S)N(H)-, -N(H)C(S)N(R)-, -N(H)C(S)O-, -N(H)C(S)S-, -S-S-, - CH2-S-, -CH2-O-, -CH2-N(H)-, -CH=CH-, and a click adduct, for example, selected from the following structures: wherein each R is independently C1-10alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl, isobutyl, butyl, 5 or hexyl); and RLa is hydrogen, C1-10alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl, isobutyl, butyl, or hexyl), C3-8cycloalkyl, 3-8 membered heterocyclyl, aryl (e.g., phenyl), or heteroaryl (e.g., 2-pyridyl). The term “click adduct”, as used herein, includes those adducts formed by a copper(I)-catalyzed azide-alkyne cycloaddition reaction, a strain-promoted azide-alkyne cycloaddition, a strain-promoted azide-trans-cycloalkene cycloaddition, and a thiol-maleimide Michael-addition reaction including, for 10 example, (i) an azide with a terminal alkyne or cycloalkyne (e.g. cyclooctyne, BCN, or DBCO); (ii) a tetrazine with a terminal alkyne or cycloalkyne (e.g. cyclooctyne); (iii) a thiol and maleimide (with or without hydrolysis of the product). In some embodiments, L and L’ are independently one of: (a) -L1-[G-L2]q-G-L3-* wherein q is 0 or an integer selected from 1-10; 15 (b) -L1-G-L2-G-L3-*; 74 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (c) -L1-G-L3-*; (d) -G-L3-*; (e) -L1-G-*; or (f) -G-*. 5 wherein in each of (a) -(f), * represent the bond to ZZ; L1 is selected from one of the following groups: (a) a bond, C(O), C(S), C(NRN), S(O)2, P(O)(OH), or P(S)(OH), wherein and RN is hydrogen or C1-6alkyl; (b) a bond, C(O), P(O)(OH), or P(S)(OH); 10 (c) a bond; (d) C(O); (e) P(O)(OH); or (f) P(S)(OH); each L2 and L3 is independently selected from one of the following groups: 15 (a) -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)O-, -OC(O)N(RN)-, - N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (b) -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-,- N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1- 20 6alkyl; or (c) -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (d) -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or 25 (e) -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; and each G is independently selected from one of the following groups: (a) C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; or 30 (b) C1-10alkyl, optionally substituted with 1, 2, or 3 R groups (e.g., 1 or 2 R groups; or 1 R group); or wherein each R group is independently selected from the group consisting of R’, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, heterocyclyl, aryl, heteroaryl, C3-8cycloalkylC1-6alkyl, heterocyclylC1-6alkyl, aryl C1-6alkyl, and heteroarylC1-6alkyl, each of which, other than R’, is 35 optionally substituted with 1, 2, or 3 R’ groups, wherein 75 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each R’ is independently halogen, cyano, azido, nitro, -N(Rb)2, -O(Ra), -S(R0), -C(O)OR0, - C(O)R0, -C(O)N(R0)2, -C(NR0)OR0, -C(NR0)R0, -C(NR0)N(R0)2, -C(S)OR0, -C(S)R0, -C(S)N(R0) 2, -S(O)2R0, -S(O)2OR0, -S(O)2N(R0)2, -N(R0)C(O)OR0, -N(R0)C(O)R0, -N(R0)C(O)N(R0)2, -N(R0 )S(O)2R0, -N(R0)S(O)2OR0, -N(R0)S(O)2N(R0)2, -OC(O)OR0, -OC(O)R0, -OC(O)N(R0)2, - 5 OS(O)2R0, -OS(O)2OR0, -OS(O)2N(R0)2, or -SC(O)R0, wherein each R0 is independently hydrogen or C1-6alkyl; each Ra is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; and each Rb is independently hydrogen, C1-6alkyl, or a nitrogen protecting group. In one embodiment of L or L’, q is 0, 1, 2, 3, 4, or 5. In another embodiment of L or L’, q is 0, 1, 10 2, 3, or 4. In another embodiment of L or L’, q is 0, 1, 2, or 3. In another embodiment of L or L’, q is 0, 1, or 2. In another embodiment of L or L’, q is 1, 2, 3, 4, or 5, In another embodiment of L or L’, q is 1, 2, 3, or 4. In another embodiment of L or L’, q is 1, 2, or 3. In another embodiment of L or L’, q is 1 or 2. In another embodiment of L or L’, q is 4. In another embodiment of L or L’, q is 3. In another embodiment of L or L’, q is 2. 15 In some embodiments, L is one of: (a) , wherein k is an integer from 1 to 10; L1 is bond, C(O), C(S), C(NRN), S(O)2, P(O)(OH), or P(S)(OH) (e.g., L1 is a bond, C(O), P(O)(OH), or P(S)(OH)); and RN is hydrogen or C1-6alkyl; (b) , wherein k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 20 3 to 10; or an integer from 4 to 10; or an integer from 5 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7; (c) , wherein t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3); (d) , wherein t is an integer from 0 to 10 (e.g., an integer from 1 to 5 or 1; or 2; or 25 3); a is an integer from 1 to 3; and s and s’ are each independently an integer from 1 to 24 (e.g., an integer from 1 to 16; an integer from 1 to 10; an integer from 3 to 10; an integer from 3 to 7; or an integer from 4 to 6); 76 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (e) wherein and s, s’, and s’’ are independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6); 5 independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); or and wherein in each of the preceding embodiments of L, * represents the bond to ZZ. 10 In some embodiments, L’ is one of: (a)*-G-L1-, wherein L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and G is C1-10alkyl, C2- 10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; or L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and G is C1-10alkyl; (b)*-G-[L2-G]q-L1-, wherein L1 is a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); each L2 is 15 independently -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-, wherein RN is hydrogen or C1-6alkyl each B is independently a bond, CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G is independently C1-10alkyl; (c) -[G-L2]q-G-L3-*, wherein each L2 is independently a bond, C(O)O, OC(O), C(O)(NRN), N(RN)C(O), OP(O)(OH)O, or OP(S)(OH)O, wherein each RN is independently hydrogen or C1- 20 6alkyl; each G is independently C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups; or each L2 is independently C(O)(NRN), N(RN)C(O), OP(O)(OH)O, or OP(S)(OH)O wherein each RN is independently hydrogen or C1-6alkyl; each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups; (d) *-L3-G-L1-, wherein L3 is -C(O)O- or C(O)N(RN)-, wherein RN is hydrogen or C1-6alkyl; L1 is -25 OP(O)(OH)O- or -OP(S)(OH)O-; and G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups (e.g., 1 or no R groups); (e) -L1-[G-L2]q-G-*, wherein L1 is a bond or -B-A-; each L2 is independently a bond, C(O)O, OC(O), C(O)(NRN), N(RN)C(O), OP(O)(OH)O, or OP(S)(OH)O, wherein each RN is independently 30 hydrogen or C1-6alkyl; and each G is independently C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups; 77 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (f) -[G-L2]q-G-*, wherein each L2 is independently C(O)(NRN) , N(RN)C(O), OP(O)(OH)O, or OP(S)(OH)O wherein each RN is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups; (g) -C2-30alkyl-*, such as -C5-20alkyl-* or -C10-20alkyl-*; 5 (h) -C1-10alkyl-*, optionally substituted with 1 or 2 R groups; (i) -C(O)-C2-30alkyl-*, such as -C(O)-C5-20alkyl-* or -C(O)-C10-20alkyl-*; wherein in each of (a) - (i), * is the bond to ZZ, and q, when present, is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, or an integer from 1 to 3; or 0, 1, 2, or 3; or 0, 1, or 2; or 0 or 1; or 0; or 1; or 2). 10 Examples of RL1 and RL3 that comprise a targeting ligand include, but are not limited to the following structures: Additional examples of RL1 and RL3 that comprise a targeting ligand include, but are not limited to the following structures: 78 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Examples of RL2 that comprise a targeting ligand include, but are not limited to the following structures: Additional examples of RL2 that comprise a targeting ligand include, but are not limited to the 5 following structure: 79 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment of any of the preceding structures comprising a targeting moiety, each RX is an integrin-receptor targeting ligand such as, 5 . In one embodiment, RL2 is . In some embodiments, targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, 10 and is of the formula -P(Y)(OH)-R3, wherein Y is O or S and R3 is: In some embodiments, targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R5, wherein Y is O or S and R5 is: 80 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . In another embodiment, multiple targeting ligands may be connected to a branched multivalent linker In certain embodiments, RL1, RL2 and RL3 can comprise a branched linking group (Δ) capable of 5 supporting multiple targeting ligands (e.g., at least 2; or 2-8; or 2-6; or 2-4; or 2; or 3). For example, in one embodiment, the mutiple targeting moieties can be connected through an RL1, RL2, and RL3 of the form, (RX-L-ZZ-)z-Δ-T- , wherein each RX is a targeting moiety; z is at least 2; or 2-8; or 2-6; or 2-4; or 2; or 3, 10 T is -L’-T’-**, wherein ** is the bond to Δ, and T’ is O, S, N(H), C(O), S(O)2, C(O)N(H), N(H)C(O), OC(O), OC(O), -P(O)(OH)-, -P(S)(OH)-, -OP(O)(OH)-, -OP(S)(OH)-, -P(O)(OH)O-, - P(S)(OH)O-, -OP(O)(OH)O-, or -OP(S)(OH)O-; and L , L’, and ZZ are each as described above, and each * represents the bond to the targeting ligand. 15 In some embodiments, T is selected from the following, wherein ** is the bond to Δ: (a) -C(O)-X1-L5-X2-C(O)-**, wherein X1 and X2 are each independently C1-10alkyl; or C2- 10alkyl; or C4-10alkyl; or C6-10alkyl; or C2-8alkyl; or C2-6alkyl; or C2-4alkyl; (b) -C(O)-C2-20alkyl-C(O)-**, such as -C(O)-C2-12alkyl-C(O)-**, (c) -C(O)-C6-20alkyl-C(O)-**, such as -C(O)-C6-12alkyl-C(O)-**, 20 (d) -C(O)-C10alkyl-C(O)-** and wherein each L5 is a bond, ZZ, or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond; or ZZ), wherein each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); 25 and ZZ is as described above (such as -C(O)N(H)-, N(H)C(O)-, -OP(O)(OH)O-, -OP(S)(OH)O-, or a click adduct). In some embodiments, T is selected from the following, ** is the bond to Δ: (f) -N(H)C(O)-C2-20alkyl-C(O)-**, (g) - N(H)C(O)-C6-20alkyl-C(O)-**, such as - N(H)C(O)-C6-12alkyl-C(O)-**, 30 (h) - N(H)C(O)-C10alkyl-C(O)-**, (i) -C(O)-C2-20alkyl-C(O)N(H)-**, (j) -C(O)-C6-20alkyl-C(O)N(H)-**, 81 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (k) -C(O)-C6-12alkyl-C(O)N(H)-**, (l) -C(O)-C10alkyl-C(O)N(H)-**, (m) -N(H)C(O)-C2-20alkyl-C(O)N(H)-**, (n) - N(H)C(O)-C6-20alkyl-C(O)N(H)-**, 5 (o) - N(H)C(O)-C6-12alkyl-C(O)N(H)-**, and (p) - N(H)C(O)-C10alkyl-C(O)N(H)-**. In some embodiments, T is selected from the following, ** is the bond to Δ: (a) -N(H)C(O)-X3-ZZ-X4-C(O)-**, (b) -C(O)-X3-ZZ-X4-C(O)N(H)-**, 10 (c) N(H)C(O)-X3-ZZ- X4-C(O)N(H)-**, wherein X3 and X4 are independently C2-12alkyl; or C4-10alkyl; or C6-10alkyl; or C4-8alkyl; and ZZ is as described above (such as -C(O)N(H)-, N(H)C(O)-, -OP(O)(OH)O-, -OP(S)(OH)O-, or a click adduct). In some embodiments, , T is selected from the following (w) -L6-[G5-O]q5-G5-L4-**, wherein L4 and L6 are independently -A1-B1-A1-, wherein each A115 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is hydrogen or C1-6alkyl; each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G5 is independently C1-10alkyl; (x) -C(O)-[CH2CH2-O]q5-G5-L4-**, L4 is -A1-B1-A1-, wherein each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is hydrogen or C1-6alkyl; each B1 is independently a 20 bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); G5 is C1-10alkyl. (y) -C(O)-[CH2CH2-O]q5-G5-L4-**, L4 is -A1-B1 or -B1-A1-, wherein each A1 is independently -O- or -N(H)-, and each B1 is independently C(O), G5 is C1-10alkyl (e.g., C2- 10alkyl or C2-6alkyl); and (z) -C(O)-[CH2CH2-O]q5- C2-10alkyl-C(O)N(H)-**; 25 wherein in each of the preceding ** is the bond to Δ, and q5, when present, is an integer selected from 1 to 20 (e.g., 1 to 10, or 2 to 10; or 2 – 8; or 1; or 2; or 3; or 4.) Examples of branched linking group (Δ) include, but are not limited to, 82 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein the broken bond is the bond to L’. Examples of -T1-Δ- include, but are not limited to, , 5 Examples of RL1 and RL3 that comprise a branched linker to a targeting ligand include, but are not limited to the following structures: 83 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Examples of branched RL1 and RL3 include, but are not limited to, the following structures: 5 84 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Examples of branched RL2 include, but are not limited to, the following structures: 5 . In some embodiments, targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R3, wherein Y is O or S and R3 is: 10 ( .g., ; . 85 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R3, wherein Y is O or S and R3 is: . In some embodiments, targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, 5 and is of the formula -P(Y)(OH)-R3, wherein Y is O or S and R3 is: . In some embodiments, targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R5, wherein Y is O or S and R5 is: wherein , r is an integer 10 selected from 1 - 10 (e.g., r is 7); and each R is: , wherein RX is a targeting ligand. In some embodiments, targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R5, wherein Y is O or S and R5 is: 15 ligand. In one embodiment of any of the preceding structures comprising a targeting moiety, each RX is an integrin-receptor targeting ligand such as, 86 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . In some embodiments, the dsRNA agent comprises a double-stranded region formed between the sense and antisense strands and optionally one or two single-stranded non-loop overhang, and wherein the one or more lipophilic moieties are conjugated to either the double-stranded region or the non-loop 5 overhang. In some embodiments, the dsRNA agent does not contain a loop (e.g., stem loop) region. In some embodiments, the dsRNA agent contains a loop (e.g., stem loop) region, and the one or more lipophilic moieties are not conjugated to the loop (e.g., stem loop) region. In some embodiments, the dsRNA agent comprises a sense strand of 10 to 53 nucleotides in length, in which the sense strand forms a duplex region with the antisense strand. For instance, the sense 10 strand may be 10 to 49, 12 to 49, 12 to 45, 12 to 42, 12 to 40, 15 to 49, 15 to 45, 15 to 42, 15 to 40, 15 to 38, or 15 to 36 nucleotides in length. In some embodiments, the duplex region is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides in length. In some embodiments, the region of complementarity to the target sequence is at least 19 contiguous nucleotides in length. In some embodiments, the sense strand comprises at its 3′-end a stem-loop set forth as: S1-L-S2, 15 in which S1 is complementary to S2, and in which L forms a loop between S1 and S2. In some embodiments, the first 17 to 25 nucleotides counting from 5’ end of the sense strand forms a duplex region with the antisense strand, and the last 11 to 28 counting from 5’ end of the sense strand forms a 3′-end a stem-loop set forth as: S1-L-S2. In some embodiments, the length of the stem loop S1-L-S2 is 11 to 28, 13 to 26, or 15 to 24 20 nucleotides in length. In one embodiment, the stem loop S1-L-S2 is 16 nucleotides in length. In some embodiments, the stem loop S1-L-S2 comprises a sequence of GCAGCCGAAAGGCUGC (SEQ ID NO: 1). In some embodiments, L is at least 3, 4, or 5 nucleotides in length. In some embodiments, L comprises a sequence of GAAA. 25 In some embodiments, the sense strand is 36 nucleotides in length, the first 20 nucleotide counting from 5’ end of the sense strand forms a duplex region with the antisense strand, and the last 16 nucleotides forms a stem loop S1-L-S2. In one embodiment, the 16-nucleotide stem loop S1-L-S2 has the sequence of GCAGCCGAAAGGCUGC (SEQ ID NO: 2), wherein L is GAAA. In some embodiments, the one or more lipophilic moieties are conjugated to a non-terminal 30 position of the sense strand. 87 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, the one or more lipophilic moieties are conjugated to one or more nucleotides of the stem loop S1-L-S2. In some embodiments, the one or more lipophilic moieties are conjugated to one or more nucleotides of the loop L. In some embodiments, S1 and S2 are complementary and contain 4-10 nucelotides, e.g., S1 and S2 5 each contain 6 complementary nucelotides. In some embodiments, S1 and S2 are complementary and contain 4-10 nucelotides and L is GAAA, e.g., S1 and S2 each contain 6 complementary nucelotides and L is GAAA. In some embodiments, the one or more lipophilic moieties containing one or more saturated or unsaturated C22 hydrocarbon chains are conjugated to one or more internal positions on at least one strand 10 of the dsRNA agent. Dual Conjugation In another embodiment, in vivo delivery enhancing moiety is connected in series with a targeting moiety, e.g., αvβ6 integrin targeting ligand, as described herein. For example, a sense or antisense strand 15 can contain a series modification at the 3’-end or 5’-end of the oligonucleotide, . such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises the targeting moiety, e.g., αvβ6 integrin targeting ligand as described herein. In one embodiment, the series modification is of the form, , wherein Q is selected 20 wherein RL2 is according to any preceding embodiment, wherein one of the broken bonds connects to a 5’-oxygen of a nucleoside or a 3’-oxygen of a nucleoside and the other connects to a 5’-terminal or 3’-terminal modification as described herein. In another example, a sense or antisense strand can contain a series modification of the form, wherein RL2 is 25 according to any preceding embodiment, each Y is independently O or S; one of the broken bonds 88 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO connects to a 5’-oxygen of a nucleoside or a 3’-oxygen of a nucleoside and the other connects any of the 5’-terminal modifications described above or 3’-terminal modifications described above. In one embodiment, a sense or antisense strand can contain a series modification of the form, , wherein the broken bond connects to the 5’-oxygen of a 5’-terminal 5 nucleoside; each Y is independently O or S; one of R51 and R52 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety) and the other comprises a second ligand moiety (e.g., a targeting moiety, such as a αvβ6 integrin targeting ligand as described herein). In another embodiment, a sense or antisense strand can contain a series modification of the form , wherein the broken bond connects to the 5’-oxygen of a 5’-terminal nucleoside; 10 each Y is independently O or S; one of R51 and R52 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety, such as any of RL or RL2 above) and the other comprises a second ligand moiety (e.g., a targeting moiety). For example an oligonucleotide may have a series modification at the 5’-end of the formula: 15 is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); and each Y is independently O or S and R510 and R520 are 89 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein each Y is independently O or S. In another embodiment, a sense or antisense strand can contain a series modification of the form, , wherein the broken bond connects to the 3’-oxygen of a 3’- 5 terminal nucleoside; each Y is independently O or S; one of R31 and R32 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety, such as any of RL2 above) and the other comprises a second ligand moiety (e.g., a targeting ligand). For example an oligonucleotide may have a series modification at the 3’-end of the formula: wherein n is selected from 7-2310 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); and each Y is independently O or S and R310 and R320 are wherein each Y is indepedently O or S. 90 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In another embodiment, can contain a single modification at the 5’-end, the 3’-end or at an internal position that contains both the in vivo delivery enhancing moiety and the targeting moiety. In one embodiment, single modification is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R3, wherein 5 wherein , wherein RL2 and RL3 comprises the in vivo delivery enhancing moiety (e.g., according to any in vivo delivery enhancing moiety embodiment of RL2 or RL3 above); RTG comprises the targeting moiety (e.g., RTG is according to Formula (X) (below), wherein R5 is -L-ZZ-L’-, where L, ZZ, and L’ are defined for Formula (X) or an embodiment thereof; E is -C(O)N(H)-(CH2)p-*, -N(H)C(O)-(CH2)p-*, -C(O)O-(CH2)p-*, -OC(O)-(CH2)p-*, - 10 -O-N(H)-(CH2)p-, wherein * is the bond to the alpha-amino acid carbon, Ph is phenyl, Y is =O or =S, p is selected from 1 – 6; RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2; and ZZ1 is a group formed by reaction of a reactive pair (e.g., a reaction between an azide and an alkyne or a cycloalkyne); 15 E1 is -O-, -S-, or -N(H)-; T is a bond or -L6-G1-[L5-G1]q1-L4-**, wherein ** is the bond to E; q1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each L4, L5, and L6 are independently a bond, -A1-B1-A1- or ZZ1; ZZ1 is a group formed by reaction of a reactive pair (e.g., a reaction between an azide and an alkyne or a cycloalkyne); each G1 is independently -D1-E1-F1-, wherein D1, E1, and F1 are independently a bond, C1-10alkyl, 20 C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups; each A1 is independently a bond, -O-, -S-, or -N(RN1)-; each B1 is independently a bond, C(O), C(S), C(NRN1), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); 25 each RN1 is independently hydrogen or C1-6alkyl, or two RN1 within an -A1-B1-A1- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl. 91 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, the single modification is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R5, wherein wherein RD is as defined above. In one embodiment of the 3’- or 5’-modification, . 5 embodiment of the 3’- or 5’-modification, r example, G1 can be C1-10alkyl. In one embodiment of the 3’- or 5’-modification, RD is 10 15 92 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO nitrogen. 5 In one embodiment of the 3’- or 5’-modification, RD is . In one embodiment of the 3’- or 5’-modification, RD is . In one embodiment of the 3’- or 5’-modification, RD is ; for example, G1 can be C2-20alkyl. In one embodiment of the 3’- or 5’- modification, wherein n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 10 21, or 22, or 23); RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2; G1 is C2-20alkyl. In one embodiment of the 3’- or 5’-modification, wherein n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23); RG is hydrogen, hydroxy, amino, -COOH, or - C(O)NH2; G1 is C2-20alkyl, and -C(O)-RTG is 93 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO broken bond is the bond between RTG and the nitrogen. 5 In another embodiment, a sense or antisense strand can contain two different ligand modifications, one at the 3'-end of the strand and the other at the 5'-end of the strand: such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises the targeting moiety, e.g., αvβ6 integrin targeting ligand as described herein. 10 In another embodiment, a sense or antisense strand can contain two different ligand modifications, one is an internal modified nuceloside or modified internucleotide linkage of the strand and the other at the 3’-end or 5'-end of the strand: such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises the 15 targeting moiety, e.g., αvβ6 integrin targeting ligand as described herein. The modified nuceloside represented by L1 can be located at a position in the strand selected from positions 2 to n-1, where the strand contains n nucleotides (e.g., n-1 is 20 when n is 21). For example, when L1 is at position 6, then segment (1) of the strand contains 5 nucleotides and segment (2) contains the remainder of the nucleotides within the strand. “Position” herein, when referring to a modified nucleotide, nucleoside, or 20 internucleotide linkage is counted from the 5’-end of the strand, for example, position 6 includes the 6th nucleotide from the 5’-end of the strand and the 6th internucleotide linkage counting from the 5’-end of the strand. 94 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, L1 comprises the in vivo delivery enhancing moiety (e.g., C10-C26 saturated or unsaturated hydrocarbon, such as a C10-C26 alkyl group, or a C14-C24 alkyl group, or a C16-C22 alkyl group, or a C16 alkyl group, or a C22 alkyl group) and a sense or antisense strand can be represented by one of: 5 wherein B is an optionally modified nucleobase (e.g., A, C, G, U, or T); RL3 or RL1 comprises the in vivo delivery enhancing moiety, L2 comprises the targeting moiety. In one embodiment, RL3 is selected from the group consisting of: , , , and wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); for example, in one embodiment, n is 13; and in another embodiment, n is 19. 10 In one embodiment, RL1 is selected from the group consisting of: a , wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); for example, in one embodiment, n is 7; and in another embodiment, n is 10. 95 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In another embodiment, RL3 is selected from the group consisting of: another embodiment, RL1 is selected from the group consisting of: . 1. Linkers/Tethers 5 Linkers/Tethers are connected to the at least one in vivo delivery enhancing moiety at a “tethering attachment point (TAP).” Linkers/Tethers may include any C1-C100 carbon-containing moiety, (e.g. C1-C75, C1-C50, C1-C20, C1-C10; C1, C2, C3, C4, C5, C6, C7, C8, C9, or C10), and may have at least one nitrogen atom. In certain embodiments, the nitrogen atom forms part of a terminal amino or amido (NHC(O)-) group on the linker/tether, which may serve as a connection point for the lipophilic moiety. 10 Non-limited examples of linkers/tethers (underlined) include TAP- nNH-; TAP-C(O)(CH2)nNH-; TAP-NR’’’’(CH2)nNH-, TAP-C(O)-(CH2)n-C(O)-; TAP-C(O)-(CH2)n-C(O)O-; TAP-C(O)-O-; TAP- C(O)-(CH2)n-NH-C(O)-; TAP-C(O)-(CH2)n-; TAP-C(O)-NH-; TAP-C(O)-; TAP-(CH2)n-C(O)-; TAP- (CH2)n-C(O)O-; TAP-(CH2)n-; or TAP-(CH2)n-NH-C(O)-; in which n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) and R’’’’ is C1-C6 alkyl. Preferably, n is 5, 6, or 11. In other 15 embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., -ONH2, or hydrazino group, -NHNH2. The linker/tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands may include, e.g., TAP-(CH2)nNH(LIGAND); TAP- C(O)(CH2)nNH(LIGAND); TAP-NR’’’’(CH2)nNH(LIGAND); TAP-(CH2)nONH(LIGAND); TAP-20 C(O)(CH2)nONH(LIGAND); TAP-NR’’’’(CH2)nONH(LIGAND); TAP-(CH2)nNHNH2(LIGAND), TAP- C(O)(CH2)nNHNH2(LIGAND); TAP-NR’’’’(CH2)nNHNH2(LIGAND); TAP-C(O)-(CH2)n- C(O)(LIGAND); TAP-C(O)-(CH2)n-C(O)O(LIGAND); TAP-C(O)-O(LIGAND); TAP-C(O)-(CH2)n-NH- C(O)(LIGAND); TAP-C(O)-(CH2)n(LIGAND); TAP-C(O)-NH(LIGAND); TAP-C(O)(LIGAND); TAP- (CH2)n-C(O) (LIGAND); TAP-(CH2)n-C(O)O(LIGAND); TAP-(CH2)n(LIGAND); or TAP-(CH2)n-NH- 25 C(O)(LIGAND). In some embodiments, amino terminated linkers/tethers (e.g., NH2, ONH2, NH2NH2) can form an imino bond (i.e., C=N) with the ligand. In some embodiments, amino terminated linkers/tethers (e.g., NH2, ONH2, NH2NH2) can acylated, e.g., with C(O)CF3. In some embodiments, the linker/ tether can terminate with a mercapto group (i.e., SH) or an olefin (e.g., CH=CH2). For example, the tether can be TAP-(CH2)n-SH, TAP-C(O)(CH2)nSH, TAP- 30 (CH2)n-(CH=CH2), or TAP-C(O)(CH2)n(CH=CH2), in which n can be as described elsewhere. The tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. The double bond can be cis or trans or E or Z. 96 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In other embodiments, the linker/tether may include an electrophilic moiety, preferably at the terminal position of the linker/tether. Exemplary electrophilic moieties include, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferred linkers/tethers (underlined) include TAP-(CH2)nCHO; TAP- 5 C(O)(CH2)nCHO; or TAP-NR’’’’(CH2)nCHO, in which n is 1-6 and R’’’’ is C1-C6 alkyl; or TAP- (CH2)nC(O)ONHS; TAP-C(O)(CH2) nC(O)ONHS; or TAP-NR’’’’(CH2) nC(O)ONHS, in which n is 1-6 and R’’’’ is C1-C6 alkyl; TAP-(CH2)nC(O)OC6F5; TAP-C(O)(CH2) nC(O) OC6F5; or TAP-NR’’’’(CH2) nC(O) OC6F5, in which n is 1-11 and R’’’’ is C1-C6 alkyl; or -(CH2)nCH2LG; TAP-C(O)(CH2)nCH2LG; or TAP-NR’’’’(CH2)nCH2LG, in which n can be as described elsewhere and R’’’’ is C1-C6 alkyl (LG can 10 be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether. In other embodiments, it can be desirable for the monomer to include a phthalimido group (K) at the terminal position of the l . 15 In other embodiments, other protected amino groups can be at the terminal position of the linker/tether, e.g., alloc, monomethoxy trityl (MMT), trifluoroacetyl, Fmoc, or aryl sulfonyl (e.g., the aryl portion can be ortho-nitrophenyl or ortho, para-dinitrophenyl). Any of the linkers/tethers described herein may further include one or more additional linking groups, e.g., -O-(CH2)n-, -(CH2)n-SS-, -(CH2)n-, or -(CH=CH)-. 20 2. Cleavable linkers/tethers In some embodiments, at least one of the linkers/tethers can be a redox cleavable linker, an acid cleavable linker, an esterase cleavable linker, a phosphatase cleavable linker, or a peptidase cleavable linker. 25 In one embodiment, at least one of the linkers/tethers can be a reductively cleavable linker (e.g., a disulfide group). In one embodiment, at least one of the linkers/tethers can be an acid cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group). In one embodiment, at least one of the linkers/tethers can be an esterase cleavable linker (e.g., an 30 ester group). In one embodiment, at least one of the linkers/tethers can be a phosphatase cleavable linker (e.g., a phosphate group). 97 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, at least one of the linkers/tethers can be a peptidase cleavable linker (e.g., a peptide bond). Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher 5 levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade 10 an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases. A cleavable linkage group, such as a disulfide bond, can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 15 5.0. Some tethers will have a linkage group that is cleaved at a preferred pH, thereby releasing the iRNA agent from a ligand (e.g., a targeting or cell-permeable ligand, such as cholesterol) inside the cell, or into the desired compartment of the cell. A chemical junction (e.g., a linking group) that links a ligand to an iRNA agent can include a disulfide bond. When the iRNA agent/ligand complex is taken up into the cell by endocytosis, the acidic 20 environment of the endosome will cause the disulfide bond to be cleaved, thereby releasing the iRNA agent from the ligand (Quintana et al., Pharm Res.19:1310-1316, 2002; Patri et al., Curr. Opin. Curr. Biol.6:466-471, 2002). The ligand can be a targeting ligand or a second therapeutic agent that may complement the therapeutic effects of the iRNA agent. A tether can include a linking group that is cleavable by a particular enzyme. The type of linking 25 group incorporated into a tether can depend on the cell to be targeted by the iRNA agent. Tethers that contain peptide bonds can be conjugated to iRNA agents target to cell types rich in peptidases. In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or 30 when in contact with other non-target tissue, e.g., tissue the iRNA agent would be exposed to when administered to a subject. Thus one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, 35 or in whole animals. It may be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds 98 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO are cleaved at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions). 5 3. Redox Cleavable Linking Groups One class of cleavable linking groups are redox cleavable linking groups that are cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular in vivo 10 delivery enhancing moiety, one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In a preferred embodiment, candidate compounds are cleaved by at most 10% in the blood. In preferred 15 embodiments, useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media. 20 4. Phosphate-Based Cleavable Linking Groups Phosphate-based linking groups are cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—25 , —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O— P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)- O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O— P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O— P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O— 30 P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S— , —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above. 5. Acid Cleavable Linking Groups 35 Acid cleavable linking groups are linking groups that are cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of 99 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, ketals, acetals, esters, and esters of amino acids. Acid 5 cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above. 10 6. Ester-Based Linking Groups Ester-based linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above. 15 7. Peptide-Based Cleaving Groups Peptide-based linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not 20 include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide cleavable linking groups have the general formula — 25 NHCHR1C(O)NHCHR2C(O)—, where R1 and R2 are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above. H. Biocleavable linkers/tethers The linkers can also include biocleavable linkers that are nucleotide and non-nucleotide linkers 30 or combinations thereof that connect two parts of a molecule. The non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides, and derivatives thereof, aliphatic, alicyclic, hetercyclic, and combinations thereof. In some embodiments, at least one of the linkers (tethers) is a bio-clevable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or 35 oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, and combinations thereof. 100 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, the bio-cleavable carbohydrate linker may have 1 to 10 saccharide units, which have at least one anomeric linkage capable of connecting two siRNA units. When two or more saccharides are present, these units can be linked via 1-3, 1-4, or 1-6 sugar linkages, or via alkyl chains. Exemplary bio-cleavable linkers include: 5 101 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 102 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . More discussion about the biocleavable linkers may be found in WO2018136620, entitled 5 “Endosomal Cleavable Linkers,” the entire contents of which are incorporated herein by reference. 8. Carriers In certain embodiments, the at least one in vivo delivery enhancing moiety is conjugated to the dsRNA agent via a carrier that replaces one or more nucleotide(s). 10 The carrier can be a cyclic group or an acyclic group. In one embodiment, the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone. 15 In some embodiments, the carrier replaces one or more nucleotide(s) in the internal position(s) of the dsRNA agent. In other embodiments, the carrier replaces the nucleotides at the terminal end of the sense strand or antisense strand. In one embodiment, the carrier replaces the terminal nucleotide on the 3’ end of the sense strand, thereby functioning as an end cap protecting the 3’ end of the sense strand. In one 20 embodiment, the carrier is a cyclic group having an amine, for instance, the carrier may be pyrrolidinyl, 103 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred 5 to herein as a ribose replacement modification subunit (RRMS). The carrier can be a cyclic or acyclic moiety and include two “backbone attachment points” (e.g., hydroxyl groups) and a ligand (e.g., the lipophilic moiety). The one or more C22 hydrocarbon chains can be directly attached to the carrier or indirectly attached to the carrier by an intervening linker/tether, as described above. 10 The ligand-conjugated monomer subunit may be the 5’ or 3’ terminal subunit of the iRNA molecule, i.e., one of the two “W” groups may be a hydroxyl group, and the other “W” group may be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand-conjugated monomer subunit may occupy an internal position, and both “W” groups may be one or more unmodified or modified ribonucleotides. More than one ligand-conjugated monomer subunit may be present in an 15 iRNA agent. (i) Sugar Replacement-Based Monomers, e.g., Ligand-Conjugated Monomers (Cyclic) Cyclic sugar replacement-based monomers, e.g., sugar replacement-based ligand-conjugated 20 monomers, are also referred to herein as RRMS monomer compounds. The carriers may have the general formula (LCM-2) provided below (in that structure preferred backbone attachment points can be chosen from R1 or R2; R3 or R4; or R9 and R10 if Y is CR9R10 (two positions are chosen to give two backbone attachment points, e.g., R1 and R4, or R4 and R9)). Preferred tethering attachment points include R7; R5 or R6 when X is CH2. The carriers are described below as an entity, which can be 25 incorporated into a strand. Thus, it is understood that the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R1 or R2; R3 or R4; or R9 or R10 (when Y is CR9R10), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone. E.g., one of the above-named R 104 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO groups can be -CH2-, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom. (LCM-2) 5 wherein: X is N(CO)R7, NR7 or CH2; Y is NR8, O, S, CR9R10; Z is CR11R12 or absent; Each of R1, R2, R3, R4, R9, and R10 is, independently, H, ORa, or (CH2)nORb, provided 10 that at least two of R1, R2, R3, R4, R9, and R10 are ORa and/or (CH2)nORb; Each of R5, R6, R11, and R12 is, independently, a ligand, H, C1-C6 alkyl optionally substituted with 1-3 R13, or C(O)NHR7; or R5 and R11 together are C3-C8 cycloalkyl optionally substituted with R14; R7 can be a ligand, e.g., R7 can be Rd , or R7 can be a ligand tethered indirectly to the 15 carrier, e.g., through a tethering moiety, e.g., C1-C20 alkyl substituted with NRcRd; or C1-C20 alkyl substituted with NHC(O)Rd; R8 is H or C1-C6 alkyl; R13 is hydroxy, C1-C4 alkoxy, or halo; R14 is NRcR7; 20 R15 is C1-C6 alkyl optionally substituted with cyano, or C2-C6 alkenyl; R16 is C1-C10 alkyl; R17 is a liquid or solid phase support reagent; L is -C(O)(CH2)qC(O)-, or -C(O)(CH2)qS-; Ra is a protecting group, e.g., CAr3; (e.g., a dimethoxytrityl group) or Si(X5’)(X5”)(X5”’) 25 in which (X5’),(X5”), and (X5”’) are as described elsewhere. Rb is P(O)(O-)H, P(OR15)N(R16)2 or L-R17; Rc is H or C1-C6 alkyl; Rd is H or a ligand; Each Ar is, independently, C6-C10 aryl optionally substituted with C1-C4 alkoxy; 30 n is 1-4; and q is 0-4. Exemplary carriers include those in which, e.g., X is N(CO)R7 or NR7, Y is CR9R10, and Z is absent; or X is N(CO)R7 or NR7, Y is CR9R10, and Z is CR11R12; or X is N(CO)R7 or NR7, Y is NR8, and Z is CR11R12; or X is N(CO)R7 or NR7, Y is O, and Z is CR11R12; or X is CH2; Y is CR9R10; Z is CR11R12, 105 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO and R5 and R11 together form C6 cycloalkyl (H, z = 2), or the indane ring system, e.g., X is CH2; Y is CR9R10; Z is CR11R12, and R5 and R11 together form C5 cycloalkyl (H, z = 1). In certain embodiments, the carrier may be based on the pyrroline ring system or the 4- hydroxyproline ring system, e.g., X is N(CO)R7 or NR7, Y is CR9R10, and Z is absent (D). 5 . OFG1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the five-membered ring (-CH2OFG1 in D). OFG2 is preferably attached directly to one of the carbons in the five-membered ring (-OFG2 in D). For the pyrroline-based carriers, -CH2OFG1 may be attached to C-2 and OFG2 may be attached to C-3; or - CH2OFG1 may be attached to C-3 and OFG2 may be attached to C-4. In certain embodiments, CH2OFG110 and OFG2 may be geminally substituted to one of the above-referenced carbons. For the 3- hydroxyproline-based carriers, -CH2OFG1 may be attached to C-2 and OFG2 may be attached to C-4. The pyrroline- and 4-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon- carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH2OFG1 and OFG2 may be cis or trans with respect to one another 15 in any of the pairings delineated above Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration 20 and the other center can have the S configuration and vice versa). The tethering attachment point is preferably nitrogen. Preferred examples of carrier D include the following: 106 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . In certain embodiments, the carrier may be based on the piperidine ring system (E), e.g., X is . 5 OFG1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group (n=1) or ethylene group (n=2), connected to one of the carbons in the six-membered ring [-(CH2)nOFG1 in E]. OFG2 is preferably attached directly to one of the carbons in the six-membered ring (-OFG2 in E). -(CH2)nOFG1 and OFG2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4. Alternatively, -(CH2)nOFG1 and 10 OFG2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CH2)nOFG1 may be attached to C-2 and OFG2 may be attached to C-3; -(CH2)nOFG1 may be attached to C-3 and OFG2 may be attached to C-2; -(CH2)nOFG1 may be attached to C-3 and OFG2 may be attached to C-4; or -(CH2)nOFG1 may be attached to C-4 and OFG2 may be attached to C- 3. The piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein 15 bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, -(CH2)nOFG1 and OFG2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the 20 monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa). The tethering attachment point is preferably nitrogen. In certain embodiments, the carrier may be based on the piperazine ring system (F), e.g., X is 25 N(CO)R7 or NR7, Y is NR8, and Z is CR11R12, or the morpholine ring system (G), e.g., X is N(CO)R7 or NR7, Y is O, and Z is CR11R12. 107 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . OFG1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the six-membered ring (-CH2OFG1 in F or G). OFG2 is preferably attached directly to one of the carbons in the six-membered rings (-OFG2 in F or G). 5 For both F and G, -CH2OFG1 may be attached to C-2 and OFG2 may be attached to C-3; or vice versa. In certain embodiments, CH2OFG1 and OFG2 may be geminally substituted to one of the above- referenced carbons.The piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH2OFG1 and OFG2 may be cis or trans with 10 respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 can both have the R configuration; or both have the S configuration; or one center 15 can have the R configuration and the other center can have the S configuration and vice versa). R’’’ can be, e.g., C1-C6 alkyl, preferably CH3. The tethering attachment point is preferably nitrogen in both F and G. In certain embodiments, the carrier may be based on the decalin ring system, e.g., X is CH2; Y is CR9R10; Z is CR11R12, and R5 and R11 together form C6 cycloalkyl (H, z = 2), or the indane ring system, 20 e.g., X is CH2; Y is CR9R10; Z is CR11R12, and R5 and R11 together form C5 cycloalkyl (H, z = 1). . OFG1 is preferably attached to a primary carbon, e.g., an exocyclic methylene group (n=1) or ethylene group (n=2) connected to one of C-2, C-3, C-4, or C-5 [-(CH2)nOFG1 in H]. OFG2 is preferably attached directly to one of C-2, C-3, C-4, or C-5 (-OFG2 in H). -(CH2)nOFG1 and OFG2 may be disposed 25 in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5. Alternatively, -(CH2)nOFG1 and OFG2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CH2)nOFG1 may be attached to C-2 and OFG2 may be attached to C-3; -(CH2)nOFG1 may be attached to C-3 and OFG2 may be attached to C- 108 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 2; -(CH2)nOFG1 may be attached to C-3 and OFG2 may be attached to C-4; or -(CH2)nOFG1 may be attached to C-4 and OFG2 may be attached to C-3; -(CH2)nOFG1 may be attached to C-4 and OFG2 may be attached to C-5; or -(CH2)nOFG1 may be attached to C-5 and OFG2 may be attached to C-4. The decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein 5 bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, -(CH2)nOFG1 and OFG2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the 10 monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa). In a preferred embodiment, the substituents at C-1 and C-6 are trans with respect to one another. The tethering attachment point is preferably C-6 or C-7. Other carriers may include those based on 3-hydroxyproline (J). 15 . Thus, -(CH2)nOFG1 and OFG2 may be cis or trans with respect to one another. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the 20 centers bearing CH2OFG1 and OFG2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa). The tethering attachment point is preferably nitrogen. Details about more representative cyclic, sugar replacement-based carriers can be found in U.S. Patent Nos.7,745,608 and 8,017,762, which are herein incorporated by reference in their entireties. 25 (ii) Sugar Replacement-Based Monomers (Acyclic) Acyclic sugar replacement-based monomers, e.g., sugar replacement-based ligand-conjugated monomers, are also referred to herein as ribose replacement monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers can have formula LCM-3 or LCM-4: 109 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . In some embodiments, each of x, y, and z can be, independently of one another, 0, 1, 2, or 3. In formula LCM-3, when y and z are different, then the tertiary carbon can have either the R or S configuration. In preferred embodiments, x is zero and y and z are each 1 in formula LCM-3 (e.g., based 5 on serinol), and y and z are each 1 in formula LCM-3. Each of formula LCM-3 or LCM-4 below can optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl. Details about more representative acyclic, sugar replacement-based carriers can be found in U.S. Patent Nos.7,745,608 and 8,017,762, which are herein incorporated by reference in their entireties. The at least one in vivo delivery enhancing moiety may be conjugated to one or more internal 10 positions on at least one strand, e.g., a sense strand or an antisense strand. Internal positions of a strand refers to the nucleotide on any position of the strand, except the terminal position from the 3’ end and 5’ end of the strand (e.g., excluding 2 positions: position 1 counting from the 3’ end and position 1 counting from the 5’ end). In one embodiment, the at least one in vivo delivery enhancing moiety may be conjugated to one 15 or more internal positions on at least one strand, which include all positions except the terminal two positions from each end of the strand (e.g., excluding 4 positions: positions 1 and 2 counting from the 3’ end and positions 1 and 2 counting from the 5’ end). In one embodiment, the at least one in vivo delivery enhancing moiety is conjugated to one or more internal positions on at least one strand, which include all positions except the terminal three positions from each end of the strand (e.g., excluding 6 positions: 20 positions 1, 2, and 3 counting from the 3’ end and positions 1, 2, and 3 counting from the 5’ end). In one embodiment, the at least one in vivo delivery enhancing moiety may be conjugated to one or more internal positions on at least one strand, except the cleavage site region of the sense strand, for instance, the at least one in vivo delivery enhancing moiety is not conjugated to positions 9-12 counting from the 5’-end of the sense strand, for example, the at least one in vivo delivery enhancing moiety is not 25 conjugated to positions 9-11 counting from the 5’-end of the sense strand. Alternatively, the internal positions exclude positions 11-13 counting from the 3’-end of the sense strand. In one embodiment, the at least one in vivo delivery enhancing moiety may be conjugated to one or more internal positions on at least one strand, which exclude the cleavage site region of the antisense strand. For instance, the internal positions exclude positions 12-14 counting from the 5’-end of the 30 antisense strand. In one embodiment, the at least one in vivo delivery enhancing moiety may be conjugated to one or more internal positions on at least one strand, which exclude positions 11-13 on the sense strand, counting from the 3’-end, and positions 12-14 on the antisense strand, counting from the 5’-end. 110 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, the at least one in vivo delivery enhancing moiety may be conjugated to one or more of the following internal positions: positions 4-8 and 13-18 on the sense strand, and positions 6- 10 and 15-18 on the antisense strand, counting from the 5’end of each strand. In one embodiment, the at least one in vivo delivery enhancing moiety may be conjugated to one 5 or more of the following internal positions: positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5’end of each strand. In one embodiment, the at least one in vivo delivery enhancing moiety may be conjugated to position 6 on the sense strand, counting from the 5’end of each strand. In some embodiments, the at least one in vivo delivery enhancing moiety may be conjugated to a 10 nucleobase, sugar moiety, or internucleosidic phosphate linkage of the dsRNA agent. In one embodiment, the at least one in vivo delivery enhancing moiety is conjugated to a sugar moiety of the dsRNA agent. In a further embodiment, the at least one in vivo delivery enhancing moiety is conjugated to the 2’ position of a ribose sugar of the dsRNA agent. 15 III. Alpha-v-Beta-6 (αvβ6) Integrin Targeting Ligands The present disclosure provides dual conjugated dsRNA agents for inhibiting expression of a target gene. In some embodiments, a dsRNA agent comprises an antisense strand and a sense strand; at least one αvβ6 integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least one 20 strand; and at least one in vivo delivery enhancing moiety conjugated to at least one strand. Integrins are cell surface receptors that, upon ligand binding, activate signal transduction pathways including signaling pathways involved in cytoskeleton organization and cell cycle regulation. Integrins are also involved in cell attachment to the extracellular matrix and the integrin ligands comprise common extracellular matrix components, including fibronectin, collagen, laminin, fibrinogen, 25 thrombospondin, and glycoproteins (e.g., tenascin C, osteopontin, and nefronectin). In humans there are at least twenty-four known integrin heterodimers composed of an alpha and a beta subunit, e.g., αvβ6. It is the combination of the alpha and beta subunits which determines the ligand specificity and function of the integrin. Nearly all cells express at least one integrin and the expression and/or activity of integrins can be influenced by other signal inducing molecules, such as cytokines or steroids. 30 The main function of αvβ6 is the activation of cytokine transforming growth factor-b1 (TGF-β1). Latent-TGF-β1 is bound to the extracellular matrix, covered by its pro-peptide latency associated peptide (LAP). αvβ6 binds LAP, and through cytoskeletal force releases TGF-β1. TGF-β1 regulates multiple processes including cell proliferation, differentiation, angiogenesis, epithelial-mesenchymal-transition (EMT) and immune suppression. These processes combine to heal wounds but when uncontrolled can 35 promote tissue pathologies. 111 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO The present disclosure provides dual conjugated dsRNA agents for inhibiting expression of a target gene that comprise an antisense strand and a sense strand; at least one αvβ6 integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least one strand; and at least one in vivo delivery enhancing moiety conjugated to at least one strand. 5 A. Alpha-v-Beta-6 (αvβ6) Integrin Targeting Ligands Conjugated to dsRNA agents In one aspect, the dsRNA agent of the present disclosure is conjugated to at least one αvβ6 integrin targeting ligand. The at least one αvβ6 integrin targeting ligand may be conjugated to the dsRNA agent via a direct attachment to the dsRNA agent, e.g., a ribosugar of the dsRNA agent. 10 Alternatively, the at least one αvβ6 integrin targeting ligand may be conjugated to the dsRNA agent via a linker or a carrier as described herein. Exemplary αvβ6 integrin targeting ligands that can conjugated to the dsRNA agents of the disclosure can be found, e.g., in U.S. Patent Nos 10,023,568, 10,450,312, 10,144,733, 10,487,080, 105,13,517, and 10,000,489, as well as in International Patent Application No. PCT/US2023/083947, 15 filed on December 14, 2023, the entire contents of each of which are incorporated herein by reference. In one embodiment, the αvβ6 integrin targeting ligand may be conjugated to the dsRNA agent via a linker comprising a compound of the structure L333 N-(aminocaproyl-DBCO)-4-hydroxyprolinol, wherein * represents the bond to the remainder of the 20 dsRNA agent. In one embodiment, the αvβ6 integrin targeting ligand comprises the structure: wherein 112 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 bond from RLig to the nitrogen, # is the bond to the integrin ligand, and * represents the bond to the remainder of the dsRNA agent. 113 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, the αvβ6 integrin targeting ligand comprises the structure: 5 In one embodiment, the αvβ6 integrin targeting ligand comprises the structure: 10 114 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 , the bond from RLig to the nitrogen, # is the bond to the integrin ligand, and * represents the bond to the remainder of the dsRNA agent. In one embodiment, the αvβ6 integrin targeting ligand comprises the structure: 10 wherein wherein 115 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 bond from RLig to the nitrogen, # is the bond to the integrin ligand, and * represents the bond to the remainder of the dsRNA agent. 116 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, the αvβ6 integrin targeting ligand comprises the structure: to the re 5 In some embodiments, the αvβ6 integrin targeting ligand or the the αvβ6 integrin targeting ligand to the dsRNA agent of the present disclosure comprises a structure selected from the table below: 117 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 118 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Z1 is a linker linking the αvβ6 integrin targeting ligand to the remainder of the dsRNA agent and wherein * represents the bond to the remainder of the dsRNA agent. 119 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO B. Alpha-v-Beta-6 (αvβ6) Integrin Targeting Ligands Conjugated to a Carrier Group In some embodiments, the alpha-v-beta-6 (αvβ6) integrin targeting ligand may be conjugated to the dsRNA agent of the disclosure, e.g., to at least one strand of the dsRNA agent of the disclosure, via a 5 carrier group. In some embodiments, the alpha-v-beta-6 (αvβ6) integrin targeting ligand conjugated to the dsRNA agent of the disclosure, e.g., to at least one strand of the dsRNA agent of the disclosure, is represented by a compound of the Formula (X): or a salt thereof, wherein: Y is O, N(H), S, or CH2; (e.g., O or CH2) 10 , wherein m is 0, 1, 2, 3, or 4; and each R2 is independently R, or two R2 groups on adjacent carbon atoms taken together with the atoms to which they are bound form a fused 4 – 8 membered ring (e.g,. cycloalkyl or heterocyclyl ring) that is optionally substituted by 1, 2, 3 or 4 groups 15 independently selected from group consisting of R and a nitrogen protecting group; A is an 5-membered heteroaryl optionally substituted with 1 or 2 substituents independently selected from methyl, ethyl, fluoro, hydroxymethyl, 2-hydroxypropan-2-yl, trifluoromethyl, difluoromethyl, and fluoromethyl; Q is -COOR1 or tetrazolyl (e.g., 1,2,3,4-tetrazol-5-yl), wherein R1 is hydrogen or C1-6alkyl (e.g., 20 methyl);and RL is -N(R3)(R4), -O(R5), -S(R5), or -R5, wherein R3 and R4 are either (i) R3 is hydrogen or C1-6alkyl and R4 is R5; or (ii) R3 and R4 taken together with the nitrogen atom to which they are 25 attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R5; and R5 is -L-ZZ-L’-RT wherein L and L’ are independently -L1-[G-L2]q-G-L3-*, wherein 120 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO * is the bond to ZZ; q is 0 or an integer selected from 1 – 25; (e.g., 1-20, or 1-15); L1 is a bond or -B-A-; each L2 is independently -A-B-A-; 5 L3 is a bond or -A-B-A-; each G is independently -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; 10 each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl, or two RN within an -A- B-A- group taken together with the atoms to which they are connected 15 from a 4-8 membered heterocyclyl; and ZZ is -A’-B’-A’- or a linking group formed by a reactive pair, wherein each A’ is independently a bond, -O-, -S-, or -N(RN3)-; each B’ is independently a bond, CH2, C(O), C(S), C(NRN3), -C=N-, S(O), 20 S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); and each RN3 is independently hydrogen or C1-6alkyl, or two RN3 within the -A’-B’- A’- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; RT is -G0-ORT1, wherein 25 G0 is absent or -D0-E0-F0-, wherein D0, E0, and F0 are independently a bond, C1- 10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; and 30 RT1 is LL-oligonucleotide, wherein LL is an oligonucleotide linking group connecting the αvβ6 integrin targeting ligand to oligonucleotide comprised in the dsRNA agent; and each R group is independently selected from the group consisting of R’, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, heterocyclyl, aryl, heteroaryl, C3-8cycloalkylC1-6alkyl, 35 heterocyclylC1-6alkyl, aryl C1-6alkyl, heteroarylC1-6alkyl, each of which, other than R’, is optionally substituted with 1, 2, or 3 R’ groups, wherein 121 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each R’ is independently halogen, cyano, azido, nitro, -N(Rb)2, -O(Ra), -S(R0), - C(O)OR0, - C(O)R0, -C(O)N(R0)2, -C(NR0)OR0, -C(NR0)R0, -C(NR0)N(R0)2, -C(S)OR0, -C(S)R0, -C( S)N(R0)2, -S(O)2R0, -S(O)2OR0, -S(O)2N(R0)2, -N(R0)C(O)OR0, -N(R0)C(O)R0, -N(R0)C( 5 O)N(R0)2, -N(R0)S(O)2R0, -N(R0)S(O)2OR0, -N(R0)S(O)2N(R0)2, -OC(O)OR0, -OC(O)R0, -OC(O)N(R0)2, -OS(O)2R0, -OS(O)2OR0, -OS(O)2N(R0)2, or -SC(O)R0, wherein each R0 is independently hydrogen or C1-6alkyl; each Ra is independently hydrogen or C1-6alkyl; and each Rb is independently hydrogen, C1-6alkyl, or a nitrogen protecting group. 10 In some embodiments, in each -D-E-F- group, at least one of D, E, and F is not a bond; and RL is not N-morpholinyl. In some embodiments of Formula (X), in -N(R3)(R4), R3 and R4 do not form a morpholino ring. In some embodiments of Formula (X), in each -A-B-A- group, B is only a bond when one of the A groups is not a bond. 15 In some embodiments, each R group is independently selected from the group consisting of R’, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, heterocyclyl, aryl, heteroaryl, C3- 8cycloalkylC1-6alkyl, heterocyclylC1-6alkyl, aryl C1-6alkyl and heteroaryl1-6alkyl, each of which, other than R’, is optionally substituted with 1, 2, or 3 R’ groups, wherein each R’ is independently halogen, cyano, azido, nitro, -N(Rb)2, -O(Ra), -S(R0), -C(O)OR0, -C(O)R0, -C(O)N(R0)2, -N(R0)C(O)R0, -OC(O)OR0, or - 20 OC(O)R0, wherein each R0 is independently hydrogen or C1-6alkyl; each Ra is independently hydrogen or C1-6alkyl; and each Rb is independently hydrogen, C1-6alkyl, or a nitrogen protecting group. In some embodiments, each R group is independently selected from the group consisting of R’, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, C2-6alkynyl, C3-6cycloalkyl, 5-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl, and benzyl, each of which, other than R’, is optionally substituted with 1, 2, or 325 R’ groups, wherein each R’ is independently halogen, cyano, azido, nitro, -N(Rb)2, -O(Ra), -S(R0), - C(O)OR0, -C(O)R0, -C(O)N(R0)2, -N(R0)C(O)R0, -OC(O)OR0, or -OC(O)R0, wherein each R0 is independently hydrogen or C1-6alkyl; each Ra is independently hydrogen or C1-6alkyl; and each Rb is independently hydrogen, C1-6alkyl, or a nitrogen protecting group. In some embodiments, each R group is independently selected from the group consisting of R’, 30 C1-6alkyl, C3-6cycloalkyl, 5-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl, and benzyl, each of which, other than R’, is optionally substituted with 1, 2, or 3 R’ groups, wherein each R’ is independently halogen, cyano, azido, nitro, -N(Rb)2, -O(Ra), -S(R0), -C(O)OR0, - C(O)R0, -C(O)N(R0)2, -N(R0)C(O)R0, -OC(O)OR0, or -OC(O)R0, wherein each R0 is independently hydrogen or C1-6alkyl; each Ra is independently hydrogen or C1-6alkyl; and each Rb is independently 35 hydrogen, C1-6alkyl, or a nitrogen protecting group. 122 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In certain embodiments, the compound of formula ( In certain embodiments, the compound of formula ( In certain embodiments, the compound of formula ( In certain embodiments, the compound of formula ( 5 A Embodiments, Formula (X) In some embodiments Formula (X), A is selected from an optionally substituted N- or a C-linked pyrazole, an optionally substituted N- or a C-linked triazole, and an optionally substituted N- or C-linked imidazole, wherein the optionally substituted N- or a C-linked pyrazole, the optionally substituted N- or a 10 C-linked triazole, and the optionally substituted N- or C-linked imidazole is optionally substituted by 1 or 2 substituents independently selected from methyl, ethyl, fluoro, hydroxymethyl, 2-hydroxypropan-2-yl, trifluoromethyl, difluoromethyl, and fluoromethyl. In some embodiments , wherein R1 is hydrogen, methyl, or ethyl; R2 is hydrogen or fluoro; and R3 is hydrogen, methyl, or ethyl. 15 In some embodiments . some embodiments A is . In some embodiments A is selected from: 123 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO RY Embodiments, Formula (X) In some embodiments of any one of Formula (X), in RY, each R2 is independently R as defined in Formula (X). 5 In some embodiments,R In some embodiments,R . In some embodiments, in RY , two R2 groups on adjacent carbon atoms taken together with the atoms to which they are bound form a fused 4 – 8 membered ring that is optionally substituted with 1, 2, 3 or 4 groups independently selected from group consisting of R and a nitrogen protecting group, wherein R is 10 as defined in Formula (IV). In some embodiments, RY is NH wherein p is 0, 1, 2, 3 or 4; and each R21 is independently selected from group consisting of R and a nitrogen protecting group, wherein R is as defined in Formula (IV). In some embodiments, RY is , wherein p is 0, 1, 2, 3 or 4; and each R21 is 15 independently selected from the group consisting of R and a nitrogen protecting group, wherein R is as defined in Formula (IV). 124 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein RP is a nitrogen protecting group. In some embodiments, 5 group. In some embodiments of any embodiment of RY wherein RP is present, then RP is 10 , wherein r is 0, 1, 2, or 3; each RP2 is independently halogen, nitro, cyano, C1- 4alkoxy, C1-4alkyl, C1-4haloalkyl; and each RP3 is independently hydrogen, methyl, or ethyl. In some embodiments, when present, RP is methoxyacetyl (mac), phenoxyacetyl (pac), 2- chlorophenoxyacetyl, 3-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2,4-dichlorophenoxyacetyl, 2- 125 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO methylphenoxyacetyl, 3-methylphenoxyacetyl, 4-methylphenoxyacetyl, 4-chloro-2- methylphenoxyacetyl, 2-nitrophenoxyacetyl, 3-nitrophenoxyacetyl, 4-nitrophenoxyacetyl, 2- isopropylphenoxyacetyl, 3-isopropylphenoxyacetyl, 4-isopropylphenoxyacetyl, 2-(t-butyl)phenoxyacetyl, 3-(t-butyl)phenoxyacetyl, 4-(t-butyl)phenoxyacetyl, 2-fluorophenoxyacetyl, 3-fluorophenoxyacetyl, 4- 5 fluorophenoxyacetyl, 2,4-difluorophenoxyacetyl, 4-(trifluoromethoxy)phenoxyacetyl, 2- phenoxypropanoyl, 2-(4-chloro-2-methylphenoxy)propanoyl, or 2-(4-chlorophenoxy)propanoyl. In some embodiments, when present, RP is methoxyacetyl (mac), phenoxyacetyl (pac), 2- chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2-methylphenoxyacetyl, 4-methylphenoxyacetyl, or 4- isopropylphenoxyacetyl. 10 In some embodiments, when present, RP is methoxyacetyl (mac). In some embodiments, when present, RP is phenoxyacetyl (pac). In some embodiments, when present, R1 is C1-6alkyl , wherein r is 0, 1, 2, or 3; each RP2 is independently halogen, nitro, cyano, C1-4alkoxy, C1-4alkyl, C1-4haloalkyl; and each RP3 is independently hydrogen, methyl, or ethyl. 15 In some embodiments or Formula (X), when present, R1 is C1-6alkyl and RP is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 3-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2,4- dichlorophenoxyacetyl, 2-methylphenoxyacetyl, 3-methylphenoxyacetyl, 4-methylphenoxyacetyl, 4- chloro-2-methylphenoxyacetyl, 2-nitrophenoxyacetyl, 3-nitrophenoxyacetyl, 4-nitrophenoxyacetyl, 2- isopropylphenoxyacetyl, 3-isopropylphenoxyacetyl, 4-isopropylphenoxyacetyl, 2-(t-butyl)phenoxyacetyl,20 3-(t-butyl)phenoxyacetyl, 4-(t-butyl)phenoxyacetyl, 2-fluorophenoxyacetyl, 3-fluorophenoxyacetyl, 4- fluorophenoxyacetyl, 2,4-difluorophenoxyacetyl, 4-(trifluoromethoxy)phenoxyacetyl, 2- phenoxypropanoyl, 2-(4-chloro-2-methylphenoxy)propanoyl, or 2-(4-chlorophenoxy)propanoyl. In some embodiments, when present, R1 is C1-6alkyl and RP is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2-methylphenoxyacetyl, 4- 25 methylphenoxyacetyl, or 4-isopropylphenoxyacetyl. In some embodiments, when present, R1 is C1-6alkyl and RP is methoxyacetyl (mac). In some embodiments, when present, R1 is C1-6alkyl and RP is phenoxyacetyl (pac). In some embodiments, when present, R1 is methyl 1, 2, or 3; each RP2 is independently halogen, nitro, cyano, C1-4alkoxy, C1-4alkyl, C1-4haloalkyl; and each 30 RP3 is independently hydrogen, methyl, or ethyl. 126 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, when present, R1 is methyl and RP is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 3-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2,4- dichlorophenoxyacetyl, 2-methylphenoxyacetyl, 3-methylphenoxyacetyl, 4-methylphenoxyacetyl, 4- chloro-2-methylphenoxyacetyl, 2-nitrophenoxyacetyl, 3-nitrophenoxyacetyl, 4-nitrophenoxyacetyl, 2- 5 isopropylphenoxyacetyl, 3-isopropylphenoxyacetyl, 4-isopropylphenoxyacetyl, 2-(t-butyl)phenoxyacetyl, 3-(t-butyl)phenoxyacetyl, 4-(t-butyl)phenoxyacetyl, 2-fluorophenoxyacetyl, 3-fluorophenoxyacetyl, 4- fluorophenoxyacetyl, 2,4-difluorophenoxyacetyl, 4-(trifluoromethoxy)phenoxyacetyl, 2- phenoxypropanoyl, 2-(4-chloro-2-methylphenoxy)propanoyl, or 2-(4-chlorophenoxy)propanoyl. In some embodiments, when present, R1 is methyl and RP is methoxyacetyl (mac), phenoxyacetyl 10 (pac), 2-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2-methylphenoxyacetyl, 4-methylphenoxyacetyl, or 4-isopropylphenoxyacetyl. In some embodiments, when present, R1 is methyl and RP is methoxyacetyl (mac). In some embodiments, when present, R1 is methyl and RP is phenoxyacetyl (pac). In some embodiments, when present, R1 is hydrogen wherein r is 15 0, 1, 2, or 3; each RP2 is independently halogen, nitro, cyano, C1-4alkoxy, C1-4alkyl, C1-4haloalkyl; and each RP3 is independently hydrogen, methyl, or ethyl. In some embodiments, when present, R1 is hydrogen and RP is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 3-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2,4- dichlorophenoxyacetyl, 2-methylphenoxyacetyl, 3-methylphenoxyacetyl, 4-methylphenoxyacetyl, 4-20 chloro-2-methylphenoxyacetyl, 2-nitrophenoxyacetyl, 3-nitrophenoxyacetyl, 4-nitrophenoxyacetyl, 2- isopropylphenoxyacetyl, 3-isopropylphenoxyacetyl, 4-isopropylphenoxyacetyl, 2-(t-butyl)phenoxyacetyl, 3-(t-butyl)phenoxyacetyl, 4-(t-butyl)phenoxyacetyl, 2-fluorophenoxyacetyl, 3-fluorophenoxyacetyl, 4- fluorophenoxyacetyl, 2,4-difluorophenoxyacetyl, 4-(trifluoromethoxy)phenoxyacetyl, 2- phenoxypropanoyl, 2-(4-chloro-2-methylphenoxy)propanoyl, or 2-(4-chlorophenoxy)propanoyl. 25 In some embodiments, when present, R1 is hydrogen and RP is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2-methylphenoxyacetyl, 4- methylphenoxyacetyl, or 4-isopropylphenoxyacetyl. In some embodiments, when present, R1 is hydrogen and RP is methoxyacetyl (mac). In some embodiments, when present, R1 is hydrogen and RP is phenoxyacetyl (pac). 30 RT Embodiments, Formula (X) In some embodiments of Formula (X), RT is RT1. 127 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments of Formula (X), RT is -G0-ORT1, wherein RT1 is as defined for Formula (X) and G0 is selected from: (a) G0 is absent or -D0-E0-F0-, wherein D0, E0, and F0 are independently a bond, C1-10alkyl, C2- 10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of 5 which is optionally substituted with 1, 2, 3, or 4 R groups; (b) G0 is -D0-E0-F0-, wherein D0 and F0 are independently a bond or C1-10alkyl optionally substituted with 1, 2, 3, or 4 R groups; and E0 is C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is 10 optionally substituted with 1, 2, 3, or 4 R groups; (c) G0 is -D0-E0-F0-, wherein D0 and F0 are independently a bond or C1-10alkyl optionally substituted with 1, 2, 3, or 4 R groups; and E0 is 3-10 membered heterocyclyl optionally substituted with 1, 2, 3, or 4 R groups; and 15 (d) G0 is 3-10 membered heterocyclyl optionally substituted with 1 or 2 R groups; examples include , s tetrahydrofuranyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, each optionally substituted with 1 or 2 R groups; examples include: 20 (e) G0 is 3-10 membered-heterocyclyl-C1-10alkyl, optionally substituted with 1, 2, 3, or 4 R groups; examples include, 128 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (f) G0 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups (e.g., 1 or 2 R groups); (g) G0 is C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups; 5 (h) G0 is C1-10alkyl optionally substituted with 1 or 2 R groups; (i) G0 is C1-10alkyl, optionally substituted with -O(Ra), wherein Ra is independently hydrogen or C1- 6alkyl; (j) G0 is C1-10alkyl, and 10 (k) G0 is absent (a bond); wherein * represents the bond to L’, the broken bond represents the bond to ORT1, and R is -C1-6alkyl- ORa or -ORa, wherein Ra is independently hydrogen or C1-6alkyl. -L’- Embodiments, Formula (X) 15 In some embodiments of Formula (X), -L’- is *-G-L1-, wherein * is the bond to ZZ; and (a) L1 is a bond or -B-A-, wherein A is a bond, -O-, -S-, or -N(RN)-; B is a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl; and 20 G is -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2- 10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; (b) L1 is a bond or -B-A-, wherein A is a bond, -O-, -S-, or -N(RN)-; 25 B is a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl; and G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; (c) L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and 30 G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; (d) L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and 129 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; or (e) L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and G is C1-10alkyl. 5 In some embodiments of Formula (X), -L’- is *-G-[L2-G]q-L1-, wherein * is the bond to ZZ; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer selected from 1 to 8, an integer selected from 1 to 5, or an integer selected from 1 to 3); and (a) L1 is a bond or -B-A-; each L2 is independently -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), 10 S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl; each G is independently -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; (b) L1 is a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); 15 each L2 is independently -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl, and each G is independently a bond, C1-10alkyl, optionally substituted with 1, 2, 3, or 4 R groups. (c) L1 is a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); 20 each L2 is independently -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-, wherein RN is hydrogen or C1-6alkyl each B is independently a bond, CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G is independently C1-10alkyl, optionally substituted with 1, 2, 3, or 4 R groups; (d) L1 is a bond, CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each L2 is independently - 25 A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-, wherein RN is hydrogen or C1-6alkyl; each B is independently a bond, CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G is independently C1-10alkyl, optionally substituted with 1, 2, 3, or 4 R groups; or 30 (e) L1 is a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); each L2 is independently -A-B- A-; each A is independently a bond, -O-, -S-, or -N(RN)-, wherein RN is hydrogen or C1- 6alkyl; each B is independently a bond, CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G is independently C1-10alkyl. 35 In some embodiments of Formula (X), -L’- is -[G-L2]q-G-L3-*, wherein * is the bond to ZZ; and 130 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (i) q is 0, 1, 2, 3, 4, or 5; each L2 is independently -A-B-A-; L3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl; each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each 5 of which is optionally substituted with 1, 2, 3, or 4 R groups; (ii) q is 0, 1, 2, or 3; each L2 is independently a bond, C(O)O, OC(O), C(O)(NRN), N(RN)C(O), SO2N(RN), N(RN)SO2, OP(O)(OH), OP(S)(OH), P(O)(OH)O, P(S)(OH)O, OP(O)(OH)O, or OP(S)(OH)O, wherein each RN is independently hydrogen or C1-6alkyl; each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted 10 with 1 or 2 R groups; (iii) q is 0, 1, 2, or 3; each L2 is independently a bond, C(O)O, OC(O), C(O)(NRN), N(RN)C(O), OP(O)(OH)O, or OP(S)(OH)O, wherein each RN is independently hydrogen or C1-6alkyl; each G is independently C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups; 15 (iv) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each L2 is independently C(O)O or OC(O); each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups; (v) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each L2 is independently C(O)(NRN) or N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl; each 20 G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups; (vi) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each L2 is independently OP(O)(OH)O, or OP(S)(OH)O (e.g., each is OP(O)(OH)O); each G is independently C1- 10alkyl, each of which is optionally substituted with 1 or 2 R groups; or 25 (vii) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each L2 is a bond; each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups. In some embodiments, -L’- is *-L3-G-L1-, wherein * is the bond to ZZ; and (a) L1 and L3 are independently -A-B-A-; each G is independently C1-10alkyl, C2-10alkenyl, C2- 10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is30 optionally substituted with 1, 2, or 3 R groups; each A is independently a bond, -O-, -S-, or - N(RN)-, wherein RN is hydrogen or C1-6alkyl; and each B is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); (b) L3 is -C(O)O- or C(O)N(RN)-, wherein RN is hydrogen or C1-6alkyl; L1 is -OP(O)(OH)O- or - OP(S)(OH)O-; and each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 35 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups; 131 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (c) L3 is -C(O)O- or C(O)N(RN)-, wherein RN is hydrogen or C1-6alkyl; L1 is -OP(O)(OH)O- or - OP(S)(OH)O-; and each G is independently C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with one R group; (d) L3 is -C(O)O- or C(O)N(RN)-, wherein RN is hydrogen or C1-6alkyl; L1 is -OP(O)(OH)O- or - 5 OP(S)(OH)O-; and each G is independently C3-10cycloalkyl or 3-10 membered heterocyclyl; or (e) L3 is -C(O)O- or C(O)N(RN)-, wherein RN is hydrogen or C1-6alkyl; L1 is -OP(O)(OH)O- or - OP(S)(OH)O-; and each G1 is independently C3-10cycloalkyl or 3-10 membered heterocyclyl. In some embodiments, - wherein X is O or S (e.g., S). 10 In some embodiments, -L’- is *-G-, wherein * is the bond to ZZ; and G is C1-10alkyl is optionally substituted with 1 or 2 R groups. In embodiments, -L’- is --L1-[G-L2]q-G-L3-*, wherein * is the bond to ZZ. In some embodiments, -L’- is -L1-G-L3-*, wherein * is the bond to ZZ. In some embodiments, -L’- is -L1-[G-L2]q-G-L3-*, wherein * is the bond to ZZ; q is 0, 1, 2, 3, 4, 15 or 5; L1 is a bond or -B-A-; each L2 is independently -A-B-A-; L3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. 20 In some embodiments, -L’- is -L1-[G-L2]q-G-*, wherein * is the bond to ZZ; q is 0, 1, 2, or 3; L1 is a bond or -B-A-; each L2 is independently a bond, C(O)O, OC(O), C(O)(NRN), N(RN)C(O), SO2N(RN), N(RN)SO2, OP(O)(OH), OP(S)(OH), P(O)(OH)O, P(S)(OH)O, OP(O)(OH)O, or OP(S)(OH)O, wherein each RN is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl, C2-10alkenyl, C2- 10alkynyl, each of which is optionally substituted with 1 or 2 R groups. 25 In some embodiments, -L’- is -L1-[G-L2]q-G-*, wherein * is the bond to ZZ; q is 0, 1, 2, or 3; L1 is a bond or -B-A-; each L2 is independently a bond, C(O)O, OC(O), C(O)(NRN), N(RN)C(O), OP(O)(OH)O, or OP(S)(OH)O, wherein each RN is independently hydrogen or C1-6alkyl; each G is independently C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups. In some embodiments, -L’- is -[G-L2]q-G-*, wherein * is the bond to ZZ; and 30 (a) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each L2 is independently C(O)O or OC(O); each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups; (b) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each L2 is independently C(O)(NRN) or N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl; and 132 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups; (c) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each L2 is independently OP(O)(OH)O, or OP(S)(OH)O (e.g., each is OP(O)(OH)O); and each G is independently 5 C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups; (d) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each L2 is a bond; and each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups. In some embodiments, -L’- is -C2-30alkyl-*, wherein * is the bond to ZZ, such as -C5-20alkyl-* or -10 C10-20alkyl-*. In some embodiments, -L’- is -C(O)-C2-30alkyl-*, wherein * is the bond to ZZ, such as -C(O)- C5-20alkyl-* or -C(O)-C10-20alkyl-*. L’-RT Embodiments In some embodiments, - , wherein * is the 15 bond to ZZ; L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and RT1 is as defined for Formula (X). In some embodiments, - wherein * is the bond to ZZ; L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and RT1 is as defined for Formula (X). In some embodiments, - , wherein * is the 20 bond to ZZ; L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); R is -OH or -C1-6alkyl-OH; and RT1 is as defined for Formula (X). 133 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, - , wherein * is the bond to ZZ; L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); R is -OH or -C1-6alkyl-OH; and RT1 is as defined for Formula (X). In some embodiments, -L’-RT is 5 wherein* is the bond to ZZ; L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); R is -OH or -C1- 6alkyl-OH; and RT1 is as defined for Form In some embodiments, -L’-RT is , wherein * is the bond to ZZ; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 10 1 to 5, or an integer from 1 to 3); each L2 is (i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -OC(O)O-, -OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, - OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(RN)-, wherein each RN is independently 15 hydrogen or C1-6alkyl; or (ii) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-,-N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (iii) is independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, - 20 OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (iv) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or 134 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (v) independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; (vi) independently selected from the group consisting of -C(O)N(RN)- and -N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl; 5 L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and RT1 is as defined for Formula (X). In some embodiments, - , wherein * is the bond to ZZ; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, or an integer from 1 to 3); each L2 is 10 (i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -OC(O)O-, -OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, - OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (ii) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - 15 N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-,-N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (iii) is independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, - OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or 20 (iv) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (v) independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; 25 (vi) independently selected from the group consisting of -C(O)N(RN)- and -N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl; L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and RT1 is as defined for Formula (X). 135 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, -L’-RT is wherein * is the bond to ZZ; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, or an integer from 1 to 3); each L2 is 5 (i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -OC(O)O-, -OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, - OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (ii) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - 10 N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-,-N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (iii) is independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, - OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or 15 (iv) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (v) independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or 20 (vi) independently selected from the group consisting of -C(O)N(RN)- and -N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl; L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and RT1 is as defined for Formula (X). In some embodiments, -L’-RT is wherein * is the bond to ZZ; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1-8; or 1-5; or 1-3); 25 each L2 is (i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -OC(O)O-, -OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, - OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or 136 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (ii) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-,-N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (iii) is independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, - 5 OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (iv) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or 10 (v) independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; (vi) independently selected from the group consisting of -C(O)N(RN)- and -N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl; L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); R is -OH or -C1-6alkyl-OH; and RT1 is as 15 defined for Formula (X). , wherein * is the bond to ZZ; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, or an integer from 1 to 3); each L2 is 20 (i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -OC(O)O-, -OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, - OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (ii) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - 25 N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-,-N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (iii) is independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, - OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or 137 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (iv) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; (v) independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or 5 (vi) independently selected from the group consisting of -C(O)N(RN)- and -N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl; L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); R is -OH or -C1-6alkyl-OH; and RT1 is as defined for Formula (X). In some embodiments, -L’-RT is 10 wherein* is the bond to ZZ; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, or an integer from 1 to 3); each L2 is (i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -15 N(RN)C(O)-, -OC(O)O-, -OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, - OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (ii) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-,-N(RN)C(O)N(RN)-, -O-, and -N(RN)-, 20 wherein each RN is independently hydrogen or C1-6alkyl; or (iii) is independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, - OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (iv) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - 25 N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (v) independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; (vi) independently selected from the group consisting of -C(O)N(RN)- and -N(RN)C(O), 30 wherein each RN is independently hydrogen or C1-6alkyl; L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); R is -O(Ra) or -C1-6alkyl-O(Ra), wherein Ra is hydrogen or a hydroxyl protecting group; and RT1 is as defined for Formula (X). 138 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, or an integer from 1 to 3); each L2 is 5 (i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -OC(O)O-, -OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, - OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (ii) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - 10 N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-,-N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (iii) is independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, - OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or 15 (iv) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, - N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (v) independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; 20 (vi) independently selected from the group consisting of -C(O)N(RN)- and -N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl; L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); R is -OH or -C1-6alkyl-OH; and RT1 is as defined for Formula (X). In some embodiments, -L’-RT is 25 wherein* is the bond to ZZ; L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); R is -O(Ra) or -C1- 6alkyl-O(Ra), wherein Ra is hydrogen; and RT1 is as defined for Formula (X). 139 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO RL Embodiments, Formula (X) In one embodiment the Formula (X), or a salt thereof, RL is -N(R3)(R4), -O(R5), -S(R5), or -R5, wherein R5 is according to one Formulae (x-a) through (x-ab): 140 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (x-ab) wherein RP3 is hydrogen and L, ZZ, L’ and RT1 are as defined for Formula (X) or any embodiment herein. In another embodiment, RL is according to one of Formulae (xi-a) through (xi-m): 141 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (xi-m) wherein RP3 is hydrogen, and L, ZZ, L’ and RT1 are as defined for Formula (X) or any embodiment herein. In another embodiment, RL is according to one of Formulae (xi-n) through (xi-z): 142 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein RP3 is hydrogen, and L, ZZ, L’ and RT1 are as defined for Formula (X) or any embodiment herein. In some embodiments, RL is -N(R3)(R4), wherein R3 is hydrogen or C1-6alkyl and R4 is R5. In some embodiments, RL is -N(R3)(R4), wherein R3 is hydrogen and R4 is R5. In some embodiments, RL is -N(R3)(R4), wherein R3 is C1-3alkyl and R4 is R5. 5 In some embodiments, RL is -N(R3)(R4), wherein R3 is methyl and R4 is R5. In some embodiments, RL is --N(R3)(R4), wherein R3 and R4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R5. In some embodiments, RL is --N(R3)(R4), wherein R3 and R4 taken together with the nitrogen 10 atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R5, provided that R3 and R4 taken together with the nitrogen atom to which they are attached do not form a morpholino group. In some embodiments, RL is --N(R3)(R4), wherein R3 and R4 taken together with the nitrogen atom to which they are attached form a group that is piperidinyl, piperazinyl, pyrrolidinyl, 15 imidazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, azetidinyl, pyrrolinyl, imidazolinyl, or pyrazolinyl, each substituted with R5. 143 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, . In one embodiment the Formula (X), or a salt thereof, RL is -N(R3)(R4). In one embodiment the Formula (X), or a salt thereof, RL is -N(R3)(R4), wherein R3 and R4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl 5 group that is substituted with R5. In some embodiments, RL is -O(R5). In some embodiments, RL is -R5. In some embodiments, RL is , wherein * is a bond to a ZZ group; s is 1 – 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an 10 integer from 4 to 6). In some embodiments, RL is , wherein * is a bond to a ZZ group; t is 0 to 10 (e.g., 1-5; or 1-3; or 1; or 2; or 3). L Embodiments, Formula (X) 15 In some embodiments of any one of Formula (X) and any embodiments thereof, L is -L1-[G-L2]q- G-L3-*, wherein * is the bond to ZZ. In another embodiment, wherein L is -L1-[G-L2]q-G-L3-*, wherein q is 0, 1, 2, 3, 4, or 5. In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 0, 1, 2, 3, or 4 In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 0, 1, 2, or 3. 20 In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 0, 1, or 2. In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 1, 2, 3, 4, or 5. In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 1, 2, 3, or 4. In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 1, 2, or 3. In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 1 or 2. 25 In another embodiment, L is -L1-[G-L2]q-G-L3-*, wherein q is 4. In another embodiment , L is - L1-[G-L2]q-G-L3-*, wherein q is 3. In another embodiment, L is -L1-[G-L2]q-G-L3-*, wherein q is 2. In another embodiment, L is -L1-G-L2-G-L3-*. In another embodiment, L is -L1-G-L3-*. In another embodiment, L is -G-L3-*. In another embodiment , L is -L1-G-*. In another embodiment, L is - G-*. 30 In another embodiment, wherein L is -[G-L2]q-G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q is 3; or q is 4; or q is 5). In some embodiments, each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)O-, -OC(O)N(RN)-, - 144 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. In some embodiments, each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-,- 5 N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. In some embodiments, each instance of A-B-A- is independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -O-, and - N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. In some embodiments, each instance of A-B-A- is independently selected from the group 10 consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. In some embodiments, each instance of A-B-A- is independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. 15 In some embodiments, D and F are each independently a bond, C1-10alkyl, C2-10alkenyl, or C2- 10alkynyl, each optionally substituted with 1, 2, 3, or 4 R groups; and E is C1-10alkyl, C2-10alkenyl, C2- 10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. In some embodiments, D and F are each independently a bond or C1-10alkyl optionally 20 substituted with 1, 2, 3, or 4 R groups; and E is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3- 10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. In some embodiments, each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted 25 with 1, 2, 3, or 4 R groups. In some embodiments, each G is independently C1-10alkyl, optionally substituted with 1, 2, or 3 R groups. In some embodiments , each G is independently C1-10alkyl, optionally substituted with 1 or 2 R groups. In some embodiments, each G is independently C1-10alkyl, optionally substituted with one R group . In some embodiments, each G is independently C1-10alkyl. 30 In some embodiments, L is -L1-G-L3-*, wherein * is the bond to ZZ ; G is -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2- 10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; L1 is -B-A-; 35 L3 is a bond or -A-B-A-; 145 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each A is independently a bond, -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. each B is independently a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); and In some embodiments, L is -L1-G-L3-*, wherein * is the bond to ZZ; 5 G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; L1 is -B-; L3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen 10 or C1-6alkyl; and each B is independently a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH; and In some embodiments, L is -L1-G-*, wherein * is the bond to ZZ; G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; 15 L1 is -B-A-, wherein, A is a bond, -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1- 6alkyl; and B is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, L is -L1-G-*, wherein * is the bond to ZZ; 20 G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1 or 2 R groups; L1 is -B-A-, wherein A is a bond, -O-, -S-, or -N(RN)-, each RN is independently hydrogen or C1-6alkyl; and B is a bond, C(O), S(O)2, P(O)(OH), or P(S)(OH). 25 In some embodiments L is -L1-G-*, wherein * is the bond to ZZ; G is C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups; L1 is bond, C(O), S(O)2, P(O)(OH), or P(S)(OH); and RN is hydrogen or C1-6alkyl. In some embodiments, L is -L1-G-*, wherein * is the bond to ZZ , G is C1-10alkyl or C2-10alkenyl, 30 and L1 is -B-A-, wherein, A is a bond, -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; and B is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, L is -L1-G-*, wherein * is the bond to ZZ , G is C1-10alkyl or C2-10alkenyl, L1 is -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. In some embodiments, L is -L1-G-*, wherein * is the bond to ZZ , G is C2-10alkyl, L1 is -O-, -S-, 35 or -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. 146 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, L is -L1-G-*, wherein * is the bond to ZZ , G is C2-10alkyl, L1 is -O- (e.g., G is C3- 10alkyl or C4-10alkyl or C5-10alkyl or C6-10alkyl or C2-8alkyl or C4-8alkyl or C4-8alkyl or C4alkyl or C5alkyl or C6alkyl). In some embodiments, L is wherein * is the bond to ZZ; k is an integer from 1 to 10; L1 is bond, C(O), C(S), C(NRN), S(O)2, P(O)(OH), or P(S)(OH); and RN is hydrogen or C1- 5 6alkyl. In some embodiments, L is wherein * is the bond to ZZ; k is an integer from 1 to 10; L1 is bond, C(O), P(O)(OH), or P(S)(OH). In some embodiments, L is , wherein * is the bond to ZZ; k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 3 to 10; or an integer from 4 to 10; or an integer from 5 10 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7. In some embodiments, L is L wherein * is the bond to ZZ; L1 is bond; and k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 3 to 10; or an integer from 4 to 10; or an integer from 5 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7. In another embodiment, wherein L is -[G-L2]q-G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q 15 is 3; or q is 4; or q is 5), each G is indepdently C1-10alkyl and L2 is O, S, or N(H). In another embodiment, wherein L is -[G-L2]q-G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q is 3; or q is 4; or q is 5), each G is indepdently C1-10alkyl and L2 is O. In another embodiment, wherein L is -[CH2CH2O]q-G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q is 3; or q is 4; or q is 5), G is indepdently C1-10alkyl. 20 In some embodiments, L is , wherein * is the bond to ZZ; t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3). In some embodiments, L is , wherein * is the bond to ZZ. In some embodiments, L is , wherein * is the bond to ZZ; t is 1 to 10 (e.g., 1-5; or 1-3; or 1; or 2; or 3); and z is 1-10 (e.g., 1-6; or 1-4; or 1; or 2; or 3 or 4 or 5 or 6). 25 In some embodiments, L is , wherein * is the bond to ZZ, and t is an integer from 1 to 5 (e.g., 1; or 2; or 3). 147 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, L is , wherein * is the bond to ZZ. In some embodiments, L is wherein * is the bond to ZZ, t is an integer from 0 to 10 (e.g., an integer from 1 to 5 or 1; or 2; or 3); a is an integer from 1 to 3; and s and s’ are each independently an integer from 1 to 24 (e.g., an integer from 1 to 16; an integer from 1 to 10; an integer from 3 to 10; an 5 integer from 3 to 7; or an integer from 4 to 6). In some embodiments, wherein * is the bond to ZZ; a is 1, 2 or 3; and each s, s’, and s” independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6). In some embodiments, L is wherein * is the bond to ZZ; and s, s’, 10 and s’’ are independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6). In some embodiments, L is , wherein * is the bond to ZZ and each s, s’, and s” independently is an integer from 1 to 24(e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6) 15 In some embodiments, L is wherein * is the bond to ZZ; s and k are independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 10 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6). 20 In some embodiments, L is wherein * is the bond to ZZ and w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6). 148 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, L is C1-20 alkyl. In some embodiments, L is C2-20 alkyl. In some embodiments, L is C6-20 alkyl. In some embodiments, L is C8-12 alkyl. In some embodiments, L is C10 alkyl. In some embodiments, L is -G-L2-G-*, wherein L2 is -A-B-A-; each A is independently a bond, 5 -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl, or two RN within an -A-B-A- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; and each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. 10 In some embodiments, L is -G10-L2-G20-*, wherein L2 is -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl, or two RN within an - A-B-A- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; G10 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, 15 aryl, or heteroaryl; and G20 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. In some embodiments, L is -G10-L2-G20-*, wherein L2 is a bond, -O-, -S-, or -N(RN)-; RN is independently hydrogen or C1-6alkyl, G10 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl; and G20 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 20 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. In some embodiments, L is -G10-L2-G20-*, wherein L2 is a bond, -O-, -S-, or -N(RN)-; RN is independently hydrogen or C1-6alkyl, G10 is aryl or heteroaryl; and G20 is C1-10alkyl or C2-10alkenyl. In some embodiments, L is -G10-L2-G20-*, wherein L2 is a bond, -O-, G10 is aryl (e.g., phenyl);25 and G20 is C1-10alkyl. In embodiments of Formula (X), including embodiments of Formulae (x-a) through (x-s) and (xi- a) throught (xi-z), RP3, when present, is hydrogen. In another embodiment, the compound of Formula (X) is according to one of Formulae (X-b) through (X-e) and (X-x) through (X-ab): 149 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (X-x) 150 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (X-aa) (X-ab) or a salt thereof, wherein RP is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group), wheren R and the remaining variables are as defined in Formula (X). In one embodiment of Formulae (X- b) through (X-e) and (X-x) through (X-ab), R1 is hydrogen. In another embodiment of Formulae (X-b) through (X-e) and (X-x) through (X-ab), R1 is C1-6alkyl (e.g., methyl or t-butyl). In another embodiment 5 of Formulae (X-b) through (X-e) and (X-x) through (X-ab), R1 is hydrogen and RP is hydrogen. In another embodiment of Formulae (X-b) through (X-e) and (X-x) through (X-ab), R1 is hydrogen and a nitrogen protecting group. In another embodiment of Formulae (X-b) through (X-e) and (X-x) through (X-ab), R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In another embodiment of Formulae (X-b) through (X-e) and (X-x) through (X-ab), R1 is C1-6alkyl (e.g., methyl or t-butyl) and a nitrogen protecting group. 10 -L-ZZ-L’-RT Embodiments In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is 151 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is (x-b). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is (x-c). 5 In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) 10 through (X-ab), -L-ZZ-L’-RT is (x-f). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is 152 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (x-g). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is (x-h). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is (x-i). 5 In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is (x-j). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is (x-k). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) 10 through (X-ab), -L-ZZ-L’-RT is 153 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is (x-m). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is (x-n). 5 In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is (x-o). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is (x-p). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through 10 (X-ab), -L-ZZ-L’-RT is 154 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (x-q). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is (x-r) In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-ab), -L-ZZ-L’-RT is (x-s). 5 RT1 Embodiments, Formula (X) Nucleotide Conjugates In embodiments of any embodiments of RL of Formula (X), Formula (X-a) through (X-e) and (X- x) through (X-ab), Formula (x-a) through (x-s), and Formula (xi-a) through (xi-z), 10 RT1 is -LL-oligonucleotide, wherein LL is a divalent linker that connects to the 3’-end of the oligonucleotide, the 5’-end of the oligonucleotide, or an internal 2’- or 3’ position on a internal nucleotide (i.e., a nucleotide that is not the 5’-terminal or 3’-terminal nucleoside). In some embodiments. LL is a bond. In some embodiments, RT1 is -LL-oligonucleotide, wherein LL is a divalent linker that connects to 15 the 3’-end of the oligonucleotide, such as one of : directly to the 3’-carbon of the 3’-terminal nucleoside; directly to the 3’-O of the 3’-terminal nucleoside; directly to the 4’-carbon of the 3’-terminal nucleoside; directly to the 2’-carbon of the 3’-terminal nucleoside; or 20 directly to the 2’-O of the 3’-terminal nucleoside. 155 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, RT1 is -LL-oligonucleotide, wherein LL is a divalent linker that connects to the 5'-end of the oligonucleotide, such as one of: directly to the 5’-carbon of the 5’-terminal nucleoside; directly to the 5’-O of the 5’-terminal nucleoside; 5 directly to the 4’-carbon of the 5’-terminal nucleoside; directly to the 2’-carbon of the 5’-terminal nucleoside; or directly to the 2’-O of the 5’-terminal nucleoside. In some embodiments, RT1 is -LL-oligonucleotide, wherein LL is a divalent linker that connects to an internal 2’- or 3’ position on an internal nucleotide. 10 In some embodiments, RT1 is -LL-oligonucleotide, wherein LL is a divalent linker that connects to an internal 2’- position on a internal nucleotide. In some embodiments, RT is RT1 , wherein RT1 is -LL-oligonucleotide, wherein LL is a divalent linker that connects to an internucleotide linkage (i.e., to an oxygen atom in a phosphodiester linkage to form a phosphotriester; or to a nitrogen when the internucleotide linkage is a phosphoroamidate). 15 In another embodiment of any of the preceding embodiments of RT1, when LL connects to a carbon atom on a nucleoside, then LL is -B3-A3-, wherein B3 is -P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH)-; and A3 is -O-, -S-, or -N(H)-. In another embodiment, when LL connects to a carbon atom on a nucleoside, then LL is -B3-A3-, wherein B3 is -P(O)(OH)- or-P(S)(OH)-; and A3 is -O-. 20 In another embodiment, when LL connects to a oxygen atom on a nucleoside, then LL is - P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH). In another embodiment, when LL connects to a oxygen atom on a nucleoside, then LL is -P(O)(OH)- or -P(S)(OH)-. In another embodiment, when LL connects to a oxygen atom on a nucleoside, then LL is -P(O)(OH)-. In another embodiment, when LL connects to a oxygen atom on a nucleoside, then LL is -P(S)(OH)-. 25 In another embodiment, when LL connects to an oxygen atom on a nucleoside, then LL is -B3-A3- LL1-A3-B3-, wherein B3 is a bond, -C(O)-, C(S)-, C(NH), S(O), S(O)2, -P(O)(OH)-, -P(S)(OH)-, or - P(S)(SH); A3 is a bond, -O-, -S-, or -N(H)- ; and LL1 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl. In another embodiment, when LL connects to a oxygen atom on a nucleoside, then LL is -B3-LL1- 30 B3-, wherein B3 is -C(O)-; and LL1 is C1-10alkyl. In another embodiment, when LL connects to a oxygen atom on a nucleoside, then LL is-a bond. In certain embodiments, RT is RT1 wherein RT1 is is -LL-oligonucleotide, when LL connects to a oxygen atom on a nucleoside, and LL is a bond, the nucleoside is of Formula (X-f), 156 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (X-f) wherein B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); L’ is according any of the preceding embodiments; and * represent the bond to ZZ. In some embodiments of Formula (X-f), -L’- is -L1-G-L3-*, wherein * is the bond to ZZ. 5 In some embodiments, -L’- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one or two R groups, and * is the bond to ZZ. In some embodiments, -L’- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one or two groups selected from the group consisting of halogen, hydroxy, C1- 6alkoxy, amino, Cl-6alkylamino, di(C1-6alkylamino), cyano, carboxy , and * is the bond to ZZ. In some embodiments, -L’- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one group selected from 10 the group consisting of halogen, hydroxy, C1-6alkoxy, amino, Cl-6alkylamino, di(C1-6alkylamino), cyano, carboxy , and * is the bond to ZZ. In some embodiments, -L’- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one group selected from the group consisting of hydroxy, amino, and carboxy , and * is the bond to ZZ. In some embodiments, -L’- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with hydroxy, and * is the bond to ZZ. 15 In some embodiments, -L’- is -C2-30alkyl-*, wherein * is the bond to ZZ. In some embodiments, - L’- is -C2-16alkyl-*, wherein * is the bond to ZZ. In some embodiments, -L’- is -C4-12alkyl-*, wherein * is the bond to ZZ. In some embodiments, -L’- is -C4-10alkyl-*, wherein * is the bond to ZZ. In some embodiments, -L’- is -C5-10alkyl-*, wherein * is the bond to ZZ. In some embodiments, -L’- is -C6alkyl-*, wherein * is the bond to ZZ. In some embodiments, -L’- is -C8alkyl-*, wherein * is the bond to ZZ. In 20 some embodiments, -L’- is -C10alkyl-*, wherein * is the bond to ZZ. In some embodiments of Formula (X-f), -L’- is -L1-[G-L2]q-G-L3-*, wherein * is the bond to ZZ; q is 0, 1, 2, 3, 4, or 5; L1 is a bond or -B-A-; each L2 is independently -A-B-A-; 25 L3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O)2, P(O)(OH), or P(S)(OH); each RN is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally 30 substituted with 1, 2, 3, or 4 R groups. 157 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, -L’- is -L1-[G-L2]q-G-*, wherein * is the bond to ZZ, q is 0, 1, 2, or 3; L1 is a bond or -B-A-; and (a) each L2 is independently a bond, C(O)O, OC(O), C(O)(NRN), N(RN)C(O), SO2N(RN), N(RN)SO2, OP(O)(OH), OP(S)(OH), P(O)(OH)O, P(S)(OH)O, OP(O)(OH)O, or 5 OP(S)(OH)O, wherein each RN is independently hydrogen or C1-6alkyl; each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1 or 2 R groups; or (b) each L2 is independently a bond, C(O)O, OC(O), C(O)(NRN), N(RN)C(O), OP(O)(OH)O, or 10 OP(S)(OH)O, wherein each RN is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups. In some embodiments, -L’- is -[G-L2]q-G-*, wherein * is the bond to ZZ, q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each G is independently C1-10alkyl, each of which is optionally 15 substituted with 1 or 2 R groups, and one of: (a) each L2 is independently C(O)O or OC(O); (b) each L2 is independently C(O)(NRN) or N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl (c) each L2 is independently OP(O)(OH)O, or OP(S)(OH)O (e.g., each is OP(O)(OH)O); 20 or (d) each L2 is a bond. In some embodiments,the compound of Formula (X) is according to one of Formulae (X-g) through (X-q): 158 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (X-q) wherein B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); each m is independently integer selected from 1-20 (e.g., 2-12, or 2-10; or 2-6; or 2; or 3; or 4; or 5; or 6). 5 In some embodiments, the compound of Formula (X) is according to one of Formulae (X-r) through (X-w): 159 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 L and ZZ are as defined in Formula (X) or in any embodiment preceding or below; 160 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); each m is independently integer selected from 1-20 (e.g., 2-12, or 2-10; or 2-6; or 2; or 3; or 4; or 5; or 6); 5 each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); RP is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group); and R1 is hydrogen or C1-6alkyl (e.g., methyl or t-butyl). In one embodiment of Formulae (X-r) through (X-w), R1 is hydrogen. In another embodiment of Formulae (X-r) through (X-w), R1 is C1-6alkyl (e.g., methyl or t-butyl). In another embodiment of 10 Formulae (X-r) through (X-w), R1 is hydrogen and RP is hydrogen. In another embodiment of Formulae (X-r) through (X-w), R1 is hydrogen and a nitrogen protecting group. In another embodiment of Formulae (X-r) through (X-w), R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In another embodiment of Formulae (X-r) through (X-w), R1 is C1-6alkyl (e.g., methyl or t-butyl) and a nitrogen protecting group. In some embodiments, two adjacent nucleosides in the oligonucleotide have one of the formula 15 according any of the preceding embodiments; and B is an optionally modified nucleobase. Therein, each nucleoside of the two adjacent nucleosides is independently according to any one of Formula (X-f) 20 through (X-q). In certain embodiments, each nucleoside is according to the same Formula. 161 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, three adjacent nucleosides in the oligonucleotide have the formula , 5 wherein each Y is independently O or S (or O; or S; or O then S or S then O, 5’ followed by 3’); represents the remainder for the oligonucleotide, L’ is according any of the preceding embodiments;and B is an optionally modified nucleobase; e.g., each Y is O . Therein, each nucleoside of the three adjacent nucleosides is independently according to any one of Formula (X-f) through (X-q). In certain embodiments, each nucleoside is according to the same Formula. 10 In some embodiments, four adjacent nucleosides in the oligonucleotide have the formula , , 162 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein each Y is independently O or S; represents the remainder for the oligonucleotide, L’ is according any of the preceding embodiments;and B is an optionally modified nucleobase; e.g., each 5 Y is O. Therein, each nucleoside of the four adjacent nucleosides is independently according to any one of Formula (X-f) through (X-q). In certain embodiments, each nucleoside is according to the same Formula. In certain embodiments, each nucleoside is according to the same Formula (X-u) or (X-w). In certain embodiments, where RT is RT1, wherein RT1 is -LL-oligonucleotide, and when LL connects to an oxygen atom or nitrogen atom in an internucleotide linkage, the internucleotide linkage 10 can be of the formula, including the 3’ and 5’ oxygen atoms of the preceding and following nucelosides, respectively, (X-pa) (X-pb) (X-pc) wherein L’ can be, for example a bond, -S(O)2- or, in for Formula (X-pc), a 5 -8 membered heterocyclyl ring optionally substituted with 1 or 2 R groups, as defined herein ; and * represent the bond to ZZ. For example, the preceding includes, 15 wherein * represent the bond to ZZ; and RN5 is hydrogen or C1-10 alkyl. 163 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO For example, the preceding includes, (X-pg) (X-ph) (X-pi) wherein * represent the bond to ZZ; m is an integer selected from 1 – 20 (e.g., 1-10, or 2-20, or 2-10, or 4-10, or 4-8; or 6-12; or 5; or 6; or 7; or 8; or 9; or 10), and RN5 is hydrogen or C1-10 alkyl. In another embodiment, the compound of Formula (X-pd) is 5 wherein Y’ is O or S, and RY, Y, R1, L, L’, and ZZ are as defined for Formula (X). In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t-butyl). In another embodiment, the compound is 10 164 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S, and RP, R1, L, L’, and ZZ are as defined for Formula (X). In another embodiment, the compound is 5 165 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein each m is an integer selected from 1 – 20 (e.g., 2-20, 2-10, 1-10, 2-16, 4-16, 4-8, or 6- 5 12), each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); Y’ is O or S, and RP, R1, L, L’, and ZZ are as defined for Formula (X). In another embodiment, the compound of Formula (X-pe) is wherein Y’ is O or S, and RY, Y, R1, L, L’, and ZZ are as defined for Formula (X). In one 10 embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 166 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen . In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 10 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen . In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and RP is hydrogen . In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound is 20 167 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S, and RP, R1, L, L’, and ZZ are as defined for Formula (X). In one embodiment, 5 Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 168 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen . In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 10 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen . In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and RP is hydrogen . In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound is 20 169 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein each m is an integer selected from 1 – 20 (e.g., 2-20, 2-10, 1-10, 2-16, 4-16, 4-8, or 6-12), Y’ is 5 O or S, and RP, R1, L, L’, and ZZ are as defined for Formula (X). In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., 10 t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 170 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen . In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 10 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen . In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and RP is hydrogen . In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). For Formula (X), when RT1 is -LL-oligonucleotide and is conjugated at the 5’-end of the 20 oligonucleotide, in one embodiment, RT1 can be represented as Formula (X-5’) wherein LL is -P(Y)(OH)-, wherein Y is O or S (e.g., S); and * represents the bond to remainder of the compound of Formula (X). In another embodiment of Formula (X), when RT1 is -LL-oligonucleotide and is conjugated at the 25 5’-end of the oligonucleotide, in one embodiment, RT1 can be represented as Formula (X-5’o) or Formula (X-5’s), wherein * represents the bond to remainder of the compound of Formula (X). 171 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In an embodiment of Formula (X-5’), (X-5’o), and Formula (X-5’s), -L-ZZ-L’-RT represents any one of Formulae (x-a) through (x-s) . In representative embodiments, -L-ZZ-L’-RT represents Formula In other embodiments-L-ZZ-L’-RT represents one of the following formulae: 5 In other embodiments of Formula (X-5’), (X-5’o), and Formula (X-5’s), L-ZZ-L’-RT represents, (x-m) In other embodiments of Formula (X-5’), (X-5’o), and Formula (X-5’s), L-ZZ-L’-RT represents, (x-s). In another embodiment, the compound of Formula (X) is 172 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S, and RY, Y, R1, L, L’, and ZZ are as defined for Formula (X) or any embodiment thereof. For Formula (X), when RT1 is -LL-oligonucleotide and is conjugated at the 3’-end of the 5 oligonucleotide, in one embodiment, RT1 can be represented as Formula (X-3’) wherein LL is -P(Y)(OH)-, wherein Y is O or S (e.g., S); and * represents the bond to remainder of the compound of Formula (X). In another embodiment of Formula (X), when RT1 is -LL-oligonucleotide and is conjugated at the 10 3’-end of the oligonucleotide, in one embodiment, RT1 can be represented as Formula (X-3’o) or Formula (X-3’s), wherein * represents the bond to remainder of the compound of Formula (X). In an embodiment of Formula (X-3’), (X-3’o), and Formula (X-3’s), -L-ZZ-L’-RT represents any 15 one of Formulae (x-a) through (x-ab) . In representative embodiments, -L-ZZ-L’-RT represents Formula (x-e), . In other embodiments-L-ZZ-L’-RT represents one of the following formulae: 173 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (x-a) (x-b) (x-c) (x-aa) (x-ab) In other embodiments of Formula (X-3’), (X-3’o), and Formula (X-3’s), L-ZZ-L’-RT represents In other embodiments of Formula (X-3’), (X-3’o), and Formula (X-3’s), L-ZZ-L’-RT represents 174 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (x-g).. In other embodiments of Formula (X-3’), (X-3’o), and Formula (X-3’s), L-ZZ-L’-RT represents (x-p). In other embodiments of Formula (X-3’), (X-3’o), and Formula (X-3’s), L-ZZ-L’-RT represents In other embodiments of Formula (X-3’), (X-3’o), and Formula (X-3’s), L-ZZ-L’-RT represents (x-r). 5 In another embodiment of Formula (X), RL is 175 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In another embodiment, the compound of Formula (X) is 5 wherein Y' is O or S, and RY, R1, Y, L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof. In one embodiment , R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). 10 In another embodiment, the compound of Formula (X) is 176 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S, and RY, Y, R1, L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof 5 In another embodiment, the compound of Formula (X) is , wherein Y’ is O or S, and RP, R1, L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. 177 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 5 In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 10 (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen 15 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is 20 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is 178 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein Y’ is O or S, and RP, R1, L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- 5 butyl). In another embodiment, -COOR1 is replaced with a carboxylic acid mimic, such as a tetrazolyl group (e.g., 1,2,3,4-tetrazol-5-yl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl 10 (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen. In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a 15 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one 20 embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen . In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 25 (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 179 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In another embodiment, the compound of Formula (X) is wherein Y’ is O or S, and RP, R1, L’, and L are as defined for Formula (X) or any embodiment thereof. 5 In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 10 In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 15 (pac)). In one embodiment, Y’ is S and RP is hydrogen . In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen 20 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one 180 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen . In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen . In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is 5 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is 10 , wherein each m is independently an integer selected from 1-10; n is 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); Y’ is O or S, and RP and R1 are as defined for Formula (X) or any embodiment thereof. In one embodiment, each m is independently an integer selected from 2-10; or 2-8, or 2-6. 15 In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 20 In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 181 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (pac)). In one embodiment, Y’ is S and RP is hydrogen . In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 5 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- 10 butyl) and RP is hydrogen . In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is 15 wherein Y’ is O or S, and RP and R1 are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- 20 butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 182 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 10 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is 20 183 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S, and RP, R1, L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). 5 In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). 10 In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen . In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 15 In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g.,20 methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 25 In another embodiment, the compound of Formula (X) is 184 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S, and RP, R1, L’, and L are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. 5 In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl 10 (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen. In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a 15 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one 20 embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), 185 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen . In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen . In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 5 (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is 10 wherein each m is independently an integer selected from 1-10; n is 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); Y’ is O or S, and RP and R1 are as defined for Formula (X) or any embodiment thereof. In one embodiment, each m is independently an integer selected from 2-10; or 2-8, or 2-6. In one embodiment, Y’ is O. In another embodiment, Y’ is S. 15 In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl 20 (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 186 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen 5 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen . In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is 10 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is 15 wherein Y’ is O or S, and RP and R1 are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). 20 In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 187 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 10 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is 20 , wherein Y’ is O or S, and RP, R1, L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). 25 In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 188 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen. In one embodiment, Y’ is O and RP is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 10 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen . In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is 20 wherein Y’ is O or S, and RP, R1, L’, and L are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). 25 In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 189 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 10 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is 20 wherein each m is independently an integer selected from 1-10; Y’ is O or S, and RP and R1, are as defined for Formula (X) or any embodiment thereof. In one embodiment, each m is independently an integer selected from 2-10; or 2-8, or 2-6. In one embodiment, Y’ is O. In another embodiment, Y’ is S. 25 In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 190 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 10 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and RP is hydrogen . In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is 20 wherein Y’ is O or S, and RP and R1 are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). 25 In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 191 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 10 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- 15 butyl) and RP is hydrogen . In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is 20 , wherein Y’ is O or S, and RP, R1, L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). 25 In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). 192 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 5 (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), 10 benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen . In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting 15 group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is wherein Y’ is O or S, and RP, R1, L’, and L are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. 20 In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl 25 (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen. In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a 193 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen 5 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is 10 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is 15 wherein each m is independently an integer selected from 1-10; Y’ is O or S, and RP and R1, are as defined for Formula (X) or any embodiment thereof. In one embodiment, each m is independently an integer selected from 2-10; or 2-8, or 2-6. In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- 20 butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S 25 and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen. In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 194 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 5 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- 10 butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is 15 wherein Y’ is O or S, and RP and R1 are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). 20 In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). 25 In one embodiment, Y’ is O and RP is hydrogen. In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 195 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), 5 benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen 10 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is , wherein Y’ is O or S, and RP, R1, L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. 15 In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl 20 (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen. In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting 25 group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), 196 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 5 (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is , wherein Y’ is O or S, and RP, R1, L’, and L are as defined for Formula (X) or any embodiment thereof. 10 In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 15 In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen. In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one 20 embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one 25 embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 197 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is , 5 wherein each m is independently an integer selected from 1-10; Y’ is O or S, and RP and R1 are as defined for Formula (X) or any embodiment thereof. In one embodiment, each m is independently an integer selected from 2-10; or 2-8, or 2-6. In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- 10 butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S 15 and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen. In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 20 In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g.,25 methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 198 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound of Formula (X) is , 5 wherein Y’ is O or S, and RP and R1 are as defined for Formula (X) or any embodiment thereof. In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group 10 (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen. In one embodiment, Y’ is O and RP is a nitrogen 15 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen 20 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is 25 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 199 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO ZZ Embodiments, Formula (X) In embodiments of Formula (X), Formula (X-a) through (X-w), including embodiments of Formulae (x-a) through (x-s) and (xi-a) through (xi-z) ZZ is a linking group formed by a reactive pair. In 5 some embodiments, ZZ comprises a group selected from the group consisting of roup or wherein RLa is hydrogen, C1-10alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl, isobutyl, butyl, or hexyl), C3-8cycloalkyl, 3-8 membered heterocyclyl, aryl (e.g., phenyl), or heteroaryl (e.g., 2-pyridyl). In some embodiments, ZZ comprises Group(1). In some embodiments, ZZ comprises Group(2). In some embodiments, ZZ comprises Group(3). In some embodiments, ZZ comprises Group(4). In some 200 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO embodiments, ZZ comprises Group(5). In some embodiments, ZZ comprises Group(6). In some embodiments, ZZ comprises Group(7). In some embodiments, ZZ comprises Group(8). In some embodiments, ZZ comprises Group(9). In some embodiments, ZZ comprises Group(10). In some embodiments, ZZ comprises Group(11). In some embodiments, ZZ comprises Group(12). 5 In some embodiments, wherein ZZ is -A’-B’-A’-. In some embodiments, ZZ is -A’-B’-A’-, wherein each A’ is independently a bond, -O-, -S-, or -N(RN3)-, wherein RN3 is independently hydrogen or C1-6alkyl and each B’ is independently CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). 10 In some embodiments, ZZ is CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, ZZ is C(O), C(S), or S(O)2. In some embodiments, ZZ is P(O)(OH), or P(S)(OH). In some embodiments, ZZ is –C(O)-. In some embodiments, ZZ is -A’-B’- or -B’-A’- wherein 15 each A’ is independently -O-, -S-, or -N(RN3)-; each B’ is independently CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and each RN3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -A’-B’- or -B’-A’- wherein each A’ is independently -O- or -N(RN3)-, wherein RN3 is independently hydrogen or C1-6alkyl. 20 each B’ is independently CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); and each RN3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -CH2O-, -OCH2-, -S-S-, -C=N-, -C=N-O-, -C=N-N(RN3)-, -N=C-, - O-N=C-, -N(RN3)-N=C-, -C(O)N(RN3)-, -N(RN3)C(O)-, -C(O)O-, -OC(O)-, -OC(O)N(RN3)-, - N(RN3)C(O)O-, -N(RN3)C(O)N(RN3)-, -S(O)2N(RN3)-, -N(RN3)S(O)2-, -OP(O)(OH)O-, -OP(S)(OH)O-, - 25 OP(O)(OH)-, -OP(S)(OH)-, -P(O)(OH)O-, or -P(S)(OH)O-. wherein RN3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -CH2O- or -OCH2-. In some embodiments, ZZ is -S-S-. In some embodiments, ZZ is -C=N-, -C=N-O-, -C=N-N(RN3)-, -N=C-, -O-N=C-, or -N(RN3)- 30 N=C-, wherein RN3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -C(O)N(RN3)-, -N(RN3)C(O)-, -C(O)O-, -OC(O)-, -OC(O)N(RN3)-, - N(RN3)C(O)O-, -N(RN3)C(O)N(RN3)-, -S(O)2N(RN3)-, or -N(RN3)S(O)2-, wherein RN3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -C(O)N(RN3)- or -N(RN3)C(O)-, wherein RN3 is independently 35 hydrogen or C1-6alkyl. In some embodiments, ZZ is -C(O)O- or -OC(O)-. 201 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, ZZ is -OC(O)N(RN3)-, -N(RN3)C(O)O-, or -N(RN3)C(O)N(RN3)-, wherein RN3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -N(RN3)C(O)N(RN3)-, wherein RN3 is independently hydrogen or C1-6alkyl. 5 In some embodiments, ZZ is -OC(O)N(RN3)- or -N(RN3)C(O)O-, wherein RN3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -OP(O)(OH)-, -OP(S)(OH)-, - P(O)(OH)O-, or -P(S)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)O- or -OP(S)(OH)O-. 10 In some embodiments, ZZ is -OP(S)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)-, -OP(S)(OH)-, -P(O)(OH)O-, or -P(S)(OH)O-. In some embodiments, ZZ is -OP(S)(OH)- or -P(S)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)- or -P(O)(OH)O-. 15 . Species of Formula (X) In another embodiment, the compound of Formula (X) is selected from the group consisting of: (X-a) (X-b) 202 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (X-c) (X-d) In another embodiment, the compound of Formula (X) is selected from the group consisting of: 203 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 204 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 205 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 206 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein * represents the bond to the remainder of the dsRNA agent. In another embodiment, the compound of Formula (X) is selected from the group consisting of: 207 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein * represents the bond to the remainder of the dsRNA agent. In another embodiment, the compound of Formula (X) is selected from the group consisting of: 208 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 209 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein * represents the bond to the remainder of the dsRNA agent. B. Alpha-v-Beta-6 (αvβ6) Integrin Targeting Ligands Conjugated to a Branched Carrier 5 Group In some embodiments, the alpha-v-beta-6 (αvβ6) integrin targeting ligand may be conjugated to the dsRNA agent of the disclosure, e.g., to at least one strand of the dsRNA agent of the disclosure, via a branched carrier group, e.g., a b. In some embodiments, the alpha-v-beta-6 (αvβ6) integrin targeting ligand conjugated to the dsRNA agent of the disclosure, e.g., to at least one strand of the dsRNA agent of 10 the disclosure, is represented by a compound of Formula (XV): (Φ-ZZ-)xΔ-T-RT (XV) or a salt thereof, wherein x is 2, 3, 4, 5, 6, 7, or 8; T is a divalent linking group; 15 Δ is a branching group; each ZZ is independently -A’-B’-A’- or a linking group formed by a first reactive pair, wherein each A’ is independently a bond, -O-, -S-, or -N(RN3)-; each B’ is independently a bond, CH2, C(O), C(S), C(NRN3), -C=N-, S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); and 20 each RN3 is independently hydrogen or C1-6alkyl, or two RN3 within the -A’-B’-A’- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; and RT is -RT1 or -G0-ORT1, wherein G0 is -D0-E0-F0-, wherein 25 D0, E0, and F0 are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; and 210 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO RT1 is -LL-oligonucleotide, wherein LL is an oligonucleotide linking group connecting the αvβ6 integrin targeting ligand to the oligonucleotide comprised in the dsRNA agent; and each Φ is a compound of the Formula 5 wherein: Y is O, N(H), S, or CH2; R1 is hydrogen, C1-6alkyl (e.g., methyl), or a carboxylic acid mimic.; RY is , wherein m is 0, 1, 2, 3, or 4; and 10 each R2 is independently R, or two R2 groups on adjacent carbon atoms taken together with the atoms to which they are bound form a fused 4 – 8 membered ring that is optionally substituted by 1, 2, 3 or 4 groups independently selected from group consisting of R and a nitrogen protecting group; A is an 5-membered heteroaryl optionally substituted with 1 or 2 substituents independently 15 selected from methyl, ethyl, fluoro, hydroxymethyl, 2-hydroxypropan-2-yl, trifluoromethyl, difluoromethyl, and fluoromethyl; Q is -COOR1 or tetrazolyl (e.g., 1,2,3,4-tetrazol-5-yl); and RL is -N(R3)(R4), -O(R5), -S(R5), or -R5, wherein 20 R3 and R4 are either (viii) R3 is hydrogen or C1-6alkyl and R4 is R5; or (ix) R3 and R4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R5; 25 and R5 is -L-* wherein L is -L1-[G-L2]q-G-L3-*, * is the bond to a ZZ; and q is 0 or an integer selected from 1 – 25; (e.g., 1-20, or 1-15); 211 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO L1 is a bond or -B-A-; each L2 is independently -A-B-A-; L3 is a bond or -A-B-A-; each G is independently -D-E-F-, wherein 5 D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2- 10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; each A is independently a bond, -O-, -S-, or -N(RN)-; 10 each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl, or two RN within an -A-B-A- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl. 15 Embodiment for the variables of Formula (XII), and (XV) that are the same as Formula (X) are as described above for Formula (X). For example, embodiments for the variable ZZ are each as described above for Formula (X); embodiments for the variable RT and L are each as described above for Formula (X). In certain embodiments, the compound of formula ( 20 In certain embodiments, the compound of formula ( In certain embodiments, the compound of formula ( T Embodiments, Formula (XV) In some embodiments, T is a bond or **-L6-G1-[L5-G1]q1-L4-, wherein ** is the bond to RT; 25 q1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each L4, L5, and L6 are independently a bond, -A1-B1-A1- or ZZ1; 212 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO ZZ1 is a group formed by reaction of a reactive pair (e.g., a reaction between an azide and an alkyne or a cycloalkyne); each G1 is independently -D1-E1-F1-, wherein D1, E1, and F1 are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of 5 which is optionally substituted with 1, 2, or 3 R groups; each A1 is independently a bond, -O-, -S-, or -N(RN1)-; each B1 is independently a bond, C(O), C(S), C(NRN1), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN1 is independently hydrogen or C1-6alkyl, or two RN1 within an -A1-B1-A1- group taken 10 together with the atoms to which they are connected from a 4-8 membered heterocyclyl. In some embodiments, T is **-L6-G1-L5-G1-L4-, wherein ** is the bond to RT; L4 and L6 are independently -A1-B1-A1-; each L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); each G1 is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered 15 heterocyclyl, aryl, or heteroaryl (e.g., C1-10alkyl or C2-10alkenyl); each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). 20 In some embodiments, T is **-L6-G1-[L5-G1]q1-L4-, wherein ** is the bond to RT; q1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each L4, L5, and L6 are independently a bond or -A1-B1-A1-, or ZZ1; ZZ1 is a group formed by reaction of an azide and alkyne or cycloalkyne; 25 each G1 is independently C1-10alkyl, each A1 is independently a bond, -O-, -S-, or -N(RN1)-; each B1 is independently a bond, C(O), C(S), C(NRN1), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); and each RN1 is independently hydrogen or C1-6alkyl. 30 In a further embodiment, one L5 is ZZ1. In a further embodiment, ZZ1 comprises: 213 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . In some embodiments, T is **-L6-G1-L5-G1-L4-, wherein ** is the bond to RT; L4 and L6 are independently -A1-B1- or -B1-A1-; 5 each L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); each G1 is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl (e.g., C1-10alkyl or C2-10alkenyl); each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl;and 10 each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, T is **-B1-G1-L5-G1-B1-, wherein ** is the bond to RT; each L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); each G1 is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered 15 heterocyclyl, aryl, or heteroaryl (e.g., C1-10alkyl or C2-10alkenyl); each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, T is **-L6-G1-L5-G1-L4-, wherein ** is the bond to RT; 20 L4 and L6 are independently -A1-B1-A1-; each L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); each G1 is independently C1-10alkyl or C2-10alkenyl; each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl; and 25 each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, T is **-L6-G1-L5-G1-L4-, wherein ** is the bond to RT; L4 and L6 are independently -B1-A1- or -A1-B1-; each L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); each G1 is independently C1-10alkyl or C2-10alkenyl; 30 each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, T is **-B1-G1-L5-G1-B1-, wherein ** is the bond to RT; 214 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); each G1 is independently C1-10alkyl or C2-10alkenyl; each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl; and 5 each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, T is **-B1-C1-10alkyl-L5-C1-10alkyl-B1-, wherein ** is the bond to RT; each L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or 10 C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, T is -L4-G1-L6-**, wherein ** is the bond to RT; L4 and L6 are independently -A1-B1-A1-; each G1 is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered 15 heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups; each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is hydrogen or C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, T is -L4-G1-L6-**, wherein ** is the bond to RT; L4 is -C(O)O- or C(O)N(RN1)-, wherein RN1 is hydrogen or C1-6alkyl; 20 L6 is -OP(O)(OH)O- or -OP(S)(OH)O-; and each G1 is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups. In some embodiments, T is one of: 25 (d) **-L6-G1-ZZ1-L5-G1-L4-, (e) **-L6-G1-L5-G1-ZZ1-G1-L4-; or (f) **-L6-G1-ZZ1-G1-L4-, 30 wherein the sum of q2 and q3 is less than q1. In certain embodiments, q2 and q3 are independently 0, 1, 2, 3 or 4. In certain embodiments, each G1 is independently C1-20 alkyl (e.g., C2- 15alkyl; or C2-12alkyl; or C2-10alkyl; or C2-8alkyl; or C2-6alkyl). In certain embodiments, each L5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, - OC(O)O-, -OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O,35 -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-. In certain embodiments, each L5 is independently - 215 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, each L5 is independently - C(O)N(H)- or -N(H)C(O)-. In certain embodiments, each G1 is independently C1-20 alkyl (e.g., C2-15alkyl; or C2-12alkyl; or C2- 10alkyl); and each L5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - 5 OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-. In certain embodiments, each G1 is independently C1-20 alkyl (e.g., C2-15alkyl; or C2-12alkyl; or C2- 10alkyl); and each L5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, L6 is a bond. In certain embodiments, L6 is -C(O)-, -S(O)2-, -P(O)(OH)-,10 or -P(S)(OH)-. In certain embodiments, L6 is -P(O)(OH)- or -P(S)(OH)-. In certain embodiments, L6 is - P(O)(OH)-. In certain embodiments, L6 is -P(S)(OH)-. In certain embodiments, L6 is -C(O)-. In certain embodiments, L4 is -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-. In certain embodiments, L4 is -P(O)(OH)O-, -OP(S)(OH)-,15 -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-. In certain embodiments, L4 is -C(O)O-, - OC(O)-, -C(O)N(H)-, or -N(H)C(O)-. In some embodiments, T is one of: 20 (d) **-L6-G1-ZZ1-L5-G1-L4-, (e) **-L6-G1-L5-G1-ZZ1-G1-L4-; or (f) **-L6-G1-ZZ1-G1-L4-, wherein the sum of q2 and q3 is less than q1; and ZZ1 is selected from the group consisting of: 216 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein RLa is hydrogen, C1-10alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl, isobutyl, butyl, or hexyl), C3-8cycloalkyl, 3-8 membered heterocyclyl, aryl (e.g., phenyl), or heteroaryl (e.g., 2-pyridyl); and the sum of q2 and q3 is less than q1 In certain embodiments, q2 and q3 are independently 0, 1, 2, 3 or 4. In certain embodiments, 5 each G1 is independently C1-20 alkyl (e.g., C2-15alkyl; or C2-12alkyl; or C2-10alkyl; or C2-8alkyl; or C2- 6alkyl). In certain embodiments, each L5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, - OC(O)O-, -OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-. In certain embodiments, each L5 is independently -10 C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, each L5 is independently - C(O)N(H)- or -N(H)C(O)-. In certain embodiments, each G1 is independently C1-20 alkyl (e.g., C2-15alkyl; or C2-12alkyl; or C2- 10alkyl); and each L5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - 15 OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-. In certain embodiments, each G1 is independently C1-20 alkyl (e.g., C2-15alkyl; or C2-12alkyl; or C2- 10alkyl); and each L5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, L6 is a bond. In certain embodiments, L6 is -C(O)-, -S(O)2-, -P(O)(OH)-, or -P(S)(OH)-. In certain embodiments, L6 is -P(O)(OH)- or -P(S)(OH)-. In certain embodiments, L6 is - 20 P(O)(OH)-. In certain embodiments, L6 is -P(S)(OH)-. In certain embodiments, L6 is -C(O)-. In certain embodiments, L4 is -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-. In certain embodiments, L4 is -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-. In certain embodiments, L4 is -C(O)O-, - 25 OC(O)-, -C(O)N(H)-, or -N(H)C(O)-. In some embodiments, T is one of: 30 (e) **-L6-C2-12alkyl-L5-C2-12alkyl-ZZ1-C2-12alkyl-L4-; or (f) **-L6-C2-12alkyl-ZZ1-C2-12alkyl-L4-, 217 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein the sum of q2 and q3 is less than q1 In certain embodiments, q2 and q3 are independently 0, 1, 2, 3 or 4. In certain embodiments, each L5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, - N(H)C(O)-, -OC(O)O-, -OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, - OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-. In certain embodiments, each L5 is 5 independently -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, each L5 is independently -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, L6 is a bond. In certain embodiments, L6 is -C(O)-, -S(O)2-, -P(O)(OH)-, or -P(S)(OH)-. In certain embodiments, L6 is -P(O)(OH)- or -P(S)(OH)-. In certain embodiments, L6 is - P(O)(OH)-. In certain embodiments, L6 is -P(S)(OH)-. In certain embodiments, L6 is -C(O)-. 10 In certain embodiments, L4 is -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-. In certain embodiments, L4 is -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-. In certain embodiments, L4 is -C(O)O-, - OC(O)-, -C(O)N(H)-, or -N(H)C(O)-. 15 In some embodiments, T is selected from the following, wherein ** is the bond to RT: (q) **-C(O)-C1-10alkyl-L5-C1-10alkylC(O)-, (r) **-C(O)-C2-10alkyl-L5-C2-10alkyl-C(O)-, (s) **-C(O)-C4-10alkyl-L5-C4-10alkyl-C(O)-, (t) **-C(O)-C6-10alkyl-L5-C6-10alkyl-C(O)-, 20 (x) **-C(O)-C2-20alkyl-C(O)-, (y) **-C(O)-C2-12alkyl-C(O)-, 25 (z) **-C(O)-C6-20alkyl-C(O)-, (aa)**-C(O)-C6-12alkyl-C(O)-, (bb) **-C(O)-C10alkyl-C(O)- and (cc)**-C(O)-CH2CH2-C(O)-, wherein each L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond), wherein each A1 is 30 independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, T is selected from the following, wherein ** is the bond to RT: (dd) **-N(H)C(O)-C2-20alkyl-C(O)-, (ee)**- N(H)C(O)-C6-20alkyl-C(O)-, 35 (ff) **- N(H)C(O)-C6-12alkyl-C(O)-, (gg) **- N(H)C(O)-C10alkyl-C(O)-, 218 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (hh) **-C(O)-C2-20alkyl-C(O)N(H)-, (ii) **-C(O)-C6-20alkyl-C(O)N(H)-, (jj) **-C(O)-C6-12alkyl-C(O)N(H)-, (kk) **-C(O)-C10alkyl-C(O)N(H)-, 5 (ll) **-N(H)C(O)-C2-20alkyl-C(O)N(H)-, (mm) **- N(H)C(O)-C6-20alkyl-C(O)N(H)-, (nn) **- N(H)C(O)-C6-12alkyl-C(O)N(H)-, and (oo) **- N(H)C(O)-C10alkyl-C(O)N(H). 10 In some embodiments, T is -L4-G1-L6-**, wherein ** is the bond to RT; L4 is -C(O)O- or C(O)N(RN1)-, wherein RN1 is hydrogen or C1-6alkyl; L6 is -OP(O)(OH)O- or -OP(S)(OH)O-; and each G1 is independently C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with one R group. 15 In some embodiments, T is -L4-G1-L6-**, wherein ** is the bond to RT; L4 is -C(O)O- or C(O)N(RN1)-, wherein RN1 is hydrogen or C1-6alkyl; L6 is -OP(O)(OH)O- or -OP(S)(OH)O-; and each G1 is independently C3-10cycloalkyl or 3-10 membered heterocyclyl. In some embodiments, T is -L4-G1-L6-**, wherein ** is the bond to RT; 20 L4 is -C(O)O- or C(O)N(RN1)-, wherein RN1 is hydrogen or C1-6alkyl; L6 is -OP(O)(OH)O- or -OP(S)(OH)O-; and each G1 is independently C3-10cycloalkyl or 3-10 membered heterocyclyl. In some embodiments, wherein ** is the bond to RT; and X is O or S (e.g., S). 25 In some embodiments, T is **-L6-[G5-O]q5-G5-L4-, wherein ** is the bond to RT; q5 is an integer selected from 1 to 20; L4 and L6 are independently -A1-B1-A1-, wherein each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is hydrogen 30 or C1-6alkyl; each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G5 is independently C1-10alkyl; and each L10 is -O-. In some embodiments, T is **-C(O)-[CH2CH2-O]q5-G5-L4-, wherein ** is the bond to RT; 219 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO q5 is an integer selected from 1 to 20; L4 is -A1-B1-A1-, wherein each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is hydrogen or C1-6alkyl; 5 each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); G5 is C1-10alkyl. In some embodiments, T is **-C(O)-[CH2CH2-O]q5-G5-L4-, wherein ** is the bond to RT; q5 is an integer selected from 1 to 20; L4 is -A1-B1 or -B1-A1-, wherein 10 each A1 is independently -O- or -N(H)-, each B1 is independently C(O), G5 is C1-10alkyl (e.g., C2-10alkyl or C2-6alkyl). In some embodiments, T is **-C(O)-[CH2CH2-O]q5- C2-10alkyl-C(O)N(H)-, wherein ** is the bond to RT, wherein q5 is an integer selected from 1 to 20 (e.g., 1 to 10, or 2 to 10; or 2 – 8; or 1; or 2; or 15 3; or 4.) In some embodiments, Formula (XV) is according to one of Formulae (XVa) through (XVc), respectively: (XVc). Δ Embodiments, Formula (XV) 20 In some embodiments, Δ is #–[G2-L7]q2-* or #–G3-([L7-G4]q3-*)y, wherein # is the bond to T; y is 1, 2, 3, 4, or 5; 25 q2 is 1, 2, 3, 4, 5, 6, 7, or 8; q3 is 0, 1, 2, 3, 4, 5, 6, 7, or 8; each G2, G3, and G4 is independently -D2-E2-F2-, wherein 220 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO D2, E2, and F2 are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 RB groups, and wherein each G2 and G4 optionally contains at least one bond to a ZZ (e.g., one bond to a 5 ZZ); or G3 is N and y is 2; each L7 is independently -A2-B2-A2-; wherein each A2 is independently a bond, -O-, -S-, or -N(RN2)-; each B2 is independently a bond, C(O), C(S), C(NRN2), S(O), S(O)2, P(O)(OH), 10 P(S)(OH), or P(S)(SH); and each RN2 is independently hydrogen, C1-6alkyl, a bond to a ZZ, or two RN2 within an -A2- B2-A2- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; and 15 each RB is independently halogen, cyano, azido, nitro, -N(R10)2, -O(R10), -S(R10), -C(O)OR10, - C(O)R10, -C(O)N(R10)2, -C(NR10)OR10, -C(NR10)R10, -C(NR10)N(R10)2, -C(S)OR10, - C(S)R10, -C(S)N(R10)2, -S(O)2R10, -S(O)2OR10, -S(O)2N(R10)2, -N(R10)C(O)OR10, -N(R10)C(O)R10 , -N(R10)C(O)N(R10)2, -N(R10)S(O)2R10, -N(R10)S(O)2OR10, -N(R10)S(O)2N(R10)2, -OC(O)OR10, - OC(O)R10, -OC(O)N(R10)2, -OS(O)2R10, -OS(O)2OR10, -OS(O)2N(R10)2, or -SC(O)R10, wherein 20 each R10 is independently hydrogen, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl.provided that Δ contains x bonds to ZZ. In some embodiments, Δ is #–[G2-L7]q2-* wherein # is the bond to T; q2 is 1, 2, 3, 4, 5, 6, 7, or 8; 25 q3 is 0, 1, 2, 3, 4, 5, 6, 7, or 8; each G2, G3, and G4 is independently -D2-E2-F2-, wherein D2, E2, and F2 are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 RB groups, and 30 wherein each G2 optionally contains at least one bond to ZZ; each L7 is independently -A2-B2-A2-; each A2 is independently a bond, -O-, -S-, or -N(RN2)-; each B2 is independently a bond, C(O), C(S), C(NRN2), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); 221 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each RN2 is independently hydrogen, C1-6alkyl, a bond to a ZZ, or two RN2 within an -A2-B2-A2- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; each RB is independently halogen, cyano, azido, nitro, -N(R10)2, -O(R10), -S(R10), - 5 C(O)OR10, -C(O)R10, -C(O)N(R10)2, -C(NR10)OR10, -C(NR10)R10, -C(NR10)N(R10)2, - C(S)OR10, - C(S)R10, -C(S)N(R10)2, -S(O)2R10, -S(O)2OR10, -S(O)2N(R10)2, -N(R10)C(O)OR10, -N(R10) C(O)R10, -N(R10)C(O)N(R10)2, -N(R10)S(O)2R10, -N(R10)S(O)2OR10, -N(R10)S(O)2N(R10)2 , -OC(O)OR10, -OC(O)R10, -OC(O)N(R10)2, -OS(O)2R10, -OS(O)2OR10, -OS(O)2N(R10)2, 10 or -SC(O)R10, wherein each R10 is independently hydrogen, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl. In one embodiment, -Δ- is #–[G2-L7]q2-*, where # is the bond to T and * is a bond to a ZZ group. For example, such embodiments include the following, 15 wherein each * is a bond to a ZZ; # is the bond to T, each G2 is independently C1-10alkyl, C2-10alkenyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 RB groups; and each L7 is independently -A2-B2-A2-, wherein each A2 is independently a bond, -O-, -S-, or - N(RN2)-; each B2 is independently a bond, C(O), S(O)2, P(O)(OH), or P(S)(OH). 20 In one embodiment, each G2 is independently C1-10alkyl, each optionally substituted with 1 or 2 RB groups. In one embodiment, each G2 is independently C1-10alkyl. In one embodiment, each L7 is independently -A2-B2-A2-, wherein each A2 is independently a bond, -O-, -S-, or -N(RN2)-; and each B2 is independently a bond, C(O) or S(O)2, provided that at least one A2 is not a bond. 25 In one embodiment, each L7 is independently -A2-B2- or - B2-A2-, wherein each A2 is independently, -O-, -S-, or -N(RN2)-; and each B2 is independently a bond, C(O) or S(O)2. For example, such embodiments include each of the following, 222 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein # is the bond to T and each * is a bond to a ZZ group. ; and each G2 is independently C1-10alkyl. For example, such embodiments include each of the following, wherein # is the bond to T and each * is a bond to a ZZ; wherein # is the bond to T and each * is a bond to a ZZ group. 5 In one embodiment, -#–G3-([L7-G4]q3-*)y,where # is the bond to T and * is a bond to a ZZ group. For example, such embodiments include each of the following, 223 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein # is the bond to T, each * is a bond to a ZZ group, and each L7 is selected from the group consisting of -O-, -S-, -N(H)-, -C(O)O-, -OC(O)-, -C(O)N(H)- , -OC(O)O-, -N(H)C(O)O-, -OC(O)N(H)-, -OP(O)(OH)O-, or -OP(S)(OH)O-; and each G4 is independently -D2-E2-F2-, wherein 5 each D2 and F2 are independently a bond or C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 RB groups, and each E2 is independently bond, C1-10alkyl, C2-10alkenyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 10 5 RB groups, provided that E2 is not a bond with D2 and F2 are each bonds. In one embodiment of -#–G3-([L7-G4]q3-*)y, each L7 is selected from the group consisting of -O-, -S-, -N(H)-, -N(H)C(O)-, -C(O)N(H)-, - OP(O)(OH)O-, and -OP(S)(OH)O-; and each G4 is independently -D2-E2-F2-, wherein 15 each D2 and F2 are independently a bond or C1-10alkyl; each E2 is independently C1-10alkyl, C2-10alkenyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 RB groups. In one embodiment of -#–G3-([L7-G4]q3-*)y, 20 each L7 is selected from the group consisting of -O-, -S-, -N(H)-, -N(H)C(O)-,-C(O)N(H)-, - OP(O)(OH)O-, and -OP(S)(OH)O-; and each G4 is independently C1-10alkyl, C2-10alkenyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 RB . In one embodiment of -#–G3-([L7-G4]q3-*)y, 25 each L7 is selected from the group consisting of -O-, -S-, -N(H)-, -N(H)C(O)-, -C(O)N(H)-, - OP(O)(OH)O-, and -OP(S)(OH)O-; and each G4 is independently C1-10alkyl which is optionally substituted with 1 or, 2 5 RB groups. In one embodiment of -#–G3-([L7-G4]q3-*)y each L7 is selected from the group consisting of -O-, -S-, -N(H)-, --N(H)C(O)-, C(O)N(H)-, - 30 OP(O)(OH)O-, and -OP(S)(OH)O-; and each G4 is independently C1-10alkyl. In one embodiment of -#–G3-([L7-G4]q3-*)y, each L7 is selected from the group consisting of --N(H)C(O)- and C(O)N(H)-; 224 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO and each G4 is independently C1-10alkyl. For example, such embodiments include each of the following, wherein each * is a bond to a ZZ; wherein # is the bond to T and each * is a bond to a ZZ group. ZZ Embodiments, Formulae (XV) and (XVa) through (XVc) 5 In embodiments of Formulae (XV) and (XVa) through (XVc), ZZ is a linking group formed by a reactive pair. In some embodiments, ZZ comprises a group selected from the group consisting of 225 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, ZZ comprises Group(1). In some embodiments, ZZ comprises Group(2). In some embodiments, ZZ comprises Group(3). In some embodiments, ZZ comprises Group(4). In some embodiments, ZZ comprises Group(5). In some embodiments, ZZ comprises Group(6). In some embodiments, ZZ comprises Group(7). In some embodiments, ZZ comprises Group(8). In some 5 embodiments, ZZ comprises Group(9). In some embodiments, ZZ comprises Group(10). In some embodiments, ZZ comprises Group(11). In some embodiments, ZZ comprises Group(12). In some embodiments, wherein ZZ is -A’-B’-A’-. In some embodiments, ZZ is -A’-B’-A’-, wherein each A’ is independently a bond, -O-, -S-, or -N(RN3)-, wherein RN3 is independently 10 hydrogen or C1-6alkyl and each B’ is independently CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, ZZ is CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, ZZ is C(O), C(S), or S(O)2. In some embodiments, ZZ is P(O)(OH), or P(S)(OH). 15 In some embodiments, ZZ is –C(O)-. In some embodiments, ZZ is -A’-B’- or -B’-A’- wherein each A’ is independently -O-, -S-, or -N(RN3)-; each B’ is independently CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and 226 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each RN3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -A’-B’- or -B’-A’- wherein each A’ is independently -O- or -N(RN3)-, wherein RN3 is independently hydrogen or C1-6alkyl. each B’ is independently CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); and 5 each RN3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -CH2O-, -OCH2-, -S-S-, -C=N-, -C=N-O-, -C=N-N(RN3)-, -N=C-, - O-N=C-, -N(RN3)-N=C-, -C(O)N(RN3)-, -N(RN3)C(O)-, -C(O)O-, -OC(O)-, -OC(O)N(RN3)-, - N(RN3)C(O)O-, -N(RN3)C(O)N(RN3)-, -S(O)2N(RN3)-, -N(RN3)S(O)2-, -OP(O)(OH)O-, -OP(S)(OH)O-, - OP(O)(OH)-, -OP(S)(OH)-, -P(O)(OH)O-, or -P(S)(OH)O-. wherein RN3 is independently hydrogen or 10 C1-6alkyl. In some embodiments, ZZ is -CH2O- or -OCH2-. In some embodiments, ZZ is -S-S-. In some embodiments, ZZ is -C=N-, -C=N-O-, -C=N-N(RN3)-, -N=C-, -O-N=C-, or -N(RN3)- N=C-, wherein RN3 is independently hydrogen or C1-6alkyl. 15 In some embodiments, ZZ is -C(O)N(RN3)-, -N(RN3)C(O)-, -C(O)O-, -OC(O)-, -OC(O)N(RN3)-, - N(RN3)C(O)O-, -N(RN3)C(O)N(RN3)-, -S(O)2N(RN3)-, or -N(RN3)S(O)2-, wherein RN3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -C(O)N(RN3)- or -N(RN3)C(O)-, wherein RN3 is independently hydrogen or C1-6alkyl. 20 In some embodiments, ZZ is -C(O)O- or -OC(O)-. In some embodiments, ZZ is -OC(O)N(RN3)-, -N(RN3)C(O)O-, or -N(RN3)C(O)N(RN3)-, wherein RN3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -N(RN3)C(O)N(RN3)-, wherein RN3 is independently hydrogen or C1-6alkyl. 25 In some embodiments, ZZ is -OC(O)N(RN3)- or -N(RN3)C(O)O-, wherein RN3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -OP(O)(OH)-, -OP(S)(OH)-, - P(O)(OH)O-, or -P(S)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)O- or -OP(S)(OH)O-. 30 In some embodiments, ZZ is -OP(S)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)-, -OP(S)(OH)-, -P(O)(OH)O-, or -P(S)(OH)O-. In some embodiments, ZZ is -OP(S)(OH)- or -P(S)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)- or -P(O)(OH)O-. 227 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . RT Embodiments, Formulae ( 5 In some embodiments of Formula (XV), RT is RT1 and is as defined for Formula (X). In some embodiments of Formula (XV), RT is -G0-ORT1, wherein RT1 is as defined for Formula (X) and G0 is selected from: (c) G0 is -D0-E0-F0-, wherein D0 and F0 are independently a bond or C1-10alkyl optionally substituted with 1, 2, 3, or 4 10 R groups; and E0 is C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; (d) G0 is -D0-E0-F0-, wherein D0 and F0 are independently a bond or C1-10alkyl optionally substituted with 1, 2, 3, or 4 15 R groups; and E0 is 3-10 membered heterocyclyl optionally substituted with 1, 2, 3, or 4 R groups; and (e) G0 is 3-10 membered heterocyclyl optionally substituted with 1 or 2 R groups; examples (f) G0 is pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, each optionally substituted with 1 20 or 2 R groups; examples include: (g) G0 is 3-10 membered-heterocyclyl-C1-10alkyl, optionally substituted with 1, 2, 3, or 4 R groups; examples include, 228 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (h) G0 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 5 3, or 4 R groups (e.g., 1 or 2 R groups); (i) G0 is C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups; (j) G0 is C1-10alkyl optionally substituted with 1 or 2 R groups; (k) G0 is C1-10alkyl, optionally substituted with -O(Ra), wherein Ra is independently hydrogen, 10 C1-6alkyl, or a hydroxyl protecting group; e.g., , (l) G0 is C1-10alkyl, and (m) G0 is absent (a bond); wherein * represents the bond to T, the broken bond represents the bond to ORT1, and R is -C1-6alkyl-ORa 15 or -ORa, wherein Ra is independently hydrogen or C1-6alkyl. Φ Embodiments In one embodiment, RY is defined in the structure of Formula (XII) according to any one of the RY Embodiments of Formula (IV). 20 In another embodiment, Formula (XII) has the structure described above for any embodiment of Formula (IV), where the variable Z is replaced by a bond to a ZZ group. In another embodiment, Formula (XII) has the structure of any one of Formula (XII-a) through (XII-h) and (XII-t): 229 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein p is0, 1, 2 or 3; each R21 is independently selected from group consisting of R and a nitrogen protecting group, and RP is a nitrogen protecting group, and R, R1, and RL are as defined above for Formula (XII). 230 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO L Embodiments Formula (XII) and (XII-a) through (XII-h) and (XII-r) In some embodiments of any one of Formula (XII) and Formulae (XII-a) through (XII-h) and (XII-r), L is -L1-[G-L2]q-G-L3-*, wherein * is the bond to a ZZ group. 5 In another embodiment, wherein L is -L1-[G-L2]q-G-L3-*, wherein q is 0, 1, 2, 3, 4, or 5. In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 0, 1, 2, 3, or 4 In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 0, 1, 2, or 3. In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 0, 1, or 2. In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 1, 2, 3, 4, or 5. 10 In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 1, 2, 3, or 4. In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 1, 2, or 3. In another embodiment , L is -L1-[G-L2]q-G-L3-*, wherein q is 1 or 2. In another embodiment, L is -L1-[G-L2]q-G-L3-*, wherein q is 4. In another embodiment , L is - L1-[G-L2]q-G-L3-*, wherein q is 3. In another embodiment, L is -L1-[G-L2]q-G-L3-*, wherein q is 2. 15 In another embodiment, L is -L1-G-L2-G-L3-*. In another embodiment nts, L is -L1-G-L3-*. In another embodiment, L is -G-L3-*. In another embodiment , L is -L1-G-*. In another embodiment, L is - G-*. In another embodiment, wherein L is -[G-L2]q-G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q is 3; or q is 4; or q is 5). 20 In some embodiments, each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)O-, -OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. In some embodiments, each instance of A-B-A- is independently selected from the group25 consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-,- N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. In some embodiments, each instance of A-B-A- is independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -O-, and - N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. 30 In some embodiments, each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. In some embodiments, each instance of A-B-A- is independently selected from the group consisting of -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen 35 or C1-6alkyl. In some embodiments, 231 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO D and F are each independently a bond, C1-10alkyl, C2-10alkenyl, or C2-10alkynyl, each optionally substituted with 1, 2, 3, or 4 R groups; and E is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. 5 In some embodiments, D and F are each independently a bond or C1-10alkyl optionally substituted with 1, 2, 3, or 4 R groups; and E is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. 10 In some embodiments, each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. In some embodiments, each G is independently C1-10alkyl, optionally substituted with 1, 2, or 3 R groups. 15 In some embodiments, each G is independently C1-10alkyl, optionally substituted with 1 or 2 R groups. In some embodiments, each G is independently C1-10alkyl, optionally substituted with one R group . In some embodiments, each G is independently C1-10alkyl. In some embodiments, L is -L1-G-L3-*, wherein * is the bond to a ZZ group ; 20 G is -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2- 10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; L1 is -B-A-; L3 is a bond or -A-B-A-; 25 each A is independently a bond, -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. each B is independently a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); and In some embodiments, L is -L1-G-L3-*, wherein * is the bond to a ZZ group; G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or 30 heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; L1 is -B-; L3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; and 35 each B is independently a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH; and In some embodiments, L is -L1-G-*, wherein * is the bond to a ZZ group; 232 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; L1 is -B-A-, wherein, A is a bond, -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1- 5 6alkyl; and B is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, L is -L1-G-*, wherein * is the bond to a ZZ group; G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1 or 2 R groups; 10 L1 is -B-A-, wherein A is a bond, -O-, -S-, or -N(RN)-, each RN is independently hydrogen or C1-6alkyl; and B is a bond, C(O), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments L is -L1-G-*, wherein * is the bond to a ZZ group; G is C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups; 15 L1 is bond, C(O), S(O)2, P(O)(OH), or P(S)(OH); and RN is hydrogen or C1-6alkyl. In some embodiments, L is wherein * is the bond to a ZZ group; k is an integer from 1 to 10; L1 is bond, C(O), C(S), C(NRN), S(O)2, P(O)(OH), or P(S)(OH); and RN is hydrogen or C1- 6alkyl. 20 In some embodiments, L is wherein * is the bond to a ZZ group; k is an integer from 1 to 10; L1 is bond, C(O), P(O)(OH), or P(S)(OH). In some embodiments, L is , wherein * is the bond to a ZZ group; k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 3 to 10; or an integer from 4 to 10; or an integer from 5 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7. 25 In some embodiments, L is , wherein * is the bond to a ZZ group; t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3). In some embodiments, 233 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein * is the bond to a ZZ group; t is an integer from 0 to 10 (e.g., an integer from 1 to 5 or 1; or 2; or 3); a is an integer from 1 to 3; and s and s’ are each independently an integer from 1 to 24 (e.g., an integer from 1 to 16; an integer from 1 to 10; an integer from 3 to 10; an integer from 3 to 7; or an integer from 4 to 6). 5 In some embodiments, wherein * is the bond to a ZZ group; a is 1, 2 or 3; and each s, s’, and s” independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6). In some embodiments, L is wherein * is the bond to a ZZ group; and s, s’, and s’’ are independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer10 from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6). In some embodiments, L is , wherein * is the bond to a ZZ group; and each s, s’, and s” independently is an integer from 1 to 24(e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6). In some embodiments, L is wherein * is the bond to ZZ; s and k 15 are independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 10 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6). In some embodiments, L is wherein * is the bond to ZZ and 20 w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6). In some embodiments, L is C1-20 alkyl. In some embodiments, L is C2-20 alkyl. In some embodiments, L is C6-20 alkyl. In some embodiments, L is C8-12 alkyl. In some embodiments, L is C10 alkyl. 234 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, L is -L1-G-*, wherein * is the bond to ZZ , G is C1-10alkyl or C2-10alkenyl, and L1 is -B-A-, wherein, A is a bond, -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; and B is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In some embodiments, L is -L1-G-*, wherein * is the bond to ZZ , G is C1-10alkyl or C2-10alkenyl, 5 L1 is -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. In some embodiments, L is -L1-G-*, wherein * is the bond to ZZ , G is C2-10alkyl, L1 is -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl. In some embodiments, L is -L1-G-*, wherein * is the bond to ZZ , G is C2-10alkyl, L1 is -O- (e.g., G is C3- 10alkyl or C4-10alkyl or C5-10alkyl or C6-10alkyl or C2-8alkyl or C4-8alkyl or C4-8alkyl or C4alkyl or C5alkyl 10 or C6alkyl). In some embodiments, L is L wherein * is the bond to ZZ; L1 is bond; and k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 3 to 10; or an integer from 4 to 10; or an integer from 5 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7. In another embodiment, wherein L is -[G-L2]q-G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q 15 is 3; or q is 4; or q is 5), each G is indepdently C1-10alkyl and L2 is O, S, or N(H). In another embodiment, wherein L is -[G-L2]q-G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q is 3; or q is 4; or q is 5), each G is indepdently C1-10alkyl and L2 is O. In another embodiment, wherein L is -[CH2CH2O]q-G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q is 3; or q is 4; or q is 5), G is indepdently C1-10alkyl. 20 In some embodiments, L is , wherein * is the bond to ZZ; t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3). In some embodiments, L is , wherein * is the bond to ZZ. In some embodiments, L is , wherein * is the bond to ZZ; t is 1 to 10 (e.g., 1-5; or 1-3; or 1; or 2; or 3); and z is 1-10 (e.g., 1-6; or 1-4; or 1; or 2; or 3 or 4 or 5 or 6). 25 In some embodiments, L is , wherein * is the bond to ZZ, and t is an integer from 1 to 5 (e.g., 1; or 2; or 3). In some embodiments, L is , wherein * is the bond to ZZ. In some embodiments, L is -G-L2-G-*, wherein L2 is -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), 30 P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl, or two RN within an -A-B-A- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; 235 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO and each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. In some embodiments, L is -G10-L2-G20-*, wherein L2 is -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, 5 P(O)(OH), P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl, or two RN within an - A-B-A- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; G10 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl; and G20 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. 10 In some embodiments, L is -G10-L2-G20-*, wherein L2 is a bond, -O-, -S-, or -N(RN)-; RN is independently hydrogen or C1-6alkyl, G10 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl; and G20 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. 15 In some embodiments, L is -G10-L2-G20-*, wherein L2 is a bond, -O-, -S-, or -N(RN)-; RN is independently hydrogen or C1-6alkyl, G10 is aryl or heteroaryl; and G20 is C1-10alkyl or C2-10alkenyl. In some embodiments, L is -G10-L2-G20-*, wherein L2 is a bond, -O-, G10 is aryl (e.g., phenyl); and G20 is C1-10alkyl. 20 RL Embodiments, Formulae (XII) and (XII-a) through h) and (XII-r) In some embodiments of any one of Formula (XII) and Formulae (XII-a) through (XII-h) and (XII-r), RL is -N(R3)(R4), wherein R3 is hydrogen or C1-6alkyl and R4 is R5. In some embodiments, RL is -N(R3)(R4), wherein R3 is hydrogen and R4 is R5. In some embodiments, RL is -N(R3)(R4), wherein R3 is C1-3alkyl and R4 is R5. 25 In some embodiments, RL is -N(R3)(R4), wherein R3 is methyl and R4 is R5. In some embodiments, RL is --N(R3)(R4), wherein R3 and R4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R5. In some embodiments, RL is --N(R3)(R4), wherein R3 and R4 taken together with the nitrogen 30 atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R5, provided that R3 and R4 taken together with the nitrogen atom to which they are attached do not form a morpholino group. In some embodiments, RL is --N(R3)(R4), wherein R3 and R4 taken together with the nitrogen atom to which they are attached form a group that is piperidinyl, piperazinyl, pyrrolidinyl, 35 imidazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, azetidinyl, pyrrolinyl, imidazolinyl, or pyrazolinyl, each substituted with R5. 236 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, RL is .In some embodiments, RL is , group; t is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); a is an integer from 1 to 3 and s and s’ are each independently an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 5 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6). In some embodiments, wherein * is a bond to a ZZ group; a is an integer from 1 to 3; and s, s’, and s’’ are each independently an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6). 10 In some embodiments, wherein * is a bond to a ZZ group; and s, s’, and s’’ are independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6. In some embodiments, RL is 15 and s’’ are independently an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and t is an integer from 1 to 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, an integer from 1 to 3, or 1, or 2, or 3). 20 In some embodiments, RL is 237 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , , wherein * is a bond to a ZZ group; s is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6). In some embodiments, RL is -O(R5). 5 In some embodiments, wherein * is a bond to a ZZ group; s is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and t is an integer from 0 to 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, an integer from 1 to 3, or 1, or 2, or 3). In some embodiments, RL is -R5. 10 In some embodiments, RL is , wherein * is a bond to a ZZ group; s is 1 – 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6). In some embodiments, RL is , wherein * is a bond to a ZZ group; t is 0 to 10 (e.g., 1-5; or 1-3; or 1; or 2; or 3). 15 In some embodiments, wherein * is a bond to a ZZ group; s and k are independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 10 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6). 20 In some embodiments, RL is , , , 238 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein * is a bond to a ZZ group and w is an integer selected from 1-10 (e.g., 2-10, 2-8, 4-8). 239 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . In some embodiments, the compound of Formula (XII) is according to one of Formulae (XII-i) through (XII-l) and (XII-s): (XII-s) 5 wherein * is a bond to a ZZ group, p is 0, 1, 2, or 3 (e.g., 0); each R21 is independently selected from group consisting of R and a nitrogen protecting group, and L is defined according to Formula (XII), or according to any of the preceding embodiments of L. 240 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment of Formulae (XII-i) through (XII-l) and (XII-s), R1 is hydrogen. In another embodiment of Formulae (XII-i) through (XII-l) and (XII-s), R1 is C1-6alkyl (e.g., methyl or t-butyl). In some embodiments, the compound of Formula (XII) is according to one of Formulae (XII-m) through (XII-q) and (XII-t): 5 or a salt thereof, wherein * is a bond to a ZZ group, p is 0, 1 or 2; each R21 is independently selected from group consisting of R; RP is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group), and L is defined according to Formula (XII), or according to any of the preceding embodiments 10 of L, wheren R and the remaining variables are as defined in Formula (XII). 241 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, the compound of Formula (XII) is according to one of Formulae (XII-u) through (XII-z): or a salt thereof, wherein * is a bond to a ZZ group, p is 0, 1 or 2; each R21 is independently selected from group consisting of R; 5 RP is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group), and L is defined according to Formula (XII), or according to any of the preceding embodiments of L, wheren R and the remaining variables are as defined in Formula (XII). In one embodiment of Formulae (XII-m) through (XII-q) and (XII-t) through (XII-z), R1 is hydrogen. In another embodiment of Formulae (XII-m) through (XII-q) and (XII-t) through (XII-z), R1 is 10 C1-6alkyl (e.g., methyl or t-butyl). In one embodiment of the compound of Formula (XV), Φ is selected from Formula (XII) and any one of Formula (XII-a) through (XII-q) and (XII-t) through (XII-z); ZZ is -CH2O-, -OCH2-, -S-S-, -C=N-, -C=N-O-, -C=N-N(RN3)-, -N=C-, -O-N=C-, -N(RN3)-N=C-, - 15 C(O)N(RN3)-, -N(RN3)C(O)-, -C(O)O-, -OC(O)-, -OC(O)N(RN3)-, -N(RN3)C(O)O-, -N(RN3)C(O)N(RN3)-, 242 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO -S(O)2N(RN3)-, -N(RN3)S(O)2-, -OP(O)(OH)O-, -OP(S)(OH)O-, -OP(O)(OH)-, -OP(S)(OH)-, - P(O)(OH)O-, or -P(S)(OH)O-. wherein RN3 is independently hydrogen or C1-6alkyl; Δ is selected from, wherein # is the bond to T and each * is a bond to a ZZ group; and 5 , , wherein ** represents the bond to T, and the remaining variables are as defined in Formula (XV), and any embodiment thereof. In one embodiment of the compound of Formula (XV), Φ is selected from Formula (XII) and any one of Formula (XII-a) through (XII-q) and (XII-t) through (XII-z); 10 ZZ is - C(O)N(RN3)-, -N(RN3)C(O)-, -C(O)O-, -OC(O)-, wherein RN3 is independently hydrogen or C1- 6alkyl; Δ is selected from, 243 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein # is the bond to T and each * is a bond to a ZZ group. each G2 is independently C1-10alkyl; and wherein ** represents the bond to T, and the remaining variables are as defined in Formula (XV), and any embodiment thereof. 5 In one embodiment of the compound of Formula (XV), Φ is selected from Formula (XII) and any one of Formula (XII-a) through (XII-q) and (XII-t) through (XII-z); ZZ is -CH2O-, -OCH2-, -S-S-, -C=N-, -C=N-O-, -C=N-N(RN3)-, -N=C-, -O-N=C-, -N(RN3)-N=C-, - C(O)N(RN3)-, -N(RN3)C(O)-, -C(O)O-, -OC(O)-, -OC(O)N(RN3)-, -N(RN3)C(O)O-, -N(RN3)C(O)N(RN3)-,10 -S(O)2N(RN3)-, -N(RN3)S(O)2-, -OP(O)(OH)O-, -OP(S)(OH)O-, -OP(O)(OH)-, -OP(S)(OH)-, - P(O)(OH)O-, or -P(S)(OH)O-. wherein RN3 is independently hydrogen or C1-6alkyl; Δ is selected from, wherein # is the bond to T and each * is a bond to a ZZ group, G3 is C1-10alkyl; 15 each G4 is independently C1-10alkyl; and 244 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , , wherein ** represents the bond to T, and the remaining variables are as defined in Formula (XV), and any embodiment thereof. In one embodiment of the compound of Formula (XV), Φ is selected from Formula (XII) and any one of Formula (XII-a) through (XII-q) and (XII-t) through 5 (XII-z); ZZ is -C(O)-, -C(O)N(RN3)-, -N(RN3)C(O)-, -C(O)O-, -OC(O)-, wherein RN3 is independently hydrogen or C1-6alkyl; Δ is selected from, , , wherein # is the bond to T and each * is a bond to a ZZ group; and 10 and the remaining variables are as defined in Formula (XV), and any embodiment thereof. In one embodiment of the compound of Formula (XV), Φ is selected from Formula (XII) and any one of Formula (XII-i) through (XII-q) and (XII-t) through (XII-z); 245 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -C(O)N(RN3)-, -N(RN3)C(O)-, -C(O)O-, -OC(O)-, wherein RN3 is independently hydrogen or C1-6alkyl; Δ is 5 T is **-L6-G1-L5-G1-L4-, wherein ** is the bond to RT; L4 and L6 are independently -B1-A1- or -A1-B1-; each L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); each G1 is independently C1-10alkyl or C2-10alkenyl; each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or 10 C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and wherein ** represents the bond to T, and the remaining variables are as defined in Formula (XV), and any embodiment thereof. In one embodiment of the compound of Formula (XV), 15 Φ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) through (XII-z); ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -C(O)N(RN3)-, -N(RN3)C(O)-, -C(O)O-, -OC(O)-, wherein RN3 is independently hydrogen or C1-6alkyl; Δ is 246 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO T is **-L6-G1-L5-G1-L4-, wherein ** is the bond to RT; L4 and L6 are independently -B1-A1- or -A1-B1-; each L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); 5 each G1 is independently C1-10alkyl or C2-10alkenyl; each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and wherein ** represents the bond to T, 10 and the remaining variables are as defined in Formula (XV), and any embodiment thereof. In one embodiment of the compound of Formula (XV), Φ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) through (XII-z); ZZ is -C(O)N(H)- or -N(H)C(O)-; 15 T is **-L6-G1-L5-G1-L4-, wherein ** is the bond to RT; L4 is -A1-B1-; L6 is -B1-; 247 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO L5 is a bond, -B1-A1-, or -A1-B1-; each G1 is independently C1-10alkyl; each A1 is independently a bond, -O- or -N(H); each B1 is C(O); and 5 , , wherein ** represents the bond to T, and the remaining variables are as defined in Formula (XV), and any embodiment thereof. In one embodiment of the compound of Formula (XV) is according to the structure, wherein RP3 is hydrogen; 10 Φ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) through (XII-z); ZZ is -C(O)N(H)- or -N(H)C(O)-; and T is **-L6-G1-L5-G1-L4-, wherein ** is the bond to RT; L4 is -A1-B1-; L6 is -B1-; 15 L5 is a bond, -B1-A1-, or -A1-B1-; each G1 is independently C1-10alkyl; each A1 is independently a bond, -O- or -N(H); each B1 is C(O); and the remaining variables are as defined in Formula (XV), and any embodiment thereof. 20 In one embodiment of the compound of Formula (XV), Φ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) through (XII-z); ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -C(O)N(RN3)-, -N(RN3)C(O)-, -C(O)O-, -OC(O)-, wherein RN3 is independently hydrogen or C1-6alkyl; 248 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Δ is T is ***-L6-[G5-O]q5-G5-L4-, wherein q5 is an integer selected from 1 to 20; L4 and L6 are independently -A1-B1-A1-, wherein 5 each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is hydrogen or C1- 6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G5 is independently C1-10alkyl; and wherein ** represents the bond to T, 10 and the remaining variables are as defined in Formula (XV), and any embodiment thereof. In one embodiment of the compound of formula (XV),is of the structure, wherein 15 Φ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) through (XII-z), wherein L is wherein * is the bond to ZZ; s and k are independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 10 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer 20 from 4 to 6); ; 249 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO q5 is an integer selected from 1 to 20; L4 and L6 are independently -A1-B1-A1-, wherein each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is hydrogen or C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), 5 or P(S)(OH); each G5 is independently C1-10alkyl; and the remaining variables are as defined in Formula (XV), and any embodiment thereof. In one embodiment of the compound of formula (XV),is of the structure, 10 wherein Φ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) through (XII-z), wherein L is wherein * is the bond to ZZ and w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6; 15 ZZ is -C(O)N(H)-, -N(H)C(O)-; T is **-C(O)-[CH2CH2-O]q5- C2-10alkyl-C(O)N(H)-, wherein ** is the bond to the pyrrolidine ring and q5 is an integer selected from 1 to 20 (e.g., 1 to 10, or 2 to 10; or 2 – 8; or 1; or 2; or 3; or 4); and the remaining variables are as defined in Formula (XV), and any embodiment thereof. 20 RT1 Embodiments, Formula (XV) In embodiments of any embodiments of RL of Formula (XV), Formula (XV-a) through (XV-k), RT1 is -LL-oligonucleotide, wherein LL is a divalent linker that connects to the 3’-end of the oligonucleotide, the 5’-end of the oligonucleotide, or an internal 2’- or 3’ position on a internal nucleotide (i.e., a nucleotide that is not the 5’-terminal or 3’-terminal nucleoside). 25 In some embodiments, RT1 is -LL-oligonucleotide, wherein LL is a divalent linker that connects to the 3’-end of the oligonucleotide, such as one of : directly to the 3’-carbon of the 3’-terminal nucleoside; directly to the 3’-O of the 3’-terminal nucleoside; directly to the 4’-carbon of the 3’-terminal nucleoside; 250 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO directly to the 2’-carbon of the 3’-terminal nucleoside; or directly to the 2’-O of the 3’-terminal nucleoside. In some embodiments, RT1 is -LL-oligonucleotide, wherein LL is a divalent linker that connects to the 5'-end of the oligonucleotide, such as one of: 5 directly to the 5’-carbon of the 5’-terminal nucleoside; directly to the 5’-O of the 5’-terminal nucleoside; directly to the 4’-carbon of the 5’-terminal nucleoside; directly to the 2’-carbon of the 5’-terminal nucleoside; or directly to the 2’-O of the 5’-terminal nucleoside. 10 In some embodiments, RT1 is -LL-oligonucleotide, wherein LL is a divalent linker that connects to an internal 2’- or 3’ position on an internal nucleotide. In some embodiments, RT1 is -LL-oligonucleotide, wherein LL is a divalent linker that connects to an internal 2’- position on a internal nucleotide. In another embodiment of any of the preceding embodiments of RT1, when LL connects to a 15 carbon aton on a nucleoside, then LL is -B3-A3-, wherein B3 is -P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH)-; and A3 is -O-, -S-, or -N(H)- . In another embodiment, when LL connects to a carbon atom on a nucleoside, then LL is -B3-A3-, wherein B3 is -P(O)(OH)- or-P(S)(OH)-; and A3 is -O-. In another embodiment, when LL connects to a oxygen atom on a nucleoside, then LL is - 20 P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH). In another embodiment, when LL connects to a oxygen atom on a nucleoside, then LL is - P(O)(OH)- or -P(S)(OH)-. In another embodiment, when LL connects to a oxygen atom on a nucleoside, then LL is - P(O)(OH)-. 25 In another embodiment, when LL connects to a oxygen atom on a nucleoside, then LL is - P(S)(OH)-. In another embodiment, when LL connects to a oxygen atom on a nucleoside, then LL is -B3-A3-LL1-A3-B3-, wherein B3 is a bond, -C(O)-, C(S)-, C(NH), S(O), S(O)2, -P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH); A3 is a bond, - 30 O-, -S-, or -N(H)- ; and LL1 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl. In another embodiment, when LL connects to a oxygen atom on a nucleoside, then LL is -B3-LL1- B3-, wherein B3 is -C(O)-; and LL1 is C1-10alkyl. In certain embodiments, where RT is RT1,wherein RT1 is -LL-oligonucleotide, when LL connects to 35 a oxygen atom on a nucleoside, and LL is a bond, the nucleoside is of Formula (XV-f), 251 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); T is according any of the preceding embodiments; and * represent the bond to ZZ. In some embodiments of Formula (XV-f), -T- is -L1-G-L3-*, wherein * is the bond to ZZ. 5 In some embodiments, -T- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one or two R groups, and * is the bond to ZZ. In some embodiments, -T- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one or two groups selected from the group consisting of halogen, hydroxy, C1- 6alkoxy, amino, Cl-6alkylamino, di(C1-6alkylamino), cyano, carboxy , and * is the bond to ZZ. In some embodiments, -T- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one group selected from 10 the group consisting of halogen, hydroxy, C1-6alkoxy, amino, Cl-6alkylamino, di(C1-6alkylamino), cyano, carboxy , and * is the bond to ZZ. In some embodiments, -T- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one group selected from the group consisting of hydroxy, amino, and carboxy , and * is the bond to ZZ. In some embodiments, -T- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with hydroxy, and * is the bond to ZZ. 15 In some embodiments, -T- is -C2-30alkyl-*, wherein * is the bond to ZZ. In some embodiments, - T- is -C2-16alkyl-*, wherein * is the bond to ZZ. In some embodiments, -T- is -C4-12alkyl-*, wherein * is the bond to ZZ. In some embodiments, -T- is -C4-10alkyl-*, wherein * is the bond to ZZ. In some embodiments, -T- is -C5-10alkyl-*, wherein * is the bond to ZZ. In some embodiments, -T- is -C6alkyl-*, wherein * is the bond to ZZ. In some embodiments, -T- is -C8alkyl-*, wherein * is the bond to ZZ. In some embodiments, 20 -T- is -C10alkyl-*, wherein * is the bond to ZZ. In some embodiments of Formula (XV-f), -T- is -L1-[G-L2]q-G-L3-*, wherein * is the bond to ZZ; q is 0, 1, 2, 3, 4, or 5; L1 is a bond or -B-A-; 25 each L2 is independently -A-B-A-; L3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O)2, P(O)(OH), or P(S)(OH); each RN is independently hydrogen or C1-6alkyl; and 30 each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. 252 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, -T- is -L1-[G-L2]q-G-*, wherein * is the bond to ZZ, q is 0, 1, 2, or 3; L1 is a bond or -B-A-; and (a) each L2 is independently a bond, C(O)O, OC(O), C(O)(NRN), N(RN)C(O), SO2N(RN), N(RN)SO2, OP(O)(OH), OP(S)(OH), P(O)(OH)O, P(S)(OH)O, OP(O)(OH)O, or OP(S)(OH)O, wherein each 5 RN is independently hydrogen or C1-6alkyl; each G is independently C1-10alkyl, C2-10alkenyl, C2- 10alkynyl, each of which is optionally substituted with 1 or 2 R groups; or (b) each L2 is independently a bond, C(O)O, OC(O), C(O)(NRN), N(RN)C(O), OP(O)(OH)O, or OP(S)(OH)O, wherein each RN is independently hydrogen or C1-6alkyl; and each G is 10 independently C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups. In some embodiments, -T- is -[G-L2]q-G-*, wherein * is the bond to ZZ, q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each G is independently C1-10alkyl, each of which is optionally 15 substituted with 1 or 2 R groups.and (c) each L2 is independently C(O)O or OC(O); (d) each L2 is independently C(O)(NRN) or N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl (e) each L2 is independently OP(O)(OH)O, or OP(S)(OH)O (e.g., each is OP(O)(OH)O); 20 or (f) each L2 is a bond. In some embodiments, the compound of Formula (XV) is according to one of Formulae (XV-g) through (XV-q): 253 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); 5 each m is independently integer selected from 1-20 (e.g., 2-12, or 2-10; or 2-6; or 2; or 3; or 4; or 5; or 6).z 254 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO or a salt thereof, wherein x is an integer selected from 2 to 8, L and ZZ are as defined in Formula (XV) or in any embodiment preceding or below; 5 B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); each m is independently integer selected from 1-20 (e.g., 2-12, or 2-10; or 2-6; or 2; or 3; or 4; or 5; or 6); 10 each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); RP is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group); and 255 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO R1 is hydrogen or C1-6alkyl (e.g., methyl or t-butyl). In one embodiment of Formulae (XV-r) through (XV-w), R1 is hydrogen. In another embodiment of Formulae (XV-r) through (XV-w), R1 is C1-6alkyl (e.g., methyl or t-butyl). In another 5 embodiment of Formulae (XV-r) through (XV-w), R1 is hydrogen and RP is hydrogen. In another embodiment of Formulae (XV-r) through (XV-w), R1 is hydrogen and a nitrogen protecting group. In another embodiment of Formulae (XV-r) through (XV-w), R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In another embodiment of Formulae (XV-r) through (XV-w), R1 is C1-6alkyl (e.g., methyl or t-butyl) and a nitrogen protecting group. 10 In certain embodiments, where RT is RT1 , wherein RT1 is -LL-oligonucleotide, when LL connects to a oxygen atom or nitrogen atom in an internucleotide linkage, the internucleotide linkage can be of the formula, including the 3’ and 5’ oxygen atoms of the preceding and following nucelosides, respectively, (XV-pa) (XV-pb) pc) wherein T can be, for example a bond, -S(O)2- or, in for Formula ( membered heterocyclyl ring optionally substituted with 1 or 2 R groups, as defined herein ; and * represent the bond to Δ. 15 For example, the preceding includes, wherein * represent the bond to Δ; and RN5 is hydrogen or C1-10 alkyl. For example, the preceding includes, (XV-pg) (XV-ph) (XV-pi) wherein * represent the bond to Δ; m is an integer selected from 1 – 20 (e.g., 1-10, or 2-20, or 2-10, or 4- 10, or 4-8; or 6-12; or 5; or 6; or 7; or 8; or 9; or 10), and RN5 is hydrogen or C1-10 alkyl. 20 In another embodiment, the compound of Formula (XV-pd) is 256 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 x is an integer selected from 2 to 8, m is an integer selected from 1 – 10; n is an integer selected from 1-6; and RY, Y, R1, RP, L, T, Δ, and ZZ are as defined for Formula (XV). In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t-butyl). In another embodiment, the compound is 257 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Y' 5 258 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each m is an integer selected from 1 – 20 (e.g., 2-20, 2-10, 1-10, 2-16, 4-16, 4-8, or 6-12), n is an integer selected from 1-6; x is an integer selected from 2 to 8, Y’ is O or S, and R1, Δ, RP and ZZ are as defined for Formula (XV). In another embodiment, the compound of Formula (XV-pe) is 5 , 259 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO x is an integer selected from 2 to 8, m is an integer selected from 1 – 10; n is an integer selected from 1-6; and RY, Y, R1, L, T , Δ, and ZZ are as defined for Formula (XV). In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- 5 butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S 10 and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and RP is hydrogen . In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 15 (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), 20 benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen . In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen . In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen 25 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, the compound is 260 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 261 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 4-16, 4-8, or 6-12), n is an integer selected from 1-6; Y’ is O or S, x is an integer selected from 2 to 8, and R1, Δ, RP, and ZZ are as defined for Formula (XV). 5 In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 10 In one embodiment, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 15 (pac)). In one embodiment, Y’ is S and RP is hydrogen . In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen 20 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen . In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- butyl) and RP is hydrogen . In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is 25 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 262 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In another embodiment, when LL connects to a oxygen atom on a nucleoside, then LL is-a bond. For Formula (XV), when RT1 is -LL-oligonucleotide and is conjugated at the 5’-end of the 5 oligonucleotide, in one embodiment, RT1 can be represented as Formula (XV-5’) wherein LL is -P(Y)(OH)-, wherein Y is O or S (e.g., S); and * represents the bond to remainder of the compound of Formula (XV). In another embodiment of Formula (XV), when RT1 is -LL-oligonucleotide and is conjugated at 10 the 5’-end of the oligonucleotide, in one embodiment, RT1 can be represented as Formula (XV-5’o) or Formula (XV-5’s), wherein * represents the bond to remainder of the compound of Formula (XV). In another embodiment, a compound of Formula (XV) can be represented by, 15 , , 263 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO ed from 2 to 8, m is an integer selected from 1-10; n is an integer selected from 1-6; and Δ, RY, Y, R1, L, T, and ZZ are as defined for Formula (XV) or any embodiment thereof. 5 For Formula (XV), when RT1 is -LL-oligonucleotide and is conjugated at the 3’-end of the oligonucleotide, in one embodiment, RT1 can be represented as Formula (XV-3’) wherein LL is -P(Y)(OH)-, wherein Y is O or S (e.g., S); and * represents the bond to remainder of the compound of Formula (XV). 10 In another embodiment of Formula (XV), when RT1 is -LL-oligonucleotide and is conjugated at the 3’-end of the oligonucleotide, in one embodiment, RT1 can be represented as Formula (XV-3’o) or Formula (XV-3’s), wherein * represents the bond to remainder of the compound of Formula (XV). 15 In another embodiment, a compound of Formula (XV) can be represented by, , 264 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 integer selected from 2 to 8, m is an integer selected from 1-10; n is an integer selected from 1-6; and Δ, RY, Y, R1, L, T, and ZZ are as defined for Formula (XV) or any embodiment thereof. In one embodiment , R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t-butyl). In another embodiment, the compound of Formula (XV) is 10 wherein Y’ is O or S; each ZZ is N(H)C(O) or C(O)N(H); 265 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each wherein m is an integer selected from 1 – 10; n in an integer selected from 1 – 6; L4 and L6 are independently -B1-A1- or -A1-B1-; each L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); 5 each G1 is independently C1-10alkyl or C2-10alkenyl; each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). 10 In another embodiment of Formula (XV-x), each m is an integer selected from 1 – 10. In another embodiment of Formula (XV-x), each wherein m is an integer selected from 1 – 10; 266 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In another embodiment of Formula (XV-x), each wherein m is an integer selected from 1 – 10; In another embodiment of Formula (XV-x), each wherein m is 5 an integer selected from 1 – 10. In another embodiment of Formula (XV-x), each wherein m is an integer selected from 1 – 10. wherein m is an integer selected from 1 – 10; and n is an integer selsected from 1 – 6. 10 267 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein m is an integer selected from 1 – 10; and n is an integer selsected from 1 – 6. In another embodiment of Formula (XV-x), each . 5 In another embodiment of Formula (XV-x), each . In another embodiment of Formula (XV-x) and each of the preceding embodiments thereof, L4 and L6 are independently -B1-A1- or -A1-B1-; L5 is a bond, -B1-A1- or -A1-B1-; each G1 is independently C1-10alkyl; each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is 10 independently hydrogen or C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In another embodiment of Formula (XV-x) and each of the preceding embodiments thereof, L4 is -B1-A1- or -A1-B1-; L6 is B1; L5 is a bond, -B1-A1- or -A1-B1-; each G1 is independently C1- 10alkyl; each A1 is independently -O- or -N(H)-, and each B1 is independently C(O), S(O)2,15 P(O)(OH), or P(S)(OH). In another embodiment of Formula (XV-x) and each of the preceding embodiments thereof, -L4-G1-L5-G1-L4- is -B1-C1-10alkyl-L5-C1-10alkyl-B1-A1-; L5 is a bond, -B1-A1- or -A1-B1-; each A1 is 268 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO independently -O- or -N(H)-, and each B1 is independently C(O), S(O)2, P(O)(OH), or P(S)(OH). In another embodiment of Formula (XV-x) and each of the preceding embodiments thereof, -L4-G1-L5-G1-L4- is -C(O)-C1-10alkyl-L5-C1-10alkyl-C(O)N(H)-, wherein L5 is a bond, -B1-A1- or -A1-B1-, 5 wherein A1 is -O- or -N(H)-, and B1 is C(O), S(O)2, P(O)(OH), or P(S)(OH). In another embodiment of Formula (XV-x) and each of the preceding embodiments thereof, -L4-G1-L5-G1-L4- is -C(O)-C2-20alkyl-C(O)N(H)- (e.g., -C(O)-C6-12alkyl-C(O)N(H)-, or -C(O)-C8-12alkyl- C(O)N(H)-, or -C(O)-C10alkyl-C(O)N(H)-). In another embodiment, the compound of Formula (XV) is 10 is an integer selected from 1 – 10; 15 n in an integer selected from 1 – 6; L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); each G1 is independently C1-10alkyl; each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl; and 20 each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In another embodiment of Formula (XV-x1), each Φ is 269 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein m is an integer selected from 1 – 10; R1 is hydrogen or C1-6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen). In another embodiment of Formula (XV-x1), each Φ is 5 , wherein m is an integer selected from 1 – 10; R1 is hydrogen or C1-6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen).. In another embodiment of Formula (XV-x1), each 10 wherein m is an integer selected from 1 – 10; R1 is hydrogen or C1-6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen). 270 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO hydrogen or a nitrogen protecting group (e.g., hydrogen). In another embodiment of Formula (XV-x1), each Φ is 5 hydrogen).. 10 In another embodiment of Formula (XV-x1), each , wherein m is an integer selected from 1 – 10; n is an integer selsected from 1 – 6; R1 is hydrogen or C1- 6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen).. 271 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In another embodiment of Formula (XV-x1), each , wherein m is an integer selected from 1 – 10; n is an integer selsected from 1 – 6; R1 is hydrogen or C1- 6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen). 5 In another embodiment of Formula (XV-x1), each , wherein R1 is hydrogen or C1-6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen).. In another embodiment of Formula (XV-x1), each , 10 wherein R1 is hydrogen or C1-6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen). In another embodiment, the compound of Formula (XV) is 272 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S; each ZZ is N(H)C(O) or C(O)N(H); each 5 is an integer selected from 1 – 10; n is an integer selected from 1-6; L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); each G1 is independently C1-10alkyl; each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or 10 C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). In another embodiment of Formula (XV-x2), each Φ is , wherein m is an integer selected from 1 – 10; R1 is hydrogen or C1-6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., 15 hydrogen). In another embodiment of Formula (XV-x2), each Φ is 273 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein m is an integer selected from 1 – 10; R1 is hydrogen or C1-6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen). 5 In another embodiment of Formula (XV-x2), each wherein m is an integer selected from 1 – 10; R1 is hydrogen or C1-6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen). 10 nitrogen protecting group (e.g., hydrogen).. In another embodiment of Formula (XV-x2), each Φ is 274 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein m is an integer selected from 1 – 10; R1 is hydrogen or C1-6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen).. In another embodiment of Formula (XV-x2), each , 5 wherein m is an integer selected from 1 – 10; n is an integer selsected from 1 – 6; R1 is hydrogen or C1- 6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen). In another embodiment of Formula (XV-x2), each , wherein m is an integer selected from 1 – 10; and n is an integer selsected from 1 – 6; R1 is hydrogen or10 C1-6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen). 275 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In another embodiment of Formula (XV-x2), each , wherein R1 is hydrogen or C1-6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen). 5 In another embodiment of Formula (XV-x2), each , wherein R1 is hydrogen or C1-6alkyl (e.g., hydrogen); and RP is hydrogen or a nitrogen protecting group (e.g., hydrogen). In one embodiment of Formula (XV-x), (XV-x1), and (XV-x2), and each of the preceding 10 embodiments thereof, Y’ is O. In another embodiment, Y’ is S. In one embodiment of Formula (XV-x), (XV-x1), and (XV-x2), and each of the preceding embodiments thereof, R1 is hydrogen. In another embodiment, R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment of Formula (XV-x), (XV-x1), and (XV-x2), and each of the preceding embodiments thereof, RP is hydrogen. In another embodiment, RP is a nitrogen protecting group (e.g., t- 15 butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment of Formula (XV-x), (XV-x1), and (XV-x2), and each of the preceding embodiments thereof, Y’ is O and R1 is hydrogen. In one embodiment, Y’ is O and R1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R1 is hydrogen. In one embodiment, Y’ is S and R1 is C1-6alkyl (e.g., methyl or t-butyl). 20 In one embodiment of Formula (XV-x), (XV-x1), and (XV-x2), and each of the preceding embodiments thereof, Y’ is O and RP is hydrogen . In one embodiment, Y’ is O and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one 276 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO embodiment, Y’ is S and RP is hydrogen. In one embodiment, Y’ is S and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment of Formula (XV-x), (XV-x1), and (XV-x2), and each of the preceding embodiments thereof ,Y’ is O, R1 is hydrogen and RP is hydrogen. In one embodiment, Y’ is S, R1 is 5 hydrogen and RP is hydrogen. In one embodiment, Y’ is O, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is hydrogen and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is hydrogen. In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t- 10 butyl) and RP is hydrogen. In one embodiment, Y’ is O, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R1 is C1-6alkyl (e.g., methyl or t-butyl) and RP is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In other embodiments of Formula (XV), the compound is of the structure: 15 277 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In other embodiments of Formula (XV), the compound is of the structure: 5 In other embodiments of Formula (XV), the compound is of the structure: 278 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . 5 In some embodiments of Formula (XV), the compound is of the structure: , 279 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein * represents the bond to the remainder of the dsRNA agent. In another embodiment, the compound of Formula (XV) is 5 wherein * represents the bond to the remainder of the dsRNA agent. 280 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In another embodiment, the compound of Formula (XV) is, 5 wherein * represents the bond to the remainder of the dsRNA agent. In another embodiment, the compound of Formula (XV) is, 10 wherein * represents the bond to the remainder of the dsRNA agent. 281 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In another embodiment, the compound of Formula (XV) is, 5 wherein * represents the bond to the remainder of the dsRNA agent. In another embodiment, the compound of Formula (XV) is, wherein B is an optionally modified nucleobase (e.g., cytosine, uracil, 5-methylcytosine, 5- methyluridine); and 282 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein * represents the bond to the remainder of the dsRNA agent. 5 III. iRNA Agents of the Disclosure Described herein are iRNA agents that inhibit the expression of a target gene in extrahepatic tissue, e.g., muscle tissue, e.g., skeletal muscle tissue and/or cardiac muscle tissue. In one embodiment, the iRNA agent includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a target gene in a skeletal and/or cardiac muscle cell or tissue, such as a cell or tissue within 10 a subject, e.g., a mammal, such as a human having a muscle disorder or disease, e.g., a skeletal and/or cardiac disorder or disease. Any target gene can be inhibited by the iRNA agents provided herein. In one embodiment, the target gene is any gene involved in a muscle disorder or disease, e.g., a skeletal and/or cardiac muscle disorder or disease. Non-limiting examples of skeletal and/or cardiac muscle target genes include any genes involved 15 in a skeletal muscle and/or a cardiac muscle disorder or disease, such as, for example, adrenoceptor beta 1 (ADRB1); calcium voltage-gated channel subunit alpha1 C (CACNA1C); calcium voltage-gated channel subunit alpha1 G (CACNA1G) (T type calcium cchannel); angiotensin II receptor type 1(AGTR1); Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1 (HCN1); Hyperpolarization Activated Cyclic Nucleotide 20 Gated Potassium Channel 4 (HCN4); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3 (HCN3); Potassium Voltage-Gated Channel Subfamily A Member 5 (KCNA5); Potassium Inwardly Rectifying Channel Subfamily J Member 3 (KCNJ3); Potassium Inwardly Rectifying Channel Subfamily J Member 4 (KCNJ4); phospholamban (PLN); calcium/calmodulin dependent protein kinase II delta (CAMK2D); Phosphodiesterase 1 (PDE1), myostatin (MSTN); Cholinergic Receptor Nicotinic 25 Alpha 1 Subunit (CHRNA1); Cholinergic Receptor Nicotinic Beta 1 Subunit (CHRNB1); Cholinergic Receptor Nicotinic Delta Subunit (CHRND); Cholinergic Receptor Nicotinic Epsilon Subunit (CHRNE); Cholinergic Receptor Nicotinic Gamma Subunit (CHRNG); Collagen Type XIII Alpha 1 Chain 283 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (COL13A1); Docking Protein 7 (DOK7); LDL Receptor Related Protein 4 (LRP4); Muscle Associated Receptor Tyrosine Kinase (MUSK); Receptor Associated Protein Of The Synapse (RAPSN); Sodium Voltage-Gated Channel Alpha Subunit 4 (SCN4A); Double Homeobox 4 (DUX4), dystrophy myotonic protein kinase (DMPK), glycogen synthase 1 (GYS1), survival of motor neuron 1 (SMN1), and alpha- 5 glucosidase (GAA). The iRNA agents provided herein comprise a sense strand and an antisense strand and at least one of the strands is modified for targeting delivery to extrahepatic tissue, e.g., muscle tissue, e.g., skeletal muscle tissue and/or cardiac muscle tissue. In one embodiment, the iRNA agents are modified by conjugation to at least one alpha-v-beta-6 (αvβ6) integrin ligand and conjugation to at least one in vivo 10 delivery enhancing moiety. By “integrin ligand” is meant any ligand that binds to an integrin or an integrin receptor. Integrin ligands comprise binding sequences that are recognized and bound by integrins or ntegrin receptors. Various ligands, including peptides and small molecules, which bind αvβ6 integrins or αvβ6 integrin receptors (αvβ6 integrin targeting ligands) have been designed and synthesized herein and in, e.g., U.S. 15 Patent 6,410,526, the entire contents of which are incorporated herein by reference. An exemplary linker to conjugate a αvβ6 integrin targeting ligand to the dsRNA agent is: 284 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 , 285 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, at least one of the strands of the iRNA agent is conjugated to at least one αvβ6 integrin targeting ligand and at least one in vivo delivery enhancing moiety. For example, the iRNA agent may be conjugated to one, two, three, four, or more αvβ6 integrin targeting ligands and one, 5 two, three, four or more in vivo delivery enhancing moieties. In some embodiments, the αvβ6 integrin targeting ligand is conjugated to the sense strand. The αvβ6 integrin targeting ligand may be conjugated to an extrernal position of sense strand, e.g., the 3’-end of the sense strand, to the 5’-end of the sense strand, or to both the 5-end and the 3’-end of the sense strand. In some embodiments, the αvβ6 integrin targeting ligand may be conjugated to an internal 10 position of the sense strand. In other embodiments, the αvβ6 integrin targeting ligand is conjugated to the antisense strand. The αvβ6 integrin targeting ligand may be conjugated to an external position of the antisense strand, e.g., the 3’-end of the antisense strand, to the 5’-end of the antisense strand, or to both the 5-end and the 3’- end of the antisense strand. In some embodiments, the αvβ6 integrin targeting ligand may be conjugated 15 to an internal position of the antisense strand. In some embodiments, the in vivo delivery enhancing moiety is conjugated to the sense strand. The in vivo delivery enhancing moiety may be conjugated to an extrernal position of sense strand, e.g., the 3’-end of the sense strand, to the 5’-end of the sense strand, or to both the 5-end and the 3’-end of the sense strand. In some embodiments, the in vivo delivery enhancing moiety may be conjugated to an 20 internal position of the sense strand. In some embodiments, the in vivo delivery enhancing moiety is not conjugated to an external position of the sense strand. In other embodiments, the αvβ6 integrin targeting ligand is conjugated to the antisense strand. The in vivo delivery enhancing moiety may be conjugated to an external position of the antisense strand, e.g., the 3’-end of the antisense strand, to the 5’-end of the antisense strand, or to both the 5-end and the 25 3’-end of the antisense strand. In some embodiments, the in vivo delivery enhancing moiety may be conjugated to an internal position of the antisense strand. In some embodiments, the in vivo delivery enhancing moiety is not conjugated to an external position of the antisense strand. In some embodiments, the αvβ6 integrin targeting ligand and the in vivo delivery enhancing moiety are both conjugated to the sense strand of the dsRNA agent. For example, the αvβ6 integrin 30 targeting ligand is conjugated to an external position of the sense strand, e.g., the 3’-end of the sense strand or the 5’-end of the sense strand, and the in vivo delivery enhancing moiety is conjugated to an internal position of the sense strand. In some embodiments, the αvβ6 integrin targeting ligand and the in vivo delivery enhancing moiety are both conjugated to the antisense strand of the dsRNA agent. For example, the αvβ6 integrin 286 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO targeting ligand is conjugated to an external position of the antisense strand, e.g., the 3’-end of the sense strand or the 5’-end of the antisense strand, and the in vivo delivery enhancing moiety is conjugated to an internal position of the antisense strand. In some embodiments, the αvβ6 integrin targeting ligand is conjugated to a sense strand, and the 5 in vivo delivery enhancing moiety is conjugated to an antisense strand. In other embodiments, the αvβ6 integrin targeting ligand is conjugated to an antisense strand, and the in vivo delivery enhancing moiety is conjugated to a sense strand. The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a target gene. The region of 10 complementarity is about 15-30 nucleotides or less in length. Upon contact with a cell expressing the target gene, the RNAi agent inhibits the expression of the target gene (e.g., a human gene, a primate gene, a non-primate gene) by at least 30% as compared to a similar cell not contacted with the RNAi agent or an RNAi agent not complementary to the target gene. Expression of the gene may be assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as 15 by immunofluorescence analysis, using, for example, western blotting or flowcytometric techniques. A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, or fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA 20 formed during the expression of a target gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides. 25 Generally, the duplex structure is 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain embodiments, the duplex structure is 30 18 to 25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24 or 24-25 base pairs in length, for example, 19-21 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure. Similarly, the region of complementarity to the target sequence is 15 to 30 nucleotides in length, 35 e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, for 287 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO example 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure. In some embodiments, the duplex structure is 19 to 30 base pairs in length. Similarly, the region of complementarity to the target sequence is 19 to 30 nucleotides in length. 5 In some embodiments, the dsRNA is 15 to 23 nucleotides in length, 19 to 23 nucleotides in length, or 25 to 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an 10 mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 15 to 36 base pairs, e.g., 15-36, 15-35, 15-34, 15-33, 15-32, 15- 15 31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, for example, 19-21 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional 20 duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an RNAi agent useful to target expression or a target gene is not generated in the target cell by cleavage of a larger dsRNA. 25 A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5'-end, 3'-end or both ends of either an antisense or sense strand of a 30 dsRNA. A dsRNA can be synthesized by standard methods known in the art. Double stranded RNAi compounds of the disclosure may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the dsRNA compound can be prepared using solution-phase or solid-phase 35 organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Similarly, single-stranded oligonucleotides of the disclosure can be prepared using solution-phase or solid-phase organic synthesis or both. 288 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one aspect, a dsRNA of the disclosure includes at least two nucleotide sequences, a sense sequence and an antisense sequence. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of a target gene. As such, in this aspect, a dsRNA will include 5 two oligonucleotides, where one oligonucleotide is described as the sense strand (passenger strand), and the second oligonucleotide is described as the corresponding antisense strand (guide strand). In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide. 10 It will be understood that, although the sequences provided herein may be described as modified or conjugated sequences, the RNA of the RNAi agent of the disclosure e.g., a dsRNA of the disclosure, may comprise any one of the sequences set forth herein that is un-modified, un-conjugated, or modified or conjugated differently than described therein. The skilled person is well aware that dsRNAs having a duplex structure of about 20 to 23 base 15 pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided herein, dsRNAs described herein can include at least one strand of a 20 length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein, and differing in their ability to inhibit the expression of a target gene by not more than 10, 15, 20, 25, 30, 35, 40, 45 or 50 % inhibition from a dsRNA 25 comprising the full sequence using the in vitro assay with, e.g., skeletal and/or cardiac muscle cells and a 10 nM concentration of the RNA agent and the PCR assay as provided in the examples herein, are contemplated to be within the scope of the present disclosure. In addition, the RNA agents described herein identify a site(s) in a target gene mRNA transcript that is susceptible to RISC-mediated cleavage. As such, the present disclosure further features RNAi 30 agents that target within this site(s). As used herein, an RNAi agent is said to “target within” a particular site of an mRNA transcript if the RNAi agent promotes cleavage of the mRNA transcript anywhere within that particular site. Such an RNAi agent will generally include at least about 15 contiguous nucleotides, preferably at least 19 nucleotides, from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a target 35 gene. III. Modifications of the RNAi Agents of the Disclosure 289 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In one embodiment, the RNA of an RNAi agent of the disclosure, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the disclosure, substantially all of the nucleotides of an RNAi agent of the disclosure are modified. In other embodiments of the disclosure, all of the nucleotides of an RNAi agent of the disclosure are modified. 5 RNAi agents of the disclosure in which “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or unmodified nucleotides. In still other embodiments of the disclosure, RNAi agents of the disclosure can include not more than 5, 4, 3, 2 or 1 modified nucleotides. The nucleic acids featured in the disclosure can be synthesized or modified by methods well 10 established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5’-end modifications (phosphorylation, conjugation, inverted linkages) or 3’-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, 15 destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2’-position or 4’-position) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or no natural internucleoside 20 linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified RNAi agent will have a phosphorus atom in its internucleoside backbone. 25 Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' 30 linkages, 2'-5'-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. In some embodiments of the disclosure, the dsRNA agents of the disclosure are in a free acid form. In other embodiments of the disclosure, the dsRNA agents of the disclosure are in a salt form. In one embodiment, the dsRNA agents of the disclosure are in a sodium salt form. In certain 35 embodiments, when the dsRNA agents of the disclosure are in the sodium salt form, sodium ions are present in the agent as counterions for substantially all of the phosphodiester and/or phosphorothioate groups present in the agent. Agents in which substantially all of the phosphodiester and/or phosphorothioate linkages have a sodium counterion include not more than 5, 4, 3, 2, or 1 phosphodiester 290 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO and/or phosphorothioate linkages without a sodium counterion. In some embodiments, when the dsRNA agents of the disclosure are in the sodium salt form, sodium ions are present in the agent as counterions for all of the phosphodiester and/or phosphorothioate groups present in the agent. Representative U.S. patents that teach the preparation of the above phosphorus-containing 5 linkages include, but are not limited to, U.S. Patent Nos.3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6, 239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 10 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and US Pat RE39464, the entire contents of each of which are hereby incorporated herein by reference. Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic 15 internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. 20 Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference. 25 In other embodiments, suitable RNA mimetics are contemplated for use in RNAi agents, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with alternate groups. The nucleobase units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, a RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA 30 compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos.5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. 35 Additional PNA compounds suitable for use in the RNAi agents of the disclosure are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500. Some embodiments featured in the disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH2--NH--CH2-, --CH2--N(CH3)-- 291 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO O--CH2--[known as a methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2-- N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- of the above-referenced U.S. Patent No.5,489,677, and the amide backbones of the above-referenced U.S. Patent No.5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced US5,034,506. The 5 native phosphodiester backbone can be represented as -O-P(O)(OH)-OCH2-. Modified RNAs can also contain one or more substituted sugar moieties. The RNAi agents, e.g., dsRNAs, featured herein can include one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O- , S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable 10 modifications include O[(CH2)nO] mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2) nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2' position: C1 to C10 alkyl, substituted alkyl, alkaryl, aralkyl, O- alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA 15 cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an RNAi agent, or a group for improving the pharmacodynamic properties of an RNAi agent, and other substituents having similar properties. In some embodiments, the modification includes a 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'- 20 dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O- dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH3)2. Further exemplary modifications include: 5’-Me-2’-F nucleotides, 5’-Me-2’-OMe nucleotides, 5’-Me-2’-deoxynucleotides, (both R and S isomers in these three families); 2’-alkoxyalkyl; and 2’-NMA (N-methylacetamide). 25 Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2’- O-hexadecyl, and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an RNAi agent, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. RNAi agents can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the 30 preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference. 35 An RNAi agent of the disclosure can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5- 292 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8- 5 thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5- bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include those disclosed in U.S. Pat. No.3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. 10 ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these modified nucleobases are particularly useful for increasing the binding 15 affinity of the oligomeric compounds featured in the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278) and are exemplary base 20 substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Patent Nos.3,687,808, 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 25 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference. An RNAi agent of the disclosure can also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A “bicyclic 30 nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the disclosure may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' 35 and 4' carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et 293 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the disclosure include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the disclosure include one or more 5 bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH2)—O-2′ (LNA); 4′-(CH2)—S-2′; 4′-(CH2)2—O-2′ (ENA); 4′- CH(CH3)—O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)—O-2′ (and analogs thereof; see, e.g., U.S. Pat. No.7,399,845); 4′-C(CH3)(CH3)—O-2′ (and analogs thereof; see e.g., US Patent No.8,278,283); 4′-CH2—N(OCH3)-2′ (and analogs thereof; see e.g., US Patent No. 10 8,278,425); 4′-CH2—O—N(CH3)-2′ (see, e.g., U.S. Patent Publication No.2004/0171570); 4′-CH2— N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No.7,427,672); 4′- CH2—C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2— C(═CH2)-2′ (and analogs thereof; see, e.g., US Patent No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference. 15 Additional representative US Patents and US Patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: US Patent Nos.6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated 20 herein by reference. Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226). An RNAi agent of the disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid comprising 25 a bicyclic sugar moiety comprising a 4'-CH(CH3)-O-2' bridge. In one embodiment, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.” An RNAi agent of the disclosure may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2’and C4’ carbons of ribose or the C3 and -C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and 30 increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering. Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US 2013/0190383; and WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference. 35 In some embodiments, an RNAi agent of the disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked "sugar" residue. In one example, UNA also encompasses monomer with bonds between C1'-C4' have been removed (i.e. the covalent carbon-oxygen- 294 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond (i.e. the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference). 5 Representative U.S. publications that teach the preparation of UNA include, but are not limited to, US8,314,227; and US Patent Publication Nos.2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference. An RNAi agent of the disclosure may also include one or more “cyclohexene nucleic acids” or (“CeNA”). CeNA are nucleotide analogs with a replacement of the furanose moiety of DNA by a 10 cyclohexene ring. Incorporation of cylcohexenyl nucleosides in a DNA chain increases the stability of a DNA/RNA hybrid. CeNA is stable against degradation in serum and a CeNA/RNA hybrid is able to activate E. Coli RNase H, resulting in cleavage of the RNA strand. (see Wang et al., Am. Chem. Soc. 2000, 122, 36, 8595–8602, hereby incorporated by reference). Potentially stabilizing modifications to the ends of RNA molecules can include N-15 (acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N- (acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4- hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"- phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in WO 2011/005861. Other potentially stabilizing modifications to the ends of RNA molecules include inverted 20 nucleotides, including inverted abasic nucleotides. In one embodiment, each end of a nucleotide comprises an inverted abasic nucleotide, e.g., a 3’->3’ linked nucleotide at the 3’-end of the oligonucleotide and a 5'->5' linked nucleotide at the 5’-end of the oligonucleotide. In each instance, independently, the inverted abasic nucleotide can be linked via a phosphodiester or phosphothioate internucleotide linkage.Other modifications of an RNAi agent of the disclosure include a 5’ phosphate or 25 5’ phosphate mimic, e.g., a 5’-terminal phosphate or phosphate mimic on the antisense strand of an RNAi agent. Suitable phosphate mimics are disclosed in, for example US 2012/0157511, the entire contents of which are incorporated herein by reference. A. Modified RNAi Agents Comprising Motifs of the Disclosure 30 In certain aspects of the disclosure, the double-stranded RNAi agents of the disclosure include agents with chemical modifications as disclosed, for example, in WO 2013/075035, the entire contents of which are incorporated herein by reference. As shown herein and in WO 2013/075035, a superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand or antisense strand of an RNAi agent, particularly at or near the cleavage 35 site. In some embodiments, the sense strand and antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand. The RNAi agent may be optionally modified with a (S)-glycol nucleic acid 295 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (GNA) modification, for instance on one or more residues of the antisense strand. The resulting RNAi agents present superior gene silencing activity. Accordingly, the disclosure provides double stranded RNAi agents capable of inhibiting the expression of a target gene in vivo. The RNAi agent comprises a sense strand and an antisense strand. 5 Each strand of the RNAi agent may be 15-30 nucleotides in length. For example, each strand may be 16- 30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. In certain embodiments, each strand is 19-23 nucleotides in length. 10 The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” The duplex region of an RNAi agent may be 15-30 nucleotide pairs in length. For example, the duplex region can be 16-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17 - 23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in 15 length, 19- 21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length. In another embodiment, the duplex region is 19-21 nucleotide pairs in length. In one embodiment, the RNAi agent may contain one or more overhang regions or capping 20 groups at the 3’-end, 5’-end, or both ends of one or both strands. The overhang can be 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1- 2 nucleotides in length. In another embodiment, the nucleotide overhang region is 2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of 25 the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers. In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2’-sugar modified, 30 such as, 2-F, 2’-O-methyl, thymidine (T), and any combinations thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The 5’- or 3’- overhangs at the sense strand, antisense strand or both strands of the RNAi agent 35 may be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3’-end of the sense strand, antisense strand, or both 296 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO strands. In one embodiment, this 3’-overhang is present in the antisense strand. In one embodiment, this 3’-overhang is present in the sense strand. The dsRNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability. For example, the single-stranded overhang 5 may be located at the 3'-terminal end of the sense strand or, alternatively, at the 3'-terminal end of the antisense strand. The RNAi may also have a blunt end, located at the 5’-end of the antisense strand (i.e., the 3’-end of the sense strand) or vice versa. Generally, the antisense strand of the RNAi has a nucleotide overhang at the 3’-end, and the 5’-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5’-end of the antisense strand and 3’-end overhang of the antisense strand favor the guide 10 strand loading into RISC process. In one embodiment, the RNAi agent is a double blunt-ended of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5’end. The antisense strand contains at least one motif of three 2’-O- methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5’end. 15 In another embodiment, the RNAi agent is a double blunt-ended of 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5’end. The antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5’end. 20 In yet another embodiment, the RNAi agent is a double blunt-ended of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5’end. The antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5’end. 25 In one embodiment, the RNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5’end; the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5’end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucleotide 30 overhang. Preferably, the 2 nucleotide overhang is at the 3’-end of the antisense strand. When the 2 nucleotide overhang is at the 3’-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three 3’-nucleotides of the antisense strand, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has 35 two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5’-end of the sense strand and at the 5’-end of the antisense strand. In one embodiment, every nucleotide in the sense strand and the antisense strand of the RNAi agent, including the nucleotides that are part of the 297 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO motifs are modified nucleotides. In one embodiment each residue is independently modified with a 2’-O- methyl or 2’-fluoro, e.g., in an alternating motif. In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5' terminal nucleotide (position 1) 5 positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3' terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1- 23 of sense strand to form a duplex; wherein at least the 3 ' terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3' terminal nucleotides are unpaired with sense strand, thereby forming a 3' single stranded overhang of 10 1-6 nucleotides; wherein the 5' terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5' overhang; wherein at least the sense strand 5' terminal and 3' terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; 15 and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2’-O-methyl modifications on 20 three consecutive nucleotides at or near the cleavage site. In one embodiment, the dsRNAi agent comprises sense and antisense strands, wherein the dsRNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2’-O- methyl modifications on three consecutive nucleotides at position 11, 12, and 13 from the 5’ end; 25 wherein the 3’ end of the first strand and the 5’ end of the second strand form a blunt end and the second strand is 1-4 nucleotides longer at its 3’ end than the first strand, wherein the duplex region which is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotide of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein dicer cleavage of the RNAi agent preferentially 30 results in an siRNA comprising the 3’ end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand. In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand. 35 In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand. 298 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO For an RNAi agent having a duplex region of 17-23 nucleotide in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5’-end. Thus the motifs of three identical modifications may occur at the 9, 10, and 11 positions; 10, 11, and 12 positions; 11, 12, and 13 positions; 12, 13, and 14 positions; or 13, 14, and 15 positions of the antisense strand, the count starting 5 from the 1st nucleotide from the 5’-end of the antisense strand, or, the count starting from the 1st paired nucleotide within the duplex region from the 5’- end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the RNAi from the 5’- end. The sense strand of the RNAi agent may contain at least one motif of three identical 10 modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide 15 overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap. In one embodiment, the sense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the 20 cleavage site of the strand and the other motifs may be a wing modification. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other, then the chemistry of the motifs are distinct from each other and when the motifs are 25 separated by one or more nucleotide than the chemistries can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif. Like the sense strand, the antisense strand of the RNAi agent may contain more than one motif of 30 three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that may be present on the sense strand. In one embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3’-end, 5’-end or both 35 ends of the strand. In another embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3’-end, 5’-end or both ends of the strand. 299 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO When the sense strand and the antisense strand of the RNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two or three nucleotides. When the sense strand and the antisense strand of the RNAi agent each contain at least two wing 5 modifications, the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, having an overlap of one, two or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two, or three nucleotides in the duplex region. 10 In one embodiment, the RNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch may occur in the overhang region or the duplex region. The base pair may be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of 15 promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings. In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs 20 within the duplex regions from the 5’- end of the antisense strand independently selected from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5’-end of the duplex. In one embodiment, the nucleotide at the 1 position within the duplex region from the 5’-end in 25 the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5’- end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5’- end of the antisense strand is an AU base pair. In another embodiment, the nucleotide at the 3’-end of the sense strand is deoxythimidine (dT). 30 In another embodiment, the nucleotide at the 3’-end of the antisense strand is deoxythimidine (dT). In one embodiment, there is a short sequence of deoxythimidine nucleotides, for example, two dT nucleotides on the 3’-end of the sense or antisense strand. In one embodiment, the sense strand sequence may be represented by Formula (I): 5' np-Na-(X X X )i-Nb-Y Y Y -Nb-(Z Z Z )j-Na-nq 3' (I) 35 wherein: i and j are each independently 0 or 1; p and q are each independently 0-6; 300 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each Na independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each Nb independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides; 5 each np and nq independently represent an overhang nucleotide; wherein Nb and Y do not have the same modification; and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2’-F modified nucleotides. In one embodiment, the Na or Nb comprise modifications of alternating pattern. 10 In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11,12 or 11, 12, 13) of - the sense strand, the count starting from the 1st nucleotide, from the 5’-end; or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5’- end. 15 In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas: 5' np-Na-YYY-Nb-ZZZ-Na-nq 3' (Ib); 5' np-Na-XXX-Nb-YYY-Na-nq 3' (Ic); or 5' np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq 3' (Id). 20 When the sense strand is represented by Formula (Ib), Nb represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. When the sense strand is represented as Formula (Ic), Nb represents an oligonucleotide sequence 25 comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. When the sense strand is represented as Formula (Id), each Nb independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5 or 6. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2- 30 15, or 2-10 modified nucleotides. Each of X, Y and Z may be the same or different from each other. In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula: 5' np-Na-YYY- Na-nq 3' (Ia). When the sense strand is represented by Formula (Ia), each Na independently can represent an 35 oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. In one embodiment, the antisense strand sequence of the RNAi may be represented by Formula (II): 5' nq’-Na′-(Z’Z′Z′)k-Nb′-Y′Y′Y′-Nb′-(X′X′X′)l-N′a-np′ 3' (II) 301 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein: k and l are each independently 0 or 1; p’ and q’ are each independently 0-6; each Na′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, 5 each sequence comprising at least two differently modified nucleotides; each Nb′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides; each np′ and nq′ independently represent an overhang nucleotide; wherein Nb’ and Y’ do not have the same modification; and X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif of three identical modifications on 10 three consecutive nucleotides. In one embodiment, the Na’ or Nb’ comprise modifications of alternating pattern. The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23nucleotidein length, the Y′Y′Y′ motif can occur at positions 9, 10, 11;10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting 15 from the 1st nucleotide, from the 5’-end; or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5’- end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13. In one embodiment, Y′Y′Y′ motif is all 2’-OMe modified nucleotides. In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1. The antisense strand can therefore be represented by the following formulas: 20 5' nq’-Na′-Z′Z′Z′-Nb′-Y′Y′Y′-Na′-np’ 3' (IIb); 5' nq’-Na′-Y′Y′Y′-Nb′-X′X′X′-np’ 3' (IIc); or 5' nq’-Na′- Z′Z′Z′-Nb′-Y′Y′Y′-Nb′- X′X′X′-Na′-np’ 3' (IId). When the antisense strand is represented by Formula (IIb), Nb ’ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na’ 25 independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. When the antisense strand is represented as Formula (IIc), Nb’ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na’ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified 30 nucleotides. When the antisense strand is represented as Formula (IId), each Nb’ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na’ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5 or 6. 35 In other embodiments, k is 0 and l is 0 and the antisense strand may be represented by the formula: 5' np’-Na’-Y’Y’Y’- Na’-nq’ 3' (Ia). 302 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO When the antisense strand is represented as Formula (IIa), each Na’ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of X′, Y′ and Z′ may be the same or different from each other. Each nucleotide of the sense strand and antisense strand may be independently modified with 5 LNA, HNA, CeNA, 2’-methoxyethyl, 2’-O-methyl, 2’-O-allyl, 2’-C- allyl, 2’-hydroxyl, or 2’-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2’-O- methyl or 2’-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2’-O-methyl modification or a 2’-fluoro modification. In one embodiment, the sense strand of the RNAi agent may contain YYY motif occurring at 9,10 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1st nucleotide from the 5’-end, or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5’- end; and Y represents 2’-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2’-OMe modification or 2’-F modification. 15 In one embodiment the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1st nucleotide from the 5’-end, or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5’- end; and Y′ represents 2’-O- methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently 20 represents a 2’-OMe modification or 2’-F modification. The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with an antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively. Accordingly, the RNAi agents for use in the methods of the disclosure may comprise a sense 25 strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by Formula (III): sense: 5' np -Na-(X X X)i -Nb- Y Y Y -Nb -(Z Z Z)j-Na-nq 3' antisense: 3' np ’-Na ’-(X’X′X′)k-Nb ’-Y′Y′Y′-Nb ’-(Z′Z′Z′)l-Na ’-nq ’ 5' (III) 30 wherein: i, j, k, and l are each independently 0 or 1; p, p′, q, and q′ are each independently 0-6; each Na and Na ’ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; 35 each Nb and Nb ’ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides; wherein 303 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each np’, np, nq’, and nq, each of which may or may not be present, independently represents an overhang nucleotide; and XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides. 5 In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1. Exemplary combinations of the sense strand and antisense strand forming an RNAi duplex include the formulas below: 10 5' np - Na -Y Y Y -Na-nq 3' 3' np ’-Na ’-Y′Y′Y′ -Na ’nq ’ 5' (IIIa) 5' np -Na -Y Y Y -Nb -Z Z Z -Na-nq 3' 3' np ’-Na ’-Y′Y′Y′-Nb ’-Z′Z′Z′-Na ’nq ’ 5' 15 (IIIb) 5' np-Na- X X X -Nb -Y Y Y - Na-nq 3' 3' np ’-Na ’-X′X′X′-Nb ’-Y′Y′Y′-Na ’-nq ’ 5' (IIIc) 5' np -Na -X X X -Nb-Y Y Y -Nb- Z Z Z -Na-nq 3' 20 3' np ’-Na ’-X′X′X′-Nb ’-Y′Y′Y′-Nb ’-Z′Z′Z′-Na-nq ’ 5' (IIId) When the RNAi agent is represented by Formula (IIIa), each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. When the RNAi agent is represented by Formula (IIIb), each Nb independently represents an 25 oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. When the RNAi agent is represented as Formula (IIIc), each Nb, Nb’ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified 30 nucleotides. When the RNAi agent is represented as Formula (IIId), each Nb, Nb’ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na, Na ’ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na’, Nb and Nb ’ independently comprises modifications of alternating pattern. 35 In one embodiment, when the RNAi agent is represented by Formula (IIId), the Na modifications are 2^-O-methyl or 2^-fluoro modifications. In another embodiment, when the RNAi agent is represented by Formula (IIId), the Na modifications are 2^-O-methyl or 2^-fluoro modifications and np′ >0 and at least one np′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet another 304 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO embodiment, when the RNAi agent is represented by Formula (IIId), the Na modifications are 2^-O- methyl or 2^-fluoro modifications , np′ >0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more ligands attached through a bivalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is 5 represented by Formula (IIId), the Na modifications are 2^-O-methyl or 2^-fluoro modifications , np′ >0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more ligands optionally attached through a bivalent or trivalent branched linker. In one embodiment, when the RNAi agent is represented by Formula (IIIa), the Na modifications 10 are 2^-O-methyl or 2^-fluoro modifications , np′ >0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more ligands attached through a bivalent or trivalent branched linker. In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented 15 by Formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites. In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more 20 duplexes represented by Formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites. In one embodiment, two RNAi agents represented by Formula (III), (IIIa), (IIIb), (IIIc), and 25 (IIId) are linked to each other at the 5’ end, and one or both of the 3’ ends and are optionally conjugated to to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites. Various publications describe multimeric RNAi agents that can be used in the methods of the disclosure. Such publications include WO2007/091269, WO2010/141511, WO2007/117686, 30 WO2009/014887, and WO2011/031520; and US 7858769, the entire contents of each of which are hereby incorporated herein by reference. In some embodiments, the dsRNA agent further comprises a phosphate or phosphate mimic at the 5’-end of the sense or antisense strand. In one embodiment, there is a phosphate or phosphate mimic at the 5’-end of the sense strand. In one embodiment, there is a phosphate or phosphate mimic at the 5’- 35 end of the antisense strand. In some embodiments, the phosphate mimic is 5’-end phosphorothioate (5’-PS), 5’-end phosphorodithioate (5’-PS2), 5’ end vinylphosphonate (5’-VP), 5’-end methylphosphonate 305 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (MePhos), or 5’-deoxy-5’-C-malonyl In one embodiment, the phosphate mimic is a 5’-vinyl phosphonate (VP). In one embodiment, the phosphate mimic is a 5’-(E)-vinyl phosphonate (VP) isomer (i.e., trans- vinylphosphate), 5’-(Z)-VP isomer (i.e., cis-vinylphosphate), or mixtures thereof. 5 In exemplary embodiments, a 5’-vinyl phosphonate modified nucleotide of the disclosure has the structure: , wherein: X is O or S (e.g., S); R is hydrogen, hydroxy, fluoro, methoxy, 2-methoxyethoxy; R5’ is =C(H)-P(O)(OH)2 and the double bond between the C5’ carbon and R5’ is in the E 10 or Z orientation (e.g., E orientation); and B is a nucleobase or a modified nucleobase, optionally where B is adenine, guanine, cytosine, thymine, or uracil. In one embodiment, R5’ is =C(H)-P(O)(OH)2 and the double bond between the C5’ carbon and R5’ is in the E orientation. In another embodiment, R is methoxy and R5’ is =C(H)- 15 P(O)(OH)2 and the double bond between the C5’ carbon and R5’ is in the E orientation. In another embodiment, X is S, R is methoxy, and R5’ is =C(H)-P(O)(OH)2 and the double bond between the C5’ carbon and R5’ is in the E orientation. In some embodiments, the -CH2OH group at the 4’-position of the 5’-terminal nucleotide is replaced with a phosphate mimic of the formula -O-CH2-P(O)(OR)2, wherein each R is 20 independently hydrogen or C1-4 alkyl (e.g., one R group is hydrogen and one R group is methyl; or both R groups are hydrogen). In one embodiment, the phosphate mimic is a 5’-cyclopropyl phosphonate (VP) (i.e., the CH2OH group at the 4’-position of the 5’-terminal nucleotide is replaced with a group of the formula -Cy-P(O)(OR)2, wherein Cy is a cyclopropyl ring and each R is independently hydrogen 25 or C1-4 alkyl (e.g., one R group is hydrogen or both R groups are hydrogen). In some exemplary embodiments, the 5’-end phosphate mimic i , 306 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , or a salt (e.g., sodium salt) thereof, wherein B is an optionally modified nucleobase (e.g., U). In some embodiments, the 5’-end phosphate mimic is part of a modified 5’-terminal nucleotide. For example, the phosphate mimic may be part of a modified 5’-terminal nucleotide 5 having the structure optionally modified nucleobase. In some embodiments, the 5’-end phosphate mimic can also include a 5’-phosphate prodrug or 5’-phosphonate prodrug. In some embodiments, the 5’-phosphate prodrug or 5’-phosphonate prodrug has a structure of formulas disclosed in WO2022/147214, which is incorporated herein 10 by reference. In some exemplary embodiments, the 5’-phosphate prodrug or 5’-phosphonate prodrug is: Pmmds ( , ((4SR,5SR)-3,3,5-trimethyl-1,2-dithiolan-4-ol) phosphodiester); cPmmds ( , ((4SR,5RS)-3,3,5-trimethyl-1,2-dithiolan-4-ol) phosphodiester (Cis Pmmds)); 15 PdAr1s -phenyl-3,3-dimethyl-1,2-dithiolan-4-ol) phosphodiester); methylphenyl)-3,3-dimethyl-1,2-dithiolan-4-ol) phosphodiester); methoxyphenyl)-3,3-dimethyl-1,2-dithiolan- 307 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 4-ol) phosphodiester); 5 In some exemplary embodiments, the 5’-phosphate prodrug or 5’-phosphonate prodrug 10 is: . The siRNA containing one of the above list of 5’ modified phosphate prodrugs generally has an activity comparable to that of the siRNA containing 5’-VP. In some exemplary embodiments, the 5’-phosphate prodrug or 5’-phosphonate prodrug is: . 308 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO The siRNA containing one of the above list of 5’ modified phosphate prodrugs can have an improved stability than that of the siRNA containing 5’-VP and better or comparable activity than that of the siRNA containing 5’-VP. In some embodiments, the 5’-end of the antisense strand of the dsRNA agent does not 5 contain a 5’-vinyl phosphonate (VP). i. Thermally Destabilizing Modifications In certain embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating thermally destabilizing modifications in the seed region of the antisense strand. As used herein “seed region” means at positions 2-9 of the 5’-end of the referenced strand. For example, 10 thermally destabilizing modifications can be incorporated in the seed region of the antisense strand to reduce or inhibit off-target gene silencing. The term “thermally destabilizing modification(s)” includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) than the Tm of the dsRNA without having such modification(s). For example, the thermally destabilizing modification(s) can decrease the Tm of the 15 dsRNA by 1 – 4 °C, such as one, two, three or four degrees Celsius. And, the term “thermally destabilizing nucleotide” refers to a nucleotide containing one or more thermally destabilizing modifications. It has been discovered that dsRNAs with an antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5’ end, 20 of the antisense strand have reduced off-target gene silencing activity. Accordingly, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5’ region of the antisense strand. In some embodiments, one or more thermally destabilizing modification(s) of the duplex is/are located in positions 2-9, or preferably positions 4-8, from the 5’-end of the antisense strand. 25 In some further embodiments, the thermally destabilizing modification(s) of the duplex is/are located at position 6, 7 or 8 from the 5’-end of the antisense strand. In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5’-end of the antisense strand. In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5 or 9 from the 5’-end of the antisense strand. 30 The thermally destabilizing modifications can include, but are not limited to, abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2’-deoxy modification , a 2’-5’ linked nucleotide (“3’-RNA’), or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA). Exemplified abasic modifications include, but are not limited to the following: 309 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein B is a modified or unmodified nucleobase. 5 Exemplified sugar modifications include, but are not limited to the following: wherein B is a modified or unmodified nucleobase. In some embodiments the thermally destabilizing modification of the duplex is selected from the 10 group consisting of: 310 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic. The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for 5 example, where any of bonds between the ribose carbons (e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or C1’-O4’) is absent or at least one of ribose carbons or oxygen (e.g., C1’, C2’, C3’, C4’ or O4’) are independently or in combination absent from the nucleotide. In some embodiments, acyclic nucleotide is modified or unmodified nucleobase, R1 and R2 independently are H, halogen, OR3, or alkyl; and R3 is H, 10 alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked "sugar" residue. In one example, UNA also encompasses monomers with bonds between C1'-C4' being removed (i.e. the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond (i.e. the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar is removed (see 15 Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are hereby incorporated by reference in their entirety). The acyclic derivative provides greater backbone flexibility without affecting the Watson-Crick pairings. The acyclic nucleotide can be linked via 2’-5’ or 3’-5’ linkage. The term ‘GNA’ refers to glycol nucleic acid which is a polymer similar to DNA or RNA but 20 differing in the composition of its “backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds: 311 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO . The thermally destabilizing modification of the duplex can be mismatches (i.e., noncomplementary base pairs) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA duplex. Exemplary mismatch base pairs include G:G, 5 G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Other mismatch base pairings known in the art are also amenable to the present disclosure. A mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from respective nucleotides independent of the modifications on the ribose sugars of the nucleotides. In certain embodiments, the dsRNA molecule 10 contains at least one nucleobase in the mismatch pairing that is a 2’-deoxy nucleobase; e.g., the 2’-deoxy nucleobase is in the sense strand. In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes nucleotides with impaired W-C H-bonding to complementary base on the target mRNA, such as: 15 . More examples of abasic nucleotide, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO 2011/133876, which is herein incorporated by reference in its entirety. 312 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO The thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications. In some embodiments, the thermally destabilizing modification of the duplex includes nucleotides with non-canonical bases such as, but not limited to, nucleobase modifications with impaired 5 or completely abolished capability to form hydrogen bonds with bases in the opposite strand. These nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010/0011895, which is herein incorporated by reference in its entirety. Exemplary nucleobase modifications are: 10 In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes one or more ^-nucleotide complementary to the base on the target mRNA, such as: wherein R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl. 15 Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are: The alkyl for the R group can be a C1-C6alkyl. Specific alkyls for the R group include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl. 20 As the skilled artisan will recognize, in view of the functional role of nucleobases is defining specificity of an RNAi agent of the disclosure, while nucleobase modifications can be performed in the various manners as described herein, e.g., to introduce destabilizing modifications into an RNAi agent of 313 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO the disclosure, e.g., for purpose of enhancing on-target effect relative to off-target effect, the range of modifications available and, in general, present upon RNAi agents of the disclosure tends to be much greater for non-nucleobase modifications, e.g., modifications to sugar groups or phosphate backbones of polyribonucleotides. Such modifications are described in greater detail in other sections of the instant 5 disclosure and are expressly contemplated for RNAi agents of the disclosure, either possessing native nucleobases or modified nucleobases as described above or elsewhere herein. In addition to the antisense strand comprising a thermally destabilizing modification, the dsRNA can also comprise one or more stabilizing modifications. For example, the dsRNA can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without 10 limitations, the stabilizing modifications all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two stabilizing modifications. The stabilizing modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the stabilizing modification can occur on every nucleotide on the sense strand or antisense strand; each stabilizing modification can occur in an alternating pattern on the sense strand or antisense strand; or the 15 sense strand or antisense strand comprises both stabilizing modification in an alternating pattern. The alternating pattern of the stabilizing modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the stabilizing modifications on the sense strand can have a shift relative to the alternating pattern of the stabilizing modifications on the antisense strand. In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, 20 seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the antisense strand can be present at any positions. In some embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5’-end. In some other embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5’-end. In still some other embodiments, the antisense comprises stabilizing modifications at positions 2, 14, and 16 25 from the 5’-end. In each of the preceding, the stabilizing modification can be a 2’-fluoro modification. In some other embodiments, the antisense comprises 2’-H modifications (DNA) at positions 2, 5, 7, 12, 14, and 16 from the 5’-end of the antisense strand. In still some other embodiments, the antisense comprises 2’-H modifications (DNA) at positions 2, 5, 7, and 12 from the 5’-end, and a 2’-fluoro modiciation at position 14 from the 5’-end of the antisense strand. In other embodiments, the antisense 30 strand is paired to a sense strand having 2’-fluoro modifications at positions 9, 10, and 11 from the 5’-end of the sense strand (or opposite to positiosn 11, 12, and 13 of the antisense strand). In other embodiments, the antisense strand is paired to a sense strand having 2’-fluoro modifications at positions 7, 9, 10, and 11 from the 5’-end of the sense strand (or opposite to positiosn 11, 12, 13, and 15 of the antisense strand). In some embodiments, the antisense strand comprises at least one stabilizing modification 35 adjacent to the destabilizing modification. For example, the stabilizing modification can be the nucleotide at the 5’-end or the 3’-end of the destabilizing modification, i.e., at position -1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing 314 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO modification at each of the 5’-end and the 3’-end of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3’-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing 5 modification. In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications (e.g., 2’-fluoro modifications). Without limitations, a stabilizing modification in the sense strand can be present at any positions. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5’- 10 end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5’-end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5’-end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications (e.g., 2’-fluoro modifications) at positions opposite or complementary to positions 11, 12, 15 13, and 15 of the antisense strand, counting from the 5’-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three, or four stabilizing modifications. In some other embodiments, the sense strand comprises stabilizing modifications (e.g., 2’-fluoro modifications) at positions opposite or complementary to positions 11, 12, and 13 of the antisense strand, counting from the 5’-end of the antisense strand. 20 In some embodiments, the sense strand does not comprise a stabilizing modification in position opposite or complementary to the thermally destabilizing modification of the duplex in the antisense strand. Exemplary thermally stabilizing modifications include, but are not limited to, 2’-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA. 25 In some embodiments, the dsRNA of the disclosure comprises at least four (e.g., four, five, six, seven, eight, nine, ten, or more) 2’-fluoro nucleotides. Without limitations, the 2’-fluoro nucleotides all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two 2’-fluoro nucleotides. The 2’-fluoro modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the 2’-fluoro modification can occur on every nucleotide on the 30 sense strand or antisense strand; each 2’-fluoro modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both 2’-fluoro modifications in an alternating pattern. The alternating pattern of the 2’-fluoro modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the 2’-fluoro modifications on the sense strand can have a shift relative to the alternating pattern of the 2’-fluoro 35 modifications on the antisense strand. In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2’-fluoro nucleotides. Without limitations, a 2’-fluoro modification in the antisense strand can be present at any positions. In some embodiments, the antisense comprises 2’-fluoro 315 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO nucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5’-end. In some other embodiments, the antisense comprises 2’-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5’-end. In still some other embodiments, the antisense comprises 2’-fluoro nucleotides at positions 2, 14, and 16 from the 5’-end. In some embodiments, the antisense strand comprises at least one 2’-fluoro nucleotide adjacent 5 to the destabilizing modification. For example, the 2’-fluoro nucleotide can be the nucleotide at the 5’- end or the 3’-end of the destabilizing modification, i.e., at position -1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a 2’-fluoro nucleotide at each of the 5’-end and the 3’-end of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification. 10 In some embodiments, the antisense strand comprises at least two 2’-fluoro nucleotides at the 3’- end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification. In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) 2’-fluoro nucleotides. Without limitations, a 2’-fluoro modification in the 15 sense strand can be present at any positions. In some embodiments, the antisense comprises 2’-fluoro nucleotides at positions 7, 10, and 11 from the 5’-end. In some other embodiments, the sense strand comprises 2’-fluoro nucleotides at positions 7, 9, 10, and 11 from the 5’-end. In some embodiments, the sense strand comprises 2’-fluoro nucleotides at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5’-end of the antisense strand. In some other 20 embodiments, the sense strand comprises 2’-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5’-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three or four 2’-fluoro nucleotides. In some embodiments, the sense strand does not comprise a 2’-fluoro nucleotide in position opposite or complementary to the thermally destabilizing modification of the duplex in the antisense 25 strand. In some embodiments, the dsRNA molecule of the disclosure comprises a 21 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense, wherein the antisense strand contains at least one thermally destabilizing nucleotide, where the at least one (e.g., only one) thermally destabilizing nucleotide occurs in the seed region of the antisense strand (i.e., at position 2-9 of the 5’-end of the 30 antisense strand), wherein one end of the dsRNA is blunt, while the other end is comprises a 2 nt overhang, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5 or 62’-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 52’-fluoro 35 modifications; (v) the sense strand comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2’-fluoro modifications; and (vii) the dsRNA comprises a blunt end at 5’-end of the antisense strand. Preferably, the 2 nt overhang is at the 3’-end of the antisense. 316 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, the dsRNA molecule of the disclosure comprising a sense and antisense strands, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5' terminal nucleotide (position 1), positions 1 to 23 of said sense strand comprise at least 8 ribonucleotides; antisense strand is 36-66 nucleotide residues in length and, starting from the 3' terminal nucleotide, at 5 least 8 ribonucleotides in the positions paired with positions 1- 23 of sense strand to form a duplex; wherein at least the 3 ' terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3' terminal nucleotides are unpaired with sense strand, thereby forming a 3' single stranded overhang of 1-6 nucleotides; wherein the 5' terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single 10 stranded 5' overhang; wherein at least the sense strand 5' terminal and 3' terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when said double stranded nucleic acid is 15 introduced into a mammalian cell; and wherein the antisense strand contains at least one thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is in the seed region of the antisense strand (i.e. at position 2-9 of the 5’-end of the antisense strand). For example, the thermally destabilizing nucleotide occurs between positions opposite or complementary to positions 14-17 of the 5’-end of the sense strand, and wherein the dsRNA optionally further has at least one (e.g., one, two, 20 three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 62’-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 52’- fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2’-fluoro modifications; and (vii) the dsRNA 25 comprises a duplex region of 12-30 nucleotide pairs in length. In some embodiments, the dsRNA molecule of the disclosure comprises a sense and antisense strands, wherein said dsRNA molecule comprises a sense strand having a length which is at least 25 and at most 29 nucleotides and an antisense strand having a length which is at most 30 nucleotides with the sense strand comprises a modified nucleotide that is susceptible to enzymatic degradation at position 11 30 from the 5’end, wherein the 3’ end of said sense strand and the 5’ end of said antisense strand form a blunt end and said antisense strand is 1-4 nucleotides longer at its 3’ end than the sense strand, wherein the duplex region which is at least 25 nucleotides in length, and said antisense strand is sufficiently complementary to a target mRNA along at least 19 nt of said antisense strand length to reduce target gene expression when said dsRNA molecule is introduced into a mammalian cell, and wherein dicer cleavage 35 of said dsRNA preferentially results in an siRNA comprising said 3’ end of said antisense strand, thereby reducing expression of the target gene in the mammal, wherein the antisense strand contains at least one thermally destabilizing nucleotide, where the at least one thermally destabilizing nucleotide is in the seed region of the antisense strand (i.e. at position 2-9 of the 5’-end of the antisense strand), and wherein the 317 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 62’-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 52’-fluoro modifications; (v) the 5 sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2’-fluoro modifications; and (vii) the dsRNA has a duplex region of 12-29 nucleotide pairs in length. In some embodiments, every nucleotide in the sense strand and antisense strand of the dsRNA molecule may be modified. Each nucleotide may be modified with the same or different modification 10 which can include one or more alteration of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2^ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose- phosphate backbone. 15 As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3’ or 5’ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in 20 the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. E.g., a phosphorothioate modification at a non- linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in 25 double strand and single strand regions, particularly at termini. The 5’ end or ends can be phosphorylated. It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5’ or 3’ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3’ or 5’ overhang may be modified, e.g., with a modification 30 described herein. Modifications can include, e.g., the use of modifications at the 2’ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2’-deoxy-2’- fluoro (2’-F) or 2’-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence. 35 In some embodiments, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, F-HNA,CeNA, 2’-methoxyethyl, 2’- O-methyl, 2’-O-allyl, 2’-C- allyl, 2’- deoxy, or 2’-fluoro. The strands can contain more than one modification. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2’-O-methyl or 2’-fluoro. 318 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO It is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand. At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2’-deoxy, 2’- O-methyl or 2’-fluoro modifications, acyclic 5 nucleotides or others. In some embodiments, the sense strand and antisense strand each comprises two differently modified nucleotides selected from 2’-O-methyl or 2’-deoxy. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2'-O-methyl nucleotide, 2’-deoxy nucleotide, 2´-deoxy-2’-fluoro nucleotide, 2'-O-N-methylacetamido (2'-O-NMA) nucleotide, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, 2'-O-aminopropyl (2'-O-AP) nucleotide, or 10 2'-ara-F nucleotide. Again, it is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand. In some embodiments, the dsRNA molecule of the disclosure comprises modifications of an alternating pattern, particular in the B1, B2, B3, B1’, B2’, B3’, B4’ regions. The term “alternating motif” or “alternative pattern” as used herein refers to a motif having one or more modifications, each 15 modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB…,” “AABBAABBAABB…,” “AABAABAABAAB…,” “AAABAAABAAAB…,” “AAABBBAAABBB…,” or “ABCABCABCABC…,” etc. 20 The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB…”, “ACACAC…” “BDBDBD…” or “CDCDCD…,” etc. 25 In some embodiments, the dsRNA molecule of the disclosure comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the 30 alternating motif in the sense strand may start with “ABABAB” from 5’-3’ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 3’-5’of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5’-3’ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 3’-5’of the strand within the duplex region, so that there is a complete or partial 35 shift of the modification patterns between the sense strand and the antisense strand. The dsRNA molecule of the disclosure may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any 319 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern. The alternating pattern of 5 the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand. In some embodiments, the dsRNA molecule comprises the phosphorothioate or 10 methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or 15 methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. Preferably, 20 these terminal three nucleotides may be at the 3’-end of the antisense strand. In some embodiments, the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and 25 the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage. In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 30 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage. 35 In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence 320 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage. In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four 5 phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either 10 phosphorothioate or methylphosphonate or phosphate linkage. In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense 15 strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage. In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 20 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage. 25 In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate 30 and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage. In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate 35 internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage. 321 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, or 4 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired 5 with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage. In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini 10 position(s) of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage at one end or both ends of the sense or antisense strand. In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the internal 15 region of the duplex of each of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate methylphosphonate internucleotide linkage at position 8-16 of the duplex region counting from the 5’-end of the sense strand; the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini position(s). 20 In some embodiments, the dsRNA molecule of the disclosure further comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 1-5 and one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 18-23 of the sense strand (counting from the 5’-end), and one to five phosphorothioate or methylphosphonate internucleotide linkage modification at positions 1 and 2 and one to five within 25 positions 18-23 of the antisense strand (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5’-end), and one phosphorothioate internucleotide linkage modification at positions 1 30 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5’-end), 35 and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). 322 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5’- end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two 5 phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5’- 10 end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate 15 internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises one 20 phosphorothioate internucleotide linkage modification within position 1-5 and one within position 18-23 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises one 25 phosphorothioate internucleotide linkage modification within position 1-5 (counting from the 5’-end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises two 30 phosphorothioate internucleotide linkage modifications within position 1-5 (counting from the 5’-end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises two 35 phosphorothioate internucleotide linkage modifications within position 1-5 and one within position 18-23 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5’-end). 323 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two 5 phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5’-end), 10 and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate15 internucleotide linkage modifications at position 20 and 21 of the sense strand (counting from the 5’- end), and one phosphorothioate internucleotide linkage modification at positions 1 and one at position 21 of the antisense strand (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate 20 internucleotide linkage modification at position 21 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 the antisense strand (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises two 25 phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 21 and 22 of the sense strand (counting from the 5’- end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5’-end). 30 In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 the antisense strand 35 (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 22 and 23 of the sense strand (counting from the 5’- 324 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5’-end). In some embodiments, the dsRNA molecule of the disclosure further comprises one 5 phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 the antisense strand (counting from the 5’-end). 10 In some embodiments, compound of the disclosure comprises a pattern of backbone chiral centers. In some embodiments, a common pattern of backbone chiral centers comprises at least 5 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 6 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 7 internucleotidic linkages 15 in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 8 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 9 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers 20 comprises at least 11 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration. In some 25 embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 30 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 19 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages in the Rp configuration. In some embodiments, a 35 common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages in the Rp configuration. In 325 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic 5 linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages which are not chiral. In some 10 embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 15 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration, and no more than 8 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic 20 linkages in the Sp configuration, and no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In 25 some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration, and no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration, and no more than 4 internucleotidic linkages which are not chiral. In some embodiments, the internucleotidic linkages in the Sp configuration are optionally contiguous or not 30 contiguous. In some embodiments, the internucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages which are not chiral are optionally contiguous or not contiguous. In some embodiments, compound of the disclosure comprises a block is a stereochemistry block. In some embodiments, a block is an Rp block in that each internucleotidic linkage of the block is Rp. In 35 some embodiments, a 5’-block is an Rp block. In some embodiments, a 3’-block is an Rp block. In some embodiments, a block is an Sp block in that each internucleotidic linkage of the block is Sp. In some embodiments, a 5’-block is an Sp block. In some embodiments, a 3’-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, 326 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each internucleotidic linkage in a natural phosphate linkage. 5 In some embodiments, compound of the disclosure comprises a 5’-block is an Sp block wherein each sugar moiety comprises a 2’-F modification. In some embodiments, a 5’-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2’-F modification. In some embodiments, a 5’-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-F 10 modification. In some embodiments, a 5’-block comprises 4 or more nucleoside units. In some embodiments, a 5’-block comprises 5 or more nucleoside units. In some embodiments, a 5’-block comprises 6 or more nucleoside units. In some embodiments, a 5’-block comprises 7 or more nucleoside units. In some embodiments, a 3’-block is an Sp block wherein each sugar moiety comprises a 2’-F modification. In some embodiments, a 3’-block is an Sp block wherein each of internucleotidic linkage is 15 a modified internucleotidic linkage and each sugar moiety comprises a 2’-F modification. In some embodiments, a 3’-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-F modification. In some embodiments, a 3’-block comprises 4 or more nucleoside units. In some embodiments, a 3’-block comprises 5 or more nucleoside units. In some embodiments, a 3’-block comprises 6 or more nucleoside units. In some embodiments, a 20 3’-block comprises 7 or more nucleoside units. In some embodiments, compound of the disclosure comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of internucleotidic linkage, e.g., natural phosphate linkage, modified internucleotidic linkage, Rp chiral internucleotidic linkage, Sp chiral internucleotidic linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In 25 some embodiments, A is followed by natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, 30 G is followed by natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp. In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages 35 between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5’-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of 327 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO the following characteristics: (i) the antisense comprises 2, 3, 4, 5 or 62’-fluoro modifications; (ii) the antisense comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 52’-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA 5 comprises at least four 2’-fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5’-end of the antisense strand. In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one 10 thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5’-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5 or 62’-fluoro modifications; (ii) the sense strand is conjugated with a ligand; (iii) the sense strand comprises 2, 3, 4 or 52’-fluoro modifications; (iv) the sense strand 15 comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2’-fluoro modifications; (vi) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5’-end of the antisense strand. In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages 20 between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5’-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5 or 62’-fluoro modifications; (ii) the antisense 25 comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 52’-fluoro modifications; (v) the sense strand comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2’- fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5’-end of the antisense strand. 30 In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the 35 duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5’-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5 or 62’-fluoro modifications; (ii) the sense strand is conjugated with a ligand; (iii) the sense strand comprises 2, 3, 4 or 328 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 52’-fluoro modifications; (iv) the sense strand comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2’-fluoro modifications; (vi) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at 5’-end of the antisense strand. 5 In some embodiments, the dsRNA molecule of the disclosure comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can 10 also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings. In some embodiments, the dsRNA molecule of the disclosure comprises at least one of the first 1, 15 2, 3, 4, or 5 base pairs within the duplex regions from the 5’- end of the antisense strand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5’-end of the duplex. In some embodiments, the nucleotide at the 1 position within the duplex region from the 5’-end 20 in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5’- end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5’- end of the antisense strand is an AU base pair. It was found that introducing 4’-modified or 5’-modified nucleotide to the 3’-end of a 25 phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage of a dinucleotide at any position of single stranded or double stranded oligonucleotide can exert steric effect to the internucleotide linkage and, hence, protecting or stabilizing it against nucleases. In some embodiments, 5’-modified nucleoside is introduced at the 3’-end of a dinucleotide at any position of single stranded or double stranded siRNA. For instance, a 5’-alkylated nucleoside may be 30 introduced at the 3’-end of a dinucleotide at any position of single stranded or double stranded siRNA. The alkyl group at the 5’ position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 5’-alkylated nucleoside is 5’-methyl nucleoside. The 5’-methyl can be either racemic or chirally pure R or S isomer. In some embodiments, 4’-modified nucleoside is introduced at the 3’-end of a dinucleotide at any 35 position of single stranded or double stranded siRNA. For instance, a 4’-alkylated nucleoside may be introduced at the 3’-end of a dinucleotide at any position of single stranded or double stranded siRNA. The alkyl group at the 4’ position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4’-alkylated nucleoside is 4’-methyl nucleoside. The 4’-methyl can be either racemic or 329 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO chirally pure R or S isomer. Alternatively, a 4’-O-alkylated nucleoside may be introduced at the 3’-end of a dinucleotide at any position of single stranded or double stranded siRNA. The 4’-O-alkyl of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4’-O-alkylated nucleoside is 4’-O- methyl nucleoside. The 4’-O-methyl can be either racemic or chirally pure R or S isomer. 5 In some embodiments, 5’-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 5’-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 5’-alkylated nucleoside is 5’- methyl nucleoside. The 5’-methyl can be either racemic or chirally pure R or S isomer. In some embodiments, 4’-alkylated nucleoside is introduced at any position on the sense strand 10 or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 4’-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4’-alkylated nucleoside is 4’- methyl nucleoside. The 4’-methyl can be either racemic or chirally pure R or S isomer. In some embodiments, 4’-O-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the 15 dsRNA. The 5’-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4’-O-alkylated nucleoside is 4’-O-methyl nucleoside. The 4’-O-methyl can be either racemic or chirally pure R or S isomer. In some embodiments, the dsRNA molecule of the disclosure can comprise 2’-5’ linkages (with 2’-H, 2’-OH and 2’-OMe and with P=O or P=S). For example, the 2’-5’ linkages modifications can be 20 used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5’ end of the sense strand to avoid sense strand activation by RISC. In another embodiment, the dsRNA molecule of the disclosure can comprise L sugars (e.g., L ribose, L-arabinose with 2’-H, 2’-OH and 2’-OMe). For example, these L sugars modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be 25 used at the 5’ end of the sense strand to avoid sense strand activation by RISC. Various publications describe multimeric siRNA which can all be used with the dsRNA of the disclosure. Such publications include WO2007/091269, US 7858769, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520 which are hereby incorporated by their entirely. 30 As described in more detail below, the RNAi agent that contains conjugations of one or more carbohydrate moieties to an RNAi agent may improve one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate 35 ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring 330 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds. The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one 5 “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides 10 a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring. 15 The RNAi agents may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group. The cyclic group can be selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalinyl. The acyclic group can be a serinol backbone or diethanolamine backbone. 20 IV. Delivery of an RNAi Agent of the Disclosure The delivery of a RNAi agent of the disclosure to a cell e.g., a skeletal muscle cell, e.g., a skeletal myocyte and/or a cardiomyocyte, e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a target gene-associated disorder, e.g., skeletal muscle 25 and/or cardiac muscle disease or disorder, can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an RNAi agent of the disclosure either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an RNAi agent, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the RNAi agent. These 30 alternatives are discussed further below. In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with a RNAi agent of the disclosure (see e.g., Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an RNAi agent include, for example, biological 35 stability of the delivered agent, prevention of non-specific effects, and accumulation of the delivered agent in the target tissue. The non-specific effects of an RNAi agent can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits 331 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the RNAi agent to be administered. For administering a RNAi agent systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of 5 the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the RNAi agent to the target tissue and avoid undesirable off-target effects (e.g., without wishing to be bound by theory, use of GNAs as described herein has been identified to destabilize the seed region of a dsRNA, resulting in enhanced preference of such dsRNAs for on-target effectiveness, relative to off-target effects, as such off-target effects are significantly weakened by such 10 seed region destabilization). Conjugation of an RNAi agent to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO. et al., (2006) Nat. Biotechnol.24:1005-1015). In an alternative embodiment, the RNAi agent can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of molecule RNAi agent 15 (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an RNAi agent by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an RNAi agent, or induced to form a vesicle or micelle (see e.g., Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116) that encases an RNAi agent. The formation of vesicles or micelles further prevents degradation of the RNAi agent when administered systemically. Methods for making 20 and administering cationic- RNAi agent complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, DR., et al. (2003) J. Mol. Biol 327:761-766; Verma, UN. et al., (2003) Clin. Cancer Res.9:1291-1300; Arnold, AS et al. (2007) J. Hypertens.25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of RNAi agents include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN. et al., (2003),25 supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS. et al., (2006) Nature 441:111-114), cardiolipin (Chien, PY. et al., (2005) Cancer Gene Ther.12:321-328; Pal, A. et al., (2005) Int J. Oncol.26:1087-1091), polyethyleneimine (Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol.71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm.3:472-487), and polyamidoamines (Tomalia, DA. et al., (2007) Biochem. Soc. Trans. 30 35:61-67; Yoo, H. et al., (1999) Pharm. Res.16:1799-1804). In some embodiments, a RNAi agent forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of RNAi agents and cyclodextrins can be found in U.S. Patent No.7, 427, 605, which is herein incorporated by reference in its entirety. Certain aspects of the instant disclosure relate to a method of reducing the expression of a target 35 gene in a skeletal muscle cell and/or a cardiac muscle cell, comprising contacting said cell with the double-stranded RNAi agent of the disclosure. In certain embodiments, the RNAi agent is taken up on one or more tissue or cell types present in organs, e.g., skeletal muscle tissue and/or cardiac muscle tissue. 332 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Another aspect of the disclosure relates to a method of reducing the expression and/or activity of a target gene in a subject, comprising administering to the subject the double-stranded RNAi agent of the disclosure. Another aspect of the disclosure relates to a method of treating a subject having a target gene- 5 associated disorder or at risk of having or at risk of developing a target gene-associated disorder, comprising administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of the disclosure, thereby treating the subject. In one embodiment, the double-stranded RNAi agent is administered subcutaneously. In one embodiment, the double-stranded RNAi agent is administered intramuscularly. 10 In one embodiment, the double-stranded RNAi agent is administered by intravenously. In one embodiment, the double-stranded RNAi agent is administered by pulmonary sytem administration, e.g., intranasal administration, or oral inhalative administration. For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to modified siRNA compounds. It may be understood, however, that these 15 formulations, compositions and methods can be practiced with other siRNA compounds, e.g., unmodified siRNA compounds, and such practice is within the disclosure. A composition that includes a RNAi agent can be delivered to a subject by a variety of routes. Exemplary routes include pulmonary system, intravenous, subcutaneous, and nasal. The RNAi agents of the disclosure can be incorporated into pharmaceutical compositions 20 suitable for administration. Such compositions typically include one or more species of RNAi agent and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except 25 insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. 30 Administration may be intratracheal, intranasal, oral, parenteral, or pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. The vascular 35 endothelial cells could be targeted by coating a balloon catheter with the RNAi agent and mechanically introducing the RNA. Compositions for pulmonary system delivery may include aqueous solutions, e.g., for intranasal or oral inhalative administration, suitable carriers composed of, e.g., lipids (liposomes, niosomes, 333 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO microemulsions, lipidic micelles, solid lipid nanoparticles) or polymers (polymer micelles, dendrimers, polymeric nanoparticles, nonogels, nanocapsules), adjuvant, e.g., for oral inhalative administration. Aqueous compositions may be sterile and may optionally contain buffers, diluents, absorbtion enhancers and other suitable additives. Such administration permits both systemic and local delivery of the double 5 stranded RNAi agents of the disclosure. Intranasal administration may include instilling or insufflating a double stranded RNAi agent into the nasal cavity with syringes or droppers by applying a few drops at a time or via atomization. Suitable dosage forms for intranasal administration include drops, powders, nebulized mists, and sprays. Nasal delivery devices include, but not limited to, vapor inhaler, nasal dropper, spray bottle, metered dose spray 10 pump, gas driven spray atomizer, nebulizer, mechanical powder sprayer, breath actuated inhaler, and insufflator. Devices for delivery deeper into the respiratory system, e.g., into the lung, include nebulizer, pressured metered-dose inhaler, dry powder inhaler, and thermal vaporization aerosol device. Devices for delivery by inhalation are available from commercial suppliers.Devices can be fixed or variable dose, single or multidose, disposable or reusable depending on, for example, the disease or disorder to be 15 prevented or treated, the volume of the agent to be delivered, the frequency of delivery of the agent, and other considerations in the art. Oral inhalative administration may include use of device, e.g., a passive breath driven or active power driven single/-multiple dose dry powder inhaler (DPI), to deliver a double stranded RNAi agent to the pulmonary system. Suitable dosage forms for oral inhalative administration include powders and 20 solutions. Suitable devices for oral inhalative administration include nebulizers, metered-dose inhalers, and dry powder inhalers. Dry powder inhalers are of the most popular devices used to deliver drugs, especially proteins to the lungs. Exemplary commercially available dry powder inhalers include Spinhaler (Fisons Pharmaceuticals, Rochester, NY) and Rotahaler (GSK, RTP, NC). Several types of nebulizers are available, namely jet nebulizers, ultrasonic nebulizers, vibrating mesh nebulizers. Jet 25 nebulizers are driven by compressed air. Ultrasonic nebulizers use a piezoelectric transducer in order to create droplets from an open liquid reservoir. Vibrating mesh nebulizers use perforated membranes actuated by an annular piezoelement to vibrate in resonant bending mode. The holes in the membrane have a large cross-section size on the liquid supply side and a narrow cross-section size on the side from where the droplets emerge. Depending on the therapeutic application, the hole sizes and number of holes 30 can be adjusted. Selection of a suitable device depends on parameters, such as nature of the drug and its formulation, the site of action, and pathophysiology of the lung. Aqueous suspensions and solutions are nebulized effectively. Aerosols based on mechanically generated vibration mesh technologies also have been used successfully to deliver proteins to lungs. Formulations for topical administration may include transdermal patches, ointments, lotions, 35 creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. 334 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and 5 talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening or flavoring agents can be added. Compositions suitable for oral administration of the agents of the disclosure are further described in PCT Application No. PCT/US20/33156, the entire contents of 10 which are incorporated herein by reference. Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes may be controlled to render the preparation isotonic. 15 In one embodiment, the administration of the siRNA compound, e.g., a double-stranded siRNA compound, is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, subcutaneous, pulmonary system, or intranasal. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery 20 are discussed in more detail below. A. Vector encoded RNAi agents of the Disclosure RNAi agents targeting the target gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; WO 00/22113, WO 00/22114, 25 and US 6,054,299). Expression can be sustained (months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292). 30 The individual strand or strands of a RNAi agent can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively, each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat 35 polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure. RNAi agent expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, such as those compatible with vertebrate cells, can be used to produce 335 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO recombinant constructs for the expression of a RNAi agent as described herein. Delivery of RNAi agent expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell. 5 Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno- associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. 10 canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells’ genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of a RNAi agent will generally require regulatory elements, e.g., promoters, 15 enhancers, etc., to ensure the expression of the RNAi agent in target cells. Other aspects to consider for vectors and constructs are known in the art. V. Pharmaceutical Compositions of the Disclosure The present disclosure also includes pharmaceutical compositions and formulations which 20 include the RNAi agents of the disclosure. In one embodiment, provided herein are pharmaceutical compositions containing an RNAi agent, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the RNAi agent are useful for treating a skeletal muscle and/or a cardiac muscle disorder, disease, or condition treatable by reduction or inhibition of the expression or activity of a target gene, e.g., a skeletal muscle and/or cardiac muscle disorder or disease. 25 In some embodiments, the pharmaceutical compositions of the disclosure are sterile. In another embodiment, the pharmaceutical compositions of the disclosure are pyrogen free. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV), intramuscular (IM), or for subcutaneous (subQ) delivery. 30 The pharmaceutical compositions of the disclosure may be administered in dosages sufficient to inhibit expression of a target gene. In general, a suitable dose of an RNAi agent of the disclosure will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. A repeat-dose regimen may include administration of a therapeutic amount of an RNAi agent on 35 a regular basis, such as monthly to once every six months. In certain embodiments, the RNAi agent is administered about once per quarter (i.e., about once every three months) to about twice per year. After an initial treatment regimen (e.g., loading dose), the treatments can be administered on a less frequent basis. 336 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In other embodiments, a single dose of the pharmaceutical compositions can be long lasting, such that subsequent doses are administered at not more than 1, 2, 3, or 4 or more month intervals. In some embodiments of the disclosure, a single dose of the pharmaceutical compositions of the disclosure is administered once per month. In other embodiments of the disclosure, a single dose of the pharmaceutical 5 compositions of the disclosure is administered once per quarter to twice per year. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single 10 treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the iRNAs encompassed by the disclosure can be made using conventional methodologies or on the basis of in vivo testing using a suitable animal model. A suitable animal model, e.g., a mouse or a rat, e.g., an animal containing a transgene expressing a skeletal muscle target gene and/or a cardiac muscle target gene, can be used to 15 determine the therapeutically effective dose and/or an effective dosage regimen for administration of an iRNA agent of the disclosure. The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be local (e.g., by intramuscular injection) or parenteral. Parenteral administration 20 includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not 25 limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like. Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other30 suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non- parenteral administration which do not deleteriously react with nucleic acids can be used. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like. 35 The compositions of the present disclosure can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain 337 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations 5 can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation. Aqueous suspensions can contain substances which increase the viscosity of the suspension 10 including, for example, sodium carboxymethylcellulose, sorbitol or dextran. The suspension can also contain stabilizers. In some embodiments, pharmaceutical compositions featured in the disclosure include (a) one or more RNAi agents and (b) one or more agents which function by a non-RNAi mechanism and which are useful in treating an muscular or pulmonary disorder or disease, e.g., DMD. 15 Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred. 20 The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the disclosure lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the disclosure, the therapeutically effective 25 dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately 30 determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. In addition to their administration, as discussed above, the RNAi agents featured in the disclosure can be administered in combination with other known agents effective in treatment of pathological processes mediated by nucleotide repeat expression. In any event, the administering physician can adjust 35 the amount and timing of RNAi agent administration on the basis of results observed using standard measures of efficacy known in the art or described herein. VI. Kits 338 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In certain aspects, the instant disclosure provides kits that include a suitable container containing a pharmaceutical formulation of a siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA 5 compound, or ssiRNA compound, or precursor thereof). Such kits include one or more dsRNA agent(s) and instructions for use, e.g., instructions for administering a prophylactically or therapeutically effective amount of a dsRNA agent(s) provided herein. The dsRNA agent may be in a vial or a pre-filled syringe. The kits may optionally further comprise means for administering the dsRNA agent (e.g., an injection device, such as a pre-filled syringe 10 ), or means for measuring the inhibition of the target gene (e.g., means for measuring the inhibition of the target gene mRNA, target gene protein, and/or target gene activity). Such means for measuring the inhibition of the target gene may comprise a means for obtaining a sample from a subject, such as, e.g., a plasma sample. The kits of the disclosure may optionally further comprise means for determining the therapeutically effective or prophylactically effective amount. 15 In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for a siRNA compound preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can 20 be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device. X. Methods of the Disclosure 25 Another aspect of the disclosure relates to a method of reducing the expression of a target gene in a cell, comprising contacting the cell with a dsRNA agent of the disclosure. The methods include contacting the cell with a dsRNA of the disclosure and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcripts of a target gene, thereby inhibiting expression of the target gene in the cell. 30 Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of a target may be determined by determining the mRNA expression level of the target gene using methods routine to one of ordinary skill in the art, e.g., northern blotting, qRT-PCR; by determining the protein level of a target protein using methods routine to one of ordinary skill in the art, such as western blotting, immunological techniques. 35 In the methods of the disclosure the extrahepatic cell, e.g., muscle cell, e.g., the skeletal muscle cell and/or cardiac muscle cell, may be contacted in vitro or in vivo, i.e., the cell may be within a subject. Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a 339 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO subject, e.g., a human subject, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting a cell are also possible. A cell suitable for treatment using the methods of the disclosure may be any cell that expresses a target gene. A cell suitable for use in the methods of the disclosure may be a mammalian cell, e.g., a 5 primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a rat cell, or a mouse cell. In one embodiment, the cell is a human cell, e.g., a human muscle cell. Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. 10 The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition. In certain embodiments, a level of inhibition, e.g., for an RNAi agent of the instant disclosure, can be assessed in cell culture conditions, e.g., wherein cells in cell culture are transfected via LipofectamineTM-mediated transfection at a concentration in the vicinity of a cell of 10 nM or less, 1 nM or less, etc. Knockdown of15 a given RNAi agent can be determined via comparison of pre-treated levels in cell culture versus post- treated levels in cell culture, optionally also comparing against cells treated in parallel with a scrambled or other form of control RNAi agent. Knockdown in cell culture of, e.g., 50% or more, can thereby be identified as indicative of “inhibiting” or “reducing”, “downregulating” or “suppressing”, etc. having occurred. It is expressly contemplated that assessment of targeted mRNA or encoded protein levels (and 20 therefore an extent of “inhibiting”, etc. caused by a RNAi agent of the disclosure) can also be assessed in in vivo systems for the RNAi agents of the instant disclosure, under properly controlled conditions as described in the art. The phrase “inhibiting expression of a target gene” or “inhibiting expression of a target,” as used herein, includes inhibition of expression of any target gene (such as, e.g., a mouse target gene, a rat target 25 gene, a monkey target gene, or a human target gene) as well as variants or mutants of a target gene that encode a target protein. Thus, the target gene may be a wild-type target gene, a mutant target gene , or a transgenic target gene in the context of a genetically manipulated cell, group of cells, or organism. “Inhibiting expression of a target gene” includes any level of inhibition of a target gene, e.g., at least partial suppression of the expression of a target gene, such as an inhibition by at least 20%. In 30 certain embodiments, inhibition is by at least 30%, at least 40%, at least 50%, at least about 60%, at least 70%, at least about 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; or to below the level of detection of the assay method. In certain method, inhibition is measured at a 10 nM concentration of the siRNA using a luciferase assay. The expression of a target gene may be assessed based on the level of any variable associated 35 with target gene expression, e.g., target mRNA level or target protein level. Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized 340 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control). In some embodiments of the methods of the disclosure, expression of a target gene is inhibited by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95%, or to below the level of detection of 5 the assay. In certain embodiments, the methods include a clinically relevant inhibition of expression of a target gene, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of a target gene. Inhibition of the expression of a target gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample 10 derived from a subject) in which a target gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with a RNAi agent of the disclosure, or by administering a RNAi agent of the disclosure to a subject in which the cells are or were present) such that the expression of a target gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s) not treated with a RNAi agent or not 15 treated with a RNAi agent targeted to the genome of interest). The degree of inhibition may be expressed in terms of: (mRNAincontrolcells) - (mRNAin treated cells) ^100 % (mRNAincontrol cells) In other embodiments, inhibition of the expression of a target gene may be assessed in terms of a reduction of a parameter that is functionally linked to a target gene expression, e.g., target protein 20 expression. Target gene silencing may be determined in any cell expressing a target gene, either endogenous or heterologous from an expression construct, and by any assay known in the art. Inhibition of the expression of a target protein may be manifested by a reduction in the level of the target protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for the assessment of genome suppression, the 25 inhibiton of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells. A control cell or group of cells that may be used to assess the inhibition of the expression of a target gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the disclosure. For example, the control cell or group of cells may be derived from an individual subject 30 (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent. The level of target gene mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing RNA expression. In one embodiment, the level of expression of target gene in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the target gene. RNA may be extracted from cells using RNA extraction 35 techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasyTM RNA preparation kits (Qiagen®) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, 341 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating target mRNA may be detected using methods the described in WO2012/177906, the entire contents of which are hereby incorporated herein by reference. In some embodiments, the level of expression of target gene is determined using a nucleic acid 5 probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific target nucleic acid or protein, or fragment thereof. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules. 10 Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of RNA levels involves contacting the isolated RNA with a nucleic acid molecule (probe) that can hybridize to target RNA. In one embodiment, the RNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated RNA on an agarose gel 15 and transferring the RNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the RNA is contacted with the probe(s), for example, in an Affymetrix® gene chip array. A skilled artisan can readily adapt known RNA detection methods for use in determining the level of target mRNA. An alternative method for determining the level of expression of target in a sample involves the 20 process of nucleic acid amplification or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, US Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase 25 (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., US Patent No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the disclosure, the level of expression of target is determined by quantitative30 fluorogenic RT-PCR (i.e., the TaqManTM System), by a Dual-Glo® Luciferase assay, or by other art- recognized method for measurement of target expression or mRNA level. The expression level of target mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See US Patent Nos.5,770,722, 35 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of target expression level may also comprise using nucleic acid probes in solution. 342 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In some embodiments, the level of RNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of this PCR method is described and exemplified in the Examples presented herein. Such methods can also be used for the detection of target nucleic acids. The level of target protein expression may be determined using any method known in the art for 5 the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent 10 assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like. Such assays can also be used for the detection of proteins indicative of the presence or replication of target proteins. In some embodiments, the efficacy of the methods of the disclosure in the treatment of a target gene-related disease is assessed by a decrease in target mRNA level (e.g, by assessment of a blood target gene level, or otherwise). 15 In some embodiments, the efficacy of the methods of the disclosure in the treatment of a target gene-related disease is assessed by a decrease in target mRNA level (e.g, by assessment of a muscle sample for target level, by biopsy, or otherwise). In some embodiments of the methods of the disclosure, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. The inhibition of expression of target may be assessed using measurements of the level or 20 change in the level of target mRNA or target protein in a sample derived from a specific site within the subject, e.g., muscle cells. In certain embodiments, the methods include a clinically relevant inhibition of expression of target, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of target gene. As used herein, the terms detecting or determining a level of an analyte are understood to mean 25 performing the steps to determine if a material, e.g., protein, RNA, is present. As used herein, methods of detecting or determining include detection or determination of an analyte level that is below the level of detection for the method used. The in vivo methods of the disclosure may include administering to a subject a composition containing a RNAi agent, where the RNAi agent includes a nucleotide sequence that is complementary to 30 at least a part of an RNA transcript of the taregt gene of the subject to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by means including, but not limited to intraperitoneal or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are 35 administered by subcutaneous injection. In certain embodiments, the compositions are administered by pulmonary delivery, e.g., oral inhalation or intranasal delivery. In some embodiments, the administration is via a depot injection. A depot injection may release the RNAi agent in a consistent way over a prolonged time period. Thus, a depot injection may reduce the 343 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of the target gene, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In certain embodiments, the depot injection is a subcutaneous injection. 5 In one embodiment, the double-stranded RNAi agent is administered by pulmonary sytem administration, e.g., intranasal administration or oral inhalative administration. Pulmonary system administration may be via a syringe, a dropper, atomization, or use of device, e.g., a passive breath driven or active power driven single/-multiple dose dry powder inhaler (DPI) device. The mode of administration may be chosen based upon whether local or systemic treatment is 10 desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting. In one aspect, the present disclosure also provides methods for inhibiting the expression of a target gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets a target gene in a cell of the mammal and maintaining the mammal for a time 15 sufficient to obtain degradation of the RNA transcript of the target gene, thereby inhibiting expression of the target gene in the cell. Reduction in genome expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. The present disclosure further provides methods of treatment of a subject in need thereof. The 20 treatment methods of the disclosure include administering an RNAi agent of the disclosure to a subject, e.g., a subject that would benefit from inhibition of target gene expression, in a therapeutically effective amount of a RNAi agent targeting a target gene or a pharmaceutical composition comprising a RNAi agent targeting a target gene. Target genes (described above), target gene-associated disorders, and subjects that would benefit 25 from a reduction or inhibition of target gene expression, e..g., those having a target gene-associated disease, subjects at risk of developing a target gene-associate disease, are described below. An RNAi agent of the disclosure may be administered as a “free RNAi agent.” A free RNAi agent is administered in the absence of a pharmaceutical composition. The naked RNAi agent may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or 30 phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the RNAi agent can be adjusted such that it is suitable for administering to a subject. In certain embodiments, the free RNAi agent may be formulated in water or normal saline. Alternatively, an RNAi agent of the disclosure may be administered as a pharmaceutical 35 composition, such as a dsRNA liposomal formulation. In one aspect, the present disclosure provides a method of treating a subject having a muscle disorder, e.g., a skeletal muscle disorder and/or a cardiac muscle disorder, comprising administering to 344 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO the subject a therapeutically effective amount of a dsRNA agent of the disclosure, thereby treating the subject. Exemplary muscle disorders include Myostatin-related muscle hypertrophy, congenital myasthenic syndrome, facioscapulohumeral muscular dystrophy (FSHD), Spinal Muscular Atrophy 5 (SMA), Myotonic Dystrophy Type 1 (DM1), Pompe disease, PLN cardiomyopathy, spasticity, obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic cardiomyopathy (FHC); Heart failure with preserved ejection fraction (HFPEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina; myocardial infarction (MI); heart failure or heart failure with reduced ejection fraction (HFREF); supraventricular tachycardia (SVT); hypertrophic cardiomyopathy (HCM); and PLN 10 cardiomyopathy. Exemplary cardiac muscle disorders include obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic cardiomyopathy (FHC); Heart failure with preserved ejection fraction (HFPEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina; myocardial infarction (MI); heart failure or heart failure with reduced ejection fraction (HFREF); supraventricular tachycardia (SVT); 15 hypertrophic cardiomyopathy (HCM); and PLN cardiomyopathy. Exemplary skeletal muscle disorders include Myostatin-related muscle hypertrophy, congenital myasthenic syndrome, facioscapulohumeral muscular dystrophy (FSHD), Spinal Muscular Atrophy (SMA), Myotonic Dystrophy Type 1 (DM1), Pompe disease, PLN cardiomyopathy, and spasticity. The dsRNA agent of the disclosure can be delivered to a subject by a variety of routes, 20 depending on the type of genes targeted and the type of disorders to be treated. In some embodiments, the dsRNA agent is administered extra-hepatically, such as intravenous, intramuscular, or subcutaneous administration. The disclosure further provides methods for the use of a RNAi agent or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction or inhibition of target 25 gene expression, e.g., a subject having a target-gene-associated disorder, in combination with other pharmaceuticals or other therapeutic methods, e.g., with known pharmaceuticals or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. Examples of the additional therapeutic agents which can be used with an RNAi agent of the disclosure include, but are not limited to, cardiovascular diseases-treating agents, anti-hyperlipemic 30 agents, hypotensive or antihypertensive agents, chemotherapeutic agents, immunotherapeutic agents, immunosuppressive agents, nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, corticosteroids, and the like. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies. 35 Anti-hyperlipemic agents include, for example, statin-based compounds which are cholesterol synthesis inhibitors (e.g., pravastatin, simvastatin, lovastatin, atorvastatin, fluvastatin, rosuvastatin and the like), squalene synthetase inhibitors or fibrate compounds having a triglyceride-lowering effect (e.g., 345 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO fenofibrate, gemfibrozil, bezafibrate, clofibrate, sinfibrate, clinofibrate and the like), niacin, PCSK9 inhibitors, triglyceride lowing agents or cholesterol sequesting agents. Hypotensive agents include, for example, angiotensin converting enzyme inhibitors (e.g., captopril, enalapril, delapril, benazepril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, 5 perindopril, quinapril, ramipril, trandolapril and the like) or angiotensin II antagonists (e.g., losartan, candesartan cilexetil, olmesartan medoxomil, eprosartan, valsartan, telmisartan, irbesartan, tasosartan, pomisartan, ripisartan forasartan, and the like) or calcium channel blockers (e.g., amlodipine) or aspirin. In addition, agents whose cachexia improving effect has been established in an animal model or at a clinical stage, such as cyclooxygenase inhibitors (e.g., indomethacin and the like), progesterone10 derivatives (e.g., megestrol acetate), glucosteroid (e.g., dexamethasone and the like), metoclopramide- based agents, tetrahydrocannabinol-based agents, lipid metabolism improving agents (e.g., eicosapentanoic acid and the like), growth hormones, IGF-1, antibodies against TNF-α, LIF, IL-6 and oncostatin M may also be employed concomitantly with an RNAi agent according to the present disclosure. Additional therapeutic agents for use in the treatment of diseases or conditions related to 15 metabolic disorders and/or impaired neurological signaling would be apparent to the skilled artisan and are within the scope of this disclosure. The RNAi agent and additional therapeutic agents may be administered at the same time or in the same combination, or the additional therapeutic agent can be administered as part of a separate composition or at separate times or by another method known in the art or described herein. 20 In one embodiment, the method includes administering a composition featured herein such that expression of the target gene is decreased, for at least one month. In some embodiments, expression is decreased for at least 2 months, 3 months, or 6 months. In certain embodiments, administration includes a loading dose administered at a higher frequency, e.g., once per day, twice per week, once per week, for an initial dosing period, e.g., 2-4 doses. 25 In some embodiments, the RNAi agents useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target gene. Compositions and methods for inhibiting the expression of these genes using RNAi agents can be prepared and performed as described herein. Administration of the dsRNA according to the methods of the disclosure may result in a 30 reduction of the severity, signs, symptoms, or markers of such diseases or disorders in a patient with a target gene-associated disorder. By “reduction” in this context is meant a statistically significant or clinically significant decrease in such level. The reduction can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%. 35 Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one 346 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of a RNAi agent targeting a target gene of interest or 5 pharmaceutical composition thereof, "effective against" a target gene-associated disorder indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating target gene-associated disorders and the related causes. 10 A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and at least 20%, 30%, 40%, 50%, or more can be indicative of effective treatment. Efficacy for a given RNAi agent drug or formulation of that drug can also be judged using an 15 experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed. Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading 20 scale. Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using a RNAi agent or RNAi agent formulation as described herein. Subjects can be administered a therapeutic amount of dsRNA, such as about 0.01 mg/kg to about 200 mg/kg. 25 The RNAi agent can be administered over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. Administration of the RNAi agent can reduce target gene levels, e.g., in a cell, tissue, blood sample or other compartment of the patient by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70,% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least about 99% or more. In one embodiment, administration of 30 the RNAi agent can reduce target gene levels, e.g., in a cell, tissue, blood sample, or other compartment of the patient by at least 50%. Before administration of a full dose of the RNAi agent, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as 35 increased cytokine (e.g., TNF-alpha or INF-alpha) levels. Alternatively, the RNAi agent can be administered by oral administration, pulmonary admistration, intravenously, i.e., by intravenous injrection, or subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired, e.g., monthly dose of RNAi agent to 347 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO a subject. The injections may be repeated over a period of time. The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimine may include administration of a therapeutic amount of RNAi agent on a regular basis, such as monthly or extending to once a quarter, twice per year, 5 once per year. In certain embodiments, the RNAi agent is administered about once per month to about once per quarter (i.e., about once every three months). XI. Target Genes and Target-Gene-Associated Diseases Specific exemplary target genes that mediate a muscle disorder, e.g., a skeletal muscle disorder 10 and/or cardiac muscle disorder, include, but are not limited to, adrenoceptor beta 1 (ADRB1); calcium voltage-gated channel subunit alpha1 C (CACNA1C); calcium voltage-gated channel subunit alpha1 G (CACNA1G) (T type calcium cchannel); angiotensin II receptor type 1(AGTR1); Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1 (HCN1); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4);15 Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3 (HCN3); Potassium Voltage- Gated Channel Subfamily A Member 5 (KCNA5); Potassium Inwardly Rectifying Channel Subfamily J Member 3 (KCNJ3); Potassium Inwardly Rectifying Channel Subfamily J Member 4 (KCNJ4); phospholamban (PLN); calcium/calmodulin dependent protein kinase II delta (CAMK2D); or Phosphodiesterase 1 (PDE1), myostatin (MSTN); Cholinergic Receptor Nicotinic Alpha 1 Subunit 20 (CHRNA1); Cholinergic Receptor Nicotinic Beta 1 Subunit (CHRNB1); Cholinergic Receptor Nicotinic Delta Subunit (CHRND); Cholinergic Receptor Nicotinic Epsilon Subunit (CHRNE); Cholinergic Receptor Nicotinic Gamma Subunit (CHRNG); Collagen Type XIII Alpha 1 Chain (COL13A1); Docking Protein 7 (DOK7); LDL Receptor Related Protein 4 (LRP4); Muscle Associated Receptor Tyrosine Kinase (MUSK); Receptor Associated Protein Of The Synapse (RAPSN); Sodium Voltage- 25 Gated Channel Alpha Subunit 4 (SCN4A); Double Homeobox 4 (DUX4), dystrophy myotonic protein kinase (DMPK), glycogen synthase 1 (GYS1), survival of motor neuron 1 (SMN1), and alpha- glucosidase (GAA). Exemplary target genes that mediate a skeletal muscle disorder include, but are not limited to, myostatin (MSTN); Cholinergic Receptor Nicotinic Alpha 1 Subunit (CHRNA1); Cholinergic Receptor 30 Nicotinic Beta 1 Subunit (CHRNB1); Cholinergic Receptor Nicotinic Delta Subunit (CHRND); Cholinergic Receptor Nicotinic Epsilon Subunit (CHRNE); Cholinergic Receptor Nicotinic Gamma Subunit (CHRNG); Collagen Type XIII Alpha 1 Chain (COL13A1); Docking Protein 7 (DOK7); LDL Receptor Related Protein 4 (LRP4); Muscle Associated Receptor Tyrosine Kinase (MUSK); Receptor Associated Protein Of The Synapse (RAPSN); Sodium Voltage-Gated Channel Alpha Subunit 4 35 (SCN4A); and Double Homeobox 4 (DUX4), dystrophy myotonic protein kinase (DMPK), glycogen synthase 1 (GYS1), survival of motor neuron 1 (SMN1), and alpha-glucosidase (GAA). Exemplary target genes that mediate a cardiac muscle disorder include, but are not limited to, adrenoceptor beta 1 (ADRB1); calcium voltage-gated channel subunit alpha1 C (CACNA1C); calcium 348 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO voltage-gated channel subunit alpha1 G (CACNA1G) (T type calcium cchannel); angiotensin II receptor type 1(AGTR1); Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1 (HCN1); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4); Hyperpolarization Activated Cyclic Nucleotide Gated 5 Potassium Channel 3 (HCN3); Potassium Voltage-Gated Channel Subfamily A Member 5 (KCNA5); Potassium Inwardly Rectifying Channel Subfamily J Member 3 (KCNJ3); Potassium Inwardly Rectifying Channel Subfamily J Member 4 (KCNJ4); phospholamban (PLN); calcium/calmodulin dependent protein kinase II delta (CAMK2D); or Phosphodiesterase 1 (PDE1). In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets 10 ADRB1 for the treatment of obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic cardiomyopathy (FHC); heart failure with preserved ejection fraction (HF-pEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina; myocardial infarction (MI); and/or heart failure or heart failure with reduced ejection fraction (HFREF). In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets 15 CACNA1C for the treatment of supraventricular tachycardia (SVT); AFIB; Angina; and/or HOCM. In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets CACNA1G for the treatment of supraventricular tachycardia (SVT); and/or angina. In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets AGTR1 for the treatment of HOCM; hypertrophic cardiomyopathy (HCM); and/or HF-pEF. 20 In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets SCN2A for the prevention and/or treatment of AFIB. In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets HCN1 for the prevention and/or treatment of AFIB; treatment, e.g., rate control, in HOCM. In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets 25 HCN4 for the prevention and/or treatment of AFIB; treatment, e.g., rate control, in HOCM. In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets HCN3 for the prevention and/or treatment of AFIB; treatment, e.g., rate control, in HOCM. In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets KCNA5 for the prevention and/or treatment of AFIB, e.g., AFIB in congestive heart failure (CHF). 30 In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets KCNJ3 for the prevention and/or treatment of AFIB. In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets KCNJ4 for the prevention and/or treatment of AFIB. In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets 35 CAMK2D for the prevention and/or treatment of heart failure and/or AFIB. In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets PLN for the prevention and/or treatment of HF-rEF, arrhythmia, and/or cardiomyopathy.In some 349 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO embodiments, the present disclosure provides a double-stranded iRNA agent that targets PDE1 for the prevention and/or treatment of CHF and/or HF-pEF. In some embodiment, , the present disclosure provides a double-stranded iRNA agent that targets myostatin for the treatment of Myostatin-related muscle dystrophy. 5 In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets CHRNA1 for the treatment of congenital myasthenic syndrome (CMS). In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets CHRNB1 for the treatment of congenital myasthenic syndrome (CMS). In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets 10 CHRBD for the treatment of congenital myasthenic syndrome (CMS). In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets CHRNE for the treatment of congenital myasthenic syndrome (CMS). In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets CHRNG for the treatment of congenital myasthenic syndrome (CMS). 15 In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets COL13A1 for the treatment of congenital myasthenic syndrome (CMS). In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets LRP4 for the treatment of congenital myasthenic syndrome (CMS). In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets 20 MUSK for the treatment of congenital myasthenic syndrome (CMS). In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets RAPSN for the treatment of congenital myasthenic syndrome. In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets SCN4A for the treatment of congenital myasthenic syndrome (CMS). 25 In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets DOK7 for the treatment of congenital myasthenic syndrome (CMS). In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets DUX4 for the treatment of Facioscapulohumeral muscular dystrophy (FSHD). In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets 30 DMPK for the treatment of myotonic dystrophy. In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets GYS1 for the treatment of glycogen storage disease. In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets SMN1 for the treatment of spinal muscular atrophy. 35 In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets GAA for the treatment of Pompe disease. In some embodiments, the present disclosure provides a double-stranded iRNA agent that targets ADRB1 for the treatment of obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic 350 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO cardiomyopathy (FHC); heart failure with preserved ejection fraction (HF-pEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina; myocardial infarction (MI); and/or heart failure or heart failure with reduced ejection fraction (HFREF). 5 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the RNAi agents and methods featured in the disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are 10 incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. This disclosure is further illustrated by the following examples which should not be construed as 15 limiting. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the informal Sequence Listing and Figures, are hereby incorporated herein by reference. 351 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO EXAMPLES Example 1: Design and Synthesis of ^v^6 Small Molecule Targeting Ligands Scheme 1 5 Compound 2: To a mixture of PPh3 (237 g, 904 mmol, 1.30 eq) and imidazole (61.6 g, 904 mmol, 1.30 eq) in DCM (700 mL) was added I2 (230 g, 904 mmol, 182 mL, 1.30 eq) portion-wise at 0-5 °C. The reaction mixture was stirred at 0-5 °C for 0.5 hrs. To this reaction mixture was added to a solution of compound 1 (140 g, 695.61 mmol, 1.00 eq) in DCM (700 mL) dropwise below 15 °C and stirred the 10 mixture overnight (12 hrs). TLC (Petroleum ether/Ethyl acetate = 0/1, Rf = 0.84) and Petroleum ether/Ethyl acetate = 5/1 indicated no compound 1 was remained, and one major new spot with lower polarity was detected (Rf = 0.43). The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=100/1 to 5/1). Compound 2 (203 g, 652 mmol, 93.8% yield) was obtained as a 15 yellow liquid. Compound 4: To a mixture of compound 2 (48.0 g, 154 mmol, 1.00 eq) and compound 3 (21.1 g, 147 mmol, 0.950 eq) in THF (740 mL) was added LiHMDS (1 M, 154 mL, 1.00 eq) drop-wise in one portion at 0 °C under N2. The mixture was stirred at 0 °C for 3 hrs. TLC (Dichloromethane/Methanol = 20/1, Rf = 0.43) indicated some of compound 2 was remained, and one major new spot with larger polarity was 20 detected. The reaction mixture was quenched by saturated solution of NH4Cl 500 mL at 25 °C, and then diluted with H2O 200 mL and extracted with EtOAc 600 mL (200 mL x 3). The combined organic layers were washed with brine 200 mL (100 mL x 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 100/1 to 3/1) and reversed-phase HPLC. Compound 4 (57.0 g, 174 25 mmol, 37.6% yield) was obtained as a brown oil with 3 batches. Compound 5: To a solution of compound 4 (28.5 g, 87.0 mmol, 1.00 eq) in DCM (50 mL) was added HCl/dioxane (4 M, 109 mL, 5.00 eq) in one portion at 25 °C. The mixture was stirred at 25 °C for 1 hr. TLC (Dichloromethane/Methanol = 20/1, Rf = 0) and LCMS indicated no compound 4 was remained, and one major new spot with larger polarity was detected. The reaction mixture was concentrated under 352 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO reduced pressure to give a residue. The crude product was used into the next step without further purification. Compound 5 (53.0 g, crude, 2HCl) was obtained as a brown solid. 1H NMR: MeOD, 400 MHz, 9.32 (dd, J = 1.7, 5.1 Hz, 1H), 9.13 (dd, J = 1.8, 8.3 Hz, 1H), 8.93 (d, J = 8.6 Hz, 1H), 8.16 - 8.05 (m, 2H), 3.56 (dd, J = 7.6, 11.6 Hz, 1H), 3.46 (ddd, J = 4.0, 8.3, 11.9 Hz, 1H), 3.36 (br d, J = 2.2 Hz, 5 1H), 3.32 - 3.23 (m, 2H), 3.03 - 2.94 (m, 1H), 2.56 - 2.42 (m, 1H), 2.41 - 2.28 (m, 1H), 2.25 - 2.02 (m, 2H), 1.79 (qd, J = 9.0, 12.9 Hz, 1H). LCMS (ESI) calculated for C14H17N3 [M+H]+ m/z = 228.14, found 228.0. Compound 6: To a mixture of compound 5 (26.5 g, 88.3 mmol, 1.00 eq, 2HCl) and compound 5B (18.2 g, 115 mmol, 1.30 eq) in DCM (265 mL) was added KOAc (30.3 g, 310 mmol, 3.50 eq) and 10 Pd(dppf)Cl2.CH2Cl2 (7.21 g, 8.83 mmol, 0.100 eq) in one portion at 25 °C under N2. The mixture was stirred at 25 °C for 2 hrs under N2. TLC (Dichloromethane/Methanol = 10/1, Rf = 0.31) indicated no compound 5 was remained, and one major new spot with lower polarity was detected. LCMS indicated desired MS. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was resolved in H2O (100 mL). The mixture was acidified by 2N HCl until PH = 4. The 15 mixture was washed by EtOAc 300 mL (100 mL x 3). The aqueous phase was basified by saturated solution of Na2CO3 until PH>8 and extracted with DCM 300 mL (100 mL x 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was used into the next step without further purification. Compound 6 (52.0 g, 160 mmol, 90.5% yield) was obtained as a brown solid.1H NMR: CDCl3, 400 MHz, 9.08 (dd, J = 2.0, 4.3 Hz, 1H), 8.16 (dd, J = 2.0, 8.1 Hz, 1H), 20 8.10 (d, J = 8.3 Hz, 1H), 7.45 (dd, J = 4.2, 8.0 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 6.99 (td, J = 6.1, 15.7 Hz, 1H), 5.99 (td, J = 1.5, 15.7 Hz, 1H), 3.74 (s, 3H), 3.31 - 3.17 (m, 2H), 3.12 - 3.00 (m, 2H), 2.92 - 2.85 (m, 1H), 2.75 - 2.66 (m, 1H), 2.48 (dt, J = 6.1, 8.7 Hz, 1H), 2.33 - 2.16 (m, 2H), 2.13 - 1.92 (m, 4H), 1.60 - 1.42 (m, 1H). LCMS (ESI) calculated for C19H23N3O2 [M+H]+ m/z = 325.18, found 326.3. Compound 7A: To a mixture of compound 6 (20.0 g, 61.5 mmol, 1.00 eq), KOH (3.62 g, 64.5 mmol, 25 1.05 eq), (R)-BINAP (3.83 g, 6.15 mmol, 0.100 eq) and compound 6B (34.1 g, 70.7 mmol, 1.15 eq) in dioxane (200 mL) and H2O (20.0 mL) was added chlororhodium;(1Z,5Z)-cycloocta-1,5-diene (1.52 g, 3.07 mmol, 0.050 eq) in one portion at 25 °C under N2. The mixture was heated to 100 °C and stirred for 4 hrs under N2. TLC (Dichloromethane/Methanol = 10/1, Rf = 0.37) indicated no compound 6 was remained, and one major new spot with lower polarity was detected. LCMS indicated desired MS. The 30 reaction mixture was concentrated under reduced pressure to remove dioxane. The residue was diluted with H2O 200 mL and extracted with DCM 450 mL (150 mL x 3). The combined organic layers were washed with brine 200 mL (100 mL x 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM/MeOH = 100/1 to 5/1) and reversed-phase HPLC. The product was further separated by SFC (condition: column:35 DAICEL CHIRALPAK AD (250mm x 50mm, 10um); mobile phase: [0.1%NH3H2O ETOH]; B%: 40%- 353 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 40%, 5.6min). Compound 7A (14.5 g, 21.3 mmol, 34.6% yield) was obtained as a yellow solid. Chiral SFC indicated its structure. LCMS (ESI) calculated for C39H51N7O4 [M+H]+ m/z = 681.4, found 682.2. Compound 7B: To a mixture of compound 6 (23.0 g, 70.7 mmol, 1.00 eq) , KOH (4.16 g, 74.2 mmol, 1.05 eq) , (S)-BINAP (4.40 g, 7.07 mmol, 0.100 eq) and compound 6B (39.2 g, 81.3 mmol, 1.15 eq) in 5 dioxane (200 mL) and H2O (20.0 mL) was added chlororhodium;(1Z,5Z)-cycloocta-1,5-diene (1.74 g, 3.53 mmol, 0.050 eq) in one portion at 25 °C under N2. The mixture was heated to 100 °C and stirred for 4 hrs under N2. TLC (Dichloromethane/Methanol=10/1, Rf = 0.43) indicated no compound 6 was remained, and one major new spot with lower polarity was detected. The reaction mixture was concentrated under reduced pressure to remove dioxane. The residue was diluted with H2O 200 mL and 10 extracted with DCM 450 mL (150 mL x 3). The combined organic layers were washed with brine 200 mL (100 mL x 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM/MeOH = 100/1 to 5/1) and revers phase HPLC. The product was further separated by SFC (condition: column: DAICEL CHIRALPAK AD (250mm x 50mm, 10um); mobile phase: [0.1%NH3H2O ETOH] B%: 40%-40% 5.2 min). 15 Compound 7B (15.0 g, 22.0 mmol, 31.1% yield) was obtained as a yellow solid. Chiral SFC indicated its structure. Scheme 2 Compound 9: To a solution of compound 7A (14.5 g, 21.3 mmol, 1.00 eq) in MeOH (150 mL) was 20 added Pd/C (3.00 g, 21.3 mmol, 10% purity, 1.00 eq) in one portion at 25 °C. The mixture was stirred at 25 °C for 12 hrs under H2 (30 psi). TLC (Dichloromethane/Methanol = 10/1, Rf = 0.27) indicated no compound 7A was remained, and one major new spot with larger polarity was detected. LCMS indicated desired MS. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC. Compound 9 (10.5 g, 15.3 mmol, 72.0% yield, 100% purity) 25 was obtained as a yellow solid.1H NMR: MeOD, 400 MHz, 7.11 (d, J = 7.3 Hz, 1H), 6.94 (s, 1H), 6.86 (s, 1H), 6.77 (s, 1H), 6.35 (d, J = 7.3 Hz, 1H), 6.06 (s, 1H), 3.57 (s, 7H), 3.41 - 3.34 (m, 3H), 3.25 - 3.17 (m, 4H), 2.93 - 2.77 (m, 3H), 2.75 - 2.66 (m, 3H), 2.65 - 2.56 (m, 2H), 2.54 - 2.38 (m, 3H), 2.26 (d, J = 6.7 Hz, 6H), 2.21 - 2.13 (m, 1H), 2.13 - 2.03 (m, 1H), 2.02 - 1.92 (m, 1H), 1.91 - 1.83 (m, 2H), 1.67 (q, J 354 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO = 7.5 Hz, 2H), 1.50 (s, 9H), 1.45 - 1.36 (m, 1H). LCMS (ESI) calculated for C39H55N7O4 [M+H]+ m/z = 685.43, found 686.3. Compound 10: To a solution of 9 (1.50 g, 2.19 mmol, 1.00 eq) in DCM (10.0 mL) was added HCl/dioxane (4 M, 2.73 mL, 5.00 eq) at 25 °C. The reaction mixture was stirred at 25 °C for 2 hrs. TLC 5 (Dichloromethane/Methanol = 10/1, Rf = 0.06) indicated no 9 was remained, and one major new spot with larger polarity was detected. The reaction mixture was concentrated under reduced pressure. The residue was resolved in water (50 mL). The aqueous phase was basified by saturated solution of Na2CO3 until PH>8 and and extracted with DCM 150 mL (50 mL x 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The crude product was used into the next step 10 without further purification. Compound 10 (1.30 g, crude) was obtained as a yellow solid. Compound 11: To a mixture of compound 10 (1.30 g, 2.22 mmol, 1.00 eq) and tetrahydropyran-2,6- dione (253 mg, 2.22 mmol, 1.00 eq) in DCM (10.0 mL) was stirred at 25 °C for 12 hrs. TLC (Dichloromethane/Methanol = 10/1, Rf = 0.01) and HPLC indicated no compound 10 was remained, and one major new spot with lower polarity was detected. The reaction mixture was concentrated under 15 reduced pressure to give a residue. Compound 11 (1.50 g, 2.14 mmol, 96.6% yield, 100% purity) was obtained as a yellow solid. 1H NMR: CDCl3, 400 MHz, 10.45 (br s, 1H), 7.18 (d, J = 7.3 Hz, 1H), 6.93 (s, 1H), 6.83 (s, 1H), 6.73 (s, 1H), 6.21 (d, J = 7.2 Hz, 1H), 5.97 (s, 1H), 3.91 - 3.81 (m, 1H), 3.77 (br d, J = 13.4 Hz, 1H), 3.73 - 3.63 (m, 3H), 3.60 (s, 3H), 3.50 - 3.41 (m, 2H), 3.37 - 3.29 (m, 1H), 3.28 - 3.19 (m, 2H), 3.18 - 3.09 (m, 1H), 2.85 - 2.52 (m, 11H), 2.39 - 2.22 (m, 9H), 2.15 - 2.02 (m, 1H), 2.01 - 1.83 20 (m, 5H), 1.68 (q, J = 7.4 Hz, 2H), 1.51 - 1.19 (m, 2H), 1.17 - 0.63 (m, 1H). LCMS (ESI) calculated for C39H53N7O5 [M+H]+ m/z = 699.41, found 700.3. Compound 13: To a solution of compound 7B (15.0 g, 22.0 mmol, 1.00 eq) in MeOH (160 mL) was added Pd/C (4.00 g, 23.5 mmol, 10% purity) in one portion at 25 °C under H2 (15 psi). The mixture was stirred at 25 °C for 12 hrs. TLC (Dichloromethane/Methanol = 10/1, Rf = 0.24) and LCMS indicated no 25 compound 7B was remained, and one major new spot with larger polarity was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition) and SFC (column: Phenomenex-Cellulose-2 (250mm x 50mm, 10um); mobile phase: [0.1%NH3H2O ETOH]; B%: 50%-50%, 10.2 min). 13 (7.00 g, 10.2 mmol, 46.4% yield, 98.8% purity) was obtained as a yellow solid. 1H NMR: MeOD, 400 MHz, 7.11 (d, J = 7.3 Hz, 1H), 30 6.94 (s, 1H), 6.86 (s, 1H), 6.78 (s, 1H), 6.33 (d, J = 7.4 Hz, 1H), 6.06 (s, 1H), 3.57 (s, 6H), 3.40 - 3.34 (m, 3H), 3.25 - 3.16 (m, 4H), 2.89 - 2.44 (m, 12H), 2.26 (d, J = 6.9 Hz, 6H), 2.14 - 2.04 (m, 2H), 2.01 - 1.84 (m, 3H), 1.66 (q, J = 7.4 Hz, 2H), 1.50 (s, 9H), 1.45 - 1.36 (m, 1H). LCMS (ESI) calculated for C39H55N7O4 [M+H]+ m/z = 685.43, found 686.3. Compound 14: To a solution of 13 (1.00 g, 1.46 mmol, 1.00 eq) in DCM (2.00 mL) was added 35 HCl/dioxane (4 M, 1.82 mL, 5.00 eq) at 25 °C. The reaction mixture was stirred at 25 °C for 2 hrs. TLC 355 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (Dichloromethane/Methanol = 10/1, Rf = 0.06) indicated no 13 was remained, and one major new spot with larger polarity was detected. The reaction mixture was concentrated under reduced pressure. The residue was resolved in H2O 50.0 mL. The aqueous phase was basified by saturated solution of Na2CO3 until PH>8 and extracted with DCM 150 mL (50.0 mL x 3), dried over Na2SO4, filtered and concentrated 5 under reduced pressure to give a residue. The crude product was used into the next step without further purification. Compound 14 (0.860 g, crude) was obtained as a yellow solid. Compound 15: To a mixture of compound 14 (860 mg, 1.47 mmol, 1.00 eq) and tetrahydropyran-2, 6- dione (168 mg, 1.47 mmol, 1.00 eq) in DCM (10 mL) was stirred at 25 °C for 12 hrs. TLC (Dichloromethane/Methanol = 10/1, Rf = 0.01) and HPLC indicated no compound 14 was remained, and 10 one major new spot with larger polarity was detected. The reaction mixture was concentrated under reduced pressure to give a residue. 15 (1.10 g, crude, 99.3% purity) was obtained as a yellow solid. 1H NMR: CDCl3, 400 MHz, 10.86 - 10.16 (m, 1H), 7.18 (d, J = 7.3 Hz, 1H), 6.88 (s, 1H), 6.82 (s, 1H), 6.70 (s, 1H), 6.19 (d, J = 7.3 Hz, 1H), 5.98 (s, 1H), 3.95 - 3.84 (m, 1H), 3.83 - 3.68 (m, 2H), 3.60 (s, 3H), 3.45 (br t, J = 5.5 Hz, 2H), 3.41 - 3.32 (m, 2H), 3.29 - 3.20 (m, 2H), 3.18 - 3.07 (m, 1H), 2.79 - 2.58 (m, 8H), 15 2.57 - 2.45 (m, 4H), 2.36 (dt, J = 3.3, 6.5 Hz, 3H), 2.28 (d, J = 10.7 Hz, 6H), 2.13 - 2.02 (m, 1H), 2.00 - 1.85 (m, 5H), 1.71 (td, J = 7.0, 13.9 Hz, 1H), 1.65 - 1.55 (m, 1H), 1.37 (br dd, J = 6.6, 12.0 Hz, 1H), 1.30 - 1.24 (m, 1H), 0.97 - 0.69 (m, 1H). LCMS (ESI) calculated for C39H53N7O5 [M+H]+ m/z = 699.41, found 700.3. Scheme 3 20 Compound 5B: KOAc (40.3 g, 410 mmol, 1.05 eq) was added to a mixture of compound 5C (70.0 g, 391 mmol, 46.0 mL, 1.00 eq) in MeCN (350 mL) at 25 °C. The mixture was stirred at 65 °C for 12 hrs. 356 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO TLC (Petroleum ether/Ethyl acetate = 5/1, Rf = 0.45) showed that the reaction was complete. The mixture was concentrated to move the solvent. The residue was diluted with water (100 mL) and extracted with EtOAc (60.0 mL x 3). The combine organic layer was dried over Na2SO4 and concentrated. Compound 5B (59.2 g, 374 mmol, 95.7% yield) was obtained as yellow oil. The product 5 was used for the next step directly. 1H NMR : CDCl3, 400 MHz, 6.95 (td, J = 4.6, 15.8 Hz, 1H), 6.04 (td, J = 2.0, 15.8 Hz, 1H), 4.75 (dd, J = 2.0, 4.6 Hz, 2H), 3.76 (s, 3H), 2.13 (s, 3H). Compound 17: Compound 16 (250 g, 996 mmol, 1.00 eq) was dissolved in dilute HCl (4:1 in water, 1.00 L) and the mixture was cooled to 0 °C. A cooled solution of NaNO2 (75.6 g, 1.10 mol, 1.10 eq) in H2O (400 mL) was added to the mixture at 0 °C and stirred for 30 minutes at that temperature. A mixture 10 of SnCl2.2H2O (674 g, 2.99 mol, 3.00 eq) in HCl (500 mL) was added to the reaction at 0 °C and stirred at 0 °C for 1 hr. LCMS showed that the reaction was complete. The mixture was filtered and the filtrate cake was washed by water (200 mL). The filtrate cake was dried under vacuum. Compound 17 (353 g, crude, 2HCl) was obtained as yellow solid. LCMS (ESI) calculated for C6H6Br2N2 [M+H]+ m/z = 263.89, found 264.9, 266.8, 268.9. 15 Compound 19: Compound 18 (199 g, 1.99 mol, 204 mL, 2.00 eq) was added to a mixture of compound 17 (337 g, 994. mmol, 1.00 eq, 2HCl) in MeCN (1.50 L) and H2O (500 mL) at 25 °C. The mixture was stirred at 80 °C for 4 hrs. TLC (Petroleum ether/Ethyl acetate = 10/1, Rf = 0.73) and LCMS showed that the reaction was complete. The mixture was concentrated to remove MeCN. The residue was diluted with water (1.00 L) and basified to pH = 7-8 by Na2CO3. It was extracted with EtOAc (1.00 L x 3), 20 organic layer was concentrated to give the crude product. The product was purified by MPLC (100-200 mesh silica gel, Petroleum ether/Ethyl acetate = 100/1-10/1). Compound 19 (272 g, 824 mmol, 82.9% yield) was obtained as yellow solid. 1H NMR: CDCl3, 400 MHz, 7.64 - 7.62 (m, 1H), 7.59 (d, J = 1.8 Hz, 2H), 6.01 (s, 1H), 2.35 (s, 3H), 2.29 (s, 3H). LCMS (ESI) calculated for C11H10Br2N2 [M+H]+ m/z = 327.92, found 328.9, 330.8, 332.9. 25 Compound 21: Pd2(dba)3 (6.65 g, 7.26 mmol, 0.02 eq) and BINAP (13.3 g, 21.4 mmol, 0.05 eq) was added to a mixture of compound 19 (133 g, 403.01 mmol, 1 eq), compound 20 (67.5 g, 362 mmol, 0.90 eq) and t-BuONa (77.5 g, 806 mmol, 2.00 eq) in anhydrous toluene (1.30 L) at 25 °C. The mixture was stirred at 65 °C for 3 hrs under N2. TLC (Petroleum ether/Ethyl acetate = 5/1, Rf = 0.28) showed that the reaction was complete. The mixture was washed by water (150 mL) and separated. The aqueous phase 30 was extracted with EtOAc (100 mL x 2). The combined organic layer was concentrated to give the crude product. The product was purified by MPLC (100-200 mesh silica gel, Petroleum ether/Ethyl acetate=100/1-5/1). Compound 21 (96.0 g, 220 mmol, 54.7% yield) was obtained as yellow solid. Compound 6B: Pd(dppf)Cl2 (9.80 g, 13.4 mmol, 0.06 eq) was added to a mixture of compound 16 (98.0 g, 225 mmol, 1.00 eq), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2- 35 dioxaborolane (60.0 g, 236 mmol, 1.05 eq) and KOAc (44.2 g, 450 mmol, 2.00 eq) in dioxane (1.00 L) at 357 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 25-30 °C. The mixture was stirred at 80 °C for 2 hrs under N2. TLC (Petroleum ether/Ethyl acetate = 3/1, Rf = 0.26) showed that the reaction was complete. The mixture was washed by water (1.00 L) and separated. The aqueous phase was extracted with EtOAc (1.00 L x 2). The combine organic layer was concentrated to give the crude product. The product was purified by MPLC (100-200 mesh silica gel, 5 Petroleum ether/Ethyl acetate = 4/1). Compound 6B (236 g, crude) was obtained as yellow solid. LCMS (ESI) calculated for C26H39BN4O4 [M+H]+ m/z = 482.3, found 483.1. Scheme 4 Compound 23: 10 To a solution of PPh3 (50.8 g, 0.19 mol) and 1H-imidazole (13.2 g, 0.19 mol) in DCM (500 mL) at 0 °C. was slowly added iodine (49.2 g, 0.19 mol). The reaction was stirred at 0° C. for 30 mins, and then a solution of 22 (30.0 g, 0.15 mol) in DCM (100 mL) was added. The reaction was stirred at room temperature overnight, then diluted with DCM (300 mL), washed with brine (200 mL X 2). The combined organic layer was dried over Na2SO4, filtered and concentrated under vacuum. The residue 15 was purified by silica gel column (EA in PE, 0% to 15%) to give 23 (45 g，96.5% yield, 144.69 mmol) as a color oil. MS((M+H):312.2. Compound 24: To the solution of 23 (20.0 g, 64.31mmol) and benzyl acrylate (16 g, 98.77 mmol) in ACN (200 mL) was added NaBH3CN (12 g, 190.48 mmol), the mixture was stirred at 85oC for 1 hour. The reaction mixture was cooled, quenched with NH4Cl(aq) (200 mL), extracted with EA (200 mL X 2). 20 The organic layer was washed with brine (200 mL X 2), dried over Na2SO4, concentrated under vacuum and purified by silica gel chromatography (EA in PE, 0% to 15%) to afford 24 (13.7 g, 61.4% yield, 39.43 mmol) as a colorless oil. MS(M+H+-56):292.4. 358 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 25: To the solution of 24 (13.7 g, 39.43 mmol) in THF (140 mL) was added LAH (1M in THF, 103 mL, 103 mmol) at 0 oC。 The mixture was stirred at 0oC for 2 hours. The reaction mixture was quenched with Na2SO4.10H2O, filtered, washed with EA (50 mL X 3). The filtrate was concentrated under vacuum and purified by silica gel chromatography (EA in PE, 0% to 50%) to afford 25 (4.4 g, 45.85% 5 yield, 18.08 mmol) as a colorless oil. MS(M+H+-56):188.2. Compound 26: To a solution of (COCl)2 (3.81 g, 30.02 mmol) in DCM (50 mL) at -70 oC under N2 was added DMSO (4.24 g, 54.27 mmol). After stirred at -70 oC for 15 min, 25 (4.4 g, 18.08 mmol) in DCM (50 mL) was added. Then the mixture was stirred at -70 oC for 1 hour, then TEA (9.15 g, 90.42 mmol) was added. The mixture was stirred at -70 oC for 30 min, 0 oC for 30 min. The reaction mixture was quenched 10 with water (50 mL), extracted with DCM (50 mL X 2), dried over Na2SO4, concentrated under vacuum. The residue was purified by silica gel chromatography (EA in PE, 0% to 25%) to afford 26 (3.24 g, 74.5% yield, 13.44 mmol) as a colorless oil. MS(M+H+-56):186.2. Compound 27: The solution of 26 (3.24 g, 13.44 mmol), 4-methylpyridin-2-amine (1.60 g, 14.81 mmol) in DCE (80 mL) was stirred at room temperature for 1 hour. To this added STAB (8.55 g, 40.33 mmol) at 15 0oC. The mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with NaHCO3 (100 mL), extracted with DCM (50 mL X 3). The organic layer was dried over Na2SO4, concentrated under vacuum. The residue was purified by silica gel chromatography (MeOH in DCM, 0% to 10%) to afford 27 (3.2 g, 71.3% yield, 9.6 mmol) as a colorless oil. MS(M+H+):334.2. Compound 28: To a solution of 27 (3.2 g, 9.6 mmol) in MeOH (10 mL) was added 4N HCl/dioxane (30 20 mL). The mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under vacuum to give 28 (crude, 9.6 mmol) as a brown oil. MS(M+H+):234.3. Compound 29: To a solution of 5B (1.52 g, 9.60 mmol) and Pd(dppf)Cl2 (0.7 g, 0.1 mmol) in DCM (30 mL) was added a solution of 28 (crude, 9.6 mmol) and TEA (4.85 g, 48.0 mmol) in DCM (20 mL), and stirred at room temperature for 5 hours under N2. The reaction mixture was diluted with 1N HCl (60 mL), 25 extracted with DCM (50 mL X 2), discard organic phase. The aqueous was adjust pH=9 with 10% NaOH at 0 oC, extracted with DCM (50 mL X 3). The organic layer was washed with brine (50 mL X 2), dried over Na2SO4, concentrated under vacuum to afford 29 (3.2 g, 100%, 9.65 mmol) as a brown oil. MS(M+H+):332.4. Compound 30: To a solution of 29 (3.0 g, 9.05 mmol) and benzyl 4-(3-(3,5-dimethyl-1H-pyrazol-1-yl)- 30 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazine-1-carboxylate (7.01 g, 13.57 mmol) in dioxane (50 mL) was added 3.6 M NaOH (5 mL, 18.0 mmol), [Rh(COD)CI]2 (0.22 g, 0.45 mmol, CAS:12092-47-6), (R)-BINAP (0.56 g, 0.90 mmol), and stirred at 90 oC for 3 hours under N2. The reaction mixture was diluted with water (100 mL), extracted with EA (100 mL X 3). The organic layer was washed with brine (60 mL X 2), dried over Na2SO4, concentrated under vacuum and purified by reverse 359 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO chromatography (ACN in 0.1% TFA/water, 5% to 50%) to afford 30 (2.0 g, 30.6 % yield, 2.77 mmol) as a brown oil. MS(M+H+):722.6. Compound 31: To a solution of 30 (2.3 g, 3.19 mmol) in EA (30 mL) and MeOH (30 mL) was added Pd/C (10%, 0.3 g), and stirred at 50 oC for 8 hours under H2. The reaction mixture was filtered and the filtrate 5 was concentrated under vacuum to give 31 (1.5 g, 2.55 mmol) as a yellow solid. MS(M+H+):588.4. Compound 32: To a solution of 31 (1.50 g, 2.55 mmol) and TEA (0.91 g, 7.65 mmol) in DCM (20 mL) was added dihydro-2H-pyran-2,6(3H)-dione (0.35 g, 3.06 mmol) at 0oC. The mixture was stirred at 0oC for 1 hour. The mixture was concentrated under vacuum and purified by prep-HPLC (ACN in 0.1% FA/water) to give 32 (0.7 g, 0.94 mmol, 36.9% yield) as a light yellow colloid.1H NMR (400 MHz, 10 CD3OD) δ 8.46 (s, 1H), 7.74 (d, J = 5.3 Hz, 1H), 7.04 (s, 1H), 6.95 (s, 1H), 6.88 (s, 1H), 6.49 (d, J = 6.2 Hz, 2H), 6.08 (s, 1H), 3.74 (s, 4H), 3.64 (d, J = 17.7 Hz, 4H), 3.60 – 3.44 (m, 4H), 3.34 (d, J = 4.4 Hz, 1H), 3.29 – 3.21 (m, 4H), 2.88 – 2.79 (m, 2H), 2.77 – 2.65 (m, 1H), 2.51 (t, J = 7.6 Hz, 2H), 2.41 – 2.32 (m, 4H), 2.30 (s, 3H), 2.26 (s, 6H), 2.18 (dt, J = 13.4, 6.5 Hz, 1H), 1.95 – 1.83 (m, 3H), 1.68 – 1.57 (m, 3H), 1.55 – 1.35 (m, 4H). MS(M+H+):702.6. 15 Scheme 5 Compound 34: To a solution of 35 (25 g, 69.1 mmol) in DMSO (200 mL) was added tert-butyl piperazine- 1-carboxylate (12.9 g, 69.3 mmol), K2CO3 (19.1 g, 138.1 mmol), Cul (2.63 g, 13.8 mmol,) and L-proline (3.2 g, 27.8 mmol). Then the reaction was stirred at 80 °C for 2 hours under N2 atmosphere. On completion, 20 the reaction mixture was quenched with H2O (150 mL) at 25 °C and then extracted with EA (100 mLx3). The combined organic layers were washed with brine (100 mL x 2), dried over Na2S04, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (0% to 10 %, EA in PE) to give 34 (16.5 g, 56.8% yield, 39.27 mmol) as a yellow solid. MS(M+H+):462.1. 25 Compound 35: The solution of 34 (16.5 g, 39.27 mmol), (3,5-dichlorophenyl)boronic acid (7.5 g, 39.29 mmol), Pd(dppf)Cl2 (2.87 g, 3.93 mmol) and Na2CO3 (8.32 g, 78.50 mmol) in dioxane (200 mL) and water (30 mL) was stirred at 80 oC for 16 hours under N2. The mixture was diluted with water (100 mL), extracted 360 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO with EA (100 mL X 3). The organic layer was washed brine (100 mL X 2), dried over Na2S04, concentrated under vacuum. The residue was purified by reverse phase chromatography (ACN in 0.1 TFA/water, 70% to 100%) to afford 35 (6.5 g, 34.2% yield, 13.43 mmol) as a brown solid. MS(M+H+):487.3. Compound 36: The solution of 35 (6.5 g, 13.43 mmol), Pin2B2 (4.09 g, 16.10 mmol), Pd(dppf)Cl2 (1.10 5 g, 1.35 mmol) and KOAc (3.95 g, 40.25 mmol) in dioxane (100 mL) was stirred at 80 oC for 16 hours under N2. The mixture was filtered and washed with EA (60 mL X 2), the filtrate was concentrated under vacuum. The residue was purified by column chromatography (EA in PE, 0% to 20%) to afford 36 (7.05 g, 13.22 mmol, 90.84% yield) as a brown oil. MS(M+H+):533.3. Compound 37: To a solution of 36 (3.46 g, 10.50 mmol) and 6 (7.05 g, 13.22 mmol) in dioxane (100 mL) 10 was added 3.6 M NaOH (5.88 mL, 20.88 mmol), [Rh(COD)CI]2 (259 mg, 0.53 mmol, CAS:12092-47-6), (R)-BINAP (654 mg, 1.05 mmol), and stirred at 90 oC for 3 hours under N2. The reaction mixture was diluted with water (100 mL), extracted with EA (100 mL X 3). The organic layer was washed with brine (60 mL X 2), dried over Na2SO4, concentrated under vacuum, dissolved in 50 mL EtOAc and stirred under a balloon with H2 and 1 gram of 10% Pd/C for 8 hours. The mixture was filtered, concentrated, and purified 15 by reverse chromatography (ACN in 0.1% TFA/water, 5% to 40%) to afford 37 (2.3 g, 30 % yield, 3.12mmol) as a yellow solid. MS(M+H+):736.7. Compound 38: To a solution of 37 (2.0 g, 2.71 mmol) in MeOH (10 mL) was added 4N HCl/dioxane (20 mL), and stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum to give 38 (2.2, crude) as a brown solid. Not purified and used directly in the next step. MS(M+H+):636.7. 20 Compound 39: To a solution of 38 (1.0 g, 1.59 mmol) and TEA (635 mg, 6.27 mmol) in DCM (10 mL) was added dihydro-2H-pyran-2,6(3H)-dione (197 mg, 1.73 mmol), and stirred at room temperature for 1 hour. The mixture was concentrated under vacuum and purified by prep-HPLC (ACN in 0.1% TFA/water) to give 39 (600 mg, 0.8 mmol, 50.4% yield) as a light yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.61 (d, J = 1.8 Hz, 2H), 7.54 (d, J = 7.3 Hz, 1H), 7.44 (t, J = 1.7 Hz, 1H), 7.14 (s, 1H), 7.11 (s, 1H), 7.04 (s, 25 1H), 6.57 (s, 1H), 3.95 – 3.53 (m, 12H), 3.51 – 3.46 (m, 2H), 3.36 – 3.32 (m, 2H), 3.29 – 2.99 (m, 3H), 2.80 (tdd, J = 23.5, 16.6, 6.7 Hz, 7H), 2.51 (d, J = 7.6 Hz, 2H), 2.42 – 2.09 (m, 4H), 1.98 – 1.71 (m, 7H). MS(M+H+):750.7. Scheme 6 361 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 41: To a stirred solution of 40 (10.0 g, 43.62 mmol) in THF (100 mL) was added 1N BH3- THF (52.3mL, 52.3 mmol) dropwise at 0 oC. The resulting mixture was stirred 3 hours at room temperature. 5 The reaction was quenched with 10% NaOH (100 mL) at 0 oC, extracted with EA (100 mL X 2). The organic layer was washed with brine (100 mL X 2), dried over Na2SO4, concentrated under vacuum to afford 41 (9.5 g, 100% yield, 44.19 mmol) as a colorless oil. MS(M+H+-56):160.2. Compound 42: To the solution of (COCl)2 (9.31 g, 73.35 mmol) in DCM (100) at -70 oC under N2 was added DMSO (10.36 g, 132.6 mmol). After stirred at -70 oC for 15 min, 41 (9.5 g, 44.19 mmol) in DCM 10 (50 mL) was added. Then stirred at -70 oC for 1 hour, then TEA (22.36 g, 220.97 mmol) was added. The mixture was stirred at -70 oC for 30 min, 0 oC for 30 min. The reaction mixture was quenched with water (100 mL), extracted with DCM (100 mL X 2), dried over Na2SO4, concentrated under vacuum. The residue was purified by silica gel chromatography (EA in PE, 0% to 40%) to afford 42 (8.6 g, 91.5% yield, 40.38 mmol) as a yellow oil. MS(M+H+-56):158.2. 15 Compound 43: To the solution of 42 (8.6 g, 40.38 mmol) and dimethyl (1-diazo-2-oxopropyl)phosphonate (7.75 g, 40.36 mmol) in MeOH (100 mL) was added K2CO3 (11.15 g, 80.8 mmol) at 0 oC, stirred at room temperature for 16 hours. The reaction mixture was diluted with EA (300 mL), washed with brine (100 mL X 3), dried over Na2SO4, concentrated under vacuum. The residue was purified by silica gel 362 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO chromatography (EA in PE, 0% to 15%) to afford 43 (7.2 g, 85.2% yield, 34.31 mmol) as a colorless oil. MS(M+H+-56):154.2. Compound 44: To a solution of 43 (7.18 g, 34.31 mmol), 2-chloro-1,8-naphthyridine (5.14 g, 31.23 mmol) in DMF (80 mL) was added Pd(PPh3)2Cl2 (2.2 g, 3.13 mmol), CuI (1.19 g, 6.25 mmol), TEA (9.48 g, 9.37 5 mmol), and stirred at 80 oC for 5 hours under N2. The reaction mixture was cooled, diluted with water (300 mL), extracted with EA (100 mL X 2). The organic layer was washed with brine (60 mL X 2), dried over Na2SO4, concentrated under vacuum. The residue was purified by silica gel chromatography (EA in PE, 0% to 100%) to afford 44 (8.8 g, 83.5% yield, 26.1 mmol) as a brown solid. MS(M+H+):338.5. Compound 45: To a solution of 44 (8.8 g, 26.1 mmol) in MeOH (100 mL) was added Raney Nickel (5 10 mL), and stirred at room temperature for 16 hours under H2 (1 atm). The reaction mixture was filtered, the filtrate was concentrated under vacuum to afford 45 (7.6 g, 85.4% yield, 22.26 mmol) as a brown solid. MS(M+H+):342.4. Compound 46: To a solution of 45 (7.6 g, 22.26 mmol) in DCM (80 mL) was added TFA (30 mL), and stirred at room temperature for 2 hours. The reaction mixture was concentrated under vacuum to give 46 15 (crude, 22.26 mmol) as a brown oil. MS(M+H+):242.0. Compound 47: To a solution of 5B (4.3 g, 27.22 mmol) and Pd(dppf)Cl2 (1.63 g, 2.23 mmol) in DCM (50 mL) was added a solution of 46 (crude, 22.26 mmol) and TEA (28.7 g, 222.5 mmol) in DCM (50 mL), and stirred at room temperature for 16 hours under N2. The reaction mixture was diluted with 1N HCl (300 mL), extracted with DCM (100 mL X 2), discard organic phase. The aqueous was adjust pH=8 with 10% 20 NaOH at 0 oC, extracted with DCM (100 mL X 3). The organic layer was washed with brine (100 mL X 2), dried over Na2SO4, concentrated under vacuum to afford 47 (6.8 g, 89.5%, 20.03 mmol) as a brown oil. MS(M+H+):340.1. Compound 48: To a solution of 47 (6.8 g, 20.03 mmol) and 6B (14.5 g, 30.06 mmol) in dioxane (100 mL) was added 3.6 M NaOH (11.1 mL, 40.0 mmol), [Rh(COD)CI]2 (0.49 g, 0.99 mmol, CAS:12092-47-6), 25 (R)-BINAP (1.25 g, 2.01 mmol), and stirred at 90 oC for 2 hours under N2. The reaction mixture was diluted with water (100 mL), extracted with EA (100 mL X 3). The organic layer was washed with brine (60 mL X 2), dried over Na2SO4, concentrated under vacuum and purified by reverse chromatography (ACN in 0.1% TFA/water, 5% to 40%) to afford 48 (5.4 g, 38.7 % yield, 7.76 mmol) as a brown oil. MS(M+H+):696.8. 30 Compound 49: To a solution of 48 (5.4 g, 7.76 mmol) in EA (100 mL) and MeOH (50 mL) was added Pd/C (10%, 1.0 g), and stirred at room temperature for 48 hours under H2. The reaction mixture was filtered and the filtrate was concentrated under vacuum to give 49 (4.8 g, 6.86 mmol) as a brown solid. MS(M+H+):700.3. 363 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 50: To a solution of 49 (2.0 g, 2.86 mmol) in MeOH (10 mL) was added 4N HCl/dioxane (20 mL), and stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum to give 50 (1.8, crude) as a brown solid. MS(M+H+):600.5. Compound 51: To a solution of 50 (1.71 g, 2.86 mmol) and TEA (1.45 g, 14.33 mmol) in DCM (30 mL) 5 was added dihydro-2H-pyran-2,6(3H)-dione (0.39 g, 3.42 mmol), and stirred at room temperature for 1 hour. The mixture was concentrated under vacuum and purified by prep-HPLC (ACN in 0.1% FA/water) to give 51 (1.1 g, 1.36 mmol, 47.6% yield) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.17 (s, 2H), 7.03 (d, J = 7.3 Hz, 1H), 6.88 (s, 1H), 6.81 (s, 1H), 6.76 (s, 1H), 6.42 (s, 1H), 6.24 (d, J = 7.3 Hz, 1H), 6.03 (s, 1H), 3.58 (d, J = 4.2 Hz, 4H), 3.50 (s, 3H), 3.28 – 3.12 (m, 7H), 2.89 – 2.78 (m, 3H), 2.77 – 2.53 10 (m, 6H), 2.39 (dd, J = 16.6, 7.7 Hz, 4H), 2.28 – 2.19 (m, 6H), 2.16 (s, 3H), 2.04 (dt, J = 15.3, 7.7 Hz, 1H), 1.92 – 1.82 (m, 1H), 1.73 (dq, J = 14.5, 7.4 Hz, 4H), 1.53 (d, J = 4.1 Hz, 2H), 1.34 – 1.22 (m, 3H). MS(M+H+):714.5. Scheme 7 15 Compound 54: To a solution of 52 (9.1 g, 33.7 mmol, 1.0 eq) and 53 (9.5 g, 40.4 mmol, 1.2 eq) in toluene (120 ml) were added Cs2CO3 (22.0 g, 67.5 mmol, 2.0 eq), t-Bu-Xphots (717.5 mg, 1.69 mmol, 0.05 eq) and Pd(OAc)2 (382 mg, 1.69 mmol, 0.05 eq) at 25 oC. The solution was stirred at 115 oC for 16 h under N2. After the reaction was completed, the solution was concentrated and the residue was purified by gel chromatography (SiO2, 100-200 mesh, DCM/MeOH = 20:1) to give a yellow oil, 54 (9.0 g, 56.8 % yield). 20 MS(M+H+):468.5. Compound 55: To a solution of 54 (9.0 g, 19.2 mmol, 1 eq) in MeOH (100 ml) was added Pd/C (10%, 1.0 g) at room temperature. The mixture solution was stirred at room temperature for 16 h under H2. After the reaction was completed. Fileted and the filtrate was concentrated to give a yellow oil, 55 (6.0 g, 93.5% yield). MS(M+H+):334.4. 364 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 56: To a solution of 55 (5.8 g, 17.4 mmol, 1.0 eq) and allylic bromide (3.18 g, 17.8 mmol, 1.02 eq) in THF (150 mL) was added TEA(3.5 g, 34.8 mmol, 2 eq) and stirred at room temperature for 16 hours under N2. After the solution was completed, brine (70 ml) and water (70 ml) were added, extracted with EA (100 mL X 2). The organic phase was dried over Na2SO4, filtered and concentrated to give a 5 yellow oil, 56 (7.48 g, 99.6 % yield). MS(M+H+):432.2. Compound 58: To a solution of 56 (5.2 g, 12.0 mmol, 1 eq) and 57 (7.43 g, 14.4 mmol, 1.2 eq) in dioxane (100 mL) was added [Rh(COD)CI]2 (296 mg, 0.6 mmol, CAS:12092-47-6), (R)-BINAP (748 g, 1.2 mmol) and 3.6 M KOH (5.7 mL, 24 mmol), and stirred at 90 oC for 3 hours under N2. The reaction mixture was diluted with water (100 mL), extracted with EA (100 mL X 3). The organic layer was washed with brine 10 (60 mL X 2), dried over Na2SO4, concentrated under vacuum and purified by reverse chromatography (ACN in 0.1% TFA/water, 10 % to 90 %) to afford 58 (5.0 g, 50.4 % yield, 6.08 mmol) as a yellow solid. MS(M+H+):822.2. Compound 59: To a solution of 58 (3.5 g, 4.0 mmol) in MeOH (100 mL) was added Pd/C (350 mg) and stirred at room temperature for 16 hours under H2. After the reaction was completed, remove Pd/C by 15 filtered. The filtrate was concentrated under vacuum to give 59 (2.4 g, 3.49 mmol, 81.9 % yield) as a yellow solid. MS(M+H+):688.3. Compound 61: To a solution of 59 (2.4 g, 3.5 mmol), 60 (550 mg, 3.85 mmol) and DIPEA (910 mg, 7 mmol) in DMF (15 mL) was added HATU (1.73 g, 4.55 mmol) and stirred at room temperature for 16 hours. The mixture was purified by reversed-phase chromatography (ACN in 0.1% TFA/water) to give 20 61 (2.6 g, 3.2 mmol, 91.4 % yield) as a yellow solid. MS(M+H+):813.2. Compound 62: To the solution of 61 (2.6 g, 3.2 mmol) in DCM (20 mL) was added TFA (10 ml) and stirred at room temperature for 2 hours. The reaction mixture was concentrated to obtain 62 (2.2 g, crude) as a yellow oil which will be used for the next step without purification. MS(M+H+):713.2. Compound 63: To a solution of 62 (2.2 g, crude) in MeOH (20 mL) and water (20 mL) was added 25 LiOH.H2O (1.3 g, 31 mmol) and stirred at room temperature for 16 hours. The reaction mixture was concentrated under vacuum and purified by prep-HPLC (ACN in 0.1%FA/water) and SFC to give Target Compd 63 (520 mg, 0.74 mmol, 24.0 %) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.17 (s, 0.46 H), 7.03 (d, J = 8.0 Hz, 1H), 6.87 (s, 1H), 6.79 (s, 1H), 6.74 (s, 1H), 6.21 (s, 1H), 6.03 (s, 1H), 5.77 (d, J = 8.0 Hz, 1H), 3.89-4.0 (m, 2H), 3.58 (br, 4H), 3.43 (br, 2 H), 3.14-3.36 (m, 8H), 2.51-2.85 (m, 8 H), 2.37- 30 2.49 (m, 4 H), 2.25 (s, 3H), 2.16 (s, 3H), 1.88 (m, 1H), 1.74 (m, 2 H), 1.58 (m, 4H).1.42 (m, 1 H). MS(M+H+):699.2 Scheme 8 365 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 65: To the solution of 64 (15 g, 68.5 mmol) and CH3I (11.7 g, 82.2 mmol) in DMF (100 mL) was added K2CO3 (11.5 g, 82.2 mmol), stirred at room temperature for 16 hours. The reaction mixture was diluted with EA (300 mL), washed with brine (100 mL X 3), H2O (100 mL X 3) and LiCl (100 mL 5 X 3), dried over Na2SO4, concentrated under vacuum. to afford 65 (14.7 g, 90% yield, 63.09 mmol) as a yellow solid. MS(M+H+):233. Compound 66: To a solution of alkyne (10.2 g, 52.3 mmol), 65 (11.1 g, 47.5 mmol) in DMF (200 mL) was added Pd(PPh3)2Cl2 (3.3 g, 4.75 mmol), CuI (1.80 g, 9.50 mmol), TEA (14.40 g, 142.50 mmol), and stirred at 80 oC for 5 hours under N2. The reaction mixture was cooled, diluted with water (300 mL), 10 extracted with EA (100 mL X 2). The organic layer was washed with LiCl (60 mL X 2), dried over Na2SO4, concentrated under vacuum. The residue was purified by silica gel chromatography (EA in PE, 0% to 30%) to afford 66 (14 g, 84.95% yield, 40.35 mmol) as a brown solid. MS(M-Na):370. Compound 67: To a solution of 66 (14 g, 40.35 mmol) in MeOH (500 mL) was added Raney Nickel (5 mL), and stirred at room temperature for 16 hours under H2 (1 atm). The reaction mixture was filtered, the 15 filtrate was concentrated under vacuum, the residue was added CH3CH2OH (1000 ml) was added Pd/C (1.4 g) and stirred at room temperature for three days under H2 (1 atm) The reaction mixture was filtered, the filtrate was concentrated under vacuum to afford 67 (11.18 g, 86% yield, 34.83 mmol) as a brown solid. MS(M+H+):322. 366 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 68: To a solution of 67 (10.0 g, 31.15 mmol) in DCM (100 mL) was added 4N HCl/dioxne (100 mL), and stirred at room temperature for 1 hour. The reaction mixture was concentrated, the residue and TEA (9.46 g, 93.49 mmol) was dissolved with DCM (100 mL) and THF (100 mL), to this added methyl (E)-4-bromobut-2-enoate (5.68 g, 31.17 mmol), and stirred at room temperature for 4 hours. The residue 5 was concentrated and diluted with EA (200 mL), washed with 1N HCl (50 mL X 3), discard organic phase. The aqueous was adjust pH=8 with 10% NaOH at 0 oC, extracted with DCM (100 mL X 3). The organic layer was washed with brine (100 mL X 2), dried over Na2SO4, concentrated under vacuum to afford 68 (6.0 g, 60.4%, 18.81 mmol) as a brown oil. MS(M+H+):320.1. Compound 69: To a solution of 68 (6.0 g, 18.81 mmol) and 6B (10.9 g, 22.59 mmol) in dioxane (100 mL) 10 was added 3.6 M NaOH (10.4 mL, 37.5 mmol), [Rh(COD)CI]2 (0.46 g, 0.94 mmol, CAS:12092-47-6), (R)-BINAP (1.17 g, 1.88 mmol), and stirred at 90 oC for 3 hours under N2. The reaction mixture was diluted with water (100 mL), extracted with EA (100 mL X 3). The organic layer was washed with brine (60 mL X 2), dried over Na2SO4, concentrated under vacuum and purified by flash (MeOH in DCM, 0% to 20%) to afford 69 (3.2 g, 25.15 % yield, 4.73 mmol) as a brown solid. MS(M+H+):676.5. 15 Compound 70: To a solution of 69 (3.2 g, 4.73 mmol) in MeOH (10 mL) was added 4N HCl/dioxane (30 mL), and stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum to give 70 (crude, 4.73 mmol) as a yellow solid. MS(M+H+):576.5. Compound 71: To a solution of 70 (crude, 4.73 mmol), 5-azidopentanoic acid (542 mg, 3.79 mmol) and DIPEA (1.83 g, 14.19 mmol) in DCM (50 mL) was added HATU (1.98 g, 5.21 mmol), and stirred at room 20 temperature for 30 min. The reaction mixture was diluted with water (100 mL), extracted with DCM (100 mL X 2). The organic layers were washed with brine (100 mL) dried over Na2SO4, concentrated to give 71 (4.56 g, crude) as a yellow solid. MS(M+H+):701.5. Compound 72: To the solution of 71 (1.5 g, 2.14 mmol) in THF (20 mL) and water (10 mL) was added LiOH (450 mg, 10.71 mmol), and stirred at room temperature for 2 hours. The reaction mixture was25 adjusted pH=7~8 with 1N HCl, extracted with DCM (50 mL X 3), concentrated and purified by prep- HPLC (ACN in 0.1% FA/water) to give Target Compd 72 (615 mg, 0.84 mmol, 39.3% yield) as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.19 (s, 1H), 6.93 – 6.84 (m, 2H), 6.80 (s, 1H), 6.75 (s, 1H), 6.33 (dd, J = 7.8, 3.4 Hz, 1H), 6.03 (s, 1H), 5.57 (d, J = 13.5 Hz, 2H), 3.72 (s, 3H), 3.59 (s, 4H), 3.18 (d, J = 21.0 Hz, 5H), 3.09 – 2.53 (m, 7H), 2.48 – 2.36 (m, 6H), 2.36 – 2.18 (m, 4H), 2.16 (s, 3H), 2.10 – 30 1.97 (m, 1H), 1.97 – 1.82 (m, 1H), 1.66 – 1.54 (m, 6H), 1.37 (dq, J = 14.9, 7.5 Hz, 1H). MS(M+H+):687.3. 367 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Scheme 8b Compound 74: To a solution of 73 (27.5 g, 201.55 mmol) in acetone (46.28 g, 398.44 mmol) was added 5 POCl3 (100 mL). The reaction mixture was then heated for 5.5 h at 80°C. Next, the reaction mixture was carefully added to 200 mL ice water, which was then basified to pH 14 with NaOH (6 M aq) and stirred for 40 min (with additional base added to maintain pH 14). The aqueous material was split into two portions. Each portion was extracted with DCM (100 mL X 6). The organic layers were combined and concentrated under vacuum to obtain 74 (crude, 201.55 mmol) as a brown solid. MS(M+H+):179.1. 10 Compound 75: To a solution of 74 (crude, 201.55 mmol) in MeOH (200 mL) was added NaOMe (24.8 mL, 403.1 mmol), and stirred at 40 °C for 16 hours. The reaction mixture was diluted with water (100 mL), extracted with EA (100 mL X 3). The organic layer was washed with brine (60 mL X 2), dried over Na2SO4, concentrated under vacuum and purified by reverse chromatography (DCM:MeOH 30:1) to afford 75 (3.3 g, 9.42% yield, 18.94 mmol) as a brown solid. MS(M+H+):175.2. 15 Compound 76: To a stirred solution of 75 (3.3 g, 18.94 mmol) and tert-butyl (R)-3-(iodomethyl) pyrrolidine-1-carboxylate (3.84 g, 12.63 mmol) in THF was cooled to 0 °C and treated under nitrogen with a solution of LiHMDS (18.9 ml, 18.9 mmol) in THF. The reaction mixture was stirred at 0 °C for 3 h. The reaction was quenched with saturated NH4Cl solution (500 mL) and water (500 ml) and ethyl acetate (1 L) was added. The layers were separated and the aqueous phase was extracted with further ethyl acetate (1 L). 20 The combined organic layers were dried (MgSO4), filtered and evaporated in vacuum. The residual brown oil was purified by chromatography (PE: EtOAc 10:1) to give 76 (1.87 g, 27.62% yield, 2.23 mmol) as a brown solid. MS(M+H+):358.2. 368 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 77: To a solution of 76 (1.87 g, 5.23 mmol) in DCM (10 mL) was added 4M HCl/dioxane (10 mL), and stirred at room temperature for 2 hours. The reaction mixture was concentrated under vacuum to give 77 (crude, 6.88 mmol) as a brown oil. MS(M+H+):258.2. Compound 78: To a solution of methyl (E)-4-bromobut-2-enoate (1.47 g, 8.24 mmol) in THF (10 mL) 5 was added a solution of 77 (1.77 g, 6.87 mmol) and TEA (2.78 g, 24.48 mmol) in THF (15 mL), and stirred at room temperature for 16 hours. The reaction mixture was diluted with 1N HCl (300 mL), extracted with DCM (100 mL X 2), discard organic phase. The aqueous was adjust pH=8 with 10% NaOH at 0 oC, extracted with DCM (100 mL X 3). The organic layer was washed with brine (100 mL X 2), dried over Na2SO4, concentrated under vacuum to afford 78 (1.32 g, 54.10%, 3.72 mmol) as a brown oil. 10 MS(M+H+):356.2. Compound 79: To a solution of 78 (4.0 g, 11.25 mmol) and 57 (7.6 g, 14.72 mmol) in dioxane (50 mL) was added [Rh(COD)CI]2 (0.28 g, 0.56 mmol, CAS:12092-47-6), (R)-BINAP (0.70 g, 1.13 mmol) and 3.6 M NaOH (6.3 mL, 22.5 mmol), and stirred at 90 oC for 3 hours under N2. The reaction mixture was diluted with water (100 mL), extracted with EA (100 mL X 3). The organic layer was washed with brine (60 mL 15 X 2), dried over Na2SO4, concentrated under vacuum and purified by reverse chromatography (ACN in 0.1% TFA/water, 5% to 40%) to afford 79 (3.3 g, 39.4 % yield, 4.43 mmol) as a brown solid. MS(M+H+):746.2. Compound 80: To a solution of 79 (3.3 g, 4.43 mmol) in MeOH (30 mL) and EA (30 mL) was added Pd/C (600 mg). The mixture was stirred at room temperature for 48 hours under H2 (1 atm). The reaction mixture 20 was filtered and concentrated under vacuum to give 80 (2.3, 3.73 mmol, 84.2% yield) as a brown solid. MS(M+H+):616.5. Compound 81: To a solution of 80 (2.3 g, 3.73 mmol), 5-azidopentanoic acid (530 mg, 3.70 mmol) and DIPEA (1.45 g, 11.24 mmol) in DCM (30 mL) was added HATU (1.85 g, 4.87 mmol). The mixture was stirred at room temperature for 1 hour. The mixture was purified by reversed-phase chromatography (5% 25 to 50%, ACN in 0.1% TFA/water) to give 81 (2.0 g, 2.70mmol, 72.4% yield) as a yellow solid. MS(M+H+):741.5. Compound 82: To the solution of 81 (2.0 g, 2.7 mmol) in THF (20 mL) and water (5 mL) was added LiOH.H2O (568 mg, 13.52 mmol), and stirred at room temperature for 2 hours. The reaction mixture was concentrated and purified by prep-HPLC (ACN in 0.1%FA/water) to give Target Compd 82 (590 mg, 0.81 30 mmol, 30% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 6.86 (s, 1H), 6.79 (s, 1H), 6.74 (s, 1H), 6.19 (s, 1H), 6.10 (d, J = 7.4 Hz, 1H), 6.03 (s, 1H), 3.73 (s, 3H), 3.58 (s, 5H), 3.27 – 3.12 (m, 8H), 2.94 – 2.86 (m, 1H), 2.79 (dd, J = 16.0, 7.5 Hz, 2H), 2.70 (dd, J = 16.0, 7.1 Hz, 1H), 2.63 – 2.52 (m, 2H), 2.48 – 2.35 (m, 8H), 2.26 (s, 3H), 2.16 (s, 3H), 2.05 (dt, J = 14.6, 7.3 Hz, 1H), 1.96 – 1.84 (m, 1H), 1.70 (dd, J = 11.1, 5.9 Hz, 2H), 1.67 – 1.53 (m, 6H), 1.36 (dd, J = 11.7, 7.1 Hz, 1H). MS(M+H+):727.3. 369 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Scheme 9 Compound 84: To a solution of 83 (18.0 g, 89.53 mmol) in DCM (300 mL) was added PPh3 (30.0 g, 114.50 mmol) and imidazole (7.9 g, 116.04 mmol). After stirring at room temperature for 20 min, iodine 5 (27.3 g, 107.56 mmol) was added at 0 °C, and stirred for 1 h at 0 °C. A saturated solution of sodium thiosulfate (50 mL) was added and the mixture was allowed to warm to room temperature. The reaction mixture was extracted with DCM (200 mL), washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduce pressure and purified by flash (PE) to give 84 (26 g, 93.4 %, 83.33 mmol) as a colorless oil.1H NMR (400 MHz, CDCl3) δ 7.40 (d, J = 7.9 Hz, 1H), 7.35 (s, 1H), 7.19 (t, J = 7.7 Hz, 1H), 10 7.12 (d, J = 7.6 Hz, 1H), 3.33 (t, J = 7.7 Hz, 2H), 3.15 (t, J = 7.7 Hz, 2H). Compound 86: The solution of 85 (12.5 g, 30.86 mmol) and K2CO3 (12.8 g, 92.75 mmol) in DMF (100 mL) was warmed to 100 oC, then to this added 84 (22 g, 70.51 mmol) in DMF (30 mL), and stirred at 100 oC for 16 hours. The reaction mixture was cooled to room temperature, diluted with EA (300 mL), washed with water (100 mL X 2), brine (100 mL X 2), dried over Na2SO4, concentrated and purified by flash (0% 15 to 40 %, EA in PE) to give 86 (7.0 g, 11.90 mmol, 38.6 % yield) as a yellow oil. MS(M+H+):590.2. Compound 87: The solution of 86 (7.0 g, 11.90 mmol) and K2CO3 (4.93 g, 35.72 mmol) in DMF (100 mL) was warmed to 100 oC, then to this added methyl 2-bromoacetate (5.5 g, 35.95 mmol), and stirred at 100 oC for 16 hours. The reaction mixture was cooled to room temperature, diluted with EA (300 mL), washed with water (100 mL X 2), brine (100 mL X 2), dried over Na2SO4, concentrated and purified by 20 flash (0% to 50 %, EA in PE) to give 87 (5.64 g, 8.55 mmol, 71.8 % yield) as a yellow oil. MS(M+H+):662.2. Compound 88: To a solution of 87 (5.64 g, 8.54 mmol), 2-methyl-1,8-naphthyridine (1.23 g, 8.53 mmol) in dioxane (60 mL) was added Pd(dba)2 (123 mg, 0.21 mmol), Xantphos (124 mg, 0.21 mmol), Cs2CO3 (5.57 g, 17.09 mmol), and stirred at 100 oC for 16 hours under N2. The reaction mixture was filtered, 25 washed with EA (100 mL), the filtrate was concentrated under vacuum. The residue was purified by silica 370 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO gel chromatography (MeOH in DCM, 0% to 3%) to afford 88 (4.69 g, 75.9% yield, 6.48 mmol) as a brown oil. MS(M+H+):724.3. Compound 89: To a solution of 88 (4.69 g, 6.48 mmol) in EtOH (50 mL) and EA (50 mL) was added Pd/C (2.0 g), and stirred at 50 oC for 16 hours under H2 (1 atm). The reaction mixture was filtered, the 5 filtrate was concentrated under vacuum to afford 89 (3.6 g, 93.5% yield, 6.06 mmol) as a brown solid. MS(M+H+):594.5. Compound 90: To a solution of 89 (3.6 g, 6.06 mmol), 5-azidopentanoic acid (0.78 mg, 5.45 mmol) and DIPEA (1.96 g, 15.19 mmol) in DCM (50 mL) was added HATU (2.76 g, 7.26 mmol), and stirred at room temperature for 1 hour. The reaction mixture was diluted with water (100 mL), extracted with DCM (100 10 mL X 2). The organic layers were washed with brine (100 mL) dried over Na2SO4, concentrated and purified by flash (ACN in 0.1%TFA/water) to give 90 (2.8 g, 64.2%, 3.89 mmol) as a yellow solid. MS(M+H+):719.4. Compound 91: To the solution of 90 (1.4 g, 1.95 mmol) in THF (20 mL) and water (10 mL) was added LiOH (410 mg, 9.76 mmol), and stirred at room temperature for 2 hours. The reaction mixture was adjusted 15 pH=3 with 1N HCl, extracted with DCM (50 mL X 3), concentrated and purified by prep-HPLC (ACN in 0.1% NH4HCO3/water) to give Target Compd 91 (500 mg, 0.71 mmol, 36.4% yield) as a white solid.1H NMR (400 MHz, DMSO-d6) δ 7.19 (t, J = 7.5 Hz, 1H), 7.14 (s, 1H), 7.12 – 6.97 (m, 3H), 6.32 (d, J = 11.5 Hz, 2H), 6.22 (d, J = 7.3 Hz, 1H), 6.09 (s, 2H), 6.01 (s, 1H), 3.99 (s, 2H), 3.73 (s, 2H), 3.55 (d, J = 17.1 Hz, 6H), 3.30 – 3.05 (m, 8H), 2.87 – 2.79 (m, 2H), 2.58 (t, J = 6.1 Hz, 2H), 2.38 (s, 2H), 2.25 (s, 3H), 2.16 20 (s, 3H), 1.78 – 1.66 (m, 2H), 1.62 – 1.50 (m, 4H). MS(M+H+):705.3. Scheme 10 371 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 93: To a mixture of 92 (7.7 g, 37 mmol) in cold water (5°C, 20 mL) was added concentrated aqueous HC1 (30 mL, 0.36 mol). The resulting solution was heated at 85°C for 16 hours. After the reaction was cooled to room temperature, EA (60 mL) was added. To the mixture was added aqueous NaOH (50 5 wt %) slowly to about pH 11, keeping the internal temperature below 25 °C. The layers were separated and the aqueous layer was extracted with EA (2 X 120 mL). The organic layers were combined and concentrated in vacuum to afford 93 (5.5 g, 91.8% yield, 34 mmol) as a bottle green oil which was used in next reaction without further purification. MS(M+H+):163.08. Compound 94: The solution of 93 (5.5 g, 33.72 mmol) and tert-butyl piperazine-1-carboxylate (6.28 g, 10 33.72 mmol) in DCE (140 mL) was stirred at room temperature for 1 hours, then added STAB (21.62 g, 102 mmol) at 0°C, the solution was stirred at room temperature for 16 hours. The reaction mixture was diluted with NaHCO3 (50 mL), extracted with EA (50 mL X 3). The organic layer was washed with brine (30mL X 2), dried over Na2SO4, concentrated under vacuum and purified by silica gel chromatography (MeOH in DCM, 0% to 5%) to afford 94 (7.8 g, 69.5 % yield, 2.34 mmol) as a blue-green solid. 15 MS(M+H+):333.1. Compound 95: To a solution of 94 (7.8 g, 2.34 mmol) in MeOH (20 mL) was added 4M HCl/dioxne (78 mL). The mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum to give 95 (crude, 2.5 mmol) as a faint yellow solid. MS(M+H+):233.17 Compound 96: To a solution of 95 (5.9 g, 2.5 mmol) and TEA (10.2 g, 10.1 mmol) in THF (150 mL) was 20 added methyl (E)-4-bromobut-2-enoate (4.48 g, 2.5 mmol), and stirred at room temperature for 16 hours. The reaction mixture was diluted with EA, and washed with 1N HCl (50 mL X 2), discard organic phase. The aqueous was adjust pH=9 with 10% NaOH at 0 oC, extracted with DCM (100 mL X 3). The organic 372 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO layer was washed with brine (100 mL X 2), dried over Na2SO4, concentrated under vacuum to afford 96 (5.3 g, 66.4% yield, 1.60 mmol) as a brown oil. MS(M+H+):331.21. Compound 97: To a solution of 96 (3.65 g, 11.05 mmol) and tert-butyl 4-(3-(3,5-dimethyl-1H-pyrazol-1- yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazine-1-carboxylate (6.4 g, 13.27 mmol) 5 in dioxane (60 mL) was added [Rh(COD)CI]2 (0.27 g, 0.55 mmol, CAS:12092-47-6), (R)-BINAP (0.69 g, 1.11 mmol) and 3.6 M NaOH (6.2 mL, 22.14 mmol), and stirred at 90 oC for 3 hours under N2. The reaction mixture was diluted with water (100 mL), extracted with EA (100 mL X 3). The organic layer was washed with brine (60 mL X 2), dried over Na2SO4, concentrated under vacuum and purified by reverse chromatography (ACN in 0.1% TFA/water, 5% to 40%) to afford 97 (3.1 g, 40.8 % yield, 4.51 mmol) as 10 a brown solid. MS(M+H+):687.5. Compound 97b: 97 (3.1 g, 4.51 mmol) was purifited by SFC to give 97b (2.45 g, 79% yield, 3.57 mmol). Compound 98: To a solution of 97b (2.45 g, 3.57 mmol) in MeOH (10 mL) was added 4N HCl/dioxane (25 mL), and stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum 15 to give 98 (2.1, crude) as a brown solid. MS(M+H+):587.5. Compound 99: To a solution of 98 (2.1 g, 3.57 mmol), 5-azidopentanoic acid (562 mg, 3.93 mmol) and DIPEA (1.38 g, 10.70 mmol) in DMF (20 mL) was added HATU (1.76 g, 4.63 mmol), and stirred at room temperature for 16 hours. The mixture was purified by reversed-phase chromatography (ACN in 0.1% TFA/water) to give 99 (1.7 g, 2.39 mmol, 66.9% yield) as a yellow solid. MS(M+H+):712.4. 20 Compound 100: To a solution of 99 (1.7 g, 2.39 mmol) in THF (20 mL) and water (5 mL) was added LiOH.H2O (402 mg, 9.57 mmol), and stirred at room temperature for 2 hours. The reaction mixture was concentrated and purified by prep-HPLC (ACN in 0.1%FA/water) to give Target Compd 100 (950 mg, 1.36 mmol, 56.9%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.17 (s, 1H), 7.07 (d, J = 7.3 Hz, 1H), 6.85 (s, 1H), 6.79 (s, 1H), 6.73 (s, 1H), 6.42 (d, J = 7.3 Hz, 1H), 6.35 (s, 1H), 6.03 (s, 1H), 3.59 (s, 25 4H), 3.35 (t, J = 6.1 Hz, 2H), 3.24 (ddd, J = 35.8, 23.2, 17.7 Hz, 10H), 2.79 (dd, J = 15.8, 6.3 Hz, 1H), 2.63 – 2.53 (m, 4H), 2.48 – 2.28 (m, 10H), 2.26 (s, 3H), 2.16 (s, 3H), 1.80 – 1.70 (m, 2H), 1.61 – 1.50 (m, 4H). MS(M+H+):698.5. Scheme 11 373 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 102: To a solution of 101 (10 g, 82.64 mmol) and 1,1-dimethoxypropan-2-one (12.7 g, 107.44 mmol) in EtOH: H2O (80 mL:10 mL) was added NaOH (10.8 mL, 53.7 mmol, 5M in H2O). The reaction mixture was stirred at 25°C for 1 hour. LCMS showed the reaction was completed. The reaction mixture 5 was diluted with water (100mL), extracted with EA (200 mL X 3). The organic layers were dried over Na2SO4, concentrated under vacuum to give 102 (17 g, crude) as a yellow oil. MS(M+H+):205.2. Compound 103: To a solution of 102 (8 g, 39.2 mmol) in MeOH: EA (40 mL: 40 mL) was added Pd/C (1.6 g), and stirred at 25 °C for 16 hours under H2 (1atm). The reaction mixture was filtered through a Celite pad, and filtrate was concentrated to give 103 (8.2 g, crude). MS(M+H+):209.0. 10 Compound 104: To a solution of 103 (8.2 g, 39.4 mmol) in cold water (40 mL) was added 0.36N HCl (40 mL), After the reaction was cooled to 13 °C, iPAC (20 mL) was added. The mixture was added aqueous NaOH (50wt%) slowly to about pH 11. Keeping the internal temperature belong 25 °C. The layers were separated and the aqueous layers was extracted with EA. The organic layers were combined and concentrated under vacuum to afford 104 (3.5 g, crude) as a brown oil. MS(M+H+):163.0. 15 Compound 105: The solution of 85b (6.4 g, 17.25 mmol), aldehyde (6.4 g, 25.88 mmol) in DCE (70 mL) was stirred at room temperature for 1 hour. To this added STAB (11 g, 51.75 mmol) at 0°C and stirred at room temperature for 16 hours. The reaction mixture was quenched with NaHCO3 (100 mL), extracted with DCM (100 mL X 2), concentrated under vacuum and purified by with flash (0%~ 40%, EA in PE) to give 105 (11.85 g, 19.68 mmol, 100% yield) as a yellow oil. MS(M+H+):603.2. 20 Compound 106: To a solution of 105 (11.85 g, 19.68 mmol), methyl 2-bromoacetate (8.98 g, 59.05 mmol) in DMF (120 mL) was added K2CO3 (8.15 g, 59.05 mmol), KI (3.3 g, 19.68 mmol). The reaction mixture was stirred at 80 °C for 16 hours. The reaction mixture was diluted with water (100 mL), extracted with EA (200 mL X 2). The organic layers were dried over Na2SO4, concentrated under vacuum and purified 374 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO by flash (0%~ 50%, EA in PE) to give 106 (7.9 g, 11.72 mmol, 59.6 % yield) as a yellow solid. MS(M+H+):675.2. Compound 107: To a solution of 106 (5.9 g, 8.74 mmol) in MeOH: EA (50 mL: 50 mL) was added Pd/C (1.2 g). The mixture was stirred at 50 °C for 16 hours under H2 (1atm). The reaction mixture was filtered 5 through a Celite pad, and filtrate was concentrated to give 107 (4.2 g, crude). MS(M+H+):541.2. Compound 108: The solution of 107 (3.5 g, 6.48 mmol), 104 (2.1 g, 12.96 mmol) in DCE (40 mL) was stirred at room temperature for 1 hour. To this added STAB (4.1 g, 19.44 mmol) at 0°C, and stirred at room temperature for 16 hours. The reaction mixture was quenched with NaHCO3 (100 mL), extracted with DCM (200 mL X 2), concentrated under vacuum and purified by flash (0%~ 40%, EA in PE) to give 108 10 (3 g, 4.37 mmol, 67.5% yield) as a yellow oil. MS(M+H+):687.4. Compound 109: To a solution of 108 (3 g, 4.37 mmol) in MeOH (10 mL) was added dropwise 4M HCl- Dioxane (30 mL) at 0°C. The reaction mixture reacted at room temperature for 2 h. The mixture was concentrated under vacuum to give 109 (3.2 g, crude). MS(M+H+):587.3. Compound 110: To a solution of 109 (2.56 g, 4.37 mmol), 5-azidopentanoic acid (500 mg, 3.5 mmol) and 15 DIPEA (2.3 g, 17.48 mmol) in DCM (30 mL) was added HATU (3.3 g, 8.74 mmol), and stirred at room temperature for 16 hours. The reaction mixture was diluted with water (100 mL), extracted with DCM (100 mL X 2). The organic layers were washed with brine (100 mL) dried over Na2SO4, concentrated to give 110 (4.8 g, crude) as a yellow solid. MS(M+H+):712.4. Compound 111: To the solution of 110 (4.8 g, 6.75 mmol) in THF (50 mL) and water (10 mL) was added 20 LiOH (1.4 g, 33.76 mmol), and stirred at room temperature for 2 hours. The reaction mixture was concentrated and purified by prep-HPLC (ACN in 0.2%NH3HCO3/water) to give Target Compd 111 (600 mg, 0.86 mmol, 12% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.07 (d, J = 7.3 Hz, 1H), 6.44 (d, J = 7.3 Hz, 1H), 6.36 (s, 1H), 6.26 (s, 1H), 6.07 (d, J = 15.9 Hz, 2H), 5.99 (s, 1H), 4.00 (s, 2H), 3.57 (s, 4H), 3.18 (dd, J = 63.8, 21.5 Hz, 12H), 2.84 (d, J = 10.5 Hz, 2H), 2.61 (t, J = 6.1 Hz, 2H), 2.38 (s, 25 2H), 2.23 (s, 3H), 2.15 (s, 3H), 1.92 (t, J = 11.1 Hz, 2H), 1.78 – 1.70 (m, 2H), 1.64 (d, J = 10.1 Hz, 3H), 1.60 – 1.52 (m, 4H), 1.29 – 1.19 (m, 2H). MS(M+H+):698.5. Scheme 12 375 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 113: To a stirred solution of 112 (12.6 g, 38.53 mmol, 1 eq) in MeOH (100 mL) was added HAc (5 ml) at room temperature with Pd hydroxide. The mixture was stirred at room temperature for 12 hours. The mixture filtered and concentrated under vacuum to give the crude 113 (10.6 g, yield 5 83.1%,32.02 mmol) as a yellow oil. LC/MS (ESI, m/z): [(M +H)] + = 332.4. Compound 114: To a stirred solution of 113 (10.6 g, 32.02 mmol, 1 eq) in HCl/MeOH (60 mL). The mixture was stirred at room temperature for 4 hours. The mixture was concentrated under vacuum. The resulting residue was purified by prep-HPLC (0.1 % FA) to give 114 (4.6 g, yield 62.16%, 19.91 mmol) as 10 a brown oil. LC/MS (ESI, m/z): [(M +H)] + = 232.2. Compound 115: To a stirred solution of 114 (5 g, 12.346 mmol, 1 eq), 6-1 (2.89 g, 14.815 mmol, 1.2 eq) and K2CO3(5.11 g, 37.037 mmol, 3 eq) in anhydrous DMF (40 mL) . The mixture was stirred at 110oC for 24 hours. The mixture was extracted with EA (300 mL) and H2O (300 mL X 2), washed with brine, dried 15 over MgSO4, filtered and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography to give 115 (3.81 g, yield 59.5%,7.341 mmol) as a brown oil. LC/MS (ESI, m/z): [(M +H)] + = 520.7. Compound 117: To a solution of 115 (3.81 g, 7.341 mmol,1eq) and 116 (2.95 g, 14.682 mmol,2eq eq) in DCM (30 mL), TEA (2.23 g, 22.023 mmol, 3 eq) was added. The mixture was stirred at room temperature 20 for 16 hours. The mixture was extracted with EA (100 mL) and H2O (100 mL) twice, washed with brine, dried over MgSO4, filtered and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography to give 117 (4.8 g, yield 95.4%,7.018 mmol) as a brown oil. 376 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO LC/MS (ESI, m/z): [(M +H)] + = 685.7. Compound 118: To a stirred solution of 117 (4.8 g, 7.018 mmol, 1 eq) and 114 (3.24 g, 14.035 mmol, 2eq) in MeCN (30 mL), TEA (2.13 g, 21.053 mmol, 3 eq) was added at room temperature. The mixture was stirred at 80oC for 16 hours. The mixture was extracted with EA (100 mL) and H2O (100 mL) twice, 5 washed with brine, dried over MgSO4, filtered and concentrated under vacuum. The resulting residue was purified by prep-HPLC (0.1 % FA) to give 118 (3.5 g, yield 64.3%,4.511 mmol) as a yellow oil. LC/MS (ESI, m/z): [(M +H)] + = 777.8. Compound 119: To a stirred solution of 118 (3.5 g, 4.511 mmol, 1 eq) and Pd/C (400 mg) in MeOH (120 mL), NH3H2O (15 ml) was added at room temperature. The mixture was stirred at room temperature for 10 16 hours under H2. The mixture was filtered and concentrated under vacuum to give the crude. The crude was purified by prep-HPLC (0.1 % NH3H2O) to give 119 (1.7 g, yield 58.82%, 2.648 mmol) as a brown solid. LC/MS (ESI, m/z): [(M +H)] + = 643.8. Compound 120: To a stirred solution of 119 (1.7 g, 2.648 mmol, 1 eq), 10-1 (378 mg, 2.648 mmol, 1 eq), 15 HUTA (2.01 g, 5.296 mmol, 2 eq) and DIEA (1.37 g,10.592 mmol, 4 eq) in DMF (15 mL). The mixture was stirred at room temperature for 2 hours. The mixture was concentrated under vacuum. The resulting residue was purified by prep-HPLC (0.1 % NH3H2O) to give 120 (2.0 g, yield 98%, 2.577 mmol) as a brown oil. LC/MS (ESI, m/z): [(M +H)] + = 768.9. 20 Compound 121: To a stirred solution of 120 (2.0 g, 2.577 mmol, 1 eq) in TFA (2 mL) and DCM (8 ml). The mixture was stirred at room temperature for 2 hours. The mixture was extracted with EA (100 mL) and H2O (100 mL) twice, washed with brine, dried over MgSO4. The resulting residue was purified by prep-HPLC (0.1 % NH3HCO3) to give 121 (500 mg, yield 27.03%, 0.703 mmol) as a white solid.1H NMR (400 MHz, DMSO-d6) δ 8.03 (s, 1H), 7.20 (d, J = 7.3 Hz, 1H), 6.69 (s, 1H), 6.63 (s, 1H), 6.54 (s, 1H), 6.32 25 (d, J = 7.2 Hz, 1H), 6.03 (s, 1H), 4.37 (d, J = 17.6 Hz, 1H), 4.04 (d, J = 17.5 Hz, 1H), 3.58 (s, 4H), 3.34 – 2.89 (m, 12H), 2.63 (t, J = 6.0 Hz, 2H), 2.46 – 2.31 (m, 4H), 2.25 (s, 3H), 2.15 (s, 3H), 1.94 – 1.70 (m, 4H), 1.61 – 1.52 (m, 4H), 1.42 (dd, J = 14.9, 6.6 Hz, 3H). LC/MS (ESI, m/z): [(M +H)] + = 712.8. Scheme 13 377 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 122: To a stirred solution of 11 (5 g, 7.14 mmol) in tetrahydrofuran (100 mL) in a round bottom flask at room temperature, phenoxyacetic anhydride (6.14 g, 21.43 mmol) was added. After the solution had stirred for 60 h the solvent was removed under vacuum leaving a residue with a mass of 11.63 5 g. This residue was taken up in DCM (100 mL), washed with sat. NH4Cl (100 mL) and brine (100 mL), dried over Na2SO4, and the solvent was removed under vacuum affording 11.15 g of crude product 122 which was used in the next step without further purification. MS (ESI+APCI), calculated for C47H59N7O7 [M+H]+ exact mass m/z = 834.45, found 834.5. Compound 354P: To a stirred solution of crude product 122 (11.15 g) in DCM (75 mL), EDC (6.85 g, 10 35.72 mmol) was added. The mixture was stirred at rt for 10 min and then pentafluorophenol (6.57 g, 35.72 mmol) and 4-(dimethylamino)pyridine (261.84 mg, 2.14 mmol) were added. The reaction was stirred at room temperature for 18 h and then water (75 mL) was added. This mixture was transferred to a separatory funnel and the layers were separated. The organic layer was washed with brine (75 mL), dried over Na2SO4, and concentrated under vacuum to a mass of 16.83 g. The crude residue was taken up in DCM and purified 15 on a 220 g silica gold column with a 5-55% THF/DCM gradient affording 4.33 g (61% yield) of pure product 354P as a white foam.1H NMR (600 MHz, CD3CN) δ 7.44 (d, J = 7.6 Hz, 1H), 7.24 (dd, J = 8.8, 7.3 Hz, 2H), 6.90 (t, J = 7.4 Hz, 2H), 6.81 (d, J = 1.5 Hz, 1H), 6.81 – 6.77 (m, 3H), 6.72 (t, J = 1.6 Hz, 1H), 5.99 (s, 1H), 5.25 (s, 2H), 3.83 (t, 2H), 3.66 (t, J = 5.3 Hz, 2H), 3.58 (t, J = 5.3 Hz, 2H), 3.51 (s, 3H), 3.28 (p, J = 7.8 Hz, 1H), 3.19 (t, J = 5.2 Hz, 2H), 3.14 (t, J = 5.3 Hz, 2H), 2.85 – 2.77 (m, 4H), 2.77 – 2.73 20 (m, 3H), 2.71 – 2.60 (m, 4H), 2.54 – 2.45 (m, 4H), 2.42 – 2.26 (m, 1H), 2.24 (s, 3H), 2.23 – 2.21 (m, 1H), 2.19 (s, 3H), 2.11 – 2.03 (m, 1H), 1.99 (p, J = 7.3 Hz, 2H), 1.94 (dt, J = 4.9, 2.5 Hz, 1H), 1.89 (dt, J = 12.2, 6.4 Hz, 3H), 1.77 – 1.67 (m, 2H), 1.43 – 1.35 (m, 1H). MS (ESI+APCI), calculated for C53H58F5N7O7 [M+H]+ exact mass m/z = 1000.44, found 1000.2. 25 Scheme 13b 378 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO To a 250 mL round-bottomed flask equipped with a stir bar was added 11 (10.19 g, 14.56 mmol, 1.00 eq). The flask was sealed with a rubber septum fit with an argon inlet needle. The flask was evacuated and 5 backfilled with argon; this process was repeated two additional times. Then, anhydrous CH2Cl2 (72 mL) was added via syringe, followed by anhydrous DIPEA (5.10 mL, 29.1 mmol, 2.00 eq) over the course of approximately two minutes. The reaction vessel was then cooled to 0 °C in an ice water bath. Once cooled, T3P (14 mL of a 50 wt% solution in ethyl acetate, 23.3 mmol, 1.60 eq) was added dropwise via syringe over the course of approximately four minutes. Then, 6-azidohexylamine (4.40 g, 31.0 mmol, 2.10 eq) was 10 added dropwise via syringe over the course of three minutes at 0 °C. Once the addition was complete, the flask was removed from the ice water bath, the argon inlet needle was removed, and the reaction mixture allowed to stir at room temperature for 17 hours. The reaction mixture was quenched with aqueous saturated sodium bicarbonate solution (100 mL) and then 15 transferred to a separatory funnel containing aqueous saturated sodium bicarbonate solution (100 mL) and EtOAc (600 mL). The organic phase was washed with deionized water (1 x 200 mL), followed by brine (2 x 200 mL). The organic phase was then dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by automated silica gel chromatography in three separate batches (RediSepGold 330g cartridge, eluting 0% to 20% EtOAc (containing 2% NEt3)/MeOH) to yield an intermediate amide as a 20 light yellow sticky foam (8.55 g, 71%). 1H NMR (600 MHz, CDCl3) δ 7.06 (d, J = 7.3 Hz, 1H), 6.81 (t, J = 2.1 Hz, 1H), 6.75 (br t, J = 1.7 Hz, 1H), 6.72 (d, J = 1.7 Hz, 1H), 6.32 (d, J = 7.3 Hz, 1H), 5.97 (s, 1H), 5.87 (br t, J = 5.0 Hz, 1H), 3.75 (t, J = 5.3 Hz, 2H), 3.63 (t, J = 5.2 Hz, 2H), 3.56 (s, 3H), 3.39 (t, J = 4.5 Hz, 2H), 3.33 – 3.15 (m, 9H), 2.88 (dd, J = 25 15.5, 6.2 Hz, 1H), 2.77 – 2.63 (m, 5H), 2.55 – 2.40 (m, 6H), 2.45 (t, J = 7.1 Hz, 3H), 2.35 (q, J = 8.5 Hz, 1H), 2.28 (s, 3H), 2.27 – 2.25 (m (overlapped singlet), 5H), 2.20 (t, J = 8.1 Hz, 1H), 2.10 (dt, J = 15.0, 7.6 Hz, 1H), 1.97 (m, 3H), 1.92 – 1.87 (m, 2H), 1.62 – 1.56 (m, 2H), 1.52 (p, J = 7.3 Hz, 2H), 1.43 – 1.31 (m, 5H). LC-MS m/z calcd for C45H65N11O4 [M+H]+: 823.52; 824.4 found. 379 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO To a 1 L round-bottomed flask equipped with a stir bar was the amide product from the previous step (8.55 g, 10.38 mmol, 1.00 eq). The flask was sealed with a rubber septum fit with an argon inlet needle. The flask was then evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous THF (54 mL) was added via syringe to give a homogenous, yellow solution. The flask was then 5 cooled to 0°C in an ice water bath. Then, KOSiMe3 (2.73 g, 21.2 mmol, 2.05 eq) was added in two portions (1.345 g, followed by 1.38 g). Once the addition was complete, the flask was removed from the ice water bath and allowed to stir at rt for 23 hours. The pH of the reaction mixture was adjusted to 3–4 by the addition of aqueous 1 M HCl. Then, the reaction 10 mixture was concentrated in vacuo. The crude residue was purified by automated reverse phase chromatography in two batches (RediSepGold 415g C18 column, eluting 5% to 35% water (0.1% TFA)/MeCN). The like fractions were combined, concentrated, and then lyophilized to yield 11Z as a sticky tan foam (11.34 g, 95%) as a TFA salt. 15 1H NMR (600 MHz, CD3OD) δ 7.56 (d, J = 7.3 Hz, 1H), 7.08 (t, J = 1.8 Hz, 1H), 6.97 (t, J = 2.0 Hz, 1H), 6.91 (t, J = 1.6 Hz, 1H), 6.61 (m, 1H), 6.12 (s, 1H), 3.87 (m, 1H), 3.83 – 3.64 (m, 5H), 3.64 – 3.44 (m, 5H), 3.34 (t, J = 5.1 Hz, 2H), 3.30 – 3.24 (m, 4H), 3.22 – 3.16 (m, 3H), 2.92 (t, J = 10.4 Hz, 1H), 2.86 – 2.62 (m, 6H), 2.47 (t, J = 7.6 Hz, 2H), 2.37 – 2.24 (m (two singlets overlapping other signals), 9H), 2.19 (m, 1H), 1.99 – 1.88 (m, 5H), 1.87 – 1.64 (m, 3H), 1.59 (p, J = 7.0 Hz, 2H), 1.52 (p, J = 7.2 Hz, 2H), 1.45 – 20 1.32 (m, 4H). LC-MS m/z calcd for C44H63N11O4 [M+H]+: 809.51; 810.6 found. Analytical HPLC RT = 7.53 min on Waters XBridge Protein BEH C4 Column (100 mm × 4.6 mm) eluting 5% to 100% water(0.1% TFA)/MeCN (0.1% TFA), flow rate 1 mL/min, detecting at 210/254/280 nm and with ELSD. Scheme 14 25 380 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 123 was synthesized by previously described methods (Carbohydrate conjugated rna agents and process for their preparation By: Nair, Jayaprakash K.; Kelin, Alexander V.; Kandasamy, Pachamuthu; Rajeev, Kallanthottahill G.; Manoharan, Muthiah United States, US20150196655 A12015- 07-16) 5 Compound 124: Compound 123 (1.03g, 1.0 mmol) is dissolved in pyridine (20 mL). NaOH (120 mg, 3 mmol) is added and the reaction mixture stir at room temperature for 18 hours. Triethylamine hydrochloride (413 mg, 3 mmol) was added, stirred for 5 minutes, then concentrated under vacuum to an oil. The crude oil was dissolved in ethyl acetate (40 mL) and washed with 22% NaCl (2 x 40 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under vacuum to give compound 124, 10 which was moved forward to the next step without further purification. Compound 125: Compound 124 (1.02 g, 1.0 mmol), HOBt monohydrate (230 mg, 1.5 mmol) and HBTU (493 mg, 1.3 mmol) were dissolved in DMF (10 mL) under an inert atmosphere and cooled to 0oC. DIPEA (0.35 mL, 2.0 mmol) was added, and the reaction was allowed to stir for 5 minutes. A solution of 3-azido-1-propanamine (130 mg, 1.3 mmol) and DMF (5 mL) was added over a period of 10 15 minutes and the reaction allowed to stir at 0oC for 4 hours. The reaction was diluted with ethyl acetate (50 mL) and washed with 5% NaCl (1 x 50 mL), 10% H3PO4, 5% NaCl (1 x 50 mL), 4% NaHCO3 (1 x 50 mL), and satd. NaCl (1 x 50 mL), dried over Na2SO4, filtered, and concentrated under vacuum to give crude compound 125. The crude residue was purified by silica gel flash chromatography to give pure compound 125. 20 Compound 126: Compound 125 (1.10 g, 1.0 mmol) was dissolved in methanol (10 mL) under an inert atmosphere. Methanesulfonic acid (84 mL, 1.3 mmol) was added, and the reaction was heated to 75oC and stirred for 18 hours. Triethylamine (0.042 mL, 0.3 mmol) was added, then the reaction was concentrated under reduced vacuum to give compound 126, which was moved forward to the next step without further purification. 25 Compound 342Z: Compound 11 (700 mg, 1.0 mmol), HOBt monohydrate (230 mg, 1.5 mmol) and HBTU (493 mg, 1.3 mmol) were dissolved in DMF (10 mL) under an inert atmosphere and cooled to 0oC. DIPEA (0.87 mL, 5.0 mmol) was added and the reaction was allowed to stir for 5 minutes. A solution of compound 126 (1.04 g, 1.3 mmol) in DMF (5 mL) was added over a period of 10 minutes and the reaction allowed to stir at 0oC for 4 hours. The reaction was diluted with ethyl acetate (50) and 30 washed with 5% NaCl (50 mL x 1), 10% H3PO4, 5% NaCl (50 mL x 1), 4% NaHCO3 (50 mL x 1), and satd. NaCl (50 mL x 1), dried over Na2SO4, filtered, and concentrated under vacuum to give crude compound 342Z. Following treatment with aq. Piperidine to effect hydrolysis, the crude residue was purified by silica gel chromatography to give pure compound 342Z. Scheme 14b 381 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 200: To a solution of compound 19 (25.0 g, 75.8 mmol, 1.00 eq) in THF (250 mL) was added i-PrMgCl (2 M, 94.7 mL, 2.50 eq) at 0 °C and stirred for 1 hr. Then B(Oi-Pr)3 (42.7 g, 227 mmol, 52.3 mL, 3.00 eq) was added drop-wised at 0 °C and stirred for 2 hrs. The reaction mixture was quenched with 5 100 mL of 2N HCl. The resulting solution was extracted with EtOAc (150 mL x 3). The combined organic layers were dried with sodium sulfate. The organic layer was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (DCM/MeOH=1:0 to 10:1). Compound 200 (30.0 g, 97.9 mmol) was obtained as a yellow solid.1H NMR: (400 MHz, DMSO- d6) δ 7.97 - 7.82 (m, 2H), 7.75 (br s, 1H), 6.27 - 6.01 (m, 1H), 2.37 - 2.28 (m, 3H), 2.24 - 2.15 (m, 3H). 10 MS(M+H+): 295.2. Compound 201: To a solution of compound 4 (32.0 g, 97.7 mmol, 1.00 eq) in MeOH (350 mL) was added Pd/C (10.4 g) under nitrogen atmosphere. The suspension was degassed and purged with hydrogen for 3 times. The mixture was stirred under hydrogen (30 Psi) at 25 °C for 12 hrs. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was used directly for 15 the next step without purification. Compound 201 (26.0 g, 78.4 mmol) was obtained as gray gum. MS(M+H+): 332.2. Compound 202: To a solution of compound 201 (26.0 g, 78.4 mmol, 1.00 eq) in DCM (200 mL) was added HCl/dioxane (4 M, 98.1 mL, 5.00 eq) at 25 °C and stirred for 2 hrs. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was used directly for the next 20 step without purification. Compound 202 (20.0 g, 65.7 mmol) was obtained as yellow gum. MS(M+H+): 232.2. Compound 203: To a solution of compound 202 (33.0 g, 108 mmol, 1.00 eq, 2HCl) in DCM (1200 mL) was added Pd(dppf)Cl2.CH2Cl2 (8.86 g, 10.9 mmol, 10% eq) and KOAc (37.3 g, 379 mmol, 3.50 eq) and 382 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO compound 5B (15.4 g, 97.6 mmol, 0.90 eq) at 25 °C under N2. And then the reaction mixture was degassed 3 times under N2. The mixture was stirred at 25 °C for 2 hrs under N2. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was resolved in water (400 mL) and acidified by 2N HCl until pH = 4. The aqueous was washed by EtOAc 750 mL (250 5 mL *3). Then the aqueous phase was basified by saturated sodium carbonate aqueous until pH > 8 and extracted with DCM 750 mL (250 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was used directly for the next step without purification. Compound 203 (26.0 g, 62.5 mmol) was obtained as a brown solid. MS(M+H+): 330.2. 10 Compound 204: To a mixture of compound 203 (20.0 g, 60.7 mmol, 1.00 eq), KOH (6.13 g, 109 mmol, 1.80 eq), (R)-BINAP (3.78 g, 6.07 mmol, 10.0% eq) and compound 200 (32.2 g, 109 mmol, 1.80 eq) in dioxane (400 mL) and H2O (40 mL) was added [Rh(COD)Cl]2 (1.50 g, 3.04 mmol, 5.0% eq) in one portion at 25 °C under N2. The reaction mixture was degassed 3 times under N2. Then the mixture was heated to 100 °C and stirred for 4 hrs under N2. The reaction mixture was concentrated under reduced pressure to 15 give a residue. The residue was resolved in water (400 mL). The aqueous was acidified by 2N HCl until pH = 4 and washed by DCM 600 mL (200 mL x 3). The aqueous phase was basified by saturated Na2CO3 aqueous until pH > 8 and extracted with DCM 600 mL (200 mL x 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by SFC. Target (11.0 g, 18.2 mmol) was obtained as red gum.1H NMR: (400 MHz, METHANOL- 20 d4) δ 7.50 (d, J = 1.8 Hz, 2H), 7.33 (s, 1H), 7.10 (d, J = 7.4 Hz, 1H), 6.44 - 6.29 (m, 1H), 6.08 (s, 1H), 3.57 (s, 3H), 3.37 (br d, J = 5.5 Hz, 2H), 2.90 - 2.75 (m, 3H), 2.71 - 2.64 (m, 4H), 2.62 - 2.56 (m, 1H), 2.52 - 2.40 (m, 3H), 2.29 (s, 3H), 2.24 (s, 3H), 2.17 - 2.11 (m, 1H), 2.09 - 2.01 (m, 1H), 2.00 - 1.91 (m, 1H), 1.86 (quin, J = 5.9 Hz, 2H), 1.65 (q, J = 7.3 Hz, 2H), 1.45 - 1.35 (m, 1H), 1.29 (s, 1H). MS(M+H+): 580.2. Scheme 14c 383 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 205: Compound 204 (410 mg, 706.22 μmol) , potassium carbonate (292.82 mg, 2.12 mmol), and XPhos PD G4 (30.38 mg, 35.31 μmol) were added to a vial and the system was flushed with Ar for 10 min. DMA (0.2 mL) was added and the resulting solution was heated to 100°C (pre-heated plate) followed 5 by addition of benzyl hex-5-ynoate (589.00 mg, 2.82 mmol, 589.00 μL). After 1.5 h, the mixture was evaporated to dryness and the residue dissolved in DCM and loaded in a silica cartridge (4g). The compound was purified via flash chromatography using 0-8% MeOH in DCM as eluant to give compound 205 (260 mg). 1H NMR (600 MHz, MeOD) δ 7.12 – 7.04 (m, 8H), 6.83 (d, J = 7.3 Hz, 1H), 6.08 (d, J = 7.3 Hz, 1H), 5.82 (s, 1H), 4.87 (s, 2H), 3.12 – 3.08 (m, 3H), 2.62 (dd, J = 15.8, 6.1 Hz, 1H), 2.58 – 2.50 10 (m, 2H), 2.46 – 2.29 (m, 8H), 2.28 – 2.13 (m, 6H), 1.92 (dd, J = 9.1, 7.4 Hz, 1H), 1.87 – 1.78 (m, 1H), 1.68 (p, J = 7.1 Hz, 3H), 1.64 – 1.54 (m, 3H), 1.41 (qt, J = 9.0, 4.2 Hz, 2H), 1.15 (ddt, J = 12.6, 8.3, 6.3 Hz, 1H). MS(M+H+): 702.4. Compound 206: To a clear solution of compound 205 (132 mg, 188.06 μmol) in MeOH (3 mL), was added 10% Palladium on activated charcoal (0.20 g) and reaction vessel was purged with H2 gas. Reaction mixture 15 was stirred at 22 °C overnight. The reaction mixture was filtered through Celite bed and the filtrate was evaporated to dryness. The residue was purified using reverse phase C18 using 10-50% MeCN in H2O (0.1% TFA) as eluent to obtain compound 206 (87 mg). 1H NMR (600 MHz, MeOD) δ 7.44 (d, J = 7.3 Hz, 2H), 7.21 (dd, J = 21.6, 1.9 Hz, 3H), 7.15 (t, J = 1.8 Hz, 1H), 6.48 (t, J = 12.7 Hz, 1H), 6.04 (s, 1H), 3.76 (s, 1H), 3.68 (s, 1H), 3.56 – 3.47 (m, 4H), 3.49 (s, 4H), 3.38 (t, J = 5.7 Hz, 4H), 3.14 (s, 1H), 3.02 (s, 20 1H), 2.79 – 2.67 (m, 4H), 2.63 (q, J = 7.9 Hz, 6H), 2.55 (s, 4H), 2.39 (s, 1H), 2.18 (t, J = 8.4 Hz, 13H), 2.06 (s, 1H), 1.83 (p, J = 6.1 Hz, 3H), 1.72 (s, 6H), 1.56 (dp, J = 36.7, 7.6 Hz, 6H), 1.33 – 1.25 (m, 3H). 614.2. MS(M-H+):579.41 384 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Scheme 14d 208 209 Compound 207: To a 4 mL reaction vial equipped with a stir bar was 204 (99 mg, 389.86 μmol, 1.00 eq), potassium acetate (105 mg, 1.07 mmol, 3.11 eq.), and (dppf)PdCl2*CH2Cl2 (14.6 mg, 17.88 μmol, 5 mol%). 5 The vial was sealed with a screw-cap fit with a septum and argon inlet needle. The vial was evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous 1,4-dioxane (0.5 mL) was added via syringe to give a deep orange-red solution with stirring. Sonicated until any solid particulates (i.e. KOAc) were of small uniform size. The solution was heated for 2 hours at 90°C, during which the color turned from red to orange. Then, the solution was cooled to room temperature and a 10 solution of sodium perborate tetrahydrate (147 mg in 6 mL of deionized water) was added. The mixture was stirred vigorously for 30 minutes at room temperature. The reaction mixture was transferred to a separatory funnel containing DCM (~50 mL). The aqueous phase was extracted with DCM (2 x ~5 mL) and then the combined organic washings dried over magnesium sulfate. The solution was then filtered and concentrated in vacuo. Purified by automated silica gel chromatography (RediSepGold 40g column, eluting 15 0% to 20% DCM/MeOH, then grading up to 100% MeOH). The product containing fractions were combined and concentrated in vacuo to yield 207 as a light yellow foam (99.6 mg). MS(M+H+): 518.3. Compound 208: To a 20 mL vial containing phenol 207 (38.9 mg, 75.15 μmol, 1.00 eq.) was added a stir bar, tert-butyl 8-bromooctanoate (21.9 mg, 78.43 μmol, 1.04 eq.), and cesium carbonate (95 mg, 291.57 μmol, 3.88 eq.). The vial was sealed with a rubber septum fit with an argon inlet needle. The vial was 20 evacuated and backfilled with argon; this process was repeated one additional time. Then, anhydrous MeCN (0.90 mL) was added via syringe to give a heterogenous yellow solution. Stirred at 45 °C in a pre- heated aluminum block for 3.5 hours. Then, the solution was cooled to room temperature, diluted with DCM and filtered through a disposable fritted funnel to remove solid particulates. The filtrate was concentrated in vacuo and the crude residue purified by automated silica gel chromatography 25 (RediSepGold 12g column, eluting 0% to 50% DCM/MeOH). The product-containing fractions were combined to yield 208 as a white film (57.6 mg). MS(M+H+): 716.3. 385 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 209: To a 20 mL vial containing 208 (26.7 mg, 37.29 μmol, 1.00 eq) was added stir bar. The vial was sealed with a rubber septum fit with an argon inlet needle. The vial was evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous 1,4-dioxane (1.00 mL) and CHCl3 (0.50 mL) were added sequentially via syringe to give a slightly hazy solution. At this time, HCl in 5 1,4-dioxane (0.80 mL of a 4M solution) was added dropwise via syringe at room temperature. The inlet needle was removed, and the solution was stirred vigorously at room temperature for 8.5 hours. Then, the reaction mixture was concentrated in vacuo to yield 209 as a tan foam without further purification (33 mg). MS(M+H+): 660.3. Scheme 14e 10 Compound 210: A mixture of 204 (25 mg), 6-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)phenyl]hexanoic acid (20.55 mg), XPhos Pd G4 (1.95 mg), and potassium carbonate (17.85 mg) was added to a vial with a septum and a stir bar. The vial was purged with argon. THF (166.01 μL) and water (41.50 μL) were added to the vial and argon was bubbled through the solution for ~10 min. The reaction 15 was heated to 60 °C and stirred for 4 hours. The reaction mixture was diluted with methanol and filtered through celite. The solvent was removed to afford a crude residue which was purified by silica gel to provide the product 210. MS(M+H+): 692.4. Scheme 14f 386 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 212: To a solution of 211 (480 g) in acetonitrile (2.88 L) was added conc. HCl (3.36 L) and NaNO2 (177 g) in water (650 mL) dropwise at -5 °C, then stirred for 19 h. Then tin (II) chloride dihydrate (1.072 kg) was dissolved in conc. HCl (750 mL) and was added to the reaction mixture. The reaction 5 mixture was stirred overnight. Then pentane-2,4-dione (475 g) was added and the mixture was stirred for 1.5 h. The reaction mixture was partitioned between water and DCM, then stirred 30 min. The product was extracted with DCM, washed with brine, dried, filtered, and concentrated. The crude product was purified on silica gel to provide 212 (415 g). 387 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Compound 213: To a solution of 212 (415 g) in DCM (4.15 L) was added boron tribromide (739 g) dropwise at 0 °C, then stirred for 18 h at room temperature. Then methanol (4 L) was added to the reaction mixture dropwise at 0 °C, then stirred for 5 h at room temperature. The reaction was concentrated to give crude product, which was partitioned between water and ethyl acetate. The product was extracted with 5 ethyl acetate, washed with brine, dried, filtered, and concentrated to provide the product 213 (300 g). Compound 215: To a solution of 214 (473 g) and 213 (260 g) in DMF (3.9 L) was added caesium carbonate (634 g), then purged with nitrogen. The reaction mixture was stirred at room temperature for 15 h. Then, the mixture was partitioned between water and ethyl acetate. The product was extracted with ethyl acetate repeatedly, then washed with brine, dried, filtered, and concentrated to give crude product. The crude was 10 purified on silica gel to provide the product 215 (400 g). Compound 216: To a solution of 215 (473 g) and in dioxane (4 L) was added potassium acetate (167 g), Bis(pinacolato)diboron (238 g), and Pd(dppf)Cl2 (34.5 g), then purged with nitrogen. The reaction mixture was heated to 90 °C for 16 h. Then, the mixture was partitioned between water and ethyl acetate. The product was extracted with ethyl acetate repeatedly, then washed with brine, dried, filtered, and 15 concentrated to give crude product. The crude was purified on silica gel to provide the product 216 (450 g). Compound 217: To a solution of 6 (160 g), 216 (253 g), and R-BINAP (30.6 g) in dioxane (4 L) was added sodium hydroxide (20.7 g) in water (0.3 L) and [Rh(COD)Cl2]2 (12 g), then purged with nitrogen. The reaction mixture was heated to 70 °C for 3 h. Then, the mixture was concentrated, partitioned between 20 water and ethyl acetate. The pH was adjusted to ~6, and the product was extracted with ethyl acetate, then washed with brine, dried, filtered, and concentrated to give crude product. The crude was purified by SFC to provide the product 217 (95 g). Compound 218: To a solution of 217 (95 g) in THF (1 L) was added nickel (285 g), then purged with hydrogen, and stirred at room temperature for 19 h. The reaction mixture was filtered, and washed with 25 THF (2 L). Then, the filtrate was concentrated to give product 218 (85 g), which was used directly without further purification. Compound 219: To a solution of 218 (85 g) in DCM (0.85 L) was added triethylamine (24 g) and phenoxyacetyl chloride (30.2 g) at -5 °C, then stirred for 18 h. The reaction mixture was washed with water, and the organic layer was dried, filtered, and concentrated. The crude was purified on silica gel to provide 30 the product 219 (60 g). Compound 220: To a solution of 219 (60 g) in DCM (0.42 L) was added trifluoroacetic acid (300 mL) at -10 °C, then stirred for 18 h. The reaction mixture was poured into a sat. sodium bicarbonate solution, and adjusted to pH ~7. The organic phase was washed with water, and the organic layer was dried, filtered, and concentrated to provide the product 220 (53 g). 388 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Example 2: Synthesis of in vivo delivery enhancing moieties (lipophilic moieties) 300 301 A solution of OBn-PEG-Glu amine 300 (2.08 g, 3.60 mmol) in DCM (20 mL) was combined 5 with 1-Methyl eicosanedioate (1.66 g, 4.68 mmol), 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (896.73 mg, 4.68 mmol), 1-Hydroxy-1H-benzotriazole hydrate (551.04 mg, 3.60 mmol), Diisopropylethylamine (1.88 mL, 10.79 mmol) and the resulting mixture was stirred overnight. DCM was added to the mixture and the organic layer was washed with 1M HCl, water and brine. The organic layer was dried over Na2SO4, filtered, and the volatiles were removed under reduced pressure. The 10 residue was purified by ISCO automated column using 0-100% (3:1 EtOAc/EtOH) in hexanes to give compound 301 (1.67 g, 52%).1H NMR (600 MHz, DMSO) δ 8.17 (d, J = 7.4 Hz, 1H), 7.88 (t, J = 5.7 Hz, 1H), 7.65 (t, J = 5.9 Hz, 1H), 7.40 – 7.32 (m, 5H), 5.14 (s, 2H), 4.19 – 4.15 (m, 3H), 3.86 (s, 2H), 3.62 – 3.51 (m, 14H), 3.44 – 3.38 (m, 4H), 3.26 (q, J = 5.9 Hz, 2H), 3.19 (q, J = 5.8 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.15 – 2.06 (m, 4H), 1.95 – 1.88 (m, 1H), 1.80 – 1.73 (m, 1H), 1.53 – 1.44 (m, 4H), 1.23 (s, 15 28H). 301 302 Compound 301 (1.67 g, 1.90 mmol) was dissolved in EtOAc (100 mL) and THF (30 mL), and warmed to 35C.10% Pd/C (200 mg, 0.19 mmol) was added to the previous solution and then the flask 20 was equipped with a three-way adapter connected to a balloon filled with Hydrogen. The flask was submitted to a sequence of vacuum-H2 refill (x3). After stirring for 24 h, the mixture was filtered through a celite pad and rinsed with warm THF (35C). The volatiles were evaporated to dryness to give compound 302 (1.5g, 100%).1H NMR (600 MHz, DMSO) δ 12.61 (s, 1H), 8.19 (d, J = 7.3 Hz, 1H), 7.90 (t, J = 5.6 Hz, 1H), 7.67 (t, J = 5.9 Hz, 1H), 4.17 (ddd, J = 9.0, 7.3, 5.4 Hz, 1H), 4.01 (s, 2H), 3.87 (s, 25 2H), 3.60 (s, 3H), 3.58 – 3.51 (m, 11H), 3.45 – 3.39 (m, 4H), 3.26 (q, J = 6.0 Hz, 2H), 3.19 (q, J = 5.8 389 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.17 – 2.06 (m, 4H), 1.95 – 1.89 (m, 1H), 1.79 – 1.71 (m, 1H), 1.52 – 1.43 (m, 4H), 1.23 (s, 28H). 303 304 5 To a solution of PEG-Acid 303 (19.69 g, 22.11 mmol) in DCM (180 mL), 1-Ethyl-3-(3′- dimethylaminopropyl)carbodiimide hydrochloride (5.09 g, 26.53 mmol) , 4-(Dimethylamino)pyridine (270.09 mg, 2.21 mmol) , and Benzyl Alcohol (3.59 g, 33.16 mmol, 3.43 mL) were added. After 12h, DCM (300 mL) and 1M HCl (180 mL) were added and the layers were separated (diffult to separated mixture - wait 1h min). The organic layer was washed with brine and dried over Na2SO4. The volatiles 10 were evaporated under reduced pressure and the residue was purified by ISCO automated column (ELSD) using 0-4%MeOH in DCM as eluant to give 304 (19.08 g, 92%).1H NMR (600 MHz, DMSO) δ 8.06 (d, J = 7.5 Hz, 1H), 7.91 (t, J = 5.6 Hz, 1H), 7.66 (t, J = 5.9 Hz, 1H), 7.40 – 7.31 (m, 5H), 5.14 (s, 2H), 4.19 (s, 2H), 4.03 (ddd, J = 9.0, 7.5, 5.3 Hz, 1H), 3.86 (s, 2H), 3.62 – 3.49 (m, 8H), 3.41 (dt, J = 16.8, 5.9 Hz, 4H), 3.25 (q, J = 6.0 Hz, 2H), 3.19 (q, J = 5.9 Hz, 2H), 2.18 – 2.06 (m, 6H), 1.88 (dtd, J = 15 13.4, 7.7, 5.3 Hz, 1H), 1.72 (ddd, J = 13.5, 8.7, 6.6 Hz, 1H), 1.46 (p, J = 7.2 Hz, 4H), 1.38 (d, J = 1.6 Hz, 18H), 1.22 (s, 24H). 304 305 TFA (120 mL) was added to a solution of PEG-OBn ester 304 (19.08 g, 20.38 mmol) in DCM 20 (360 mL). After 6h, the volatiles were removed under reduced pressure, co-evaporated with DCM and MeCN. The crude residue was dissolved in DCM (200 mL) and combined with 1-Ethyl-3-(3′- dimethylaminopropyl)carbodiimide hydrochloride (11.72 g, 61.14 mmol) , 4-(Dimethylamino)pyridine (746.94 mg, 6.11 mmol), and methanol (7.84 g, 244.56 mmol, 9.91 mL) . The mixture was stirred overnight, then evaporated to dryness. The residue was re-dissolved in DCM (150 mL) and washed with 25 1M HCL (50 mLx2), water and brine. The volatiles were evaporated under reduced pressure and the residue was purified by ISCO automated column using 0 - 5% MeOH in DCM as eluant to give 390 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO compound 305 (14.06 g, 80% over two steps).1H NMR (600 MHz, DMSO) δ 8.17 (d, J = 7.4 Hz, 1H), 7.88 (t, J = 5.6 Hz, 1H), 7.65 (t, J = 5.9 Hz, 1H), 7.40 – 7.32 (m, 5H), 5.15 (s, 2H), 4.19 (s, 3H), 3.87 (s, 2H), 3.62 – 3.51 (m, 14H), 3.44 – 3.39 (m, 4H), 3.26 (q, J = 6.0 Hz, 2H), 3.19 (q, J = 5.8 Hz, 2H), 2.27 (t, J = 7.4 Hz, 2H), 2.18 – 2.06 (m, 4H), 1.92 (dtd, J = 13.4, 7.8, 5.4 Hz, 1H), 1.77 (ddd, J = 14.0, 8.9, 6.9 5 Hz, 1H), 1.53 – 1.44 (m, 4H), 1.23 (s, 24H). 305 306 Compound 305 (14.06 g, 16.50 mmol) was dissolved in EtOAc (500 mL) and THF (100 mL) and mixed with 10% Pd/C (Deggusa type) (1.7 g). The mixture was warmed to 30 °C, then the flask was 10 degassed under vacuum and refilled with hydrogen (x3). A balloon filled with hydrogen was connected to the flask and the reaction was stirred overnight. The sample was diluted with EtOAc and filtered though a celite pad. The filtride was evaporated to dryness to give compound 306 (9.46 g, 75%). The product was used without further purification.1H NMR (600 MHz, DMSO) δ 12.58 (s, 1H), 8.17 (d, J = 7.4 Hz, 1H), 7.88 (t, J = 5.6 Hz, 1H), 7.65 (t, J = 5.9 Hz, 1H), 4.18 (ddd, J = 9.0, 7.4, 5.4 Hz, 1H), 4.01 (s, 15 2H), 3.87 (s, 2H), 3.61 – 3.51 (m, 14H), 3.45 – 3.39 (m, 4H), 3.26 (q, J = 5.9 Hz, 2H), 3.19 (q, J = 5.8 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.17 – 2.08 (m, 4H), 1.92 (dtd, J = 13.4, 7.8, 5.4 Hz, 1H), 1.76 (ddt, J = 13.8, 9.1, 7.5 Hz, 1H), 1.54 – 1.44 (m, 4H), 1.23 (s, 24H). 307 308 20 To a solution of Palmitic acid (1.59 g, 6.21 mmol) in DCM (60 mL) were added 4- Methylmorpholine (1.88 g, 18.62 mmol, 2.05 mL) , 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (1.19 g, 6.21 mmol) , and 1-Hydroxybenzotriazole hydrate (950.26 mg, 6.21 mmol) at 0°C. The reaction mixture was stirred at 0 °C for 1 h. Then compound 307 (1 g, 6.21 mmol) was added to the previous mixture. The reaction mixture was stirred at room temperature under Ar for 48h. The 25 reaction was quenched by addition of 1M HCl (30 mL). The organic layer was separated and the aqueous layer was extracted with more DCM. The combined organic layers were washed with water and brine and adsorbed in celite. The residue was purified by ISCO automated column using a 3:1 EtOAc/EtOH mixture in hexanes 4:1 hexanes as eluant to give compound 308 (471 mg, 20%).1H NMR (600 MHz, DMSO) δ 12.16 (s, 1H), 8.17 (d, J = 7.5 Hz, 1H), 4.24 (ddd, J = 9.3, 7.5, 5.3 Hz, 1H), 3.61 (s, 3H), 2.27 391 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (t, J = 7.5 Hz, 2H), 2.09 (td, J = 7.3, 2.0 Hz, 2H), 1.97 – 1.90 (m, 1H), 1.76 (ddt, J = 14.0, 9.3, 7.1 Hz, 1H), 1.47 (q, J = 7.1 Hz, 2H), 1.23 (s, 24H), 0.85 (t, J = 7.0 Hz, 3H). LRMS (ESI) calculated for C22H42NO5 [M+H]+ m/z = 400.3063 , found 400.4. 5 307 309 To a solution of stearic acid (1.77 g, 6.21 mmol) in DCM (60.00 mL) were added 4- Methylmorpholine (1.88 g, 18.62 mmol, 2.05 mL), 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (1.19 g, 6.21 mmol) , and 1-Hydroxybenzotriazole hydrate (950.26 mg, 6.21 mmol) at 0°C. The reaction mixture was stirred at 0 °C for 1 h. Then compound (1 g, 6.21 mmol) was added. The 10 reaction mixture was stirred at room temperature under Ar for 48h. The mixture was diluted with DCM and washed combined with 1 M HCl (40 mL). The organic layer was combined with celite, evaporated to dryness and the residue purified by ISCO automated column using 0-10% MeOH in DCM to give compound 309 (871 mg, 32%).1H NMR (600 MHz, DMSO) δ 12.14 (s, 1H), 8.17 (d, J = 7.5 Hz, 1H), 4.24 (ddd, J = 9.3, 7.5, 5.3 Hz, 1H), 3.61 (s, 3H), 2.27 (t, J = 7.5 Hz, 2H), 2.09 (td, J = 7.3, 2.0 Hz, 2H), 15 1.93 (dtd, J = 13.2, 7.8, 5.3 Hz, 1H), 1.76 (ddt, J = 14.0, 9.3, 7.0 Hz, 1H), 1.47 (q, J = 7.0 Hz, 2H), 1.23 (s, 28H), 0.85 (t, J = 7.0 Hz, 3H). LRMS (ESI) calculated for C24H46NO5 [M+H]+ m/z = 428.3376 , found 428.4. 307 310 20 To a solution of arachidic acid (1.94 g, 6.21 mmol, 2.06 mL) in DCM (59.82 mL) were added 4- Methylmorpholine (1.88 g, 18.62 mmol, 2.05 mL), 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (1.19 g, 6.21 mmol) , and 1-Hydroxybenzotriazole hydrate (950.26 mg, 6.21 mmol) at 0°C. The reaction mixture was stirred at 0 °C for 1 h. Then compound 307 (1 g, 6.21 mmol) was added. The reaction mixture was stirred at room temperature under nitrogen for 48h. The mixture was diluted 25 with DCM and washed combined with 1 M HCl. The organic layer was combined with celite, evaporated to dryness and the residue purified by ISCO automated column using 0-10% MeOH in DCM. The system suffered from overpressure during the run, and most of the product/mixture remained in the column with only a fraction of the desired compound 310 was obtained (172 mg, 6%).1H NMR (600 MHz, DMSO) δ 12.14 (s, 1H), 8.17 (d, J = 7.5 Hz, 1H), 4.24 (ddd, J = 9.2, 7.5, 5.3 Hz, 1H), 3.61 (s, 3H), 2.27 (t, J = 7.5 30 Hz, 2H), 2.09 (td, J = 7.3, 2.0 Hz, 2H), 1.93 (dtd, J = 13.2, 7.8, 5.3 Hz, 1H), 1.76 (ddt, J = 14.0, 9.3, 7.1 392 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Hz, 1H), 1.50 – 1.44 (m, 2H), 1.23 (s, 32H), 0.85 (t, J = 6.9 Hz, 3H). LRMS (ESI) calculated for C26H50NO5 [M+H]+ m/z = 456.3689, found 456.4. 307 311 5 To a solution of behenic acid (2.11 g, 6.21 mmol, 2.25 mL) in DCM (60 mL) were added 4- Methylmorpholine (1.88 g, 18.62 mmol, 2.05 mL) , 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (1.19 g, 6.21 mmol) , and 1-Hydroxybenzotriazole hydrate (950.26 mg, 6.21 mmol) at 0°C. The reaction mixture was stirred at 0 °C for 1 h. Then compound 307 (1 g, 6.21 mmol) was added. The reaction mixture was stirred at room temperature under nitrogen for 48h. The mixture was diluted 10 with DCM and washed combined with 1 M HCl (40 mL). The organic layer was combined with celite, evaporated to dryness and the residue purified by ISCO automated column using 0-10% MeOH in DCM to give compound 311 (232 mg, 7%).1H NMR (600 MHz, DMSO) δ 12.16 (s, 1H), 8.17 (d, J = 7.5 Hz, 1H), 4.26 – 4.21 (m, 1H), 3.61 (s, 3H), 2.27 (t, J = 7.5 Hz, 2H), 2.09 (td, J = 7.3, 1.9 Hz, 2H), 1.93 (td, J = 13.4, 7.7 Hz, 1H), 1.76 (ddd, J = 16.3, 14.0, 7.3 Hz, 1H), 1.47 (d, J = 7.4 Hz, 2H), 1.23 (s, 36H), 0.85 15 (t, J = 6.9 Hz, 3H). LRMS (ESI) calculated for C28H54NO5 [M+H]+ m/z = 484.4002, found 484.4. 312 313 TFA (5 mL) was added to a solution of compound 312 (1 g, 2.85 mmol) in DCM (10 mL). After 3h, the volatiles were removed under reduced pressure and co-evaporated with DCM and acetonitrile. 20 The residual oil was dissolved in DCM (10 mL) and combined with 1-Hydroxybenzotriazole hydrate (479.39 mg, 3.13 mmol), 1-Methyl hexadecanedioate (894.70 mg, 2.99 mmol), 1-Ethyl-3-(3′- dimethylaminopropyl)carbodiimide hydrochloride (709.21 mg, 3.70 mmol) and Diisopropylethylamine (1.49 mL, 8.54 mmol) at 0°C. The mixture was allowed to reach r.t. and stirred for 24h. The reaction mixture was diluted with chloroform (50 mL) and washed with 1M HCl, water, brine. The residue was 25 purified by ISCO automated column using 0-30% EtOAc in hexanes as eluant to give compound 313 (468 mg, 30% over two steps).1H NMR (600 MHz, DMSO) δ 8.19 (d, J = 7.6 Hz, 1H), 7.39 – 7.30 (m, 5H), 5.08 (d, J = 1.2 Hz, 2H), 4.27 (ddd, J = 9.3, 7.5, 5.2 Hz, 1H), 3.60 (s, 3H), 3.57 (s, 3H), 2.43 (td, J = 7.2, 1.7 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.09 (td, J = 7.3, 1.8 Hz, 2H), 2.00 (dtd, J = 13.2, 7.8, 5.3 Hz, 393 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 1H), 1.82 (dddd, J = 14.0, 9.4, 7.9, 6.5 Hz, 1H), 1.53 – 1.44 (m, 4H), 1.22 (d, J = 7.8 Hz, 20H). LRMS (ESI) calculated for C30H48NO7 [M+H]+ m/z = 534.3431, found 534.4. 313 314 5 Compound 313 (468 mg, 876.91 μmol) was dissolved in EtOAc (20 mL) and mixed with 10% Pd/C (Deggusa type) (93 mg). The flask was degassed under vacuum and refilled with hydrogen (x3). A balloon filled with Hydrogen was connected to the flask and the reaction was stirred overnight. The mixture was diluted with DCM and filtered through a celite pad, then the volatiles were evaporated to dryness to give compound 314 (383 mg, 98%).1H NMR (600 MHz, DMSO) δ 12.16 (s, 1H), 8.17 (d, J = 10 7.5 Hz, 1H), 3.61 (s, 3H), 3.57 (s, 3H), 2.27 (td, J = 7.5, 5.8 Hz, 4H), 2.09 (td, J = 7.2, 2.1 Hz, 2H), 1.93 (dtd, J = 13.2, 7.8, 5.3 Hz, 1H), 1.76 (ddd, J = 13.9, 9.2, 6.8 Hz, 1H), 1.54 – 1.44 (m, 4H), 1.23 (s, 20H). LRMS (ESI) calculated for C23H42NO7 [M+H]+ m/z = 444.2961, found 444.2. 312 315 15 TFA (5mL) was added to s solution of compound 312 (1 g, 2.85 mmol) in DCM (10 mL). After 2h, the volatiles were removed under reduced pressure and co-evaporated with DCM and acetonitrile. The crude residue was dissolved in DCM (10mL) and combined with 1-Methyl octadecanedioate (978.54 mg, 2.99 mmol) , 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (709.21 mg, 3.70 mmol) , 1- Hydroxybenzotriazole hydrate (479.39 mg, 3.13 mmol) , and Diisopropylethylamine (1.10 g, 8.54 mmol, 20 1.49 mL) at 0°C. The mixture was allowed to reach r.t. and stirred for 24h. The reaction mixture was diluted with chloroform (50 mL) and washed with 1M HCl, water, bicarb. The residue was purified by ISCO automated column using 0-30% EtOAc in hexanes as eluant to give compound 315 (510 mg, 32%). 1H NMR (600 MHz, DMSO) δ 8.19 (d, J = 7.6 Hz, 1H), 7.39 – 7.30 (m, 5H), 5.08 (d, J = 1.1 Hz, 2H), 4.27 (ddd, J = 9.3, 7.5, 5.2 Hz, 1H), 3.60 (s, 3H), 3.57 (s, 3H), 2.45 – 2.42 (m, 2H), 2.28 (t, J = 7.4 Hz, 25 2H), 2.09 (td, J = 7.3, 1.9 Hz, 2H), 2.04 – 1.96 (m, 1H), 1.85 – 1.79 (m, 1H), 1.54 – 1.44 (m, 4H), 1.22 (d, J = 2.9 Hz, 24H). LRMS 394 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 315 316 Compound 315 (510 mg, 907.88 μmol) was dissolved in EtOAc (20 mL) and mixed with 10% Pd/C (Deggusa type) (95 mg). The flask was degassed under vacuum and refilled with hydrogen (x3). A balloon filled with hydrogen was connected to the flask and the reaction was stirred for overnight. The 5 mixture was diluted with DCM and filtered through a celite pad then the volatiles were evaporated to dryness to give compound 316 (421 mg, 98%).1H NMR (600 MHz, DMSO) δ 12.15 (s, 1H), 8.17 (d, J = 7.5 Hz, 1H), 4.24 (ddd, J = 9.2, 7.5, 5.3 Hz, 1H), 3.61 (s, 3H), 3.57 (s, 3H), 2.27 (td, J = 7.5, 5.1 Hz, 4H), 2.09 (td, J = 7.3, 2.3 Hz, 2H), 1.97 – 1.90 (m, 1H), 1.76 (ddt, J = 14.0, 9.3, 7.1 Hz, 1H), 1.53 – 1.43 (m, 4H), 1.23 (s, 24H). LRMS (ESI) calculated for C25H46NO7 [M+H]+ m/z = 472.3274, found 472.4. 10 312 317 TFA (5mL) was added to a solution of compound 312 (1 g, 2.85 mmol) in DCM (10 mL). After 3h, the volatiles were removed under reduced pressure and co-evaporated with DCM and acetonitrile. The crude residue was dissolved in DCM (10mL) and combined with 1-Methyl eicosanedioate (1.06 g, 15 2.99 mmol) , 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (709.21 mg, 3.70 mmol), 1-Hydroxybenzotriazole hydrate (479.39 mg, 3.13 mmol) and Diisopropylethylamine (1.49 mL, 8.54 mmol) at 0C. The mixture was allowed to reach r.t. and stirred for 24h. The reaction mixture was diluted with chloroform (50 mL) and washed with 1M HCl, water, brine. The residue was purified by ISCO automated column using 0-30% EtOAc in hexanes as eluant to give compound 317 (446 mg, 31%).1H 20 NMR (600 MHz, DMSO) δ 8.19 (d, J = 7.6 Hz, 1H), 7.42 – 7.28 (m, 5H), 5.08 (d, J = 1.1 Hz, 2H), 4.27 (ddd, J = 9.3, 7.6, 5.2 Hz, 1H), 3.60 (s, 3H), 3.57 (s, 3H), 2.43 (td, J = 7.2, 1.7 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.09 (d, J = 3.5 Hz, 2H), 2.03 – 1.97 (m, 1H), 1.86 – 1.79 (m, 1H), 1.54 – 1.43 (m, 4H), 1.22 (d, J = 4.2 Hz, 28H). LRMS 25 317 318 Compound 317 (446 mg, 0.756 mmol) was dissolved in EtOAc (20 mL) and mixed with 10% Pd/C (Deggusa type) (93 mg). The flask was degassed under vacuum and refilled with hydrogen (x3). A balloon filled with hydrogen was connected to the flask and the reaction was stirred for overnight. The mixture was diluted with DCM and filtered through a celite pad then the volatiles were evaporated to 395 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO dryness to give compound 318 (370 mg, 97%).1H NMR (600 MHz, DMSO) δ 12.16 (s, 1H), 8.17 (d, J = 7.5 Hz, 1H), 4.24 (ddd, J = 9.2, 7.4, 5.2 Hz, 1H), 3.61 (s, 3H), 3.57 (s, 3H), 2.27 (td, J = 7.5, 5.3 Hz, 4H), 2.09 (td, J = 7.2, 2.0 Hz, 2H), 1.93 (dtd, J = 13.2, 7.8, 5.3 Hz, 1H), 1.76 (ddt, J = 14.0, 9.4, 7.2 Hz, 1H), 1.53 – 1.43 (m, 4H), 1.23 (s, 28H). LRMS (ESI) calculated for C27H50NO7 [M+H]+ m/z = 500.3587, 5 found 500.4. 312 319 TFA (5mL) was added to a solution of compound 312 (1 g, 2.85 mmol) in DCM (10 mL). After 3h, the volatiles were removed under reduced pressure and co-evaporated with DCM and acetonitrile. 10 The crude residue was dissolved in DCM (10mL) and combined with 1-Methyl docosanedioate (1.15 g, 2.99 mmol) , Diisopropylethylamine (1.10 g, 8.54 mmol, 1.49 mL) , 1-Ethyl-3-(3′- dimethylaminopropyl)carbodiimide hydrochloride (709.21 mg, 3.70 mmol) , and 1- Hydroxybenzotriazole hydrate (479.39 mg, 3.13 mmol) at 0C. The mixture was allowed to reach r.t. and stirred for 24h. The reaction mixture was diluted with chloroform (50 mL) and washed with 1M HCl, 15 water, brine. The residue was purified by ISCO automated column using 0-4% MeOH in DCM as eluant to give compound 319 (1.37 g, 77%).1H NMR (600 MHz, DMSO) δ 8.19 (d, J = 7.5 Hz, 1H), 7.39 – 7.30 (m, 5H), 5.08 (s, 2H), 4.27 (ddd, J = 9.3, 7.6, 5.3 Hz, 1H), 3.60 (s, 3H), 3.57 (s, 3H), 2.45 – 2.41 (m, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.09 (td, J = 7.2, 1.9 Hz, 2H), 2.04 – 1.96 (m, 1H), 1.87 – 1.79 (m, 1H), 1.53 – 1.43 (m, 4H), 1.22 (d, J = 5.4 Hz, 32H). LRMS 20 319 320 Compound 319 (950 mg, 1.61 mmol) was dissolved in a 1:1 mixture of EtOAc/THF (200 mL) and mixed with 10% Pd/C (Deggusa type) (93 mg). The flask was degassed under vacuum and refilled with hydrogen (x3). A balloon filled with hydrogen was connected to the flask and the reaction was 25 stirred for overnight. The mixture was diluted with DCM and filtered through a celite pad then the volatiles were evaporated to dryness to give compound 320 (326 mg, 40%).1H NMR (600 MHz, DMSO) δ 12.14 (s, 1H), 8.17 (d, J = 7.5 Hz, 1H), 4.27 – 4.20 (m, 1H), 3.61 (s, 3H), 3.57 (s, 3H), 2.27 (td, J = 7.5, 5.2 Hz, 4H), 2.09 (td, J = 7.3, 1.9 Hz, 2H), 1.97 – 1.90 (m, 1H), 1.79 – 1.71 (m, 1H), 1.55 – 1.43 (m, 4H), 1.23 (s, 32H). LRMS 396 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 321 322 HBTU (7.17 g, 18.90 mmol) and N-ethyl-N-isopropyl-propan-2-amine (6.11 g, 47.25 mmol, 8.23 mL) were added to a stirred solution of Bis-Lys acid (10.67 g, 18.90 mmol) in DMF (100 mL). After 5 stirring for 5 min, compound 321 (10.17 g, 15.75 mmol) was added in a single portion and the resulting solution was stirred overnight. The mixture was evaporated to dryness and the residue dissolved in EtOAc (200 mL) and washed with bicarb, water and brine. The organic layer was dryed over na2so4, filtered and evaporated to dryness. The residue was purified by ISCO automated column using 0-12% MeOH in DCM as eluant to give compound 322 (13.25 g, 70%).1H NMR (600 MHz, DMSO) δ 11.38 (d, 10 J = 2.1 Hz, 1H), 9.48 (d, J = 7.9 Hz, 1H), 9.40 (t, J = 5.7 Hz, 1H), 8.08 (t, J = 5.6 Hz, 1H), 7.75 – 7.67 (m, 3H), 7.40 – 7.35 (m, 2H), 7.32 (dd, J = 8.6, 7.0 Hz, 2H), 7.26 – 7.23 (m, 4H), 6.93 – 6.87 (m, 4H), 5.79 (d, J = 3.6 Hz, 1H), 5.28 (dd, J = 8.1, 2.1 Hz, 1H), 5.13 (d, J = 6.7 Hz, 1H), 4.25 – 4.14 (m, 2H), 3.96 (ddd, J = 7.0, 4.4, 2.7 Hz, 1H), 3.89 (dd, J = 5.2, 3.7 Hz, 1H), 3.74 (s, 6H), 3.63 – 3.50 (m, 2H), 3.30 (dd, J = 10.8, 4.6 Hz, 1H), 3.23 (dd, J = 10.7, 2.7 Hz, 1H), 3.19 (s, 4H), 3.08 – 2.93 (m, 6H), 2.01 (t, J = 15 7.5 Hz, 4H), 1.72 – 1.62 (m, 2H), 1.53 – 1.17 (m, 24H). LRMS (ESI) calculated for C58H74F6N7O13 [M-H]- m/z = 1190.5249, found 1190.2. 322 323 1H-imidazole (1.68 g, 24.66 mmol) and tert-Butyldimethylsilyl chloride (2.30 g, 14.80 mmol, 20 97% purity) were added sequentially to a stirred solution of compound 322 (11.76 g, 9.86 mmol) in DMF (100 mL). After stirring for 12h, the volatiles were evaporated to dryness and the residue dissolved partitioned in EtOAc and sat bicarb. The layers were separated, and the organic layer was washed with water, brine and dried over Na2SO4. The solids were removed by filtration and the solution was evaporated to dryness. The residue was purified by ISCO automated column using a 50-100% gradiend 25 of EtOAc:EtOH (3:1) in hexanes to give compound 323 (9.79 g, 75%).1H NMR (600 MHz, DMSO) δ 11.39 (d, J = 2.2 Hz, 1H), 9.48 (d, J = 7.9 Hz, 1H), 9.40 (t, J = 5.8 Hz, 1H), 8.08 (t, J = 5.6 Hz, 1H), 7.86 (d, J = 8.1 Hz, 1H), 7.70 (dt, J = 9.0, 5.6 Hz, 2H), 7.39 – 7.30 (m, 4H), 7.28 – 7.20 (m, 5H), 6.92 – 6.86 (m, 4H), 5.77 (d, J = 2.8 Hz, 1H), 5.28 (dd, J = 8.1, 2.2 Hz, 1H), 4.27 (dd, J = 7.0, 5.1 Hz, 1H), 4.22 (td, 397 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO J = 8.4, 5.8 Hz, 1H), 3.91 (td, J = 5.8, 3.3 Hz, 2H), 3.73 (s, 6H), 3.59 (dt, J = 9.5, 6.4 Hz, 1H), 3.46 – 3.37 (m, 2H), 3.22 – 3.09 (m, 3H), 3.09 – 2.95 (m, 6H), 2.03 – 1.99 (m, 4H), 1.68 (ddd, J = 13.1, 6.8, 3.6 Hz, 2H), 1.50 – 1.42 (m, 8H), 1.40 – 1.18 (m, 16H), 0.75 (s, 9H), 0.03 (s, 3H), -0.07 (s, 3H). LRMS (ESI) calculated for C64H88F6N7O13Si [M-H]- m/z = 1304.6114, found 1304.6. 5 323 324 DMAP (1.83 g, 14.97 mmol), triethylamine (2.09 mL, 14.97 mmol), and 2,4,6-tris(1- methylethyl)benzenesulfonyl chloride (4.53 g, 14.97 mmol) were added sequentially to a stirred solution of compound 323 (9.78 g, 7.49 mmol) in MeCN (100 mL). After stirring for 2h, ammonium hydroxide, 10 28% solution (8 M, 46.79 mL) was added and stirred for additional 2h followed by removal of the volatiles under reduced pressure. The crude residue was combined with EtOAc (300 mL) and water (50 mL), the layers were separated, and the organic layer was washed with, sat NH4Cl, water and brine. The crude yellow foam was dissolved in DMF (100 mL) and combined with acetic anhydride (2.29 g, 22.46 mmol, 2.12 mL) and stirred overnight. The volatiles were removed under reduced pressure, and the 15 residue dissolved in ethyl acetate, washed with sat. sodium bicarbonate, water, brine, and dried over anhyd. sodium sulfate. The solids were removed by filtration and the solvent was removed under vacuum. The residue was purified by ISCO automated column using 10-80% (3:1 EtOAc/EtOH) in hexanes as eluant to give compound 324 (7.27 g, 72%).1H NMR (600 MHz, DMSO) δ 10.93 (s, 1H), 9.48 (d, J = 7.9 Hz, 1H), 9.40 (t, J = 5.8 Hz, 1H), 8.47 (d, J = 7.5 Hz, 1H), 8.08 (t, J = 5.6 Hz, 1H), 7.70 20 (td, J = 5.6, 2.3 Hz, 2H), 7.39 – 7.31 (m, 4H), 7.29 – 7.21 (m, 5H), 6.99 (d, J = 7.5 Hz, 1H), 6.91 – 6.87 (m, 4H), 5.83 (s, 1H), 4.34 (dd, J = 8.8, 4.7 Hz, 1H), 4.22 (td, J = 8.4, 5.8 Hz, 1H), 4.04 – 3.99 (m, 2H), 3.85 – 3.78 (m, 2H), 3.74 (s, 6H), 3.55 – 3.45 (m, 2H), 3.24 – 3.09 (m, 3H), 3.09 – 2.90 (m, 7H), 2.09 (s, 3H), 2.01 (t, J = 7.5 Hz, 4H), 1.72 – 1.61 (m, 2H), 1.53 – 1.42 (m, 9H), 1.39 – 1.19 (m, 17H), 0.71 (s, 9H), -0.12 (s, 3H). LRMS (ESI) calculated for C66H92F6N8NaO13Si [M+Na]+ m/z =1369.6355, found 25 1369.6. 324 325 398 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Triethylamine (4.51 mL, 32.37 mmol) and Triethylamine trihydrofluoride, ca 37% HF (2.64 mL, 16.18 mmol) were added to a solution of nucleoside 324 (7.27 g, 5.39 mmol) in THF (50 mL). After 20h, more NEt3 (5 mL) and NEt3HF (2.5 mL) were added and the solution was heated to 45C for 3h. The volatiles were then removed under reduced pressure and the residue was dissolved in EtOAc and washed 5 with sat bicarb, and brine. The solution was dried over Na2SO4, the solids were removed by filtration and the solvent was removed under vacuum. The residue was purified by ISCO automated column using 0-80% EtOAc in hexanes as eluant to give product 325 (5.68 g, 85%). LRMS (ESI) calculated for C60H78F6N8NaO13 [M+Na]+ m/z =1255.5490, found 1255.4 10 325 326 1H-Tetrazole (0.45 M, 9.80 mL) and 3-bis[bis(1-methylethyl)amino]phosphanyloxypropanenitrile (5.32 mL, 16.76 mmol) were added to a solution of nucleoside 325 (5.44 g, 4.41 mmol) in MeCN (15 mL) at 0°C. The cold bath was removed, and the mixture stirred for 2h. The volatiles were removed under reduced pressure and the residue dissolved in EtOAc (150 mL), washed with sat bicarb, brine and water. 15 The solution was dried over Na2SO4, the solids were removed by filtration and the solvent was removed under vacuum. The residue was purified by ISCO automated column using 0-80% (3:1 EtOAc/EtOH) in hexanes to give compound 326 (5.88 g, 92%).1H NMR (600 MHz, DMSO) δ 10.89 (s, 1H), 9.48 (d, J = 7.9 Hz, 1H), 9.40 (t, J = 5.9 Hz, 1H), 8.41 – 8.33 (m, 1H), 8.08 (t, J = 5.6 Hz, 1H), 7.70 (q, J = 5.8 Hz, 2H), 7.44 – 7.21 (m, 8H), 6.96 – 6.85 (m, 4H), 5.84 (dd, J = 13.3, 1.1 Hz, 1H), 4.47 (td, J = 9.0, 4.6 Hz, 20 1H), 4.35 (dt, J = 8.7, 4.9 Hz, 1H), 4.24 – 4.12 (m, 2H), 4.00 – 3.93 (m, 1H), 3.74 (d, J = 4.2 Hz, 6H), 3.72 – 3.41 (m, 5H), 3.34 (d, J = 0.9 Hz, 16H), 3.21 – 2.95 (m, 7H), 2.73 (t, J = 6.4 Hz, 1H), 2.66 – 2.53 (m, 1H), 2.08 (d, J = 1.4 Hz, 2H), 2.01 (t, J = 7.6 Hz, 3H), 1.67 (ddt, J = 11.8, 8.9, 4.5 Hz, 2H), 1.56 – 1.19 (m, 19H), 1.14 – 1.05 (m, 8H), 0.95 (d, J = 6.7 Hz, 2H).31P NMR (243 MHz, DMSO) δ 148.70, 147.65.19F NMR (565 MHz, DMSO) δ -74.43, -74.98. LRMS (ESI) calculated for 25 C69H95F6N10NaO14P [M+Na]+ m/z =1455.6569, found 1455.8 Scheme 15 399 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Step 1: 2M solution of AlMe3 (50 mL, 0.10 mol) was added slowly for ca.15 min to a stirred suspension of 1-docosanol (108 g, 0.33 mol) in anhyd. diglyme (90 mL) under Ar atmosphere in a 2-neck 1L flask fitted with a magnetic stirring bar, and an outlet with a gas bubbler over a reflux condenser. After 5 completion of the addition, the mixture was heated to 100 °C until the end of evolution of gas through the bubbler (30 min). The mixture was cooled to 65 °C and diluted with anhyd. AcOEt (150 mL) and anhyd. ACN (150 mL). The mixture was cooled down to rt in the bath overnight, the white residue formed was filtered through a 600 mL glass filtering funnel under cushion of Ar, and washed with a 1:1 mixture of anhyd. AcOEt and anhyd. ACN (400 mL x 2) under cushion of Ar. The residue was dried on the funnel 10 in reverse flow of nitrogen, transferred to a flask and dried in high vacuum for 24 h to afford 107.4 g of the alkoxide 327 of ca.93% purity containing ca.7% of 1-docosanol that was used in the next step without of further purification. The product was stored under Ar atmosphere. Step 2.5’-OTBDPS-2’-O-docosyl-uridine (328): A mixture of 5'-TBDPS-protected anhydro-uridine (18.6 g, 40 mmol), aluminum alkoxide 327 15 (~93%, 47.6 g, 44 mmol) and anhyd. diglyme (60 mL) was heated to 145 °C bath temperature in a flask fitted with a magnetic stirring bar and a reflux condenser under slight positive pressure of Ar using a balloon for 48 h. The mixture was cooled down to 70 °C in the bath, diluted with AcOEt (200 mL), further cooled down 30 °C and quenched by addition of 10% H3PO4 (200 mL). A suspension thus formed was stirred at rt overnight, filtered through a 600 mL glass filtering funnel, and the solids were 20 washed thoroughly with water (ca.50 mL) and AcOEt (ca.300 mL) mixture (Note 1). Thoroughly compressed solid residue was dried in warm air to afford 25.4 g (55%) of recovered 1-docosanol. The 400 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO filtrate was transferred to a separatory funnel, the organic layer was separated, washed with 1% NaCl (500 mL x 2), saturated NaCl (200 mL) and dried over anhyd. Na2SO4. The solvent was removed in vacuo, the residue was co-evaporated with additional portion of AcOEt (300 mL) to afford 61.8 g of crude residue. The latter was dissolved in 190 mL of AcOEt-hexanes 1:4 mixture and liquid-loaded on a 5 standard 330 g column of silicagel. The column was eluted with isocratic 20 % AcOEt in hexanes followed by gradient of 20 to 40% of AcOEt in hexanes, the fractions containing product were pulled, evaporated in vacuum, co-evaporated twice with ACN-diethyl ether mixture, and dried in high vacuum to afford 11.4 g (36%) of pure product 328. H1 NMR (600 MHz, Acetone-d6) δ 10.08 (s, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.80 – 7.76 (m, 2H), 7.76 - 7.73 (m, 2H), 7.53-7.44 (m, 6H); 6.00 (d, J = 3.0 Hz, 1H), 5.27 10 (d, J = 8.4 Hz, 1H), 4.46 (q, J = 5.4 Hz, 1H), 4.12 (dd, J = 12.0, 2.4 Hz, 1H), 4.08 – 4.04 (m, 2H), 3.98 (dd, J = 11.4, 2.4 Hz, 1H), 3.95 (d, J = 7.2 Hz, 1H), 3.78 (dt, J = 9.6, 6.6 Hz, 1H), 3.69 (dt, J = 9.6, 6.6 Hz, 1H), 1.65 – 1.59 (m, 2H), 1.43 - 1.36 (m, 2H), 1.35 - 1.25 (m, 36H), 1.13 (s, 9H), 0.89 (t, J = 6.6 Hz, 3H). MS (ESI+APCI), calculated for C47H74N2O6Si [M+H]+ exact mass m/z = 791.54, found 791.7. 15 Step 3.2’-O-Docosyl-uridine (329): A mixture of TBDPS-protected nucleoside 328 (11.4 g, 14.4 mmol), anhyd. THF (50 mL), and triethylamine trihydrofluoride (9.7 mL, 60 mmol) was heated at 50 °C under Ar atmosphere for 24 h. Heptane (200 mL) followed by water (200 mL) were added, the heating bath was removed, the mixture was stirred overnight at rt, filtered, and washed thoroughly by water-heptane mixture. The solid was 20 dried in warm air until constant mass to afford 3 as a white solid. Subsequent reaction of DMT-tritylation of 329 thus obtained indicated presence of one equivalent of crystallization water, yield: 8.07 g (98%) as a monohydrate. H1 NMR (600 MHz, Acetone-d6) δ 10.01 (s, 1H), 7.88 (d, J = 8.4 Hz, 1H), 5.97 (d, J = 4.2 Hz, 1H), 5.60 (d, J = 8.4 Hz, 1H), 4.37 (s, 1H), 4.35-4.29 (m, 1H), 4.06 (t, J = 4.8 Hz, 1H), 4.00 (dt, J = 5.4, 2.4 Hz, 1H), 3.93 – 3.87 (m, 1H), 3.84 (d, J = 6.6 Hz, 1H), 3.83 - 3.78 (m, 1H), 3.74 - 3.63 (m, 25 2H), 1.64 - 1.56 (m, 2H), 1.43 - 1.23 (m, 38H), 0.89 (t, J = 6.6 Hz, 3H). MS (ESI+APCI), calculated for C31H56N2O6 [M+H]+ exact mass m/z = 553.42, found 553.5. Step 4.5’-DMTr-2’-O-Docosyl-uridine (330): Triethylamine (4.3 mL, 31 mmol) was added to a solution of nucleoside 329 monohydrate (8.07 30 g, 14.1 mmol) and DMTrCl (10.5 g, 31 mmol) in anhyd. pyridine (50 mL) under Ar atmosphere. The mixture was stirred at rt overnight, diluted with ACN (150 mL), and quenched by addition of MeOH (0.7 mL). The solvents were evaporated in vacuo at 25 °C, the residue was co-evaporated with ACN (150 mL) containing TEA (2 mL) at 25 °C and partitioned between AcOEt (150 mL) and 5% NaCl (200 mL). The organic phase was separated, washed with sat. NaCl, and dried over anhyd. Na2SO4. The solvent 401 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO was removed in vacuo, and the residue was co-evaporated with ACN-AcOEt (2:1) mixture (300 mL) to afford 19.0 g of crude product containing no pyridine. The latter was purified by chromatography over a standard 220 g column of silica gel with isocratic 25% of AcOEt in hexanes followed by gradient of 25 to 45% of AcOEt in hexanes. Fractions contained product were pulled evaporated in vacuum, co- 5 evaporated twice with ACN-DCM 5:1 mixture, and dried in high vacuum to afford 10.8 g (90%) of product 330 as a yellowish foam. H1 NMR (600 MHz, Acetone-d6) δ 10.08 (s, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.52 - 7.48 (m, 2H), 7.40 – 7.33 (m, 6H), 7.29 – 7.26 (m, 1H), 6.93 (d split, J = 9.0 Hz, 4H), 5.96 (d, J = 2.4 Hz, 1H), 5.27 (d, J = 7.8 Hz, 1H), 4.52 – 4.48 (m, 1H), 4.12 - 4.07 (m, 2H), 3.94 (d, J = 7.8 Hz, 1H), 3.85 - 3.77 (m, 1H), 3.81 (s, 6H), 3.73 – 3.68 (m, 1H), 3.52 (dd, J = 10.8, 3.6 Hz, 1H), 3.46 (dd, J = 10 10.8, 3.0 Hz, 1H), 1.67 – 1.59 (m, 2H), 1.43 – 1.56 (m, 2H), 1.36 – 1.23 (m, 36H), 0.89 (t, J = 6.6 Hz, 3H). MS (ESI+APCI), calculated for C52H74N2O8 [M-H]- exact mass m/z = 853.54, found 853.4. Step 5.5’-DMTr-2’-O-Docosyl-uridine 3’-phosphoramidite (331): DIPEA (1.74 mL, 10 mmol) followed by 2-cyanoethyl N,N-diisopropylchlorophosphoramidite 15 (2.20 mL, 9.8 mmol) were added to a cooled to 0 °C solution of compound 330 (6.45 g, 7.5 mmol) in anhyd. AcOEt (40 mL) under Ar. The cooling bath was removed, the mixture was stirred at rt for 19 h, cooled to 0 °C, diluted with AcOEt (40 mL) and quenched by addition of sat. NaHCO3. The organic phase was separated, washed with sat. NaCl, and dried over anhyd. sodium sulfate. Crude material (8.79 g) was purified over a standard 120 g flash column of silica gel with isocratic 40% of AcOEt containing 20 0.5% of TEA in hexanes to afford 6.96 g (88%) of 331 as a white foam. Ratio of phosphorous diastereomers ~1:1. (Note 1). H1 NMR (600 MHz, acetone-d6) δ 10.04 (s, 1H), 7.98 (d, J = 8.4 Hz, 0.5H), 7.92 (d, J = 8.4 Hz, 0.5H), 7.55 – 7.48 (m, 2H), 7.43 – 7.33 (m, 6.5H), 7.33 – 7.26 (m, 1.5H), 6.97 – 6.90 (m, 4H), 5.99 (d, J = 3.0 Hz, 0.5H), 5.97 (d, J = 3.6 Hz, 0.5H), 5.23 (d, J = 1.2 Hz, 0.5H), 5.22 (d, J = 1.8 Hz, 0.5H), 4.68 – 4.63 (m, 0.5H), 4.61 – 4.55 (m, 0.5H), 4.30 – 4.26 (m, 0.5H), 4.21 (dd, J = 4.8, 25 3.0 Hz, 0.5H), 4.18 (dd, J = 4.8, 3.0 Hz, 0.5H), 4.01 – 3.95 (m, 0.5H), 3.94 – 3.83 (m, 1H), 3.83 – 3.80 (m, 0.5H), 3.82 (s, 3H), 3.81 (s, 3H), 3.79 – 3.73 (m, 2.5H), 3.72 – 3.64 (m, 2.5H), 3.58 – 3.51 (m, 1.5H), 3.49 (dd, J = 10.8, 3.6 Hz, 0.5H), 2.83 – 2.76 (m, 1H), 2.70 – 2.61 (m, 1H), 1.68 – 1.58 (m, 2H), 1.47 – 1.37 (m, 2H), 1.37 – 1.25 (m, 36H), 1.25 – 1.19 (m, 10H), 1.11 (d, J = 6.6 Hz, 3H), 0.89 (t, J = 6.6 Hz, 3H). P31 NMR (243 MHz, acetone-d6) δ 149.68, 149.29. MS (ESI+APCI), calculated for 30 C61H91N4O9P [M-H]- exact mass m/z = 1053.65, found 1053.6. Scheme 15b 402 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO To a 500 mL round-bottomed flask equipped with a stir bar was added carboxylic acid 552 (10.08 g, 25.56 mmol, 1.02 eq.). The flask was sealed with a rubber septum fit with an argon inlet needle. The flask was evacuated and backfilled with argon gas; this process was repeated two additional times. Then, 5 anhydrous DMF (200 mL) was added by opening a new SureSeal bottle (2 x 100 mL) and decanting in the solvent under a counterflow of argon. Then, HATU (10.72 g, 28.19 mmol, 1.10 eq) was added in one portion under a counterflow of argon, followed by HOBt monohydrate (4.34 g, 28.34 mmol, 1.11 eq.). The homogenous, slightly brown solution was cooled to 0°C in an ice water bath and then DIPEA (5.3 mL, 30.43 mmol, 1.19 eq.) was added via syringe over the course of approximately 3 minutes. Allowed 10 to stir at 0 degrees for 15 minutes, at which time tert-butyl (5-aminopentyl)carbamate (5.09 g, 25.16 mmol, 1.00 eq.) was added dropwise via syringe over the course of approximately two minutes. The homogenous, light orange reaction mixture was removed from the cold bath and allowed to stir at room temperature for 17.5 hours. At this time, the solvent was removed in vacuo at 45 °C to remove most of the DMF. The crude residue was diluted with DCM (325 mL) and transferred to a separatory funnel. The 15 organic layer was washed sequentially with saturated ammonium chloride solution (3 x 50 mL), saturated sodium bicarbonate solution (3 x 50 mL), DI water (100 mL), and brine (2 x 50 mL). The organic fraction was dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude residue was purified in two batches via automated silica gel chromatography (RediSepGold 330g column, eluting 10% EtOAc/hexanes to 100% EtOAc, loading in DCM) to afford 553 as a glossy white solid (12.12 g, 20 82%). 1H NMR (600 MHz, CDCl3) δ 7.77 (d, J = 7.6 Hz, 2H), 7.59 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 6.01 (s, 1H), 5.37 (s, 1H), 4.58 (s, 1H), 4.48 – 4.37 (m, 2H), 4.21 (t, J = 6.8 Hz, 1H), 4.09 (s, 1H), 3.36 – 3.19 (m, 4H), 3.15 – 3.02 (m, 2H), 1.94 – 1.80 (m, 1H), 1.69 – 1.60 (m, 3H), 403 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 1.55 – 1.46 (m, 5H), 1.44 (s, 11H; overlapping signals), 1.33 (d, J = 7.2 Hz, 2H). LC-MS (ESI+APCI) m/z calcd for C31H43N6O5 [M+H]+: 579.32; 579.2 found. To a 100 mL reaction vial equipped with a stir bar was added Fmoc amide (4.64 g, 8.02 mmol, 1.00 eq.). 5 The flask was sealed with a rubber septum fit with an argon inlet needle. The flask was evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous EtOAc (11.5 mL) and anhydrous DMF (5.0 mL) were added via syringe, followed by piperidine (2.90 mL). The solution was stirred at room temperature for 19 hours. At this time, the solvent was removed in vacuo. The crude residue was purified in over two runs using automated silica gel chromatography (RediSepGold 330g 10 column, eluting 0% to 40% DCM/MeOH) to afford 554 as a viscous yellow oil (2.15 g, 75%). 1H NMR (600 MHz, CDCl3) δ 7.29 (t, J = 6.1 Hz, 1H), 4.56 (s, 1H), 3.37 (s, 1H), 3.29 (t, J = 6.8 Hz, 2H), 3.25 (q, J = 6.7 Hz, 2H), 3.10 (q, J = 6.9 Hz, 2H), 1.91 – 1.82 (m, 1H), 1.67 – 1.59 (m, 2H), 1.56 – 1.45 (m, 7H), 1.44 (s, 9H), 1.34 (p, J = 8.3 Hz, 2H). LC-MS (ESI+APCI) m/z calcd for C16H33N6O3 [M+H]+: 356.25; 357.2 found. 15 To a 200 mL round-bottomed flask equipped with a stir bar was added docosanoic acid (2.08 g, 6.11 mmol, 1.00 eq.). The flask was sealed with a rubber septum fit with an argon inlet needle. The flask was evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous DMF (55 mL) was added followed by HATU (2.56 g, 6.74 mmol, 1.10 eq.) and HOBt monohydrate 20 (1.07 g, 6.99 mmol, 1.14 eq.). The slightly cloudy solution was then cooled to 0 °C in an ice water bath. Once cooled, DIPEA (1.30 mL, 7.46 mmol, 1.22 eq.) was added dropwise via syringe over the course of approximately 15 seconds. The solution turned bright yellow and was allowed to stir at 0 °C for 30 minutes. At this time, amine 554 (2.52 g, 7.04 mmol, 1.15 eq.) was added as a solution in DMF (8 mL), followed by anhydrous DCM (45 mL). The resulting solution was allowed to stir at room temperature for 25 17.5 hours. At this time, the solvents were removed in vacuo. The crude residue was dissolved in CHCl3 (150 mL) and transferred to a separatory funnel. Washed the organic phase sequentially with aq. saturated sodium bicarbonate solution (2 x 100 mL) and brine (1 x ~50 mL). The organic phase was then dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude material was purified by automated silica gel chromatography (RediSepGold 220g column, eluting 15% to 100% EtOAc/hexanes, 30 then eluting 0% to 50% EtOAc/MeOH; sample loaded in CHCl3) to afford 555 as a white solid (1.79 g, 43%). 1H NMR (600 MHz, CDCl3) δ 6.16 (s, 1H), 6.07 (s, 1H), 4.61 (s, 1H), 4.36 (q, J = 7.3 Hz, 1H), 3.35 – 3.19 (m, 4H), 3.10 (s, 2H), 2.20 (dd, J = 8.4, 6.8 Hz, 2H), 1.91 – 1.83 (m, 1H), 1.67 – 1.56 (m, 11H), 1.56 404 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO – 1.47 (m, 5H), 1.45 (s, 10H), 1.41 (p, J = 8.0 Hz, 2H), 1.25 (d, J = 2.9 Hz, 39H), 0.88 (t, J = 7.0 Hz, 3H). LC-MS (ESI+APCI) m/z calcd for C38H74N6NaO4 [M+Na]+: 701.57; 701.6 found. To a 40 mL reaction vial equipped with a stir bar was added Boc amine 555 (1.03 g, 1.52 mmol, 1.00 5 eq.). The vial was sealed with a screw-cap fit with a septum and argon inlet needle. The vial was evacuated and backfilled with argon; this process was repeated two additional times. Then, CHCl3 (13 mL) was added via syringe to give a slightly cloudy solution. The solution was cooled to 0°C and then TFA (2.40 mL, 30.34 mmol, 20 eq.) was added dropwise over the course of approximately 1.5 minutes. The solution was allowed to stir at room temperature for 16 hours. Once the reaction was complete by 10 LCMS, the solvents were removed in vacuo to afford 556 (988 mg, 94%) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 7.91 – 7.84 (m, 2H), 7.61 (s, 3H), 4.18 (td, J = 8.6, 5.5 Hz, 1H), 3.29 (t, J = 6.8 Hz, 2H), 3.11 – 2.97 (m, 2H), 2.79 – 2.71 (m, 2H), 2.18 – 2.02 (m, 2H), 1.65 – 1.56 (m, 1H), 1.56 – 1.43 (m, 7H), 1.39 (p, J = 7.2 Hz, 2H), 1.23 (s, 43H), 0.85 (t, J = 7.0 Hz, 3H). LC-MS (ESI+APCI) m/z calcd for C33H67N6O2 [M+H]+: 579.52; 579.6 found. 15 To a 20 mL reaction vial equipped with a stir bar was added 11 (500 mg, 0.714 mmol, 1.00 eq.). The vial was sealed with a screw-cap fit with a septum and argon inlet needle. The vial was evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous DCM (3.48 mL) was added via syringe. The solution was cooled to 0°C in an ice water bath, and then DIPEA (0.37 mL, 20 2.14 mmol, 3.00 eq.) was added via syringe. Then, T3P (0.70 mL of a 50 wt% solution in EtOAc = 374 mg T3P, 1.18 mmol, 1.65 eq.) was added dropwise via syringe over the course of approximately 40 seconds. Then, amine 556 (904 mg, 1.30 mmol) was added in one portion under a counterflow of argon, followed by anhydrous CHCl3 (3 mL) and anhydrous DMF (4 mL). The solution was allowed to stir at 45 oC for 17 hours, at which time an additional portion of T3P (0.70 mL of a 50 wt% solution in EtOAc 25 = 374 mg T3P, 1.18 mmol, 1.65 eq.) and DIPEA (0.15 mL, 0.86 mmol, 1.20 eq.) were added via syringe. The resulting solution was allowed to stir at 65 oC for 1.5 hours. Once the reaction was complete by LCMS, the reaction mixture was cooled to room temperature and transferred to a separatory funnel containing EtOAc (75 mL). The organic phase was washed with aq. saturated sodium bicarbonate solution (1 x 100 mL). The organic fraction was dried over magnesium sulfate, filtered, and concentrated 30 in vacuo. The crude residue was purified by automated silica gel chromatography (RediSepGold 220g column, eluting 0% to 30% EtOAc (2% TEA)/MeOH, loading in CHCl3/MeOH) to afford 557 as a yellowish solid (153.4 mg, 17%). 1H NMR (600 MHz, DMSO-d6) δ 7.85 (t, J = 6.3 Hz, 2H), 7.74 (t, J = 5.6 Hz, 1H), 7.00 (s, 1H), 6.85 (s, 1H), 6.79 (s, 1H), 6.73 (s, 1H), 6.23 (s, 2H), 6.03 (s, 1H), 4.22 – 4.14 (m, 1H), 3.64 – 3.53 (m, 5H), 3.53 405 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO – 3.43 (m, 4H), 3.31 – 3.26 (m, 8H), 3.25 – 3.08 (m, 10H), 3.06 – 2.95 (m, 5H), 2.85 – 2.78 (m, 1H), 2.64 – 2.56 (m, 4H), 2.55 – 2.53 (m, 4H), 2.43 – 2.35 (m, 6H), 2.33 (t, J = 7.4 Hz, 4H), 2.25 (s, 5H), 2.16 (s, 5H), 2.14 – 2.04 (m, 6H), 1.91 (s, 1H), 1.77 (s, 1H), 1.75 – 1.69 (m, 5H), 1.65 – 1.53 (m, 4H), 1.53 – 1.42 (m, 7H), 1.37 (t, J = 7.3 Hz, 5H), 1.27 – 1.20 (m, 4H). LC-MS (ESI+APCI) m/z calcd for 5 C72H118N13O6 [M+H]+: 1260.93; 1260.8 found. To a 20 mL reaction vial equipped with a stir bar was added methyl ester 557 (153.4 mg, 0.122 mmol, 1.00 eq. The vial was sealed with a rubber septum fit with an argon inlet needle. The vial was evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous THF (1.0 10 mL) was added via syringe to give a slightly heterogenous solution with stirring. Then, KOTMS (33 mg, 0.257 mmol, 2.11 eq.) was added in one portion under a counterflow of argon. The resulting yellow- orange solution was stirred at room temperature for 17 hours and gradually grew more homogenous. At this time, an additional portion of KOTMS (30 mg, 0.233 mmol, 1.9 eq.) was added, followed by additional anhydrous THF (4 mL). The solution was stirred for 22 hours at room temperature. Once the 15 reaction was complete by LCMS, all volatiles were removed in vacuo. The crude residue was dissolved in DMSO/AcOH until pH ~ 3 (as judged by pH paper) and purified by automated reverse phase chromatography (RediSepGold 30 g C18 column, eluting 30% to 100% water (0.1% TFA)/MeCN (0.1% TFA)) to afford 506 as a white powder after lyophilization (119.1 mg, 78%). 1H NMR (600 MHz, MeOD-d4) δ 7.56 (d, J = 7.3 Hz, 1H), 7.06 (t, J = 1.8 Hz, 1H), 6.96 (t, J = 2.0 Hz, 20 1H), 6.90 (t, J = 1.6 Hz, 1H), 6.60 (d, J = 7.2 Hz, 1H), 6.09 (s, 1H), 4.26 (dd, J = 8.8, 5.6 Hz, 1H), 3.92 – 3.78 (m, 1H), 3.75 (t, J = 5.3 Hz, 2H), 3.71 (q, J = 5.1 Hz, 3H), 3.65 – 3.53 (m, 3H), 3.50 (t, J = 5.7 Hz, 2H), 3.34 (t, J = 4.7 Hz, 2H), 3.30 – 3.27 (m, 2H; partial overlap with solvent), 3.22 – 3.12 (m, 5H), 2.97 – 2.89 (m, 1H), 2.81 (t, J = 6.3 Hz, 3H), 2.77 – 2.65 (m, 3H), 2.47 (t, J = 7.6 Hz, 2H), 2.30 (s, 3H), 2.28 – 2.22 (m, 7H; overlapping peaks), 1.98 – 1.87 (m, 5H), 1.86 – 1.73 (m, 3H), 1.68 – 1.56 (m, 6H), 1.56 – 25 1.48 (m, 4H), 1.48 – 1.35 (m, 4H), 1.29 (d, J = 1.7 Hz, 39H), 0.90 (t, J = 7.1 Hz, 3H). LC-MS (ESI+APCI) m/z calcd for C71H116N13O6 [M+H]+: 1245.91; 1246.8 found. Analytical HPLC RT 10.74 min on Waters XBridge Protein BEH C4 Column (100 mm × 4.6 mm) eluting 5% to 100% water(0.1% TFA)/MeCN (0.1% TFA), flow rate 1 mL/min, detecting at 210/254/280 nm and with ELSD. 30 Scheme 15c 406 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO To a 200 mL round-bottomed flask equipped with a stir bar was added carboxylic acid 552 (5.09 g, 12.90 mmol, 1.00 eq.). The flask was sealed with a rubber septum fit with an argon inlet needle. The flask was evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous 5 DMF (100 mL) was added by opening a new SureSeal bottle and decanting into the flask under a counterflow of argon. Then, HATU (5.48 g, 14.41 mmol, 1.12 eq.) and HOBt monohydrate (2.20 g, 14.37 mmol, 1.11 eq.) were sequentially added in solid portions under a counterflow of argon. Then, diisopropylethylamine (2.00 g, 15.49 mmol, 2.70 mL, 1.20 eq.) was added dropwise via syringe over the course of approximately 1 minute to give an orange-brown homogenous solution. The reaction mixed10 was allowed to stir for 15 minutes and then it was cooled to 0 °C in an ice water bath. Then, tert-butyl (2- (2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate (4.25 g, 14.54 mmol, 1.13 eq.) was added dropwise via syringe at 0 °C over the course of approximately 1.5 minutes. Once the addition was complete, the solution was allowed to stir at room temperature for 16.5 hours. At this time, the reaction mixture was concentrated in vacuo at 45°C to remove the majority of the DMF. The resulting residue was 15 then diluted with DCM (200 mL) and transferred to a separatory funnel. The organic phase was washed sequentially with aq. saturated ammonium chloride solution (2 x 30 mL), aq. saturated sodium bicarbonate solution (2 x 30 mL), DI water (1 x 40 mL), and brine (1 x 40 mL). The organic phase was then dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by automated silica gel chromatography (RediSepGold 330g column, eluting 10% to 90% hexanes/EtOAc- 20 EtOH (3:1 solution); loading in DCM) to afford 558 as a pale yellow gum (7.35 g, 85%). 1H NMR (600 MHz, DMSO-d6) δ 7.95 (t, J = 6.3 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.73 (dd, J = 7.5, 4.7 Hz, 2H), 7.46 (d, J = 8.3 Hz, 1H), 7.42 (t, J = 7.7 Hz, 2H), 7.32 (tt, J = 7.4, 1.4 Hz, 2H), 6.74 (t, J = 5.8 Hz, 1H), 4.32 – 4.25 (m, 1H), 4.25 – 4.18 (m, 2H), 3.96 (td, J = 8.7, 5.3 Hz, 1H), 3.52 – 3.47 (m, 7H), 407 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 3.46 – 3.42 (m, 1H), 3.40 (t, J = 6.0 Hz, 2H), 3.36 (t, J = 6.1 Hz, 2H), 3.31 – 3.28 (m, 2H), 3.28 – 3.14 (m, 2H), 3.05 (q, J = 6.1 Hz, 2H), 1.66 – 1.58 (m, 1H), 1.58 – 1.48 (m, 3H), 1.36 (s, 9H), 1.34 – 1.26 (m, 2H). LC-MS (ESI+APCI) m/z calcd for C34H49N6O8 [M+H]+: 669.35; 669.4 found. 5 To a 100 mL round-bottomed flask equipped with a stir bar and under argon atmosphere was added a solution of 558 (3.60 g, 5.38 mmol, 1.00 eq.) in anhydrous EtOAc (16 mL). Then, piperidine (2.00 mL) was added dropwise via syringe over the course of approximately 1 minute. The solution gradually grew slightly heterogeneous and was allowed to stir at room temperature for 17 hours. At this time, the solvent was removed in vacuo. The crude residue was purified by automated silica gel chromatography 10 (RediSepGold 330g column, eluting 0% to 40% DCM/MeOH, loading in DCM) to afford 559 as a yellow oil (2.30 g, 96%). 1H NMR (600 MHz, CDCl3) δ 7.51 (s, 1H), 5.13 (s, 1H), 3.69 – 3.61 (m, 8H), 3.61 – 3.57 (m, 2H), 3.55 (t, J = 5.1 Hz, 3H), 3.52 – 3.38 (m, 3H), 3.35 – 3.25 (m, 4H), 2.46 – 2.03 (m, 3H), 1.84 (s, 1H), 1.67 – 15 1.55 (m, 3H), 1.53 – 1.40 (m, 12H; overlapping signals). LC-MS (ESI+APCI) m/z calcd for C19H39N6O6 [M+H]+: 447.29; 447.4 found. To a 200 mL round-bottomed flask equipped with a stir bar was added docosanoic acid (1.40 g, 4.10 mmol, 1.00 eq.). The flask was sealed with a rubber septum and argon inlet needle. The flask was 20 evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous DMF (25 mL) and anhydrous chloroform (27 mL) were added via syringe to give a homogenous, colorless solution. Then, HATU (1.72 g, 4.53 mmol, 1.10 eq.) and HOBt (693 mg 4.53 mmol, 1.10 eq.) were added sequentially in solid portions under a counterflow of argon. Then, the solution was cooled to 0°C in an ice water bath and diisopropylethylamine (667.80 mg, 5.17 mmol, 0.90 mL, 1.26 eq.) was 25 added dropwise via syringe over the course of approximately 30 seconds, causing the solution to turn a bright yellow color. The solution was stirred at 0 °C for 15 minutes, at which time amine 559 (2.20 g, 4.94 mmol, 1.20 eq.) was added as a solution in anhydrous DMF (8 mL). The yellow solution was allowed to stir at room temperature for 15 hours over which time it gradually grew cloudy. At this time, the solvents were removed in vacuo at 45°C. Then, the crude material was dissolved in CHCl3 (250 mL) 30 and transferred to a separatory funnel. The organic layer was washed sequentially with aq. saturated sodium bicarbonate solution (2 x 75 mL) and brine (1 x 75 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by automated silica chromatography (RediSepGold 220g column, eluting 5% to 50% hexanes/EtOAc-EtOH (3:1 solution), loading in CHCl3) to afford 560 as a waxy white solid (2.74 g, 86%). 408 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 1H NMR (600 MHz, CDCl3) δ 6.66 (s, 1H), 6.24 (s, 1H), 5.17 (s, 1H), 4.43 (d, J = 7.2 Hz, 1H), 3.65 – 3.50 (m, 12H), 3.47 – 3.41 (m, 2H), 3.34 – 3.20 (m, 4H), 2.18 (td, J = 7.7, 1.9 Hz, 2H), 1.88 – 1.75 (m, 1H), 1.67 – 1.52 (m, 5H), 1.47 – 1.33 (m, 11H), 1.30 – 1.20 (m, 36H), 0.92 – 0.82 (m, 3H). LC-MS 5 (ESI+APCI) m/z calcd for C41H80N6NaO7 [M+Na]+: 791.60; 791.6 found. To a 40 mL reaction vial equipped with a stir bar was added Boc amine 560 (1.91 g, 2.48 mmol, 1.00 eq.). The vial was sealed with a screw-cap fit with a septum and argon inlet needle. The vial was evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous 10 DCM (12 mL) was added via syringe with vigorous stirring until homogenous. The solution was cooled to 0 °C in an ice water bath and then TFA (3.9 mL, 49.6 mmol, 20.0 eq.) was added dropwise via syringe. The homogenous solution was allowed to stir at room temperature for 15 hours. At this time, the solvents were removed in vacuo. The crude material was purified by automated silica gel chromatography (RediSepGold 80g column, loading in DCM/CHCl3, eluting 0% to 40% to 100% DCM (1% 15 TEA)/MeOH) to afford 561 as a white solid (855 mg, 51%). 1H NMR (600 MHz, CDCl3) δ 8.45 (s, 1H), 6.29 (s, 1H), 4.36 (q, J = 7.4 Hz, 1H), 3.87 – 3.79 (m, 1H), 3.79 – 3.71 (m, 2H), 3.71 – 3.53 (m, 12H), 3.27 (t, J = 6.8 Hz, 2H), 3.23 – 3.10 (m, 2H), 3.10 – 2.89 (m, 2H), 2.30 – 2.13 (m, 2H), 1.88 – 1.76 (m, 1H), 1.69 – 1.56 (m, 5H), 1.56 – 1.34 (m, 2H), 1.32 – 1.18 (m, 20 37H), 0.88 (t, J = 7.0 Hz, 3H). LC-MS (ESI+APCI) m/z calcd for C36H73N6O5 [M+H]+: 669.56; 669.6 found. To a 20 mL reaction vial equipped with a stir bar was added 11 (148.5 mg, 0.212 mmol, 1.00 eq.). The vial was sealed with a screw-cap fit with a septum and argon inlet needle. The vial was evacuated and 25 backfilled with argon; this process was repeated two additional times. Then, anhydrous DCM (1 mL) was added via syringe to give a homogenous, yellow-orange solution. Then, diisopropylethylamine (81.62 mg, 0.632 mmol, 0.110 mL, 3.00 eq.) was added dropwise via syringe and the solution subsequentially cooled to 0 °C in an ice water bath. Then, T3P (0.20 mL of a 50 wt% solution in EtOAc, 107 mg, 0.336 mmol, 1.58 eq.) was added dropwise via syringe. Then, amine 561 (257 mg, 0.384 mmol, 1.81 eq.) was 30 added in one solid portion at 0 °C followed by anhydrous CHCl3 (1.4 mL). The yellow-brown solution was stirred at room temperature for 17 hours. At this time, the reaction was diluted with CHCl3 (150 mL) and transferred to a separatory funnel. The organic phase was washed sequentially with aq. saturated sodium bicarbonate solution (2 x 50 mL), followed by brine (1 x 30 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by 409 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO automated silica gel chromatography (RediSepGold 80g column, eluting 0% to 20% EtOAc (2% TEA)/MeOH, loading in CHCl3) to afford 562 as a light yellow solid (119 mg, 41%). 1H NMR (600 MHz, DMSO-d6) δ 8.36 (s, 1H), 7.93 (t, J = 5.7 Hz, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.84 (d, 5 J = 5.7 Hz, 1H), 6.99 (d, J = 7.3 Hz, 1H), 6.84 (dd, J = 76.7, 1.9 Hz, 1H), 6.78 (t, J = 2.0 Hz, 1H), 6.73 (t, J = 1.9 Hz, 1H), 6.23 (d, J = 7.1 Hz, 2H), 6.02 (s, 1H), 4.22 (td, J = 8.7, 5.3 Hz, 1H), 3.61 – 3.54 (m, 4H), 3.52 – 3.46 (m, 11H), 3.39 (q, J = 6.1 Hz, 5H), 3.24 – 3.08 (m, 13H), 2.81 (dd, J = 15.5, 6.2 Hz, 1H), 2.73 – 2.64 (m, 2H), 2.64 – 2.54 (m, 4H), 2.43 – 2.29 (m, 8H), 2.25 (s, 3H), 2.16 (s, 3H), 2.14 – 2.02 (m, 6H), 1.98 – 1.92 (m, 1H), 1.88 – 1.77 (m, 2H), 1.77 – 1.68 (m, 4H), 1.63 – 1.52 (m, 3H), 1.52 – 1.42 (m, 5H), 10 1.35 – 1.20 (m, 42H), 0.85 (t, J = 6.9 Hz, 4H). LC-MS (ESI+APCI) m/z calcd for C75H124N13O9 [M+H]+: 1350.96; 1350.8 found. To a 20 mL reaction vial equipped with a stir bar was added methyl ester 562 (119 mg, 0.088 mmol, 1.00 eq.). Then, the vial was sealed with a rubber septum fit with an argon inlet needle. The vial was 15 evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous THF (1.00 mL) was added via syringe to give a mostly homogenous, yellow solution. KOTMS (31.4 mg, 0.245 mmol, 2.78 mmol) was added in one solid portion at rt under a counterflow of argon. The homogenous reaction mixture was stirred at room temperature for 18.5 hours, at which time an additional portion of KOTMS wad added (23 mg, 0.179 mmol, 2 eq) and the solution stirred for an additional 19.5 20 hours. At this time, the solvent was removed in vacuo and the resulting crude residue dissolved in DMSO/AcOH. The crude material was purified by automated reverse phase chromatography (RediSepGold 30g C18 column, eluting 30% to 100% water (0.1% TFA)/MeCN) to afford 507 as a fluffy white solid after lyophilization (71.2 mg, 56%). 25 1H NMR (600 MHz, MeOD-d4) δ 7.57 (d, J = 7.4 Hz, 1H), 7.06 (t, J = 1.9 Hz, 1H), 6.96 (t, J = 2.0 Hz, 1H), 6.90 (t, J = 1.6 Hz, 1H), 6.61 (d, J = 7.3 Hz, 1H), 6.09 (s, 1H), 4.31 (dd, J = 8.8, 5.5 Hz, 1H), 3.75 (t, J = 5.4 Hz, 2H), 3.72 (t, J = 4.7 Hz, 3H), 3.65 – 3.59 (m, 9H), 3.59 – 3.53 (m, 6H), 3.50 (t, J = 5.7 Hz, 2H), 3.43 – 3.33 (m, 6H), 3.30 – 3.28 (m, 2H; overlap with solvent), 2.81 (t, J = 6.2 Hz, 3H), 2.76 – 2.63 (m, 3H), 2.48 (t, J = 7.6 Hz, 2H), 2.31 (s, 3H), 2.29 (t, J = 7.1 Hz, 2H), 2.26 (s, 3H), 2.24 (td, J = 7.3, 2.9 30 Hz, 2H), 1.98 – 1.89 (m, 5H), 1.88 – 1.74 (m, 3H), 1.69 – 1.53 (m, 6H), 1.50 – 1.37 (m, 2H), 1.32 – 1.25 (m, 38H), 0.90 (t, J = 7.1 Hz, 3H).LC-MS (ESI+APCI) m/z calcd for C74H122N13O9 [M+H]+: 1336.94; 1336.8 found. Analytical HPLC RT = 10.66 min on Waters XBridge Protein BEH C4 Column (100 mm × 4.6 mm) eluting 5% to 100% water(0.1% TFA)/MeCN (0.1% TFA), flow rate 1 mL/min, detecting at 210/254/280 nm and with ELSD. 410 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Scheme 15d To a 200 mL round-bottomed flask equipped with a stir bar was added hexadecanoic acid (1.58 g, 6.16 mmol, 1.00 eq.). The flask was sealed with a screw-cap fit with a septum and argon inlet needle. The 5 flask was evacuated and backfilled with argon; this process was repeated two additional times. Then, a SureSealed bottle of DMF was opened with a bottle opener and 50 mL decanted into the flask under a counterflow of argon. This gave a homogenous solution with stirring. Then, HATU (2.58 g, 6.80 mmol, 1.10 eq.) was added in one portion under a counterflow of argon, followed by HOBt monohydrate (1.04 g, 6.78 mmol, 1.10 eq.). The reaction mixture was cooled to 0 °C in an ice water bath, and then 10 diisopropylethylamine (964.60 mg, 7.46 mmol, 1.30 mL, 1.21 eq.) was added via syringe over the course of approximately 30 seconds. The resulting bright yellow, heterogeneous solution was allowed to stir for 20 minutes at 0 oC. Then, amine 554 (2.64 g, 7.42 mmol, 1.20 eq.) was added as a solution in anhydrous DMF (10 mL) via syringe, followed by additional anhydrous DCM (15 mL). The solution was stirred at 35 oC in an aluminum block for 14.5 hours. At this time, the reaction mixture was concentrated in 15 vacuo. The crude residue was dissolved in CHCl3 (175 mL) and transferred to a separatory funnel. The organic phase was washed sequentially with aq. saturated sodium bicarbonate solution (2 x 50 mL) followed by brine (1 x 50 mL). The organic phase was dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude residue was purified via automated silica gel chromatography (RediSepGold 220g column, loading in DCM/CHCl3, eluting 10% to 60% hexanes/EtOAc-EtOH (3:1 20 solution) to afford 563 as a white solid (3.41 g, 93%). 411 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 1H NMR (600 MHz, CDCl3) δ 6.19 (s, 1H), 6.07 (s, 1H), 4.62 (s, 1H), 4.37 (q, J = 7.3 Hz, 1H), 3.34 – 3.19 (m, 4H), 3.15 – 3.03 (m, 2H), 2.19 (dd, J = 8.5, 6.8 Hz, 2H), 1.92 – 1.81 (m, 1H), 1.66 – 1.61 (m, 3H; overlaps with water), 1.59 – 1.56 (m, 1H), 1.55 – 1.47 (m, 4H), 1.44 (s, 9H), 1.43 – 1.37 (m, 2H), 1.36 – 1.21 (m, 27H), 0.88 (t, J = 7.0 Hz, 3H). LC-MS (ESI+APCI) m/z calcd for C32H63N6O4 5 [M+H]+: 595.48; 595.6 found. To a 100 mL round-bottomed flask equipped with a stir bar was added Boc amine 563 (2.03 g, 3.41 mmol, 1.00 eq.). The flask was sealed with a rubber septum and argon inlet needle. The flask was evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous 10 DCM (15 mL) was added via syringe to give a homogenous solution. The reaction mixture was cooled to 0 °C in an ice water bath and then TFA (5.3 mL, 68.8 mmol, 20.2 eq.) was added dropwise via syringe over the course of approximately 2 minutes. The solution was allowed to stir at room temperature for 15 hours. At this time, the volatiles were removed in vacuo to afford 564 as an off-white solid (1.94 g, 93%). 15 1H NMR (600 MHz, DMSO-d6) δ 7.95 – 7.81 (m, 2H), 7.64 (s, 3H), 4.18 (td, J = 8.6, 5.5 Hz, 1H), 3.29 (t, J = 6.8 Hz, 2H), 3.09 – 2.96 (m, 2H), 2.79 – 2.70 (m, 2H), 2.18 – 2.03 (m, 2H), 1.66 – 1.56 (m, 1H), 1.56 – 1.42 (m, 7H), 1.39 (p, J = 7.3 Hz, 2H), 1.33 – 1.16 (m, 29H), 0.85 (t, J = 6.9 Hz, 3H). LC-MS (ESI+APCI) m/z calcd for C27H55N6O2 [M+H]+: 495.43; 495.4 found. 20 To a 100 mL round-bottomed flask equipped with a stir bar was added 11 (1.20 g, 1.71 mmol, 1.00 eq.). The vial was sealed with a rubber septum fit with an argon inlet needle. The flask was evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous DCM (6 mL) and DIPEA (0.90 mL, 5.14 mmol, 3.00 eq.) were added via syringe to give a homogenous yellow-brown solution with stirring. The flask was cooled to 0 °C in an ice water bath. Once cold, T3P (1.70 mL of a 50 25 wt% solution in EtOAc, 908 mg, 2.86 mmol, 1.67 eq.) was added dropwise via syringe over the course of approximately 1 minute. Then, the amine 564 (1.89 g, 3.09 mmol, 1.80 eq.) was added in one solid portion under a counterflow of argon at 0 °C, followed by anhydrous DMF (5 mL) via syringe. Once the addition was complete, the resulting red-brown solution was allowed to stir at 35 °C for 31 hours. At this time, the reaction mixture 30 was concentrated in vacuo. The crude residue was dissolved in CHCl3 (125 mL) and transferred to a separatory funnel. The organic phase was washed sequentially with aq. saturated sodium bicarbonate solution (2 x 50 mL) followed by brine (1 x 50 mL). The organic phase was then dried over magnesium sulfate, filtered, and concentrated with the aid of a rotary evaporator. The crude material was purified by 412 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO automated silica gel chromatography (RediSepGold 220g column, eluting 0 % to 20% EtOAc (2% TEA)/MeOH) to yield 565 as a yellowish solid (292 mg, 14%). 1H NMR (600 MHz, DMSO-d6) δ 7.89 – 7.79 (m, 3H), 7.76 – 7.65 (m, 1H), 6.99 (d, J = 7.3 Hz, 1H), 5 6.84 (t, J = 1.8 Hz, 1H), 6.79 (t, J = 2.0 Hz, 1H), 6.73 (t, J = 1.5 Hz, 1H), 6.23 (s, 1H), 6.22 (s, 1H), 6.02 (s, 1H), 4.23 – 4.11 (m, 2H), 3.62 – 3.53 (m, 4H), 3.49 (s, 3H), 3.28 (t, J = 6.8 Hz, 2H), 3.24 – 3.11 (m, 8H), 3.07 – 2.96 (m, 5H), 2.81 (dd, J = 15.5, 6.2 Hz, 1H), 2.71 – 2.65 (m, 2H), 2.62 – 2.52 (m, 5H), 2.42 – 2.29 (m, 7H), 2.27 – 2.23 (m, 3H), 2.16 (s, 3H), 2.13 – 2.04 (m, 5H), 1.96 (p, J = 7.6 Hz, 1H), 1.89 – 1.78 (m, 1H), 1.76 – 1.68 (m, 4H), 1.65 – 1.54 (m, 4H), 1.53 – 1.42 (m, 7H), 1.40 – 1.34 (m, 5H), 1.30 – 10 1.20 (m, 36H), 0.85 (t, J = 7.1 Hz, 3H). LC-MS (ESI+APCI) m/z calcd for C66H106N13O6 [M+H]+: 1176.83; 1176.8 found. To a 20 mL reaction vial equipped with a stir bar was added methyl ester 565 (292 mg, 0.248 mmol, 1.00 eq.). The vial was sealed with a screw-cap fit with a septum and argon inlet needle. The vial was 15 evacuated and backfilled with argon; this process was repeated two additional times. Then, anhydrous THF (1.0 mL) was added via syringe to give a homogenous solution with sonication and stirring. Then, KOTMS (68 mg, 0.530 mmol, 2.14 eq.) was added as a solid in one portion under a counterflow of argon. The sides of the vial were washed with anhydrous THF (0.4 ml). The solution was allowed to stir at room temperature for 17.5 hours. At this time, the reaction mixture was concentrated in vacuo. The 20 crude residue was dissolved in DMSO/AcOH and purified by automated reverse phase chromatography (RediSepGold 30g C18 column, eluting 30% to 100% water (0.1 % TFA)/MeCN) to afford 508 as a fluffy white solid after lyophilization (317.6 mg, 68%). 1H NMR (600 MHz, MeOD-d4) δ 7.57 (d, J = 7.3 Hz, 1H), 7.05 (t, J = 2.1 Hz, 1H), 6.96 (t, J = 2.0 Hz, 25 1H), 6.89 (t, J = 1.6 Hz, 1H), 6.61 (d, J = 7.3 Hz, 1H), 6.08 (s, 1H), 4.26 (dd, J = 8.8, 5.6 Hz, 1H), 3.75 (t, J = 5.3 Hz, 2H), 3.72 (t, J = 5.2 Hz, 3H), 3.65 – 3.53 (m, 3H), 3.49 (t, J = 5.7 Hz, 3H), 3.35 – 3.33 (m, 2H), 3.28 (t, J = 6.7 Hz, 3H; overlap with solvent), 3.22 – 3.12 (m, 5H), 2.81 (t, J = 6.3 Hz, 3H), 2.76 – 2.65 (m, 3H), 2.47 (t, J = 7.6 Hz, 2H), 2.30 (s, 3H), 2.28 – 2.21 (m, 8H; overlapping signals), 1.99 – 1.88 (m, 4H), 1.88 – 1.70 (m, 4H), 1.67 – 1.57 (m, 5H), 1.54 – 1.49 (m, 4H), 1.48 – 1.21 (m, 31H), 0.90 (t, J = 30 7.0 Hz, 3H). LC-MS (ESI+APCI) m/z calcd for C65H104N13O6 [M+H]+: 1162.82; 1162.8 found. Analytical HPLC RT = 9.89 min on Waters XBridge Protein BEH C4 Column (100 mm × 4.6 mm) eluting 5% to 100% water(0.1% TFA)/MeCN (0.1% TFA), flow rate 1 mL/min, detecting at 210/254/280 nm and with ELSD. Compound L520 was made in an analogous manner to 508. 413 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Example 3: Design and Synthesis of αvβ6 targeting siRNA Conjugates This Example describes methods for the design, synthesis, and conjugation of siRNA agents. Nucleic acid sequences provided herein are represented using standard nomenclature. See the abbreviations of Table 1. 5 General Conditions for Solid-Phase Oligonucleotide Synthesis: All oligonucleotides were synthesized on a MerMade-12 DNA/RNA synthesizer. Solvents/reagents purchased commercially were used as received.500-Å controlled pore glass (CPG) solid supports were obtained from Prime Synthesis 2’-OMe, 2’-F nucleoside and 2′-O-C16 cytidine 3’- phosphoramidites were purchased from Hogene. The 2’-OMe-uridine-5’-bis-POM-(E)- vinyl phosphate 10 (VP) 3’-phosphoramidite was obtained from Wuxi AppTec.2’-O-C22 cytidine phosphoramidite was obtained from Nublocks.2’-aminohexyl cytidine 3’-phosphoramidite was purchased from Innovassynth. 0.15 M phosphoramidite solutions were prepared in anhydrous acetonitrile with 20% DMF as a co- solvent for 2’-OMe uridine and 50% DCM for 2’-O-C22 cytidine. Standard coupling conditions were employed on the synthesizer. The oxidizing reagent was 0.02 M I2 in THF/pyridine/water. Xanthine 15 hydride, 0.20 M in 100% pyridine, was used as the sulfurizing reagent. The detritylation reagent was 3% dichloroacetic acid (DCA) in dichloromethane (DCM). After the synthesis, the oligonucleotides were cleaved from the support and deprotected using 28-30% ammonium hydroxide solution containing 5% (v/v) diethylamine (DEA) at 60 °C for 5h (O’Shea et al, Tetrahedron, 2018). For amino containing oligonucleotides, the CPG solid support was 20 incubated with a mixture of ammonia and methylamine (50%-50%, v/v, AMA) 35 °C for 3 h. Purification was performed by ion-exchange chromatography with TSKGel SuperQ-5PW(20) (Sigma) using an appropriate gradient of mobile phase (buffer A: 20 mM sodium phosphate, 15% ACN, pH 8.5; buffer B: 1 M NaBr, 20 mM sodium phosphate, 15% ACN, pH 8.5). The purified oligonucleotides were then desalted by size exclusion chromatography (SEC) using a custom packed 25 with Sephadex G25 (GE Healthcare) and water as an eluent. The purity and molecular weight were determined using reverse phase HPLC and mass spectrometry, respectively. The lyophilized sense and antisense strands were first resuspended in distilled and deionized water. The concentrations of each strand were determined by determining the absorbance at 260 nm. Equimolar amounts of both strands were mixed and heated at 90oC, followed by slow cooling to form the 30 duplex. The duplexes were then analyzed by mass spectrometry and tested for endotoxin as well as osmolality. Conjugation of integrin ligands and/or lipophilic moieties to the sense strand is carried out through various chemical reactions illustrated and described below. Sequences for sense and antisense strands can be found in Table 2. 414 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Synthesis of Sense Strand Conjugates A-3340763 The C22-modified oligonucleotide was synthesized on a MerMade-12 DNA/RNA synthesizer using 5 standard conditions described above. Similar purification and desalting procedures were followed. The purity and molecular weight of the conjugate were determined using reverse phase HPLC and mass spectrometry, respectively. A-3748001 10 In a typical synthesis, the lyophilized oligonucleotide precursor 332 (one mole equivalent) containing three amine groups was resuspended in distilled and deionized water, followed by the addition of 0.25 volume equivalent of 1M pH 8.5 NaHCO3 buffer. To this solution, 10 mole equivalents of PFP- ester 354P that was initially dissolved in acetonitrile were added. The resulting mixture was then incubated at room temperature overnight (~16 hrs.). Protecting groups were removed by adding 2 equivalent volumes 15 of 20% (v/v) aqueous piperidine and incubating the solution at 37oC overnight. After confirming the complete deprotection via mass spectrometry, piperidine from the crude solution was removed by rotary evaporation. Preparative reverse phase chromatography (XBridge C18 column) using an appropriate gradient of mobile phase (buffer A: 20 mM sodium acetate, 3% ACN, pH 7; buffer B: 20 mM sodium 415 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO acetate, 80% ACN, pH 7) was used to purify 354. This was followed by SEC for desalting. The purity and molecular weight of the conjugate were determined using reverse phase HPLC and mass spectrometry, respectively. Synthesis and conjugation of A-3748012, A-4124027, A-5336259, A-6178434, A-6178435, A- 5 6178436, A-5336260, and A-5336261 were performed in analogous fashion to A-3748001. A-4503380 In a typical synthesis, 334 (one mole equivalent) was first brought in a pH 7 buffered solution using 1M pH 7.22-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer. In a separate 10 container, 2 mole equivalents of 11Z was dissolved in 4:1 NMP:HEPES buffer. The copper catalyst solution was prepared by combining 4 mole equivalents of tetrakis(acetonitrile)copper(I) hexafluorophosphate and 12 mole equivalents of tris-hydroxypropyltriazolylmethylamine (THPTA) in 1mL of anhydrous NMP and incubating the solution at room temperature for five minutes. The oligonucleotide and 11Z solutions were first combined followed by the addition of the copper catalyst 15 solution. The reaction mixture was then purged with Ar gas for 10 minutes prior to incubation at 37oC for 16 hrs. After verifying the completion of reaction via mass spectrometry, copper was removed by adding copper chelating resin (Chelex 100) that was initially resuspended in 0.1 M HEPES buffer. Preparative reverse phase chromatography (XBridge C18 column) using an appropriate gradient of mobile phase (buffer A: 20 mM sodium acetate, 3% ACN, pH 7; buffer B: 20 mM sodium acetate, 80% ACN, pH 7) was 20 used to purify 358. This was followed by SEC for desalting. The purity and molecular weight of the conjugate were determined using reverse phase HPLC and mass spectrometry, respectively. Synthesis and conjugation of A-4184750, A-4517857, A-4549515, A-5031127, A-5031128, A-5031129, and A-4517957 were performed in analogous fashion to A-4503380. A-4148942 416 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In a typical synthesis, 334b (one mole equivalent) containing an amine and an alkyne was diluted with 2-3 mL of NMP. In a separate container, 3 mole equivalents of 306, 3.1 mole equivalents of (7- Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) and 9 mole 5 equivalents of N,N-Diisopropylethylamine (DIPEA) were combined in 2 mL of anhydrous NMP. The two solutions were then combined and incubated at room temperature for five minutes. After verifying the 334c via mass spectrometry, deprotection of 334c was performed by adding 2 equivalent volumes of 20% (v/v) 417 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO aqueous piperidine and incubating the solution at 37oC overnight. Piperidine from the crude solution was removed by rotary evaporation prior to desalting by SEC. The desalted 334c was resuspended in distilled and deionized water, followed by the addition of 0.50 volume equivalent of 1M pH 7.2 HEPES buffer. In a separate container, 2 mole equivalents of 342Z 5 was dissolved in 1:1 NMP:HEPES buffer. The copper catalyst solution was prepared by combining 4 mole equivalents of tetrakis(acetonitrile)copper(I) hexafluorophosphate and 12 mole equivalents of tris- hydroxypropyltriazolylmethylamine (THPTA) in 1mL of anhydrous NMP and incubating the solution at room temperature for five minutes. The oligonucleotide and 342Z solutions were first combined followed by the addition of the copper catalyst solution. The reaction mixture was then purged with Ar gas for 10 10 minutes prior to incubation at 37oC for 16 hrs. After verifying the completion of reaction via mass spectrometry, copper was removed by adding copper chelating resin (Chelex 100) that was initially resuspended in 0.1 M HEPES buffer. Preparative reverse phase chromatography (XBridge C8 column) using an appropriate gradient of mobile phase (buffer A: 20 mM sodium acetate, 3% ACN, pH 7; buffer B: 20 mM sodium acetate, 80% ACN, pH 7) was used to purify 342. This was followed by SEC for 15 desalting. The purity and molecular weight of the conjugate were determined using reverse phase HPLC and mass spectrometry, respectively. Synthesis and conjugation of A-4148937, A-4148945, A-4148941, A-4212889, and A-4148935 were performed in analogous fashion to A-4148942. A-4251955 20 In a typical synthesis, the precursor oligonucleotide (one mole equivalent) containing two amines was first resuspended in 0.5 mL distilled and deionized water before adding 1 mL of NMP. The activated 11 was prepared by mixing 6 mole equivalents of 11, 6.2 mole equivalents of (7-Azabenzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) and 18 mole equivalents of N,N- Diisopropylethylamine (DIPEA) in 2 mL of anhydrous NMP. The two solutions were then combined, and 25 the resulting mixture was incubated at room temperature for five minutes. After confirming the completion of reaction via mass spectrometry, deprotection was performed by adding 2 equivalent volumes of 20% (v/v) aqueous piperidine and incubating the solution at 37oC overnight. After deprotection, piperidine from the crude solution was removed by rotary evaporation. Preparative reverse phase chromatography (XBridge C18 column) using an appropriate gradient of mobile phase (buffer A: 20 mM sodium acetate, 30 3% ACN, pH 7; buffer B: 20 mM sodium acetate, 80% ACN, pH 7) was used to purify the conjugate. This was followed by SEC for desalting. The purity and molecular weight of the conjugate were determined using reverse phase HPLC and mass spectrometry, respectively. A-4525634 418 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 5 A mixture of 354P (15 g, 15 mmol) and primary amine (7.61 g, 14.28 mmol) was dissolved in DCM (80 mL). After the solution was stirred at rt for 23 h the solvent was removed under vacuum. The crude residue (25.3 g) was solid loaded onto a 330 g silica column that had been equilibrated with DCM containing 1% TEA. The column was eluted with a 0-10% MeOH/DCM gradient. Pure fractions were pulled and dried affording 15.2 g (78.9%) of 354HYP as a yellow solid.1H NMR (600 MHz, DMSO) δ 10 7.75 (t, J = 5.5 Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.33 – 7.26 (m, 5H), 7.25 – 7.22 (m, 2H), 7.21 – 7.14 (m, 5H), 6.97 (d, J = 7.6 Hz, 1H), 6.89 (d, J = 7.3 Hz, 2H), 6.88 – 6.84 (m, 4H), 6.82 (s, 1H), 6.78 (d, J = 7.7 Hz, 3H), 6.71 (s, 1H), 6.02 (s, 1H), 5.22 (s, 2H), 4.94 (d, 1H), 4.33 (d, 1H), 4.18 – 4.06 (m, 1H), 3.78 (t, 2H), 3.72 (s, 6H), 3.62 – 3.55 (m, 3H), 3.54 (s, 2H), 3.46 (s, 3H), 3.20 – 3.13 (m, 4H), 3.12 (t, J = 5.3 Hz, 2H), 3.03 – 2.96 (m, 3H), 2.78 (dd, J = 15.5, 6.2 Hz, 1H), 2.73 (t, J = 6.4 Hz, 2H), 2.67 (t, J = 9.3 Hz, 15 2H), 2.63 – 2.60 (m, 2H), 2.57 (d, J = 7.2 Hz, 1H), 2.53 (d, J = 8.4 Hz, 1H), 2.39 (t, J = 11.6 Hz, 1H), 419 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 2.35 (s, 1H), 2.32 (t, J = 7.5 Hz, 2H), 2.23 (s, 3H), 2.20 (t, J = 7.5 Hz, 1H), 2.15 (s, 3H), 2.13 (d, J = 8.2 Hz, 1H), 2.11 – 2.07 (m, 2H), 2.00 (dq, J = 14.3, 6.8 Hz, 2H), 1.96 – 1.88 (m, 1H), 1.84 (m, 4H), 1.73 (q, J = 7.5 Hz, 2H), 1.66 (dd, J = 7.6, 2.8 Hz, 2H), 1.47 (m, 2H), 1.38 (m, 2H), 1.35 – 1.19 (m, 4H), 1.18 – 1.01 (m, 1H). MS (ESI+APCI), calculated for C79H98N9O11 [M+H]+ exact mass m/z = 1348.74, found 5 1350.2. A solution of 354HYP (1.0 g, 741.47 μmol), succinic anhydride (222.60 mg, 2.22 mmol), and dimethylaminopyridine (272 mg, 2.22 mmol) in DCM (5.7 mL) was stirred at rt for 16 h. The reaction was diluted with DCM (50 mL), washed with 0.1 M triethylammonium bicarbonate buffer (3x50 mL), dried over Na2SO4, and dried under vacuum. The crude residue (0.95 g) was taken up in DCM and 10 injected onto a 12 g silica column that had been equilibrated with 5% TEA in DCM. The column was eluted with a 0-20% MeOH/DCM gradient. Pure fractions were pulled and dried affording 1.02 g (88.8%) of 352S as an off white solid.1H NMR (600 MHz, DMSO) δ 7.76 (t, J = 5.9 Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.30 (dq, J = 15.6, 7.5 Hz, 4H), 7.23 (d, J = 7.9 Hz, 2H), 7.22 – 7.15 (m, 5H), 6.97 (d, J = 7.6 Hz, 1H), 6.92 – 6.84 (m, 5H), 6.82 (s, 1H), 6.78 (d, J = 8.7 Hz, 3H), 6.71 (s, 1H), 6.01 (s, 1H), 5.76 15 (s, 8H), 5.35 (s, 1H), 5.31 (s, 3H), 5.22 (s, 2H), 4.18 (s, 1H), 3.80 – 3.77 (m, 2H), 3.72 (s, 6H), 3.57 (s, 2H), 3.56 – 3.52 (m, 3H), 3.46 (s, 4H), 3.37 (q, J = 7.3 Hz, 13H), 3.16 (s, 3H), 3.12 (s, 2H), 3.04 – 2.98 (m, 3H), 2.82 – 2.71 (m, 9H), 2.67 (t, J = 8.4 Hz, 2H), 2.61 (td, J = 8.2, 2.8 Hz, 2H), 2.57 (d, J = 7.4 Hz, 1H), 2.53 (d, J = 8.4 Hz, 1H), 2.47 (q, J = 3.4 Hz, 2H), 2.45 (d, J = 2.9 Hz, 1H), 2.41 – 2.36 (m, 1H), 2.32 (q, J = 6.9 Hz, 3H), 2.23 (s, 3H), 2.20 (dd, J = 13.4, 5.5 Hz, 2H), 2.15 (s, 3H), 2.10 (dt, J = 10.7, 7.5 Hz, 20 3H), 2.05 – 1.93 (m, 2H), 1.91 (s, 2H), 1.84 (m, 3H), 1.73 (q, J = 7.4 Hz, 2H), 1.70 – 1.62 (m, 2H), 1.46 (q, J = 10.1 Hz, 2H), 1.39 (m, 2H), 1.33 – 1.25 (m, 3H), 1.23 (t, J = 7.2 Hz, 14H), 1.06 (t, J = 7.2 Hz, 8H). MS (ESI+APCI), calculated for C83H102N9O14 [M+H]+ exact mass m/z = 1448.75, found 1448.6. A mixture of 352S (0.50 g, 345 μmol) and DIPEA (0.45 g, 3.45 mmol, 600 μL) was dissolved in ACN (250 mL) in a round-bottom flask with a reinforced neck. To this solution was added HBTU (157 25 mg, 414 μmol) and the reaction was stirred for ~10 min. To the reaction mixture was added CPG (3.451 g, 153 μmol/g). The reaction was put on the mechanical shaker for 16 h. The mixture was filtered, washed with ACN (200 mL), MeOH (200 mL), ACN (200 mL), diethyl ether (200 mL) and dried on the high vacuum for 4 h. The CPG was transferred to a round-bottom flask with a reinforced neck and a 1:1 mixture of cap A & B (400 mL) was added. (Cap Mix A = 5% Phenoxyacetic Anhydride in THF/Pyridine Glen30 Research pn# 40-4210-52) (Cap Mix B = 10% 1-Methylimidazole in THF Glen Research pn# 40-4120- 57). This mixture was put back on the mechanical shaker for 3 h. The reaction mixture was filtered, and the obtained CPG was washed with 10% H2O/THF (100 mL), MeOH (100 mL), 10% H2O/THF (100 mL), MeOH (100 mL), ACN (100 mL), and diethyl ether (100 mL) then dried on the high vacuum for 60 h affording 3.22 g of 352L with a loading of 58.53 μmol/g. 420 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO The C22 lipid modification was introduced to sense strand through 331 and conjugate ligand via the solid support 352L, during the solid phase synthesis. Standard coupling conditions were employed during the synthesis. Afterwards, the oligo-containing CPGs were treated with 10% piperidine in ACN for 30 minutes under room temperature to remove cyanoethyl protecting groups. The CPG was washed with 5 ACN thrice before incubating with 20% aqueous piperidine at 37oC overnight. The CPG was then filtered, and the filtrate was subjected to vacuum centrifugation to remove the piperidine. Ammonia with 5% DEA was added to the concentrated filtrate and the resulting solution was incubated overnight at 40oC to complete the deprotection of the bases. The crude oligonucleotide solution was purified and desalted using ion-exchange chromatography (IEX) and size exclusion chromatography (SEC), respectively. The purity 10 and molecular weight of 352 were determined using reverse phase HPLC and mass spectrometry, respectively. Synthesis and conjugation of A-4996054, A-4985653, A-4996055, A-4996056, A-4996057, A-4996058, A-4996059, A-4996060, A-4996061, A-4996062, A-4996063, A-4996064, A-4996065, A-4996066, A- 5892371 and A-4996068 were performed in analogous fashion to A-4525634. 15 A-5336258 421 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO To a solution of 220 (105 g) in DCM (1 L) was added triethylamine (26.6 g), DMAP (1.6 g), HATU (60 g), and primary amine (102 g). The mixture was stirred at room temperature for 17 h. Then, the mixture was concentrated. Ethyl acetate (2 L) and water (1 L) was added, and the product was 5 extracted with ethyl acetate. The organic phase was dried, filtered, and concentrated to provide a residue which was purified on silica gel to provide 221 (117 g). This was dissolved in THF (1.17 L), and then 1 M TBAF in THF (164 mL) was added. It was stirred at room temperature for 6 h. Then, the mixture was concentrated. It was partitioned between water and ethyl acetate, and the product was extracted with ethyl acetate. The organic layers were dried, filtered, concentrated and 222 (97.5 g) was used in the next step 10 without further purification. Compound 222 (97.5 g) was dissolved in DCM (975 mL) and to this was added DIEA (19.2 g), DMAP (0.9 g), and succinic anhydride (22.3 g). The reaction was stirred at room temperature for 6 h, then washed with water and sat. sodium bicarbonate. The organic layer was dried, filtered, and concentrated. The residue was purified on silica gel to provide 223 (80 g). 15 Compound 223 (1 g) and DIPEA (1 g) was dissolved in ACN (500 mL) in a round-bottom flask with a reinforced neck. To this solution was added HBTU (300 mg) and the reaction was stirred for ~10 min. To the reaction mixture was added CPG (7 g, 153 μmol/g). The reaction was put on the mechanical shaker for 16 h. The mixture was filtered, washed with ACN (200 mL), MeOH (200 mL), ACN (200 mL), diethyl ether (200 mL) and dried on the high vacuum for 4 h. The CPG was transferred to a round- 20 bottom flask with a reinforced neck and a 1:1 mixture of cap A & B (800 mL) was added. (Cap Mix A = 5% Phenoxyacetic Anhydride in THF/Pyridine Glen Research pn# 40-4210-52) (Cap Mix B = 10% 1- Methylimidazole in THF Glen Research pn# 40-4120-57). This mixture was put back on the mechanical shaker for 3 h. The reaction mixture was filtered, and the obtained CPG was washed with 10% H2O/THF (100 mL), MeOH (100 mL), 10% H2O/THF (100 mL), MeOH (100 mL), ACN (100 mL), and diethyl 25 ether (100 mL) then dried on the high vacuum for 54 h affording 6.5 g of 224 with a loading of 61.74 μmol/g. The C22 lipid modification was introduced to sense strand through 331C and conjugate ligand via the solid support 224, during the solid phase synthesis. Standard coupling conditions were employed during the synthesis. Afterwards, the oligo-containing CPGs were treated with 10% piperidine in ACN for 30 422 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO minutes under room temperature to remove cyanoethyl protecting groups. The CPG was washed with ACN thrice before incubating with 20% aqueous piperidine at 37oC overnight. The CPG was then filtered, and the filtrate was subjected to vacuum centrifugation to remove the piperidine. Ammonia with 5% DEA was added to the concentrated filtrate and the resulting solution was incubated overnight at 40oC to 5 complete the deprotection of the bases. The crude oligonucleotide solution was purified and desalted using ion-exchange chromatography (IEX) and size exclusion chromatography (SEC), respectively. The purity and molecular weight of 493 were determined using reverse phase HPLC and mass spectrometry, respectively. 10 Scheme 16 Alternatively, trivalent conjugates can be synthesized through loading of the ligand onto solid support, followed by oligonucleotide synthesis, cleavage from the support, and deprotection. 423 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 424 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Table A. Sense strand sequences and mass data for αvβ6 integrin targeting siRNA conjugates. 425 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 426 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Table 2. siRNA antisense (AS) and sense strand (SS) sequences of dsRNA agents targeting SOD1 or HPRT1 used for in vitro and/or in vivo studies to evaluate pharmacodynamic and/or pharmacologic activity of αvβ6 integrin targeting siRNA conjugates. 427 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Annealing of siRNA Duplexes The lyophilized sense and antisense strands were first resuspended in distilled and deionized water. The concentrations of each strand were determined by determining the absorbance at 260 nm. Equimolar 5 amounts of both strands were mixed and heated at 90oC, followed by slow cooling to form the duplex. The duplexes were then analyzed by mass spectrometry and tested for endotoxin as well as osmolality. Example 4: Evaluation of αvβ6 integrin targeting siRNA conjugates 428 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Table 3. siRNA antisense (AS) and sense strand (SS) sequences of dsRNA agents targeting SOD1 or HPRT1 used for in vitro and/or in vivo studies to evaluate pharmacodynamic and/or pharmacologic activity of αvβ6 integrin targeting siRNA conjugates. 429 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In vivo Study - Mouse Female C57BL/6 mice (8-10 weeks of age) (N=3) were dosed with a single intravenous bolus or subcutaneous injection at given dose diluted in 1x PBS. Terminal tissues were collected on day 7 or 21 5 after injection. Levels of SOD1 or HPRT1 mRNA were quantified from powdered whole tissue by RT- qPCR. Reduction of target mRNA was measured, normalized to GAPDH mRNA for each sample, and reported as relative reduction compared to control animals treated with PBS alone. mRNA quantification by RT-qPCR: Tissue powders were homogenized and lysed in 750 μL of QIAzol Lysis Reagent for 5 minutes x2 at 25 Hz using a TissueLyser. Chloroform (150 μL) was 10 thoroughly mixed with each sample and lysates were centrifugated at full speed for 15 minutes at 4 °C. Total RNA was isolated from the supernatant using the RNeasy® 96 Universal Tissue Kit. Resulting 430 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO RNA (1500 ng) was used for cDNA synthesis using the Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor. For qPCR reactions, 2uL cDNA was used per 10 ul reaction. RT-qPCR was performed using the LightCycler® 480 Probes Master in the LightCycler® 480 Instrument system using gene specific TaqMan assays for each target mouse-specific SOD1, HPRT1 and 5 Gapdh were used for RT-qPCR analysis. Each sample was analyzed in duplicate by RT-qPCR. Data were analyzed using the ΔΔCt method normalizing to control animals treated with 1X PBS alone. FIG.1 is a bar graph showing AD-1812376 mediated SOD1 knockdown in quadriceps and gastrocnemius of a mouse model at Day 21 following a single IV dose of 2 mg/kg. PBS as used for the control group. 10 FIG.3: is a bar graph showing AD-2032892 mediated SOD1 knockdown in liver, quadriceps and gastrocnemius of a mouse model at Day 21 following administration of a single IV dose of 1 mg/kg, 3 mg/kg or 9 mg/kg. PBS was used for the control group. FIG.7: is a bar graph showing AD-2432777, AD-2902547, AD-2889354, AD-2889355, AD- 2889356, and and AD-2889357 mediated SOD1 knockdown in quadriceps of a mouse model at Day 7 15 following administration of a single subcutaneous dose of 1 mg/kg. PBS was used for the control group. FIG.8: is a bar graph showing AD-2432777, AD-2902547, AD-2889354, AD-2889355, AD- 2889356, and and AD-2889357 mediated SOD1 knockdown in heart of a mouse model at Day 7 following administration of a single subcutaneous dose of 1 mg/kg. PBS was used for the control group. FIG.9: is a bar graph showing AD-2640041, AD-2700084, AD-2700085, AD-2700086, AD-20 2700087, AD-2700088, AD-2700089, AD-2700090, AD-2700091, AD-2700092, AD-2700093, AD- 2700094, AD-2700095, AD-2700096, AD-2700097, and AD-2700099 mediated SOD1 knockdown in quadricep of a mouse model at Day 7 following administration of a single IV dose of 0.3 mg/kg. PBS was used for the control group. FIG.10: is a bar graph showing AD-2640041, AD-2700084, AD-2700085, AD-2700086, AD-25 2700087, AD-2700088, AD-2700089, AD-2700090, AD-2700091, AD-2700092, AD-2700093, AD- 2700094, AD-2700095, AD-2700096, AD-2700097, and AD-2700099 mediated SOD1 knockdown in gastrocnemius of a mouse model at Day 7 following administration of a single IV dose of 0.3 mg/kg. PBS was used for the control group. FIG.11: is a bar graph showing AD-2640041, AD-2700084, AD-2700085, AD-2700086, AD-30 2700087, AD-2700088, AD-2700089, AD-2700090, AD-2700091, AD-2700092, AD-2700093, AD- 2700094, AD-2700095, AD-2700096, AD-2700097, and AD-2700099 mediated SOD1 knockdown in heart of a mouse model at Day 7 following administration of a single IV dose of 0.3 mg/kg. PBS was used for the control group. FIG.12: is a bar graph showing AD-3214512 mediated HPRT1 knockdown in quadricep and 35 heart of a mouse model at Day 7 following administration of a single subcutaneous dose of 1 mg/kg. PBS was used for the control group. The results presented in FIGs.1, 3, and 7-12 indicate that dsRNA agents of the disclosure can effectively knock down gene expression in mice. 431 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO In vivo Study - Cynomolgus Monkey Female cynomolgus monkeys (N=3) were dosed at given dose diluted in 1x PBS. Terminal skeletal tissues indicated were collected specified days after injection. Tissues were homogenized by 5 TissueLyser plus ball bearing (50 cycles/sec for 2 mins) in a guanidine-based lysis buffer. RNA was extracted using Qiagen's RNeasy Fibrous Tissue Mini Kit Catalog # 7470, using manual columns and including a proteinase K digestion at 55°C for 10 minutes. Levels of SOD1 mRNA were quantified from powdered whole tissue by RT-qPCR, using Invitrogen SuperScript IV VILO Master Mix, Applied Biosystems ViiA7 RT-PCR system, and Applied Biosystems TaqMan Fast Advanced Master Mix . 10 Target mRNA reduction was normalized to PPIB mRNA for each sample and reported as relative reduction (ΔΔCT method) compared to control animals treated with PBS alone. FIG.2: is a bar graph showing AD-1812376 mediated SOD1 knockdown in quadriceps, gastrocnemius and liver of non-human primates at Days 29 and 57 following a single IV dose of 1 mg/kg, 3 mg/kg or 9 mg/kg. PBS was used for the control group. 15 FIG.4: is a bar graph showing AD-2032892 mediated NHP SOD1 knockdown in gastrocnemius, quadriceps and liver of non-human primates following administration of a single IV dose of 10 mg/kg. PBS was used for the control group. FIG.5: is a bar graph showing NHP SOD1 knockdown mediated by AD-2432777, AD-2241090, AD-2315832, AD-2315873 and AD-2315833 in biceps, gastrocnemius and quadriceps of non-human 20 primates at Day 29 following administration of a single IV dose of 10 mg/kg. PBS was used for the control group. FIG.6: is a bar graph showing NHP SOD1 knockdown mediated by AD-2432777 and AD- 2640041 in biceps, gastrocnemius and soleus of non-human primates at Day 28 following administration of a single SC 10 mg/kg dose. PBS was used for the control group. 25 FIG.13 is a bar graph showing NHP DMPK knockdown mediaded by AD-3100656 in heart, gastrocnemius, and quadriceps of non-human primates at Day 32 following a single SC dose of 3 mg/kg. The control group was the average of animals treated with non-DMPK targeting siRNAs. The DMPK mRNA % remaining is relative to two housekeeping genes (PPIB and ADD1). The DMPK-targeting siRNA duplex used in this study (AD-3100656) comprised (i) a sense strand having 21 nucleotides, an in 30 vivo delivery enhancing moiety at sense position 6 counting from the 5’ end, and an αvβ6 integrin targeting ligand at the 3’end of the sense strand having the chemical structure of L493 as shown herein; and (ii) an antisense strand having 23 nucleotides. The results presented in FIGs.2, 4-6 and 13 indicate that dsRNA agents of the disclosure can effectively knock down gene expression in non-human primates. 35 In vitro study – ELISA Displacement Assay ELISA plates were coated with 0.6 ug/mL recombinant human LAP/TGF1b (R&D Systems 246LP025) in carbonate buffer (pH 9.6) overnight at 4 C. Plates were washed with PBST and blocked for 1 432 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO hour at room temperature with assay buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 2 mM MnCl2, 0.5% BSA, pH 7.4). Serial dilutions of competitor compound in assay buffer were added to plate, as well as 1.0 ug/mL of purified recombinant human αvb6 (R&D Systems 3817AV050), and samples were incubated for 1 hour at room temperature. Plates were washed with PBS-T, then incubated with 2.5 5 ug/mL of anti-integrin av primary antibody (Sigma MAB1978) for 1 hour at room temperature. Plates were washed with PBS-T, then incubated with 2.0 ug/mL of HRP-conjugated secondary antibody for 1 hour at room temperature. Samples were incubated with TMB substrate (Thermo 34024) for 15 minutes, reaction was quenched with 3 M sulfuric acid, and absorbance was measured at 450 nm. Percent binding inhibition was calculated by normalizing to a maximum value obtained from no-competitor samples and a minimum value 10 obtained from no-competitor-no-integrin samples. EC50 values are reported in the following ranges: “A” EC50 >100 nM; “B” EC5010-100 nM; “C” EC50 <10 nM. Table 4. αvb6 integrin binding efficiencies of duplexes as measured by ELISA displacement assay. 15 Table 5. Tissue AUC (ng/g) for siRNA conjugates in NHP following 10 mg/kg IV dose 433 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Table 6. Mouse SOD1 KD at a dose of 1 mg/kg versus PBS control group at Day 7 following single IV dose. 434 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Table 7. NHP SOD1 KD in cardiac tissue at a dose of 10 mg/kg versus PBS control group at Day 29 following single IV dose. Table 8. Mouse SOD1 KD in cardiac tissue at a dose of 1 mg/kg versus PBS control group at Day 29 following single IV dose. Table 1. Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 3'^5’phosphodiester bonds; and it is understood that when the nucleotide contains a 2’-fluoro modification, then the fluoro replaces the hydroxy at that position in the parent nucleotide (i.e., it is a 2’-deoxy-2’- fluoronucleotide). The abbreviations are understood to omit the 3’-phosphate (i.e. they are 3’-OH) when placed at the 3’-terminal position of an oligonucleotide (unless the 3’-end is linked to a ligand). 435 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 436 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 437 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 438 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 439 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 440 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 441 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 442 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 443 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 444 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 445 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 446 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 447 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 448 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 449 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 450 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 451 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 452 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 453 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 454 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 455 ME1\53466565.v1

Claims

Atty. Docket No.121301-23820/ALN-523-WO CLAIMS What is claimed is: 1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a target gene, comprising: an antisense strand which is complementary to the target gene; a sense strand which is complementary to the antisense strand and forms a double stranded region with the antisense strand; at least one alpha-v-beta-6 (αvβ6) integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least one strand; and at least one in vivo delivery enhancing moiety conjugated to one or more positions on at least one strand. 2. The dsRNA agent of claim 1, wherein the in vivo delivery enhancing moiety comprises at least one C10-C26 hydrocarbon chain. 3. The dsRNA agent of claim 2, wherein the in vivo delivery enhancing moiety comprises at least one C22 hydrocarbon chain. 4. The dsRNA agent of claim 2, wherein the in vivo delivery enhancing moiety comprises at least one C17 hydrocarbon chain. 5 The dsRNA agent of claim 2, wherein the in vivo delivery enhancing moiety comprises at least one C11 hydrocarbon chain. 6. The dsRNA agent of any one of claims 2-5, wherein the at least one C10-C26 hydrocarbon chain is unsubstituted or substituted with at least one functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. 7. The dsRNA agent of claim 6, wherein the at least one C10-C26 hydrocarbon chains is substituted with a carboxylic acid group. 8. The dsRNA of any one of claims 2-5, wherein the at least one C10-C26 hydrocarbon chain is covalently attached to the following moiety: 456 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein is a bond connecting the moiety to the at least one C10-C26 hydrocarbon chain. 9. The dsRNA agent of any one of claims 1-8, wherein the in vivo delivery enhancing moiety is attached to the dsRNA agent via a linker or via a carrier or via an internucleotide phosphate linkage. 10. The dsRNA agent of claim 9, wherein the in vivo delivery enhancing moiety is attached to the dsRNA agent via a linker. 11. The dsRNA agent of claim 10, wherein the linker comprises an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, or carbamate. 12. The dsRNA agent of claim 10, wherein the linker is selected from the group consisting of - (CH2)nNH-; -C(O)(CH2)nNH-; -NR’’’’(CH2)nNH-, -C(O)-(CH2)n-C(O)-; -C(O)-(CH2)n-C(O)O-; -C(O)-O- ; -C(O)-(CH2)n-NH-C(O)-; -C(O)-(CH2)n-; -C(O)-NH-; -C(O)-; -(CH2)n-C(O)-; -(CH2)n-C(O)O-; -(CH2)n- ; and -(CH2)n-NH-C(O)-; wherein n is a number from 1 to 20; and R’’’’ is C1-C6 alkyl. 13. The dsRNA agent of claim 10, wherein the linker comprises –(CH2)n-NH-C(O)- or –(CH2)n-NH- C(O)-(CH2)2-C(COOH)-NH-C(O)-, wherein n is a number from 1 to 20. 14. The dsRNA agent of claim any one of claims 10-13, wherein the linker comprises –(CH)2-O- (CH2CH2)-(O)-(CH2CH2)-NH-C(O)-. 15. The dsRNA agent of any one of claims 1-14, wherein the in vivo delivery enhancing moiety is represented by the following structure: , , , 457 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , . wherein a broken bond represents a bond to the remainder of the dsRNA agent. 16. The dsRNA agent of any one of claims 1-14, wherein the in vivo delivery enhancing moiety is represented by the following structure: , wherein m is selected from 1-6, n is selected from 7-23, RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2; and E is -C(O)N(H)-(CH2)p-*, -N(H)C(O)-(CH2)p-*, -C(O)O-(CH2)p-*, -OC(O)-(CH2)p-*, - OP(Y)(OH)O-(CH2)p-*, -O-(CH2)p-, -N(H)-(CH2)p, -S-(CH2)p-, -N(H)-O-(CH2)p-, -O-N(H)-(CH2)p-, N(H)N(H)-(CH2)p-, or -S-S-(CH2)p-*, -Ph-(CH2)p-, -OPh-(CH2)p-, or -ZZ1-(CH2)p-; wherein * is the bond to the alpha-amino acid carbon, Ph is phenyl, Y is =O or =S, p is selected from 1 – 6; and 458 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO ZZ1 is a group formed by reaction of a reactive pair. 17. The dsRNA agent of claim 16, wherein the in vivo delivery enhancing moiety is represented by the following structure: , wherein m is selected from 1-6 and n is selected from 7-23. 18. The dsRNA agent of any one of claims 1-17, wherein the in vivo delivery enhancing moiety is conjugated to the 5’-end of the at least one strand. 19. The dsRNA agent of claim 18, wherein the in vivo delivery enhancing moiety is conjugated to the 5’-oxygen of the 5’-terminal nucleotide of the at least one strand. 20. The dsRNA agent of any one of claims 1-17, wherein the in vivo delivery enhancing moiety is conjugated to the 3’-end of the at least one strand. 21. The dsRNA agent of any one of claims 1-17, wherein the in vivo delivery enhancing moiety is conjugated to the 3’-oxygen of the 3’-terminal nucleotide of the at least one strand. 22. The dsRNA agent of any one of claims 1-21, wherein the in vivo delivery enhancing moiety is attached to the remainder of the dsRNA agent at a 2’ position on an internal nucleotide. 23. The dsRNA agent of any one of claims 1-22, wherein the αvβ6 integrin targeting ligand comprises a structure represented by Formula (X): or a salt thereof, wherein: Y is O, N(H), S, or CH2; , wherein m is 0, 1, 2, 3, or 4; and 459 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each R2 is independently R, or two R2 groups on adjacent carbon atoms taken together with the atoms to which they are bound form a fused 4 – 8 membered ring that is optionally substituted by 1, 2, 3 or 4 groups independently selected from group consisting of R and a nitrogen protecting group; A is an 5-membered heteroaryl optionally substituted with 1 or 2 substituents independently selected from methyl, ethyl, fluoro, hydroxymethyl, 2-hydroxypropan-2-yl, trifluoromethyl, difluoromethyl, and fluoromethyl; Q is -COOR1 or tetrazolyl, wherein R1 is hydrogen or R1-6alkyl; and RL is -N(R3)(R4), -O(R5), -S(R5), or -R5, wherein R3 and R4 are either (i) R3 is hydrogen or C1-6alkyl and R4 is R5; or (ii) R3 and R4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R5; and R5 is -L-ZZ-L’-RT wherein L and L’ are independently -L1-[G-L2]q-G-L3-*, wherein * is the bond to ZZ; q is 0 or an integer selected from 1 to 25; L1 is a bond or -B-A-; each L2 is independently -A-B-A-; L3 is a bond or -A-B-A-; each G is independently -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); 460 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each RN is independently hydrogen or C1-6alkyl, or two RN within an -A- B-A- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; and ZZ is -A’-B’-A’- or a linking group formed by a reactive pair, wherein each A’ is independently a bond, -O-, -S-, or -N(RN3)-; each B’ is independently a bond, CH2, C(O), C(S), C(NRN3), -C=N-, S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); and each RN3 is independently hydrogen or C1-6alkyl, or two RN3 within the -A’-B’- A’- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; RT is RT1 or -G0-ORT1, wherein G0 is absent or -D0-E0-F0-, wherein D0, E0, and F0 are independently a bond, C1- 10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; RT1 is LL-oligonucleotide, wherein LL is an oligonucleotide linking group connecting the αvβ6 integrin targeting ligand to oligonucleotide comprised in the dsRNA agent; and each R group is independently selected from the group consisting of R’, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, heterocyclyl, aryl, heteroaryl, C3-8cycloalkylC1-6alkyl, heterocyclylC1-6alkyl, aryl C1-6alkyl, heteroarylC1-6alkyl, each of which, other than R’, is optionally substituted with 1, 2, or 3 R’ groups, wherein each R’ is independently halogen, cyano, azido, nitro, -N(Rb)2, -O(Ra), -S(R0), - C(O)OR0, C(O)R0, -C(O)N(R0)2, -C(NR0)OR0, -C(NR0)R0, -C(NR0)N(R0)2, -C(S)OR0, -C(S)R0, -C( S)N(R0)2, -S(O)2R0, -S(O)2OR0, -S(O)2N(R0)2, -N(R0)C(O)OR0, -N(R0)C(O)R0, -N(R0)C( O)N(R0)2, -N(R0)S(O)2R0, -N(R0)S(O)2OR0, -N(R0)S(O)2N(R0)2, -OC(O)OR0, -OC(O)R0, -OC(O)N(R0)2, -OS(O)2R0, -OS(O)2OR0, -OS(O)2N(R0)2, or -SC(O)R0, wherein each R0 is independently hydrogen or C1-6alkyl; each Ra is independently hydrogen or C1-6alkyl; and each Rb is independently hydrogen, C1-6alkyl, or a nitrogen protecting group 461 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO provided that in each -D-E-F- group, at least one of D, E, and F is not a bond; and RL is not N- morpholinyl. 24. The dsRNA agent of claim 23, wherein RY is , wherein p is 0, 1, 2, 3 or 4; and each R21 is independently selected from the group consisting of R and a nitrogen protecting group. 25. The dsRNA agent of claim 23, wherein , wherein RP is a nitrogen protecting group. 26. The dsRNA agent of any one of claims 23-25, wherein -L’-RT is wherein* is the bond to ZZ; L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and R is -OH or -C1-6alkyl-OH. 27. The dsRNA agent of any one of claims 23-26, wherein -L’- is -L1-G-L3-*, wherein * is the bond to ZZ. 28. The dsRNA agent of any one of claims 23-27, wherein the αvβ6 integrin targeting ligand comprises the following structure: 462 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (X-b), (X-c), 463 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (X-aa), or (X-ab). 29. The dsRNA agent of any one of claims 23-28, wherein L is -L1-[G-L2]q-G-L3-*, wherein q is 0, 1, 2, 3, 4, or 5. 30. The dsRNA agent of any one of claims 23-28, wherein L is -L1-G-*, wherein * is the bond to ZZ; G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; L1 is -B-A-, wherein, A is a bond, -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1- 6alkyl; and B is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). 31. The dsRNA agent of any one of claims 23-28, wherein L is wherein * is the bond to ZZ; k is an integer from 1 to 10; L1 is bond, C(O), C(S), C(NRN), S(O)2, P(O)(OH), or P(S)(OH); and RN is hydrogen or C1-6alkyl. 32. The dsRNA agent of any one of claims 23-28, wherein L is a group selected from: n integer from 1 to 3; and s and s’ are each independently an integer from 1 to 24; (c) wherein * is the bond to ZZ; a is 1, 2 or 3; and each s, s’, and s” independently is an integer from 1 to 24; 464 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein * is the bond to ZZ; and s, s’, and s’’ are independently is an integer from 1 to 24; wherein * is the bond to ZZ and each s, s’, and s” independently is an integer from 1 to 24; and (f) wherein * is the bond to ZZ; s and k are independently is an integer from 1 to 20; and w is an integer from 1 to 10. 33. The dsRNA agent of any one of claims 23-28, wherein L is wherein * is the bond to ZZ and w is an integer from 1 to 20. 34. The dsRNA agent of any one of claims 23-28, wherein L is , wherein * is the bond to ZZ; k is an integer from 1 to 10. 35. The dsRNA agent of any one of claims 23-28, wherein L is L is -L1-G-*, wherein * is the bond to ZZ , G is C1-10alkyl or C2-10alkenyl, and L1 is -B-A-, wherein, A is a bond, -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; and B is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). 36. The dsRNA agent of any one of claims 23-28, wherein L is -[G-L2]q-G-*, wherein q is an integer from 1 to 5, each G is independently C1-10alkyl and L2 is O, S, or N(H). 37. The dsRNA agent of any one of claims 23-28, wherein L is -G-L2-G-*, wherein L2 is -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl, or two RN within an -A-B-A- group taken together with the atoms to which they are connected form a 4-8 membered heterocyclyl; and each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; 465 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein each R group is independently selected from the group consisting of R’, C1-6alkyl, C1- 6haloalkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, heterocyclyl, aryl, heteroaryl, C3- 8cycloalkylC1-6alkyl, heterocyclylC1-6alkyl, aryl C1-6alkyl, heteroarylC1-6alkyl, each of which, other than R’, is optionally substituted with 1, 2, or 3 R’ groups, wherein each R’ is independently halogen, cyano, azido, nitro, -N(Rb)2, -O(Ra), -S(R0), -C(O)OR0, - C(O)R0, -C(O)N(R0)2, -C(NR0)OR0, -C(NR0)R0, -C(NR0)N(R0)2, -C(S)OR0, -C(S)R0, -C(S)N (R0)2, -S(O)2R0, -S(O)2OR0, -S(O)2N(R0)2, -N(R0)C(O)OR0, -N(R0)C(O)R0, -N(R0)C(O)N(R0 )2, -N(R0)S(O)2R0, -N(R0)S(O)2OR0, -N(R0)S(O)2N(R0)2, -OC(O)OR0, -OC(O)R0, - OC(O)N(R0)2, -OS(O)2R0, -OS(O)2OR0, -OS(O)2N(R0)2, or -SC(O)R0, wherein each R0 is independently hydrogen or C1-6alkyl; and each Ra is independently hydrogen or C1- 38. The dsRNA agent of any one of claims 23-37, wherein ZZ comprises a group selected from the group consisting of: Group( 466 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO Group Group 39. The dsRNA agent of any one of claims 23-38, wherein LL is a divalent linker that connects to the 3’-end of the oligonucleotide , the 5’-end of the oligonucleotide, a 2’ position on an internal nucleotide or a 3’ position on an internal nucleotide. 40. The dsRNA agent of claim 39, wherein LL connects to an oxygen atom on a nucleotide of the oligonucleotide, and is -P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH)-. 41. The dsRNA agent of any one of claims 23-40, wherein RT is RT1, wherein RT1 is -LL- oligonucleotide, wherein LL connects to an oxygen atom on a nucleotide of the oligonucleotide, LL is a bond, and the nucleoside is represented by Formula (X-f): (X-f) wherein B is an optionally modified nucleobase and * represents the bond to ZZ. 42. The dsRNA agent of claim 41, wherein -L’- is -C4-10alkyl-*, wherein * is the bond to ZZ. 43. The dsRNA agent of claim 41, wherein the nucleoside is of a formula selected from the group consisting of, (X-g) (X-h) 467 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO B is an optionally modified nucleobase; each n is independently 0 or an integer selected from 1-10; and each m is independently integer selected from 1-20. 468 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 44. The dsRNA agent of any one of claims 1-43, wherein the αvβ6 integrin targeting ligand is conjugated at the 5’-end of the sense strand or at the 5’-end of the antisense strand. 45. The dsRNA agent of claim 44, wherein the sense strand or the antisense strand conjugated to the αvβ6 integrin targeting ligand comprised in the dsRNA agent is represented by the following formula: wherein Y’ is O or S; and represents the remainder for the sense strand or the antisense strand. 46. The dsRNA agent of any one of claims 1-43, wherein the αvβ6 integrin targeting ligand is conjugated at the 3’-end of the sense strand or the 3’ end of the antisense strand. 47. The dsRNA agent of claim 46, wherein sense strand or the antisense strand conjugated to the αvβ6 integrin targeting ligand comprised in the dsRNA agent is represented by the formula selected from the group consisting of: , 469 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , , , 470 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S, R1 is hydrogen or C1-6alkyl, RP is hydrogen or a nitrogen protecting group and represents the remainder for the sense strand or the antisense strand. 48. The dsRNA agent of claim 47, wherein RP is hydrogen and R1 is hydrogen. 49. The compound of claim 47 or 48, wherein Y’ is O. 50. The compound of claim 47 or 48, wherein Y’ is S. 51. The dsRNA agent of any one of claims 1-22, wherein the αvβ6 integrin targeting ligand comprises a compound represented by Formula (XV): (Φ-ZZ-)xΔ-T-RT (XV) or a salt thereof, wherein 471 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO x is 2, 3, 4, 5, 6, 7, or 8; T is a divalent linking group; Δ is a branching group; each ZZ is independently -A’-B’-A’- or a linking group formed by a first reactive pair, wherein each A’ is independently a bond, -O-, -S-, or -N(RN3)-; each B’ is independently a bond, CH2, C(O), C(S), C(NRN3), -C=N-, S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); and each RN3 is independently hydrogen or C1-6alkyl, or two RN3 within the -A’-B’-A’- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; RT is -G0-ORT1, wherein G0 is -D0-E0-F0-, wherein D0, E0, and F0 are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3- 10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; and RT1 is LL-oligonucleotide, wherein LL is an oligonucleotide linking group connecting the αvβ6 integrin targeting ligand to the oligonucleotide comprised in the dsRNA agent; and each Φ is a compound of the Formula (XII), wherein: Y is O, N(H), S, or CH2; R1 is hydrogen or C1-6alkyl; , wherein m is 0, 1, 2, 3, or 4; and 472 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each R2 is independently R, or two R2 groups on adjacent carbon atoms taken together with the atoms to which they are bound form a fused 4 – 8 membered ring that is optionally substituted by 1, 2, 3 or 4 groups independently selected from group consisting of R and a nitrogen protecting group; A is an 5-membered heteroaryl optionally substituted with 1 or 2 substituents independently selected from methyl, ethyl, fluoro, hydroxymethyl, 2-hydroxypropan-2-yl, trifluoromethyl, difluoromethyl, and fluoromethyl; Q is -COOR1 or tetrazolyl (e.g., 1,2,3,4-tetrazol-5-yl);and RL is -N(R3)(R4), -O(R5), -S(R5), or -R5, wherein R3 and R4 are either (i) R3 is hydrogen or C1-6alkyl and R4 is R5; or (ii) R3 and R4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R5; and R5 is -L-* wherein L is -L1-[G-L2]q-G-L3-*, * is the bond to a ZZ; and q is 0 or an integer selected from 1 – 25; (e.g., 1-20, or 1-15); L1 is a bond or -B-A-; each L2 is independently -A-B-A-; L3 is a bond or -A-B-A-; each G is independently -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); and each RN is independently hydrogen or C1-6alkyl, or two RN within an -A-B-A- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl. 52. The dsRNA agent of claim 51, wherein the αvβ6 integrin targeting ligand is selected from the group consisting of, 473 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (XVa) (XVb) and (XVc). 53. The dsRNA agent of claim 51 or 52, wherein RY is , wherein p is 0, 1, 2, 3 or 4; and each R21 is independently selected from the group consisting of R and a nitrogen protecting group. 54. The dsRNA agent of any one of claims 51-53, wherein RY is , wherein RP is a nitrogen protecting group. 55. The dsRNA agent of any one of claims 51-54, wherein RT is -G0-ORT1, wherein G0 is -D0-E0-F0-, wherein D0 and F0 are independently a bond or C1-10alkyl optionally substituted with 1, 2, 3, or 4 R groups; and E0 is C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups. 56. The dsRNA agent of claim 55, wherein G0 is 3-10 membered heterocyclyl optionally substituted with 1 or 2 R groups. 57. The dsRNA agent of claim 56, wherein wherein * represents the bond to T, the broken bond represents the bond to ORT1, and R is -C1-6alkyl-ORa or -ORa, wherein Ra is independently hydrogen or C1-6alkyl; 474 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 58. The dsRNA agent of claim 56, wherein wherein * represents the bond to T, the broken bond represents the bond to ORT1, and R is -C1-6alkyl-ORa or -ORa, wherein Ra is independently hydrogen or C1-6alkyl. 59. The dsRNA agent of any one of claims 51-58, wherein T is a bond or **-L6-G1-[L5-G1]q1-L4-, wherein ** is the bond to RT; q1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each L4, L5, and L6 are independently a bond or -A1-B1-A1-, or ZZ1; ZZ1 is a group formed by reaction of a reactive pair; each G1 is independently -D1-E1-F1-, wherein D1, E1, and F1 are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups; each A1 is independently a bond, -O-, -S-, or -N(RN1)-; each B1 is independently a bond, C(O), C(S), C(NRN1), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN1 is independently hydrogen or C1-6alkyl, or two RN1 within an -A1-B1-A1- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl. 60. The dsRNA agent of any one of claims 51-59, wherein T is **-L6-G1-[L5-G1]q1-L4-, wherein ** is the bond to RT; q1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each L4, L5, and L6 are independently a bond or -A1-B1-A1-, or ZZ1; ZZ1 is a group formed by reaction of an azide and alkyne or cycloalkyne; each G1 is independently C1-10alkyl, each A1 is independently a bond, -O-, -S-, or -N(RN1)-; each B1 is independently a bond, C(O), C(S), C(NRN1), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN1 is independently hydrogen or C1-6alkyl. 475 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 61. The dsRNA agent of claim 60, wherein one L5 is ZZ1. 62. The dsRNA agent of claim 61, wherein ZZ1 comprises: . 63. The dsRNA agent of any one of claims 51-62, wherein T is **-C(O)-C2-20alkyl-C(O)N(H)- wherein ** is the bond to RT: 64. The dsRNA agent of any one of claims 51-62, wherein T is T is **-C(O)-[CH2CH2-O]q5-G5-L4-, wherein ** is the bond to RT; q5 is an integer selected from 1 to 20; L4 is -A1-B1-A1-, wherein each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is hydrogen or C1- 6alkyl; each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and G5 is C1-10alkyl. 65. The dsRNA agent of any one of claims 51-64, wherein Δ is #–[G2-L7]q2-* or #–G3-([L7-G4]q3-*)y, wherein # is the bond to T; 476 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO y is 1, 2, 3, 4, or 5; q2 is 1, 2, 3, 4, 5, 6, 7, or 8; q3 is 0, 1, 2, 3, 4, 5, 6, 7, or 8; each G2, G3, and G4 is independently -D2-E2-F2-, wherein D2, E2, and F2 are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 RB groups, and wherein each G2 and G4 optionally contains at least one bond to a ZZ; or G3 is N and y is 2; each L7 is independently -A2-B2-A2-; wherein each A2 is independently a bond, -O-, -S-, or -N(RN2)-; each B2 is independently a bond, C(O), C(S), C(NRN2), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); and each RN2 is independently hydrogen, C1-6alkyl, a bond to a ZZ, or two RN2 within an -A2-B2-A2- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; and each RB is independently halogen, cyano, azido, nitro, -N(R10)2, -O(R10), -S(R10), -C(O)OR10, - C(O)R10, -C(O)N(R10)2, -C(NR10)OR10, -C(NR10)R10, -C(NR10)N(R10)2, -C(S)OR10, - C(S)R10, -C(S)N(R10)2, -S(O)2R10, -S(O)2OR10, -S(O)2N(R10)2, -N(R10)C(O)OR10, -N(R10)C(O)R10, -N(R10 )C(O)N(R10)2, -N(R10)S(O)2R10, -N(R10)S(O)2OR10, -N(R10)S(O)2N(R10)2, -OC(O)OR10, -OC(O)R10, - OC(O)N(R10)2, -OS(O)2R10, -OS(O)2OR10, -OS(O)2N(R10)2, or -SC(O)R10, wherein each R10 is independently hydrogen, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl.provided that Δ contains x bonds to ZZ. 66. The dsRNA agent of claim 65, wherein Δ is selected from the group consisting of, wherein each * is a bond to a ZZ; # is the bond to T, 477 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO each G2 is independently C1-10alkyl, C2-10alkenyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 RB groups; and each L7 is independently -A2-B2-A2-, wherein each A2 is independently a bond, -O-, -S-, or - N(RN2)-; each B2 is independently a bond, C(O), S(O)2, P(O)(OH), or P(S)(OH). 67. The dsRNA agent of claim 66, wherein each G2 is independently C1-10alkyl, each optionally substituted with 1 or 2 RB groups. 68. The dsRNA agent of any one of claims 65-67, wherein Δ is selected from the group consisting of, wherein # is the bond to T and each * is a bond to a ZZ group. ; and each G2 is independently C1-10alkyl. 69. The dsRNA agent of any one of claims 65-67, wherein Δ is selected from the group consisting of, 478 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein # is the bond to T and each * is a bond to a ZZ group. 70. The dsRNA agent of claim 65, wherein Δ is -#–G3-([L7-G4]q3-*)y, wherein # is the bond to T and * is a bond to a ZZ group. 71. The dsRNA agent of claim 70, wherein Δ is selected from the group consisting of, wherein # is the bond to T, each * is a bond to a ZZ group, and each L7 is selected from the group consisting of -O-, -S-, -N(H)-, -C(O)O-, -OC(O)-, -C(O)N(H)- , -OC(O)O-, -N(H)C(O)O-, -OC(O)N(H)-, -OP(O)(OH)O-, or -OP(S)(OH)O-; and each G4 is independently -D2-E2-F2-, wherein each D2 and F2 are independently a bond or C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 RB groups, and each E2 is independently bond, C1-10alkyl, C2-10alkenyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 RB groups, provided that E2 is not a bond with D2 and F2 are each bonds. 479 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 72. The dsRNA agent of claim 65, wherein Δ is selected from the group consisting of, wherein # is the bond to T and each * is a bond to a ZZ group. 73. The dsRNA agent of any one of claims 51-72, wherein ZZ is -A’-B’-A’-, wherein each A’ is independently a bond, -O-, -S-, or -N(RN3)-, wherein RN3 is independently hydrogen or C1-6alkyl and each B’ is independently CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). 74. The dsRNA agent of any one of claims 51-72, wherein ZZ is -CH2O-, -OCH2-, -S-S-, -C=N-, - C=N-O-, -C=N-N(RN3)-, -N=C-, -O-N=C-, -N(RN3)-N=C-, -C(O)N(RN3)-, -N(RN3)C(O)-, -C(O)O-, - OC(O)-, -OC(O)N(RN3)-, -N(RN3)C(O)O-, -N(RN3)C(O)N(RN3)-, -S(O)2N(RN3)-, -N(RN3)S(O)2-, - OP(O)(OH)O-, -OP(S)(OH)O-, -OP(O)(OH)-, -OP(S)(OH)-, -P(O)(OH)O-, or -P(S)(OH)O-. wherein RN3 is independently hydrogen or C1-6alkyl. 75. The dsRNA agent of any one of claims 51-74, wherein Formula (XII) is selected from the group consisting of: 480 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (XII-r) (XII-s) 481 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (XII-t) wherein p is 0, 1, 2 or 3; each R21 is independently selected from group consisting of R and a nitrogen protecting group, and RP is a nitrogen protecting group. 76. The dsRNA agent of any one of claims 51-75, wherein L is -L1-[G-L2]q-G-L3-*, wherein q is 0, 1, 2, 3, 4, or 5. 77. The dsRNA agent of any one of claims 51-75, wherein L is -L1-G-*, wherein * is the bond to ZZ; G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; L1 is -B-A-, wherein, A is a bond, -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1- 6alkyl; and B is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). 78. The dsRNA agent of any one of claims 51-75, wherein L is wherein * is the bond to ZZ; k is an integer from 1 to 10; L1 is bond, C(O), C(S), C(NRN), S(O)2, P(O)(OH), or P(S)(OH); and RN is hydrogen or C1-6alkyl. 79. The dsRNA agent of any one of claims 51-75, wherein L is a group selected from: (a) , wherein * is the bond to ZZ; t is an integer from 0 to 10; (b) L is wherein * is the bond to ZZ, t is an integer from 0 to 10; a is an integer from 1 to 3; and s and s’ are each independently an integer from 1 to 24; 482 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (c) wherein * is the bond to ZZ; a is 1, 2 or 3; and each s, s’, and s” independently is an integer from 1 to 24; an integer from 1 to 24; and (f) wherein * is the bond to ZZ; s and k are independently is an integer from 1 to 20; and w is an integer from 1 to 10. 80. The dsRNA agent of any one of claims 51-75, wherein L is wherein * is the bond to ZZ and w is an integer from 1 to 20. 81. The dsRNA agent of any one of claims 51-75, wherein L is , wherein * is the bond to ZZ; k is an integer from 1 to 10. 82. The dsRNA agent of any one of claims 51-75, wherein L is L is -L1-G-*, wherein * is the bond to ZZ , G is C1-10alkyl or C2-10alkenyl, and L1 is -B-A-, wherein, A is a bond, -O-, -S-, or -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; and B is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). 83. The dsRNA agent of any one of claims 51-75, wherein L is -[G-L2]q-G-*, wherein q is an integer from 1 to 5, each G is independently C1-10alkyl and L2 is O, S, or N(H). 84. The dsRNA agent of any one of claims 51-75, wherein L is -G-L2-G-*, wherein L2 is -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-; each B is independently a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each RN is independently hydrogen or C1-6alkyl, or two RN within an -A-B-A- group taken together with the atoms to which they are connected form a 4-8 483 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO membered heterocyclyl; and each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.; wherein each R group is independently selected from the group consisting of R’, C1-6alkyl, C1- 6haloalkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, heterocyclyl, aryl, heteroaryl, C3- 8cycloalkylC1-6alkyl, heterocyclylC1-6alkyl, aryl C1-6alkyl, heteroarylC1-6alkyl, each of which, other than R’, is optionally substituted with 1, 2, or 3 R’ groups, wherein each R’ is independently halogen, cyano, azido, nitro, -N(Rb)2, -O(Ra), -S(R0), -C(O)OR0, - C(O)R0, -C(O)N(R0)2, -C(NR0)OR0, -C(NR0)R0, -C(NR0)N(R0)2, -C(S)OR0, -C(S)R0, -C(S)N (R0)2, -S(O)2R0, -S(O)2OR0, -S(O)2N(R0)2, -N(R0)C(O)OR0, -N(R0)C(O)R0, -N(R0)C(O)N(R0 )2, -N(R0)S(O)2R0, -N(R0)S(O)2OR0, -N(R0)S(O)2N(R0)2, -OC(O)OR0, -OC(O)R0, - OC(O)N(R0)2, -OS(O)2R0, -OS(O)2OR0, -OS(O)2N(R0)2, or -SC(O)R0, wherein each R0 is independently hydrogen or C1-6alkyl; and each Ra is independently hydrogen or C1- 6alkyl; and each Rb is independently hydrogen, C1-6alkyl, or a nitrogen protecting group. 85. The dsRNA agent of any one of claims 51-84, wherein LL is a divalent linker that connects to the 3’-end of the oligonucleotide, the 5’-end of the oligonucleotide, a 2’position on an internal nucleotide, a 3’ position on an internal nucleotide, or a internucleotide linkage. 86. The dsRNA agent of claim 85, wherein LL connects to an oxygen atom on a nucleotide, and is P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH). 87. The dsRNA agent of any one of claims 51-86, wherein RT is RT1 wherein RT1 is -LL- oligonucleotide, wherein LL connects to an oxygen atom on a nucleoside of the oligonucleotide, LL is a bond, and the nucleoside is represented by Formula (XV-f): (XV-f) wherein B is an optionally modified nucleobase and * represents the bond to Δ. 88. The dsRNA agent of claim 87, wherein -T- is -C4-10alkyl-*, wherein * is the bond to Δ. 89. The dsRNA agent of claim 87, wherein the nucleoside is of a formula selected from the group consisting of, 484 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO (XV-q) wherein 485 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO B is an optionally modified nucleobase; each n is independently 0 or an integer selected from 1-10; and each m is independently integer selected from 1-20. 90. The dsRNA agent of any one of claims 51-84, wherein the αvβ6 integrin targeting ligand is conjugated at the 5’-end of the oligonucleotide. 91. The dsRNA agent of claim 90, wherein oligonucleotide conjugated to the αvβ6 integrin targeting ligand comprised in the dsRNA agent is represented by the following formula: wherein Y’ is O or S and represents the remainder for the oligonucleotide. 92. The dsRNA agent of any one of claims 51-84, wherein the αvβ6 integrin targeting ligand is conjugated at the 3’-end of the oligonucleotide. 93. The dsRNA agent of claim 92, wherein oligonucleotide conjugated to the αvβ6 integrin targeting ligand comprised in the dsRNA agent is represented by the formula: wherein Y' is O or S. 94. The dsRNA agent of claim 93, wherein oligonucleotide conjugated to the αvβ6 integrin targeting ligand comprised in the dsRNA agent is represented by Formula (XV-x): 486 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S; each ZZ is N(H)C(O) or C(O)N(H); each wherein m is an integer selected from 1 – 10; L4 and L6 are independently -B1-A1- or -A1-B1-; each L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); each G1 is independently C1-10alkyl or C2-10alkenyl; each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). 95. The dsRNA agent of claim 94, wherein oligonucleotide conjugated to the αvβ6 integrin targeting ligand comprised in the dsRNA agent is represented by Formula (XV-x1): 487 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO wherein Y’ is O or S; each ZZ is N(H)C(O) or C(O)N(H); each wherein m is an integer selected from 1 – 10; L5 is a bond or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond); each G1 is independently C1-10alkyl; each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl; and each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH). 96. The dsRNA agent of claim 95, wherein each Φ is selected from the group consisting of: , 488 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , wherein m is an integer selected from 1 – 10. 97. The dsRNA agent of claim 96, wherein RP is hydrogen and R1 is hydrogen. 98. The dsRNA agent of claim 97, wherein Y’ is O. 99. The dsRNA agent of claim 97, wherein Y’ is S. 100. The dsRNA agent of any one of claims 1-22, wherein the αvβ6 integrin targeting ligand comprises the following structure: 489 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , , 490 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , 491 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , , 492 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , 493 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO , or any combination thereof, wherein * represents the bond to the remainder of the dsRNA agent. 101. The dsRNA agent of any one of claims 1-100, wherein the at least one αvβ6 integrin targeting ligand is conjugated to the sense strand. 102. The dsRNA agent of claim 101, wherein the at least one αvβ6 integrin targeting ligand is conjugated to the 3’-end of the sense strand. 103. The dsRNA agent of claim 101, wherein the at least one αvβ6 integrin targeting ligand is conjugated to the 5’-end of the sense strand. 104. The dsRNA agent of claim 101, wherein the at least one αvβ6 integrin targeting ligand is conjugated to both the 5’-end and the 3’-end of the sense strand. 105. The dsRNA agent of claim 101, wherein the at least one αvβ6 integrin targeting ligand is conjugated to an internal position of the sense strand. 494 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 106. The dsRNA agent of any one of claims 1-100, wherein the at least one αvβ6 integrin targeting ligand is conjugated to the antisense strand. 107. The dsRNA agent of claim 106, wherein the at least one αvβ6 integrin targeting ligand is conjugated to the 3’ end antisense strand. 108. The dsRNA agent of claim 106, wherein the at least one αvβ6 integrin targeting ligand is conjugated to the 5’ end of the antisense strand. 109. The dsRNA agent of claim 106, wherein the at least one αvβ6 integrin targeting ligand is conjugated to both the 5’-end and the 3’-end of the antisense strand. 110. The dsRNA agent of claim 106, wherein the at least one αvβ6 integrin targeting ligand is conjugated to an internal position of the antisense strand. 111. The dsRNA agent of any one of claims 1-110, wherein the at least one in vivo delivery enhancing moiety is conjugated to an internal position or an external position of the sense strand. 112. The dsRNA agent of claim 111, wherein the at least one in vivo delivery enhancing moiety is conjugated to one or more of the following internal positions: positions 4-8 and 13-18 on the sense strand, counting from the 5’ end. 113. The dsRNA agent of claim 112, wherein the at least one in vivo delivery enhancing moiety is conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, 16 and 17 on the sense strand, counting from the 5’-end. 114. The dsRNA agent of claim 113, wherein the at least one in vivo delivery enhancing moiety is conjugated to position 6 on the sense strand, counting from the 5’-end. 115. The dsRNA agent of any one of claims 1-110, wherein the at least one in vivo delivery enhancing moiety is conjugated to an internal position or external position of the antisense strand. 116. The dsRNA agent of claim 115, wherein the at least one in vivo delivery enhancing moiety is conjugated to one or more of the following internal positions: positions 6-10 and 15-18 on the antisense strand, counting from the 5’-end. 117. The dsRNA agent of claim 116, wherein the at least one in vivo delivery enhancing moiety is conjugated to one or more of the following internal positions: positions 15, 16 and 17 on the antisense strand, counting from the 5’-end. 118. The dsRNA agent of any one of claims 1-100, wherein the at least one αvβ6 integrin targeting ligand and the at least one in vivo delivery enhancing moiety are both conjugated to the sense strand. 119. The dsRNA agent of any one of claims 1-100, wherein the at least one αvβ6 integrin targeting ligand and the at least one in vivo delivery enhancing moiety are both conjugated to the antisense strand. 495 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 120. The dsRNA agent of any one of claims 1-100, wherein the at least one αvβ6 integrin targeting ligand is conjugated to the sense strand and the at least one in vivo delivery enhancing moiety is conjugated to the antisense strand. 121. The dsRNA agent of any one of claims 1-100, wherein the at least one αvβ6 integrin targeting ligand is conjugated to the antisense strand and the at least one in vivo delivery enhancing moiety is conjugated to the sense strand. 122. The dsRNA agent of any one of claims 1-121, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length; 19 to 25 nucleotides in length; or 21 to 23 nucleotides in length. 123. The dsRNA agent of any one of claims 1-122, wherein the target gene is selected from the group consisting of adrenoceptor beta 1 (ADRB1); calcium voltage-gated channel subunit alpha1 C (CACNA1C); calcium voltage-gated channel subunit alpha1 G (CACNA1G) (T type calcium channel); angiotensin II receptor type 1(AGTR1); Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1 (HCN1); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3 (HCN3); Potassium Voltage-Gated Channel Subfamily A Member 5 (KCNA5); Potassium Inwardly Rectifying Channel Subfamily J Member 3 (KCNJ3); Potassium Inwardly Rectifying Channel Subfamily J Member 4 (KCNJ4); phospholamban (PLN); calcium/calmodulin dependent protein kinase II delta (CAMK2D); and Phosphodiesterase 1 (PDE1); myostatin (MSTN); Cholinergic Receptor Nicotinic Alpha 1 Subunit (CHRNA1); Cholinergic Receptor Nicotinic Beta 1 Subunit (CHRNB1); Cholinergic Receptor Nicotinic Delta Subunit (CHRND); Cholinergic Receptor Nicotinic Epsilon Subunit (CHRNE); Cholinergic Receptor Nicotinic Gamma Subunit (CHRNG); Collagen Type XIII Alpha 1 Chain (COL13A1); Docking Protein 7 (DOK7); LDL Receptor Related Protein 4 (LRP4); Muscle Associated Receptor Tyrosine Kinase (MUSK); Receptor Associated Protein Of The Synapse (RAPSN); Sodium Voltage-Gated Channel Alpha Subunit 4 (SCN4A); Double Homeobox 4 (DUX4); dystrophy myotonic protein kinase (DMPK); glycogen synthase 1 (GYS1); survival of motor neuron 1 (SMN1), and alpha-glucosidase (GAA). 124. A cell containing the dsRNA agent of any one of claims 1-123. 125. A pharmaceutical composition for inhibiting expression of the target gene, comprising the dsRNA agent of any one of claims 1-123. 126. A method of inhibiting expression of a target gene in a muscle cell, said method comprising contacting the cell with the dsRNA agent of any one of claims 1-123 or the pharmaceutical composition of claim 124, thereby inhibiting expression of the target gene in the muscle cell. 496 ME1\53466565.v1 Atty. Docket No.121301-23820/ALN-523-WO 127. The method of claim 126, wherein the muscle cell is a skeletal muscle cell or a cardiac muscle cell. 128. The method of claim 126 or 127, wherein the cell is within a subject. 129. A method of treating a subject having a muscle disorder, said method comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1-123 or the pharmaceutical composition of claim 125, thereby treating the subject. 130. The method of claim 129, wherein the muscle disorder is a skeletal muscle disorder or a cardiac muscle disorder. 131. The method of claim 130, wherein the skeletal muscle disorder or cardiac muscle disorder is selected from the group consisting of myostatin-related muscle hypertrophy, congenital myasthenic syndrome, facioscapulohumeral muscular dystrophy (FSHD), Spinal Muscular Atrophy (SMA), Myotonic Dystrophy Type 1 (DM1), Pompe disease, PLN cardiomyopathy, spasticity, obstructive hypertrophic cardiomyopathy (HOCM); familial hypertrophic cardiomyopathy (FHC); heart failure with preserved ejection fraction (HFPEF); atrial fibrillation (AFIB); ventricular fibrillation (VFIB); angina; myocardial infarction (MI); heart failure or heart failure with reduced ejection fraction (HFREF); supraventricular tachycardia (SVT); hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmia, and congestive heart failure (CHF). 132. The method of any one of claims 129-131, wherein the dsRNA agent is administered to the subject subcutaneously. 133. The method of any one of claims 129-131, wherein the dsRNA agent is administered to the subject intramuscularly. 134. The method of any one of claims 129-131, wherein the dsRNA agent is administered to the subject intravenously. 135. The method of any one of claims 128-134, wherein the subject is a human. 136. An RNA-induced silencing complex (RISC) comprising an antisense strand of any of the dsRNA agents of claims 1-123. 497 ME1\53466565.v1
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