WO2023081823A1 - Lipid conjugation for targeting astrocytes of the central nervous system - Google Patents
Lipid conjugation for targeting astrocytes of the central nervous system Download PDFInfo
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- WO2023081823A1 WO2023081823A1 PCT/US2022/079302 US2022079302W WO2023081823A1 WO 2023081823 A1 WO2023081823 A1 WO 2023081823A1 US 2022079302 W US2022079302 W US 2022079302W WO 2023081823 A1 WO2023081823 A1 WO 2023081823A1
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- oligonucleotide
- sense strand
- conjugated
- lipid
- nucleotide
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N2310/31—Chemical structure of the backbone
- C12N2310/312—Phosphonates
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- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
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- C12N2310/35—Nature of the modification
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- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/32—Special delivery means, e.g. tissue-specific
Definitions
- the disclosure relates to oligonucleotides linked to lipid moieties useful in the inhibition of target genes in astrocytes of the central nervous system.
- the present disclosure relates to oligonucleotide-lipid conjugates, methods to prepare them, their chemical configuration and methods to modulate (e.g., inhibit or reduce) the expression of a target gene in an astrocyte of the central nervous system (abbreviated “CNS” hereinafter) (e.g., tissue, or region of the CNS) using the conjugated nucleic acids and oligonucleotides according to the description provided herein.
- CNS central nervous system
- the disclosure also provides pharmaceutically acceptable compositions comprising the conjugates of the present description and methods of using said compositions in the treatment of various diseases or disorders.
- oligonucleotide or nucleic acid-based therapeutics have been under the clinical investigation, including antisense oligonucleotides (ASO), short interfering RNA (siRNA), double-stranded nucleic acids (dsNA), aptamers, ribozymes, exon-skipping and splice-altering oligonucleotides, immunomodulatory oligonucleotides, mRNAs, and CRISPR.
- ASO antisense oligonucleotides
- siRNA short interfering RNA
- dsNA double-stranded nucleic acids
- aptamers aptamers
- ribozymes ribozymes
- exon-skipping and splice-altering oligonucleotides immunomodulatory oligonucleotides
- mRNAs mRNAs
- CRISPR CRISPR
- RNAi oligonucleotide-based therapeutics comprising siRNAs or double-stranded nucleic acids (dsNAs) offer the potential for considerable expansion of the druggable target space and the possibility for treating orphan diseases that may be therapeutically unapproachable by other drug modalities (e.g., antibodies and/or small molecules).
- RNAi oligonucleotide-based therapeutics that inhibit or reduce expression of specific target genes in the liver have been developed and are currently in clinical use (Sehgal et al., (2013) JOURNAL OF HEPATOLOGY 59: 1354-59).
- RNAi oligonucleotides in extrahepatic cells, tissues, and organs (e.g., the CNS).
- Therapeutic gene silencing mediated by RNAi oligonucleotide-based therapeutics in the CNS is of particular interest to treat neurological diseases (Boudreau & Davidson (2010) BRAIN RESEARCH 1338: 112-21).
- the mammalian CNS is a complex system of tissues, including cells, fluids and chemicals that interact in concert to enable a wide variety of functions, including movement, navigation, cognition, speech, vision, and emotion.
- diseases and disorders of the CNS are known (e.g., neurological disorders) and affect or disrupt some or all of these functions.
- treatments for diseases and disorders of the CNS have been limited to small molecule drugs, antibodies and/or to adaptive or behavioral therapies. There exists an ongoing need to develop treatment of diseases and disorders of the CNS associated with inappropriate gene expression.
- the present disclosure is based, at least in part, on the discovery of lipid-conjugated RNAi oligonucleotides that effectively reduce target gene expression in astrocytes of the CNS.
- Exemplary lipid-conjugated RNAi oligonucleotides provided herein have demonstrated reduction of target gene expression of astrocyte-specific mRNA in the CNS following a single administration.
- exemplary lipid-conjugated RNAi oligonucleotides provided herein have demonstrated pharmacological activity in multiple regions throughout the CNS, including difficult to reach areas such as the hippocampus and frontal cortex.
- the hydrophobic moiety facilitates delivery and distribution of the lipid- conjugated RNAi oligonucleotides into the CNS, thereby increasing efficacy and durability of gene knockdown in astrocytes.
- the disclosure provides methods of treating a disease or disorder by modulating expression of an astrocyte gene in the CNS using the lipid- conjugated RNAi oligonucleotides, and pharmaceutically acceptable compositions thereof, described herein.
- the disclosure further provides methods of using the lipid-conjugated RNAi oligonucleotides in the manufacture of a medicament for treating a disease or disorder by modulating expression of an astrocyte gene in the CNS.
- the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 15-30 nucleotides in length and a sense strand of 15-50 nucleotides in length, wherein the antisense and sense strands form a duplex region of 15-30 base pairs, wherein the antisense strand comprises a region of complementarity to an astrocyte mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide of the sense strand.
- the lipid moiety is selected from
- the lipid moiety is a hydrocarbon chain. In some aspects, the hydrocarbon chain is a C8-C30 hydrocarbon chain. In some aspects, the hydrocarbon chain is a C16 hydrocarbon chain. In some aspects, the C16 hydrocarbon chain is represented by In any of the foregoing or related aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide.
- the oligonucleotide is blunt ended. In some aspects, the oligonucleotide is blunt ended at the 3’ terminus of the oligonucleotide. In some aspects, the oligonucleotide comprises a blunt end. In some aspects, the blunt end comprises the 3’ terminus of the sense strand. In some aspects, the sense strand is 20-22 nucleotides. In some aspects, the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 8, position 12, position 13, position 18, or position 20 of the sense strand, wherein positions are numbered 5’ to 3’.
- the astrocyte mRNA target is expressed in the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 8, position 12, position 13, position 18 or position 20 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the astrocyte mRNA target is expressed in the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 8, position 12, position 13, position 18 or position 20 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the astrocyte mRNA target is expressed in the cerebellum, wherein the at least one lipid moiety is conjugated to a nucleotide at position 4, position 12, position 13, position 18 or position 20 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the astrocyte mRNA target is expressed in the hypothalamus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 12, position 13, position 18 or position 20 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the astrocyte mRNA target is expressed in the frontal cortex, wherein the at least one lipid moiety is conjugated to a nucleotide at position 4 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the sense strand comprises a stem-loop at the 3 ’end, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2.
- the sense strand is 36-38 nucleotides.
- the astrocyte mRNA target is expressed in the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 8, position 12, position 13, position 18, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the astrocyte mRNA target is expressed in the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 18, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the astrocyte mRNA target is expressed in the cerebellum, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 23, position 28, or position 29 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the astrocyte mRNA target is expressed in the hypothalamus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 12, position 13, position 18, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the astrocyte mRNA target is expressed in the frontal cortex, wherein the at least one lipid moiety is conjugated to a nucleotide at position 23 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the antisense strand is 22-24 nucleotides. In some aspects, the duplex region is 20-22 base pairs.
- the antisense strand comprises a 1-4 nucleotide overhang at the 3’ terminus.
- the overhang comprises purine nucleotides.
- the overhang sequence is 2 nucleotides in length.
- the overhang is selected from AA, GG, AG, and GA.
- the overhang is GG or AA.
- the overhang is GG.
- the region of complementarity is complementary to at least 15 consecutive nucleotides of the astrocyte mRNA target sequence. In some aspects, the region of complementarity is complementary to 19 consecutive nucleotides of the astrocyte mRNA target sequence. In some aspects, the region of complementarity is fully complementary to the astrocyte mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the astrocyte mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the astrocyte mRNA target sequence. In some aspects, the region of complementarity comprises up to four mismatches to the astrocyte mRNA target sequence.
- the oligonucleotide comprises at least one modified nucleotide.
- the modified nucleotide comprises a 2'- modification.
- each of the nucleotides of the sense strand and the antisense strand comprise a 2'-modification except the nucleotide of the sense strand conjugated to the at least one lipid moiety.
- the 2 '-modification is a modification selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid.
- nucleotides of the sense strand comprise a 2'-fluoro modification.
- about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the nucleotides of the antisense strand comprise a 2'-fluoro modification.
- about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the nucleotides of the oligonucleotide comprise a 2'-fluoro modification.
- the sense strand comprises 20 nucleotides with positions 1-20 from 5' to 3', wherein each of positions 8-11 comprise a 2'-fluoro modification. In some aspects, the sense strand comprises 20 nucleotides with positions 1-20 from 5' to 3', wherein each of positions 9-11 comprise a 2'-fluoro modification. In some aspects, the sense strand comprises 36 nucleotides with positions 1-36 from 5' to 3', wherein each of positions 8-11 comprise a 2'-fluoro modification. In some aspects, the sense strand comprises 36 nucleotides with positions 1-36 from 5' to 3', wherein each of positions 9-11 comprise a 2'-fluoro modification.
- the antisense strand comprises 22 nucleotides with positions 1-22 from 5' to 3', and wherein each of positions 2, 3, 4, 5, 7, 10 and 14 comprise a 2'-fluoro modification.
- the remaining nucleotides comprise a 2'-O-methyl modification except the nucleotide of the sense strand conjugated to the at least one lipid moiety.
- the oligonucleotide comprises at least one modified internucleotide linkage.
- the at least one modified internucleotide linkage is a phosphorothioate linkage.
- the antisense strand comprises a phosphorothioate linkage (i) between positions 1 and 2, and between positions 2 and 3; or (ii) between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, wherein positions are numbered 1-4 from 5’ to 3’.
- the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a phosphorothioate linkage between positions 20 and 21 and between positions 21 and 22, wherein positions are numbered 1-22 from 5' to 3'.
- the sense strand comprises a phosphorothioate linkage between position 1 and 2, wherein positions are numbered 1-2 from 5' to 3'.
- the sense strand is 20 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1- 22 from 5' to 3'.
- the antisense strand comprises a phosphorylated nucleotide at the 5’ terminus, wherein the phosphorylated nucleotide is selected from uridine and adenosine.
- the phosphorylated nucleotide is uridine.
- the 4'-carbon of the sugar of the 5 '-nucleotide of the antisense strand comprises a phosphate analog.
- the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate.
- the region of complementary is fully complementary to the astrocyte mRNA target sequence at nucleotide positions 2-8 of the antisense strand, wherein nucleotide positions are numbered 5’ to 3’. In some aspects, the region of complementary is fully complementary to the astrocyte mRNA target sequence at nucleotide positions 2-11 of the antisense strand, wherein nucleotide positions are numbered 5’ to 3’.
- the oligonucleotide is a Dicer substrate.
- the oligonucleotide is a Dicer substrate that, upon endogenous Dicer processing, yields double-stranded nucleic acids of 19-21 nucleotides in length capable of reducing an astrocyte mRNA expression in a mammalian cell.
- the astrocyte mRNA target sequence is located in a region of the central nervous system (CNS).
- the region of the CNS is selected from the spinal cord, lumbar spinal cord, thoracic spinal cord, cervical spinal cord, medulla, hippocampus, cerebellum, hypothalamus, frontal cortex, and a combination thereof.
- the oligonucleotide reduces expression of a target mRNA in an astrocyte or population of astrocytes in vitro and/or in vivo.
- the disclosure provides a pharmaceutical composition
- a pharmaceutical composition comprising an oligonucleotide described herein, and a pharmaceutically acceptable carrier, delivery agent or excipient.
- the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of an astrocyte mRNA, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein, thereby treating the subject.
- the disclosure provides a method of delivering an oligonucleotide to an astrocyte or a population of astrocytes in a subject, the method comprising administering a pharmaceutical composition described herein to the subject.
- the astrocyte or a population of astrocytes is located in a region of the CNS.
- the region of the CNS is selected from the spinal cord, lumbar spinal cord, thoracic spinal cord, cervical spinal cord, medulla, hippocampus, cerebellum, hypothalamus, frontal cortex, and a combination thereof.
- the disclosure provides a method for reducing expression of an astrocyte mRNA in a cell, a population of cells or a subject, the method comprising the step of: i. contacting the cell or the population of cells with an oligonucleotide or pharmaceutical composition described herein, optionally wherein the cell or population of cells is an astrocyte or a population of astrocytes; or ii. administering to the subject an oligonucleotide or pharmaceutical composition described herein.
- reducing expression of the astrocyte mRNA comprises reducing an amount or level of mRNA, an amount or level of protein, or both.
- the subject has a disease, disorder or condition associated with expression of the astrocyte mRNA.
- the cell or population of cells is located in a region of the CNS.
- the region of the CNS is selected from the spinal cord, lumbar spinal cord, thoracic spinal cord, cervical spinal cord, medulla, hippocampus, cerebellum, hypothalamus, frontal cortex, and a combination thereof.
- the methods comprise administering via intrathecal administration.
- the disclosure provides a method of reducing expression of a target mRNA expressed in an astrocyte in a tissue of the CNS of a subject, comprising administering to the subject a double-stranded oligonucleotide comprising an antisense strand of 15-30 nucleotides in length and a sense strand of 15-50 nucleotides in length, wherein the antisense and sense strands form a duplex region of 15-30 base pairs, wherein the antisense strand comprises a region of complementarity to a target sequence in the target mRNA, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide of the sense strand.
- the lipid moiety is a C16 hydrocarbon.
- the oligonucleotide is blunt ended at the 3’ terminus of the oligonucleotide.
- the oligonucleotide comprises a blunt end.
- the blunt end comprises the 3’ terminus of the sense strand.
- the sense strand is 22-24 nucleotides.
- the tissue is the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 8, position 12, position 13, position 18 or position 20 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the tissue is the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 8, position 12, position 13, position 18 or position 20 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the tissue is the cerebellum, wherein the at least one lipid moiety is conjugated to a nucleotide at position 4, position 12, position 13, position 18 or position 20 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the tissue is the hypothalamus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 12, position 13, position 18 or position 20 of the sense strand, and wherein positions are numbered 5’ to 3.
- the tissue is the frontal cortex, wherein the at least one lipid moiety is conjugated to a nucleotide at position 4 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the sense strand comprises a stem-loop at the 3 ’end, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2.
- the sense strand is 36-38 nucleotides.
- the tissue is the spinal cord, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 8, position 12, position 13, position 18, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the tissue is the medulla, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 18, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the tissue is the cerebellum, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 23, position 28, or position 29 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the tissue is the hypothalamus, wherein the at least one lipid moiety is conjugated to a nucleotide at position 1, position 4, position 12, position 13, position 18, position 20, position 23, position 28, position 29 or position 30 of the sense strand, and wherein positions are numbered 5’ to 3’.
- the tissue is the frontal cortex, wherein the at least one lipid moiety is conjugated to a nucleotide at position 23 of the sense strand, and wherein positions are numbered 5’ to 3’.
- a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 23 weeks, at least 26 weeks, or at least 29 weeks. In some aspects, a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for at least 4 weeks. In some aspects, a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for at least 8 weeks. In some aspects, a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for at least 12 weeks.
- a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for at least 23 weeks. In some aspects, a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for at least 26 weeks. In some aspects, a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for at least 29 weeks.
- a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for up to 4 weeks, up to 8 weeks, up to 12 weeks, up to 23 weeks, up to 26 weeks, or up to 29 weeks.
- a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for up to 4 weeks.
- a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for up to 8 weeks.
- a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for up to 12 weeks.
- a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for up to 23 weeks. In some aspects, a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for up to 26 weeks. In some aspects, a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for up to 29 weeks.
- a single dose of the oligonucleotide or pharmaceutical composition reduces expression of the astrocyte mRNA for up to one year.
- the disclosure provides a kit comprising an oligonucleotide described herein, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder or condition associated with expression of an astrocyte mRNA.
- the package insert comprises instructions for intrathecal administration.
- the disclosure provides use of an oligonucleotide or pharmaceutical composition described herein, in the manufacture of a medicament for the treatment of a disease, disorder or condition associated with expression of an astrocyte mRNA.
- the disclosure provides an oligonucleotide or pharmaceutical composition described herein for use, or adaptable for use, in the treatment of a disease, disorder or condition associated with expression of an astrocyte mRNA.
- FIGs. 1A-1F provide graphs depicting the concentration response of a GalNAc- conjugated oligonucleotide targeting the astrocyte-specific gene GFAP.
- mice were treated with 10, 32, 100, 320, or 1000 pg the GalNAc- conjugated Gfap oligonucleotide formulated in artificial cerebrospinal fluid (aCSF) via intrathecal injection into the lumbar spine. Seven (7) days following intrathecal injection, the level of Gfap mRNA was normalized to Ribosomal Protein L23 (RPL23) mRNA and overall expression was determined between tissue types relative to control mice treated with aCSF.
- aCSF cerebrospinal fluid
- FIG. 2 provides a graph depicting the average percent (%) of murine Gfap mRNA remaining in different tissues of the central nervous system (CNS) based on the results in FIGs. 1A-1F and the resulting ECso (EDso) calculated for each CNS tissue.
- FIGs. 3A-3D provide graphs depicting the concentration- response of a GalNAc- conjugated Gfap oligonucleotide.
- Mice were treated with 10, 32, 100, or 300 pg of the oligonucleotide formulated in artificial cerebrospinal fluid (aCSF) via intracerebroventricular (i.c.v) injection. Seven (7) days following i.c.v. injection, the level of Gfap mRNA was normalized to Ribosomal Protein L23 (RPL23) mRNA and overall expression was determined between tissue types relative to control mice treated with aCSF.
- RPL23
- FIG. 4 provides a graph depicting the average percent (%) of murine Gfap mRNA remaining in different tissues of the CNS based on the results in FIGs. 3A-3D and the resulting ECso (EDso) calculated for each CNS tissue.
- FIGs. 5A-5B provide graphs depicting the percent (%) of rat Gfap mRNA remining in tissues of the central nervous system of rats after treatment with C16 lipid-conjugated (FIG. 5A) or GalN Ac-conjugated “GalXC” (FIG. 5B) Gfap tetraloop oligonucleotides.
- Rats were treated with 1000 pg of the indicated Gfap oligonucleotides in Table 3 formulated in artificial cerebrospinal fluid (aCSF) via intrathecal injection to the lumbar spine.
- aCSF artificial cerebrospinal fluid
- the level of Gfap mRNA was normalized to peptidylprolyl Isomerase B (Ppib) mRNA and overall expression was determined between tissue types relative to control rats treated with aCSF.
- Ppib peptidylprolyl Isomerase B
- FIG. 6 provides a graph depicting the percent (%) of rat Gfap mRNA remining in tissues of the central nervous system of rats after treatment with a C16 lipid-conjugated Gfap tetraloop oligonucleotide.
- Rats were treated with 1000 pg of the indicated Gfap lipid- conjugated tetraloop oligonucleotide in Table 3 formulated in artificial cerebrospinal fluid (aCSF) via intracisternal magna injection.
- aCSF cerebrospinal fluid
- the level of Gfap mRNA was normalized to peptidylprolyl Isomerase B (Ppib) mRNA and overall expression was determined between tissue types relative to control rats treated with aCSF.
- Ppib peptidylprolyl Isomerase B
- FIGs. 7A-7B provide graphs depicting the percent (%) of rat Gfap mRNA remining in tissues of the central nervous system of rats after treatment with a C16 lipid-conjugated Gfap tetraloop oligonucleotide.
- Rats were treated with 1000 pg of the indicated Gfap lipid- conjugated tetraloop oligonucleotide in Table 3 formulated in artificial cerebrospinal fluid (aCSF) via intracisternal magna injection.
- aCSF cerebrospinal fluid
- the level of Gfap mRNA was normalized to peptidylprolyl Isomerase B (Ppib) mRNA and overall expression was determined between tissue types relative to control rats treated with aCSF.
- Ppib peptidylprolyl Isomerase B
- FIGs. 8A-8F provide graphs depicting the percent (%) of murine Gfap mRNA remaining in lumbar spinal cord (FIG. 8A), medulla (FIG. 8B), cerebellum (FIG. 8C), hypothalamus (FIG. 8D), hippocampus (FIG. 8E), and frontal cortex (FIG. 8F) of mice after treatment with Gfap tetraloop oligonucleotides having a lipid conjugated at a nucleotide position indicated on the x-axis.
- mice were treated with 300 pg of the indicated Gfap lipid- conjugated tetraloop oligonucleotides in Table 4 formulated in artificial cerebrospinal fluid (aSCF) via intrathecal (i.t.) injection. Seven (7) days post dose, the level of Gfap mRNA was normalized to Ribosomal Protein L23 (RPL23) mRNA and overall expression was determined between tissue types relative to control mice treated with aCSF.
- aSCF cerebrospinal fluid
- FIGs. 9A-9F provide graphs depicting the percent (%) of murine Gfap mRNA remaining in lumbar spinal cord (FIG. 9A), medulla (FIG. 9B), cerebellum (FIG. 9C), hypothalamus (FIG. 9D), hippocampus (FIG. 9E), and frontal cortex (FIG. 9F) of mice after treatment with Gfap blunt-end or tetraloop oligonucleotides having a lipid conjugated at a nucleotide position indicated on the x-axis.
- mice were treated with 300 pg of the indicated C16 lipid-conjugated Gfap oligonucleotides in Table 5 formulated in artificial cerebrospinal fluid (aCSF) via intrathecal injection into the lumbar spine. Seven (7) days following intrathecal injection, the level of Gfap mRNA was normalized to Ribosomal Protein L23 (RPL23) mRNA and overall expression was determined between tissue types relative to control mice treated with aCSF.
- aCSF cerebrospinal fluid
- FIGs. 10A-10F provide graphs depicting the percent (%) of murine Gfap mRNA remaining in lumbar spinal cord (FIG. 10A), medulla (FIG. 10B), cerebellum (FIG. 10C), hypothalamus (FIG. 10D), hippocampus (FIG. 10E), and frontal cortex (FIG. 10F) of mice after treatment with lipid-conjugated Gfap blunt-end or tetraloop oligonucleotides as assessed in FIGs 8A-8F and 9A-9F.
- Experiment 1 represents the oligonucleotides assessed in FIGs. 8A-8F.
- Experiment 2 represents the tetraloop oligonucleotides assessed in FIGs. 9A-9F.
- Experiment 3 represents the blunt-end oligonucleotides assessed in FIGs. 9A-9F.
- FIGs. 11A-11B provide graphs depicting the concentration response of Cl 6- conjugated Gfap blunt-end oligonucleotides.
- Mice were treated with 3, 10, 30, 100, or 300 pg of the indicated oligonucleotide formulated in artificial cerebrospinal fluid (aCSF) via intrathecal injection into the lumbar spine. Seven (7) days (FIG. 11 A) and 28-days (FIG. 11B) following intrathecal injection, the level of Gfap mRNA was normalized to Ribosomal Protein L23 (RPL23) mRNA and overall expression and EDso was determined between tissue types relative to control mice treated with aCSF.
- RPL23 Ribosomal Protein L23
- the disclosure provides oligonucleotide-lipid conjugates (e.g., RNAi oligonucleotide-lipid conjugates) that reduce expression of a target gene expressed in astrocytes in the central nervous system (CNS).
- the disclosure provides methods of treating a disease or disorder associated with expression of an astrocyte mRNA (e.g., a disease of the CNS).
- the disclosure provides methods of treating a disease or disorder (e.g., a neurological disease and/or by inappropriate gene expression) associated with expression of an astrocyte mRNA using the lipid-conjugated RNAi oligonucleotides, or pharmaceutically acceptable compositions thereof, described herein.
- the disclosure provides methods of using the lipid-conjugated RNAi oligonucleotides described herein in the manufacture of a medicament for treating a disease or disorder associated with expression of an astrocyte mRNA.
- the lipid- conjugated RNAi oligonucleotides provided herein are used to treat a neurological disease or disorder by modulating (e.g., inhibiting or reducing) expression of an astrocyte target gene associated with the neurological disease or disorder in the CNS.
- the disclosure provides methods of treating a neurological disease or disorder by reducing expression of an astrocyte target gene associated with the neurological disease or disorder in the CNS (e.g., in cells, tissues or regions of the CNS).
- RNAi oligonucleotides e.g., RNAi oligonucleotide-lipid conjugates
- a lipid-conjugated RNAi oligonucleotide provided by the disclosure is targeted to an mRNA encoding the target gene.
- Messenger RNA (mRNA) that encodes a target gene and is targeted by a lipid-conjugated RNAi oligonucleotide of the disclosure is referred to herein as “target mRNA”.
- the lipid-conjugated RNAi oligonucleotide is targeted to a target sequence comprising a target astrocyte mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotide is targeted to a target sequence within a target astrocyte mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotide, or a portion, fragment, or strand thereof (e.g., an antisense strand or a guide strand of a double-stranded oligonucleotide) binds or anneals to a target sequence comprising a target astrocyte mRNA, thereby reducing target gene expression.
- the lipid-conjugated RNAi oligonucleotide is targeted to a target sequence comprising target astrocyte mRNA for the purpose of reducing expression of an astrocyte target gene in vivo.
- the amount or extent of reduction of target gene expression by an lipid-conjugated RNAi oligonucleotide targeted to a specific astrocyte target sequence correlates with the potency of the lipid-conjugated RNAi oligonucleotide.
- the amount or extent of reduction of target gene expression by an lipid-conjugated RNAi oligonucleotide targeted to a specific astrocyte target sequence correlates with the amount or extent of therapeutic benefit in a subject or patient having a disease, disorder or condition associated with target gene expression treated with the lipid-conjugated RNAi oligonucleotide.
- nucleotide sequence of mRNAs encoding target genes including mRNAs of multiple different species (e.g., human, cynomolgus monkey, mouse, and rat) and as a result of in vitro and in vivo testing, it has been discovered that certain nucleotide sequences and certain systemic modifications to those oligonucleotides are more amenable than others to RNAi oligonucleotide-mediated reduction and are thus useful as part of oligonucleotides that are otherwise targeted to specific gene target sequences.
- mRNAs of multiple different species e.g., human, cynomolgus monkey, mouse, and rat
- a sense strand of a lipid-conjugated RNAi oligonucleotide, or a portion or fragment thereof, described herein comprises a nucleotide sequence that is similar (e.g., having no more than 4 mismatches) or is identical to a target sequence comprising an astrocyte target mRNA.
- a portion or region of the sense strand of a double-stranded oligonucleotide described herein comprises a target sequence comprising an astrocyte target mRNA.
- the astrocyte mRNA target sequence is associated with acute or chronic pain. In some embodiments, the astrocyte mRNA target sequence is associated with a neurological disorder. In some embodiments, the astrocyte mRNA target sequence is an mRNA expressed in astrocytes of at least one region of the CNS. In some embodiments, the astrocyte mRNA target sequence is an mRNA expressed in astrocytes of the spinal cord. In some embodiments, the astrocyte mRNA target sequence is an mRNA expressed in astrocytes of the lumbar spinal cord. In some embodiments, the astrocyte mRNA target sequence is an mRNA expressed in astrocytes of the thoracic spinal cord.
- the astrocyte mRNA target sequence is an mRNA expressed in astrocytes of the cervical spinal cord. In some embodiments, the astrocyte mRNA target sequence is an mRNA expressed in astrocytes of the hypothalamus. In some embodiments, the astrocyte mRNA target sequence is an mRNA expressed in astrocytes of the medulla. In some embodiments, the astrocyte mRNA target sequence is an mRNA expressed in astrocytes of the hippocampus. In some embodiments, the astrocyte mRNA target sequence is an mRNA expressed in astrocytes of the cerebellum. In some embodiments, the astrocyte mRNA target sequence is an mRNA expressed in astrocytes of the frontal cortex. In some embodiments, the astrocyte mRNA target sequence is an mRNA associated with a disease, disorder or condition of the CNS.
- the oligonucleotides herein have regions of complementarity to GFAP mRNA (e.g., astrocyte mRNA target sequence is a GFAP sequence.
- the target sequence is within a target sequence of GFAP mRNA) for purposes of targeting the mRNA in cells and inhibiting its expression.
- the oligonucleotides herein comprise a GFAP targeting sequence (e.g., an antisense strand or a guide strand of a double-stranded oligonucleotide) having a region of complementarity that binds or anneals to a GFAP target sequence by complementary (Watson-Crick) base pairing.
- a portion or region of the sense strand of a double-stranded oligonucleotide described herein comprises a GFAP target sequence.
- a GFAP target sequence comprises, or consists of, a nucleotide sequence of any one of SEQ ID NOs: 28-31.
- the lipid-conjugated RNAi oligonucleotides provided by the disclosure comprise a targeting sequence.
- targeting sequence refers to a nucleotide sequence having a region of complementarity to a specific nucleotide sequence comprising an mRNA (e.g., an astrocyte target mRNA).
- the lipid- conjugated RNAi oligonucleotides provided by the disclosure comprise a gene targeting sequence having a region of complementarity to a nucleotide sequence comprising a target sequence of a target mRNA.
- the targeting sequence is an astrocyte mRNA target sequence.
- the targeting sequence imparts the lipid-conjugated RNAi oligonucleotide with the ability to specifically target an mRNA by binding or annealing to a target sequence comprising a target mRNA by complementary (Watson-Crick) base pairing.
- the lipid-conjugated RNAi oligonucleotides herein (or a strand thereof, e.g., an antisense strand or a guide strand of a double-stranded oligonucleotide) comprise a targeting sequence having a region of complementarity that binds or anneals to a target sequence comprising an astrocyte target mRNA by complementary (Watson-Crick) base pairing.
- the lipid- conjugated RNAi oligonucleotides herein (or a strand thereof, e.g., an antisense strand or a guide strand of a double-stranded oligonucleotide) comprise a targeting sequence having a region of complementarity that binds or anneals to a target sequence within an astrocyte target mRNA by complementary (Watson-Crick) base pairing.
- the targeting sequence is generally of suitable length and base content to enable binding or annealing of the lipid-conjugated RNAi oligonucleotide (or a strand thereof) to a specific target mRNA (e.g., astrocyte mRNA) for purposes of inhibiting target gene expression.
- the targeting sequence is at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29 or at least about 30 nucleotides in length.
- the targeting sequence is at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 nucleotides.
- the targeting sequence is about 12 to about 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides in length.
- the targeting sequence is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the targeting sequence is 18 nucleotides in length. In some embodiments, the targeting sequence is 19 nucleotides in length. In some embodiments, the targeting sequence is 20 nucleotides in length. In some embodiments, the targeting sequence is 21 nucleotides in length. In some embodiments, the targeting sequence is 22 nucleotides in length. In some embodiments, the targeting sequence is 23 nucleotides in length. In some embodiments, the targeting sequence is 24 nucleotides in length.
- the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is fully complementary to a target sequence comprising an astrocyte target mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is fully complementary to a target sequence within an astrocyte target mRNA. In some embodiments, the targeting sequence is partially complementary to a target sequence comprising a target mRNA. In some embodiments, the targeting sequence is partially complementary to a target sequence within an astrocyte target mRNA. In some embodiments, the targeting sequence comprises a region of contiguous nucleotides comprising the antisense strand.
- the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising an astrocyte target mRNA, wherein the contiguous sequence of nucleotides is about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 20, 12 to 18, 12 to 16, 14 to 22, 16 to 20, 18 to 20 or 18 to 19 nucleotides in length).
- the lipid-conjugated RNAi oligonucleotides comprise a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising an astrocyte target mRNA, wherein the contiguous sequence of nucleotides is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotides comprise a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising a target mRNA, wherein the contiguous sequence of nucleotides is 15 nucleotides in length.
- the lipid-conjugated RNAi oligonucleotides comprise a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising a target mRNA, wherein the contiguous sequence of nucleotides is 19 nucleotides in length.
- the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising an astrocyte target mRNA, wherein the contiguous sequence of nucleotides is 15 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising an astrocyte target mRNA, wherein the contiguous sequence of nucleotides is 19 nucleotides in length.
- a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an astrocyte target mRNA and comprises the entire length of an antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an astrocyte target mRNA and comprises a portion of the entire length of an antisense strand.
- a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an astrocyte target mRNA and comprises 10 to 20 nucleotides of the antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an astrocyte target mRNA and comprises 15 to 19 nucleotides of the antisense strand.
- a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an astrocyte target mRNA and comprises 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, or 22 nucleotides of the antisense strand.
- a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an astrocyte target mRNA and comprises 19 nucleotides of the antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising an astrocyte target mRNA and comprises 20 nucleotides of the antisense strand.
- a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g. , having no more than 4 mismatches) to a target sequence comprising an astrocyte target mRNA and comprises the entire length of an antisense strand.
- a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g, having no more than 4 mismatches) to a target sequence comprising an astrocyte target mRNA and comprises a portion of the entire length of an antisense strand.
- a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g, having no more than 4 mismatches) to a target sequence comprising an astrocyte target mRNA and comprises 10 to 20 nucleotides of the antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising an astrocyte target mRNA and comprises 15 to 19 nucleotides of the antisense strand.
- a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising an astrocyte target mRNA and comprises 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, or 22 nucleotides of the antisense strand.
- a targeting sequence of a lipid- conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising an astrocyte target mRNA and comprises 19 nucleotides of the antisense strand. In some embodiments, a targeting sequence of a lipid- conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising an astrocyte target mRNA and comprises 20 nucleotides of the antisense strand.
- a lipid-conjugated RNAi oligonucleotide herein comprises a targeting sequence having one or more base pair (bp) mismatches with the corresponding target sequence comprising an astrocyte target mRNA.
- the targeting sequence has a 1 bp mismatch, a 2 bp mismatch, a 3 bp mismatch, a 4 bp mismatch, or a 5 bp mismatch with the corresponding target sequence comprising an astrocyte target mRNA provided that the ability of the targeting sequence to bind or anneal to the target sequence under appropriate hybridization conditions and/or the ability of the lipid-conjugated RNAi oligonucleotide to inhibit or reduce target gene expression is maintained (e.g., under physiological conditions).
- the targeting sequence comprises no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 bp mismatches with the corresponding target sequence comprising an astrocyte target mRNA provided that the ability of the targeting sequence to bind or anneal to the target sequence under appropriate hybridization conditions and/or the ability of the lipid-conjugated RNAi oligonucleotide to inhibit or reduce target gene expression is maintained.
- the lipid- conjugated RNAi oligonucleotide comprises a targeting sequence having 1 mismatch with the corresponding target sequence.
- the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having 2 mismatches with the corresponding target sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having 3 mismatches with the corresponding target sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having 4 mismatches with the corresponding target sequence. In some embodiments, the lipid- conjugated RNAi oligonucleotide comprises a targeting sequence having 5 mismatches with the corresponding target sequence.
- the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, wherein at least 2 (e.g., all) of the mismatches are positioned consecutively (e.g., 2, 3, 4, 5 or more mismatches in a row), or wherein the mismatches are interspersed in any position throughout the targeting sequence.
- mismatch e.g., 2, 3, 4, 5 or more mismatches
- the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, wherein at least 2 (e.g., all) of the mismatches are positioned consecutively (e.g., 2, 3, 4, 5 or more mismatches in a row), or wherein at least one or more non-mismatched base pair is located between the mismatches, or a combination thereof.
- mismatch e.g., 2, 3, 4, 5 or more mismatches
- RNAi oligonucleotide types and/or structures are useful for reducing target gene expression (e.g., reducing expression of a target gene expressed in an astrocyte) in the methods herein.
- target gene expression e.g., reducing expression of a target gene expressed in an astrocyte
- Any of the RNAi oligonucleotide types described herein or elsewhere are contemplated for use as a framework to incorporate a targeting sequence herein for the purposes of inhibiting or reducing corresponding target gene expression in an astrocyte in the CNS.
- the lipid-conjugated RNAi oligonucleotides herein inhibit target gene expression by engaging with RNA interference (RNAi) pathways upstream or downstream of Dicer involvement.
- RNAi RNA interference
- RNAi oligonucleotides have been developed with each strand having sizes of about 19-25 nucleotides with at least one 3' overhang of 1 to 5 nucleotides (see, e.g., US Patent No. 8,372,968). Longer oligonucleotides also have been developed that are processed by Dicer to generate active RNAi products (see, e.g., US Patent No. 8,883,996).
- RNAi oligonucleotides conjugates herein engage with the RNAi pathway downstream of the involvement of Dicer (e.g., Dicer cleavage).
- the oligonucleotides described herein are Dicer substrates.
- double-stranded nucleic acids of 19-23 nucleotides in length capable of reducing expression of an astrocyte target mRNA are produced.
- the lipid-conjugated RNAi oligonucleotide has an overhang (e.g., of 1, 2, or 3 nucleotides in length) in the 3' end of the sense strand.
- the lipid-conjugated RNAi oligonucleotide (e.g., siRNA conjugate) comprises a 21 -nucleotide guide strand that is antisense to an astrocyte target mRNA and a complementary passenger strand, in which both strands anneal to form a 19-bp duplex and 2 nucleotide overhangs at either or both 3' ends.
- oligonucleotide designs also are contemplated including oligonucleotides having a guide strand of 23 nucleotides and a passenger strand of 21 nucleotides, where there is a blunt end on the right side of the molecule (3' end of passenger strand/5' end of guide strand) and a two nucleotide 3 '-guide strand overhang on the left side of the molecule (5' end of the passenger strand/3' end of the guide strand). In such molecules, there is a 21 bp duplex region. See, e.g., US Patent Nos. 9,012,138; 9,012,621 and 9,193,753.
- the RNAi oligonucleotides conjugates disclosed herein comprise sense and antisense strands that are both in the range of about 17 to 26 (e.g, 17 to 26, 20 to 25 or 21-23) nucleotides in length.
- the lipid-conjugated RNAi oligonucleotides disclosed herein comprise a sense and antisense strand that are both in the range of about 19-22 nucleotides in length.
- the sense and antisense strands are of equal length.
- the lipid-conjugated RNAi oligonucleotides disclosed herein comprise sense and antisense strands, such that there is a 3 '-overhang on either the sense strand or the antisense strand, or both the sense and antisense strand.
- a 3' overhang on the sense, antisense, or both sense and antisense strands is 1 or 2 nucleotides in length.
- an lipid-conjugated RNAi oligonucleotide has a guide strand of 22 nucleotides and a passenger strand of 20 nucleotides, where there is a blunt end on the right side of the molecule (3' end of passenger strand/5' end of guide strand) and a 2 nucleotide 3 '-guide strand overhang on the left side of the molecule (5' end of the passenger strand/3' end of the guide strand). In such molecules, there is a 20 bp duplex region.
- RNAi oligonucleotide designs for use with the compositions and methods herein include: 16-mer siRNAs (see, e.g., Nucleic Acids in Chemistry and Biology, Blackbum (ed.), ROYAL SOCIETY OF CHEMISTRY, 2006), shRNAs (e.g., having 19 bp or shorter stems; see, e.g., Moore et al. (2010) METHODS MOL. BIOL. 629: 141-58), blunt siRNAs (e.g., of 19 bps in length; see, e.g., Kraynack & Baker (2006) RNA 12: 163-76), asymmetrical siRNAs (aiRNA; see, e.g., Sun etal.
- siRNAs see, e.g., Nucleic Acids in Chemistry and Biology, Blackbum (ed.), ROYAL SOCIETY OF CHEMISTRY, 2006
- shRNAs e.g., having 19 bp or shorter
- siRNA small internally segmented interfering RNA
- oligonucleotide structure that may be used in some embodiments to reduce or inhibit the expression of a target gene are microRNA (miRNA), short hairpin RNA (shRNA) and short siRNA (see, e.g., Hamilton etal. (2002) EMBO J. 21 :4671-79; see also, US Patent Application Publication No. 2009/0099115).
- miRNA microRNA
- shRNA short hairpin RNA
- siRNA see, e.g., Hamilton etal. (2002) EMBO J. 21 :4671-79; see also, US Patent Application Publication No. 2009/0099115.
- an antisense strand of a lipid-conjugated RNAi oligonucleotide is referred to as a “guide strand.”
- a guide strand an antisense strand that engages with RNA-induced silencing complex (RISC) and binds to an Argonaute protein such as Ago2, or engages with or binds to one or more similar factors, and directs silencing of a target gene
- RISC RNA-induced silencing complex
- Ago2 Argonaute protein
- a sense strand complementary to a guide strand is referred to as a “passenger strand.”
- a lipid-conjugated RNAi oligonucleotide herein comprises an antisense strand of up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, up to 15, or up to 12 nucleotides in length).
- a lipid-conjugated RNAi oligonucleotide herein comprises an antisense strand of at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35 or at least 38 nucleotides in length).
- a herein comprises an antisense strand in a range of about 12 to about 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 30, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40 or 32 to 40) nucleotides in length.
- a lipid-conjugated RNAi oligonucleotide herein comprises an antisense strand of 15 to 30 nucleotides in length.
- an antisense strand of any one of the lipid-conjugated RNAi oligonucleotide disclosed herein is of 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 or 40 nucleotides in length.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 19-23 nucleotides in length.
- a lipid- conjugated RNAi oligonucleotide comprises an antisense strand of 19 nucleotides in length.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 20 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 21 nucleotides in length. In some embodiments, a lipid- conjugated RNAi oligonucleotide comprises an antisense strand of 22 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 23 nucleotides in length.
- a lipid-conjugated RNAi oligonucleotide disclosed herein comprises a sense strand (or passenger strand) of up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17 or up to 12 nucleotides in length).
- a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36 or at least 38 nucleotides in length).
- a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand in a range of about 12 to about 50 (e.g., 12 to 50, 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40 or 32 to 40) nucleotides in length.
- a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand 15 to 50 nucleotides in length.
- a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand 18 to 36 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 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, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 17-21 nucleotides in length.
- a lipid- conjugated RNAi oligonucleotide herein comprises a sense strand of 17 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 18 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 19 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 20 nucleotides in length.
- a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 21 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 22 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 23 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 24 nucleotides in length.
- a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 25 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 26 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 27 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 28 nucleotides in length.
- a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 29 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 30 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 31 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 32 nucleotides in length.
- a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 33 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 34 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 35 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 36 nucleotides in length.
- a sense strand comprises a stem-loop structure at its 3' end. In some embodiments, the stem-loop is formed by intrastrand base pairing. In some embodiments, a sense strand comprises a stem-loop structure at its 5' end. In some embodiments, a stem is a duplex of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 2 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 3 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 4 nucleotides in length.
- the stem of the stem-loop comprises a duplex of 5 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 6 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 7 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 8 nucleotides in length. In some embodiments, the stem of the stemloop comprises a duplex of 9 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 10 nucleotides in length.
- the stem of the stem-loop comprises a duplex of 11 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 12 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 13 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 14 nucleotides in length.
- a stem-loop provides the lipid-conjugated RNAi oligonucleotide protection against degradation (e.g., enzymatic degradation), facilitates or improves targeting and/or delivery to a target cell, tissue, or organ, or both.
- the loop of a stem-loop provides nucleotides comprising one or more modifications that facilitate, improve, or increase targeting to a target mRNA (e.g., a target mRNA expressed in the CNS), inhibition of target gene expression, and/or delivery to a target cell, tissue, or organ (e.g., the CNS), or a combination thereof.
- the stemloop itself or modification(s) to the stem-loop do not substantially affect the inherent gene expression inhibition activity of the lipid-conjugated RNAi oligonucleotide, but facilitates, improves, or increases stability (e.g., provides protection against degradation) and/or delivery of the lipid-conjugated RNAi oligonucleotide to a target cell, tissue, or organ (e.g., the CNS).
- a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand comprising (e.g, at its 3' end) a stem-loop set forth as: S1-L-S2, in which SI is complementary to S2, and in which L forms a single-stranded loop between SI and S2 of up to about 10 nucleotides in length (e.g, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length).
- the loop (L) is 3 nucleotides in length. In some embodiments, the loop (L) is 4 nucleotides in length.
- the tetraloop comprises the sequence 5’-GAAA-3’.
- the stem loop comprises the sequence 5’-GCAGCCGAAAGGCUGC-3’ (SEQ ID NO: 32).
- a loop (L) of a stem-loop having the structure S1-L-S2 as described above is a triloop.
- the triloop comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof.
- a loop (L) of a stem-loop having the structure S1-L-S2 as described above is a tetraloop (e.g., within a nicked tetraloop structure).
- the tetraloop comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof.
- a loop (L) of a stem-loop having the structure S1-L-S2 as described above is a tetraloop as described in US Patent No. 10,131,912, incorporated herein by reference (e.g., within a nicked tetraloop structure).
- Duplex Length is a tetraloop as described in US Patent No. 10,131,912, incorporated herein by reference (e.g., within a nicked tetraloop structure).
- a duplex formed between a sense and antisense strand is at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, a duplex formed between a sense and antisense strand is in the range of 12-30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30 or 21 to 30 nucleotides in length). In some embodiments, a duplex formed between a sense and antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
- a duplex formed between a sense and antisense strand is 15-30 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 17-21 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 17 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 18 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 19 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 20 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 21 base pairs in length.
- a duplex formed between a sense and antisense strand does not span the entire length of the sense strand and/or antisense strand. In some embodiments, a duplex between a sense and antisense strand spans the entire length of either the sense or antisense strands. In some embodiments, a duplex between a sense and antisense strand spans the entire length of both the sense strand and the antisense strand.
- a lipid-conjugated RNAi oligonucleotide disclosed herein comprises sense and antisense strands, such that there is a 3 ’-overhang on either the sense strand or the antisense strand, or both the sense and antisense strand.
- a lipid-conjugated RNAi oligonucleotide herein has one 5 ’end that is thermodynamically less stable compared to the other 5’ end.
- an asymmetric lipid-conjugated RNAi oligonucleotide conjugate is provided that includes a blunt end at the 3 ’end of a sense strand and overhang at the 3’ end of the antisense strand.
- a 3’ overhang on an antisense strand is 1-4 nucleotides in length (e.g., 1, 2, 3, or 4 nucleotides in length).
- the 3’-overhang is about one (1) to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length).
- the 3’ overhang is about one (1) to nineteen (19), one (1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen (16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) to thirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1) to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven (7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one (1) to three (3), or about one (1) to two (2) nucleotides in length.
- the 3 ’-overhang is (1) nucleotide in length. In some embodiments, the 3 ’-overhang is two (2) nucleotides in length. In some embodiments, the 3 ’-overhang is three (3) nucleotides in length. In some embodiments, the 3 ’-overhang is four (4) nucleotides in length. In some embodiments, the 3 ’-overhang is five (5) nucleotides in length. In some embodiments, the 3 ’-overhang is six (6) nucleotides in length. In some embodiments, the 3 ’-overhang is seven (7) nucleotides in length. In some embodiments, the 3 ’-overhang is eight (8) nucleotides in length.
- the 3’-overhang is nine (9) nucleotides in length. In some embodiments, the 3’-overhang is ten (10) nucleotides in length. In some embodiments, the 3’-overhang is eleven (11) nucleotides in length. In some embodiments, the 3 ’-overhang is twelve (12) nucleotides in length. In some embodiments, the 3’-overhang is thirteen (13) nucleotides in length. In some embodiments, the 3’-overhang is fourteen (14) nucleotides in length. In some embodiments, the 3’-overhang is fifteen (15) nucleotides in length. In some embodiments, the 3’-overhang is sixteen (16) nucleotides in length.
- the 3’-overhang is seventeen (17) nucleotides in length. In some embodiments, the 3 ’-overhang is eighteen (18) nucleotides in length. In some embodiments, the 3 ’-overhang is nineteen (19) nucleotides in length. In some embodiments, the 3 ’-overhang is twenty (20) nucleotides in length.
- an oligonucleotide for RNAi has a two (2) nucleotide overhang on the 3 ’ end of the antisense (guide) strand.
- an overhang is a 3’ overhang comprising a length of between one and four nucleotides, optionally one to four, one to three, one to two, two to four, two to three, or one, two, three, or four nucleotides.
- the overhang is a 5’ overhang comprising a length of between one and four nucleotides, optionally one to four, one to three, one to two, two to four, two to three, or one, two, three, or four nucleotides.
- an oligonucleotide herein comprises a sense strand and an antisense strand, wherein the 5’ terminus of either or both strands comprise a 5 ’-overhang comprising one or more nucleotides. In some embodiments, an oligonucleotide herein comprises a sense strand and an antisense strand, wherein the sense strand comprises a 5’- overhang comprising one or more nucleotides. In some embodiments, an oligonucleotide herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a 5 ’-overhang comprising one or more nucleotides.
- an oligonucleotide herein comprises a sense strand and an antisense strand, wherein both the sense strand and the antisense strand comprises a 5 ’-overhang comprising one or more nucleotides.
- the 5’-overhang is about one (1) to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length).
- the 5’ overhang is about one (1) to nineteen (19), one (1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen (16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) to thirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1) to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven (7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one (1) to three (3), or about one (1) to two (2) nucleotides in length.
- the 5’-overhang is (1) nucleotide in length. In some embodiments, the 5’-overhang is two (2) nucleotides in length. In some embodiments, the 5 ’-overhang is three (3) nucleotides in length. In some embodiments, the 5 ’-overhang is four (4) nucleotides in length. In some embodiments, the 5 ’-overhang is five (5) nucleotides in length. In some embodiments, the 5 ’-overhang is six (6) nucleotides in length. In some embodiments, the 5 ’-overhang is seven (7) nucleotides in length. In some embodiments, the 5 ’-overhang is eight (8) nucleotides in length.
- the 5’-overhang is nine (9) nucleotides in length. In some embodiments, the 5’-overhang is ten (10) nucleotides in length. In some embodiments, the 5’-overhang is eleven (11) nucleotides in length. In some embodiments, the 5’-overhang is twelve (12) nucleotides in length. In some embodiments, the 5’-overhang is thirteen (13) nucleotides in length. In some embodiments, the 5’-overhang is fourteen (14) nucleotides in length. In some embodiments, the 5’-overhang is fifteen (15) nucleotides in length. In some embodiments, the 5’-overhang is sixteen (16) nucleotides in length.
- the 5’-overhang is seventeen (17) nucleotides in length. In some embodiments, the 5’-overhang is eighteen (18) nucleotides in length. In some embodiments, the 5 ’-overhang is nineteen (19) nucleotides in length. In some embodiments, the 5 ’-overhang is twenty (20) nucleotides in length.
- one or more (e.g., 2, 3, or 4) terminal nucleotides of the 3’ end or 5’ end of a sense and/or antisense strand are modified.
- one or two terminal nucleotides of the 3’ end of the antisense strand are modified.
- the last nucleotide at the 3’ end of an antisense strand is modified, e.g., comprises 2’ modification, e.g., a 2’-O-methoxyethyl.
- the last one or two terminal nucleotides at the 3’ end of an antisense strand are complementary with the target.
- the last one or two nucleotides at the 3’ end of the antisense strand are not complementary with the target.
- an RNAi oligonucleotide conjugate disclosed herein comprises a stem-loop structure at the 3’ end of the sense strand and comprises two terminal overhang nucleotides at the 3’ end of the antisense strand.
- an RNAi oligonucleotide conjugate herein comprises a nicked tetraloop structure, wherein the 3’ end of the sense strand comprises a stem-tetraloop structure and comprises two terminal overhang nucleotides at the 3’ end of the antisense strand.
- the overhang is selected from AA, GG, AG, and GA. In some embodiments, the overhang is AA. In some embodiments, the overhang is AG. In some embodiments, the overhang is GA. In some embodiments, the two terminal overhang nucleotides are GG. Typically, one or both of the two terminal GG nucleotides of the antisense strand are not complementary with the target.
- the 5’ end and/or the 3 ’end of a sense or antisense strand has an inverted cap nucleotide.
- one or more (e.g., 2, 3, 4, 5, 6) modified intemucleotide linkages are provided between terminal nucleotides of the 3’ end or 5’ end of a sense and/or antisense strand.
- modified internucleotide linkages are provided between overhang nucleotides at the 3’ end or 5’ end of a sense and/or antisense strand.
- an RNAi oligonucleotide conjugate disclosed herein comprises one or more modifications.
- Oligonucleotides e.g., RNAi oligonucleotides
- the modification is a modified sugar. In some embodiments, the modification is a 5 ’-terminal phosphate group. In some embodiments, the modification is a modified internucleoside linkage. In some embodiments, the modification is a modified base. In some embodiments, an oligonucleotide described herein can comprise any one of the modifications described herein or any combination thereof. For example, in some embodiments, an oligonucleotide described herein comprises at least one modified sugar, a 5’- terminal phosphate group, at least one modified internucleoside linkage, and at least one modified base.
- oligonucleotide e.g., an RNAi oligonucleotide
- oligonucleotides may be delivered in vivo by conjugating them to or encompassing them in a lipid nanoparticle (LNP) or similar carrier.
- LNP lipid nanoparticle
- an oligonucleotide is not protected by an LNP or similar carrier, it may be advantageous for at least some of the nucleotides to be modified. Accordingly, in some embodiments, all or substantially all of the nucleotides of an oligonucleotides are modified.
- the sugar moiety of all nucleotides comprising the oligonucleotide is modified at the 2’ position. In some embodiments, the sugar moiety of all nucleotides comprising the oligonucleotide is modified at the 2’ position, except for the nucleotide conjugated to a lipid (e.g., the 5 ’-terminal nucleotide of the sense strand). The modifications may be reversible or irreversible.
- an oligonucleotide as disclosed herein has a number and type of modified nucleotides sufficient to cause the desired characteristics (e.g., protection from enzymatic degradation, capacity to target a desired cell after in vivo administration, and/or thermodynamic stability).
- a nucleotide modification in a sugar comprises a 2'- modification.
- a 2'-modification may be 2'-O-propargyl, 2'-O- propylamin, 2'-amino, 2'-ethyl, 2'-fluoro (2'-F), 2'-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'- O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA) or 2'-deoxy-2'- fluoro-P-d-arabinonucleic acid (2'-FANA).
- a modification in a sugar comprises a modification of the sugar ring, which may comprise modification of one or more carbons of the sugar ring.
- a modification of a sugar of a nucleotide may comprise a 2'-oxygen of a sugar is linked to a 1 '-carbon or 4'-carbon of the sugar, or a 2'-oxygen is linked to the 1 '-carbon or d'carbon via an ethylene or methylene bridge.
- a modified nucleotide has an acyclic sugar that lacks a 2'-carbon to 3 '-carbon bond.
- a modified nucleotide has a thiol group, e.g., in the 4' position of the sugar.
- a lipid-conjugated RNAi oligonucleotide described herein comprises at least about 1 modified nucleotide e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or more).
- the sense strand of the lipid-conjugated RNAi oligonucleotide comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or more).
- the antisense strand of the lipid-conjugated RNAi oligonucleotide comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more).
- all the nucleotides of the sense strand of the lipid-conjugated RNAi oligonucleotide are modified. In some embodiments, all the nucleotides of the antisense strand of the lipid-conjugated RNAi oligonucleotide are modified. In some embodiments, all the nucleotides of the lipid-conjugated RNAi oligonucleotide (i.e., both the sense strand and the antisense strand) are modified.
- the modified nucleotide comprises a 2'-modification (e.g., a 2'-F or 2'-OMe, 2'-M0E, and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid).
- a 2'-modification e.g., a 2'-F or 2'-OMe, 2'-M0E, and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid.
- the disclosure provides lipid-conjugated RNAi oligonucleotides having different modification patterns.
- the modified lipid-conjugated RNAi oligonucleotides comprise a sense strand sequence having a modification pattern as set forth in the Examples and Sequence Listing and an antisense strand having a modification pattern as set forth in the Examples and Sequence Listing.
- a lipid-conjugated RNAi oligonucleotide disclosed herein comprises an antisense strand having nucleotides that are modified with 2'-F. In some embodiments, an lipid-conjugated RNAi oligonucleotide disclosed herein comprises an antisense strand comprises nucleotides that are modified with 2'-F and 2'-OMe. In some embodiments, a lipid-conjugated RNAi oligonucleotide disclosed herein comprises a sense strand having nucleotides that are modified with 2'-F. In some embodiments, a lipid-conjugated RNAi oligonucleotide disclosed herein comprises a sense strand comprises nucleotides that are modified with 2'-F and 2'-OMe.
- an oligonucleotide described herein comprises a sense strand with about 10-25%, 10%, 11%, 12%, 13%, 14% 15%, 16%, 17%, 18%, 19% or 20% of the nucleotides of the sense strand comprising a 2’ -fluoro modification.
- about 11% of the nucleotides of the sense strand comprise a 2-fluoro modification.
- about 20% of the nucleotides of the sense strand comprise a 2-fluoro modification.
- an oligonucleotide described herein comprises an antisense strand with about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the nucleotides of the antisense strand comprising a 2’ -fluoro modification. In some embodiments, about 32% of the nucleotides of the antisense strand comprise a 2’-fluoro modification. In some embodiments, the oligonucleotide has about 15-25%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of its nucleotides comprising a 2’-fluoro modification.
- nucleotides in the oligonucleotide comprise a 2’ -fluoro modification. In some embodiments, about 26% of the nucleotides in the oligonucleotide comprise a 2’ -fluoro modification.
- one or more of positions 8, 9, 10 or 11 of the sense strand is modified with a 2'-F group.
- the sugar moiety at each of nucleotides not modified with a 2’-F group or conjugated to a lipid in the sense strand is modified with a 2'-OMe.
- the sugar moiety at each of nucleotides at positions 1-7 and 12-20 in the sense strand is modified with a 2'-OMe.
- the sugar moiety at each of nucleotides at positions 2-7 and 12-20 in the sense strand is modified with a 2'-OMe. In some embodiments, for these oligonucleotides, the sugar moiety at each of nucleotides at positions 1-6 and 12-20 in the sense strand is modified with a 2'-0Me. In some embodiments, for these oligonucleotides, the sugar moiety at each of nucleotides at positions 1-3, 5-7, and 12-20 in the sense strand is modified with a 2'-0Me.
- the sugar moiety at each of nucleotides at positions 1-7 and 13-20 in the sense strand is modified with a 2'-0Me. In some embodiments, for these oligonucleotides, the sugar moiety at each of nucleotides at positions 1-7, 12, and 14-20 in the sense strand is modified with a 2'-0Me. In some embodiments, for these oligonucleotides, the sugar moiety at each of nucleotides at positions 1-7, 12-17, and 19-20 in the sense strand is modified with a 2'-0Me.
- the sugar moiety at each of nucleotides at positions 1-7 and 12-19 in the sense strand is modified with a 2'-0Me. In some embodiments, for these oligonucleotides the sugar moiety at each of nucleotides at positions 1-7 and 12-36 in the sense strand is modified with a 2’-0Me. In some embodiments, for these oligonucleotides the sugar moiety at each of nucleotides at positions 2-7 and 12-36 in the sense strand is modified with a 2’-0Me.
- the sugar moiety at each of nucleotides at positions 2-7 and 12-36 in the sense strand is modified with a 2’-0Me. In some embodiments, for these oligonucleotides the sugar moiety at each of nucleotides at positions 1-3, 5-7, and 12-36 in the sense strand is modified with a 2’- OMe. In some embodiments, for these oligonucleotides the sugar moiety at each of nucleotides at positions 1-7 and 12-36 in the sense strand is modified with a 2’-0Me.
- the sugar moiety at each of nucleotides at positions 1-7 and 13-36 in the sense strand is modified with a 2’-0Me. In some embodiments, for these oligonucleotides the sugar moiety at each of nucleotides at positions 1-7 and 12, and 14-36 in the sense strand is modified with a 2’-OMe. In some embodiments, for these oligonucleotides the sugar moiety at each of nucleotides at positions 1-7 and 12-17 and 19-36 in the sense strand is modified with a 2’-0Me.
- the sugar moiety at each of nucleotides at positions 1-7, 12-19, and 21-36 in the sense strand is modified with a 2’-0Me. In some embodiments, for these oligonucleotides the sugar moiety at each of nucleotides at positions 1-7, 12-22, and 24-36 in the sense strand is modified with a 2’-0Me. In some embodiments, for these oligonucleotides the sugar moiety at each of nucleotides at positions 1-7, 12-27, and 29-36 in the sense strand is modified with a 2’-0Me.
- the sugar moiety at each of nucleotides at positions 1-7, 12-28, and 30-36 in the sense strand is modified with a 2’-0Me. In some embodiments, for these oligonucleotides the sugar moiety at each of nucleotides at positions 1-7, 12-29, and 31-36 in the sense strand is modified with a 2’-0Me.
- the sense strand comprises at least one 2’-F modified nucleotide wherein the remaining nucleotides not modified with a 2’-F group or conjugated to a lipid are modified with a 2’-0Me.
- the antisense strand has 7 nucleotides that are modified at the 2’ position of the sugar moiety with a 2’-F. In some embodiments, the sugar moiety at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand are modified with a 2’-F. In some embodiments, the antisense strand has 14 nucleotides that are modified at the 2’ position of the sugar moiety with a 2’-0Me. In some embodiments, the sugar moiety at positions 6, 8, 9, 11, 12, 13, 15, 16,
- the sense strand has 4 nucleotides that are modified at the 2’ position of the sugar moiety with a 2’-F. In some embodiments, the sugar moiety at positions 2, 3, 8, 9, 10, and 11 of the sense strand are modified with a 2’-F. In some embodiments, the sense strand has 15 nucleotides that are modified at the 2’ position of the sugar moiety with a 2’-0Me. In some embodiments, the sugar moiety at positions 6, 8, 9, 11, 12, 13, 15, 16, 17,
- the antisense strand has 3 nucleotides that are modified at the 2'-position of the sugar moiety with a 2'-F.
- the sugar moiety at positions 2, 5 and 14 and optionally up to 3 of the nucleotides at positions 1, 3, 7 and 10 of the antisense strand are modified with a 2'-F.
- the sugar moiety at each of the positions at positions 2, 5 and 14 of the antisense strand is modified with the 2'-F.
- the sugar moiety at each of the positions at positions 1, 2, 5 and 14 of the antisense strand is modified with the 2'-F.
- the sugar moiety at each of the positions at positions 2, 4, 5 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 1, 2, 3, 5, 7 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 7 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 1, 2, 3, 5, 10 and 14 of the antisense strand is modified with the 2'-F.
- the sugar moiety at each of the positions at positions 2, 3, 4, 5, 10 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 5, 7, 10 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the antisense strand has 9 nucleotides that are modified at the 2'-position of the sugar moiety with a 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19 of the antisense strand is modified with the 2'-F.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 5, and 14 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2’-O-methyl (2'-0Me), 2’-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'- 0-NMA), and 2’-deoxy-2’-fluoro-P-d-arabinonucleic acid (2'-FANA).
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'- aminoethyl (EA), 2’-O-methyl (2'-0Me), 2’-O-methoxyethyl (2'-M0E), 2'-O-[2- (methylamino)-2-oxoethyl] (2'-0-NMA), and 2’-deoxy-2’-fluoro-P-d-arabinonucleic acid (2'- FANA).
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 1, 2, 5, and 14 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'- 0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 1, 2, 3, 5, 7, and 14 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2- oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).
- a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions
- an lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'- aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2- (methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'- FANA).
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2- oxoethyl] (2 -0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).
- a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with 2'-F.
- a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with 2'-0Me.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2’-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'- 0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).
- a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 8-11 modified with 2'-F. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 3, 5, 8, 10, 12, 13, 15 and 17 modified with 2'-F. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 1-7 and 12-17 or 12-20 modified with 2’0Me.
- a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 2-7 and 12-17 or 12-20 modified with 2’0Me. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 1-6 and 12-17 or 12-20 modified with 2’0Me. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 1, 2, 4, 6, 7, 9, 11, 14, 16 and 18-20 modified with 2’OMe.
- a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety of each of the nucleotides at positions 1-7 and 12-17 or 12-20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2’-aminoethyl (EA), 2'-O- methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O- NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).
- a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety of each of the nucleotides at positions 2-7 and 12-17 or 12-20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O- propylamin, 2'-amino, 2'-ethyl, 2’-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O- methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy-2'- fluoro-P-d-arabinonucleic acid (2'-FANA).
- a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety of each of the nucleotides at positions 1-6 and 12-17 or 12-20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2’-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2- (methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'- FANA).
- a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety at positions 1, 2, 4, 6, 7, 9, 11, 14, 16 and 18- 20 of the sense strand modified with a modification selected from the group consisting of 2'- O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2’ -aminoethyl (EA), 2'-O-methyl (2'-0Me), 2 '-O-m ethoxy ethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy- 2'-fluoro-P-d-arabinonucleic acid (2 -FANA).
- a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 modified with 2'-F.
- a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 modified with 2'-0Me.
- a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 modified with a modification selected from the group consisting of 2'-O- propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2’-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'- O-methoxy ethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy-2'- fluoro-P
- a lipid-conjugated RNAi oligonucleotide described herein comprises a 5’-terminal phosphate.
- the 5'-terminal phosphate groups of the lipid-conjugated RNAi oligonucleotide enhance the interaction with Ago2.
- oligonucleotides comprising a 5 '-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which can limit their bioavailability in vivo.
- an lipid-conjugated RNAi oligonucleotide herein comprises analogs of 5' phosphates that are resistant to such degradation.
- the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate, or a combination thereof.
- the 5' end of a lipid-conjugated RNAi oligonucleotide strand is attached to chemical moiety that mimics the electrostatic and steric properties of a natural 5'- phosphate group (“phosphate mimic”).
- a lipid-conjugated RNAi oligonucleotide herein has a phosphate analog at a 4'-carbon position of the sugar (referred to as a “4'-phosphate analog”). See, e.g., Inti. Patent Application Publication No. WO 2018/045317.
- a lipid- conjugated RNAi oligonucleotide herein comprises a 4'-phosphate analog at a 5'-terminal nucleotide.
- a phosphate analog is an oxymethyl phosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety e.g., at its 4'-carbon) or analog thereof.
- a 4'-phosphate analog is a thiomethyl phosphonate or an aminomethyl phosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the amino methyl group is bound to the 4'-carbon of the sugar moiety or analog thereof.
- a 4'-phosphate analog is an oxymethyl phosphonate.
- an oxymethyl phosphonate is represented by the formula -O-CH2-PO(OH)2,- O-CH2-PO(OR)2, or -O-CH2-POOH(R), in which R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, ClhOCIhCIhSi (CH3)3 or a protecting group.
- the alkyl group is CH2CH3. More typically, R is independently selected from H, CH3 or CH2CH3.
- R is CH3.
- the 4’- phosphate analog is 5’-methoxyphosphonate-4’-oxy. In some embodiments, the 4’-phosphate analog is 4’-oxymethylphosphonate.
- a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand comprising a 4'-phosphate analog at the 5 '-terminal nucleotide, wherein 5’-terminal nucleotide comprises the following structure:
- a lipid-conjugated RNAi oligonucleotide herein comprises a modified internucleotide linkage.
- phosphate modifications or substitutions result in an oligonucleotide that comprises at least about 1 (e.g., at least 1, at least 2, at least 3 or at least 5) modified internucleotide linkage.
- any one of the oligonucleotides disclosed herein comprises about 1 to about 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3 or 1 to 2) modified internucleotide linkages.
- any one of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modified internucleotide linkages.
- a modified internucleotide linkage may be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonate linkage or a boranophosphate linkage.
- at least one modified internucleotide linkage of any one of the oligonucleotides as disclosed herein is a phosphorothioate linkage.
- a lipid-conjugated RNAi oligonucleotide provided herein has a phosphorothioate linkage between one or more of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.
- the oligonucleotide described herein has a phosphorothioate linkage between each of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.
- the oligonucleotide described herein has a phosphorothioate linkage between each of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.
- the oligonucleotide described herein has a phosphorothioate linkage between each of positions 1 and 2 of the sense strand, positions 18 and 19 of the sense strand, positions 19 and 20 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.
- an oligonucleotide conjugate described herein comprises a peptide nucleic acid (PNA).
- PNAs are oligonucleotide mimics in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton, composed of N-(2- aminoethyljglycine units. Nucleobases are linked to this skeleton through a two-atom carboxymethyl spacer.
- an oligonucleotide conjugate described herein comprises a morpholino oligomer (PMO) comprising an internucleotide linkage backbone of methylene morpholine rings linked through phosphorodiamidate groups.
- PMO morpholino oligomer
- a lipid-conjugated RNAi oligonucleotide herein comprises one or more modified nucleobases.
- modified nucleobases also referred to herein as base analogs
- a modified nucleobase is a nitrogenous base.
- a modified nucleobase does not contain nitrogen atom. See, e.g., US Patent Application Publication No. 2008/0274462.
- a modified nucleotide comprises a universal base.
- a modified nucleotide does not contain a nucleobase (abasic).
- a universal base is a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a duplex, can be positioned opposite more than one type of base without substantially altering structure of the duplex.
- a reference single-stranded nucleic acid e.g., oligonucleotide
- a single-stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower Tm than a duplex formed with the complementary nucleic acid.
- the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher Tm than a duplex formed with the nucleic acid comprising the mismatched base.
- Non-limiting examples of universal-binding nucleotides include, but are not limited to, inosine, l-P-D-ribofuranosyl-5-nitroindole and/or l-P-D-ribofuranosyl-3 -nitropyrrole (see, US Patent Application Publication No. 2007/0254362; Van Aerschot et al. (1995) NUCLEIC ACIDS RES. 23:4363-4370; Loakes et al. (1995) NUCLEIC ACIDS RES. 23:2361-66; and Loakes & Brown (1994) NUCLEIC ACIDS RES. 22:4039-43).
- a lipid-conjugated RNAi oligonucleotide comprises at least one nucleotide (e.g.,
- nucleotides 1, 2, 3, 4, 5, 6 or more nucleotides conjugated to one or more targeting ligand(s).
- 1 or more (e.g., 1, 2, 3, 4, 5 or 6) nucleotides of a lipid- conjugated RNAi oligonucleotide disclosed herein are each conjugated to a separate targeting ligand.
- 1 nucleotide of a lipid-conjugated RNAi oligonucleotide herein is conjugated to a separate targeting ligand.
- 2 to 4 nucleotides of a lipid-conjugated RNAi oligonucleotide herein are each conjugated to a separate targeting ligand.
- targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g. , targeting ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5 ' or 3 ' end of the sense or antisense strand) such that the targeting ligands resemble bristles of a toothbrush and the lipid-conjugated RNAi oligonucleotide resembles a toothbrush.
- a lipid-conjugated RNAi oligonucleotide may comprise a stem-loop at either the 5' or 3' end of the sense strand and 1, 2, 3 or 4 nucleotides of the loop of the stem may be individually conjugated to a targeting ligand.
- a lipid-conjugated RNAi oligonucleotide provided by the disclosure comprises a stem-loop at the 3' end of the sense strand, wherein the loop of the stem-loop comprises a triloop or a tetraloop, and wherein the 3 or 4 nucleotides comprising the triloop or tetraloop, respectfully, are individually conjugated to a targeting ligand.
- GalNAc is a high affinity ligand for the ASGPR, which is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalizing and subsequent clearing circulating glycoproteins that contain terminal galactose or GalNAc residues (asialoglycoproteins). Conjugation (either indirect or direct) of GalNAc moieties to oligonucleotide of the instant disclosure can be used to target these oligonucleotides to the ASGPR expressed on cells.
- an oligonucleotide of the instant disclosure is conjugated to at least one or more GalNAc moieties, wherein the GalNAc moieties target the oligonucleotide to an ASGPR expressed on human liver cells (e.g., human hepatocytes).
- the GalNAc moiety target the oligonucleotide to the liver.
- an oligonucleotide of the instant disclosure is conjugated directly or indirectly to a monovalent GalNAc. In some embodiments, the oligonucleotide is conjugated directly or indirectly to more than one monovalent GalNAc (i.e., is conjugated to
- an oligonucleotide is conjugated to one or more bivalent GalNAc, trivalent GalNAc or tetravalent GalNAc moieties.
- 1 or more (e.g., 1, 2, 3, 4, 5 or 6) nucleotides of an oligonucleotide are each conjugated to a GalNAc moiety.
- 2 to 4 nucleotides of a tetraloop are each conjugated to a separate GalNAc.
- 1 to 3 nucleotides of a triloop are each conjugated to a separate GalNAc.
- targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5' or 3' end of the sense or antisense strand) such that the GalNAc moieties resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush.
- GalNAc moieties are conjugated to a nucleotide of the sense strand.
- four (4) GalNAc moieties can be conjugated to nucleotides in the tetraloop of the sense strand where each GalNAc moiety is conjugated to 1 nucleotide.
- the tetraloop is any combination of adenine and guanine nucleotides.
- a lipid-conjugated RNAi oligonucleotide herein comprises a monovalent GalNAc attached to a guanine nucleotide referred to as [ademG-GalNAc] or 2'- aminodiethoxymethanol-Guanine-GalNAc, as depicted below:
- a lipid-conjugated RNAi oligonucleotide herein comprises a monovalent GalNAc attached to an adenine nucleotide, referred to as [ademA-GalNAc] or 2'- aminodi ethoxymethanol- Adenine-GalNAc, as depicted below:
- a targeting ligand is conjugated to a nucleotide using a click linker.
- an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal- based linkers are disclosed, for example, in Inti. Patent Application Publication No. WO 2016/100401.
- the linker is a labile linker. However, in other embodiments, the linker is stable.
- a loop comprising from 5' to 3' the nucleotides GAAA, in which GalNAc moi eties are attached to nucleotides of the loop using an acetal linker.
- Such a loop may be present, for example, at positions 27-30 of the sense strand.
- a targeting ligand is conjugated to a nucleotide using a click linker.
- an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in Inti. Patent Application Publication No. WO 2016/100401.
- the linker is a labile linker. However, in other embodiments, the linker is a stable linker.
- a duplex extension (e.g., of up to 3, 4, 5 or 6 bp in length) is provided between a targeting ligand (e.g., a GalNAc moiety) and a lipid-conjugated RNAi oligonucleotide.
- a targeting ligand e.g., a GalNAc moiety
- a lipid-conjugated RNAi oligonucleotide herein does not have a GalNAc conjugated thereto.
- any of the lipid moieties described herein are conjugated to a nucleotide of the sense strand of the oligonucleotide.
- a lipid moiety is conjugated to a terminal position of the oligonucleotide.
- the lipid moiety is conjugated to the 5’ terminal nucleotide of the sense strand.
- the lipid moiety is conjugated to the 3’ terminal nucleotide of the sense strand.
- the lipid moiety is conjugated to an internal nucleotide on the sense strand.
- An internal position is any nucleotide position other than the two terminal positions from each end of the sense strand.
- the lipid moiety is conjugated to one or more internal positions of the sense strand.
- the lipid moiety is conjugated to position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 of a sense strand.
- the lipid moiety is conjugated to position 1 of the sense strand.
- the lipid moiety is conjugated to position 4 of the sense strand.
- the lipid moiety is conjugated to position 8 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 12 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 13 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 18 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 20 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 23 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 28 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 29 of the sense strand.
- the lipid moiety is conjugated to position 30 of the sense strand.
- a lipid-conjugated RNAi oligonucleotide described herein comprises at least one nucleotide conjugated with one or more lipid moieties.
- the one or more lipid moieties are conjugated to the same nucleotide.
- the one or more lipid moieties are conjugated to different nucleotides.
- one, two, three, four, five, or six lipid moieties are conjugated to the oligonucleotide.
- one or more lipid moieties are conjugated to an adenine nucleotide.
- one or more lipid moieties are conjugated to a guanine nucleotide. In some embodiments, one or more lipid moieties are conjugated toa cytosine nucleotide. In some embodiments, one or more lipid moieties are conjugated to a thymine nucleotide. In some embodiments, one or more lipid moieties are conjugated to a uracil nucleotide.
- the lipid moiety is a hydrocarbon chain. In some embodiments, the hydrocarbon chain is saturated. In some embodiments, the hydrocarbon chain is unsaturated. In some embodiments, the hydrocarbon chain is branched. In some embodiments, the hydrocarbon chain is straight. In some embodiments, the lipid moiety is a C8-C30 hydrocarbon chain.
- the lipid moiety is a C8:0, C10:0, Cl 1 :0, C12:0, C14:0, C16:0, C17:0, C18:0, C18:l, C18:2, C22:5, C22:0, C24:0, C26:0, C22:6, C24:l, diacyl C16:0 or diacyl C18: l.
- the lipid moiety is a C 16 hydrocarbon chain.
- the lipid moiety is conjugated to the oligonucleotide via a linker.
- a nucleotide of the lipid-conjugated oligonucleotide is represented by formula Il-b or II-c:
- L 1 is a covalent bond, a monovalent or a bivalent saturated or unsaturated, straight or branched Ci-50 hydrocarbon chain, wherein 0-10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, - R 4 is hydrogen, R A , or a suitable amine protection group; and
- R 5 is adamantyl, or a saturated or unsaturated, straight, or branched C 1-50 hydrocarbon chain, wherein 0-10 methylene units of the hydrocarbon chain are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) 2 -, -P(O)OR-, or -P(S)OR.
- R 5 is selected from
- lipid-conjugated RNAi oligonucleotide In certain embodiments of the lipid-conjugated RNAi oligonucleotide,
- R 5 is selected from In some embodiments, R 5 is selected from
- a nucleotide of the lipid-conjugated RNAi oligonucleotide is represented by formula Il-Ib or II-Ic:
- B is a nucleobase or hydrogen; m is 1-50;
- X 1 is -O-, or -S-;
- Y is hydrogen
- R 3 is hydrogen, or a suitable protecting group
- X 2 is O, or S
- X 3 is -O-, -S-, or a covalent bond
- Y 1 is a linking group attaching to the 2'- or 3 '-terminal of a nucleoside, a nucleotide, or an oligonucleotide
- Y 2 is hydrogen, a phosphoramidite analogue, an internucleotide linking group attaching to the 5 '-terminal of a nucleoside, a nucleotide, or an oligonucleotide, or a linking group attaching to a solid support;
- R 5 is adamantyl, or a saturated or unsaturated, straight, or branched C 1-50 hydrocarbon chain, wherein 0-10 methylene units of the hydrocarbon chain are independently replaced by -
- R is hydrogen, a suitable protecting group, or an optionally substituted group selected from Ci- 6 aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated heterocyclic having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
- the lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
- the oligonucleotide of the oligonucleotide-ligand conjugate is a double-stranded molecule.
- the oligonucleotide is an RNAi molecule.
- the double stranded oligonucleotide comprises a stem loop.
- the stem loop is set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2.
- the ligand is conjugated to any of the nucleotides in the loop of the stem loop.
- the ligand is conjugated to any of the nucleotides in the stem of the stem loop.
- the ligand is conjugated to the first nucleotide from 5’ to 3’ in the loop. In some embodiments, the ligand is conjugated to the second nucleotide from 5’ to 3’ in the loop. In some embodiments, the ligand is conjugated to the third nucleotide from 5’ to 3’ in the loop. In some embodiments, the ligand is conjugated to the fourth nucleotide from 5’ to 3’ in the loop. In some embodiments, the ligand is conjugated to one, two, three, or four of the nucleotides in the loop. In some embodiments, the ligand is conjugated to three of the nucleotides in the stem loop.
- the stem loop is 16 nucleotides in length.
- the ligand is conjugated to the third nucleotide from 5’ to 3’ in the stem loop. In some embodiments, the ligand is conjugated to the eighth nucleotide from 5’ to 3’ in the stem loop. In some embodiments, the ligand is conjugated to the ninth nucleotide from 5’ to 3’ in the stem loop. In some embodiments, the ligand is conjugated to the tenth nucleotide from 5’ to 3’ in the stem loop.
- the lipid-conjugated RNAi oligonucleotide comprises a sense strand of 20 nucleotides with positions numbered 1-20 from 5’ to 3’. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 1 of a 20- nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 4 of a 20-nucleotide sense strand.
- the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 8 of a 20-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 12 of a 20-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 13 of a 20-nucleotide sense strand.
- the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 18 of a 20-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a lipid conjugated to position 20 of a 20-nucleotide sense strand.
- the lipid-conjugated RNAi oligonucleotide comprises a sense strand of 36 nucleotides with positions numbered 1-36 from 5’ to 3’. In some embodiments, the lipid-conjugated RNAi oligonucleotide- comprises a lipid conjugated to position 1 of a 36- nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide- comprises a lipid conjugated to position 4 of a 36-nucleotide sense strand.
- the lipid-conjugated RNAi oligonucleotide- comprises a lipid conjugated to position 8 of a 36-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide- comprises a lipid conjugated to position 12 of a 36-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide- comprises a lipid conjugated to position 13 of a 36-nucleotide sense strand.
- the lipid- conjugated RNAi oligonucleotide- comprises a lipid conjugated to position 18 of a 36- nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide- comprises a lipid conjugated to position 20 of a 36-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide- comprises a lipid conjugated to position 23 of a 36-nucleotide sense strand.
- the lipid-conjugated RNAi oligonucleotide- comprises a lipid conjugated to position 28 of a 36-nucleotide sense strand. In some embodiments, the lipid-conjugated RNAi oligonucleotide- comprises a lipid conjugated to position 29 of a 36-nucleotide sense strand. In some embodiments, the lipid- conjugated RNAi oligonucleotide- comprises a lipid conjugated to position 30 of a 36- nucleotide sense strand.
- the lipid-conjugated RNAi oligonucleotide comprises a nucleotide conjugated with a fatty acid.
- the fatty acid is a saturated fatty acid.
- the fatty acid is an unsaturated fatty acid.
- lipid-conjugated RNAi oligonucleotide comprises a nucleotide conjugated with a lipid.
- the lipid is a carbon chain.
- the carbon chain is saturated.
- the carbon chain is unsaturated.
- the lipid- conjugated RNAi oligonucleotide comprises a nucleotide conjugated with a 16-carbon (Cl 6) lipid.
- the C16 lipid comprises at least one double bond.
- the oligonucleotide of the lipid-conjugated RNAi oligonucleotide is conjugated to a C16 lipid as shown in:
- the lipid-conjugated RNAi oligonucleotide comprises a sense strand of 20 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises an anti-sense strand of 22 nucleotides in length. In some embodiments, the sense strand is 20 nucleotides in length and the antisense strand in 22 nucleotides in length.
- lipid-conjugated RNAi oligonucleotide comprises a sense strand of 20 nucleotides in length and an antisense strand in 22 nucleotides in length, wherein the sense and antisense strands form a duplex region of 20 base pairs.
- the 3’ end of the sense strand is a blunt end.
- the 5’ end of the antisense strand is a blunt end.
- the 3’ end of the antisense strand comprises an overhang.
- the overhang is 2 nucleotides in length. In some embodiments the overhang is GG.
- the lipid-conjugated RNAi oligonucleotide comprises one or more 2’ modifications.
- the 2’ modifications are selected from 2’-fluoro and 2 ’-methyl.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3 ’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one hydrocarbon chain conjugated to an internal nucleotide of the sense strand (e.g., nucleotide at position 7).
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand and a sense strand described herein, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to an internal nucleotide of the sense strand (e.g., nucleotide at position 7).
- not all internal nucleotides are suitable for lipid conjugation for delivery of an RNAi oligonucleotide to an astrocyte of the CNS.
- conjugation at positions 9 or 10 of a sense strand numbered from 5’ to 3’ is not suitable for delivery of an RNAi oligonucleotide to an astrocyte of the CNS.
- lipid conjugation at an internal position of a sense strand numbered from 5’ to 3’ excludes positions 9 and 10.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one hydrocarbon chain conjugated to an internal nucleotide of the sense strand (e.g., nucleotide at position 7).
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 22 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to an internal nucleotide of the sense strand (e.g., nucleotide at position 7).
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one hydrocarbon chain conjugated to an internal nucleotide of the sense strand (e.g., nucleotide at position 7).
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22- 24 nucleotides and a sense strand of 20-22 nucleotides described herein, wherein the antisense and sense strands form an asymmetric duplex region of 20-22 base pairs having an overhang on the 3’ end of the antisense strand and a blunt-end at the 3’ end of the oligonucleotide, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to an internal nucleotide of the sense strand (e.g., nucleotide at position 7).
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- Antisense Strand 5’ - [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX]
- the lipid-conjugated RNAi oligonucleotide comprises a sense strand of 36 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises an anti-sense strand of 22 nucleotides in length. In some embodiments, the sense strand is 36 nucleotides in length and the antisense strand in 22 nucleotides in length.
- a lipid-conjugated RNAi oligonucleotide comprises a sense strand of 36 nucleotides in length and an antisense strand in 22 nucleotides in length, wherein the sense and antisense strands form a duplex region of 20 base pairs.
- the 3’ end of the sense strand comprises a stem-loop. In some embodiments, the 3’ end of the sense strand comprises a tetraloop. In some embodiments, the 3’ end of the sense strand comprises a stem-loop comprising the sequence of SEQ ID NO: 32. In some embodiments, the 3’ end of the antisense strand comprises an overhang. In some embodiments, the overhang is 2 nucleotides in length. In some embodiments the overhang is GG.
- a lipid-conjugated RNAi oligonucleotide comprises a sense strand comprising a stem-loop at its 3’ end and at least one hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises a sense strand comprising a stem-loop at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide of the sense strand.
- a lipid-conjugated RNAi oligonucleotide comprises a sense strand comprising a stem-loop at its 3’ end and at least one C16 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises a sense strand comprising a tetraloop and at least one C16 hydrocarbon chain conjugated to a nucleotide of the tetraloop.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C 16 hydrocarbon chain conjugated to the first nucleotide (Position 1 from 5’ > 3’) of the sense strand.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20- 36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the fourth nucleotide (Position 4 from 5’ > 3’) of the sense strand.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22- 24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the eight nucleotide (Position 8 from 5’ > 3’) of the sense strand.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C 16 hydrocarbon chain conjugated to the twelth nucleotide (Position 12 from 5’ > 3’) of the sense strand.
- a lipid- conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one Cl 6 hydrocarbon chain conjugated to the thirteenth nucleotide (Position 13 from 5’ > 3’) of the sense strand.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the eighteenth nucleotide (Position 18 from 5’ > 3’) of the sense strand.
- a lipid- conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the twentieth nucleotide (Position 20 from 5’ > 3’) of the sense strand.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the twenty -third nucleotide (Position 23 from 5’ > 3’) of the sense strand.
- a lipid- conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the twenty-eighth nucleotide (Position 28 from 5’ > 3’) of the sense strand.
- a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the twenty-ninth nucleotide (Position 29 from 5’ > 3’) of the sense strand.
- a lipid- conjugated RNAi oligonucleotide comprises an antisense strand of 22-24 nucleotides and a sense strand of 20-36 nucleotides described herein, wherein the antisense and sense strands form a duplex region of 20-22 base pairs, wherein the sense strand comprises at least one C16 hydrocarbon chain conjugated to the thirtieth nucleotide (Position 30 from 5’ > 3’) of the sense strand.
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- Antisense Strand 5’ - [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX]
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of Sense Strand: 5’- [mXs][mX][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][fX][fX]
- Antisense Strand 5’ - [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX]
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte target gene comprises the modification pattern of
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3 ’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 2, Position 3, Position 6, Position 13, Position 14, Position 15, Position 19, Position 20, Position 23, Position 28, Position 29, or Position 30 of the sense strand, wherein positions are numbered 5’ to 3’.
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stemloop at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 1, Position 4, Position 8, Position 12, Position 13, Position 18, Position 20, Position 23, Position 28, Position 29, or Position 30 of the sense strand, wherein positions are numbered 5’ to 3’.
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a stem-loop at its 3 ’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 1, Position 4, Position 23, Position 28, Position 29, or Position 30 of the sense strand, wherein positions are numbered 5’ to 3’.
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3 ’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 1, Position 4, Position 12, Position 13, Position 18, Position 20, Position 23, Position 28, Position 29, or Position 3 Oof the sense strand, wherein positions are numbered 5’ to 3’.
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3 ’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 23 of the sense strand, wherein positions are numbered 5’ to 3’.
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the spinal cord comprises a sense strand comprising a blunt-end at its 3 ’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 1, Position 4, Position 8, Position 12, Position 13, Position 18, or Position 20 of the sense strand, wherein positions are numbered 5’ to 3’.
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt- end at its 3’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 1, Position 4, Position 8, Position 12, Position 13, Position 18, or Position 20 of the sense strand, wherein positions are numbered 5’ to 3’.
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a blunt-end at its 3 ’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 4, Position 12, Position 13, Position 18, or Position 20 of the sense strand, wherein positions are numbered 5’ to 3’.
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3 ’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 1, Position 4, Position 12, Position 13, Position 18, or Position 20 of the sense strand, wherein positions are numbered 5’ to 3’.
- a lipid-conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the frontal cortex comprises a sense strand comprising a blunt-end at its 3 ’ end and at least one hydrocarbon chain conjugated to a nucleotide at Position 4 of the sense strand, wherein positions are numbered 5’ to 3’.
- position numbers described throughout are based on numbering from the 5’ end to the 3’ end, for example, the terminal nucleotide at the 5’ end is Position 1.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 1 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 4 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 8 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 12 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 13 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 18 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 20 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 28 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 29 of the sense strand,.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 30 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 1 of the sense strand In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 4 of the sense strand,.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla a sense strand comprising a stemloop at its 3’ end and a lipid conjugated to Position 18 of the sense strand In some embodiments, a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 19 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 20 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stemloop at its 3’ end and a lipid conjugated to Position 23 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 28 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 29 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stemloop at its 3’ end and a lipid conjugated to Position 30 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 1 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 4 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 28 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 29 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 1 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 4 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 12 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 13 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 18 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 20 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 28 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 29 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 30 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 1 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 4 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 8 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 12 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 13 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 18 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 20 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 28 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 29 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 30 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stemloop at its 3’ end and a lipid conjugated to Position 1 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 4 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 8 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 12 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 13 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 18 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 20 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 28 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 29 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 30 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 1 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 4 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 28 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 29 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 1 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 4 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 12 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 13 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 18 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3 ’ end and a lipid conjugated to Position 20 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3 ’ end and a lipid conjugated to Position 28 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 29 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 30 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the frontal cortex comprises a sense strand comprising a stem-loop at its 3’ end and a lipid conjugated to Position 23 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 1 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 4 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord a sense strand comprising a blunt-end at its 3 ’ end and a lipid conjugated to Position 8 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 12 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 13 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 18 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 20 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt- end at its 3’ end and a lipid conjugated to Position 1 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 4 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 8 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 12 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 13 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 18 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 20 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 4 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum a sense strand comprising a blunt-end at its 3 ’ end and a lipid conjugated to Position 12 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum a sense strand comprising a blunt- end at its 3’ end and a lipid conjugated to Position 13 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum a sense strand comprising a blunt-end at its 3 ’ end and a lipid conjugated to Position 18 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum a sense strand comprising a blunt- end at its 3’ end and a lipid conjugated to Position 20 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 1 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 4 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 12 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 13 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 18 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 20 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the frontal cortex comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 4 of the sense strand.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 1 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 4 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 8 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 12 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 13 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 18 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the lumbar spinal cord comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 20 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt- end at its 3’ end and a lipid conjugated to Position 1 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 4 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt- end at its 3’ end and a lipid conjugated to Position 8 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 12 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt- end at its 3’ end and a lipid conjugated to Position 13 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 18 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the medulla comprises a sense strand comprising a blunt- end at its 3’ end and a lipid conjugated to Position 20 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 4 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 12 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 13 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 18 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the cerebellum comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 20 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 1 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 4 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 12 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 13 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 18 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the hypothalamus comprises a sense strand comprising a blunt-end at its 3 ’ end and a lipid conjugated to Position 20 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- a lipid conjugated RNAi oligonucleotide for reducing expression of an astrocyte mRNA in the frontal cortex comprises a sense strand comprising a blunt-end at its 3’ end and a lipid conjugated to Position 4 of the sense strand, and wherein the oligonucleotide comprises at least one modified nucleotide.
- nucleic acids and analogues thereof comprising lipid conjugate described herein can be made using a variety of synthetic methods known in the art, including standard phosphoramidite methods. Any phosphoramidite synthesis method can be used to synthesize the provided nucleic acids of this disclosure.
- phosphoramidites are used in a solid phase synthesis method to yield reactive intermediate phosphite compounds, which are subsequently oxidized using known methods to produce phosphonate-modified oligonucleotides, typically with a phosphodiester or phosphorothioate intemucleotide linkages.
- the oligonucleotide synthesis of the present disclosure can be performed in either direction: from 5' to 3' or from 3' to 5' using art known methods.
- the method for synthesizing a provided nucleic acid comprises (a) attaching a nucleoside or analogue thereof to a solid support via a covalent linkage; (b) coupling a nucleoside phosphoramidite or analogue thereof to a reactive hydroxyl group on the nucleoside or analogue thereof of step (a) to form an internucleotide bond there between, wherein any uncoupled nucleoside or analogue thereof on the solid support is capped with a capping reagent; (c) oxidizing said internucleotide bond with an oxidizing agent; and (d) repeating steps (b) to (c) iteratively with subsequent nucleoside phosphoramidites or analogue thereof to form a nucleic acid or analogue thereof, wherein at least the nucleoside or analogue thereof of step (a), the nucleoside phosphoramidite or analogue thereof of step (b) or at least one of the subsequent nucleoside or
- an oligonucleotide is prepared comprising 1-3 nucleic acid or analogues thereof comprising lipid conjugates units on a tetraloop.
- nucleic acids, and analogues thereof of the present disclosure are generally prepared according to Scheme A, Scheme Al and Scheme B set forth below:
- a nucleic acid or analogue thereof of formula 1-1 is conjugated with one or more ligand/lipophilic compound to form a compound of formula I or la comprising one more ligand/lipid conjugates.
- conjugation is performed through an esterification or amidation reaction between a nucleic acid or analogue thereof of formula 1-1 or I-la and one or more adamantyl and/or lipophilic compound (e.g., fatty acid) in series or in parallel by known techniques in the art.
- nucleic acid or analogue thereof of formula I or la can then be deprotected to form a compound of formula 1-2 or I-2a and protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula 1-3 or I-3a.
- a suitable hydroxyl protecting group e.g., DMTr
- nucleic acid-ligand conjugates of formula 1-3 or I-3a can be covalently attached to a solid support (e.g., through a succinic acid linking group) to form a solid support nucleic acid-ligand conjugate or analogue thereof of formula 1-4 or I-4a comprising one or more adamantyl and/or lipid conjugate.
- a nucleic acidligand conjugates of formula 1-3 or I-3a can react with a P(III) forming reagent (e.g., 2- cyanoethyl M -di-i sopropylchlorophosphoramidite) to form a nucleic acid or analogue thereof of formula 1-5 or I-5a comprising a P(III) group.
- a nucleic acid-ligand conjugate or analogue thereof of formula 1-5 or I-5a can then be subjected to oligomerization forming conditions preformed using known and commonly applied processes to prepare oligonucleotides in the art.
- the compound of formula 1-5 or I-5a is coupled to a solid supported nucleic acid-ligand conjugate or analogue thereof bearing a 5’-hydroxyl group.
- Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and/or cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, including one or more lipid conjugate nucleotide units represented by a compound of formula II-l or Il-Ia.
- Each of B, E, L, ligand, LC, n, PG 1 , PG 2 , PG 4 , R 1 , R 2 , R 3 , X, X 1 , X 2 , X 3 , and Z is as defined above and described herein.
- a nucleic acid or analogue thereof of formula 1-1 can be deprotected to form a compound of formula 1-6, protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula 1-7, and reacted with a P(III) forming reagent (e.g., 2-cyanoethyl A,A-di-isopropylchlorophosphoramidite) to form a nucleic acid or analogue thereof of formula 1-8 comprising a P(III) group.
- a suitable hydroxyl protecting group e.g., DMTr
- P(III) forming reagent e.g., 2-cyanoethyl A,A-di-isopropylchlorophosphoramidite
- a nucleic acid or analogue thereof of formula 1-8 is subjected to oligomerization forming conditions preformed using known and commonly applied processes to prepare oligonucleotides in the art.
- the compound of formula 1-8 is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group.
- Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and/or cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths represented by a compound of formula II-2.
- An oligonucleotide of formula II-2 can then be conjugated with one or more ligands e.g., adamantyl, or lipophilic compound (e.g, fatty acid) to form a compound of formula II-l comprising one or more ligand conjugates.
- ligands e.g., adamantyl, or lipophilic compound (e.g, fatty acid)
- conjugation is performed through an esterification or amidation reaction between a nucleic acid or analogue thereof of formula II-2 and one or more adamantyl or fatty acid in series or in parallel by known techniques in the art.
- nucleic acids, and analogues thereof of the present disclosure are prepared according to Scheme C and Scheme D set forth below:
- nucleic acid or analogue thereof of formula Cl is protected to form a compound of formula C2.
- Nucleic acid or analogue thereof of formula C2 is then alkylated (e.g., using DMSO and acetic acid via the Pummerer rearrangement) to form a monothioacetal compound of formula C3.
- nucleic acid or analogue thereof of formula C3 is coupled with C4 under appropriate conditions (e.g., mild oxidizing conditions) to form a nucleic acid or analogue thereof of formula C5.
- Nucleic acid or analogue thereof of formula C5 can then be deprotected to form a compound of formula C6 and coupled with a ligand (adamantyl or lipophilic compound (e.g., a fatty acid)) of formula C7 under appropriate amide forming conditions (e.g., HATU, DIPEA), to form a nucleic acid-ligand conjugate or analogue thereof of formula I-b comprising a lipid conjugate of the disclosure.
- Nucleic acid-ligand conjugate or analogue thereof of formula I-b can then be deprotected to form a compound of formula C8 and protected with a suitable hydroxyl protecting group e.g., DMTr) to form a compound of formula C9.
- nucleic acid, or analogue thereof of formula C9 can be covalently attached to a solid support e.g., through a succinic acid linking group) to form a solid support nucleic acid-ligand conjugate or analogue thereof of formula CIO comprising a ligand conjugate (adamantyl or lipid moiety) of the disclosure.
- a nucleic acid-ligand conjugate or analogue thereof of formula C9 can reacted with a P(III) forming reagent e.g., 2-cyanoethyl A,A-di-isopropylchlorophosphoramidite) to form a nucleic acidligand conjugate or analogue thereof of formula Cll comprising a P(III) group.
- a nucleic acidligand conjugate or analogue thereof of formula Cll can then be subjected to oligomerization forming conditions preformed using known and commonly applied processes to prepare oligonucleotides in the art.
- the compound of formula Cll is coupled to a solid supported nucleic acid-ligand conjugate or analogue thereof bearing a 5 ’-hydroxyl group.
- Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and/or cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, including one or more adamantyl and/or lipid conjugate nucleotide units represented by a compound of formula II-b-3.
- Each of B, E, L 2 , PG 1 , PG 2 , PG 3 , PG 4 , R 1 , R 2 , R 3 , R 4 , R 5 , X 1 , X 2 , X 3 , V, W, and Z is as defined above and described herein.
- Each of B, E, L 2 , PG 1 , PG 2 , PG 3 , PG 4 , R 1 , R 2 , R 3 , R 4 , R 5 , X 1 , X 2 , X 3 , V, W, and Z is as defined above and described herein.
- a nucleic acid or analogue thereof of formula C5 can be selectively deprotected to form a compound of formula DI, protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula D2, and reacted with a P(III) forming reagent (e.g., 2-cyanoethyl A,A-di- isopropylchlorophosphoramidite) to form a nucleic acid or analogue thereof of formula D3.
- a nucleic acid or analogue thereof of formula D3 is subjected to oligomerization forming conditions preformed using known and commonly applied processes to prepare oligonucleotides in the art.
- the compound of formula D3 is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group. Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and/or cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, represented by a compound of formula D4.
- An oligonucleotide of formula D4 can then be deprotected to form a compound of formula D5 and coupled with a hydrophobic ligand (e.g., adamantyl or a lipophilic moiety) to form a compound of formula C7 (e.g., adamantyl or a fatty acid) under appropriate amide forming conditions (e.g., HATU, DIPEA), to form an oligonucleotide of formula II-b-3 comprising a ligand (e.g, adamantyl or a fatty acid) conjugate of the disclosure.
- a hydrophobic ligand e.g., adamantyl or a lipophilic moiety
- C7 e.g., adamantyl or a fatty acid
- appropriate amide forming conditions e.g., HATU, DIPEA
- nucleic acid or analogues thereof of the disclosure such as aliphatic groups, alcohols, carboxylic acids, esters, amides, aldehydes, halogens, and nitriles can be interconverted by techniques well known in the art including, but not limited to reduction, oxidation, esterification, hydrolysis, partial oxidation, partial reduction, halogenation, dehydration, partial hydration, and hydration. See for example, “MARCH’S ADVANCED ORGANIC CHEMISTRY”, (5 th Ed., Ed.: Smith, M.B.
- the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugate, said lipid conjugate unit represent by formula II-a-1: or a pharmaceutically acceptable salt thereof, comprising the steps of:
- oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art.
- the compound of formula I-5a is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group.
- Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, represented by a compound of formula Il-la comprising a lipid conjugate of the disclosure.
- the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugate, further comprising preparing a nucleic acid or analogue thereof of formula I-5a:
- nucleic acid or analogue thereof of formula I-3a with a P(III) forming reagent to form a nucleic acid or analogue thereof of formula I-5a, wherein each of B, E, L, LC, n, PG 4 , R 1 , R 2 , R 3 , X, X 1 , X 2 , X 3 , E, and Z is as defined above and described herein.
- PG 1 and PG 2 of a compound of formula la comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anion.
- reagents providing fluoride anion for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-A-butylammonium fluoride, and the like.
- a compound of formula I-2a is protected with a suitable hydroxyl protecting group.
- the protecting group PG 4 used for protection of the 5 ’-hydroxyl group of a compound of formula I-2a includes an acid labile protecting group such as trityl, 4-methyoxytrityl, 4,4’-dimethyoxytrityl, 4,4’,4”-trimethyoxytrityl, 9-phenyl- xanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like.
- the acid labile protecting group is suitable for deprotection during both solutionphase and solid-phase synthesis of acid-sensitive nucleic acids or analogues thereof using for example, dichloroacetic acid or trichloroacetic acid.
- a P(III) forming reagent is a phosphorus reagent that is reacted to for a phosphorus (III) compound.
- the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate.
- the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite.
- step (d) above is preformed using 7V,7V-dimethylphosphoramic dichloride as a P(V) forming reagent.
- the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugates, further comprising preparing a nucleic acid-lipid conjugate or analogue thereof of formula la: or a salt thereof, comprising the steps of:
- a nucleic acid or analogue thereof of formula I-la is conjugated with one or more lipophilic compounds to form a compound of formula la comprising one more lipid conjugates of the disclosure.
- conjugation is performed through an esterification or amidation reaction between a nucleic acid or analogue thereof of formula I-la and one or more fatty acids in series or in parallel by known techniques in the art.
- conjugation is performed under suitable amide forming conditions to afford a compound of formula I comprising one more lipid conjugates.
- Suitable amide forming conditions can include the use of an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
- an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
- conjugation of a lipophilic compound can be accomplished by any one of the cross-coupling technologies described in Table A herein.
- the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugate, said lipid conjugate unit represent by formula II-l: or a pharmaceutically acceptable salt thereof, comprising the steps of:
- step (b) conjugating one or more lipophilic compounds to an oligonucleotide of formula II-2 to form an oligonucleotide of formula II-l comprising one or more lipid conjugates.
- an oligonucleotide of formula II-2 is conjugated with one or more lipophilic compounds to form an oligonucleotide of formula II-l comprising one more lipid conjugates of the disclosure.
- conjugation is performed through an esterification or amidation reaction between an oligonucleotide of formula II-2 and one or more fatty acids in series or in parallel by known techniques in the art.
- conjugation is performed under suitable amide forming conditions to afford an oligonucleotide of formula II-l comprising one more lipid conjugates.
- suitable amide forming conditions can include the use of an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
- conjugation of a lipophilic compound can be accomplished by any one of the cross-coupling technologies described in Table A herein.
- the present disclosure provides a method for preparing an oligonucleotide comprising a unit represent by formula II-2: or a pharmaceutically acceptable salt thereof, comprising the steps of:
- oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art.
- the compound of formula 1-8 is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group.
- Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, represented by a compound of formula II-2.
- the present disclosure provides a method for preparing a nucleic acid or analogue thereof comprising one or more lipid conjugate, further comprising preparing a nucleic acid or analogue thereof of formula 1-8:
- PG 1 and PG 2 of a compound of formula 1-1 comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anion.
- reagents providing fluoride anion for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-A-butylammonium fluoride, and the like.
- a compound of formula 1-6 is protected with a suitable hydroxyl protecting group.
- the protecting group PG 4 used for protection of the 5 ’-hydroxyl group of a compound of formula 1-6 includes an acid labile protecting group such as trityl, 4-methyoxytrityl, 4,4’-dimethyoxytrityl, 4,4’,4”-trimethyoxytrityl, 9-phenyl- xanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like.
- the acid labile protecting group is suitable for deprotection during both solutionphase and solid-phase synthesis of acid-sensitive nucleic acids or analogues thereof using for example, dichloroacetic acid or trichloroacetic acid.
- a P(III) forming reagent is a phosphorus reagent that is reacted to for a phosphorus (III) compound.
- the P(III) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate.
- the P(III) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite.
- step (d) above is preformed using 7V,7V-dimethylphosphoramic dichloride as a P(V) forming reagent.
- the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising one or more adamantyl and/or lipid moieties, said conjugate unit represented by formula II-b-3:
- oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art.
- the compound of formula Cll is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group.
- Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and cleavage from the solid support to provide an oligonucleotide-ligand conjugate of various nucleotide lengths, with one or more nucleic acid-ligand conjugate units, wherein each unit is represented by a compound of formula II-b-3 comprising an adamantyl or lipid moiety of the disclosure.
- the method for preparing an oligonucleotide of formula II-b-3 comprising one or more lipid conjugate further comprises preparing a nucleic acid-ligand conjugate or analogue thereof of formula Cll: or a salt thereof, comprising the steps of:
- step (d) treating said nucleic acid-ligand conjugate or analogue thereof of formula C9 with a P(III) forming reagent to form a nucleic acid or analogue thereof of formula Cll.
- PG 1 and PG 2 of a compound of formula I-b comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anion.
- reagents providing fluoride anion for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-A- butylammonium fluoride, and the like.
- a compound of formula C8 is protected with a suitable hydroxyl protecting group.
- the protecting group PG 4 used for protection of the 5 ’-hydroxyl group of a compound of formula C8 includes an acid labile protecting group such as trityl, 4-methyoxytrityl, 4,4’-dimethyoxytrityl, 4,4’,4”-trimethyoxytrityl, 9-phenyl- xanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like.
- the acid labile protecting group is suitable for deprotection during both solutionphase and solid-phase synthesis of acid-sensitive nucleic acids or analogues thereof using for example, dichloroacetic acid or trichloroacetic acid.
- a P(III) forming reagent is a phosphorus reagent that is reacted to for a phosphorus (III) compound.
- the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate.
- the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite.
- step (d) above is preformed using 7V,7V-dimethylphosphoramic dichloride as a P(V) forming reagent.
- the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate of formula II-b-3 comprising one or more nucleic acid-ligand conjugate units each comprising one or more adamantyl or lipid moieties, further comprising preparing a nucleic acid-ligand conjugate or analogue thereof of formula I-b:
- step (b) conjugating a lipophilic compound to a nucleic acid or analogue thereof of formula C6 to form a nucleic acid-ligand conjugate or analogue thereof of formula I-b comprising one or more adamantyl and/or lipid conjugates.
- conjugation is performed under suitable amide forming conditions to afford a compound of formula I-b comprising an adamantyl and/or lipid conjugate.
- Suitable amide forming conditions can include the use of an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
- an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
- the amide forming conditions comprise HATU and DIPEA or TEA.
- a nucleic acid-ligand conjugate or analogue thereof of formula C6 is provided in salt form (e.g., a fumarate salt) and is first converted to the free base (e.g., using sodium bicarbonate) before preforming the conjugation step.
- salt form e.g., a fumarate salt
- free base e.g., sodium bicarbonate
- the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate of formula II-b-3 comprising one or more nucleic acid-ligand conjugate units, further comprises preparing a nucleic acid-ligand conjugate or analogue thereof of formula C6: C6 or a salt thereof, comprising the steps of:
- step (e) deprotecting said nucleic acid or analogue thereof of formula C5 to form a nucleic acidligand conjugate or analogue thereof of formula C6.
- step (b) PG 1 and PG 2 groups of formula C2 are taken together with their intervening atoms to form a cyclic diol protecting group, such as a cyclic acetal or ketal.
- Such groups include methylene, ethylidene, benzylidene, isopropylidene, cyclohexylidene, and cyclopentylidene, silylene derivatives such as di-t-butylsilylene and 1,1,3,3-tetraisopropylidisiloxanylidene, a cyclic carbonate, a cyclic boronate, and cyclic monophosphate derivatives based on cyclic adenosine monophosphate (i.e., cAMP).
- the cyclic diol protection group is 1, 1,3,3- tetraisopropylidisiloxanylidene prepared from the reaction of a diol of formula Cl and 1,3- dichloro-l,l,3,3-tetraisopropyldisiloxane under basic conditions.
- a nucleic acid or analogue thereof of formula C2 is alkylated with a mixture of DMSO and acetic anhydride under acidic conditions.
- a mixture of DMSO and acetic anhydride in the presence of acetic acid forms (methylthio)methyl acetate in situ via the Pummerer rearrangement which then reacts with the hydroxyl group of the nucleic acid or analogue thereof of formula C2 to provide a monothioacetal functionalized fragment nucleic acid or analogue thereof of formula C3.
- step (d) above substitution of the thiomethyl group of a nucleic acid or analogue thereof of formula C3 using a nucleic acid or analogue thereof of formula C4 affords a nucleic acid or analogue thereof of formula C4.
- substitution occurs under mild oxidizing and/or acidic conditions.
- V is oxygen.
- the mild oxidation reagent includes a mixture of elemental iodine and hydrogen peroxide, urea hydrogen peroxide complex, silver nitrate/silver sulfate, sodium bromate, ammonium peroxodi sulfate, tetrabutylammonium peroxy di sulfate, Oxone®, Chloramine T, Selectfluor®, Selectfluor® II, sodium hypochlorite, or potassium iodate/sodium periodiate.
- the mild oxidizing agent includes N-iodosuccinimide, N- bromosuccinimide, N-chlorosuccinimide, l,3-diiodo-5,5-dimethylhydantion, pyridinium tribromide, iodine monochloride or complexes thereof, etc.
- Acids that are typically used under mild oxidizing condition include sulfuric acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, methanesulfonic acid, and trifluoroacetic acid.
- the mild oxidation reagent includes a mixture of N-iodosuccinimide and trifluoromethanesulfonic acid.
- step (e) above removal of PG 3 and optionally R 4 (when R 4 is a suitable amine protecting group) of a nucleic acid-ligand conjugate or analogue thereof of formula C5 affords a nucleic acid-ligand conjugate or analogue thereof of formula C6 or a salt thereof.
- PG 3 and/or R 4 comprise carbamate derivatives that can be removed under acidic or basic conditions.
- the protecting groups e.g., both PG 3 and R 4 or either of PG 3 or R 4 independently
- the protecting groups are removed by acid hydrolysis.
- a salt of formula C6 thereof is formed upon acid hydrolysis of the protecting groups of a nucleic acid-ligand conjugate or analogue thereof of formula C5, a salt of formula C6 thereof is formed.
- an acid-labile protecting group of a nucleic acid-ligand conjugate or analogue thereof of formula C5 is removed by treatment with an acid such as hydrochloric acid, then the resulting amine compound would be formed as its hydrochloride salt.
- acids are useful for removing amino protecting groups that are acid-labile and therefore a wide variety of salt forms of a nucleic acid or analogue thereof of formula C6 are contemplated.
- the protecting groups e.g., both PG 3 and R 4 or either of PG 3 or R 4 independently
- the protecting groups are removed by base hydrolysis.
- Fmoc and trifluoroacetyl protecting groups can be removed by treatment with base.
- bases are useful for removing amino protecting groups that are base-labile.
- a base is piperidine.
- a base is 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU).
- a nucleic acid-ligand conjugate or analogue thereof of formula C5 is deprotected under basic conditions followed by treating with an acid to form a salt of formula C6.
- the acid is fumaric acid
- the salt of formula C6 is the fumarate.
- the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising one or more nucleic acid-ligand conjugate, said nucleic acid-ligand conjugate unit represented by formula II-b-3: or a pharmaceutically acceptable salt thereof, comprising the steps of:
- step (b) conjugating one or more adamantyl or lipophilic compounds to an oligonucleotide of formula D5 to form an oligonucleotide-ligand conjugate of formula II-b-3 comprising one or more nucleic acid-ligand conjugate units.
- conjugation is performed under suitable amide forming conditions to afford a compound of formula D5 comprising an adamantyl or lipid conjugate.
- suitable amide forming conditions can include the use of an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC,
- EDC EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU,
- the amide forming conditions comprise HATU and DIPEA or TEA.
- the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising a unit represent by formula D5:
- step (b) deprotecting said compound of formula D4 to form a compound of formula D5.
- removal of PG 3 and optionally R 4 (when R 4 is a suitable amine protecting group) of an oligonucleotide of formula D4 affords an oligonucleotide-ligand conjugate of formula D5 or a salt thereof.
- PG 3 and/or R 4 comprise carbamate derivatives that can be removed under acidic or basic conditions.
- the protecting groups (e.g., both PG 3 and R 4 or either of PG 3 or R 4 independently) of an oligonucleotide- ligand conjugate of formula D4 are removed by acid hydrolysis.
- the protecting groups e.g., both PG 3 and R 4 or either of PG 3 or R 4 independently
- the protecting groups are removed by base hydrolysis.
- Fmoc and trifluoroacetyl protecting groups can be removed by treatment with base.
- bases are useful for removing amino protecting groups that are base-labile.
- a base is piperidine.
- a base is 1,8- diazabicy clo[5.4.0]undec-7-ene (DBU) .
- the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising one or more nucleic acid-ligand conjugate unit with one or more adamantyl and/or lipid moiety, said conjugate unit represented by formula D4: or a pharmaceutically acceptable salt thereof, comprising the steps of:
- oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art.
- the nucleic acid or analogue thereof of formula D3 is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group.
- Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, represented by a compound of formula D4 comprising an adamantyl or lipid conjugate of the disclosure.
- the present disclosure provides a method for preparing a nucleic acid or analogue thereof comprising one or more lipid conjugate, further comprising preparing a nucleic acid or analogue thereof of formula D3: or a salt thereof, comprising the steps of:
- PG 1 and PG 2 of a nucleic acid or analogue thereof of formula C5 comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anion.
- reagents providing fluoride anion for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-A- butylammonium fluoride, and the like.
- a nucleic acid or analogue thereof of formula DI is protected with a suitable hydroxyl protecting group.
- the protecting group PG 4 used for protection of the 5 ’-hydroxyl group of a compound of formula DI includes an acid labile protecting group such as trityl, 4-methy oxytrityl, 4,4’ -dimethy oxytrityl, 4,4’ ,4”- trimethyoxytrityl, 9-phenyl-xanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like.
- the acid labile protecting group is suitable for deprotection during both solution-phase and solid-phase synthesis of acid-sensitive nucleic acids or analogues thereof using for example, di chloroacetic acid or trichloroacetic acid.
- a nucleic acid or analogue thereof of formula D2 is treated with a P(III) forming reagent to afford a compound of formula D3.
- a P(III) forming reagent is a phosphorus reagent that is reacted to for a phosphorus (III) compound.
- the P(III) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate.
- the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite.
- step (d) above is preformed using 7V,7V-dimethylphosphoramic dichloride as a P(V) forming reagent.
- oligonucleotides e.g., lipid-conjugated RNAi oligonucleotides
- a formulation that minimizes degradation, facilitates delivery and/or uptake, or provides another beneficial property to the oligonucleotides in the formulation e.g., lipid-conjugated RNAi oligonucleotides
- compositions comprising oligonucleotides reduce the expression of a target mRNA (e.g., a target mRNA expressed in an astrocyte of the CNS).
- compositions can be suitably formulated such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient portion of the oligonucleotides enter the cell to reduce target gene expression.
- oligonucleotide formulations can be used to deliver oligonucleotides for the reduction of astrocyte target gene expression as disclosed herein.
- an oligonucleotide is formulated in buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids.
- the formulations herein comprise an excipient.
- an excipient confers to a composition improved stability, improved absorption, improved solubility and/or therapeutic enhancement of the active ingredient.
- an excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil).
- a buffering agent e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide
- a vehicle e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil.
- an oligonucleotide is lyophilized for extending its shelf-life and then made into a solution before use (e.g., administration to a subject).
- an excipient in a composition comprising any one of the oligonucleotides described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, FicollTM or gelatin).
- a lyoprotectant e.g., mannitol, lactose, polyethylene glycol or polyvinylpyrrolidone
- a collapse temperature modifier e.g., dextran, FicollTM or gelatin.
- the oligonucleotides herein may be provided in the form of their free acids.
- a pharmaceutical composition is formulated to be compatible with its intended route of administration.
- routes of administration include parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, intrathecal), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal and rectal administration.
- a pharmaceutical composition is formulated for delivery to the central nervous system (e.g., intrathecal, epidural).
- a pharmaceutical composition is formulated for delivery to the eye (e.g., ophthalmic, intraocular, subconjunctival, intravitreal, retrobulbar, intracam eral).
- compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
- isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
- Sterile injectable solutions can be prepared by incorporating the oligonucleotides in a required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
- a composition may contain at least about 0.1% of the therapeutic agent (e.g., an lipid-conjugated RNAi oligonucleotide herein) or more, although the percentage of the active ingredient(s) may be between about 1% to about 80% or more of the weight or volume of the total composition.
- the therapeutic agent e.g., an lipid-conjugated RNAi oligonucleotide herein
- the percentage of the active ingredient(s) may be between about 1% to about 80% or more of the weight or volume of the total composition.
- Factors such as solubility, bioavailability, biological halflife, route of administration, product shelflife, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
- the disclosure provides methods for contacting or delivering to a cell or population of cells an effective amount of any of the lipid-conjugated RNAi oligonucleotides herein to reduce expression of a target gene in an astrocyte in the CNS.
- expression of an astrocyte target gene is reduced in a region of the CNS.
- regions of the CNS include, but are not limited to, cerebrum, prefrontal cortex, frontal cortex, motor cortex, temporal cortex, parietal cortex, occipital cortex, somatosensory cortex, hippocampus, caudate, striatum, globus pallidus, thalamus, midbrain, tegmentum, substantia nigra, pons, brainstem, cerebellar white matter, cerebellum, dentate nucleus, medulla, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, cervical dorsal root ganglion, thoracic dorsal root ganglion, lumbar dorsal root ganglion, sacral dorsal root ganglion, nodose ganglia, femoral nerve, sciatic nerve, sural nerve, amygdala, hypothalamus, putamen, corpus callosum, and cranial nerve.
- the region of the CNS is selected from the lumbar spinal cord, cerebellum, medulla, hippocampus, hypothalamus, frontal cortex, and a combination thereof. In some embodiments, the region of the CNS is selected from the spinal cord, lumbar spinal cord, thoracic spinal cord, cervical spinal cord, medulla, hippocampus, hypothalamus, cerebellum, frontal cortex, and a combination thereof. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an astrocyte in the spinal cord.
- a lipid- conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an astrocyte in the lumbar spinal cord. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an astrocyte in the thoracic spinal cord. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an astrocyte of the cervical spinal cord. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an astrocyte in hypothalamus.
- a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an astrocyte in the medulla. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an astrocyte in the hippocampus. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an astrocyte in the cerebellum. In some embodiments, a lipid-conjugated RNAi oligonucleotide described herein reduces expression of a target gene in an astrocyte in the frontal cortex
- a reduction of target gene expression is determined by measuring a reduction in the amount or level of target mRNA, protein encoded by the target mRNA, or target gene (mRNA or protein) activity in a cell.
- the methods include those described herein and known to one of ordinary skill in the art.
- the disclosure provides methods for contacting or delivering to a cell or population of cells an effective amount of any of the oligonucleotides (e.g. RNAi oligonucleotides) herein to reduce GFAP expression.
- a reduction of GFAP expression is determined by measuring a reduction in the amount or level of GFAP mRNA, GFAP protein, or GFAP activity in a cell. The methods include those described herein and known to one of ordinary skill in the art.
- the disclosure provides methods for reducing GFAP expression in the central nervous system.
- the central nervous system comprises the brain and spinal cord.
- GFAP expression is reduced in at least one region of the brain.
- regions of the brain include spinal cord, lumbar spinal cord, thoracic spinal cord, cervical spinal cord, medulla, cerebellum, hypothalamus, hippocampus, and frontal cortex.
- a method for reducing expression of a target gene in an astrocyte of the spinal cord in a subject comprises administering to the subject a lipid-conjugated RNAi oligonucleotide comprising a blunt-end and at least one lipid moiety conjugated at Position 1, Position 4, Position 8, Position 12, Position 13, Position 18, or Position 20 of the sense strand.
- a method for reducing expression of a target gene in an astrocyte of the medulla in a subject comprises administering to the subject a lipid-conjugated RNAi oligonucleotide comprising a blunt-end and at least one lipid moiety conjugated at Position 4, Position 8, Position 12, Position 13, Position 18, or Position 20 of the sense strand.
- a method for reducing expression of a target gene in an astrocyte of the cerebellum in a subject comprises administering to the subject a lipid-conjugated RNAi oligonucleotide comprising a blunt-end and at least one lipid moiety conjugated at Position 4, Position 12, Position 13, Position 18, or Position 20 of the sense strand.
- a method for reducing expression of a target gene in an astrocyte of the hypothalamus in a subject comprises administering to the subject a lipid-conjugated RNAi oligonucleotide comprising a blunt-end and at least one lipid moiety conjugated at Position 1, Position 4, Position 12, Position 13, Position 18, or Position 20 of the sense strand.
- a method for reducing expression of a target gene in an astrocyte of the frontal cortex in a subject comprises administering to the subject a lipid-conjugated RNAi oligonucleotide comprising a blunt-end and at least one lipid moiety conjugated at Position 4 of the sense strand.
- a method for reducing expression of a target gene in an astrocyte of the spinal cord in a subject comprises administering to the subject a lipid-conjugated RNAi oligonucleotide comprising a stem-loop and at least one lipid moiety conjugated at Position 1, Position 4, Position 8, Position 12, Position 143, Position 18, Position 20, Position 23, Position 28, Position 29, or Position 30 of the sense strand.
- a method for reducing expression of a target gene in an astrocyte of the medulla in a subject comprises administering to the subject a lipid-conjugated RNAi oligonucleotide comprising a stem-loop and at least one lipid moiety conjugated at Position 1, Position 4, Position 18, Position 20, Position 23, Position 28, Position 29, or Position 30 of the sense strand.
- cerebellum in a subject comprises administering to the subject a lipid-conjugated RNAi oligonucleotide comprising a stem-loop and at least one lipid moiety conjugated at Position 1, Position 4, Position 23, Position 28, or Position 29 of the sense strand.
- a method for reducing expression of a target gene in an astrocyte of the hypothalamus in a subject comprises administering to the subject a lipid-conjugated RNAi oligonucleotide comprising a blunt-end and at least one lipid moiety conjugated at Position 1, Position 1, Position 4, Position 12, Position 13, Position 18, Position 20, Position 23, Position 28, Position 29, or Position 30 of the sense strand.
- a method for reducing expression of a target gene in an astrocyte of the frontal cortex in a subject comprises administering to the subject a lipid-conjugated RNAi oligonucleotide comprising a blunt-end and at least one lipid moiety conjugated at Position 23 of the sense strand.
- a single dose of oligonucleotide or pharmaceutical compositions described herein reduces expression of an astrocyte mRNA for at least 2, 3, 4, 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 or 35 weeks. In some embodiments, a single dose of oligonucleotide or pharmaceutical compositions described herein reduces expression of an astrocyte mRNA for at least 4 weeks. In some embodiments, a single dose of oligonucleotide or pharmaceutical compositions described herein reduces expression of an astrocyte mRNA for at least 8 weeks.
- a single dose of oligonucleotide or pharmaceutical compositions described herein reduces expression of an astrocyte mRNA for at least 12 weeks. In some embodiments, a single dose of oligonucleotide or pharmaceutical compositions described herein reduces expression of an astrocyte mRNA for at least 23 weeks. In some embodiments, a single dose of oligonucleotide or pharmaceutical compositions described herein reduces expression of an astrocyte mRNA for at least 26 weeks. In some embodiments, a single dose of oligonucleotide or pharmaceutical compositions described herein reduces expression of an astrocyte mRNA for at least 29 weeks.
- a single dose of oligonucleotide or a pharmaceutical composition herein reduces expression of an astrocyte mRNA for up to 1 month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 7 months, up to 8 months, up to 9 months, up to 10 months, up to 11 months, up to 12 months, up to 13, months, up to 14 months, up to 15 months, or up to 16 months.
- a single dose of oligonucleotide or a pharmaceutical composition herein reduces expression of an astrocyte mRNA for up to 12 months.
- a reduction of target gene expression is determined by measuring a reduction in the amount or level of target mRNA, protein encoded by the target mRNA, or target gene (mRNA or protein) activity in a cell.
- the methods include those described herein and known to one of ordinary skill in the art.
- a cell is any cell that expresses the astrocyte target mRNA.
- the cell is a primary astrocyte cell obtained from a subject.
- the primary cell has undergone a limited number of passages such that the cell substantially maintains is natural phenotypic properties.
- a cell to which the oligonucleotide is delivered is ex vivo or in vitro (i.e., can be delivered to a cell in culture or to an organism in which the cell resides).
- the lipid-conjugated RNAi oligonucleotides disclosed herein are delivered to a cell or population of cells (e.g., astrocytes ) using a nucleic acid delivery method known in the art including, but not limited to, injection of a solution or pharmaceutical composition containing the lipid-conjugated RNAi oligonucleotide, bombardment by particles covered by the lipid-conjugated RNAi oligonucleotide, exposing the cell or population of cells to a solution containing the lipid-conjugated RNAi oligonucleotide, or electroporation of cell membranes in the presence of the lipid-conjugated RNAi oligonucleotide.
- Other methods known in the art for delivering oligonucleotides to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and others.
- reduction of target gene expression is determined by an assay or technique that evaluates one or more molecules, properties or characteristics of a cell or population of cells associated with target gene expression, or by an assay or technique that evaluates molecules that are directly indicative of target gene expression in a cell or population of cells (e.g., target mRNA or protein).
- the extent to which a lipid- conjugated RNAi oligonucleotide provided herein reduces target gene expression in an astrocyte is evaluated by comparing target gene expression in an astrocyte or population of astrocytes contacted with the lipid-conjugated RNAi oligonucleotide to a control cell or population of cells (e.g., an astrocyte or population of astrocytes not contacted with the lipid- conjugated RNAi oligonucleotide or contacted with a control lipid-conjugated RNAi oligonucleotide).
- a control cell or population of cells e.g., an astrocyte or population of astrocytes not contacted with the lipid- conjugated RNAi oligonucleotide or contacted with a control lipid-conjugated RNAi oligonucleotide.
- a control amount or level of target gene expression in a control cell or population of cells is predetermined, such that the control amount or level need not be measured in every instance the assay or technique is performed.
- the predetermined level or value can take a variety of forms.
- a predetermined level or value can be single cut-off value, such as a median or mean.
- contacting or delivering a lipid-conjugated RNAi oligonucleotide described herein to an astrocyte or a population of astrocytes results in a reduction in expression of an astrocyte target gene.
- the reduction in target gene expression is relative to a control amount or level of target gene expression in cell or population of cells not contacted with the lipid-conjugated RNAi oligonucleotide or contacted with a control lipid-conjugated RNAi oligonucleotide.
- the reduction in target gene expression is about 1% or lower, about 5% or lower, about 10% or lower, about 15% or lower, about 20% or lower, about 25% or lower, about 30% or lower, about 35% or lower, about 40% or lower, about 45% or lower, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower, or about 90% or lower relative to a control amount or level of target gene expression.
- the control amount or level of target gene expression is an amount or level of target mRNA and/or protein in a cell or population of cells that has not been contacted with a lipid-conjugated RNAi oligonucleotide herein.
- the effect of delivery of a lipid-conjugated RNAi oligonucleotide to a cell or population of cells according to a method herein is assessed after any finite period or amount of time (e.g., minutes, hours, days, weeks, months).
- target gene expression is determined in a cell or population of cells at least about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours; or at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days or more after contacting or delivering the lipid-conjugated RNAi oligonucleotide to the cell or population of cells.
- target gene expression is determined in a cell or population of cells at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months or more after contacting or delivering the lipid-conjugated RNAi oligonucleotide to the cell or population of cells.
- the disclosure provides oligonucleotides for use as a medicament, in particular for use in a method for the treatment of diseases, disorders, and conditions associated with the CNS.
- the disclosure also provides lipid-conjugated RNAi oligonucleotide s for use, or adaptable for use, to treat a subject (e.g., a human) having a disease, disorder or condition associated with expression of an astrocyte target gene that would benefit from reducing expression of the astrocyte target gene.
- the disclosure provides lipid-conjugated RNAi oligonucleotides for use, or adapted for use, to treat a subject having a disease, disorder or condition associated with astrocyte target gene expression.
- the disclosure also provides lipid- conjugated RNAi oligonucleotides for use, or adaptable for use, in the manufacture of a medicament or pharmaceutical composition for treating a disease, disorder or condition associated with expression of an astrocyte target gene.
- a subject having a disease, disorder or condition associated with expression of an astrocyte target gene or is predisposed to the same is selected for treatment with a lipid-conjugated RNAi oligonucleotide herein.
- the method comprises selecting an individual having a marker (e.g., a biomarker) for a disease, disorder or condition associated with expression of an astrocyte target gene, or predisposed to the same, such as, but not limited to, target mRNA, protein, or a combination thereof.
- some embodiments of the methods provided by the disclosure include steps such as measuring or obtaining a baseline value for a marker of expression of an astrocyte target gene, and then comparing such obtained value to one or more other baseline values or values obtained after the subject is administered the lipid- conjugated RNAi oligonucleotide to assess the effectiveness of treatment.
- the disclosure also provides methods of treating a subject having, suspected of having, or at risk of developing a disease, disorder or condition associated with expression of an astrocyte target gene with a lipid-conjugated RNAi oligonucleotide provided herein.
- the disclosure provides methods of treating or attenuating the onset or progression of a disease, disorder or condition associated with expression of an astrocyte target gene using the lipid-conjugated RNAi oligonucleotides provided herein.
- the disclosure provides methods to achieve one or more therapeutic benefits in a subject having a disease, disorder or condition associated with expression of an astrocyte target gene using the lipid-conjugated RNAi oligonucleotides provided herein.
- the subject is treated by administering a therapeutically effective amount of any one or more of the lipid-conjugated RNAi oligonucleotides provided herein.
- treatment comprises reducing expression of an astrocyte target gene (e.g., in the CNS).
- the subject is treated therapeutically.
- the subject is treated prophylactically.
- a lipid-conjugated RNAi oligonucleotide provided herein, or a pharmaceutical composition comprising the lipid-conjugated RNAi oligonucleotide is administered to a subject having a disease, disorder or condition associated with expression of an astrocyte target gene such that target gene expression is reduced in the subject, thereby treating the subject.
- a subject having a disease, disorder or condition associated with expression of an astrocyte target gene such that target gene expression is reduced in the subject, thereby treating the subject.
- an amount or level of target mRNA is reduced in the subject.
- an amount or level of protein encoded by the target mRNA is reduced in the subject.
- a lipid-conjugated RNAi oligonucleotide provided herein, or a pharmaceutical composition comprising the lipid-conjugated RNAi oligonucleotide is administered to a subject having a disease, disorder or condition associated with expression of an astrocyte target gene such that target gene expression is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99% when compared to target gene expression prior to administration of the lipid-conjugated RNAi oligonucleotide or pharmaceutical composition.
- expression of an astrocyte target gene is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99% when compared to target gene expression in a subject (e.g., a reference or control subject) not receiving the lipid-conjugated RNAi oligonucleotide or pharmaceutical composition or receiving a control lipid-conjugated RNAi oligonucleotide, pharmaceutical composition or treatment.
- a subject e.g., a reference or control subject
- an lipid-conjugated RNAi oligonucleotide herein, or a pharmaceutical composition comprising the lipid-conjugated RNAi oligonucleotide is administered to a subject having a disease, disorder or condition associated with expression of an astrocyte target gene such that an amount or level of target mRNA is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99% when compared to the amount or level of target mRNA prior to administration of the lipid-conjugated RNAi oligonucleotide or pharmaceutical composition.
- an amount or level of target mRNA is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99% when compared to an amount or level of target mRNA in a subject e.g., a reference or control subject) not receiving the lipid-conjugated RNAi oligonucleotide or pharmaceutical composition or receiving a control lipid-conjugated RNAi oligonucleotide, pharmaceutical composition or treatment.
- a subject e.g., a reference or control subject
- a lipid-conjugated RNAi oligonucleotide herein, or a pharmaceutical composition comprising the lipid-conjugated RNAi oligonucleotide is administered to a subject having a disease, disorder or condition associated with expression of an astrocyte target gene such that an amount or level of protein encoded by the astrocyte target gene is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99% when compared to the amount or level of protein encoded by the target gene prior to administration of the lipid- conjugated RNAi oligonucleotide or pharmaceutical composition.
- an amount or level of protein encoded by an astrocyte target gene is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99% when compared to an amount or level of protein encoded by the target gene in a subject (e.g., a reference or control subject) not receiving the lipid-conjugated RNAi oligonucleotide or pharmaceutical composition or receiving a control lipid-conjugated RNAi oligonucleotide, pharmaceutical composition or treatment.
- a subject e.g., a reference or control subject
- a lipid-conjugated RNAi oligonucleotide herein, or a pharmaceutical composition comprising the lipid-conjugated RNAi oligonucleotide is administered to a subject having a disease, disorder or condition associated with expression of an astrocyte target gene such that an amount or level of target gene activity is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99% when compared to the amount or level of target gene activity prior to administration of the lipid-conjugated RNAi oligonucleotide or pharmaceutical composition.
- an amount or level of target gene activity is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99% when compared to an amount or level of target gene activity in a subject (e.g., a reference or control subject) not receiving the lipid-conjugated RNAi oligonucleotide or pharmaceutical composition or receiving a control lipid-conjugated RNAi oligonucleotide, pharmaceutical composition or treatment.
- a subject e.g., a reference or control subject
- Suitable methods for determining target gene expression, an amount or level of target mRNA, an amount or level of protein encoded by the target gene, and/or an amount or level of target gene activity, in the subject, or in a sample from the subject, are known in the art. Further, the Examples set forth herein illustrate exemplary methods for determining target gene expression.
- target gene expression, an amount or level of target gene mRNA, an amount or level of protein encoded by a target gene, an amount or level of target gene activity, or any combination thereof is reduced in a cell (e.g., an astrocyte), a population or a group of cells (e.g., an organoid), an organ (e.g., CNS), blood or a fraction thereof (e.g., plasma), a tissue (e.g., brain tissue), a sample (e.g., CSF sample or a brain biopsy sample), or any other biological material obtained or isolated from the subject.
- a cell e.g., an astrocyte
- a population or a group of cells e.g., an organoid
- an organ e.g., CNS
- blood or a fraction thereof e.g., plasma
- a tissue e.g., brain tissue
- sample e.g., CSF sample or a brain biopsy sample
- expression of an astrocyte target gene, an amount or level of target gene mRNA, an amount or level of protein encoded by the target gene, an amount or level of target gene activity, or any combination thereof is reduced in more than one type of cell (e.g., astrocyte), more than one groups of cells, more than one organ (e.g., brain and one or more other organ(s)), more than one fraction of blood (e.g., plasma and one or more other blood fraction(s)), more than one type of tissue (e.g., brain tissue and one or more other type(s) of tissue), more than one type of sample e.g., a brain biopsy sample and one or more other type(s) of biopsy sample) obtained or isolated from the subject.
- astrocyte e.g., astrocyte
- organ e.g., brain and one or more other organ(s)
- fraction of blood e.g., plasma and one or more other blood fraction(s)
- tissue e.g., brain tissue and one or more other type(s
- expression of an astrocyte target gene is reduced in one or more of the spinal cord, lumbar spinal cord, thoracic spinal cord, cervical spinal cord, medulla, hippocampus, hypothalamus, somatosensory cortex, cerebellum, or frontal cortex.
- expression of an astrocyte target gene is reduced in the spinal cord.
- expression of an astrocyte target gene is reduced in the lumbar spinal cord.
- expression of an astrocyte target gene is reduced in the thoracic spinal cord.
- expression of an astrocyte target gene is reduced in the cervical spinal cord.
- expression of an astrocyte target gene is reduced in hypothalamus.
- expression of an astrocyte target gene is reduced in the medulla. In some embodiments, expression of an astrocyte target gene is reduced in the hippocampus. In some embodiments, expression of an astrocyte target gene is reduced in the somatosensory cortex. In some embodiments, expression of an astrocyte target gene is reduced in the cerebellum. In some embodiments, expression of an astrocyte target gene is reduced in the frontal cortex.
- Examples of a disease, disorder or condition associated with expression of an astrocyte target gene include, but are not limited to, Spinocerebellar Ataxia 1, Spinocerebellar Ataxia 2 Spinocerebellar ataxia 3 , Prion Disease, Alexander’s’ Disease, MECP2 Duplication Syndrome, Huntington’s Disease, Parkinson’s Disease, Alzheimer’s Disease, Progressive Supranuclear Palsy (PSP), Corticobasal degeneration (CBD), Argyrophilic grain disease (AGD), Globular glial tauopathy (GGT), Ageing-related tau astrogliopathy (ARTAG), Familial Frontotemporal Dementia 17 (FTD-17), Tauopathy with Respiratory Failure, Dementia with Seizures, Pick’s disease, Myotonic dystrophy 1 or 2 (MD1 or MD2), Down’s syndrome, Spastic Paraplegia (SP), Niemann-Pick disease type C, Dementia with Lewy bodies (DLB), Lewy body dysphag
- Examples of a disease, disorder or condition associated with expression of an astrocyte target gene include, but are not limited to, Spinocerebellar Ataxia 1, Spinocerebellar Ataxia 2, Spinocerebellar ataxia 3 , Prion Disease, Alexander’s’ Disease, MECP2 Duplication Syndrome, Huntington’s Disease, Parkinson’s Disease, and Alzheimer’s Disease.
- the astrocyte target gene may be a target gene from any mammal, such as a human. Any astrocyte gene may be silenced according to the method described herein.
- Methods described herein are typically involve administering to a subject a therapeutically effective amount of a lipid-conjugated RNAi oligonucleotide herein, that is, an amount capable of producing a desirable therapeutic result.
- a therapeutically acceptable amount may be an amount that can therapeutically treat a disease or disorder.
- the appropriate dosage for any one subject will depend on certain factors, including the subject's size, body surface area, age, the composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently.
- a subject is administered any one of the compositions herein either enterally (e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intraarterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intracerebroventricular injection, intrathecal), topically (e.g., epicutaneous, inhalational, via eye drops, or through a mucous membrane), or by direct injection into a target organ (e.g., the brain of a subject).
- enterally e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy or rectally
- parenterally e.g., subcutaneous injection, intravenous injection or infusion, intraarterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intracerebroventricular injection, intrathe
- a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof is administered intrathecally into cerebrospinal fluid (CSF) (e.g., injection or infusion into the fluid within the subarachnoid space).
- CSF cerebrospinal fluid
- intrathecal administration of a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof is performed as a bolus injection into the subarachnoid space.
- intrathecal administration of a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof is performed as an infusion into the subarachnoid space.
- intrathecal administration of a herein, or a composition thereof is performed via a catheter into the subarachnoid space. In some embodiments, intrathecal administration of a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof, is performed via a pump. In some embodiments, intrathecal administration of a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof, is performed via an implantable pump. In some embodiments, administration is performed via an implantable device that operates or functions a reservoir.
- a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof is administered intrathecally into the cerebellomedullary cistern (also referred to as the cisterna magna). Intrathecal administration into the cistema magna is referred to as “intraci sternal administration” or “intraci sternal magna (i.c.m.) administration). In some embodiments, a lipid-conjugated RNAi oligonucleotide herein, or composition thereof, is administered intrathecally into the subarachnoid space of the lumbar spinal cord.
- Intrathecal administration into the subarachnoid space of the lumbar spinal cord is referred to as “lumbar intrathecal (i.t.) administration”.
- a lipid-conjugated RNAi oligonucleotide herein, or composition thereof is administered intrathecally into the subarachnoid space of the cervical spinal cord.
- Intrathecal administration into the subarachnoid space of the cervical spinal cord is referred to as “cervical intrathecal (i.t.) administration”.
- a lipid-conjugated RNAi oligonucleotide herein, or composition thereof is administered intrathecally into the subarachnoid space of the thoracic spinal cord.
- Intrathecal administration into the subarachnoid space of the thoracic spinal cord is referred to as “thoracic intrathecal (i.t.) administration”.
- a lipid-conjugated RNAi oligonucleotide herein, or composition thereof is administered by intracerebroventricular injection or infusion into the cerebral ventricles.
- Intracerebroventricular administration into the ventricular space is referred to as “intracerebroventricular (i.c.v.) administration”.
- an Ommaya reservoir is used to administer a lipid-conjugated RNAi oligonucleotide herein, or composition thereof, by intracerebroventricular injection or infusion.
- a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof is administered once every year, once every 6 months, once every 4 months, quarterly (once every three months), bi-monthly (once every two months), monthly or weekly.
- a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof is administered every week or at intervals of two, or three weeks.
- a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof is administered daily.
- a subject is administered one or more loading doses of a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof, followed by one or more maintenance doses of the lipid-conjugated RNAi oligonucleotide, or a composition thereof.
- the subject to be treated is a human or non-human primate or other mammalian subject.
- Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.
- the disclosure provides a kit comprising a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof, described herein, and instructions for use.
- the kit comprises a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof, described herein, and a package insert containing instructions for use of the kit and/or any component thereof.
- the kit comprises, in a suitable container, a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof, described herein, one or more controls, and various buffers, reagents, enzymes and other standard ingredients well known in the art.
- the container comprises at least one vial, well, test tube, flask, bottle, syringe, or other container means, into which the lipid- conjugated RNAi oligonucleotide herein, or a composition thereof, is placed, and in some instances, suitably aliquoted.
- the kit contains additional containers into which this component is placed.
- the kits can also include a means for containing a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof, and any other reagent in close confinement for commercial sale.
- Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
- Containers and/or kits can include labeling with instructions for use and/or warnings.
- a kit comprises a lipid-conjugated RNAi oligonucleotide herein, or a composition thereof, described herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the lipid-conjugated RNAi oligonucleotide and instructions for treating or delaying progression of a disease, disorder or condition associated with expression of an astrocyte target gene in a subject in need thereof.
- the term “and/or” includes any and all combinations of one or more of the associated listed items. Further, the singular forms and the articles “a”, “an” and “the” are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms: includes, comprises, including and/or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Further, it will be understood that when an element, including component or subsystem, is referred to and/or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present.
- PCR polymerase chain reaction
- LCR ligase chain reach on(LCR)
- Q.beta.-replicase amplification RNA polymerase mediated techniques
- NASBA RNA polymerase mediated techniques
- homologous nucleic acids of the disclosure are found in Berger, Sambrook, and Ausubel, as well as in Mullis et al., (1987) U.S. Pat. No. 4,683,202; Innis et al., eds. (1990); PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS (Academic Press Inc.
- Ranges can be expressed herein as from “about” one value, and/or to "about” another value. When such a range is expressed, another embodiment includes from the one value and/or to the other value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are several values disclosed herein, and that each value is also herein disclosed as “about” that value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
- complementary refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that permits the two nucleotides to form base pairs with one another.
- a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another.
- complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes.
- two nucleic acids may have regions of multiple nucleotides that are complementary with each other to form regions of complementarity, as described herein.
- deoxyribonucleotide refers to a nucleotide having a hydrogen in place of a hydroxyl at the 2' position of its pentose sugar when compared with a ribonucleotide.
- a modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions of atoms other than at the 2' position, including modifications or substitutions in or of the sugar, phosphate group or base.
- double-stranded RNA refers to an RNA oligonucleotide that is substantially in a duplex form.
- the complementary base-pairing of duplex region(s) of a dsRNA oligonucleotide is formed between antiparallel sequences of nucleotides of covalently separate nucleic acid strands.
- complementary base-pairing of duplex region(s) of a dsRNA formed between antiparallel sequences of nucleotides of nucleic acid strands that are covalently linked.
- complementary base-pairing of duplex region(s) of a dsRNA is formed from single nucleic acid strand that is folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that base pair together.
- a dsRNA comprises two covalently separate nucleic acid strands that are fully duplexed with one another.
- a dsRNA comprises two covalently separate nucleic acid strands that are partially duplexed (e.g., having overhangs at one or both ends).
- a dsRNA comprises antiparallel sequence of nucleotides that are partially complementary, and thus, may have one or more mismatches, which may include internal mismatches or end mismatches.
- duplex in reference to nucleic acids (e.g., oligonucleotides), refers to a structure formed through complementary base pairing of two antiparallel sequences of nucleotides.
- excipient refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute to a desired consistency or stabilizing effect.
- loop refers to an unpaired region of a nucleic acid (e.g., oligonucleotide) that is flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to one another, such that under appropriate hybridization conditions (e.g. , in a phosphate buffer, in a cells), the two antiparallel regions, which flank the unpaired region, hybridize to form a duplex (referred to as a “stem”).
- GFAP refers to Glial Fibrillary Acidic Protein. GFAP is found in astrocytes of the central nervous system (CNS). GFAP is an intermediate filament protein which provides support for cell structure.
- “reduction of GFAP expression” refers to a decrease in the amount or level of GFAP mRNA, GFAP protein and/or GFAP activity in a cell, a population of cells, a sample or a subject when compared to an appropriate reference (e.g., a reference cell, population of cells, sample, or subject).
- modified intemucleotide linkage refers to an internucleotide linkage having one or more chemical modifications when compared with a reference intemucleotide linkage comprising a phosphodiester bond.
- a modified nucleotide is a non-naturally occurring linkage.
- a modified intemucleotide linkage confers one or more desirable properties to a nucleic acid in which the modified intemucleotide linkage is present.
- a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc.
- modified nucleotide refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide and thymidine deoxyribonucleotide.
- a modified nucleotide is a non-naturally occurring nucleotide.
- a modified nucleotide has one or more chemical modification in its sugar, nucleobase and/or phosphate group. In some embodiments, a modified nucleotide has one or more chemical moieties conjugated to a corresponding reference nucleotide. Typically, a modified nucleotide confers one or more desirable properties to a nucleic acid in which the modified nucleotide is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc.
- astrocyte mRNA and “astrocyte gene” refers to any gene, mRNA, and/or protein encoded/expressed by a gene in astrocytes of the central nervous system.
- Astrocytes are glial cells which perform several functions throughout the CNS such as synaptic support and neurotransmission.
- RNAi oligonucleotide that is characterized by separate sense (passenger) and antisense (guide) strands, in which the sense strand has a region of complementarity with the antisense strand, and in which at least one of the strands, generally the sense strand, has a tetraloop configured to stabilize an adjacent stem region formed within the at least one strand.
- oligonucleotide refers to a short nucleic acid (e.g., less than about 100 nucleotides in length).
- An oligonucleotide may be single stranded (ss) or ds.
- An oligonucleotide may or may not have duplex regions.
- an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide, short siRNA or ss siRNA.
- a double-stranded (dsRNA) is an RNAi oligonucleotide.
- lipid-conjugated RNAi oligonucleotide and “oligonucleotide-ligand conjugate” are used interchangeably and refer to an oligonucleotide comprising one or more nucleotides conjugated with one or more targeting ligands (e.g., lipid).
- overhang refers to terminal non-base pairing nucleotide(s) resulting from one strand or region extending beyond the terminus of a complementary strand with which the one strand or region forms a duplex.
- an overhang comprises one or more unpaired nucleotides extending from a duplex region at the 5' terminus or 3' terminus of a dsRNA.
- the overhang is a 3' or 5' overhang on the antisense strand or sense strand of a dsRNA.
- phosphate analog refers to a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group.
- a phosphate analog is positioned at the 5' terminal nucleotide of an oligonucleotide in place of a 5'- phosphate, which is often susceptible to enzymatic removal.
- a 5' phosphate analog contains a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5' methylene phosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'- VP).
- an oligonucleotide has a phosphate analog at a 4'-carbon position of the sugar (referred to as a “4'-phosphate analog”) at a 5'- terminal nucleotide.
- a 4'-phosphate analog is oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4'-carbon) or analog thereof. See, e.g., US Provisional Patent Application Nos. 62/383,207 (filed on 2 September 2016) and 62/393,401 (filed on 12 September 2016).
- Other modifications have been developed for the 5' end of oligonucleotides (see, e.g., Inti.
- RNA transcript e.g., target mRNA
- protein encoded by the target gene e.g., protein encoded by the target gene
- an appropriate reference e.g., a reference cell, population of cells, sample, or subject
- the act of contacting a cell with an oligonucleotide or conjugate herein may result in a decrease in the amount or level of target mRNA, protein encoded by a target gene, and/or target gene activity (e.g., via inactivation and/or degradation of target mRNA by the RNAi pathway) when compared to a cell that is not treated with the double-stranded oligonucleotide.
- reducing expression refers to an act that results in reduced expression of a target gene.
- region of complementarity refers to a sequence of nucleotides of a nucleic acid (e.g., a dsRNA) that is sufficiently complementary to an antiparallel sequence of nucleotides to permit hybridization between the two sequences of nucleotides under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cell, etc.).
- an oligonucleotide herein comprises a targeting sequence having a region of complementary to a mRNA target sequence.
- ribonucleotide refers to a nucleotide having a ribose as its pentose sugar, which contains a hydroxyl group at its 2' position.
- a modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than at the 2' position, including modifications or substitutions in or of the ribose, phosphate group or base.
- RNAi oligonucleotide refers to either (a) a dsRNA having a sense strand (passenger) and antisense strand (guide), in which the antisense strand or part of the antisense strand is used by the Argonaute 2 (Ago2) endonuclease in the cleavage of a target mRNA or (b) a ss oligonucleotide having a single antisense strand, where that antisense strand (or part of that antisense strand) is used by the Ago2 endonuclease in the cleavage of a target mRNA.
- Ago2 Argonaute 2
- strand refers to a single, contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages or phosphorothioate linkages). In some embodiments, a strand has two free ends (e.g., a 5' end and a 3' end).
- “subject” means any mammal, including mice, rabbits, and humans. In one embodiment, the subject is a human or NHP. Moreover, “individual” or “patient” may be used interchangeably with “subject.”
- “synthetic” refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine (e.g., a solid-state nucleic acid synthesizer)) or that is otherwise not derived from a natural source (e.g., a cell or organism) that normally produces the molecule.
- targeting ligand refers to a molecule or “moiety” (e.g., a carbohydrate, amino sugar, cholesterol, polypeptide, or lipid) that selectively binds to a cognate molecule (e.g., a receptor) of a tissue or cell of interest and/or that is conjugatable to another substance for purposes of targeting the other substance to the tissue or cell of interest.
- a targeting ligand may be conjugated to an oligonucleotide for purposes of targeting the oligonucleotide to a specific tissue or cell of interest.
- a targeting ligand selectively binds to a cell surface receptor.
- a targeting ligand when conjugated to an oligonucleotide facilitates delivery of the oligonucleotide into a particular cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of the complex comprising the oligonucleotide, targeting ligand and receptor.
- a targeting ligand is conjugated to an oligonucleotide via a linker that is cleaved following or during cellular internalization such that the oligonucleotide is released from the targeting ligand in the cell.
- tetraloop refers to a loop that increases stability of an adjacent duplex formed by hybridization of flanking sequences of nucleotides.
- the increase in stability is detectable as an increase in melting temperature (Tm) of an adjacent stem duplex that is higher than the Tm of the adjacent stem duplex expected, on average, from a set of loops of comparable length consisting of randomly selected sequences of nucleotides.
- Tm melting temperature
- a tetraloop can confer a Tm of at least about 50°C, at least about 55°C, at least about 56°C, at least about 58°C, at least about 60°C, at least about 65°C or at least about 75°C in 10 mM NaHPOi to a hairpin comprising a duplex of at least 2 base pairs (bp) in length.
- a tetraloop may stabilize a bp in an adjacent stem duplex by stacking interactions.
- a tetraloop comprises or consists of 3 to 6 nucleotides and is typically 4 to 5 nucleotides.
- a tetraloop comprises or consists of 3, 4, 5 or 6 nucleotides, which may or may not be modified (e.g., which may or may not be conjugated to a targeting moiety).
- a tetraloop consists of 4 nucleotides. Any nucleotide may be used in the tetraloop and standard IUPAC-IUB symbols for such nucleotides may be used as described in Cornish- Bowden ((1985) NUCLEIC ACIDS RES. 13:3021-3030).
- N may be used to mean that any base may be in that position
- R may be used to show that A (adenine) or G (guanine) may be in that position
- “B” may be used to show that C (cytosine), G (guanine), or T (thymine) may be in that position.
- tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al., (1990) PROC. NATL. ACAD. SCI. USA 87:8467-71; Antao e/ a/., (1991) NUCLEIC ACIDS RES. 19:5901-05).
- UUCG UUCG
- GNRA GNRA family of tetraloops
- CUUG tetraloop Wiese et al., (1990) PROC. NATL. ACAD. SCI. USA 87:8467-71; Antao e/ a/., (1991) NUCLEIC ACIDS RES. 19:5901-05.
- DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA), the d(GNRA)) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)).
- d(GNNA) family of tetraloops e.g., d(GTTA), the d(GNRA) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)).
- d(GNNA) d(GTTA)
- d(GNRA) d(GNAB) family of tetraloops
- treat refers to the act of providing care to a subject in need thereof, for example, by administering a therapeutic agent (e.g., an oligonucleotide herein) to the subj ect, for purposes of improving the health and/or well-being of the subj ect with respect to an existing condition (e.g., a disease, disorder) or to prevent or decrease the likelihood of the occurrence of a condition.
- a therapeutic agent e.g., an oligonucleotide herein
- treatment involves reducing the frequency or severity of at least one sign, symptom or contributing factor of a condition (e.g., disease, disorder) experienced by a subject.
- RNAi oligonucleotides The oligonucleotides (RNAi oligonucleotides) described in the foregoing Examples were chemically synthesized using methods described herein. Generally, RNAi oligonucleotides were synthesized using solid phase oligonucleotide synthesis methods as described for 19-23mer RNAi oligonucleotides (see, e.g., Scaringe et al. (1990) NUCLEIC ACIDS RES. 18:5433-41 and Usman et al. (1987) J. AM. CHEM. SOC. 109:7845-45; see also, US Patent Nos.
- RNAi oligonucleotides having a 19mer core sequence were formatted into constructs having a 36mer sense strand and a 22mer antisense strand to allow for processing by the RNAi machinery.
- the 19mer core sequence is complementary to a region in the GFAP mRNA.
- RNA oligonucleotides were synthesized and HPLC purified according to standard methods (Integrated DNA Technologies; Coralville, IA). For example, RNA oligonucleotides were synthesized using solid phase phosphorami di te chemistry, deprotected and desalted on NAP-5 columns (Amersham Pharmacia Biotech; Piscataway, NJ) using standard techniques (Damha & Olgivie (1993) METHODS MOL. BIOL. 20:81-114; Wincott et al. (1995) NUCLEIC ACIDS RES. 23:2677-84) and the phosphorami dite synthesis as shown below:
- the oligomers were purified using ion-exchange high performance liquid chromatography (IE-HPLC) on an Amersham Source 15Q column (1.0 cm z 25 cm; Amersham Pharmacia Biotech) using a 15 min step-linear gradient. The gradient varied from 90: 10 Buffers A:B to 52:48 Buffers A:B, where Buffer A is 100 mM Tris pH 8.5 and Buffer B is 100 mM Tris pH 8.5, 1 M NaCl. Samples were monitored at 260 nm and peaks corresponding to the full-length oligonucleotide species were collected, pooled, desalted on NAP-5 columns, and lyophilized.
- IE-HPLC ion-exchange high performance liquid chromatography
- each oligomer was determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc.; Fullerton, CA).
- the CE capillaries have a 100 pm inner diameter and contain ssDNA 100R Gel (Beckman-Coulter).
- ssDNA 100R Gel (Beckman-Coulter)
- about 0.6 nmole of oligonucleotide was injected into a capillary, run in an electric field of 444 V/cm, and was detected by UV absorbance at 260 nm.
- Denaturing Tris-Borate-7 M-urea running buffer was purchased from Beckman-Coulter. Oligoribonucleotides were obtained that were at least 90% pure as assessed by CE for use in experiments described below.
- RNA oligomers Single strand RNA oligomers were resuspended (e.g., at 100 pM concentration) in duplex buffer consisting of 100 mM potassium acetate, 30 mM HEPES, pH 7.5. Complementary sense and antisense strands were mixed in equal molar amounts to yield a final solution of, for example, 50 pM duplex. Samples were heated to 100°C for 5' in RNA buffer (IDT) and were allowed to cool to room temperature before use. The RNAi oligonucleotides were stored at -20° C. Single strand RNA oligomers were stored lyophilized or in nuclease-free water at -80° C.
- duplex buffer consisting of 100 mM potassium acetate, 30 mM HEPES, pH 7.5.
- Complementary sense and antisense strands were mixed in equal molar amounts to yield a final solution of, for example, 50 pM duplex. Samples were heated
- Example 2 GalNAc-Conjugated GFAP RNAi Oligonucleotides Inhibit Mouse Gfap In Vivo in a Concentration Dependent Manner Via Intrathecal and Intracerebroventricular Administration
- Glial fibrillary acidic protein encodes intermediate filament proteins primarily found in astrocytes of the CNS. Sufficient knockdown of GFAP indicates ability of oligonucleotides described herein to reduce expression of target genes expressed in astrocytes.
- RNAi oligonucleotides synthesized as described in Example 1 were used to generate doublestranded RNAi oligonucleotides comprising a nicked tetraloop GalNAc-conjugated structure (referred to herein as “GalNAc-conjugated GFAP oligonucleotides” or “GalNAc-GVHE oligonucleotides”) having a 36-mer passenger strand and a 22-mer guide strand.
- the nucleotide sequences comprising the passenger strand and guide strand have a distinct pattern of modified nucleotides and phosphorothioate linkages.
- Three of the nucleotides comprising the tetraloop were each conjugated to a GalNAc moiety (CAS#14131-60-3). The modification pattern of each strand is illustrated below: Sense Strand: 5’
- GFAP-1477 as shown in Table 2, was assessed for the ability to reduce murine Gfap in the central nervous system (CNS) via intrathecal (i.t.) administration. Specifically, mice were administered 10, 32, 100, 320, or 1000 pg of GFAP-1477 formulated in artificial cerebrospinal fluid (aCSF) via i.t. lumbar injections. Animals were sacrificed 7 days following intrathecal injection.
- CNS central nervous system
- aCSF artificial cerebrospinal fluid
- Gfap expression was reduced by about 50% or greater in samples from the cervical spinal cord, thoracic spinal cord, lumbar spinal cord, and cerebellum as shown in FIGs. 1A, IB, 1C, and IF, respectively. Less reduction of Gfap expression was observed in the frontal cortex and hippocampus as shown in FIGs. ID and IE, respectively. The average percent (%) of mRNA remaining is shown in FIG. 2, along with the EDso values determined for each region of the brain based on the percent mRNA remaining in the tissue at each concentration of oligonucleotide. Together, this data demonstrates GFAP targeting oligonucleotides having a GalNAc- conjugated tetraloop inhibit GFAP expression following i.t. administration in a dosedependent manner in several tissues of the CNS.
- GFAP-1477 was also assessed for the ability to reduce murine Gfap in the central nervous system (CNS) via intracerebroventricular (i.c.v) administration. Specifically, mice were administered 10, 32, 100, or 300 pg of GFAP-1477 formulated in aCSF via i.c.v. Animals were sacrificed 7 days following i.c.v. injection. RNA was extracted from CNS tissue as described above. Reduction of Gfap expression was observed in a dose-dependent manner across tissues of the CNS. Specifically, Gfap expression was reduced by about 50% or greater in samples of the frontal cortex, brain stem, hippocampus, and lumbar spinal cord as shown in FIGs. 3A-3D, respectively.
- the average percent (%) of mRNA remaining is shown in FIG. 4, along with the EDso values determined for each region of the brain based on the percent mRNA remaining in the tissue at each concentration of oligonucleotide.
- this data demonstrates GFAP targeting oligonucleotides having a GalNAc- conjugated tetraloop inhibit GFAP expression in several tissues of the CNS via i.c.v administration in a dose-dependent manner.
- Example 3 Synthesis of Tetraloop RNAi Oligonucleotide-Lipid Conjugates
- Lipid-conjugated tetraloop oligonucleotides described herein can be synthesized using post-synthetic methods described in detail in PCT application No. PCT/US2021/42469. Specifically, the oligonucleotides can be synthesized using a post-synthetic conjugation approach such as that depicted below.
- Scheme 1 Synthesis of RNAi oligonucleotide-lipid conjugates with mono-lipid
- R1COOH group represents fatty acid C8:0, C10:0, Cl 1 :0, C12:0, C14:0, C16:0,
- Synthesis Sense 1 and Antisense 1 were prepared by solid-phase synthesis. Synthesis of Conjugated Sense la-li.
- Conjugated Sense la was synthesized through post-syntenic conjugation approach.
- Eppendorf tube 1 a solution of octanoic acid (0.58 mg, 4 umol) in DMA (0.75 mL) was treated with HATU (1.52 mg, 4 umol) at rt.
- Eppendorf tube 2 a solution of oligo Sense 1 (10.00 mg, 0.8 umol) in H2O (0.25 mL) was treated with DIPEA (1.39 uL, 8 umol). The solution in Eppendorf tube 1 was added to the Eppendorf tube 2 and mixed using ThermoMixer at rt.
- reaction mixture was diluted with 5 mL of water and purified by revers phase XB ridge Cl 8 column using a 5-95% gradient of 100 mM TEAA in ACN and H2O.
- the product fractions were concentrated under reduced pressure using Genevac.
- the combined residual solvent was dialyzed against water (1 X), saline (1 X), and water (3 X) using Amicon® Ultra- 15 Centrifugal (3K).
- Amicon membrane was washed with water (3 X 2 mL) and the combined solvents were then lyophilized to afford an amorphous white solid of Conjugated Sense la (6.43 mg, 64% yield).
- Conjugated Sense Ib-li were prepared using similar procedures as described for the synthesis of Conjugated Sense la and obtained in 42%-69% yields.
- Conjugated Sense la (10 mg, measured by weight) was dissolved in 0.5 mL deionized water to prepare a 20 mg/mL solution.
- Antisense 1 (10 mg, measured by OD) was dissolved in 0.5 mL deionized water to prepare a 20 mg/mL solution, which was used for the titration of the conjugated sense and quantification of the duplex amount. Based on the calculation of molar amounts of both conjugated sense and antisense, a proportion of required Antisense 1 was added to the Conjugated Sense la solution. The resulting mixture was stirred at 95 °C for 5 min and allowed to cool down to rt. The annealing progress was monitored by ion-exchange HPLC.
- Duplex Ib-li were prepared using the same procedures as described for the annealing of Duplex la (C8).
- Sense IB and Antisense IB were prepared by solid-phase synthesis.
- Eppendorf tube 1 a solution of oligo (10.00 mg, 0.8 umol) in a 3: 1 mixture of DMA/ H2O (0.5 mL) was treated with the lipid linker azide (11.26 mg, 4 umol).
- Eppendorf tube 2 CuBr dimethyl sulfide (1.64 mg, 8 umol) was dissolved in ACN (0.5 mL). Both solutions were degassed for 10 min by bubbling N2 through them. The ACN solution of CuBrSMe2 was then added into tube 1 and the resulting mixture was stirred at 40 °C.
- reaction mixture was diluted with 0.5 M EDTA (2 mL) and dialyzed against water (2 X) using a Amicon® Ultra- 15 Centrifugal (3K).
- the reaction crude was purified by revers phase XBridge Cl 8 column using a 5-95% gradient of 100 mM TEAA in ACN (with 30% IPA spiked in) and H2O.
- the product fractions were concentrated under reduced pressure using Genevac.
- the combined residual solvent was dialyzed against water (1 X), saline (1 X), and water (3 X) using Amicon® Ultra- 15 Centrifugal (3K).
- the Amicon membrane was washed with water (3 X 2 mL) and the combined solvents were lyophilized to afford an amorphous white solid of Conjugated Sense Ij (6.90 mg, 57% yield).
- Duplex Ij (PEG2K-diacyl C18) was prepared using the same procedures as described for the annealing of Duplex la (C8).
- the following Scheme 1-3 depicts the synthesis of Nicked tetraloop GalXC conjugates with di-lipid on the loop using post-synthetic conjugation approach.
- Sense 2 and Antisense 2 were prepared by solid-phase synthesis.
- Conjugated Sense 2a and 2b were prepared using similar procedures as described for the synthesis of Conjugated Sense la but with 10 eq of lipid, 10 eq of HATU, and 20 eq of DIPEA.
- Duplex 2a (2XC11) and 2b (2XC22) were prepared using the same procedures as described for the annealing of Duplex la (C8).
- the following Scheme 1-4 depicts the synthesis of GalXC of fully phosphorothioated stem-loop conjugated with mono-lipid using post-synthetic conjugation approach.
- Sense 3 and Antisense 3 were prepared by solid-phase synthesis.
- Conjugated Sense 3a was prepared using similar procedures as described for the synthesis of Conjugated Sense la and obtained in a 65% yield.
- Duplex 3a (PS-C22) was prepared using the same procedures as described for the annealing of Duplex la (C8).
- Sense 4 and Antisense 4 were prepared by solid-phase synthesis.
- Conjugated Sense 4a was prepared using similar procedures as described for the synthesis of Conjugated Sense la and obtained in a 74% yield.
- Duplex 4a (SS-C22) was prepared using the same procedures as described for the annealing of Duplex la (C8).
- the following scheme 1-6 depicts an example of solid phase synthesis of Nicked tetraloop GalXC conjugated with lipid(s) on the loop.
- Conjugated Sense 6 was prepared by solid-phase synthesis using a commercial oligo synthesizer.
- the oligonucleotides were synthesized using 2’-modified nucleoside phosphoramidites, such as 2’-F or 2’-0Me, and 2'-diethoxymethanol linked fatty acid amide nucleoside phosphoramidites.
- Oligonucleotide synthesis was conducted on a solid support in the 3’ to 5’direction using a standard oligonucleotide synthesis protocol.
- 5-ethylthio-lH- tetrazole (ETT) was used as an activator for the coupling reaction. Iodine solution was used for phosphite triester oxidation.
- Duplex 6 was prepared using the same procedures as described for the annealing of
- Conjugated Sense 10a was obtained using the same method or a substantially similar method to the synthesis of Conjugated Sense 5.
- Duplex 10a was obtained using the same method or a substantially similar method to the synthesis of Duplex 5.
- Example 4 G7 1C-Targeting Oligonucleotides Demonstrate Long-term Duration of Action via Intrathecal and Intracisterna Magna Administration
- duration of action was determined using oligonucleotides with different targeting ligands. Specifically, a GalN Ac-conjugated Gfap oligonucleotide as described in Example 2, and a C16-conjugated Gfap oligonucleotide generated by methods described in Example 1, the sequences of which are provided in Table 3 were administered to rats for long term-studies. A C16 lipid moiety was conjugated at position 28 of the sense strand.
- the GalN Ac-conjugated and lipid-conjugated Gfap oligonucleotides described above were administered to rats via intrathecal (i.t.) injection to assess long-term duration of reduction of Gfap expression.
- intrathecal (i.t.) injection to assess long-term duration of reduction of Gfap expression.
- Sprague-Dawley rats (250 g) were administered a single 1000 pg dose of an oligonucleotide in Table 3 formulated in aCSF vis i.t. injection.
- Target knock-down was assessed 8, 12, and 23 weeks after i.t. injection.
- the levels of murine Gfap mRNA were determined using PrimeTimeTM qPCR Probe Assays (IDT),
- IDT PrimeTimeTM qPCR Probe Assays
- the Cl 6- conjugated oligonucleotide demonstrated about 50% or greater reduction of Gfap expression in the brainstem and SC1-SC8 over the course of the study (i.e., at 8, 12, and 23 weeks) (FIG. 5A).
- FIG. 5B This data demonstrates the long-term sustained inhibition of astrocyte target mRNA (e.g., GFAP) in the central nervous system following a single intrathecal injection.
- astrocyte target mRNA e.g., GFAP
- the C16-conjugated GFAP oligonucleotide described in Table 3 was administered to rats intracistema magna (i.c.m.) injection to assess long-term duration of reduction of Gfap expression.
- rats intracistema magna (i.c.m.) injection were administered to rats intracistema magna (i.c.m.) injection to assess long-term duration of reduction of Gfap expression.
- Sprague-Dawley rats were administered a single 1000 pg dose of oligonucleotide formulated in aCSF via i.c.m injection.
- Gfap expression was assessed 4 and 12 weeks after injection.
- RNA was extracted from tissue samples of the prefrontal cortex, somatosensory cortex, hippocampus, hypothalamus, cerebellum, brain stem, cervical spinal cord, and lumbar spinal cord to determine rat Gfap mRNA levels by qPCR as described in Example 2.
- RNAi oligonucleotide-lipid conjugates comprising a tetraloop to reduce mRNA expression in astrocytes of the CNS was evaluated in vivo.
- C16-conjugated Gfap oligonucleotides were generated as described in Example 1. Specifically, a C16 lipid was conjugated at one of positions (P) 1, 4, 8, 12, 13, 18, 20, 23, 28, 29, and 30 in the sense strand as shown in the modification patterns below.
- the unmodified sense and antisense strands are provided in SEQ ID NOs: 3 and 4, respectively, and the modified strands are shown in Table 4
- Antisense Strand [MePhosphonate-40- mXs] [fXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [mX]
- RNAi oligonucleotide formulated in artificial cerebrospinal fluid via intrathecal (i.t.) lumbar injection. Target expression was assessed 7 days after injection.
- the following schematic depicts the synthesis of a blunt end oligonucleotide with a C 16-lipid at the 5 ’-end.
- Lipid-conjugated blunt-ended oligonucleotides described herein can be synthesized using post-synthetic methods described in detail in PCT application No. PCT/US2021/42469. Specifically, the oligonucleotides can be synthesized using a postsynthetic conjugation approach such as that depicted below.
- Eppendorf tube 1 a solution of palmitic acid in DMA was treated with HATU at rt.
- Eppendorf tube 2 a solution of oligo sense strand in H2O was treated with DIPEA .
- the solution in Eppendorf tube 1 was added to the Eppendorf tube 2 and mixed using ThermoMixer at rt. After the reaction was completed indicated by LC-MS analysis, the reaction mixture was diluted with 5 mL of water and purified by reverse phase XBridge Cl 8 column using a 5-95% gradient of 100 mM TEAA in ACN and H2O. The product fractions were concentrated under reduced pressure using Genevac. The combined residual solvent was dialyzed against water (1 X), saline (1 X), and water (3 X) using Amicon® Ultra-15 Centrifugal (3K). The Amicon membrane was washed with water (3 X 2 mL) and the combined solvents were then lyophilized to afford an amorphous white solid.
- RNAi oligonucleotides conjugated to a lipid at various positions of the sense strand were evaluated for their ability to reduce an astrocyte target expression in the CNS.
- RNAi oligonucleotides conjugated to a C16 lipid were generated as described above. Specifically, oligonucleotides having a blunt-end at the 3 ’terminus and a 2-nucleotide overhang at the 5’terminus were generated with a C16 lipid conjugated at positions (P) 1, 4, 8, 12, 13, 18, or 20 of the sense strand as shown in the modification patterns below.
- Select lipid-conjugated tetraloop oligonucleotides from Example 5 were included for comparison (Pl, P4, P23, and P28).
- the unmodified sense and antisense strands are provided in SEQ ID NOs: 19 and 4, respectively, and the modified strands are shown in Table 5.
- Antisense Strand [MePhosphonate-40- mXs] [fXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [mX]
- mRNA expression was reduced in the cerebellum for oligonucleotides with a C16 lipid conjugated at P4, P12, P13, P18, or P20 (FIG. 9C).
- expression was reduced by oligonucleotides with a C16 lipid conjugated at Pl, P4, P12, P13, P18, or P20 (FIG. 9D).
- Minimal inhibition of Gfap mRNA was observed in the hippocampus and frontal cortex (FIGs. 9E and 9F, respectively).
- the lipid-conjugated tetraloop oligonucleotides tested demonstrated similar expression reduction as that shown in Example 5. Overall, several positions of lipid- conjugation across blunt-end oligonucleotide are successful inhibitors of target mRNA in the CNS.
- Example 5 The percent remaining mRNA from the experiments described in Example 5 and the present example was compared. Specifically, the data is summarized in FIGs. 10A-10F to demonstrate potency across brain regions and lipid-positions in tetraloop and blunt-end oligonucleotides.
- Example 8 C16-Conjugated GFAP Blunt-End Oligonucleotides Inhibit Mouse Gfap In Vivo in a Concentration Dependent Manner
- a blunt-end oligonucleotide with a C16 lipid conjugated at position 1 was assessed for the ability to reduce murine Gfap in the central nervous system (CNS) via i.t. administration in a concentration-dependent manner.
- mice were administered 3, 10, 30, 100, or 300 pg of GFAP- 1477 (SEQ ID NO: 20 (sense strand) and SEQ ID NO: 18 (antisense strand)) formulated in aCSF via i.t. injection. Animals were sacrificed 7- or 28- days following i.t. injection.
- RNA was extracted from liver tissue as described in Example 2. Reduction of Gfap expression was observed in a concentration-dependent manner across several tissues of the CNS.
- FIG. 11 A Specifically, at 7 days following injection, expression was reduced in the hypothalamus, cerebellum, brain stem, and lumbar spinal cord (FIG. 11 A). The reduction was maintained out to 28-days in the same tissues, indicating long-term inhibition after a single administration of blunt-end oligonucleotide (FIG. 11B). Together, this data demonstrates the long-term potency of lipid-conjugated blunt-end oligonucleotides to inhibit astrocyte target mRNA.
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