EP4598544A1 - Plasminogen (plg)-irna-zusammensetzungen und verfahren zur verwendung davon - Google Patents

Plasminogen (plg)-irna-zusammensetzungen und verfahren zur verwendung davon

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Publication number
EP4598544A1
EP4598544A1 EP23875804.9A EP23875804A EP4598544A1 EP 4598544 A1 EP4598544 A1 EP 4598544A1 EP 23875804 A EP23875804 A EP 23875804A EP 4598544 A1 EP4598544 A1 EP 4598544A1
Authority
EP
European Patent Office
Prior art keywords
nucleotides
ome
nucleotide
plg
strand
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23875804.9A
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English (en)
French (fr)
Inventor
Martina SLINGSBY
Benjamin HOLMES
Elane FISHILEVICH
Jeffrey ZUBER
Eleftheria Maratos-Flier
John GANSNER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alnylam Pharmaceuticals Inc
Original Assignee
Alnylam Pharmaceuticals Inc
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Publication date
Application filed by Alnylam Pharmaceuticals Inc filed Critical Alnylam Pharmaceuticals Inc
Publication of EP4598544A1 publication Critical patent/EP4598544A1/de
Pending legal-status Critical Current

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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
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    • C12N2310/3533—Halogen

Definitions

  • formula (III) is represented by formula (IIId): sense: 5' n p -N a –X X X- N b -Y Y Y -N b -Z Z Z -N a - n q 3' antisense: 3' n p′ -N a′ - X′X′X′- N b′ -Y′Y′Y′-N b′ -Z′Z′Z′- N a′ - n q′ 5' (IIId) wherein each N b and N b ′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides and each N a and N a ′ independently represents an oligonucleotide sequence comprising 2- 10 modified nucleotides.
  • n p ′ is linked to a neighboring nucleotide via a phosphorothioate linkage. In another embodiment, wherein all n p ′ are linked to neighboring nucleotides via phosphorothioate linkages. In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification. In one embodiment, the ligand is conjugated to the 3’ end of the sense strand of the dsRNA agent. In one embodiment, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached through a monovalent, bivalent, or trivalent branched linker.
  • GalNAc N-acetylgalactosamine
  • the ligand is In one embodiment, the dsRNA agent is conjugated to the ligand as shown in the following schematic and, wherein X is O or S. In one embodiment, the X is O. In one aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of plasminogen (PLG) in a cell.
  • dsRNA double stranded ribonucleic acid
  • the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of plasminogen (PLG) in a cell.
  • the dsRNA agent includes a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding PLG, wherein each strand is about 14 to about 30 nucleotides in length, wherein the dsRNA agent is represented by formula (III): sense: 5' n p -N a -(X X X) i -N b -Y Y Y -N b -(Z Z Z) j -N a - n q 3' antisense: 3' n p ′-N a ′-(X′X′X′) k -N b ′-Y′Y′Y′-N b ′-(Z′Z′Z′) l -N
  • the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of plasminogen (PLG) in a cell.
  • the dsRNA agent includes a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding PLG, wherein each strand is about 14 to about 30 nucleotides in length, wherein the dsRNA agent is represented by formula (III): sense: 5' n p -N a -(X X X) i -N b -Y Y Y -N b -(Z Z Z) j -N a - n q 3' antisense: 3' n p ′-N a ′-(X′X′X′) k -N b ′-Y′Y′Y′-N b ′-(Z′Z′Z′) l -N
  • the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of plasminogen (PLG) in a cell.
  • the dsRNA agent includes a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding PLG, wherein each strand is about 14 to about 30 nucleotides in length, wherein the dsRNA agent is represented by formula (III): sense: 5' n p -N a -(X X X) i -N b -Y Y Y -N b -(Z Z Z) j -N a - n q 3' antisense: 3' n p ′-N a ′-(X′X′X′) k -N b ′-Y′Y′Y′-N b ′-(Z′Z′Z′) l -N
  • the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of plasminogen (PLG) in a cell.
  • the dsRNA agent includes a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding PLG, wherein each strand is about 14 to about 30 nucleotides in length, wherein the dsRNA agent is represented by formula (III): sense: 5' n p -N a -Y Y Y - N a - n q 3' antisense: 3' n p ′-N a ′- Y′Y′Y′- N a ′- n q ′ 5' (IIIa) wherein: each n p , n q , and n q ′, each of which may or may not be present, independently represents an overhang nucleotide; p,
  • the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of plasminogen (PLG) in a cell.
  • the dsRNA agent includes a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 or 3 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:2 or 4, wherein substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2’-O-methyl modification and a 2’-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5’-terminus
  • the dsRNA agent includes a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of SEQ ID NO:1 or 3 and the antisense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of SEQ ID NO:2 or 4, wherein substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2’-O-methyl modification and a 2’-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5’-terminus, wherein substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2’-O-methyl modification and a 2’-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleot
  • all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.
  • the region of complementarity comprises any one of the antisense sequences listed in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B.
  • the sense strand and the antisense strand comprise nucleotide sequences selected from the group consisting of the nucleotide sequences of any one of the agents listed in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B.
  • the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of plasminogen (PLG) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region.
  • the sense strand comprises a nucleotide sequence of any one of the agents in Tables 3, 4, 5, 6, 7, 8A, or 8B and the antisense strand comprises a nucleotide sequence of any one of the agents in Tables 3, 4, 5, 6, 7, 8A, or 8B.
  • the dsRNA agent targets a hotspot region of an mRNA encoding PLG.
  • the present invention provides a dsRNA agent that targets a hotspot region of a plasminogen (PLG) mRNA.
  • PLG plasminogen
  • the present invention also provides cells, vectors, and pharmaceutical compositions which include any of the dsRNA agents of the invention.
  • the dsRNA agents may be formulated in an unbuffered solution, e.g., saline or water, or in a buffered solution, e.g., a solution comprising acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.
  • the buffered solution is phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the present invention provides a method of inhibiting plasminogen (PLG) expression in a cell. The method includes contacting the cell with a dsRNA agent or a pharmaceutical composition of the invention, thereby inhibiting expression of PLG in the cell.
  • the cell may be within a subject, such as a human subject.
  • the PLG expression is inhibited by at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or to below the level of detection of PLG expression. In another embodiment the PLG expression is inhibited by about 50% or less. In a specific embodiment, the PLG expression is inhibited by about 50%.
  • the human subject suffers from a PLG-associated disease, disorder, or condition.
  • the PLG-associated disease, disorder, or condition is a bleeding disorder, such as hereditary hemorrhagic telangiectasia (HHT).
  • HHT hereditary hemorrhagic telangiectasia
  • a symptom of the bleeding disorder is heavy menstrual bleeding (HMB).
  • the bleeding disorder is selected from the group consisting of PAI-1 deficiency, hereditary hemorrhagic telangiectasia (HHT), heavy menstrual bleeding (HMB), von Willebrand disease, low factor XI levels, platelet defects, hemophilia A, hemophilia B, afibrinogenemia, parahemophilia, low factor VIII levels, low factor IX levels, low factor VII levels, low factor XIII levels, low factor X levels, low factor V levels, low factor II levels, nose bleeds, bleeding gums, easy bruising, postpartum bleeding, and excessive bleeding following surgery.
  • the bleeding disorder is hereditary hemorrhagic telangiectasia (HHT).
  • the PLG-associated disease, disorder, or condition is a surgical procedure such as cardiac surgery, oral and maxillo-facial surgery, liver surgery, nephrolithotomy, orthopedic surgery, gynecologic surgery, trauma intervention, tooth extraction, or dermatologic procedures.
  • the subject is treated pre-surgery to prevent excessive bleeding.
  • the PLG-associated disease, disorder, or condition is melasma or hyperpigmentation of the skin.
  • the present invention provides a method of inhibiting the expression of PLG in a subject. The methods include administering to the subject a therapeutically effective amount of a dsRNA agent or a pharmaceutical composition of the invention, thereby inhibiting the expression of PLG in the subject.
  • the present invention provides a method of treating a subject suffering from a PLG- associated disease, disorder, or condition.
  • the method includes administering to the subject a therapeutically effective amount of a dsRNA agent or a pharmaceutical composition of the invention, thereby treating the subject suffering from a PLG-associated disease, disorder, or condition.
  • the present invention provides a method of preventing at least one symptom in a subject having a disease, disorder or condition that would benefit from reduction in expression of a PLG gene.
  • the method includes administering to the subject a prophylactically effective amount of the agent of a dsRNA agent or a pharmaceutical composition of the invention, thereby preventing at least one symptom in a subject having a disease, disorder or condition that would benefit from reduction in expression of a PLG gene.
  • the administration of the dsRNA agent or the pharmaceutical composition to the subject causes a decrease in PLG activity, a decrease in PLG protein accumulation, and/or a decrease in excessive bleeding in a subject.
  • the PLG-associated disease, disorder, or condition is a bleeding disorder.
  • a symptom of the bleeding disorder is heavy menstrual bleeding (HMB).
  • the bleeding disorder is hereditary hemorrhagic telangiectasia (HHT).
  • HHT hereditary hemorrhagic telangiectasia
  • HMB heavy menstrual bleeding
  • PAI-1 deficiency von Willebrand disease
  • von Willebrand disease low factor XI levels
  • platelet defects hemophilia A, hemophilia B, afibrinogenemia, parahemophilia
  • low factor VIII levels low factor IX levels, low factor VII levels, low factor XIII levels, low factor X levels, low factor V levels, low factor II levels
  • nose bleeds bleeding gums, easy bruising, postpartum bleeding, and excessive bleeding following surgery.
  • the PLG-associated disease, disorder, or condition is a surgical procedure such as cardiac surgery, oral and maxillo-facial surgery, liver surgery, nephrolithotomy, orthopedic surgery, gynecologic surgery, trauma intervention, tooth extraction, or dermatologic procedures.
  • the PLG-associated disease, disorder, or condition is melasma or hyperpigmentation of the skin.
  • the subject is treated pre-surgery to prevent excessive bleeding.
  • the bleeding disorder is a mucocutaneous bleeding disorder (MCB).
  • the mucocutaneous bleeding disorder is selected from the group consisting of inherited platelet disorders (IPD), hereditary hemorrhagic telangiectasia (HHT), hypermobility spectrum disorders (HSD), Ehlers-Danlos syndromes (EDS), and von Willebrand disease (VWD).
  • the methods and uses of the invention further include administering an additional therapeutic to the subject.
  • the dsRNA agent is administered to the subject at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.
  • the agent may be administered to the subject intravenously, intramuscularly, or subcutaneously. In one embodiment, the agent is administered to the subject subcutaneously.
  • the methods and uses of the invention further include determining, the level of PLG in the subject.
  • the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of plasminogen (PLG) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence of any one of the agents in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B, and the antisense strand comprises a nucleotide sequence of any one of the agents in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B, wherein substantially all of the nucleotide of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the dsRNA agent is conjugated to a ligand.
  • dsRNA agent is conjugated to a ligand.
  • the RNAi agent is a pharmaceutically acceptable salt thereof.
  • “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, and mixtures thereof.
  • 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).
  • the iRNAs of the invention targeting PLG may include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 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 nucleotides in length, which region is substantially complementary to at least
  • one or both of the strands of the double stranded RNAi agents of the invention is up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA transcript of a PLG gene.
  • such iRNA agents having longer length antisense strands may include a second RNA strand (the sense strand) of 20-60 nucleotides in length wherein the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.
  • RNAi RNA interference
  • compositions including these iRNAs are useful for treating a subject who would benefit from inhibiting or reducing the expression of a PLG gene, e.g., a subject that would benefit from a reduction of bleeding, e.g., a subject suffering or prone to suffering from a PLG-associated disease disorder, or condition, such as a subject suffering or prone to suffering from bleeding disorder, e.g., a subject suffering from hereditary hemorrhagic telangiectasia (HHT), heavy menstrual bleeding (HMB), PAI-1 deficiency, von Willebrand disease, low factor XI levels, platelet defects, hemophilia A, hemophilia B, afibrinogenemia, parahemophilia, low factor VIII levels, low factor IX levels, low factor VII levels, low factor XIII levels, low factor X levels, low factor V levels, low factor II levels, nose bleeds, bleeding gums, easy bruising, postpartum bleeding, or excessive bleeding following surgery.
  • Exemplary nucleotide and amino acid sequences of PLG can be found, for example, at GenBank Accession No. NM_000301.5 (SEQ ID NO: 1; reverse complement SEQ ID NO: 2) and GenBank Accession No. NM_001168338.1 (SEQ ID NO: 3; reverse complement SEQ ID NO: 4) for Homo sapiens PLG; GenBank Accession No. XM_005551498.2 (SEQ ID NO: 685; reverse complement SEQ ID NO: 686) for Macaca fascicularis PLG; GenBank Accession No. NM_008877.3 (SEQ ID NO: 687; reverse complement SEQ ID NO: 688) for Mus musculus PLG; and GenBank Accession No.
  • target sequence refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a PLG 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 iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a PLG gene.
  • the target sequence of a PLG gene may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length.
  • 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.
  • 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, or a surrogate replacement moiety (see, e.g., Table 2).
  • guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety.
  • a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil.
  • 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
  • iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
  • RNAi RNA interference
  • the iRNA modulates, e.g., inhibits, the expression of PLG gene in a cell, e.g., a cell within a subject, such as a mammalian subject.
  • an RNAi agent of the invention includes a single stranded RNA that interacts with a target RNA sequence, e.g., a PLG target mRNA sequence, to direct the cleavage of the target RNA.
  • a target RNA sequence e.g., a PLG target mRNA sequence
  • Dicer Type III endonuclease
  • 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 RNAi agents 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 which are hereby incorporated herein by reference.
  • an “iRNA” for use in the compositions and methods of the invention is a double-stranded RNA and is referred to herein as a “double stranded RNAi agent,” “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 PLG gene.
  • a double-stranded RNA dsRNA triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.
  • each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or 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, and/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 invention include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims.
  • 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 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 least 2, at least 3, 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.
  • 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.
  • an RNAi agent of the invention is a dsRNA, each strand of which comprises less than 30 nucleotides, e.g., 17-27, 19-27, 17-25, 19-25, or 19-23, that interacts with a target RNA sequence, e.g., a PLG target mRNA sequence, to direct the cleavage of the target RNA.
  • an RNAi agent of the invention is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence, e.g., a PLG target mRNA sequence, to direct the cleavage of the target RNA.
  • a target RNA sequence e.g., a PLG target mRNA sequence
  • the sense strand is 21 nucleotides in length.
  • the antisense strand is 23 nucleotides in length.
  • nucleotide overhang refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA.
  • a dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can 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.
  • 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., 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides or 10-15 nucleotides in length.
  • an extended overhang is on the sense strand of the duplex.
  • an extended overhang is present on the 3’end of the sense strand of the duplex.
  • an extended overhang is present on the 5’end of the sense strand of the duplex.
  • 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 of the duplex. In certain embodiments, one or more of the nucleotides in the extended overhang is replaced with a nucleoside thiophosphate.
  • the terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang.
  • materials which can serve as pharmaceutically-acceptable carriers 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 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; (1
  • a “sample derived from a subject” refers to blood or plasma drawn from the subject.
  • iRNAs of the Invention Described herein are iRNAs which inhibit the expression of a target gene.
  • the iRNAs inhibit the expression of a PLG gene.
  • the iRNA agent includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a PLG gene in a cell, such as a liver cell, such as a liver cell within a subject, e.g., a mammal, such as a human having a bleeding disorder or condition.
  • dsRNA double stranded ribonucleic acid
  • the duplex structure is between 15 and 30 base pairs in length, e.g., between, 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 region of complementarity to the target sequence is between 15 and 30 nucleotides in length, e.g., between 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
  • 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.
  • a dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.
  • iRNA compounds of the invention 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 siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both.
  • RNA interference within such “hotspot regions” means that iRNA agents targeting the region will likely have higher efficacy in inducing iRNA interference than iRNA agents which target other regions of the target RNA.
  • the accessibility of a target region of a target RNA may influence the efficacy of iRNA agents which target that region, with some hotspot regions having increased accessibility.
  • Secondary structures, for instance, that form in the RNA target may affect the ability of the iRNA agent to bind the target region and induce RNA interference.
  • an iRNA agent may be designed to target a hotspot region of any of the target RNAs described herein, including any identified portions of a target RNA (e.g., a particular exon).
  • a hotspot region may refer to an approximately 19-200, 19-150, 19-100, 19- 75, 19-50, 21-200, 21-150, 21-100, 21-75, 21-50, 50-200, 50-150, 50-100, 50-75, 75-200, 75-150, 75-100, 100-200, or 100-150 nucleotide region of a target RNA sequence for which targeting using RNAi agents provides an observably higher probability of efficacious silencing relative to targeting other regions of the same target RNA.
  • a hotspot region may comprise a limited region of the target RNA, and in some cases, a substantially limited region of the target, including for example, less than half of the length of the target RNA, such as about 5%, 10%, 15%, 20%, 25%, or 30% of the length of the target RNA.
  • the other regions against which a hotspot is compared may cumulatively comprise at least a majority of the length of the target RNA.
  • the other regions may cumulatively comprise at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% of the length of the target RNA.
  • RNAi agents targeting various regions that span a target RNA may be compared for frequency of efficacious iRNA agents (e.g., the amount by which target gene expression is inhibited, such as measured by mRNA expression or protein expression) that bind each region.
  • a hotspot can be recognized by observing clustering of multiple efficacious RNAi agents that bind to a limited region of the RNA target.
  • a hotspot may be sufficiently characterized as such by observing efficacy of iRNA agents which cumulatively span at least about 60% of the target region identified as a hotspot, such as about 70%, about 80%, about 90%, or about 95% or more of the length of the region, including both ends of the region (i.e. at least about 60%, 70%, 80%, 90%, or 95% or more of the nucleotides within the region, including the nucleotides at each end of the region, were targeted by an iRNA agent).
  • iRNA agents which cumulatively span at least about 60% of the target region identified as a hotspot, such as about 70%, about 80%, about 90%, or about 95% or more of the length of the region, including both ends of the region (i.e. at least about 60%, 70%, 80%, 90%, or 95% or more of the nucleotides within the region, including the nucleotides at each end of the region, were targeted by an iRNA agent).
  • an iRNA agent which demonstrates at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% inhibition over the region (e.g., no more than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% mRNA remaining) may be identified as efficacious. Amenability to targeting of RNA regions may also be assessed using quantitative comparison of inhibition measurements across different regions of a defined size (e.g, 25, 30, 40, 50, 60, 70, 80, 90, or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nts).
  • a defined size e.g, 25, 30, 40, 50, 60, 70, 80, 90, or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nts.
  • an average level of inhibition may be determined for each region and the averages of each region may be compared.
  • the average level of inhibition within a hotspot region may be substantially higher than the average of averages for all evaluated regions.
  • the average level of inhibition in a hotspot region may be at least about 10%, 20%, 30%, 40%, or 50% higher than the average of averages.
  • the average level of inhibition in a hotspot region may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.51.6, 1.7, 1.8.1.9, or 2.0 standard deviations above the average of averages.
  • the average level of inhibition may be higher by a statistically significant (e.g., p ⁇ 0.05) amount.
  • a standard for evaluating a hotspot may comprise various combinations of the above standards where compatible (e.g., an average level of inhibition of at least about a first amount and having no inhibition measurements below a threshold level of a second amount, lesser than the first amount). It is therefore expressly contemplated that any iRNA agent, including the specific exemplary iRNA agents described herein, which targets a hotspot region of a target RNA, may be preferably selected for inducing RNA interference of the target mRNA as targeting such a hotspot region is likely to exhibit a robust inhibitory response relative to targeting a region which is not a hotspot region.
  • RNAi agents targeting target sequences that substantially overlap e.g., by at least about 70%, 75%, 80%, 85%, 90%, 95% of the target sequence length) or, preferably, that reside fully within the hotspot region may be considered to target the hotspot region.
  • Hotspot regions of the RNA target(s) of the instant invention may include any region for which the data disclosed herein demonstrates higher frequency of targeting by efficacious RNAi agents, including by any of the standards described elsewhere herein, whether or not the range(s) of such hotspot region(s) are explicitly specified.
  • a dsRNA agent of the present invention targets a hotspot region of an mRNA encoding PLG. III.
  • the sense strand comprises no more than 4 nucleotides comprising 2'-fluoro modifications (e.g., no more than 32'-fluoro modifications, or no more than 22'-fluoro modifications).
  • the antisense strand comprises no more than 6 nucleotides comprising 2'-fluoro modifications (e.g., no more than 52'-fluoro modifications, no more than 42'-fluoro modifications, no more than 42'-fluoro modifications, or no more than 22'-fluoro modifications).
  • all of the nucleotides of an iRNA of the invention are modified and the iRNA agents comprise no more than 10 nucleotides comprising 2’-fluoro modifications (e.g., no more than 92-fluoro modifications, no more than 82-fluoro modifications, no more than 72-fluoro modifications, no more than 62'-fluoro modifications, no more than 52'-fluoro modifications, no more than 42'-fluoro modifications, no more than 52'-fluoro modifications, no more than 42'-fluoro modifications, no more than 3 2'-fluoro modifications, or no more than 22'-fluoro modifications).
  • 2’-fluoro modifications e.g., no more than 92-fluoro modifications, no more than 82-fluoro modifications, no more than 72-fluoro modifications, no more than 62'-fluoro modifications, no more than 52'-fluoro modifications, no more than 42'-fluoro modifications, no more than 52'-fluoro modifications, no
  • the double stranded RNAi agent of the invention further comprises a 5’- phosphate or a 5’-phosphate mimic at the 5’ nucleotide of the antisense strand.
  • the double stranded RNAi agent further comprises a 5’-phosphate mimic at the 5’ nucleotide of the antisense strand.
  • the 5’-phosphate mimic is a 5’-vinyl phosphonate (5’-VP).
  • the phosphate mimic is a 5’-cyclopropyl phosphonate.
  • the 5’-end of the antisense strand of the double-stranded iRNA agent does not contain a 5’-vinyl phosphonate (VP).
  • at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy- modified nucleotide, a glycol modified nucleotide (GNA), e.g., Ggn, Cgn, Tgn, or Agn, a nucleotide with a 2’ phosphate, e.g., G2p, C2p, A2p or U2p, and, a vinyl-phosphonate nucleotide; and combinations thereof.
  • each of the duplexes of Tables 3, 4, 5, 6, 7, 8A, or 8B may be particularly modified to provide another double-stranded iRNA agent of the present disclosure.
  • the 3’-terminus of each sense duplex may be modified by removing the 3’-terminal L96 ligand and exchanging the two phosphodiester internucleotide linkages between the three 3’-terminal nucleotides with phosphorothioate internucleotide linkages.
  • the sense sequence: gsuscaacAfaCfAfUfccugggauuuL96 (SEQ ID NO: 279) may be replaced with gsuscaacAfaCfAfUfccugggaususu (SEQ ID NO: 684) while the antisense sequence remains unchanged to provide another double-stranded iRNA agent of the present disclosure.
  • the nucleic acids featured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al.
  • 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 iRNA will have a phosphorus atom in its internucleoside backbone.
  • 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 and/or phosphorothioate linkages without a sodium counterion.
  • sodium ions are present in the agent as counterions for all of the phosphodiester and/or phosphorothiotate groups present in the agent.
  • Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, 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.
  • RNA of an iRNA can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat.
  • RNA of the invention can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • 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-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-thiol, 8-thioalkyl, 8-hydroxyl anal other 8- substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substitute
  • nucleobases include those disclosed in U.S. Pat. No.3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. 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.
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention.
  • These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 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.
  • An iRNA of the invention can also be modified to include one or more bicyclic sugar moities.
  • a “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms.
  • a “bicyclic 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.
  • an agent of the invention 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' 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 invention include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms.
  • the antisense polynucleotide agents of the invention include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge.
  • RNA molecules can include N- (acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N- (acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-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 PCT Publication No.
  • an RNAi agent of the present invention is an agent that inhibits the expression of a PLG gene which is selected from the group of agents listed in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B. Any of these agents may further comprise a ligand.
  • the double stranded RNAi agents of the invention include agents with chemical modifications as disclosed, for example, in WO 2013/075035, filed on November 16, 2012, the entire contents of which are incorporated herein by reference. Accordingly, the invention provides double stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., a PLG gene) in vivo.
  • the RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may range from 12-30 nucleotides in length.
  • each strand may be between 14-30 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.
  • the sense strand is 21 nucleotides in length.
  • the antisense strand is 23 nucleotides in length.
  • 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 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 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 strands. In one embodiment, this 3’-overhang is present in the antisense strand.
  • the RNAi agent is a double ended bluntmer 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, 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, 13 from the 5’end.
  • the RNAi agent is a double ended bluntmer 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, 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, 13 from the 5’end.
  • the RNAi agent is a double ended bluntmer 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, 11 from the 5’end.
  • the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.
  • the RNAi agent comprises sense and antisense strands, wherein the RNAi 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, 13 from the 5’ end; 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
  • the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting 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.
  • 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.
  • the antisense strand of the RNAi agent may contain more than one motifs of 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.
  • 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 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. 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.
  • Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens and/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.
  • 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.
  • the sense strand when paired with the antisense strand in the dsRNA duplex the 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 5’-3’of the strand within the duplex region.
  • alternating pattern of 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.
  • a double-stranded RNAi agent comprises 6-8phosphorothioate internucleotide linkages.
  • each N a independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
  • the antisense strand sequence of the RNAi may be represented by formula (II): 5' n q’ -N a ′-(Z’Z′Z′) k -N b ′-Y′Y′Y′-N b ′-(X′X′X′) l -N′ a -n p ′ 3' (II) wherein: k and l are each independently 0 or 1; p’ and q’ are each independently 0-6; each N a ′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b ′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides; each
  • 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.
  • the RNAi agent of the invention contains 6 nucleotides with a 2’-fluoro modification, e.g., 4 nucleotides with a 2’-fluoro modification in the sense strand and 2 nucleotides with a 2’-fluoro modification in the antisense strand.
  • an RNAi agent of the invention may contain an ultra-low number of nucleotides containing a 2’-fluoro modification, e.g., 2 or fewer nucleotides containing a 2’-fluoro modification.
  • the RNAi agent may contain 2, 1 of 0 nucleotides with a 2’-fluoro modification.
  • the RNAi agent may contain 2 nucleotides with a 2’-fluoro modification, e.g., 0 nucleotides with a 2-fluoro modification in the sense strand and 2 nucleotides with a 2’-fluoro modification in the antisense strand.
  • Various publications describe multimeric RNAi agents that can be used in the methods of the invention. Such publications include WO2007/091269, US Patent No.7858769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520 the entire contents of each of which are hereby incorporated herein by reference.
  • B1, B2, B3, B1’, B2’, B3’, and B4’ each contain 2’-OMe or 2’-F modifications. In certain embodiments, at least one of B1, B2, B3, B1’, B2’, B3’, and B4’ contain 2'-O-N-methylacetamido (2'-O-NMA) modification.
  • C1 is a thermally destabilizing nucleotide placed at a site opposite to the seed region of the antisense strand (i.e., at positions 2-8 of the 5’-end of the antisense strand).
  • C1 is at a position of the sense strand that pairs with a nucleotide at positions 2-8 of the 5’-end of the antisense strand. In one example, C1 is at position 15 from the 5’-end of the sense strand.
  • C1 nucleotide bears the thermally destabilizing modification which can include abasic modification; mismatch with the opposing nucleotide in the duplex; and sugar modification such as 2’-deoxy modification or acyclic nucleotide e.g., unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA).
  • UNA unlocked nucleic acids
  • GNA glycerol nucleic acid
  • the thermally destabilizing modification in C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; and optionally, at least one nucleobase in the mismatch pair is a 2’-deoxy nucleobase.
  • the thermally destabilizing modification in C1 is GNA or T1, T1’, T2’, and T3’ each independently represent a nucleotide comprising a modification providing the nucleotide a steric bulk that is less or equal to the steric bulk of a 2’-OMe modification.
  • n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 1, B3’ is 2’-OMe or 2’-F, q 5 is 6, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5’-end of the sense strand), 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 of the antisense strand).
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 4 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at
  • the dsRNA agent also comprises a 5’-PS.
  • B1 is 2 -OMe or 2 -F
  • n 8
  • T1 is 2 F
  • n 3
  • B2 is 2 -OMe
  • n 7,
  • B3 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q 4 is 0, B3’ is 2’-OMe or 2’-F
  • q 5 is 7, T3’ is 2’-F
  • q 7 1
  • the RNAi agent also comprises a 5’-P.
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q 4 is 0,
  • B3’ is 2’-OMe or 2’-F
  • q 5 7, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide
  • the RNAi agent also comprises a 5’- PS 2 .
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q 4 is 0,
  • B3’ is 2’-OMe or 2’-F
  • q 5 7, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleo
  • the RNAi agent also comprises a 5’- P.
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, T2’ is 2’-F, q 4 is 2,
  • B3’ is 2’-OMe or 2’-F
  • q 5 5
  • T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand),
  • the RNAi agent also comprises a 5’- PS 2 .
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 4 2,
  • B3’ is 2’-OMe or 2’-F
  • q 5 5
  • T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense
  • the RNAi agent also comprises a 5’- P.
  • B1 is 2’-OMe or 2’-F
  • n 1 8 T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q is 0, B3 is 2 -OMe or 2 -F, q is 7, T3 is 2 -F
  • q 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2
  • the RNAi agent also comprises a 5’- VP.
  • the 5’-VP may be 5’-E-VP, 5’-Z-VP, or combination thereof.
  • B1 is 2’-OMe or 2’-F
  • n 1 is 8
  • T1 is 2’F
  • n 2 is 3
  • B2 is 2’-OMe
  • n 3 is 7,
  • n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3,
  • B1’ is 2’-OMe or 2’-F
  • q 1 9, T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q 4 is 0,
  • B3’ is 2’-OMe or 2’-F
  • q 5 is 7, T3’ is 2’-F
  • q 6 is 1
  • B4’ is 2’-F
  • q 7 is 1; with two phosphorothioate internucleotide linkage modifications
  • the RNAi agent also comprises a 5’- PS 2 .
  • B1 is 2’-OMe or 2’-F
  • n 1 is 8
  • T1 is 2’F
  • n 2 is 3
  • B2 is 2’-OMe
  • n 3 is 7,
  • n 4 is 0,
  • B3 is 2’-OMe
  • n 5 is 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9, T1’ is 2’-F
  • q 3 4
  • q 4 is 0,
  • B3’ is 2’-OMe or 2’-F
  • q 5 is 7, T3’ is 2’-F
  • q 6 is 1
  • B4’ is 2’-F
  • q 7 is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 4 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications
  • the RNAi agent also comprises a 5’-PS and a targeting ligand.
  • the 5’-PS is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 4 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 4 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications
  • the RNAi agent also comprises a 5’- PS 2 and a targeting ligand.
  • the 5’-PS 2 is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 4 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications
  • the RNAi agent also comprises a 5’-P and a targeting ligand.
  • the 5’-P is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8 T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q 4 is 0, B3’ is 2’-OMe or 2’-F
  • q 5 7, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucle
  • the RNAi agent also comprises a 5’-PS and a targeting ligand.
  • the 5’-PS is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8 T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q 4 is 0, B3’ is 2’-OMe or 2’-F
  • q 5 7, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucle
  • the RNAi agent also comprises a 5’-VP (e.g., a 5’-E-VP, 5’-Z-VP, or combination thereof) and a targeting ligand.
  • a 5’-VP e.g., a 5’-E-VP, 5’-Z-VP, or combination thereof
  • the 5’-VP is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2 -OMe or 2 -F
  • n 8
  • T1 is 2 F
  • n 3
  • B2 is 2 -OMe
  • n 7, n is 0,
  • B3 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q 4 is 0, B3’ is 2’-OMe or 2’-F
  • q 5 7
  • T3’ 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide
  • the RNAi agent also comprises a 5’-PS 2 and a targeting ligand.
  • the 5’-PS 2 is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8 T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q 4 is 0, B3’ is 2’-OMe or 2’-F
  • q 5 7, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucle
  • the RNAi agent also comprises a 5’-deoxy-5’-C-malonyl and a targeting ligand.
  • the 5’-deoxy-5’-C-malonyl is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 4 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at
  • the RNAi agent also comprises a 5’-P and a targeting ligand.
  • the 5’-P is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 4 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at
  • the RNAi agent also comprises a 5 -PS and a targeting ligand.
  • the 5 -PS is at the 5 -end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 4 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at
  • the RNAi agent also comprises a 5’-VP (e.g., a 5’-E-VP, 5’-Z-VP, or combination thereof) and a targeting ligand.
  • a 5’-VP e.g., a 5’-E-VP, 5’-Z-VP, or combination thereof
  • the 5’-VP is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 4 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at
  • the RNAi agent also comprises a 5’-PS 2 and a targeting ligand.
  • the 5’-PS 2 is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8
  • T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 4 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at
  • the RNAi agent also comprises a 5’-deoxy-5’-C-malonyl and a targeting ligand.
  • the 5’-deoxy-5’-C-malonyl is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8 T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q 4 is 0, B3’ is 2’-OMe or 2’-F
  • q 5 7, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothio
  • the RNAi agent also comprises a 5’-P and a targeting ligand.
  • the 5’-P is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8 T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q 4 is 0, B3’ is 2’-OMe or 2’-F
  • q 5 7, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothio
  • the RNAi agent also comprises a 5’- PS and a targeting ligand.
  • the 5’-PS is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8 T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q 4 is 0, B3’ is 2’-OMe or 2’-F
  • q 5 7, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothio
  • the RNAi agent also comprises a 5’- VP (e.g., a 5’-E-VP, 5’-Z-VP, or combination thereof) and a targeting ligand.
  • a 5’-VP e.g., a 5’-E-VP, 5’-Z-VP, or combination thereof
  • the 5’-VP is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8 T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q 4 is 0, B3’ is 2’-OMe or 2’-F
  • q 5 7, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothio
  • the RNAi agent also comprises a 5’- PS 2 and a targeting ligand.
  • the 5’-PS 2 is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • B1 is 2’-OMe or 2’-F
  • n 1 8 T1 is 2’F
  • n 2 3
  • B2 is 2’-OMe
  • n 3 7, n 4 is 0,
  • B3 is 2’-OMe
  • n 5 3
  • B1’ is 2’-OMe or 2’-F
  • q 1 9
  • T1’ is 2’-F
  • q 2 1, B2’ is 2’-OMe or 2’-F
  • q 3 4, q 4 is 0, B3’ is 2’-OMe or 2’-F
  • q 5 7, T3’ is 2’-F
  • q 7 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothio
  • the RNAi agent also comprises a 5’-deoxy-5’-C-malonyl and a targeting ligand.
  • the 5’-deoxy-5’-C-malonyl is at the 5’-end of the antisense strand
  • the targeting ligand is at the 3’-end of the sense strand.
  • an RNAi agent of the present invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; and (iii) 2’-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19, and 21, and 2’-OMe modifications at positions 2, 4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21, and 23, and 2’F modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (counting from the 5’ end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 21 and 22, and between nu
  • an RNAi agent of the present invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19, and 21, and 2’-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5’ end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2’F modifications at positions 2, 4, 6, 8, 10,
  • an RNAi agent of the present invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, 2’-F modifications at positions 7, and 9, and a desoxy-nucleotide (e.g.
  • RNAi agents have a two-nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at
  • an RNAi agent of the present invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2 -OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2 -F modifications at positions 7, 9, 11, 13, and 15; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21 to 23, and 2’-F modifications at positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5’ end
  • an RNAi agent of the present invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 9, and 12 to 21, and 2’-F modifications at positions 10, and 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2’-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5’ end); and (iii)
  • a RNAi agent of the present invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-F modifications at positions 1, 3, 5, 7, 9 to 11, and 13, and 2’-OMe modifications at positions 2, 4, 6, 8, 12, and 14 to 21; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-OMe modifications at positions 1, 3, 5 to 7, 9, 11 to 13, 15, 17 to 19, and 21 to 23, and 2’-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5
  • RNAi agents have a four-nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand.
  • a RNAi agent of the present invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2’-F modifications at positions 7, and 9 to 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to 13, 15, and 17 to 23, and 2’-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5’ end); and (iii)
  • a RNAi agent of the present invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2’-F modifications at positions 7, and 9 to 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 23, and 2’- F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5’ end); and (iii)
  • the iRNA for use in the methods of the invention is an agent selected from agents listed in Tables 3, 4, 5, 6, 7, 8A, or 8B. These agents may further comprise a ligand.
  • IV. iRNAs Conjugated to Ligands Another modification of the RNA of an iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., (1989) Proc. Natl. Acid. Sci.
  • Acids Res., 20:533-538 an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl.
  • a phospholipid e.g., di-hexadecyl-rac-glycerol
  • Acids Res., 18:3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta,1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp.
  • a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated.
  • a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.
  • Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.
  • Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or a lipid.
  • the ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid.
  • polyamino acids examples include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L- glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine.
  • PLL polylysine
  • poly L-aspartic acid poly L- glutamic acid
  • styrene-maleic acid anhydride copolymer poly(L-lactide-co-glycolied) copolymer
  • divinyl ether-maleic anhydride copolymer divinyl ether-
  • polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
  • Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
  • a targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.
  • ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g.
  • intercalating agents e.g. acridines
  • cross-linkers e.g. psoralene, mitomycin C
  • porphyrins TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
  • artificial endonucleases e.g.
  • EDTA lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino, alkyl,
  • Biotin can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell.
  • Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose.
  • the ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF- ⁇ B.
  • the ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell’s cytoskeleton, e.g., by disrupting the cell’s microtubules, microfilaments, and/or intermediate filaments.
  • the drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
  • a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator).
  • PK modulator pharmacokinetic modulator
  • PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc.
  • exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.
  • Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands).
  • ligands e.g. as PK modulating ligands
  • aptamers that bind serum components are also suitable for use as PK modulating ligands in the embodiments described herein.
  • Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below).
  • This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.
  • the oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis.
  • the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.
  • the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.
  • the ligand or conjugate is a lipid or lipid-based molecule.
  • a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA).
  • HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body.
  • the target tissue can be the liver, including parenchymal cells of the liver.
  • Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used.
  • a lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
  • a lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
  • the lipid based ligand binds HSA.
  • it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue.
  • the affinity not be so strong that the HSA-ligand binding cannot be reversed.
  • the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney.
  • Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
  • the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell.
  • a target cell e.g., a proliferating cell.
  • vitamins include vitamin A, E, and K.
  • Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).
  • B low density lipoprotein
  • the ligand is a cell-permeation agent, preferably a helical cell-permeation agent.
  • the agent is amphipathic.
  • An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo- peptide linkages, and use of D-amino acids.
  • the helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
  • the ligand can be a peptide or peptidomimetic.
  • a peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide.
  • the attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption.
  • the peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • a peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe).
  • the peptide moiety can be a dendrimer peptide, constrained peptide or cross-linked peptide.
  • the peptide moiety can include a hydrophobic membrane translocation sequence (MTS).
  • An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 5).
  • An RFGF analogue e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 6) containing a hydrophobic MTS can also be a targeting moiety.
  • the peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes.
  • sequences from the HIV Tat protein GRKKRRQRRRPPQ (SEQ ID NO: 7) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 8) have been found to be capable of functioning as delivery peptides.
  • a peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991).
  • OBOC one-bead-one-compound
  • Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic.
  • a peptide moiety can range in length from about 5 amino acids to about 40 amino acids.
  • the peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.
  • An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glyciosylated or methylated, to facilitate targeting to a specific tissue(s).
  • RGD- containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics.
  • a “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell.
  • a microbial cell-permeating peptide can be, for example, a ⁇ -helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., ⁇ - defensin, ⁇ -defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR- 39 or indolicidin).
  • a cell permeation peptide can also include a nuclear localization signal (NLS).
  • a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).
  • MPG nuclear localization signal
  • C. Carbohydrate Conjugates In some embodiments of the compositions and methods of the invention, an iRNA oligonucleotide further comprises a carbohydrate.
  • carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein.
  • “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom.
  • Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums.
  • Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).
  • a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:
  • a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide.
  • the monosaccharide is an N-acetylgalactosamine, such as Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,
  • the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker.
  • the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent, e.g., the 3’ or 5’end of the sense strand of a dsRNA agent as described herein.
  • the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) of GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.
  • each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.
  • the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.
  • Additional carbohydrate conjugates (and linkers) suitable for use in the present invention include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.
  • D. Linkers In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.
  • linker or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound.
  • Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO 2 , SO 2 NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl,
  • the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8- 16 atoms.
  • a cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together.
  • the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
  • Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules.
  • cleavage agents are more prevalent or found at higher 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 an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
  • 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
  • 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 5.0.
  • Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.
  • a linker can include a cleavable linking group that is cleavable by a particular enzyme.
  • cleavable linking group incorporated into a linker can depend on the cell to be targeted.
  • a liver- targeting ligand can be linked to a cationic lipid through a linker that includes an ester group.
  • Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich.
  • Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.
  • Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.
  • 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 when in contact with other non-target tissue.
  • a degradative agent or condition
  • the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue.
  • the evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals.
  • useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 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).
  • a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation.
  • An example of 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 targeting agent
  • 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. In one, candidate compounds are cleaved by at most about 10% in the blood.
  • useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 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.
  • a cleavable linker comprises a phosphate-based cleavable linking group.
  • a phosphate-based cleavable linking group is 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-, -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-, -O-P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, - O-P(S)(Rk)-O-, -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-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(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.
  • a cleavable linker comprises an acid cleavable linking group.
  • An acid cleavable linking group is a linking group that is cleaved under acidic conditions.
  • acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 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, esters, and esters of amino acids.
  • 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.
  • a cleavable linker comprises an ester-based cleavable linking group.
  • An ester-based cleavable linking group is 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.
  • v. Peptide-based cleaving groups In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group.
  • a peptide-based cleavable linking group is 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 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- based cleavable linking groups have the general formula – NHCHRAC(O)NHCHRBC(O)- , where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.
  • an iRNA of the invention is conjugated to a carbohydrate through a linker.
  • Non- limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,
  • a ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.
  • a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of Formula XLIV – XLVII: wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH 2 , CH 2 NH or CH 2 O; Q 2A , q
  • Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula XLIII: wherein L 5A , L 5B and L 5C represent a monosaccharide, such as GalNAc derivative.
  • suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.
  • RNA conjugates include, but are not limited to, U.S. Pat. Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,26
  • iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression.
  • RNA of an iRNA can be modified by a non-ligand group.
  • a number of non- ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature.
  • Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S- tritylthiol (Manoharan et al., Ann. N.Y. Acad.
  • lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manohara
  • Acids Res., 1990, 18:3777 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).
  • RNA conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate. V.
  • an iRNA of the invention to a cell 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 bleeding disorder) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, 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 iRNA. These alternatives are discussed further below.
  • any method of delivering a nucleic acid molecule can be adapted for use with an iRNA of the invention (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).
  • factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue.
  • the non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation.
  • VEGF dsRNA intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ. et al., (2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ. et al. (2003) Mol.
  • RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo- nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation.
  • an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of an iRNA 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).
  • the iRNA 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 an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell.
  • Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, 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 iRNA.
  • vesicles or micelles further prevents degradation of the iRNA when administered systemically.
  • Methods for making and administering cationic- iRNA 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).
  • DOTAP Disposon-based lipid particle
  • Oligofectamine "solid nucleic acid lipid particles”
  • cardiolipin Choen, 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.
  • Patent No.7, 427, 605 which is herein incorporated by reference in its entirety.
  • A. Vector encoded iRNAs of the Invention iRNA targeting the PLG 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; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type.
  • iRNA expressing vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA 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.
  • 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.
  • pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g.
  • the constructs can include viral sequences for transfection, if desired.
  • the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors.
  • Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are known in the art. VI.
  • compositions of the invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention.
  • a pharmaceutical composition comprising a double stranded ribonucleic acid (dsRNA) agent that inhibits expression of plasminogen (PLG) in a cell, such as a liver cell, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1 or 3, and said antisense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO: 2 or 4; and a pharmaceutically acceptable carrier.
  • dsRNA double stranded ribonucleic acid
  • PLG plasminogen
  • compositions comprising a dsRNA agent that inhibits expression of PLG in a cell, such as a liver cell, wherein the dsRNA agent comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from any one of the antisense sequences listed in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B; and a pharmaceutically acceptable carrier.
  • the dsRNA agent comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides from any one of the antisense sequences listed in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B.
  • the pharmaceutical compositions containing the iRNA of the invention are useful for treating a disease or disorder associated with the expression or activity of a PLG gene, e.g., a bleeding disorder.
  • 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 delivery.
  • compositions that are formulated for direct delivery into the liver, e.g., by infusion into the liver, such as by continuous pump infusion.
  • the pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a PLG gene.
  • a suitable dose of an iRNA of the invention 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 suitable dose of an iRNA of the invention will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg.
  • a repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as every other day to once a year.
  • the iRNA is administered about once per week, once every 7-10 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once per month, once every 2 months, once every 3 months (once per quarter), once every 4 months, once every 5 months, or once every 6 months.
  • the treatments can be administered on a less frequent basis.
  • treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments.
  • Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein. Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as a PLG-associated disease, disorder, or condition that would benefit from reduction in the expression of PLG.
  • Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose.
  • Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose.
  • Suitable mouse models are known in the art and include, for example, mouse thrombosis models, mouse stroke models, hemophilia A mouse models (FVIII deficient mice).
  • Non-limiting examples of such models for in vivo testing of iRNA include Strilchuk et al., Int. Society on Throm. and Haemost. abstract 2021; Batty, et al., Int. Society on Throm. and Haemost. abstract 2021; and Singh et al., 2016. J. Thromb.
  • compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.
  • the iRNA can be delivered in a manner to target a particular cell or tissue, such as the liver (e.g., the hepatocytes of the liver).
  • the pharmaceutical compositions of the invention are suitable for intramuscular administration to a subject.
  • the pharmaceutical compositions of the invention are suitable for intravenous administration to a subject.
  • the pharmaceutical compositions of the invention are suitable for subcutaneous administration to a subject, e.g., using a 29g or 30g needle.
  • the pharmaceutical compositions of the invention may include an RNAi agent of the invention in an unbuffered solution, such as saline or water, or in a buffer solution, such as a buffer solution comprising acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.
  • the pharmaceutical compositions of the invention e.g., such as the compositions suitable for subcutaneous administration, comprise an RNAi agent of the invention in phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Suitable concentrations of PBS include, for example, 1mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 6.5 mM, 7 mM, 7.5.mM, 9 mM, 8.5 mM, 9 mM, 9.5 mM, or about 10 mM PBS.
  • a pharmaceutical composition of the invention comprises an RNAi agent of the invention dissolved in a solution of about 5 mM PBS (e.g., 0.64 mM NaH 2 PO 4 , 4.36 mM Na 2 HPO 4 , 85 mM NaCl).
  • the pH of the pharmaceutical compositions of the invention may be between about 5.0 to about 8.0, about 5.5 to about 8.0, about 6.0 to about 8.0, about 6.5 to about 8.0, about 7.0 to about 8.0, about 5.0 to about 7.5, about 5.5 to about 7.5, about 6.0 to about 7.5, about 6.5 to about 7.5, about 5.0 to about 7.2, about 5.25 to about 7.2, about 5.5 to about 7.2, about 5.75 to about 7.2, about 6.0 to about 7.2, about 6.5 to about 7.2, or about 6.8 to about 7.2.
  • the osmolality of the pharmaceutical compositions of the invention may be suitable for subcutaneous administration, such as no more than about 400 mOsm/kg, e.g., between 50 and 400 mOsm/kg, between 75 and 400 mOsm/kg, between 100 and 400 mOsm/kg, between 125 and 400 mOsm/kg, between 150 and 400 mOsm/kg, between 175 and 400 mOsm/kg, between 200 and 400 mOsm/kg, between 250 and 400 mOsm/kg, between 300 and 400 mOsm/kg, between 50 and 375 mOsm/kg, between 75 and 375 mOsm/kg, between 100 and 375 mOsm/kg, between 125 and 375 mOsm/kg, between 150 and 375 mOsm/kg, between 175 and 375 mOsm
  • compositions of the invention comprising the RNAi agents of the invention, may be present in a vial that contains about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or about 2.0 mL of the pharmaceutical composition.
  • the concentration of the RNAi agents in the pharmaceutical compositions of the invention may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 130, 125, 130, 135, 140, 145, 150, 175, 180, 185, 190, 195, 200, 205, 210, 215, 230, 225, 230, 235, 240, 245, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 330, 325, 330, 335, 340, 345, 350, 375, 380, 385, 390, 395, 400, 405, 410, 415, 430, 425, 430, 435, 440, 445, 450, 475, 480, 485, 490, 495, or about 500 mg/mL.
  • the concentration of the RNAi agents in the pharmaceutical compositions of the invention is about 100 mg/mL. Values intermediate to the above recited ranges and values are also intended to be part of this invention.
  • the pharmaceutical compositions of the invention may comprise a dsRNA agent of the invention in a free acid form. In other embodiments of the invention, the pharmaceutical compositions of the invention may comprise a dsRNA agent of the invention in a salt form, such as a sodium salt form. In certain embodiments, when the dsRNA agents of the invention are in the sodium salt form, sodium ions are present in the agent as counterions for substantially all of the phosphodiester and/or phosphorothiotate 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 and/or phosphorothioate linkages without a sodium counterion.
  • sodium ions are present in the agent as counterions for all of the phosphodiester and/or phosphorothiotate groups present in the agent.
  • Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).
  • neutral e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline
  • negative e.g., dimyristoylphosphatidyl glycerol DMPG
  • cationic e.g., dioleoyltetramethylaminopropyl DOTAP and
  • iRNA Formulations Comprising Membranous Molecular Assemblies
  • An iRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle.
  • a membranous molecular assembly e.g., a liposome or a micelle.
  • surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery.
  • Non-cationic liposomes although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
  • lipid vesicles In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores. Liposomes are useful for the transfer and delivery of active ingredients to the site of action.
  • a liposome containing an iRNA agent can be prepared by a variety of methods.
  • the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component.
  • the lipid component can be an amphipathic cationic lipid or lipid conjugate.
  • the detergent can have a high critical micelle concentration and may be nonionic.
  • pH can also be adjusted to favor condensation.
  • Methods for producing stable polynucleotide delivery vehicles which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference.
  • Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; US Patent No.4,897,355; US Patent No. 5,171,678; Bangham, et al. M. Mol. Biol.23:238, 1965; Olson, et al. Biochim.
  • Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging iRNA agent preparations into liposomes.
  • Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res.
  • Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH- sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
  • One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine.
  • Neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).
  • Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.
  • PC phosphatidylcholine
  • Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol.
  • Non-ionic liposomal formulations comprising Novasome TM I (glyceryl dilaurate/cholesterol/polyoxyethylene-10- stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).
  • liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated iRNAs in their internal compartments from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p.245).
  • Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.
  • cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (TransfectamTM, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5- carboxyspermyl-amide (“DPPES”) (see, e.g., US Patent No.5,171,678).
  • DOGS 5-carboxyspermylglycine dioctaoleoylamide
  • DPES dipalmitoylphosphatidylethanolamine 5- carboxyspermyl-amide
  • Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC- Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X.
  • cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.
  • Liposomal formulations are particularly suited for topical administration; liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA agent into the skin.
  • liposomes are used for delivering iRNA agent to epidermal cells and also to enhance the penetration of iRNA agent into dermal tissues, e.g., into skin.
  • the liposomes can be applied topically.
  • Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol.2,405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276.1987; Nicolau, C. et al. Meth. Enz.149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz.101:512-527, 1983; Wang, C.
  • Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol.
  • Non-ionic liposomal formulations comprising Novasome TM I (glyceryl dilaurate/cholesterol/polyoxyethylene-10- stearyl ether) and Novasome TM II (glyceryl distearate/ cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin.
  • Such formulations with iRNA agent are useful for treating a dermatological disorder.
  • Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome.
  • transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNAs can be delivered, for example, subcutaneously by infection in order to deliver iRNAs to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient.
  • transferosomes due to the lipid properties, these transferosomes can be self- optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self- loading.
  • Other formulations amenable to the present invention are described in WO 2008/042973.
  • Transfersomes are yet another type of liposomes and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet.
  • Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading.
  • surface edge-activators usually surfactants
  • Transfersomes have been used to deliver serum albumin to the skin.
  • the transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
  • Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes.
  • Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters.
  • Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class.
  • the polyoxyethylene surfactants are the most popular members of the nonionic surfactant class. If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic.
  • Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class. If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides. The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p.285).
  • the micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate.
  • Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.
  • a first micellar composition is prepared which contains the RNAi and at least the alkali metal alkyl sulphate.
  • the first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition.
  • the micellar composition is prepared by mixing the RNAi, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.
  • Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether.
  • HFA 134a (1,1,1,2 tetrafluoroethane) may be used.
  • concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.
  • the term SPLP refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle.
  • LNPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683.
  • the particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic.
  • the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.
  • the cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I -(2,3- dioleoyloxy)propyl)- N,N,N-trimethylammonium chloride (DOTAP), N-(I -(2,3- dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3- dioleyloxy)propylamine (DODMA), 1,2- DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylamino
  • the cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.
  • the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- dioxolane is described in United States provisional patent application number 61/107,998 filed on October 23, 2008, which is herein incorporated by reference.
  • the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4- dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0 ⁇ 20 nm and a 0.027 siRNA/Lipid Ratio.
  • the non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoy
  • the non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
  • the conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG- dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof.
  • the PEG- DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci 2 ), a PEG-dimyristyloxypropyl (Ci 4 ), a PEG-dipalmityloxypropyl (Ci 6 ), or a PEG- distearyloxypropyl (C] 8 ).
  • the conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.
  • the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.
  • LNP01 the lipidoid ND98 ⁇ 4HCl (MW 1487) (see U.S. Patent Application No. 12/056,230, filed 3/26/2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (e.g., LNP01 particles).
  • Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml.
  • the ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio.
  • the combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM.
  • Lipid-dsRNA nanoparticles typically form spontaneously upon mixing.
  • the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc).
  • a thermobarrel extruder such as Lipex Extruder (Northern Lipids, Inc).
  • the extrusion step can be omitted.
  • Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration.
  • Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
  • PBS phosphate buffered saline
  • LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference. Additional exemplary lipid-dsRNA formulations are provided in the following Table 1.
  • Table 1 Exemplary lipid formulations DSPC: distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine
  • PEG-DMG PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)
  • PEG-DSG PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)
  • PEG-cDMA PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)
  • SNALP l,
  • WO2009/127060 filed April 15, 2009, which is hereby incorporated by reference.
  • XTC comprising formulations are described, e.g., in U.S. Provisional Serial No.61/148,366, filed January 29, 2009; U.S. Provisional Serial No.61/156,851, filed March 2, 2009; U.S. Provisional Serial No.61/185,712, filed June 10, 2009; U.S. Provisional Serial No.61/228,373, filed July 24, 2009; U.S. Provisional Serial No.61/239,686, filed September 3, 2009, and International Application No. PCT/US2010/022614, filed January 29, 2010, which are hereby incorporated by reference.
  • MC3 comprising formulations are described, e.g., in U.S.
  • ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on November 10, 2009, which is hereby incorporated by reference.
  • C12-200 comprising formulations are described in U.S. Provisional Serial No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.
  • Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No.5,213,804 and European Patent No. EP 0496 813 B1).
  • Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No.5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.).
  • Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other.
  • emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety.
  • w/o water-in-oil
  • o/w oil-in-water
  • Emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion.
  • a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
  • Emulsions are characterized by little or no thermodynamic stability.
  • the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation.
  • Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams.
  • Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion.
  • Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).
  • Synthetic surfactants also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p.199).
  • polar inorganic solids such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
  • non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions.
  • Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
  • polysaccharides for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth
  • cellulose derivatives for example, carboxymethylcellulose and carboxypropylcellulose
  • synthetic polymers for example, carbomers, cellulose ethers, and
  • emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives.
  • preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.
  • Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.
  • microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte.
  • microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
  • Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants.
  • Surfactants are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced.
  • these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene- 9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M.
  • bile salts includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.
  • Chelating agents can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced.
  • chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339).
  • Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A.
  • EDTA disodium ethylenediaminetetraacetate
  • citric acid e.g., citric acid
  • salicylates e.g., sodium salicylate, 5-methoxysalicylate and homovanilate
  • N-acyl derivatives of collagen e.g., laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A.
  • non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33).
  • This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621- 626).
  • Agents that enhance uptake of iRNAs at the cellular level can also be added to the pharmaceutical and other compositions of the present invention.
  • cationic lipids such as lipofectin (Junichi et al, U.S.
  • transfection reagents examples include, for example LipofectamineTM (Invitrogen; Carlsbad, CA), Lipofectamine 2000TM (Invitrogen; Carlsbad, CA), 293fectinTM (Invitrogen; Carlsbad, CA), CellfectinTM (Invitrogen; Carlsbad, CA), DMRIE-CTM (Invitrogen; Carlsbad, CA), FreeStyleTM MAX (Invitrogen; Carlsbad, CA), LipofectamineTM 2000 CD (Invitrogen; Carlsbad, CA), LipofectamineTM (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA), OligofectamineTM (Invitrogen; Carlsbad, CA), OptifectTM (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOT
  • a nucleic acid and a carrier compound can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.
  • the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano- stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res.
  • compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations 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 and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/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 including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension can also contain stabilizers.
  • the therapeutically effective 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 IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma can be measured, for example, by high performance liquid chromatography.
  • the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by PLG expression.
  • the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
  • Synthesis of cationic lipids Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles featured in the invention may be prepared by known organic synthesis techniques. All substituents are as defined below unless indicated otherwise.
  • Alkyl means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.
  • Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
  • alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2- pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
  • Alkynyl means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons.
  • Heterocycle means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring.
  • the heterocycle may be attached via any heteroatom or carbon atom.
  • Heterocycles include heteroaryls as defined below.
  • Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
  • Halogen means fluoro, chloro, bromo and iodo.
  • the methods featured in the invention may require the use of protecting groups.
  • Protecting group methodology is well known to those skilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T.W. et al., Wiley-Interscience, New York City, 1999).
  • protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group.
  • a protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group.
  • an “alcohol protecting group” is used.
  • the cationic lipid is XTC (2,2-Dilinoleyl-4- dimethylaminoethyl-[1,3]-dioxolane).
  • the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.
  • Lipid A where R 1 and R 2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R 3 and R 4 are independently lower alkyl or R 3 and R 4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1.
  • Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art.
  • ketal 3 Treatment of ketal 3 with amine 4 yields lipids of formula A.
  • the lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.
  • the ketone 1 starting material can be prepared according to Scheme 2.
  • Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.
  • Synthesis of ALNY-100 Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3: Synthesis of 515: To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1L), was added a solution of 514 (10g, 0.04926mol) in 70 mL of THF slowly at 00C under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 00C and quenched with careful addition of saturated Na2SO4 solution.
  • Reaction mixture was diluted with DCM (300 mL) and washed with water (2 x 100 mL) followed by saturated NaHCO3 (1 x 50 mL) solution, water (1 x 30 mL) and finally with brine (1x 50 mL).
  • Organic phase was dried over Na 2 SO 4 and solvent was removed in vacuum.
  • Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC.
  • formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay.
  • the present invention also provides methods of using an iRNA of the invention and/or a composition of the invention to reduce and/or inhibit PLG expression in a cell, such as a cell in a subject, e.g., a hepatocyte.
  • the methods include contacting the cell with an RNAi agent or pharmaceutical composition comprising an iRNA agent of the invention.
  • the cell is maintained for a time sufficient to obtain degradation of the mRNA transcript of a PLG gene. Reduction in gene expression can be assessed by any methods known in the art.
  • a reduction in the expression of PLG may be determined by determining the mRNA expression level of PLG using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR; by determining the protein level of PLG using methods routine to one of ordinary skill in the art, such as Western blotting, ELISA, or immunological techniques.
  • a reduction in the expression of PLG may also be assessed indirectly by measuring a decrease in biological activity of PLG, e.g., a decrease in the serine protease activity of PLG.
  • the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.
  • the cell is a human cell, e.g., a human liver cell.
  • PLG expression is inhibited in the cell by at least about 5, 6, 7, 8, 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, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100%.
  • the in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the PLG gene of the mammal to be treated.
  • the in vivo methods of the invention may include administering to a subject a composition containing a first iRNA agent and a second iRNA agent, where the first iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the PLG gene of the mammal to be treated and the second iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of a second gene of the mammal to be treated.
  • the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration.
  • intracranial e.g., intraventricular, intraparenchymal and intrathecal
  • intravenous intramuscular
  • subcutaneous e.g., transdermal
  • airway aerosol
  • nasal rectal
  • topical including buccal and sublingual
  • the compositions are administered by intravenous infusion or injection.
  • the compositions are administered by subcutaneous injection.
  • the administration is via a depot injection.
  • a depot injection may release the iRNA in a consistent way over a prolonged time period.
  • a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of PLG, 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.
  • the depot injection is a subcutaneous injection.
  • the administration is via a pump.
  • the pump may be an external pump or a surgically implanted pump.
  • the pump is a subcutaneously implanted osmotic pump.
  • the pump is an infusion pump.
  • An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions.
  • the infusion pump is a subcutaneous infusion pump.
  • the pump is a surgically implanted pump that delivers the iRNA to the liver.
  • An iRNA of the invention may be present in a pharmaceutical composition, such as in a suitable buffer solution.
  • the buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.
  • the buffer solution is phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.
  • an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.
  • the mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated.
  • the route and site of administration may be chosen to enhance targeting.
  • the present invention also provides methods for inhibiting the expression of a PLG gene in a mammal.
  • the methods include administering to the mammal a composition comprising a dsRNA that targets a PLG gene in a cell of the mammal, thereby inhibiting expression of the PLG gene in the cell.
  • the methods include administering to the mammal a composition comprising a dsRNA that targets a PLG gene in a cell of the mammal, thereby inhibiting expression of the PLG gene in the cell.
  • the methods include administering to the mammal a pharmaceutical composition comprising a dsRNA agent that targets a PLG gene in a cell of the mammal.
  • a pharmaceutical composition comprising a dsRNA agent that targets a PLG gene in a cell of the mammal.
  • the present invention provides use of an iRNA agent or a pharmaceutical composition of the invention for inhibiting the expression of a PLG gene in a mammal.
  • the present invention provides use of an iRNA agent of the invention targeting a PLG gene or a pharmaceutical composition comprising such an agent in the manufacture of a medicament for inhibiting expression of a PLG gene in a mammal.
  • the present invention also provides use of a therapeutically effective amount of an iRNA agent of the invention or a pharmaceutical composition comprising a dsRNA that inhibits expression of PLG for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of PLG expression, e.g., a PLG- associated disease, e.g., a bleeding disorder.
  • a subject e.g., a subject that would benefit from a reduction and/or inhibition of PLG expression, e.g., a PLG- associated disease, e.g., a bleeding disorder.
  • the present invention provides use of an iRNA agent, e.g., a dsRNA, of the invention targeting a PLG gene or a pharmaceutical composition comprising an iRNA agent targeting a PLG gene in the manufacture of a medicament for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of PLG for expression, e.g., a PLG-associated disease.
  • the present invention also provides use of a prophylactically effective amount of an iRNA agent of the invention or a pharmaceutical composition comprising a dsRNA that inhibits expression of PLG for preventing at least one symptom in a subject having a disorder that would benefit from reduction in PLG expression, e.g., a bleeding disorder.
  • the present invention provides use of an iRNA agent, e.g., a dsRNA, of the invention targeting a PLG gene or a pharmaceutical composition comprising an iRNA agent targeting a PLG gene in the manufacture of a medicament for preventing at least one symptom in a subject having a disorder that would benefit from reduction in PLG expression, e.g., a bleeding disorder.
  • an iRNA agent e.g., a dsRNA
  • the present invention provides methods of treating a subject having a disorder that would benefit from reduction in PLG expression, e.g., a PLG-associated disease, such as a bleeding disorder (e.g., heavy menstrual bleeding).
  • the bleeding disorder is hereditary hemorrhagic telangiectasia (HHT), heavy menstrual bleeding (HMB), PAI-1 deficiency, von Willebrand disease, low factor XI levels, platelet defects, hemophilia A, hemophilia B, afibrinogenemia, parahemophilia, low factor VIII levels, low factor IX levels, low factor VII levels, low factor XIII levels, low factor X levels, low factor V levels, low factor II levels, nose bleeds, bleeding gums, easy bruising, postpartum bleeding, or excessive bleeding following surgery.
  • HHT hereditary hemorrhagic telangiectasia
  • HMB heavy menstrual bleeding
  • the first and second dsRNA agents may be formulated in the same composition or different compositions and may be administered to the subject in the same composition or in separate compositions.
  • an “iRNA” for use in the methods of the invention is a “dual targeting RNAi agent.”
  • the term “dual targeting RNAi agent” refers to a molecule comprising a first dsRNA agent comprising 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 first target RNA, i.e., a PLG gene, covalently attached to a molecule comprising a second dsRNA agent comprising 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 second target RNA.
  • a dual targeting RNAi agent triggers the degradation of the first and the second target RNAs, e.g., mRNAs, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.
  • the dsRNA agent may be administered to the subject at a dose of about 0.1 mg/kg to about 50 mg/kg. Typically, a suitable dose will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg.
  • the iRNA can be administered by intravenous infusion 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.
  • the iRNA is administered about once per week, once every 7-10 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once per month, once every 2 months, once every 3 months, once per quarter), once every 4 months, once every 5 months, or once every 6 months.
  • the method includes administering a composition featured herein such that expression of the target PLG gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours.
  • a bleeding 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 bleeding disorders and the related causes.
  • 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.
  • a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment.
  • the invention provides methods for the use of a iRNA agent or a pharmaceutical composition of the invention and an iRNA agent targeting a second target, e.g., for treating a subject that would benefit from reduction and/or inhibition of PLG expression and expression of a second target, e.g., a subject having a PLG-associated disease disorder, or condition (e.g., bleeding disorder), in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders.
  • a second target e.g., a subject having a PLG-associated disease disorder, or condition (e.g., bleeding disorder)
  • other pharmaceuticals and/or other therapeutic methods e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders.
  • an iRNA agent or pharmaceutical composition of the invention is administered in combination with, e.g., tranexamic acid (TXA), anti-angiogenic drugs, such as drugs that target the VEGF pathway, oral contraceptive pills (OCP), intrauterine devices (IUDs), such as Mirena IUD and Kyleena IUD, other progestin approaches, endometrial ablation, total or subtotal hysterectomy, myomectomy, uterine artery embolization, human plasma-donor derived Glu-plasminogen, inhibitors of the plasminogen pathway, or a combination of any of the foregoing.
  • TXA tranexamic acid
  • anti-angiogenic drugs such as drugs that target the VEGF pathway
  • OCP oral contraceptive pills
  • IUDs intrauterine devices
  • Mirena IUD and Kyleena IUD other progestin approaches, endometrial ablation, total or subtotal hysterectomy, myomectomy, uterine
  • kits for performing any of the methods of the invention include one or more RNAi agent(s) and instructions for use, e.g., instructions for inhibiting expression of a PLG in a cell by contacting the cell with an RNAi agent or pharmaceutical composition of the invention in an amount effective to inhibit expression of the PLG.
  • kits may optionally further comprise means for contacting the cell with the RNAi agent (e.g., an injection device), or means for measuring the inhibition of PLG (e.g., means for measuring the inhibition of PLG mRNA and/or PLG protein).
  • Such means for measuring the inhibition of PLG may comprise a means for obtaining a sample from a subject, such as, e.g., a blood sample.
  • the kits of the invention may optionally further comprise means for administering the RNAi agent(s) to a subject or means for determining the therapeutically effective or prophylactically effective amount.
  • L96 The chemical structure of L96 is as follows: 2The chemical structure of uL96 is as follows: Experimental Methods This Example describes methods for the design, synthesis, and selection of PLG iRNA agents.
  • Transcripts A set of siRNAs targeting the human plasminogen gene (PLG; human NCBI refseqID NM_000301.5; NCBI GeneID: 5340; and human NCBI refseqID NM_001168338.1) were designed using custom R and Python scripts.
  • PLG Multi-Dose Screens in Primary Human Hepatocytes PLG Multi-Dose Screens in Primary Cyno Hepatocytes (PCH)
  • PBX mice were administered an exemplary siRNA duplex, AD-2315878, at either 1 mg/kg or 3 mg/kg subcutaneous in a single dose at day 0. Blood was obtained and plasma hPLG levels were determined at days 0, 7, and 14 as described above. The results in FIG.3 are presented as percent plasma PLG remaining relative to the baseline (pre-dose) control. PBX mice were administered exemplary siRNA duplexes AD-2137018 or AD-2136718 at 0.3 mg/kg, 1 mg/kg, or 3 mg/kg subcutaneous in a single dose at day 0. Blood was obtained and plasma hPLG levels were determined at days 0, 7, 14, 21, 28, 35, 42, 49, 57, and 63 as described above. The percent plasma PLB remaining was determined. The results for AD-2137018 are presented in FIG.4 and the results for AD-2136718 are presented in FIG.5. PLG Sequences

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