Apolipoprotein E (ApoE) iRNA agent compositions and methods of use thereof

Abstract

The present disclosure relates to double-stranded ribonucleic acid (dsRNAi) agents and compositions that target the APOE gene, and methods of using such dsRNAi agents and compositions to inhibit expression of the APOE gene and to treat subjects with APOE-associated neurodegenerative diseases or disorders, such as Alzheimer's disease and Parkinson's disease.

Description

Technical field

[0001] Related application This application claims priority to US Provisional Patent Application No. 63 / 015,867, filed April 27, 2020, the entire contents of which are incorporated herein by reference.

[0002] sequence listing The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 20, 2021, is named 121301_11220_SL.txt and is 200,944 bytes in size. [Background technology]

[0003] The apolipoprotein E gene encodes the apolipoprotein E (APOE) protein, a glycoprotein composed of 299 amino acids after cleavage of the 18 amino acid signal peptide. APOE exists in three common isoforms, APOE2, APOE3, and APOE4, encoded by corresponding alleles. Three APOE isoforms, ApoE ε2 (APOE2), ApoE ε3 (APOE3), ApoE ε4 (APOE4), differ from each other only at amino acid positions 112 and 158, APOE2 having Cys112 and Cys158 and APOE3 having Cys112 and Arg158. and APOE4 has Arg112 and Arg158. APOE is widely expressed, but peripherally expressed primarily in hepatocytes of the liver and glial cells in the central nervous system (CNS).

[0004] In the periphery, APOE functions in lipid homeostasis. These lipoprotein particles cannot cross the blood-brain barrier. Studies have shown that apoE-containing particles released from astrocytes and microglia are the major source of brain apoE [Bjorkhem I, et al. (1998) J Lipid Research 39(8). ): 1594-1600; Pitas RE, et al. (1987) Biochimica Biophysica Acta. 13;917(1):148-161; Krasemann S, et al. (2017) Immunity. e9. doi:10.1016 / j.immuni.2017.08.008]. In the brain, APOE modulates multiple pathways including lipid transport, synaptic integrity and plasticity, glucose metabolism, neuroinflammation, and cerebrovascular integrity. For example, when APOE is secreted from the cell, several transporters (e.g., the ATP-binding cassette (cssestte) transporter) transfer cholesterol and phospholipids to nascent APOE to form lipoprotein particles, which It is then distributed to neurons through binding to APOE receptors such as LDL receptor (LDLR) family members. Furthermore, it has been observed that the recipient serum APOE phenotype is fully converted to the donor phenotype after liver transplantation, whereas the cerebrospinal fluid (CSF) ApoE phenotype is not. In addition, astrocytes produce APOE in high-density lipoprotein (HDL)-like particles, which have properties distinct from APOE from other sources [e.g., Morikawa, et al. al., Neurobiol Dis.. Jun-Jul 2005;19(1-2):66-76]. Therefore, APOE in CSF cannot be obtained from plasma pools and must therefore be synthesized locally [Linton MF, et al. (1991) J Clin Invest. 88(1):270-281. doi: 10.1172 / JCI115288].

[0005] Polymorphisms in the APOE gene have been associated with multiple proteinopathies. The most established association between APOE polymorphisms and disease is between APOE genotype and Alzheimer's disease (AD), which is a risk determinant of late-onset Alzheimer's disease with symptom onset after age 65 years. It has been shown that In addition, a recent study from the Haltzman lab showed that carrying the ε4 allele significantly accelerated disease progression (p=0.02), with one ε4 allele increasing the rate of progression by 14% compared to non-carriers. an increase of 23% with two ε4 alleles [Holtzman, et al. (2017) Nature 549:523]. AD is the leading cause of dementia in the elderly and its pathological hallmarks include the deposition of extracellular amyloid-β (Aβ) aggregates as amyloid plaques and intracellular hyperphosphorylation as neurofibrillary tangles. Tau aggregates, as well as neuronal loss and glial activation. Since individuals with late-onset AD make up over 95% of the total AD population, various efforts are underway to elucidate the role of APOE in AD.

[0006] More specifically, subjects with one copy of APOE4 have a >3-fold risk of developing AD and subjects with two copies of APOE4 have a >12-fold increased risk of developing AD. In contrast, two copies of APOE2 have been shown to be protective against developing AD in subjects [Reiman EM, et al. (2020) Nature Communications 11 (1); 667]. In addition, although there are three human cases of APOE knockout that have been reported, none of these subjects (40-60 years old) experienced dementia at presentation [Ghiselli, et al. 1981) Science 214(4526):1239; Mak, et al. (2014) JAMA Neurol 71:1228; and Lohse, et al. (1992) J Lipid Res. (11):1583]. In one of the three cases (a 40-year-old man), MRI and cerebrospinal fluid (CSF) biomarker studies showed no signs of neurodegeneration, intact brain structures, and tau and p-tau levels. Levels were demonstrated to be in the normal range. Moreover, recent studies show that the Christchurch mutation in ApoE3 contributes to pyresenilin-1-driven dementia, as evidenced by conserved cognitive function and limited tauopathy by PET. It has been shown that it can be protective against When the Christchurch mutation is present, ApoE3 loses its ability to bind to HSPG and LDL receptors, and the patient has hyperlipoproteinemia type III but no cardiovascular disease [Arboleda-Velasquez, et al. (2019) Nature Medicine 25:1680].

[0007] Increased ApoE4 expression accelerates amyloid accumulation and neuritic dystrophy in an ApoE-induced amyloid mouse model [Liu, et al. (2017) Neuron 96:1024] and Huynh, et al. [Neuron (2017) ) 96:1013], as well as that antisense inhibition of APOE4 is protective in amyloid precursor protein (APP) / presenilin 1 (PS1-21) transgenic mice. In addition, deletion of ApoE4 was protective against neurodegeneration in a mouse model of tauopathy [Holtzman, et al. (2017) Nature 549:523], and APOE4-expressing but APOE-null showed that re-introduction of APOE4 expression in human neurons derived from induced pluripotent stem cells obtained toxic effects from APOE4 [wang, et al. (2018) Nat Medicine 24:647]. there is Furthermore, it has been shown that restricting APOE4 expression to the liver in mice can affect cognitive performance, impair the blood-brain barrier, and increase neuroinflammation (alzforum .org / news / research-news / apoe-has-hand-Alzheimer's-beyond-av-beyond-brain).

[0008] Currently, there are no curative or prophylactic treatments for treating subjects with APOE-related neurodegenerative diseases such as AD, and supportive and symptomatic care is the mainstay of treatment. Accordingly, there is a need in the art for compositions and methods for treating subjects with or at risk of developing a neurodegenerative disease. [Outline of the invention]

[0009] The present disclosure provides RNAi agent compositions that effect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the apolipoprotein E (APOE) gene. The APOE gene may be intracellular, eg, in a cell within a subject, eg, a human. The present disclosure is also directed to subjects, e.g., APOE-associated neurodegeneration, for which inhibition or reduction of expression of the APOE gene, e.g., the pathogenic APOE allele, i.e., APOE4, would be beneficial. Also provided are methods of using the RNAi agent compositions of the present disclosure to treat a subject suffering from or predisposed to suffering from a disease, such as an amyloid-beta-mediated disease or a tau-mediated disease. In particular, the RNAi agent compositions herein can affect specific APOE expression by astrocytes within the CNS to treat APOE-related neurodegenerative diseases.

[0010] Accordingly, in one aspect, the present disclosure provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting expression of an apolipoprotein E (APOE) gene, the RNAi agent comprising a sense strand and an antisense strand, The antisense strand differs from any one of the antisense sequences listed in any one of Tables 2-5 and 7-10 by no more than 3 nucleotides (i.e., 3, 2, 1, or Provided are RNAi agents comprising regions of complementarity comprising at least 15 contiguous nucleotides (different by 0 nucleotides). In certain embodiments, the antisense strand has a region of complementarity comprising at least 15 contiguous nucleotides of any one of the antisense sequences listed in any one of Tables 2-5 and 7-10. including. In certain embodiments, the antisense strand comprises a region of complementarity comprising at least 15 contiguous nucleotides of any one of the antisense sequences listed in any one of Tables 7 and 8. In certain embodiments, the antisense strand comprises a region of complementarity comprising at least 15 contiguous nucleotides of any one of the antisense sequences listed in any one of Tables 9 and 10. In certain embodiments, the antisense strand differs from any one of the antisense sequences listed in any one of Tables 2-5 and 7-10 by no more than 3 nucleotides (i.e., 3, regions of complementarity comprising at least 19 contiguous nucleotides (different by 2, 1, or 0 nucleotides). In certain embodiments, the antisense strand comprises any one of the antisense sequences listed in any one of Tables 7 and 8 (i.e., 3, 2, 1, or 0 nucleotides). differ), including regions of complementarity comprising at least 19 contiguous nucleotides. In certain embodiments, the antisense strand comprises any one of the antisense sequences listed in any one of Tables 9 and 10 (i.e., 3, 2, 1, or 0 nucleotides). differ), including regions of complementarity comprising at least 19 contiguous nucleotides. In certain embodiments, the antisense strand has a region of complementarity comprising at least 19 contiguous nucleotides of any one of the antisense sequences listed in any one of Tables 2-5 and 7-10. including. In certain embodiments, the antisense strand comprises a region of complementarity comprising at least 19 contiguous nucleotides of any one of the antisense sequences listed in any one of Tables 7 and 8. In certain embodiments, the antisense strand comprises a region of complementarity comprising at least 19 contiguous nucleotides of any one of the antisense sequences listed in any one of Tables 9 and 10. In certain embodiments, thymine to uracil or uracil to thymine differences between the aligned (compared) sequences are not counted as different nucleotides between the aligned (compared) sequences.

[0011] In some embodiments, the agent comprises one or more lipophilic moieties conjugated to one or more internal nucleotide positions, optionally via a linker or carrier.

[0012] In other embodiments, the agent further comprises a targeting ligand that targets liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier. .

[0013] In yet other embodiments, the agent comprises one or more lipophilic moieties conjugated to one or more internal nucleotide positions, optionally via a linker or carrier, and a double-stranded Further included are targeting ligands that target liver tissue, eg, one or more GalNAc derivatives, optionally conjugated to the RNAi agent.

[0014] In certain embodiments, the double-stranded RNAi agent inhibits expression of APOE2, APOE3, and APOE4 alleles. In other embodiments, the double-stranded RNAi agent inhibits expression of APOE4 but does not substantially inhibit expression of APOE2 and APOE3, e.g., expression of APOE2 and APOE3 is inhibited by about 10% or less. .

[0015] Another aspect of the present disclosure is a double-stranded RNAi agent for inhibiting expression of the apolipoprotein E (APOE) gene, wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprising: Table 2 At least 15 contiguous sequences that differ by no more than 3 nucleotides (i.e., differ by 3, 2, 1, or 0 nucleotides) from any one of the sense strand sequences presented in ~5 and 7-10 nucleotides, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense strand nucleotide sequences presented in Tables 2-5 and 7-10. Provide RNAi agents. In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides of any one of the sense strand sequences presented in Tables 2-5 and 7-10, and the antisense strand comprises any one of Tables 2-5 and 7-10. Contains at least 15 contiguous nucleotides of any one of the antisense strand nucleotide sequences presented in 7-10. In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides of any one of the sense strand sequences presented in Tables 7 and 8, and the antisense strand comprises the antisense sequences presented in Tables 7 and 8. Contains at least 15 contiguous nucleotides of any one of the strand nucleotide sequences. In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides of any one of the sense strand sequences presented in Tables 9 and 10, and the antisense strand comprises the antisense sequences presented in Tables 9 and 10. Contains at least 15 contiguous nucleotides of any one of the strand nucleotide sequences. In certain embodiments, the sense strand is any one of the sense strand sequences presented in Tables 2-5 and 7-10 (i.e., differs by 3, 2, 1, or 0 nucleotides). Comprising at least 19 contiguous nucleotides, the antisense strand comprises at least 19 contiguous nucleotides of any one of the antisense strand nucleotide sequences presented in Tables 2-5 and 7-10 (i.e., 3 , differ by 2, 1, or 0 nucleotides). In certain embodiments, the sense strand comprises at least 19 (i.e., differing by 3, 2, 1, or 0 nucleotides) of any one of the sense strand sequences presented in Tables 7 and 8. Containing contiguous nucleotides, the antisense strand comprises at least 19 nucleotides of any one of the antisense strand nucleotide sequences presented in Tables 7 and 8 (i.e., differing by 3, 2, 1, or 0 nucleotides). contains consecutive nucleotides. In certain embodiments, the sense strand comprises at least 19 nucleotides of any one of the sense strand sequences presented in Tables 9 and 10 (i.e., differing by 3, 2, 1, or 0 nucleotides). Comprising contiguous nucleotides, the antisense strand comprises at least 19 nucleotides of any one of the antisense strand nucleotide sequences presented in Tables 9 and 10 (i.e., differing by 3, 2, 1, or 0 nucleotides). contains consecutive nucleotides.

[0016] In some embodiments, the agent comprises one or more lipophilic moieties conjugated to one or more internal nucleotide positions, optionally via a linker or carrier.

[0017] In other embodiments, the agent further comprises a targeting ligand that targets liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier. .

[0018] In yet other embodiments, the agent comprises one or more lipophilic moieties conjugated to one or more internal nucleotide positions, optionally via a linker or carrier, and a double-stranded Further included are targeting ligands that target liver tissue, eg, one or more GalNAc derivatives, optionally conjugated to the RNAi agent.

[0019] In certain embodiments, the double-stranded RNAi agent inhibits expression of APOE2, APOE3, and APOE4 alleles. In other embodiments, the double-stranded RNAi agent inhibits expression of APOE4 but does not substantially inhibit expression of APOE2 and APOE3, e.g., expression of APOE2 and APOE3 is inhibited by about 10% or less. .

[0020] A further aspect of the present disclosure is a double-stranded RNAi agent for inhibiting expression of the apolipoprotein E (APOE) gene, wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprising SEQ ID NO:1 , 3, 5, 7, or 9, or at least 90% nucleotide sequence identity to the entire nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, 7, or 9, e.g. , 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical nucleotide sequences, and differ by no more than 3 nucleotides (i.e., 3, 2, 1 1, 3, 5, 7, and 9 (compare aligned sequences if) is any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, and 9, or at least 90% of the entire nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, 7, or 9 , e.g., nucleotide sequences with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, differ by no more than 3 nucleotides (i.e., 3, 2, 1, or 0 nucleotide differences), and the antisense strand is any of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, or 10. or one, or the entire nucleotide sequence of any one of SEQ ID NOs: 2, 4, 6, 8, or 10, e.g., 90, 91, 92, 93, 94, 95, 96 , a nucleotide sequence having 97, 98, 99, or 100% identity, and differing by no more than 3 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides) at least 15 Substitutions of thymine to uracil (when comparing aligned sequences) in any of SEQ ID NOs: 2, 4, 6, 8, and 10 containing contiguous nucleotides are SEQ ID NOs: 2, 4, 6, 8, and 10 nucleotide sequences, or at least 90% nucleotide sequence identity to the entire nucleotide sequence of any one of SEQ ID NOs: 2, 4, 6, 8, or 10, e.g., 90, 91, 92, A nucleotide sequence with 93, 94, 95, 96, 97, 98, 99, or 100% identity differs by no more than 3 nucleotides (i.e., 3, 2, 1, or 0 nucleotides) at least one of the sense and antisense strands are conjugated to one or more internal nucleotide positions via a linker or carrier as appropriate. A double-stranded RNAi agent is provided that includes multiple lipophilic moieties.

[0021] In one embodiment, the APOE-targeting double-stranded RNAi agent differs by no more than 3 nucleotides from the nucleotide sequence of the sense strand nucleotide sequences of the duplexes set forth in Tables 2-5 and 7-10 (i.e., a sense strand comprising at least 15 contiguous nucleotides that differ by 3, 2, 1, or 0 nucleotides. In one embodiment, the APOE-targeting double-stranded RNAi agent differs by no more than 3 nucleotides from the nucleotide sequence of the sense strand nucleotide sequences of the duplexes set forth in Tables 7 and 8 (i.e., 3, 2 a sense strand comprising at least 15 contiguous nucleotides (different by 1, 1, or 0 nucleotides). Double-stranded RNAi agents that target APOE differ from the nucleotide sequence of the sense strand nucleotide sequences of the duplexes listed in Tables 9 and 10 by no more than 3 nucleotides (i.e., 3, 2, 1, a sense strand comprising at least 15 contiguous nucleotides (or differing by 0 nucleotides).

[0022] In one embodiment, the APOE-targeting double-stranded RNAi agent comprises the antisense nucleotide sequence of any one of the duplexes described in one of Tables 2-5 and 7-10 and no more than 3 nucleotides. Includes antisense strands that include at least 15 contiguous nucleotides that differ (ie, differ by 3, 2, 1, or 0 nucleotides). In one embodiment, the APOE-targeting double-stranded RNAi agent differs from the double-stranded antisense nucleotide sequence of one of Tables 7 and 8 by no more than 3 nucleotides (i.e., 3, 2 and an antisense strand that comprises at least 15 contiguous nucleotides (different by 1, 1, or 0 nucleotides). In one embodiment, the APOE-targeting double-stranded RNAi agent differs from the double-stranded antisense nucleotide sequence of one of Tables 9 and 10 by no more than 3 nucleotides (i.e., 3, 2 and an antisense strand that comprises at least 15 contiguous nucleotides (different by 1, 1, or 0 nucleotides).

[0023] In one embodiment, the double-stranded RNAi agent targeting APOE is nucleotides 50-113, 59-97, 59-90, 107-177, 107-153, 124-153, 198-240 of SEQ ID NO:1, 203~240, 209~240, 283~378, 283~312, 307~378, 322~369, 330~357, 394~419, 568~600, 568~594, 841~879, 900~926, 997~ differs from any one of the nucleotide sequences 1055, 1002-1044, 1014-1044, 1019-1044, 1120-1166, 1130-1166, 1130-1155 by no more than 3 nucleotides (i.e., 3, 2, 1 a sense strand comprising at least 15 contiguous nucleotides (different by 1, or 0 nucleotides), and an antisense strand comprising at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:2.

[0024] In one embodiment, the double-stranded RNAi agent targeting APOE is any one and three of the nucleotide sequences of nucleotides 59-90, 330-357, 568-594, 1019-1044, 1130-1155 of SEQ ID NO:1. a sense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides (i.e., differ by 3, 2, 1, or 0 nucleotides), and at least from the corresponding nucleotide sequence of SEQ ID NO:2; Includes an antisense strand containing 15 contiguous nucleotides.

[0025] In one embodiment, the double-stranded RNAi agent targeting APOE is nucleotides 57-79, 62-84, 75-97, 86-108, 207-229, 213-235, 218-240 of SEQ ID NO:1, at least 15 that differ by no more than 3 nucleotides from any one of the nucleotide sequences 898-920, 1128-1150, 637-659 (i.e., differ by 3, 2, 1, or 0 nucleotides) A sense strand comprising contiguous nucleotides and an antisense strand comprising at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:2.

[0026] In one embodiment, the double-stranded RNAi agent that targets APOE is any one of nucleotides 57-79, 62-84, 207-229, 1128-1150 of SEQ ID NO: 1 and no more than 3 nucleotides. (i.e., differ by 3, 2, 1, or 0 nucleotides) and at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:2 includes an antisense strand containing nucleotides that

[0027] In one embodiment, the double-stranded RNAi agent that targets APOE is Any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of -1204711, AD-1204712, and AD-1204713 differs by no more than 3 nucleotides (i.e., 3, 2, 1 , or differ by 0 nucleotides), including an antisense strand comprising at least 15 contiguous nucleotides.

[0028] In one embodiment, the APOE-targeting double-stranded RNAi agent comprises a double-stranded antisense strand nucleotide sequence selected from the group consisting of AD-1204704, AD-1204705, AD-1204708, and AD-1204712. Includes antisense strands comprising at least 15 contiguous nucleotides that differ from any one by no more than 3 nucleotides (ie, differ by 3, 2, 1, or 0 nucleotides).

[0029] In some embodiments, the agent further comprises a targeting ligand that targets liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier. include.

[0030] In certain embodiments of the invention, the double-stranded RNAi agent inhibits expression of APOE2, APOE3, and APOE4 alleles. In other embodiments, the double-stranded RNAi agent inhibits expression of APOE4 but does not substantially inhibit expression of APOE2 and APOE3, e.g., expression of APOE2 and APOE3 is inhibited by about 10% or less. .

[0031] A double-stranded RNAi agent may comprise at least one modified nucleotide.

[0032] In certain embodiments, logK ow The lipophilicity of the lipophilic moiety as measured by is greater than zero.

[0033] In some embodiments, the hydrophobicity of the double-stranded RNAi agent as measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent is greater than 0.2. In a related embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0034] In certain embodiments, substantially all of the nucleotides of the sense strand are modified nucleotides. All of the nucleotides of the sense strand may be modified nucleotides.

[0035] In some embodiments, substantially all of the nucleotides of the antisense strand are modified nucleotides. All of the nucleotides of the antisense strand may be modified nucleotides.

[0036] All of the nucleotides of the sense strand and all of the nucleotides of the antisense strand may be modified nucleotides.

[0037] In one embodiment, at least one of the modified nucleotides is a deoxynucleotide, a 3' terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2' -deoxy modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyly modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phospho Luamidate, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, 5' -nucleotides containing a methylphosphonate group, nucleotides containing a 5' phosphate or 5' phosphate mimetic, nucleotides containing a vinyl phosphonate, nucleotides containing adenosine-glycol nucleic acid (GNA), thymidine-glycol nucleic acid (GNA) S-isomers a nucleotide containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, a nucleotide containing 2'-deoxythymidine-3'phosphate, a nucleotide containing 2'-deoxyguanosine-3'-phosphate, or a cholesteryl derivative or dodecanoic acid It is the terminal nucleotide linked to the bisdecylamide group.

[0038] In related embodiments, the modified nucleotides are 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 3' terminal deoxythymidine nucleotides (dT), locked nucleotides, Base nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, or nucleotides containing non-natural bases.

[0039] In one embodiment the modified nucleotides comprise a short sequence of 3' terminal deoxythymidine nucleotides (dT).

[0040] In another embodiment, modifications on nucleotides are 2'-O-methyl, 2'fluoro, and GNA modifications.

[0041] In a further embodiment, the double-stranded RNAi agent comprises at least one phosphorothioate internucleotide linkage. A double-stranded RNAi agent may comprise 6-8 (eg, 6, 7, or 8) phosphorothioate internucleotide linkages.

[0042] In certain embodiments, the regions of complementarity are at least 17 nucleotides in length. The regions of complementarity may be 19-23 nucleotides long. The region of complementarity may be 19 nucleotides long.

[0043] In one embodiment, each strand is 30 nucleotides or less in length.

[0044] In another embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide. At least one strand may include a 3' overhang of at least 2 nucleotides.

[0045] In certain embodiments, the double-stranded RNAi agent further comprises a lipophilic ligand, e.g., C16 ligand, conjugated by a monovalent or branched bivalent or trivalent linker to the 3' end of the sense strand. . In certain embodiments, the double-stranded RNAi agent further comprises a lipophilic ligand, eg, a C16 ligand, conjugated to an internal nucleotide position, eg, by a monovalent or branched bivalent or trivalent linker.

[0046] In one embodiment, the ligand is

[0047] [formation] (wherein B is a nucleotide base or nucleotide base analog, B may be adenine, guanine, cytosine, thymine, or uracil) is.

[0048] In other embodiments, the agent further comprises a targeting ligand that targets liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier. .

[0049] In yet other embodiments, the agent is a lipophilic ligand, e.g., a C16 ligand, conjugated to an internal nucleotide position, e.g., by a monovalent or branched bivalent or trivalent linker, and 3' of the sense strand. Further included are targeting ligands that target liver tissue, eg, one or more GalNAc derivatives, conjugated at the ends by monovalent or branched bivalent or trivalent linkers.

[0050] In another embodiment, the region of complementarity to APOE comprises any one of the antisense sequences set forth in any one of Tables 2-5 and 7-10. In certain embodiments, the region of complementarity to APOE comprises any one of the antisense sequences set forth in any one of Tables 7 and 8. In certain embodiments, the region of complementarity to APOE comprises any one of the antisense sequences set forth in any one of Tables 9 and 10.

[0051] In further embodiments, the region of complementarity to APOE is of any one of the antisense sequences set forth in any one of Tables 2-5 and 7-10. In certain embodiments, the region of complementarity to APOE is of any one of the antisense sequences listed in any one of Tables 7 and 8. In some embodiments, internal nucleotide positions include all but two positions from each end of the strand to the end. In certain embodiments, the region of complementarity to APOE is of any one of the antisense sequences listed in any one of Tables 9 and 10. In some embodiments, internal nucleotide positions include all but two positions from each end of the strand to the end.

[0052] In a related embodiment, internal positions include all but the three positions from each end of the chain to the end. Internal positions may exclude the cleavage site region of the sense strand.

[0053] In some embodiments, internal positions exclude positions 9-12 counting from the 5' end of the sense strand. In certain embodiments, the sense strand is 21 nucleotides long.

[0054] In other embodiments, internal positions exclude positions 11-13 counting from the 3' end of the sense strand. Internal positions may exclude the cleavage site region of the antisense strand. In certain embodiments, the sense strand is 21 nucleotides long.

[0055] In some embodiments, internal positions exclude positions 12-14 counting from the 5' end of the antisense strand. In certain embodiments, the antisense strand is 23 nucleotides in length.

[0056] In another embodiment, internal positions exclude positions 11-13 counting from the 3' end on the sense strand and positions 12-14 counting from the 5' end on the antisense strand. In certain embodiments, the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

[0057] In a further embodiment, the one or more lipophilic moieties are located at the following internal positions: positions 4-8 and 13-18 in the sense strand and position 6 in the antisense strand, counting from the 5' end of each strand. conjugated to one or more of ~10 and positions 15-18. The one or more lipophilic moieties are located at the following internal positions: positions 5, 6, 7, 15, and 17 in the sense strand and positions 15 and 17 in the antisense strand, counting from the 5' end of each strand. It may be conjugated to one or more. In certain embodiments, the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

[0058] In certain embodiments, the lipophilic moiety is an aliphatic compound, cycloaliphatic compound, or polycycloaliphatic compound. Lipophilic moieties include lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O (hexadecyl)glycerol, geranyloxyhexanol (hexyanol), hexadecylglycerol, borneol. , menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0059] In some embodiments, the lipophilic moiety is a saturated or unsaturated C 4 ~C 30 It contains a hydrocarbon chain and suitable functional groups selected from hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, or alkyne.

[0060] In certain embodiments, the lipophilic moiety is saturated or unsaturated C 6 ~C 18 Contains hydrocarbon chains. The lipophilic moiety can be saturated or unsaturated C 16 It may contain hydrocarbon chains. In a related embodiment, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides at internal positions. In certain embodiments, the carrier is pyrrolidinyl, pyrazolidinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, A cyclic group that is pyridazinonyl, tetrahydrofuranyl, or decalinyl, or an acyclic moiety based on a serinol or diethanolamine backbone.

[0061] In one embodiment, the lipophilic moiety is via linkers containing ethers, thioethers, ureas, carbonates, amines, amides, maleimide-thioethers, disulfides, phosphodiesters, sulfonamide linkages, products of click reactions, or carbamates. and conjugated to a double-stranded RNAi agent.

[0062] In one embodiment, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleoside linkage.

[0063] In another embodiment, the double-stranded RNAi agent further comprises a phosphate or phosphate mimetic at the 5' end of the antisense strand. A phosphate mimetic may be a 5'-vinylphosphonate (VP).

[0064] In certain embodiments, the double-stranded RNAi agent further comprises a targeting ligand, eg, a hydrophilic ligand, that targets receptors that mediate delivery to CNS tissues. In certain embodiments, the targeting ligand is C16 ligand.

[0065] In some embodiments, the double-stranded RNAi agent further comprises a targeting ligand that targets brain tissue, eg, the striatum.

[0066] In some embodiments, the double-stranded RNAi agent further comprises a targeting ligand that targets liver tissue, eg, hepatocytes.

[0067] In one embodiment, the lipophilic moiety or targeting ligand is a functionalized mono- or oligosaccharide of DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose, or a combination thereof. conjugated via a sexual linker.

[0068] In a related embodiment, the 3' end of the sense strand is protected via an endcap which is a cyclic group with an amine, the cyclic group being pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1, 3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

[0069] In one embodiment, the RNAi agent is a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a glycol nucleic acid (GNA) containing nucleotide, or a vinyl phosphonate containing nucleotide, at least one Contains modified nucleotides. The RNAi agent contains at least each of the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, glycol nucleic acid (GNA) containing nucleotides, and vinyl phosphonate containing nucleotides. May contain one.

[0070] In another embodiment, the RNAi agent comprises a pattern of modified nucleotides as shown in Tables 2-5 and 7-10 below, where 2'-C16, 2'-O-methyl, GNA, The location of the phosphorothioate and 2'-fluoro modifications is irrelevant to the individual nucleotide base sequences of the indicated RNAi agents. In one embodiment, the RNAi agent comprises a pattern of modified nucleotides as shown in Tables 7 and 8 below, where 2'-C16, 2'-O-methyl, GNA, phosphorothioate, and 2' -The location of the fluoro modification is independent of the individual nucleotide base sequence of the indicated RNAi agent. In one embodiment, the RNAi agent comprises a pattern of modified nucleotides as shown in Tables 9 and 10 below, where 2'-C16, 2'-O-methyl, GNA, phosphorothioate, and 2' -The location of the fluoro modification is independent of the individual nucleotide base sequence of the indicated RNAi agent.

[0071] Another aspect of the present disclosure is a double-stranded RNAi agent for inhibiting expression of the APOE gene, the double-stranded RNAi agent comprising a sense strand complementary to the antisense strand, and contains a region complementary to a portion of the mRNA encoding APOE, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III): Sense: 5'n p -N a -(X X X) i -N b -Y Y Y-N b -(Z Z Z) j -N a -n q 3' Antisense: 3'n p ’-N a ’-(X’X’X’) k -N b '-Y'Y'Y'-N b ’-(Z’Z’Z’) l -N a ’-n q '5' (III) (In the formula, i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6, each N a and N a ' independently represents an oligonucleotide sequence containing 0-25 nucleotides, which may be modified or unmodified, or a combination thereof, each sequence comprising at least two differently modified containing a nucleotide each N b and N b 'independently represents an oligonucleotide sequence comprising 0-10 nucleotides, which may be modified or unmodified, or a combination thereof; each n p , n p ’, n q , and n q ' each independently represents an overhanging nucleotide, which may or may not be present, XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides; N. b The modification above differs from the modification on Y in that N b ' is different from the modification on Y') is represented by the sense strand is conjugated to at least one ligand; A double-stranded RNAi agent is provided.

[0072] In one embodiment, i is 0, j is 0, i is 1, j is 1, i and j are both 0, or i and j are both are both 1.

[0073] In another embodiment, k is 0, l is 0, k is 1, l is 1, k and l are both 0, or k and l are both are 1.

[0074] In certain embodiments, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z' Complementary.

[0075] In certain embodiments, the double-stranded RNAi agent inhibits expression of APOE2, APOE3, and APOE4 alleles. In other embodiments, the double-stranded RNAi agent inhibits expression of APOE4 but does not substantially inhibit expression of APOE2 and APOE3, e.g., expression of APOE2 and APOE3 is inhibited by about 10% or less. .

[0076] In another embodiment, the YYY motif occurs at or near the cleavage site of the sense strand.

[0077] In a further embodiment, the Y'Y'Y' motif occurs at positions 11, 12, and 13 from the 5' end of the antisense strand. Y' may be 2'-O-methyl.

[0078] In some embodiments, Formula (III) is Formula (IIIa): Sense: 5'n p -N a -Y Y Y-N a -n q 3' Antisense: 3'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 5' (IIIa) represented by

[0079] In another embodiment, formula (III) is represented by formula (IIIb): Sense: 5'n p -N a -Y Y Y-N b -Z Z Z-N a -n q 3' Antisense: 3'n p’ -N a’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5' (IIIb) (where each N b and N b 'independently represents an oligonucleotide sequence containing 1-5 modified nucleotides) represented by

[0080] In a further embodiment, formula (III) is represented by formula (IIIc): Sense: 5'n p -N a -X X X-N b -Y Y Y-N a -n q 3' Antisense: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N a’ -n q’ 5’ (IIIc) (where each N b and N b 'independently represents an oligonucleotide sequence containing 1-5 modified nucleotides) represented by

[0081] In certain embodiments, formula (III) is represented by formula (IIId): Sense: 5'n p -N a -X X X-N b -Y Y Y-N b -Z Z Z-N a -n q 3' Antisense: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5’ (IIId) (where each N b and N b ’ independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, N a and N a 'independently represents an oligonucleotide sequence containing 2-10 modified nucleotides) represented by

[0082] In another embodiment, the double-stranded region is 15-30 nucleotide pairs in length. The double-stranded region may be 17-23 nucleotide pairs in length.

[0083] In certain embodiments, the double-stranded region is 17-25 nucleotide pairs in length. The double-stranded region may be 23-27 nucleotide pairs in length.

[0084] In some embodiments, the double-stranded region is 19-21 nucleotide pairs in length. The double-stranded region may be 21-23 nucleotide pairs in length.

[0085] In certain embodiments, each strand has 15-30 nucleotides. Each strand may have 19-30 nucleotides. Each strand may have 19-23 nucleotides.

[0086] In certain embodiments, the double-stranded region is 19-21 nucleotide pairs in length, with each strand having 19-23 nucleotides.

[0087] In another embodiment, the nucleotide modifications of the RNAi agent are LNA, glycol nucleic acid (GNA), HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'- C-allyl, 2'-fluoro, 2'-deoxy, or 2'-hydroxyl, and combinations thereof. Modifications on nucleotides include 2'-O-methyl, 2'-fluoro, or GNA, and combinations thereof. In related embodiments, the modification on the nucleotide is a 2'-O-methyl or 2'-fluoro modification.

[0088] In one embodiment, the RNAi agent comprises a ligand that is or includes one or more lipophilic, eg, C16, moieties attached by bivalent or trivalent branched linkers.

[0089] In other embodiments, the agent further comprises a targeting ligand that targets liver tissue, eg, one or more GalNAc derivatives.

[0090] In still other embodiments, the agent is a lipophilic ligand, e.g., a C16 ligand, conjugated by a monovalent or branched bivalent or trivalent linker to the 3' end of the sense strand, and a 3' Further included are targeting ligands that target liver tissue, eg, one or more GalNAc derivatives, conjugated at the ends by monovalent or branched bivalent or trivalent linkers.

[0091] In certain embodiments, the ligand is attached to the 3'end of the sense strand.

[0092] In some embodiments, the RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In a related embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand. The strand may be the antisense strand. In another embodiment, the strand is the sense strand. In a related embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand. The strand may be the antisense strand. In another embodiment, the strand is the sense strand.

[0093] In another embodiment, phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand. The strand may be the antisense strand. In another embodiment, the strand is the sense strand.

[0094] In a further embodiment, the base pair at position 1 of the 5' end of the antisense strand of the RNAi agent duplex is an A:U base pair.

[0095] In certain embodiments, a Y nucleotide contains a 2'-fluoro modification.

[0096] In some embodiments, the Y'nucleotides contain 2'-O-methyl modifications.

[0097] In certain embodiments, p'>0. It may be p'=2.

[0098] In some embodiments, q'=0, p=0, q=0 and the overhanging nucleotides of p' are complementary to the target mRNA.

[0099] In certain embodiments, q'=0, p=0, q=0 and the overhanging nucleotides of p' are non-complementary to the target mRNA.

[0100] In one embodiment, the sense strand of the RNAi agent has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0101] In another embodiment, at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages. all n p ' may be linked to adjacent nucleotides via phosphorothioate linkages.

[0102] In certain embodiments, the APOE RNAi agents of this disclosure are one of those listed in Tables 2-5 and 7-10. In certain embodiments, the APOE RNAi agents of this disclosure are one of those listed in Tables 7 and 8. In some embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise modifications. In certain embodiments, the APOE RNAi agents of this disclosure are one of those listed in Tables 9 and 10. In some embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise modifications.

[0103] Another aspect of the present disclosure is a double-stranded RNAi agent for inhibiting expression of an APOE gene in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand and anti- The sense strand contains a region complementary to a portion of the mRNA encoding the APOE gene, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III): Sense: 5'n p -N a -(X X X) i -N b -Y Y Y-N b -(Z Z Z) j -N a -n q 3' Antisense: 3'n p ’-N a ’-(X’X’X’) k -N b '-Y'Y'Y'-N b ’-(Z’Z’Z’) l -N a ’-n q '5' (III) (In the formula, i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6, each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which may be modified or unmodified, or a combination thereof, each sequence having at least two different modifications. containing a nucleotide each N b and N b 'independently represents an oligonucleotide sequence comprising 0-10 nucleotides, which may be modified or unmodified, or a combination thereof; each n p , n p ’, n q , and n q ' each independently represents an overhanging nucleotide, which may or may not be present, XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, and the modification is a 2'-O-methyl or 2'-fluoro modification, N. b The modification above differs from the modification on Y in that N b ' is different from the modification on Y') is represented by The sense strand is conjugated to at least one ligand, which may be one or more lipophilic, e.g., C16 ligands, and / or one or more GalNAc derivatives, A double-stranded RNAi agent is provided.

[0104] In certain embodiments, the double-stranded RNAi agent inhibits expression of APOE2, APOE3, and APOE4 alleles. In other embodiments, the double-stranded RNAi agent inhibits expression of APOE4 but does not substantially inhibit expression of APOE2 and APOE3, e.g., expression of APOE2 and APOE3 is inhibited by about 10% or less. .

[0105] A further aspect of the present disclosure is a double-stranded RNAi agent for inhibiting expression of an APOE gene in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand, The strands contain a region complementary to a portion of the mRNA encoding APOE, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III): Sense: 5'n p -N a -(X X X) i -N b -Y Y Y-N b -(Z Z Z) j -N a -n q 3' Antisense: 3'n p ’-N a ’-(X’X’X’) k -N b '-Y'Y'Y'-N b ’-(Z’Z’Z’) l -N a ’-n q '5' (III) [In the formula, i, j, k, and l are each independently 0 or 1; each n p , n p ’, n q , and n q ' each independently represents an overhanging nucleotide, which may or may not be present, p, q, and q' are each independently 0 to 6, n p ’>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which may be modified or unmodified, or a combination thereof, each sequence having at least two different modifications. containing a nucleotide each N b and N b 'independently represents an oligonucleotide sequence comprising 0-10 nucleotides, which may be modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, and the modification is a 2'-O-methyl, glycol nucleic acid (GNA), or 2'-fluoro modification; N. b The modification above differs from the modification on Y in that N b modification on ' is different from modification on Y'] is represented by The sense strand is conjugated to at least one ligand, which may be one or more lipophilic, e.g., C16 ligands, and / or one or more GalNAc derivatives, A double-stranded RNAi agent is provided.

[0106] In certain embodiments, the double-stranded RNAi agent inhibits expression of APOE2, APOE3, and APOE4 alleles. In other embodiments, the double-stranded RNAi agent inhibits expression of APOE4 but does not substantially inhibit expression of APOE2 and APOE3, e.g., expression of APOE2 and APOE3 is inhibited by about 10% or less. .

[0107] Another aspect of the present disclosure is a double-stranded RNAi agent for inhibiting expression of an APOE gene in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand and anti- The sense strand has at least 90% nucleotide sequence identity with the mRNA encoding APOE (SEQ ID NO: 1, or the entire nucleotide sequence of SEQ ID NO: 1, e.g., 90, 91, 92, 93, 94, 95, 96, 97, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula ( III): Sense: 5'n p -N a -(X X X) i -N b -Y Y Y-N b -(Z Z Z) j -N a -n q 3' Antisense: 3'n p ’-N a ’-(X’X’X’) k -N b '-Y'Y'Y'-N b ’-(Z’Z’Z’) l -N a’-n q '5' (III) (In the formula, i, j, k, and l are each independently 0 or 1; each n p , n p ’, n q , and n q ' each independently represents an overhanging nucleotide, which may or may not be present, p, q, and q' are each independently 0 to 6, n p ’>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which may be modified or unmodified, or a combination thereof, each sequence having at least two different modifications. containing a nucleotide each N b and N b 'independently represents an oligonucleotide sequence comprising 0-10 nucleotides, which may be modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, and the modification is a 2'-O-methyl or 2'-fluoro modification, N. b The modification above differs from the modification on Y in that N b ' is different from the modification on Y') is represented by The sense strand is conjugated to at least one ligand, which may be one or more lipophilic, e.g., C16 ligands, and / or one or more GalNAc derivatives, A double-stranded RNAi agent is provided.

[0108] In certain embodiments, the double-stranded RNAi agent inhibits expression of APOE2, APOE3, and APOE4 alleles. In other embodiments, the double-stranded RNAi agent inhibits expression of APOE4 but does not substantially inhibit expression of APOE2 and APOE3, e.g., expression of APOE2 and APOE3 is inhibited by about 10% or less. .

[0109] A further aspect of the present disclosure is a double-stranded RNAi agent for inhibiting expression of an APOE gene in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand, The strand has at least 90% nucleotide sequence identity with the mRNA encoding APOE (SEQ ID NO: 1, or the entire nucleotide sequence of SEQ ID NO: 1, e.g. , 99, or 100% identical nucleotide sequences), each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent having the formula (III ): Sense: 5'n p -N a -(X X X) i -N b -Y Y Y-N b -(Z Z Z) j -N a -n q 3' Antisense: 3'n p ’-N a ’-(X’X’X’) k -N b '-Y'Y'Y'-N b ’-(Z’Z’Z’) l -N a ’-n q '5' (III) (In the formula, i, j, k, and l are each independently 0 or 1; each n p , n p ’, n q , and n q ' each independently represents an overhanging nucleotide, which may or may not be present, p, q, and q' are each independently 0 to 6, n p ’>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which may be modified or unmodified, or a combination thereof, each sequence having at least two different modifications. containing a nucleotide each N b and N b 'independently represents an oligonucleotide sequence comprising 0-10 nucleotides, which may be modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, and the modification is a 2'-O-methyl or 2'-fluoro modification, N. b The modification above differs from the modification on Y in that N b ' is different from the modification on Y') is represented by the sense strand contains at least one phosphorothioate linkage, The sense strand is conjugated to at least one ligand, which may be one or more lipophilic, e.g., C16 ligands, and / or one or more GalNAc derivatives, A double-stranded RNAi agent is provided.

[0110] In certain embodiments, the double-stranded RNAi agent inhibits expression of APOE2, APOE3, and APOE4 alleles. In other embodiments, the double-stranded RNAi agent inhibits expression of APOE4 but does not substantially inhibit expression of APOE2 and APOE3, e.g., expression of APOE2 and APOE3 is inhibited by about 10% or less. .

[0111] Another aspect of the present disclosure is a double-stranded RNAi agent for inhibiting expression of an APOE gene in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand and anti- The sense strand has at least 90% nucleotide sequence identity with the mRNA encoding APOE (SEQ ID NO: 1, or the entire nucleotide sequence of SEQ ID NO: 1, e.g., 90, 91, 92, 93, 94, 95, 96, 97, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula ( III): Sense: 5'n p -N a -Y Y Y-N a -n q 3' Antisense: 3'n p ’-N a '-Y'Y'Y'-N a ’-n q '5' (IIIa) (In the formula, each n p , n p ’, n q , and n q ' each independently represents an overhanging nucleotide, which may or may not be present, p, q, and q' are each independently 0 to 6, n p ’>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which may be modified or unmodified, or a combination thereof, each sequence having at least two different modifications. containing a nucleotide YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications) is represented by the sense strand contains at least one phosphorothioate linkage, The sense strand is conjugated to at least one ligand, which may be one or more lipophilic, e.g., C16 ligands, and / or one or more GalNAc derivatives, A double-stranded RNAi agent is provided.

[0112] In certain embodiments, the double-stranded RNAi agent inhibits expression of APOE2, APOE3, and APOE4 alleles. In other embodiments, the double-stranded RNAi agent inhibits expression of APOE4 but does not substantially inhibit expression of APOE2 and APOE3, e.g., expression of APOE2 and APOE3 is inhibited by about 10% or less. .

[0113] A further aspect of the present disclosure is a double-stranded RNAi agent for inhibiting expression of the APOE gene, wherein the double-stranded RNAi agent targeting APOE comprises a sense strand and an antisense strand forming a double-stranded region. and the sense strand comprises any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, and 9, or the entire nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, 7, or 9 and differs by no more than 3 nucleotides from a nucleotide sequence having at least 90% nucleotide sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity comprising at least 15 contiguous nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides), the antisense strand comprising the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, and 10 or any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, and 10, for example, 90, 91, 92, 93, Nucleotide sequences with 94, 95, 96, 97, 98, 99, or 100% identity differ by no more than 3 nucleotides (i.e., differ by 3, 2, 1, or 0 nucleotides). ) replacement of any of the thymines with uracil in the sequences shown in SEQ ID NOs: 1-10 comprising at least 15 contiguous nucleotides (when comparing aligned sequences) is shown in SEQ ID NOs: 1-10 is not counted as a difference contributing to no more than 3 nucleotides different from any one of the nucleotide sequences shown, and substantially all of the nucleotides in the sense strand are 2'-O-methyl modifications, GNAs, or 2 Including modifications that are '-fluoro modifications, the sense strand contains two phosphorothioate internucleotide linkages at the 5' end, and substantially all of the nucleotides in the antisense strand are 2'-O-methyl modifications and 2'- The antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises one or more is conjugated to a lipophilic, e.g., C16, ligand of C16, and may further comprise a liver-targeting ligand, e.g., a ligand comprising one or more GalNAc derivatives. .

[0114] In certain embodiments, the double-stranded RNAi agent inhibits expression of APOE2, APOE3, and APOE4 alleles. In other embodiments, the double-stranded RNAi agent inhibits expression of APOE4 but does not substantially inhibit expression of APOE2 and APOE3, e.g., expression of APOE2 and APOE3 is inhibited by about 10% or less. .

[0115] Another aspect of the present disclosure is a double-stranded RNAi agent for inhibiting expression of the APOE gene, wherein the double-stranded RNAi agent targeting APOE comprises a sense strand and an antisense strand forming a double-stranded region. wherein the sense strand is any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, and 9, or the entire nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, 7, or 9 a nucleotide sequence having at least 90% nucleotide sequence identity, such as 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, with comprising at least 15 contiguous nucleotides that differ (i.e., differ by 3, 2, 1, or 0 nucleotides), the antisense strand comprising the nucleotides of SEQ ID NOs: 2, 4, 6, 8, and 10 at least 90% nucleotide sequence identity to the entire nucleotide sequence of any one of the sequences, or any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, and 10, e.g., 90, 91, 92, 93 , 94, 95, 96, 97, 98, 99, or 100% identical nucleotide sequences differ by no more than 3 nucleotides (i.e., 3, 2, 1, or 0 nucleotides different) containing at least 15 contiguous nucleotides and any substitution of thymine for uracil in the sequences shown in SEQ ID NOs: 1-10 (when comparing aligned sequences) is shown in SEQ ID NOs: 1-10. Not counted as a difference contributing to a difference of no more than 3 nucleotides from any one of the nucleotide sequences shown, the sense strand containing at least one 3' terminal deoxythymidine nucleotide (dT) and the antisense strand provide a double-stranded RNAi agent that includes a 3' terminal deoxythymidine nucleotide (dT).

[0116] In certain embodiments, the double-stranded RNAi agent inhibits expression of APOE2, APOE3, and APOE4 alleles. In other embodiments, the double-stranded RNAi agent inhibits expression of APOE4 but does not substantially inhibit expression of APOE2 and APOE3, e.g., expression of APOE2 and APOE3 is inhibited by about 10% or less. .

[0117] In one embodiment, all of the nucleotides on the sense strand and all of the nucleotides on the antisense strand are modified nucleotides.

[0118] In another embodiment, each strand has 19-30 nucleotides.

[0119] In certain embodiments, the antisense strand of the RNAi agent comprises at least one thermally destabilizing modification (or precursor thereof) of the duplex within the first 9 nucleotide positions of the 5' region. Thermally destabilizing modifications of the duplex are

[0120] [formation] (Wherein, B is a nucleobase) may be one or more of

[0121] Another aspect of the disclosure provides a cell containing a double-stranded RNAi agent of the disclosure.

[0122] A further aspect of the disclosure provides a pharmaceutical composition for inhibiting expression of the APOE gene comprising a double-stranded RNAi agent of the disclosure.

[0123] In one embodiment, the double-stranded RNAi agent is administered in an unbuffered solution. The unbuffered solution may be saline or water.

[0124] In another embodiment, the double-stranded RNAi agent is administered with a buffered solution. Buffered solutions may include acetates, citrates, prolamines, carbonates, or phosphates, or any combination thereof. In another embodiment, the buffer solution is phosphate buffered saline (PBS).

[0125] Another aspect of the disclosure provides a pharmaceutical composition comprising a double-stranded RNAi agent of the disclosure and a lipid formulation.

[0126] In one embodiment, the lipid formulation comprises lipid nanoparticles (LNPs).

[0127] A further aspect of the disclosure is a method of inhibiting expression of an APOE gene in a cell comprising (a) contacting the cell with a double-stranded RNAi agent of the disclosure or a pharmaceutical composition of the disclosure; and (b) ) maintaining the cells produced in step (a) for a time sufficient to result in degradation of the APOE gene mRNA transcripts, thereby inhibiting expression of the APOE gene in the cells. do.

[0128] In one embodiment, the cell is within a subject. A subject may be a human.

[0129] In certain embodiments, the subject is a rhesus monkey, cynomolgus monkey, mouse, or rat.

[0130] In certain embodiments, the human subject has an APOE-related neurodegenerative disease, e.g., an amyloid-beta mediated disease, e.g., Alzheimer's disease, Down's syndrome, and cerebral amyloid angiopathy, or a tau-mediated disease, For example, primary tauopathies such as frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), bulbar gliata. Globular glial tauopathy (GGT), frontotemporal dementia with parkinsonism (FTDP, FTDP-17), chronic traumatic encephalopathy (CTE), boxer dementia, frontotemporal lobar degeneration (FTLD) ), argyria granulopathy (AGD), and primary age-related tauopathy (PART), or secondary tauopathies such as AD, Creutzfeldt-Jakob disease, Down syndrome, and familial British dementia .

[0131] In certain embodiments, the method further comprises administering to the subject an additional therapeutic agent, such as a cholinesterase inhibitor and / or memantine.

[0132] In certain embodiments, the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0133] In some embodiments, the double-stranded RNAi agent is administered intrathecally to the subject.

[0134] In one embodiment, the method reduces APOE gene expression in brain (eg, striatum) or spinal tissue. The brain or spinal tissue may be striatal, cortical, cerebellar, cervical, lumbar, or thoracic.

[0135] In some embodiments, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0136] In one embodiment, the method reduces APOE gene expression in the liver.

[0137] In other embodiments, the method reduces expression of the APOE gene in liver and brain.

[0138] Another aspect of the disclosure is a method of inhibiting expression of APOE in a subject, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of the disclosure or a pharmaceutical composition of the disclosure, thereby A method is provided comprising inhibiting the expression of APOE in .

[0139] A further aspect of the disclosure is a method for treating or preventing an APOE-associated neurodegenerative disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of the disclosure or a pharmaceutical composition of the disclosure administering, thereby treating or preventing an APOE-related neurodegenerative disease or disorder in a subject.

[0140] In certain embodiments, the APOE-related neurodegenerative disease is an amyloid-beta mediated disease, e.g., an amyloid-beta mediated disease selected from the group consisting of Alzheimer's disease, Down's syndrome, and cerebral amyloid angiopathy. .

[0141] In certain embodiments, the APOE-related neurodegenerative disease is a tau-mediated disease, such as primary or secondary tauopathy.

[0142] In certain embodiments, the primary tauopathy is Frontotemporal Dementia (FTD), Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Pick's Disease (PiD), Pulmogliata Woopathy (GGT), frontotemporal dementia with parkinsonism (FTDP, FTDP-17), chronic traumatic encephalopathy (CTE), boxer dementia, frontotemporal lobar degeneration (FTLD), ginorrhea granules disease (AGD), and primary age-related tauopathy (PART).

[0143] In certain embodiments, the secondary tauopathy is selected from the group consisting of AD, Creutzfeldt-Jakob disease, Down's syndrome, and familial British dementia.

[0144] Another aspect of the disclosure provides a) a double-stranded RNAi agent of the disclosure, b) instructions for use, and c) optionally, a device for administering the double-stranded RNAi agent to a subject. Kits are provided for practicing the methods of the present disclosure, comprising:

[0145] A further aspect of the disclosure is a double-stranded ribonucleic acid (RNAi) agent for inhibiting expression of the APOE gene, wherein the RNAi agent has a sense strand and an antisense strand, the antisense strand comprising at least 15 contiguous sequences that differ from any one of the ~5 and 7-10 antisense strand nucleobase sequences by no more than 3 nucleotides (i.e., differ by 3, 2, 1, or 0 nucleotides) e.g., at least 15 nucleotides (i.e., differ by 3, 2, 1, or 0 nucleotides), at least 19 nucleotides (i.e., 3, 2, 1, or 0 RNAi agents are provided that include regions of complementarity that differ by a single nucleotide. In one embodiment, the RNAi agent has the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C-alkyl modified nucleotides, glycol nucleic acid (GNA), phosphorothioate (PS), and nucleotides including vinyl phosphonate (VP). RNAi agents have the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C-alkyl modified nucleotides, glycol nucleic acids (GNA), phosphorothioates (PS), and a nucleotide containing vinyl phosphonate (VP).

[0146] In certain embodiments, the double-stranded RNAi agent inhibits expression of APOE2, APOE3, and APOE4 alleles. In other embodiments, the double-stranded RNAi agent inhibits expression of APOE4 but does not substantially inhibit expression of APOE2 and APOE3, e.g., expression of APOE2 and APOE3 is inhibited by about 10% or less. .

[0147] In another embodiment, the RNAi agent contains 4 or more PS modifications, may contain 6 to 10 PS modifications, and may contain 8 PS modifications.

[0148] In a further embodiment, each of the sense and antisense strands of the RNAi agent has a 5' end and a 3' end, and the RNAi agent has a 3' end and a 5' end of each of the RNAi agent sense and antisense strands. ' contains 8 PS modifications located at each of the penultimate and last internucleotide linkages of each end.

[0149] In another embodiment, each of the sense and antisense strands of the RNAi agent comprises a 5' end and a 3' end, and the RNAi agent comprises only one nucleotide comprising GNA. The GNA-containing nucleotide may be located on the antisense strand at the 7th nucleobase residue from the 5' end of the antisense strand.

[0150] In further embodiments, each of the sense and antisense strands of the RNAi agent comprises a 5' end and a 3' end and the RNAi agent comprises 1-4 2'-C-alkyl modified nucleotides. A 2'-C-alkyl modified nucleotide may be a 2'-C16 modified nucleotide. An RNAi agent may comprise a single 2'-C-alkyl, eg, C16 modified nucleotide. A single 2'-C-alkyl, eg, C16 modified nucleotide may be placed on the sense strand at the sixth position from the 5' end of the sense strand.

[0151] In another embodiment, each of the sense and antisense strands of the RNAi agent comprises a 5' end and a 3' end and the RNAi agent comprises two or more 2'-fluoro modified nucleotides. Each of the sense and antisense strands of the RNAi agent may contain two or more 2'-fluoro modified nucleotides. The 2'-fluoro modified nucleotides are nucleobase positions 7, 9, 10, and 11 on the sense strand from the 5' end of the sense strand and nucleobases on the antisense strand from the 5' end of the antisense strand. may be placed at positions 2, 14, and 16 of the

[0152] In further embodiments, each of the sense and antisense strands of the RNAi agent comprises a 5' end and a 3' end, and the RNAi agent comprises one or more VP modifications. RNAi agents may contain a single VP modification at the 5' end of the antisense strand.

[0153] In another embodiment, each of the sense and antisense strands of the RNAi agent comprises a 5' end and a 3' end and the RNAi agent comprises two or more 2'-O-methyl modified nucleotides. RNAi agents may include 2'-O-methyl modified nucleotides at all nucleobase positions not modified by 2'-fluoro, 2'-alkyl, or glycol nucleic acid (GNA). The two or more 2'-O-methyl modified nucleotides are positioned on the sense strand from the 5' end of the sense strand at positions 1, 2, 3, 4, 5, 8, 12, 13, 14, 15, 16, on the antisense strand at positions 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, from the 5' end of the antisense strand, at 17, 18, 19, 20, and 21; May be placed at 17, 18, 19, 20, 21, 22, and 23.

[0154] In one aspect, the invention provides a method of inhibiting expression of the APOE gene in astrocytes. The method comprises contacting astrocytes with a dsRNA agent or pharmaceutical composition of the invention; and maintaining the produced astrocytes for a time sufficient to allow degradation of the APOE gene mRNA transcripts. , thereby inhibiting the expression of the APOE gene in astrocytes.

[0155] In certain embodiments, the cell is within a subject, eg, within a human subject.

[0156] In some embodiments, contacting the astrocytes is by intrathecal administration of the pharmaceutical composition.

[0157] In certain embodiments, the antisense strand of the dsRNA agent is any one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1204704, AD-1204705, AD-1204708, and AD-1204712. contain at least 15 contiguous nucleotides that differ from each other by no more than 3 nucleotides; [Brief description of the drawing]

[0158] [Fig. 1-1]FIG. 1A depicts the right hemisphere (BRH) of the brain of homozygous humanized APOE knock-in mice that received a single 300 μg dose of the indicated duplex or artificial CSF (aCSF) control by intracerebroventricular injection (ICV). , is a graph showing the percentage of APOE mRNA remaining 14 days after dosing. FIG. 1B depicts livers of homozygous humanized APOE knock-in mice administered a single 300 μg dose of the indicated duplex or artificial CSF (aCSF) control by intracerebroventricular injection (ICV), 14 days after dosing. Figure 10 is a graph showing the percentage of APOE mRNA remaining in . [Fig. 1-2] FIG. 1A depicts the right hemisphere (BRH) of the brain of homozygous humanized APOE knock-in mice that received a single 300 μg dose of the indicated duplex or artificial CSF (aCSF) control by intracerebroventricular injection (ICV). , is a graph showing the percentage of APOE mRNA remaining 14 days after dosing. FIG. 1B depicts livers of homozygous humanized APOE knock-in mice administered a single 300 μg dose of the indicated duplex or artificial CSF (aCSF) control by intracerebroventricular injection (ICV), 14 days after dosing. Figure 10 is a graph showing the percentage of APOE mRNA remaining in .

Figure 2

[0159] The present disclosure provides RNAi compositions that effect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of genes. The APOE gene can be intracellular, eg, intracellular within a subject, such as a human. The present disclosure also provides the benefits of inhibiting expression of the APOE gene or disorders, such as APOE-associated neurodegenerative diseases, where inhibition or reduction of expression of the APOE gene, e.g., the pathogenic APOE allele, i.e., APOE4, would be beneficial. For example, methods of using the RNAi compositions of the disclosure to treat a subject with an amyloid-beta-mediated disease or a tau-mediated disease are provided.

[0160] RNAi agents of the present disclosure are about 30 nucleotides or less in length, e.g. , 15~22, 15~21, 15~20, 15~19, 15~18, 15~17, 18~30, 18~29, 18~28, 18~27, 18~26, 18~25, 18 ~24, 18~23, 18~22, 18~21, 18~20, 19~30, 19~29, 19~28, 19~27, 19~26, 19~25, 19~24, 19~23 , 19~22, 19~21, 19~20, 20~30, 20~29, 20~28, 20~27, 20~26, 20~25, 20~24, 20~23, 20~22, 20 RNA strands with regions ~21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length ( antisense strand), and this region is at least part of the mRNA transcript of the APOE gene. In certain embodiments, an RNAi agent of the disclosure has a region that is about 21-23 nucleotides in length, and the region is substantially complementary to at least a portion of the mRNA transcript of the APOE gene. , containing the RNA strand (antisense strand).

[0161] In certain embodiments, RNAi agents of the present disclosure are longer lengths having a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of the mRNA transcript of the APOE gene. an RNA strand (antisense strand), which can comprise, for example, up to 66 nucleotides, e.g. include. Those RNAi agents with longer antisense strand lengths preferably comprise a second RNA strand (sense strand) 20-60 nucleotides in length, where the sense and antisense strands are 18 to 60 nucleotides long. Forms a duplex of 30 consecutive nucleotides.

[0162] The use of these RNAi agents allows targeted degradation of APOE gene mRNA in mammals. Thus, methods and compositions comprising these RNAi agents are useful in subjects, e.g., APOE-related neurodegenerative diseases, e.g. useful for treating a subject with

[0163] The detailed description below provides methods of making and using compositions containing RNAi agents to inhibit expression of the APOE gene, as well as subjects having a disease or disorder that would benefit from inhibition or reduction of expression of the gene. Disclosed are compositions or methods for treating

[0164] I. Definition In order that this disclosure may be more readily understood, certain terms will first be defined. In addition, whenever a value or range of values ​​for a parameter is recited, it is intended that values ​​and ranges intermediate to the recited values ​​are also intended to be part of the disclosure.

[0165] The articles "a" and "an" are used herein to mean one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element, eg, a plurality of elements.

[0166] The term "including" is used herein to mean the phrase "including but not limited to" and is interchangeable with the phrase used for The term "or" is used herein to mean the term "and / or" and is used interchangeably with such terms, unless the context clearly dictates otherwise.

[0167] The term "about" is used herein to mean within the typical intersections in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. It will be understood that when about precedes a series of numbers or ranges, "about" can modify each number or range in the series.

[0168] The terms "at least," "greater than," or "or more" preceding a number or series of numbers refer to the number adjacent to the term "at least," and to all that may be logically included as is clear from the context. It is understood to include trailing digits or integers. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21 nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. It will be understood that when the term at least precedes a series of numbers or ranges, "at least" can modify each of the numbers and ranges in the series.

[0169] As used herein, "less than or equal to" or "less than" is to be understood from the value adjacent to the term and a value or integer logically less than that value to zero where logical from the context. be. For example, a duplex with an overhang of "2 nucleotides or less" has an overhang of 2, 1, or 0 nucleotides. It will be understood that when "less than or equal to" precedes a series of numbers or ranges, "less than or equal to" can modify each number or range in the series.

[0170] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0171] In the event of a mismatch between the indicated target site and the nucleotide sequence for the sense or antisense strand, the indicated sequence prevails.

[0172] If the chemical structure and chemical name conflict, the chemical structure controls.

[0173] The term "APOE" or "APOE", also known as "apolipoprotein E", "Alzheimer's disease 2", "LPG" and "LDLCQ5", refers to the well-known gene encoding the protein APOE. APOE is synthesized throughout the body, primarily in the liver, functions as a lipid transport protein and is the major ligand for the low-density lipoprotein (LDL) receptor. APOE has been shown to play a role in cholesterol metabolism and cardiovascular disease, and more recently has emerged as a major risk factor for Alzheimer's disease, as well as other neurodegenerative disease pathologies. Associated.

[0174] The nucleotide and amino acid sequences of APOE can be found, for example, in GenBank Accession No. NM_000041.4 [Human (Homo sapiens) APOE, SEQ ID NO: 1, reverse complement, SEQ ID NO: 2]; GenBank Accession No. NM_001270681.1 [Rattus norvegicus) APOE , SEQ ID NO: 3; reverse complement, SEQ ID NO: 4]; GenBank Accession No. NM_001305843.1 [Mus musculus APOE, SEQ ID NO: 5, reverse complement, SEQ ID NO: 6]; GenBank Accession No. XM_028839202.1 [Rhesus monkey ( Macaca mulatta) APOE, SEQ ID NO: 7, reverse complement, SEQ ID NO: 8]; and GenBank Accession No. XM_005589554.2 [Cynomolgus monkey (Macaca fascicularis) APOE, SEQ ID NO: 9; .

[0175] Additional examples of APOE sequences can be found in publicly available databases such as GenBank, OMIM, and UniProt. Additional information regarding APOE can be found, for example, at www.ncbi.nlm.nih.gov / gene / 348.

[0176] The term APOE, as used herein, refers to the APOE gene, including variants of human APOE provided in the SNP database, e.g., ncbi.nlm.nih.gov / clinvar / ?term=APOE[gene] Also refers to a variant of

[0177] The human APOE gene contains two single nucleotide polymorphisms, these being the three most common variants: APOE2 (also called APOE*ε2 or ε2; Cys112, Cys158), APOE3 (APOE*ε3 or ε3 Cys112, Arg158) and APOE4 (also called APOE*ε4 or ε4; Arg112, Arg158). GenBank Accession No. NM_000041.4 (human APOE, SEQ ID NO:1, reverse complement, SEQ ID NO:2) is the nucleotide sequence of the APOE*ε3 (APOE3) variant; Having a single nucleotide change at nucleotide 595C>T of 1, the APOE*ε4 (APOE4) mutant has a single nucleotide change at nucleotide 457T>C of SEQ ID NO:1.

[0178] Unless specified herein, the terms "APOE," "ApoE," etc. should be understood to refer to any one or more of the three APOE variants or alleles. For example, as used herein, the term "APOE gene" refers to APOE2 alleles, APOE3 alleles, and / or APOE4 alleles, whereas terms such as "APOE4 allele" refer to APOE4 alleles only. Point.

[0179] As used herein, a "target sequence" is a continuous portion of a nucleotide sequence such as an mRNA molecule formed upon transcription of the APOE gene, e.g., an mRNA that is a product of RNA processing of the primary transcript. means. In one embodiment, the target portion of the sequence is at least long enough to serve as a substrate for RNAi-dependent cleavage at or near a portion of the nucleotide sequence of the mRNA molecule formed upon transcription of the APOE gene. would be

[0180] The target sequence is approximately 15-30 nucleotides in length. For example, the target sequence is about 15-30 nucleotides long, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15 ~20, 15~19, 15~18, 15~17, 18~30, 18~29, 18~28, 18~27, 18~26, 18~25, 18~24, 18~23, 18~22 , 18~21, 18~20, 19~30, 19~29, 19~28, 19~27, 19~26, 19~25, 19~24, 19~23, 19~22, 19~21, 19 ~20, 20~30, 20~29, 20~28, 20~27, 20~26, 20~25, 20~24, 20~23, 20~22, 20~21, 21~30, 21~29 , 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 19-23 nucleotides in length, optionally 21-23 nucleotides in length. Ranges and lengths intermediate to those recited above are also contemplated to be part of this disclosure.

[0181] As used herein, the term "sequence-comprising strand" means an oligonucleotide comprising a strand of nucleotides described by a referenced sequence using standard nucleotide nomenclature.

[0182] "G", "C", "A", "T", and "U" are generally guanine, cytosine, adenine, thymidine as bases, respectively, in the context of modified or unmodified nucleotides , and represent nucleotides containing uracil. However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, or surrogate replacement moieties (see, e.g., Table 1), as described in more detail below. . One skilled in the art will appreciate that guanine, cytosine, adenine, thymidine, and uracil can be replaced with other moieties without substantially altering the base-pairing properties of oligonucleotides containing nucleotides with such replacement moieties. fully aware of For example, but not limited to, a nucleotide containing inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine can be replaced with nucleotides containing, for example, inosine in the nucleotide sequences of the dsRNAs featured in this disclosure. In another example, adenine and cytosine in either of the oligonucleotides can be replaced with guanine and uracil, respectively, to form G-UWobble base-pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in this disclosure.

[0183] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interfering agent," as used interchangeably herein, contain RNA as the terms are defined herein and It refers to those agents that mediate targeted cleavage in RNA transcription via the silencing complex (RISC) pathway. RNA interference (RNAi) is the process that directs the sequence-specific degradation of mRNA. RNAi modulates, eg, inhibits, expression of APOE in a cell, eg, a cell within a subject, eg, a mammalian subject.

[0184] In one embodiment, an RNAi agent of the disclosure comprises a single-stranded RNAi that interacts with a target RNA sequence, eg, an APOE target mRNA sequence, to direct cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNAs introduced into cells are catalyzed by a type III endonuclease known as Dicer to cleave small double-stranded molecules, including the sense and antisense strands. It is thought to be degraded into RNA (siRNA) [Sharp et al. (2001) Genes Dev. 15:485]. Dicer, a ribonuclease III-like enzyme, processes these dsRNAs into small interfering RNAs of 19-23 base pairs with a characteristic two-base 3' overhang [Bernstein, et al., (2001). Nature 409:363]. These siRNAs are then introduced into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex and the complementary antisense strand induces target recognition. [Nykanen, et al., (2001) Cell 107:309]. Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing [Elbashir, et al., (2001) Genes Dev. 15: 188]. Thus, in one aspect, the present disclosure provides single-stranded RNA (ssRNA) (siRNA duplexes) produced in cells to promote formation of RISC complexes, thereby silencing a target gene, namely the APOE gene. strand antisense strand). Accordingly, the term "siRNA" is used herein to also mean RNAi as described above.

[0185] In another embodiment, an RNAi agent can be single-stranded RNA introduced into a cell or organism to inhibit target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNA is described in U.S. Pat. No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. be Any of the antisense nucleotide sequences described herein can be synthesized as single-stranded siRNAs described herein or by the methods described in Lima et al., (2012) Cell 150:883-894. can be used as a single-stranded siRNA that is selectively modified.

[0186] In another embodiment, the "RNAi agent" for use in the compositions and methods of the present disclosure is double-stranded RNA, herein referred to as "double-stranded RNAi agent", "double-stranded RNA (dsRNA ) molecule”, “dsRNA agent”, or “dsRNA”. The term "dsRNA" refers to a target RNA, i.e., a duplex structure comprising two antiparallel, substantially complementary nucleic acid strands, said to have a "sense" or "antisense" orientation with respect to the APOE gene. A complex of ribonucleic acid molecules with In some embodiments of the present disclosure, double-stranded RNA (dsRNA) induces target RNA, eg, mRNA, degradation by a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0187] In general, dsRNA molecules can contain ribonucleotides, but as detailed herein, each strand or both strands may contain one or more ribonucleotides, e.g., deoxyribonucleotides, modified Nucleotides can also be included. Additionally, as used herein, an "RNAi agent" can include ribonucleotides with chemical modifications; an RNAi agent can include substantial modifications at multiple nucleotides. As used herein, the term "modified nucleotide" means a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase. Thus, the term modified nucleotides encompasses substitutions, additions, or removals of, for example, functional groups or atoms, to internucleoside linkages, sugar moieties, or nucleobases. Modifications suitable for use in the medicaments of the present disclosure encompass all types of modifications disclosed herein or known in the art. Any such modifications used in siRNA-type molecules are encompassed by "RNAi agents" for the purposes of this specification and claims.

[0188] In certain embodiments of the disclosure, the inclusion of deoxynucleotides, which are recognized as naturally occurring forms of nucleotides when present in RNAi agents, can be considered to constitute modified nucleotides.

[0189] The double-stranded region can be of any length that allows specific degradation of the desired target RNA by the RISC pathway, and is about 15-36 base pairs long, e.g., about 15, 16, 17, 18. , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, e.g., about 15-30, 15~29, 15~28, 15~27, 15~26, 15~25, 15~24, 15~23, 15~22, 15~21, 15~20, 15~19, 15~18, 15~ 17, 18~30, 18~29, 18~28, 18~27, 18~26, 18~25, 18~24, 18~23, 18~22, 18~21, 18~20, 19~30, 19~29, 19~28, 19~27, 19~26, 19~25, 19~24, 19~23, 19~22, 19~21, 19~20, 20~30, 20~29, 20~ 28, 20~27, 20~26, 20~25, 20~24, 20~23, 20~22, 20~21, 21~30, 21~29, 21~28, 21~27, 21~26, It can range in length from 21-25, 21-24, 21-23, or 21-22 base pairs. In certain embodiments, the double-stranded region is 19-21 base pairs long, eg, 21 base pairs long. Ranges and lengths intermediate to those recited above are also contemplated to be part of this disclosure.

[0190] The two strands that form the duplex structure can be different parts of one larger RNA molecule, or they can be separate RNA molecules. Two strands are part of one larger molecule and are therefore interrupted between the 3' end of one strand and the 5' end of each other strand forming a duplex structure. are connected by strands of non-nucleotides, in which case the connecting RNA strands are called "hairpin loops." A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides or the target site of the dsRNA. can include nucleotides not intended for In some embodiments, the hairpin loop can be 10 nucleotides or less. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 unpaired nucleotides.

[0191] The two substantially complementary strands of dsRNA are contained in separate RNA molecules, and those molecules can, but need not, be covalently linked. two strands are covalently linked between the 3' end of one strand and the 5' end of each other strand forming a duplex structure by means other than a strand of uninterrupted nucleotides In certain embodiments, connecting structures are referred to as "linkers" (although certain other structures defined elsewhere herein may also be referred to as "linkers"). RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, RNAi may contain one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one strand comprises a 3' overhang of at least 1 nucleotide. In another embodiment, at least one strand has a 3' overhang of at least 2 nucleotides, such as 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. including. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least 1 nucleotide. In certain embodiments, at least one strand has a 5' overhang of at least 2 nucleotides, e.g. Including hang. In still other embodiments, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

[0192] In one embodiment, an RNAi agent of the disclosure is a dsRNA, each strand of which independently interacts with a target RNA sequence, e.g., an APOE target mRNA sequence, to induce cleavage of the target RNA. Contains ~23 nucleotides.

[0193] As used herein, the term "nucleotide overhang" means at least one unpaired nucleotide overhanging the duplex structure of an RNAi agent, eg, dsRNA. For example, if the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, and vice versa, there is a nucleotide overhang. The dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang comprises at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, or more nucleotides. can be done. Nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs such as deoxynucleotides / nucleosides. Overhangs can be on the sense strand, the antisense strand, or any combination thereof. Moreover, overhanging nucleotides can be present at the 5' end, 3' end, or both of either the antisense or sense strands of the dsRNA.

[0194] In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' or 5' end, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. overhang. In one embodiment, the sense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' or 5' end, e.g. have overhangs. In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphates.

[0195] In certain embodiments, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' or 5' end, e.g., 0-3, 1-3, 2-4, 2-5, 4-10 , with 5-10 nucleotide overhangs, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhangs. In one embodiment, the sense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' or 5' end, e.g. have overhangs. In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphates.

[0196] In certain embodiments, the overhangs on the sense or antisense strand are longer than 10 nucleotides, e.g., 1-30 nucleotides in length, 2-30 nucleotides in length, 10-30 nucleotides in length, or 10-15 nucleotides in length. , may include the stretched length. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present at the 3'end of the sense strand of the duplex. In certain embodiments, an extended overhang is present at the 5'end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present at the 3'end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present at the 5' end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhangs are replaced with nucleoside thiophosphates. In certain embodiments, the overhangs contain self-complementary portions such that the overhangs can form stable hairpin structures under physiological conditions.

[0197] The term "blunt" or "blunt ended" as used herein with respect to dsRNA is the absence of unpaired nucleotides or nucleotide analogues at a given end of the dsRNA, i.e., nucleotide overhangs. does not exist. One or both ends of the dsRNA can be blunt. A dsRNA is said to be blunt-ended if both ends of the dsRNA are blunt. For the sake of clarity, a "blunt-ended" dsRNA is a dsRNA that is blunt at both ends, ie, has no nucleotide overhangs at either end of the molecule. In most cases, such molecules will be double-stranded over their entire length.

[0198] The terms "antisense strand" or "guide strand" refer to a strand of an RNAi agent, eg, dsRNA, that contains a region substantially complementary to a target sequence, eg, APOE mRNA.

[0199] As used herein, the term "region of complementarity" is substantially complementary to a sequence, e.g., a target sequence, e.g., an APOE nucleotide sequence, as defined herein. A region on the antisense strand. Where the regions of complementarity are not perfectly complementary to the target sequence, the mismatches can be in internal or terminal regions of the molecule. In general, the most acceptable mismatches are in terminal regions, eg, within 5, 4, 3, or 2 nucleotides of the 5'or 3'end of the RNAi agent.

[0200] The terms "sense strand" or "passenger strand" as used herein include a region that is substantially complementary to a region of the antisense strand as the term is defined herein. A strand of an RNAi agent.

[0201] As used herein, the term "cleavage region" means the region located immediately adjacent to the cleavage site. A cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases immediately adjacent at either end of the cleavage site. In some embodiments, the cleavage region comprises two bases immediately adjacent at either end of the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bounded by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

[0202] As used herein, unless otherwise specified, the term "complementary" is used to describe a first nucleotide sequence in the context of a second nucleotide sequence, as understood by those of skill in the art. When used, it means the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a duplex with an oligonucleotide or polynucleotide comprising a second nucleotide sequence under certain conditions. do.

[0203] Complementary sequences within an RNAi agent, e.g., within a dsRNA described herein, direct a first nucleotide sequence to an oligonucleotide or polynucleotide, including a second nucleotide sequence, over the entire length of one or both nucleotide sequences. including base pairing of oligonucleotides or polynucleotides comprising. Such sequences can be referred to herein as being "fully complementary" with respect to each other. However, when a first sequence is considered herein to be "substantially complementary" to a second sequence, the two sequences may be fully complementary or may form one or more, but generally no more than 5, 4, 3, or 2, mismatched base pairs upon hybridization for a duplex of up to 30 base pairs, whereas and retain the ability to hybridize under conditions most relevant to their ultimate application, eg, inhibition of gene expression by the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs are not considered mismatches for purposes of determining complementarity. not. For example, a dsRNA comprising one oligonucleotide that is 21 nucleotides in length and another that is 23 nucleotides in length, wherein the longer oligonucleotide is a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide. Such dsRNAs can still be considered "fully complementary" for the purposes described herein.

[0204] "Complementary" sequences, as used herein, are non-Watson-Crick base pairs, or base pairs formed from non-naturally modified nucleotides, as long as the above requirements regarding their ability to hybridize are met. may also include or be formed entirely therefrom. Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogsteen base pairs.

[0205] The terms "complementary," "completely complementary," and "substantially complementary," as used herein, are understood from the context in which they are used between two oligonucleotides or polynucleotides. , for example, in connection with base matching between the sense and antisense strands of a dsRNA, or between the antisense strand and a target sequence of an RNAi agent.

[0206] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding APOE). A polynucleotide that is substantially complementary to a polynucleotide. For example, a polynucleotide is complementary to at least a portion of an APOE mRNA if the sequence is substantially complementary to the uninterrupted portion of the mRNA encoding APOE.

[0207] Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target APOE sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to a target APOE sequence and are SEQ ID NOS: 1, 3, 5, 7 or 9 of APOE, or APOE at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of a fragment of SEQ ID NO: 1, 3, 5, 7 or 9 of SEQ ID NO: 1, 3, 5, 7 or 9, e.g. %, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary , contains a contiguous nucleotide sequence.

[0208] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target APOE sequence and have a sense sequence in any one of APOE Tables 2-5 and 7-10. at least about 80% complementary over its entire length to any one of the strand nucleotide sequences, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about Includes contiguous nucleotide sequences that are 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0209] In one embodiment, an RNAi agent of this disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is the same as the target APOE sequence, wherein the sense strand polynucleotides are SEQ ID NOS: 2, 4, 6, 8 and 10, or at least about 80%, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99 Contains contiguous nucleotide sequences that are % complementary.

[0210] In one embodiment, the at least partial suppression of APOE gene expression is assessed by a reduction in the amount of APOE mRNA, such mRNA indicating that the APOE gene is transcribed and that APOE gene expression is inhibited. can be isolated from a first cell or group of cells that has been or is being treated with, or is detected in such a cell or group of cells and is substantially identical to the first cell or group of cells are compared to a second cell or group of cells (control cells) that have been so treated or not. The degree of inhibition can be expressed in units of:

[0211]

number

[0212] The phrase "contacting a cell with an RNAi agent," such as dsRNA, as used herein includes contacting a cell by any possible means. Contacting the cell with the RNAi agent includes contacting the cell with the RNAi agent in vitro or contacting the cell with the RNAi agent in vivo. Contacting can be done directly or indirectly. Thus, for example, an RNAi agent may be brought into physical contact with a cell by performing the method separately, or the RNAi agent may subsequently allow or cause contact with the cell. can be placed

[0213] Contacting the cells in vitro can be done, for example, by incubating the cells with the RNAi agent. Contacting a cell in vivo can be accomplished by, for example, injecting an RNAi agent into or near the tissue in which the cell is located, or to another area, such as the central nervous system (CNS). Optionally, by injecting the RNAi agent by intrathecal injection, intravitreal injection, or other injection, or so that the agent subsequently reaches the tissue where the cells to be contacted are located. This can be done by injecting the RNAi agent into the subcutaneous space. For example, an RNAi agent can be a ligand that directs or stabilizes the RNAi agent to a site of interest, e.g., the CNS, e.g., described below and further in e.g. It may contain or be coupled to a lipophilic moiety as detailed. In some embodiments, the RNAi agent comprises a ligand, e.g., one or more GalNAc derivatives described below, that directs or otherwise stabilizes the RNAi agent to a site of interest, e.g., the liver. may contain or be coupled thereto. In other embodiments, an RNAi agent may contain or be coupled to a lipophilic moiety and one or more GalNAc derivatives. A combination of in vitro and in vivo methods for contacting is also possible. For example, cells may be contacted with an RNAi agent in vitro and then transferred to a subject.

[0214] In one embodiment, contacting a cell with an RNAi agent includes "introducing" or "delivering the RNAi agent into the cell" by promoting or effecting uptake or absorption into the cell. Absorption or uptake of an RNAi agent can occur by spontaneous diffusive or active cellular processes or by auxiliary agents or devices. Introducing the RNAi agent into the cell can be in vitro or in vivo. For example, for in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Additional approaches are described herein below or known in the art.

[0215] The term "lipophilic" or "lipophilic moiety" broadly refers to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is the octanol-water partition coefficient logK ow by and in this case K ow is the ratio of the octanol phase chemical concentration to the aqueous phase chemical concentration in a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property in substances. However, it can also be predicted by using coefficients attributable to the chemical's structural composition calculated using first principles or empirical methods [e.g. , Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001)]. It provides a thermodynamic measure of a substance's propensity to prefer a non-aqueous or oily environment rather than water (ie, hydrophilic / lipophilic balance). As a rule, chemicals have logK ow is greater than 0, it is lipophilic in nature. Typically, the lipophilic moiety has a logK greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow have For example, the logK of 6-aminohexanol ow is expected to be approximately 0.7. Using the same method, the logK of cholesteryl N-(hexane-6-ol)carbamate ow is expected to be 10.7.

[0216] The lipophilicity of a molecule can be altered with respect to the functional groups it possesses. For example, by adding hydroxyl or amine groups at the ends of the lipophilic moiety, the partition coefficient of the lipophilic moiety (e.g., log K ow ) value can be increased or decreased.

[0217] Alternatively, the hydrophobicity of a double-stranded RNAi agent conjugated to one or more lipophilic moieties can be measured by its protein binding properties. For example, in certain embodiments, the unbound fraction of a plasma protein binding assay of a double-stranded RNAi agent can be positively correlated with the silencing activity of the double-stranded RNAi agent. It can be determined to correlate positively with hydrophobicity.

[0218] In one embodiment, the plasma protein binding assay that is determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol for this binding assay is described in detail, for example, in PCT / US2019 / 031170. The hydrophobicity of the double-stranded RNAi agent, as measured by the fraction of unbound dsRNA in the binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35 for enhanced in vivo delivery of dsRNA. greater than, greater than 0.4, greater than 0.45, or greater than 0.5.

[0219] Thus, conjugation of lipophilic moieties to internal positions of double-stranded RNAi agents provides optimal hydrophobicity for enhanced in vivo delivery of siRNAs.

[0220] The term "lipid nanoparticle" or "LNP" is a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, such as an RNAi agent or a plasmid from which an RNAi agent is transcribed. LNPs are described, for example, in US Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0221] As used herein, a "subject" is an animal, e.g., mammal, e.g., primate (e.g., human, non-human primate, e.g., monkey, chimpanzee, etc.), or non-primate (e.g., rat or mouse). In preferred embodiments, the subject is a human, e.g., a human being treated or evaluated for a disease, disorder, or condition in which reduction in APOE expression would be beneficial; a disease in which reduction in APOE expression would be beneficial; humans at risk for a disorder, or condition; humans with a disease, disorder, or condition that would benefit from reduced APOE expression; or diseases that would benefit from reduced APOE expression as described herein; A human being treated for a disorder or condition.

[0222] As used herein, the terms "treating" or "treatment" refer to beneficial or desired results, including one or more symptoms or symptoms associated with APOE gene expression or APOE protein production. Conditions such as APOE-related neurodegenerative diseases such as amyloid-β mediated diseases such as Alzheimer's disease, Down's syndrome, and cerebral amyloid angiopathy, or tau-mediated diseases such as primary tauopathies such as frontotemporal type dementia, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), chronic traumatic encelopathy (CTE), frontotemporal type Dementia (FTD, FTDP-17), frontotemporal lobar degeneration (FTLD), argyria granulopathy (AGD), primary age-related tauopathy (PART), and globular glial tauopathy (GGT), or secondary Alleviation or amelioration of tauopathies such as AD, Creutzfeldt-Jakob disease, Down's syndrome, familial British dementia, and boxer dementia, without limitation. "Treatment" can also mean prolonging survival as compared to expected survival if not treating.

[0223] The term "lower" in reference to levels of APOE or disease markers or symptoms in a subject means a statistically significant decrease in such levels. The reduction is, for example, at least 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, It can be 85%, 90%, 95% or more. In certain embodiments, the reduction is at least 20%. In certain embodiments, the reduction is at least a 50% reduction in disease marker, eg, protein or gene expression level. "Reduce" in the context of the level of APOE in a subject preferably to a level accepted as within the normal range in individuals without such disorders. In certain embodiments, "reduce" is a reduction in the difference between the level of a marker or symptom in a subject suffering from a disease and a level accepted within the normal range by an individual, e.g., an obese individual It is the level of weight loss between weight loss and an individual with an acceptable body weight within the normal range. As used herein, reducing can refer to reducing or predominantly reducing the level of APOE gene mRNA having a nucleotide repeat expansion.

[0224] As used herein, "prevention" or "preventing" when used in reference to diseases, disorders, or conditions where reduction in APOE gene expression or APOE protein production would be beneficial: It refers to a reduction in the likelihood that a subject will develop symptoms associated with such diseases, disorders, or conditions, eg, symptoms of APOE-related neurodegenerative diseases. Absence of the disease, disorder, or condition, or reduction in the development of symptoms associated with such disease, disorder, or condition (e.g., at least about 10% of the clinically acceptable magnitude for the disease or disorder) ), or a delayed presentation of symptoms (eg, a delay of days, weeks, months, or years) is considered effective prophylaxis.

[0225] As used herein, the term "APOE-associated neurodegenerative disease" or "APOE-associated neurodegenerative disorder" is understood as any disease or disorder in which a reduction in the expression and / or activity of APOE would be beneficial. . Exemplary APOE-related neurodegenerative diseases include amyloid-β mediated diseases such as Alzheimer's disease, Down's syndrome, and cerebral amyloid angiopathy, and tau-mediated diseases such as primary tauopathies such as frontotemporal Dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), globular glial tauopathy (GGT), frontotemporal dementia with parkinsonism (FTDP, FTDP-17), chronic traumatic encephalopathy (CTE), boxer dementia, frontotemporal lobar degeneration (FTLD), argyrophilia granulopathy (AGD), and primary age-related tauopathy (PART), and Secondary tauopathies such as AD, Creutzfeldt-Jakob disease, Down's syndrome, and familial British dementia.

[0226] As used herein, the term “amyloid-β-mediated disease” is a disorder resulting from extracellular accumulation of amyloid-β, which is associated with the formation of amyloid plaques within brain tissue. bring. Exemplary amyloid-β mediated diseases include Alzheimer's disease, Down's syndrome, and cerebral amyloid angiopathy (CAA).

[0227] As used herein, the term "tau-mediated disease" is a disorder that results from aggregation of tau protein, resulting in neurofibrillary tangles (tangles). Tangles are formed by hyperphosphorylation of tau, causing the protein to dissociate from microtubules and from aggregates. Tauopathies can be divided into ``primary tauopathies,'' in which the pathology is driven primarily by tau aggregation, and ``secondary tauopathies,'' in which another factor drives the disease (e.g., amyloid-β plaques in Alzheimer's disease). The presence of tauopathy exacerbates disease progression. Examples of primary tauopathies include frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), glial tauopathy (GGT). ), frontotemporal dementia with parkinsonism (FTDP, FTDP-17), chronic traumatic encephalopathy (CTE), boxer dementia, argyria granulopathy (AGD), and primary age-related tauopathy (PART) is mentioned. Examples of secondary tauopathies include AD, Creutzfeldt-Jakob disease, Down's syndrome, and familial British dementia.

[0228] APOE polymorphisms are associated with numerous tauopathies. The APOE4 allele was found to accelerate neurodegeneration and reduce the age at onset of frontotemporal dementia (FTD) in patients with MAPT mutations [Koriath, C. et al. (2019 ) Alzheimers Dement 11:277-280]. In addition, the presence of APOE4 has been demonstrated in brain anatomy of football players exposed to low frequency repetitive head impact [Verscaj, C. et al. (2017) Neurology 88 (16) Supplement S9.001], and It correlated with more advanced chronic traumatic encephalopathy (CTE) in the brains of boxers [Jordan, B.D. et al. (1997) JAMA 278(2): 136-140]. The presence of the APOE4 allele is also associated with increased risk of Creutzfeldt-Jakob disease (CJD), while the presence of the APOE3 allele is associated with protection against susceptibility to Creutzfeldt-Jakob disease (CJD). [Wei, Y. et al. (2013) J Clinical Neuroscience 21(3): 390-394].

[0229] Furthermore, Shi et al. describe a marked increase in tau levels, brain atrophy and neuroinflammation when APOE4 is present compared to when APOE2, APOE3 or APOE knockout are present in the P301S mouse model of FTD. [Shi et al., (2017) Nature 549: 523-527]. In another study using a mouse model expressing human tau with the P301L mutation found in FTD with parkinsonism, hyperphosphorylated tau, tau aggregation and behavioral abnormalities were exacerbated against the APOE2 background [Zhao, N. et al., (2018) Nat Commun 9:4388]. Zhao et al. further identified the relationship between APOEε2 / ε2 genotype and tauopathy risk in confirmed cases of progressive supranuclear palsy (PSP) and corticobasal degeneration, and APOE2 is protective in the presence of amyloid pathology. suggesting that APOE2 is associated with increased severity of tau pathology in the absence of amyloid pathology.

[0230] "Alzheimer's disease" ("AD") is a chronic neurodegenerative disorder that usually begins slowly and worsens gradually over time. The most common early symptom is difficulty remembering recent events. As the disease progresses, symptoms may include language problems, disorientation (including being easily lost), mood swings, loss of motivation, inability to self-manage, and behavioral problems. As the person's condition declines, they often withdraw from family and society. A gradual loss of bodily functions, which eventually leads to death.

[0231] Neuropathologically, AD is characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical regions. This loss results in marked atrophy of the affected areas, including degeneration in the temporal and parietal lobes, and portions of the frontal cortex and cingulate gyrus. Degeneration is also present in brainstem nuclei such as the locus coeruleus. Studies using MRI and PET show specific brain regions in people with AD as they progress from mild cognitive impairment to Alzheimer's disease and compared with similar images from healthy older adults. A reduction in the magnitude of is recorded.

[0232] Both amyloid plaques and neurofibrillary tangles are clearly visible microscopically in the brains of people with AD. Plaques are dense, mostly insoluble deposits of beta-amyloid peptide and cellular material outside and around neurons. Tangles (neurofibrillary tangles) are aggregates of the microtubule-associated protein tau that hyperphosphorylate and accumulate within cells as such. Although many older people develop some plaques and tangles as a result of aging, the brains of people with AD have more of them in certain brain regions, such as the temporal lobe. Lewy bodies are not uncommon in the brains of people with AD.

[0233] The National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) and the Alzheimer's Disease and Related Disorders Association (ADRDA, now known as the Alzheimer's Association) are the most The commonly used NINCDS-ADRDA Alzheimer's disease criteria were established in 1984 and extensively updated in 2007. These criteria require that the presence of cognitive impairment and suspected cognitive syndrome be confirmed by neuropsychological testing for a clinical diagnosis of probable or probable AD. Definitive diagnosis requires histopathological confirmation, including microscopic examination of brain tissue. Excellent statistical reliability and validity have been demonstrated between diagnostic criteria and definitive histopathological confirmation. Eight intellectual domains are most commonly impaired in AD: memory, language, perceptual skills, attention, motor skills, orientation, problem-solving, and executive functioning. These domains are equivalent to the NINCDS-ADRDA Alzheimer's disease criteria listed in the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR) published by the American Psychiatric Association.

[0234] Currently available drugs for treating AD patients include cholinesterase inhibitors and memantine. These drugs can improve a patient's quality of life, for example, by treating symptoms related to memory, thinking, and language; however, they alter the rate of disease progression or decline. do not.

[0235] The etiology of AD is poorly understood, but as discussed above, the presence of APOE4 is a major risk determinant of late-onset Alzheimer's disease (AD) with symptom onset after age 65 years. It has been shown that many studies in non-human animal models of amyloid-β-mediated disease (AD) and tau-mediated disease suggest that inhibiting APOE, e.g., APOE4, may improve amyloid plaque formation and cognitive function. have demonstrated beneficial effects on

[0236] "Down's syndrome" ("DS"), also known as trisomy 21, is a genetic order caused by the presence of all or part of the third copy of chromosome 21. DS is a lifelong condition that is associated with intellectual disability, characteristic facial features, and weakness of muscle tone in childhood, and those with DS often experience a gradual decline in cognitive function. The third, chromosome 21, has an extra amyloid precursor protein (APP) gene, and excessive amyloid production is associated with the accumulation of amyloid-β plaques and consequent early-onset Alzheimer's disease (AD) risk in 50%. Provides super increase. Another gene that is tripled in DS is DYRK1A, which affects alternative splicing of tau, stimulates tau to aberrant hyperphosphorylation, and promotes neurofibrillary degeneration [ Hartley D. et al. (2016) Alzheimers Dement 11(6): 700-709]. DS individuals with AD have neuropathological changes similar to common AD patients, including amyloid plaques, tau neurofibrillary tangles, oxidative damage, and neuronal loss. High levels of both amyloid and tau are found in the cerebrospinal fluid of DS individuals [Lee, N.C. et al. (2017) Neurology and Therapy 6: 69-81].

[0237] "Cerebral amyloid angiopathy" ("CAA") is a form of angiopathy in which amyloid plaques are deposited in the walls of small to medium blood vessels and in certain areas of the brain. Amyloid plaques damage brain cells and impair various parts of the brain. In addition, amyloid deposits within blood vessels replace the muscle and elastic fibers that give them flexibility, making them more susceptible to breakage. CAA can cause dementia, intracranial hemorrhage, and transient neurological events. CAA is recognized as one of the morphological hallmarks of Alzheimer's disease. Mutations in the amyloid-beta precursor protein (APP) gene are the most common cause of hereditary CAA [Desimone C.V. et al. (2017) J Am Coll Cardiol 70(9): 1173-1182].

[0238] "Frontotemporal dementia" ("FTD") includes diseases such as Pick's disease, progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). FTD is a common form of dementia in patients under the age of 65 and encompasses a group of neurodegenerative diseases characterized by progressive decline in behavior, executive function, or language. In FTD, neurons in the frontal and temporal lobes of the brain are lost, hence FTD is also called frontotemporal lobar degeneration (FTLD). Mutations in the microtubule-associated protein tau (MAPT) gene and accumulation of tau are associated with several subtypes of FTD, including Pick's disease, progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). found.

[0239] "Pick's disease" is characterized by marked, well-demarcated atrophy of the frontal, temporal, and cingulate gyrus, with better preservation of the parietal lobes.

[0240] "Corticobasal degeneration" ("CBD") is characterized by predominant loss of cells in the dorsal prefrontal cortex, supplementary motor cortex, peri-rolandic cortex, and subcortical nucleus.

[0241] "Progressive supranuclear palsy" ("PSP") is associated with atrophy of the ridges of the frontal arch; subcortical atrophy is severe at the level of the globus pallidus, subthalamic nucleus, and brainstem nucleus [Olney, N.T. et al. (2017) Neurol Clin 35(2): 339-374].

[0242] "Globular glial tauopathy" ("GGT") is a rare frontotemporal lobar degeneration of the type with extensive globular inclusions in astrocytes and oligodendrocytes that contain 4 repeat tau isoforms. (FLD). These cases are associated with a range of clinical presentations that correlate with the severity and distribution of underlying tau pathology and neurodegeneration [Ahmed, Z. et al. (2013) Acta Neuropathol 126(4): 537- 544].

[0243] "Frontotemporal dementia with parkinsonism" ("FTDP") is a less common form of FTD that also affects movement. Chromosome 17 was found to be linked to FTDP (FTDP-17), and mutations in the microtubule-associated protein tau (MAPT) on chromosome 17 were associated with many families with familial FTDP-17. found in FTDP-17 for mutations in MAPT begins between ages 25 and 65 with penetrance approaching 100%. Symptoms include executive dysfunction and personality and behavioral changes, with aphasia and parkinsonism occurring in many individuals [Boeve, B.F. et al. (2008) Arch Neurol 65(4): 460-464].

[0244] "Chronic Traumatic Encephalopathy" ("CTE") is a debilitating neurodegenerative disease resulting from repetitive mild traumatic brain injuries found in many athletes, especially football players. Neuropathological signatures of CTE include accumulation of phosphorylated tau in sulcus and perivascular regions, microgliosis, and astrocytosis; It can progress to late-stage global brain atrophy. CTE can progress from mild symptoms, such as short-term memory deficits and mild aggression, to advanced language deficits and psychotic symptoms, including delusions and parkinsonism, over years [Fesharaki-Zadeh, A. (2019) Front Neurol 10:713].

[0245] “Fighter dementia” is a form of CTE involving profound impairment of cognitive and motor function due to repetitive blows to the head from boxing [Castellani. R.J et al. (2017) J Alzheimers Dis 60 (4): 1209-1221].

[0246] "Argyrophilic granulopathy" ("AGD") is a very frequent sporadic tauopathy and in some studies the most common neurodegenerative disease after Alzheimer's disease. AGD is a late-onset neurodegenerative disease characterized by small, spindle-shaped or tortoise-shaped, silver-staining-positive lesions in neuronal processes called argyrid granules (AG). Phosphorylated tau is a major component of AG. The most common AGD findings are slowly progressive amnesia and mild cognitive impairment, with a high prevalence of concomitant neuropsychiatric symptoms. Lacking prominent clinical features, AGD is often diagnosed only postmortem on the basis of three pathologic features: AG, oligodendrocyte coil bodies, and neural tangles [Rodriguez, R.D. et al. (2015) Dement Neuropsychol 9(1): 2-8].

[0247] "Primary age-related tauopathy" ("PART") is a pathology commonly observed postmortem in the brains of older individuals with normal or only mildly impaired cognitive function. PART brains have tau neurofibrillary tangles indistinguishable from those of Alzheimer's disease, but no amyloid-β plaques [Crary, J.F. et al. (2014) Acta Neuropathol 128(6): 755-66].

[0248] "Creutzfeldt-Jakob disease" ("CJD") belongs to a family of human and animal diseases known as transmissible spongiform encephalopathies (TSE) or prion diseases. Prions, a term derived from "protein" and "infectious," cause CJD in humans and TSE in animals. Spongy refers to the characteristic appearance of the infected brain, which becomes filled with holes to the point of being sponge-like when viewed under a microscope. CJD is a rare degenerative and fatal brain disorder that usually presents late and has a rapid course. Typical onset of symptoms occurs at about age 60, and about 70 percent of individuals die within a year. In the early stages of the disease, people may have forgetfulness, behavioral changes, lack of coordination, and visual disturbances. As the disease progresses, mental deterioration becomes evident and involuntary movements, blindness, limb weakness, and coma can occur. In addition to prion plaques, tau pathology has also been observed in several brain regions of CJD patients, and the cerebrospinal fluid of patients with extensive tau pathology also has elevated total tau protein [Kovacs, G.G et al. (2017) Brain Pathol 3: 332-344].

[0249] "Familiar British dementia" ("FBD") was originally diagnosed in affected members of a large British family with a clinical picture that included dementia, spastic tetreparesis, and cerebellar ataxia. A reported form of cerebral amyloid angiopathy. FBD is caused by mutations in the BRI2 gene. Amyloid plaques in FBD consist of amyloid-Bri, and tau-positive neurofibrillary tangles are found in areas affected by amyloid-Bri lesions. Tau immunoblotting in FBD is similar to the tau pattern in Alzheimer's disease [Holton J.L. et al. (2001) Am J Patho 2: 515-526].

[0250] A “therapeutically effective amount,” as used herein, is the treatment of the disease (e.g., the reduction of an existing disease or one or more symptoms of the disease) when administered to a subject with an APOE-associated neurodegenerative disease. (by reducing, improving, or maintaining). A "therapeutically effective amount" includes an RNAi agent, how the agent is administered, the disease and its severity, as well as medical history, age, weight, family history, genetic make-up, type of prior or concurrent treatment, as present. It may vary, if any, depending on other individual characteristics of the subject being treated.

[0251] A "prophylactically effective amount," as used herein, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease when administered to a subject with an APOE-associated neurodegenerative disorder. It is intended to include amounts of RNAi agents. Amelioration of a disease includes slowing the course of the disease or reducing the severity of later-onset disease. A "prophylactically effective amount" includes the RNAi agent, how the agent is administered, the degree of risk of disease, as well as medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, as present. It may vary, if any, depending on other individual characteristics of the patient being treated.

[0252] A "therapeutically effective amount" or "prophylactically effective amount" also encompasses an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agents used in the disclosed methods can be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0253] The phrase "pharmaceutically acceptable" means within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio, without undue toxicity, irritation, allergic reaction, or other problems or complications. is used herein to mean any compound, material, composition, or dosage form suitable for use in contact with tissue of human and animal subjects.

[0254] The phrase "pharmaceutically acceptable carrier," as used herein, is involved in carrying or transporting a subject compound from one organ, or part of the body, to another organ, e.g., part of the body. pharmaceutically acceptable materials, compositions, or vehicles such as liquid or solid fillers, diluents, excipients, manufacturing aids such as lubricants, magnesium talc, calcium stearate or zinc stearate , or stearic acid), or a solvent-encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, and not deleterious to the subject being treated. Some examples of materials that can serve as pharmaceutically acceptable carriers include (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, lauryl (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut, cottonseed, safflower, sesame, olive, corn, and soybean oils; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (20) pH buffer solutions; (21) polyesters, polycarbonates, or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL, and LDL, etc.; and (22) other non-toxic affinity substances used in pharmaceutical formulations.

[0255] The term "sample," as used herein, encompasses a collection of similar bodily fluids, cells, or tissues isolated from a subject as well as bodily fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, spinal fluid, ocular fluid, lymph, urine, saliva, and the like. Tissue samples can include samples from tissues, organs, or localized areas. For example, samples can be obtained from particular organs, portions of organs, or fluids or cells within those organs. In certain embodiments, the sample is the brain (e.g., the whole brain or a segment of the brain, e.g., the striatum, or certain types of cells in the brain, e.g., neurons and glial cells (astrocytes, oligodendrocytes). , microglial cells)). In other embodiments, "sample obtained from a subject" refers to liver tissue (or subcomponents thereof) obtained from a subject. In some embodiments, "sample obtained from a subject" refers to blood obtained from a subject or plasma or serum obtained therefrom. In a further embodiment, "a sample obtained from a subject" refers to brain tissue (or subcomponents thereof) or retinal tissue (or subcomponents thereof) obtained from a subject.

[0256] II. RNAi Agents of the Disclosure Described herein are RNAi agents that inhibit expression of the APOE gene. In some embodiments, RNAi agents provided herein inhibit expression of APOE2, APOE3, and APOE4 alleles. In other embodiments, an RNAi agent provided herein inhibits expression of an APOE4 allele, e.g., the RNAi agent does not substantially inhibit expression of an APOE2 allele or an APOE3 allele, e.g. , the inhibition of APOE2 and / or APOE3 expression is about 10% or less. In one embodiment, the RNAi agent is used in cells, e.g., subjects [e.g., mammals, e.g., APOE-associated neurodegenerative diseases, e.g., amyloid-beta mediated diseases, e.g., Alzheimer's disease, Down's syndrome, and cerebral amyloid angiopathy, as well as tau-mediated diseases such as primary tauopathies, such as frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), spherocytosis Gliatauopathy (GGT), frontotemporal dementia with parkinsonism (FTDP, FTDP-17), chronic traumatic encephalopathy (CTE), boxer dementia, frontotemporal lobar degeneration (FTLD), addiction Aggranular disease (AGD), and primary age-related tauopathy (PART), or secondary tauopathies, e.g., AD, Creutzfeldt-Jakob disease, Down's syndrome, and familial British dementia]. comprising a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of the APOE gene in . The dsRNA includes an antisense strand that has a region of complementarity that is complementary to at least a portion of the mRNA formed upon expression of the APOE gene. A region of complementarity is about 15-30 nucleotides in length or less. Upon contact with a cell expressing the APOE gene, the RNAi agent inhibits expression of the APOE gene (e.g., human gene, primate gene, non-primate gene) by, e.g., PCR or branched DNA (bDNA)-based methods, or Inhibit at least 50% as assayed by protein-based methods, such as Western blotting or immunofluorescence analysis using flow cytometry techniques. In one embodiment, the level of knockdown is assayed at 10 nM concentration of siRNA in human neuroblastoma BE(2)-C cells using the dual luciferase assay method provided in Example 1 below. be done.

[0257] A dsRNA contains two RNA strands that are complementary and hybridize to form a duplex structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a region of complementarity that is substantially complementary, generally perfectly complementary to the target sequence. A target sequence can be obtained from the sequence of the mRNA formed upon expression of the APOE gene. The other strand (the sense strand) contains a region complementary to the antisense strand, whereby the two strands hybridize to form a duplex structure when combined under suitable conditions. do. As described elsewhere herein and known in the art, the complementary sequences of the dsRNA are contained as self-complementary regions of a single nucleic acid molecule to face each other on separate oligonucleotides. can also

[0258] Generally, the duplex structure is 15 to 30 base pairs in length, e.g. , 15~21, 15~20, 15~19, 15~18, 15~17, 18~30, 18~29, 18~28, 18~27, 18~26, 18~25, 18~24, 18 ~23, 18~22, 18~21, 18~20, 19~30, 19~29, 19~28, 19~27, 19~26, 19~25, 19~24, 19~23, 19~22 , 19~21, 19~20, 20~30, 20~29, 20~28, 20~27, 20~26, 20~25, 20~24, 20~23, 20~22, 20~21, 21 ~30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain preferred embodiments the duplex structure is 18 to 25 base pairs in length, e.g. ~25, 19~24, 19~23, 19~22, 19~21, 19~20, 20~25, 20~24, 20~23, 20~22, 20~21, 21~25, 21~24 , 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, or 24-25 base pairs in length, e.g., 19-21 base pairs in length. be. Ranges and lengths intermediate to those recited above are also contemplated to be part of this disclosure.

[0259] Similarly, the region of complementarity to the target sequence is 15 to 30 nucleotides long, e.g. 22, 15~21, 15~20, 15~19, 15~18, 15~17, 18~30, 18~29, 18~28, 18~27, 18~26, 18~25, 18~24, 18~23, 18~22, 18~21, 18~20, 19~30, 19~29, 19~28, 19~27, 19~26, 19~25, 19~24, 19~23, 19~ 22, 19~21, 19~20, 20~30, 20~29, 20~28, 20~27, 20~26, 20~25, 20~24, 20~23, 20~22, 20~21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides long, such as 19-23 nucleotides long or 21-23 Nucleotide length. Ranges and lengths intermediate to those recited above are also contemplated to be part of this disclosure.

[0260] In some embodiments, the dsRNA is 15 to 23 nucleotides long, or 25 to 30 nucleotides long. Generally the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can function as substrates for Dicer. As those skilled in the art will also appreciate, the regions of RNA targeted for cleavage are most often part of longer RNA molecules, often mRNA molecules. As relevant, a "portion" of an mRNA target is a contiguous sequence of the mRNA target of sufficient length to allow it to be a substrate for RNAi-dependent cleavage (i.e., cleavage by the RISC pathway). .

[0261] Those skilled in the art will appreciate that the duplex region is a primary functional portion of the dsRNA, e.g., 15 to 36 base pairs, e.g. 15~30, 15~29, 15~28, 15~27, 15~26, 15~25, 15~24, 15~23, 15~22, 15~21, 15~20, 15~19, 15~ 18, 15~17, 18~30, 18~29, 18~28, 18~27, 18~26, 18~25, 18~24, 18~23, 18~22, 18~21, 18~20, 19~30, 19~29, 19~28, 19~27, 19~26, 19~25, 19~24, 19~23, 19~22, 19~21, 19~20, 20~30, 20~ 29, 20~28, 20~27, 20~26, 20~25, 20~24, 20~23, 20~22, 20~21, 21~30, 21~29, 21~28, 21~27, It will also be appreciated that the duplex region is 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, eg, 19-21 base pairs. Thus, in one embodiment, a duplex region of greater than 30 base pairs that targets the desired RNA for cleavage, e.g., 15-30 base pairs, as long as it is processed into a functional duplex. is a dsRNA. Accordingly, those skilled in the art will recognize that in one embodiment the miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, RNAi agents useful for targeting APOE expression are not generated in target cells by cleavage of larger dsNRAs.

[0262] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs of, eg, 1, 2, 3, or 4 nucleotides. Nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs such as deoxynucleotides / nucleosides. Overhangs can be on the sense strand, on the antisense strand, or any combination thereof. Moreover, overhanging nucleotides can be present on either the 5' end, the 3' end, or both of the antisense or sense strands of the dsRNA.

[0263] dsRNA can be synthesized by standard methods known in the art.

[0264] In one aspect, the dsRNA of this disclosure comprises at least two nucleotide sequences, a sense strand and an antisense strand. The sense strand sequence for APOE can be selected from the group of sequences provided in any one of Tables 2-5, and 7-10, and the corresponding nucleotides of the sense strand, the antisense strand are shown in Tables 2-5. 5, and any one sequence of 7-10. In this embodiment, one of the two sequences is complementary to the other of the two sequences, where one of the sequences is substantially relative to the sequence of the mRNA produced upon expression of the APOE gene. Complementary. Therefore, in this embodiment, the dsRNA is described where one oligonucleotide is the sense strand (passenger strand) in any one of Tables 2-5 and 7-10 for APOE, and the second oligonucleotide is described as the It will contain two oligonucleotides, described as the corresponding antisense strand (guide strand) to the sense strand in any one of 2-5 and 7-10.

[0265] In one embodiment, the sequences substantially complementary to the dsRNA are contained in separate oligonucleotides. In another embodiment, the sequences substantially complementary to the dsRNA are contained in a single oligonucleotide.

[0266] The sequences in Tables 3, 5, 8 and 10 are described as modified or conjugated sequences and the sequences in Tables 2, 4, 7 and 9 are described as unmodified sequences, although the RNAi agent RNAs, e.g., dsRNAs of the present disclosure, are unmodified, unconjugated, or modified or conjugated differently than described therein, Tables 2-5 and 7-10 can include any one of the sequences set forth in any one of. One or more lipophilic ligands and / or one or more GalNAc ligands can be included in any of the RNAi agent positions provided in this application.

[0267] Those skilled in the art are fully aware that dsRNAs having a duplex structure of about 20 to 23 base pairs, e.g., 21 base pairs, are hailed as particularly effective in inducing RNA interference [Elbashir et al. et al., (2001) EMBO J., 20:6877-6888]. However, others have found that shorter or longer RNA duplex structures may also be effective [Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226]. In the embodiments described above, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein can comprise at least one strand with a minimum length of 21 nucleotides. It can reasonably be expected that shorter duplexes minus a few nucleotides at one or both ends would be equally effective compared to the dsRNA described above. Thus, a dsRNA having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein, Their ability to inhibit expression of the APOE gene when using an in vitro assay with Cos7 and an RNA agent at a concentration of 10 nM and a PCR assay, as provided in the Examples of 10, 15, 20, A dsRNA that differs from the dsRNA containing the full-length sequence by no more than 25 or 30% inhibition is contemplated within the scope of this disclosure.

[0268] In addition, the RNAs described herein identify sites in APOE transcripts that are susceptible to RISC-mediated cleavage. As such, the disclosure further features RNAi agents that target within this site. As used herein, an RNAi agent is said to target within a particular site of an RNA transcript if it promotes cleavage of the transcript anywhere within that particular site. Such RNAi agents generally comprise at least about 15 nucleotides from one of the sequences provided herein coupled to additional nucleotide sequences taken from regions flanking the selected sequence in the APOE gene. , preferably will contain at least 19 nucleotides.

[0269] Thus, the RNAi agents described herein can contain one or more mismatches to the target sequence. In one embodiment, the RNAi agents described herein contain no more than 3 mismatches (ie, 3, 2, 1, or 0 mismatches). In one embodiment, the RNAi agents described herein contain no more than two mismatches. In one embodiment, the RNAi agents described herein contain no more than one mismatch. In one embodiment, the RNAi agents described herein contain 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the mismatches are limited to within the last 5 nucleotides from the 5' or 3' end of the region of complementarity, as appropriate. You can also For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand complementary to the region of the APOE gene generally does not contain any mismatches within the middle 13 nucleotides. Determining whether an RNAi agent containing mismatches to a target sequence is effective in inhibiting expression of the APOE gene by using methods described herein or known in the art can be done. In particular, if specific regions of complementarity in the APOE gene are known to have polymorphic sequence variations within the population, the effectiveness of mismatched RNAi agents in inhibiting expression of the APOE gene can be evaluated. It is important to consider

[0270] III. Modified RNAi Agents of the Disclosure In one embodiment, the RNA, eg, dsRNA, of an RNAi agent of the present disclosure is unmodified, eg, free of chemical modifications or conjugations known in the art and described herein. In preferred embodiments, the RNA, eg, dsRNA, of the RNAi agents of the present disclosure are chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of this disclosure, substantially all of the nucleotides of the RNAi agents of this disclosure are modified. In other embodiments of this disclosure, all of the nucleotides of the RNAi agents of this disclosure are modified. An RNAi agent of the disclosure in which "substantially all of the nucleotides are modified" can be predominantly but not entirely modified and can contain 5, 4, 3, 2, 1, or unmodified nucleotides. In still other embodiments of the present disclosure, RNAi agents of the present disclosure may contain 5, 4, 3, 2 or 1 modified nucleotides.

[0271] Nucleic acids featured in this disclosure can be prepared using methods well established in the art, such as "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Edrs.), which is incorporated herein by reference. , John Wiley & Sons, Inc., New York, NY, USA. Modifications include, for example, terminal modifications, such as 5′ end modifications (phosphorylation, conjugation, reverse ligation) or 3′ end modifications (conjugation, DNA nucleotides, reverse ligation, etc.), base modifications, such as stabilizing bases. , replacement of destabilizing bases or bases that base-pair with partners of the expanded repertoire, removal of bases (abasic nucleotides) or conjugated bases, sugar modifications (e.g., at the 2' or 4' positions) ) or sugar replacements, or backbone modifications including phosphodiester bond modifications or replacements. Specific examples of RNAi agents useful in embodiments described herein include, but are not limited to, RNAs that contain modified backbones or do not contain natural internucleoside linkages. RNAs with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, modified RNAs that do not have a phosphorus atom in their internucleoside backbone, as sometimes referred to in the art, can also be considered oligonucleosides. In some embodiments, a modified RNAi agent has a phosphorus atom in its internucleoside backbone.

[0272] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates, including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, 3 phosphoramidates, including '-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and with regular 3'-5' linkages Boranophosphates, their 2′-5′ linked analogs and adjacent pairs of nucleoside units linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′ and those having reversed polarities. Various salts such as sodium salts, mixed salts and free acid forms are also included.

[0273] Representative U.S. patents teaching the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5 ,476,925, 5,519,126, 5,536,821 , 5,541,316, 5,550,111, 5,563,253, 5,571,799, 5,587,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, No. 6,172,209, No. 6,239,265, No. 6,277,603, 6,326,199, 6,346,614, 6,444,423, 6,531,590, 6,534,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6 ,878,805, 7,015,315, 7,041,816 , 7,273,933, 7,321,029 and RE39464, the entire contents of each of which are incorporated herein by reference.

[0274] Modified RNA backbones that do not contain a phosphorus atom in them may have short alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages or one or more short heteroatoms or heterocyclic nucleosides. It has a skeleton formed by interlinkages. These include morpholino linkages (some formed from nucleoside sugar moieties), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones. , sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, those with amide backbones and mixed N, O, S and CH 2 Others with constituent parts are included.

[0275] Representative U.S. patents teaching the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; , 64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,6 02,240, 5,608,046, 5,610,289 Nos. 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437 and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0276] In other embodiments, RNA mimetics suitable for use in RNAi agents are contemplated in which both the sugar and internucleoside linkages of the nucleotide units, ie the backbone, are replaced with novel groups. Base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimetic known to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and attached directly or indirectly to the aza nitrogen atoms of the amide moieties of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, United States Patent Nos. 5,539,082, 5,714,331 and 5,719,262, the entire contents of each of which are incorporated herein by reference. incorporated into. Additional PNA compounds suitable for use in the RNAi agents of the present disclosure are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0277] Some embodiments featured in this disclosure include RNAs with phosphorothioate backbones and heteroatom backbones, particularly --CH of U.S. Pat. No. 5,489,677, referenced above. 2 --NH--CH 2 -, --CH 2 --N(CH 3 )--O--CH 2 --[known as methylene (methylimino) or MMI backbone], --CH 2 --O--N(CH 3 )--CH 2 --, --CH 2 --N(CH 3 )--N(CH 3 )--CH 2 -- and --N(CH 3 )--CH 2 --CH 2 -- and oligonucleosides with an amide backbone of US Pat. No. 5,602,240, referenced above. In some embodiments, the RNA featured herein has the morpholino backbone structure of US 5,034,506 referenced above. The natural phosphodiester backbone is O-P(O)(OH)-OCH 2 - can be expressed as

[0278] Modified RNAs may also contain one or more substituted sugar moieties. RNAi agents, e.g., dsRNA, featured herein have the following at the 2' position: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; - or N-alkynyl or O-alkyl-O-alkyl, where alkyl, alkenyl and alkynyl are substituted or unsubstituted C 1 ~C 10 alkyl or C 2 ~C 10 It can be alkenyl and alkynyl. An exemplary suitable modification is O[(CH 2 ) n O] m CH 3 , O(CH 2 ). n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 wherein n and m are from 1 to about 10. In other embodiments, the dsRNA has the following at the 2' position: C 1 ~C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, interfering substance, group for improving the pharmacokinetic properties of an RNAi agent or the pharmacokinetics of an RNAi agent Including one of the groups to improve properties and other substituents with similar properties. In some embodiments, the modification is 2'-O--CH, also known as 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE). 2 CH 2 OCH 3 ) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), ie, alkoxy-alkoxy groups. As another exemplary modification, 2'-dimethylaminooxyethoxy, i.e. O(CH 2 ) 2 ON(CH 3 ) 2 and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH 2 --O--CH 2 --N(CH 2 ) 2 There is Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (R and S isomers in these three families). both), 2'-alkoxyalkyl and 2'-NMA (N-methylacetamide).

[0279] Other modifications include 2'-methoxy (2'-OCH 3 ), 2'-aminopropoxy (2'-OCH 2 CH 2 CH2 NH 2 ), 2'-O-hexadecyl and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the RNAi agent, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of the 5' terminal nucleotide. can be done. RNAi agents may also have sugar mimetics, eg, cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873 , No. 5,646,265, No. 5,658,873, 5,670,633 and 5,700,920, certain of which are commonly owned with this application. The entire contents of each of the foregoing are incorporated herein by reference.

[0280] The RNAi agents of the present disclosure may also include nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), the pyrimidine bases thymine (T), cytosine (C) and uracil ( U) are included. Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and guanine. 6-methyl and other alkyl derivatives, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil , cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, In particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613 and Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in this disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca. Raton, 1993, pp. 276-278), even more particularly exemplary base substitutions when combined with 2'-O-methoxyethyl sugar modifications.

[0281] Representative U.S. patents that teach the preparation of certain of the above modified nucleobases as well as other modified nucleobases include, but are not limited to, the above U.S. Pat. 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5 ,552,540, 5,587,469, 5,594,121 , No. 5,596,091, No. 5,614,617, No. 5,681,941, No. 5,750,692, No. 6,015,886, No. 6,147,200, No. 6,166,197, No. 6,222,025, No. 6,235,887, No. 6,380,368, No. 6,528,640, No. 6,639,062, No. 6,617,438, 7,045,610, 7,427,672 and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0282] In some embodiments, RNAi agents of this disclosure can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a ring formed by bridging two carbons, whether adjacent or not. A "bicyclic nucleoside" ("BNA") comprises rings formed by bridging two carbons of a sugar ring, whether adjacent or not, thereby forming a bicyclic ring structure. is a nucleoside having a sugar moiety that In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring, optionally via a 2'-acyclic oxygen atom. Thus, in some embodiments, agents of the invention may comprise one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with modified ribose moieties in which the ribose moieties contain an additional bridge connecting the 2' and 4' carbons. In other words, the LNA is the 4'-CH 2 Nucleotides containing bicyclic sugar moieties containing -O-2' bridges. This structure effectively "locks" the ribose into the 3'-end structural conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects [Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447 et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193]. Examples of bicyclic nucleosides for use in the polynucleotides of the invention include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, antisense polynucleotide agents of the invention include one or more bicyclic nucleosides comprising a 4' to 2' bridge.

[0283] Locked nucleosides have the following structures (stereochemistry omitted):

[0284] [formation] where B is a nucleobase or modified nucleobase and L is a linking group connecting the 2'-carbon to the 4'-carbon of the ribose ring. Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH 2 )--O-2'(LNA), 4'-(CH 2 )--S-2', 4'-(CH 2 ) 2 --O-2'(ENA), 4'-CH(CH 3 )--O-2' (also called "constrained ethyl" or "cEt") and 4'-CH(CH 2 OCH 3 )--O-2' (and analogues thereof, see, e.g., U.S. Pat. No. 7,399,845), 4'-C(CH 3 )(CH 3 )--O-2' (and analogues thereof, see, e.g., U.S. Pat. No. 8,278,283), 4'-CH 2 -N(OCH 3 )-2′ (and analogues thereof, see, e.g., U.S. Pat. No. 8,278,425), 4′-CH 2 --O--N(CH 3 )-2′ (see, e.g., U.S. Patent Publication No. 2004 / 0171570), 4′-CH 2 -N(R)--O-2' (wherein R is H, C1-C12 alkyl or a nitrogen protecting group) (see, e.g., U.S. Patent No. 7,427,672), 4'-CH 2 -C(H)(CH 3 )-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134) and 4′-CH 2--C(=CH 2 )-2' (and analogs thereof, see, eg, US Pat. No. 8,278,426). The entire contents of each of the foregoing are incorporated herein by reference.

[0285] Further representative U.S. patents and U.S. patent publications teaching the preparation of locked nucleic acid nucleotides include, but are not limited to: U.S. Pat. Nos. 6,268,490; 6,998,484, 7,053,207, 7,034,133, 7,084,125, 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8 , 278,425, 8,278,426, 8,278,283 US2008 / 0039618 and US2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0286] For example, any of the above bicyclic nucleosides with one or more stereochemical sugar configurations can be prepared, including α-L-ribofuranose and β-D-ribofuranose (see WO99 / 14226 ).

[0287] The RNAi agents of this disclosure can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-0-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as "S-cEt."

[0288] RNAi agents of the present disclosure may also include one or more "conformation-restricting nucleotides" ("CRNs"). CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose or the C3 and -C5' carbons of ribose. CRN locks the ribose ring into a stable conformation, increasing hybridization affinity for mRNA. The linker is of sufficient length to place the oxygen in the optimal position for stability and affinity, resulting in less ribose ring puckering.

[0289] Representative publications teaching the preparation of certain of the above CRNs include, but are not limited to, US2013 / 0190383 and WO2013 / 036868, the entire contents of each of which are incorporated herein by reference. incorporated.

[0290] In some embodiments, RNAi agents of this disclosure comprise one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is a non-locked acyclic nucleic acid in which any sugar linkages have been removed, forming a non-locked "sugar" residue. In one example, UNA also includes monomers in which the bond between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) is removed [herein incorporated by reference, Nuc. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039].

[0291] Representative U.S. publications teaching the preparation of UNA include, but are not limited to, US Pat. The entire contents of each of are incorporated herein by reference.

[0292] Potential stabilizing modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp- C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6- amino), 2-docosanoyl-uridine-3″-phosphate, reverse base dT (idT) and others. A disclosure of this modification can be found in WO2011 / 005861.

[0293] Other modifications of the RNAi agents of the present disclosure include 5' phosphates or 5' phosphate mimetics, such as 5' terminal phosphates or phosphate mimetics on the antisense strand of the RNAi agent. Suitable phosphate mimetics are disclosed, for example, in US2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0294] A. Modified RNAi Agents Containing Motifs of the Disclosure In certain aspects of the disclosure, double-stranded RNAi agents of the disclosure include agents having chemical modifications, eg, as disclosed in WO2013 / 075035, the entire contents of which are incorporated herein by reference. As shown herein and in WO2013 / 075035, one or more motifs of three identical modifications on three consecutive nucleotides are introduced in the sense or antisense strand of an RNAi agent at or near the cleavage site. excellent results can be obtained by introducing In some embodiments, the sense and antisense strands of the RNAi agent may be otherwise fully modified. Introduction of these motifs, if present, interferes with the pattern of modification of the sense or antisense strand. An RNAi agent may be conjugated to a lipophilic ligand, eg, a C16 ligand, eg, on the sense strand. RNAi agents may be modified, for example, with (S)-glycol nucleic acid (GNA) modifications at one or more residues of the antisense strand. The resulting RNAi agent exhibits excellent gene silencing activity.

[0295] Accordingly, the present disclosure provides double-stranded RNAi agents capable of inhibiting target gene (ie, APOE gene) expression in vivo. RNAi agents include a sense strand and an antisense strand. Each strand of the RNAi agent can be 15-30 nucleotides in length. For example, each strand is 16-30 nucleotides long, 17-30 nucleotides long, 25-30 nucleotides long, 27-30 nucleotides long, 17-23 nucleotides long, 17-21 nucleotides long, 17-19 nucleotides long, 19-30 nucleotides long, It can be 25 nucleotides long, 19-23 nucleotides long, 19-21 nucleotides long, 21-25 nucleotides long or 21-23 nucleotides long. In certain embodiments, each strand is 19-23 nucleotides in length.

[0296] The sense and antisense strands typically form a double-stranded double-stranded RNA (“dsRNA”), also referred to herein as the “RNAi agent”. The duplex region of an RNAi agent can be 15-30 nucleotide pairs in length. For example, the duplex region may be 16-30 nucleotide pairs long, 17-30 nucleotide pairs long, 27-30 nucleotide pairs long, 17-23 nucleotide pairs long, 17-21 nucleotide pairs long. length, 17-19 nucleotide pairs long, 19-25 nucleotide pairs long, 19-23 nucleotide pairs long, 19-21 nucleotide pairs long, 21-25 nucleotide pairs long or 21-25 nucleotide pairs long It can be 23 nucleotide pairs long. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 nucleotides in length. In preferred embodiments, the double-stranded region is 19-21 nucleotide pairs in length.

[0297] In one embodiment, an RNAi agent may contain one or more overhanging regions or capping groups at the 3' end, 5' end or both ends of one or both strands. The overhang is 1-6 nucleotides long, e.g., 2-6 nucleotides long, 1-5 nucleotides long, 2-5 nucleotides long, 1-4 nucleotides long, 2-4 nucleotides long, 1-3 nucleotides long, 2-6 nucleotides long, It can be 3 nucleotides long or 1-2 nucleotides long. In preferred embodiments, the nucleotide overhang region is 2 nucleotides in length. Overhangs can be the result of one strand being longer than the other or the result of two strands of the same length being twisted. The overhang can form a mismatch with the target mRNA, can be complementary to the targeted gene sequence, or can be another sequence. The first and second strands can also be joined by additional bases, eg, to form hairpins, or by other non-basic linkers.

[0298] In one embodiment, each nucleotide in the overhang region of the RNAi agent is independently 2'-sugar modified, e.g., 2-F, 2'-O-methyl, thymidine (T), and any combination thereof. It may be a modified or unmodified nucleotide, including but not limited to.

[0299] For example, TT can be an overhanging sequence at either end on either strand. The overhangs can form mismatches with the target mRNA, can be complementary to the targeted gene sequence, or can be another sequence.

[0300] The 5'- or 3'-overhangs of the sense strand, the antisense strand, or both strands of the RNAi agent can be phosphorylated. In some embodiments, the overhang region(s) contain two nucleotides with a phosphorothioate between the two nucleotides, and the two nucleotides can be the same or different. In one embodiment, overhangs are present on the 3' ends of the sense strand, the antisense strand, or both strands. In one embodiment, this 3'-overhang is present in the antisense strand. In one embodiment, this 3'-overhang is present in the sense strand.

[0301] An RNAi agent may contain only a single overhang that can enhance the interfering activity of RNAi without affecting its overall stability. For example, a single-stranded overhang can be located at the 3' end of the sense strand or at the 3' end of the antisense strand. RNAi can also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. Generally, the antisense strand of RNAi has a nucleotide overhang at the 3'end and is blunt at the 5'end. Without wishing to be bound by theory, the asymmetrical blunt end of the 5' antisense strand and the 3' overhang of the antisense strand favor guide strand loading into the RISC process.

[0302] In one embodiment, the RNAi agent is a 19-nucleotide long double ended bluntmer, where the sense strand is 3 contiguous nucleotides at positions 7, 8, 9 from the 5' end. contains at least one motif of two 2'-F modifications. The antisense strand contains at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

[0303] In another embodiment, the RNAi agent is blunt-ended at both ends and is 20 nucleotides long, and the sense strand comprises three 2'- It contains at least one motif of F modification. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

[0304] In yet another embodiment, the RNAi agent is blunt-ended at both ends and is 21 nucleotides in length, and the sense strand comprises three 2' nucleotides on three consecutive nucleotides at positions 9, 10, 11 from the 5' end. Contains at least one motif of -F modification. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

[0305] In one embodiment, the RNAi agent comprises a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides, the sense strand comprising three 2'-F sequences on three consecutive nucleotides at positions 9, 10, 11 from the 5' end. containing at least one motif of modification, the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5' end, and RNAi One end of the agent is blunt and the other end contains an overhang of 2 nucleotides. Preferably, the 2 nucleotide overhang is at the 3' end of the antisense strand. If a two nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides and two of the three nucleotides A hang nucleotide, the third nucleotide is the next paired nucleotide of the overhang nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, any nucleotide in the sense and antisense strands of the RNAi agent that contains a nucleotide that is part of the motif is a modified nucleotide. In one embodiment, each residue is independently modified with, for example, 2'-O-methyl or 2'-fluoro in alternating motifs. RNAi agents may further comprise ligands (eg, lipophilic ligands, optionally C16 ligands).

[0306] In one embodiment, the RNAi agent comprises a sense and an antisense strand, the sense strand being 25-30 nucleotide residues in length, starting at the 5' terminal nucleotide (position 1) and continuing from the first strand. contains at least 8 ribonucleotides and the antisense strand is 36-66 nucleotide residues long and is paired with positions 1-23 of the sense strand, starting from the 3' terminal nucleotide. comprising at least 8 ribonucleotides at forming positions to form a duplex, at least the 3' terminal nucleotide of the antisense strand not pairing with the sense strand, and up to 6 contiguous 3' terminal nucleotides of the sense strand; strand unpaired, thereby forming a 3′ single-stranded overhang of 1-6 nucleotides, the 5′ end of the antisense strand comprising 10-30 contiguous nucleotides unpaired with the sense strand; thereby forming a single-stranded 5' overhang of 10-30 nucleotides, wherein at least the 5' and 3' terminal nucleotides of the sense strand are aligned for maximum complementarity when the sense and antisense strands are aligned for maximum complementarity. A base that pairs with a nucleotide of the antisense strand, thereby forming a substantially double-stranded region between the sense and antisense strands, the antisense strand being at least as long as the antisense strand. Sufficiently complementary to the target RNA along 19 ribonucleotides to reduce target gene expression when the double-stranded nucleic acid is introduced into mammalian cells, and the sense strand consists of three 2's on 3 consecutive nucleotides. It contains at least one motif of the '-F modification, at least one of which occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0307] In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the RNAi agent has a first strand having a length that is at least 25 and at most 29 nucleotides and a length that is at most 30 nucleotides. and a second strand having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand forms a blunt end, the second strand being 1-4 nucleotides longer at its 3' end than the first strand, and the double-stranded region being at least 25 nucleotides long. and the second strand is sufficiently complementary to the target mRNA along a second strand length of at least 19 nucleotides to inhibit target gene expression when the RNAi agent is introduced into a mammalian cell. Dicer cleavage of the RNAi agent preferentially results in an siRNA containing the 3' end of the second strand, thereby reducing target gene expression in the mammal. Optionally, the RNAi agent may further comprise a ligand.

[0308] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs occurring at the cleavage site in the sense strand.

[0309] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs being the cleavage site in the antisense strand. occur at or near

[0310] For RNAi agents with duplex regions 17-23 nucleotides in length, the antisense strand cleavage sites are usually at approximately positions 10, 11 and 12 from the 5' end. Thus, the three identical modification motifs are at positions 9, 10, 11, positions 10, 11, 12, positions 11, 12, 13, positions 12, 13, 14 or positions 13, 14, 15 of the antisense strand. As may occur, the numbers start at the first nucleotide from the 5' end of the antisense strand, or the numbers start at the first paired nucleotide within the duplex region from the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the RNAi duplex region from the 5' end.

[0311] The sense strand of the RNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the site of strand cleavage, the antisense strand on three consecutive nucleotides at or near the site of strand cleavage. can have at least one motif of three identical modifications of When the sense and antisense strands form a dsRNA duplex, combine the sense and antisense strands with one motif of three nucleotides on the sense strand and one motif of three nucleotides on the antisense strand. has at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand base-pairs with at least one of the three nucleotides of the motif in the antisense strand. can. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

[0312] In one embodiment, the sense strand of the RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the site of the strand break and the other motif may be a wing modification. As used herein, the term "wing modification" refers to motifs that occur in different parts of the strand away from the motif at or near the cleavage site on the same strand. The wing modifications are adjacent to or separated from the first motif by at least one or more nucleotides. The chemistry of the motifs is distinct from each other when the motifs are immediately adjacent to each other, and may be the same or different when the motifs are separated by one or more nucleotides. . There may be more than one wing modification. For example, if two wing modifications are present, each wing modification can occur one end to the first motif at or near the cleavage site, or on either side of the lead motif.

[0313] Similar to the sense strand, the antisense strand of the RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides, at least one of the motifs being at or at the cleavage site of the strand. occur nearby. The antisense strand may also contain one or more wing modifications in sequence similar to the wing modifications that may be present on the sense strand.

[0314] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0315] In another embodiment, the wing modifications on the sense or antisense strand of the RNAi agent are usually paired at the 3′ end, 5′ end or both ends of the strand, the first one or two within the duplex region. does not contain nucleotides that

[0316] When the sense and antisense strands of the RNAi agent each contain at least one wing modification, the wing modifications may fall at the same end of the duplex region, overlapping by 1, 2 or 3 nucleotides. have

[0317] When the sense or antisense strand of the RNAi agent each contains at least two wing modifications, the sense and antisense strands are combined with the two modifications from one strand each representing one of the duplex regions. Two modifications from one strand each enter the other end of the double-stranded region and have an overlap of 1, 2 or 3 nucleotides. With overlap, two modified strands can be arranged on each side of the lead motif, with an overlap of 1, 2 or 3 nucleotides in the duplex region.

[0318] In one embodiment, the RNAi agent contains mismatch(es) or a combination thereof within the duplex with the target. Mismatches can occur in overhang regions or duplex regions. Base pairs can be ranked based on their propensity to promote dissociation or melting (e.g., the free energy of association or dissociation for a particular pairing; the simplest approach is to examine the pairs on the basis of individual pairs). but the next neighbor analysis or similar analysis could also be used). In terms of promotion of dissociation: A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical pairings or anything other than canonical pairings (as described elsewhere herein), canonical (A:T, A:U, G:C) Preferred over formation, pairing involving universal bases is preferred over canonical pairing.

[0319] In one embodiment, the RNAi agent comprises A:U, G:U, I:C and mismatched pairs, such as non-canonical pairs, to promote dissociation of the antisense strand at the 5' end of the duplex. The first 1, 2, 3, 4 or 5 within the duplex region from the 5' end of the antisense strand, independently selected from the group of pairings that include non-canonical pairing or universal bases contain at least one of the three base pairs.

[0320] In one embodiment, the nucleotide at position 1 within the duplex region from the 5' end in the antisense strand is selected from the group consisting of A, dA, dU, U and dT. Alternatively, at least one of the first 1, 2 or 3 base pairs in the duplex region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the duplex region from the 5' end of the antisense strand is an AU base pair.

[0321] In another embodiment, the nucleotide at the 3'-end of the sense strand is deoxy-thymidine (dT). In another embodiment, the nucleotide at the 3'-end of the antisense strand is deoxy-thymidine (dT). In one embodiment, there is a short sequence of deoxy-thymine nucleotides, eg, two dT nucleotides at the 3' end of the sense or antisense strand.

[0322] In one embodiment, the sense strand sequence has formula (I): 5'n p -N a -(X X X) i -N b -Y Y -N b -(Z Z )Z j -N a -n q 3' (I) [In the formula, i and j are each independently 0 or 1, p and q are each independently 0 to 6, each N a independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least 2 differently modified nucleotides, each N b independently represents an oligonucleotide sequence containing 0-10 modified nucleotides, each n p and n q independently represent an overhanging nucleotide, Nb and Y do not have the same modification, and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides] can be represented by Preferably YYY are all 2'-F modified nucleotides.

[0323] In one embodiment, N a or N b contains alternating pattern modifications.

[0324] In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 17-23 nucleotides long, the YYY motif can occur at or near the cleavage site of the sense strand (e.g., positions 6, 7, 8, 7, 8 , 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13), the number starts at the first nucleotide from the 5' end, or optionally the number is 5 The ' end may start at the first paired nucleotide in the duplex region.

[0325] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 1. Thus, the sense strand has the formula: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib), 5'n p -N a -XXX-N b -YYY-N a -n q 3' (Ic), or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' (Id) can be represented by

[0326] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides.

[0327] each N a can independently represent an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

[0328] When the sense strand is represented as formula (Ic), N b represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. each N a may independently represent an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

[0329] When the sense strand is represented by the formula (Id), each N b independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. preferably N bis 0, 1, 2, 3, 4, 5 or 6. each N a may independently represent an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

[0330] Each of X, Y and Z may be the same or different from each other.

[0331] In other embodiments, i is 0, j is 0, and the sense strand has the formula: 5'n p -N a -YYY-N a -n q 3' (Ia) can be represented by

[0332] When the sense strand is represented by formula (Ia), each N a can independently include oligonucleotide sequences containing 2-20, 2-15 or 2-10 modified nucleotides.

[0333] In one embodiment, the RNAi antisense strand sequence has the formula (II): 5'n q’ -N a ’-(Z’Z’Z’) k -N b '-Y'Y'Y'-N b ’-(X’X’X’) l -N' a -n p '3' (II) [In the formula, k and l are each independently 0 or 1, p' and q' are each independently 0 to 6, each N a ' independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least 2 differently modified nucleotides, each N b 'independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ’ and n q ' independently represents an overhanging nucleotide, N. b ' and Y' do not have the same modification, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides] can be represented by

[0334] In one embodiment, N a ’ or N b ' includes alternating pattern modifications.

[0335] A Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand. For example, if the RNAi agent has a double-stranded region that is 17-23 nucleotides long, the Y'Y'Y' motifs are located at positions 9, 10, 11, 10, 11, 12, and 12 of the antisense strand. may occur at positions 11, 12, 13, positions 12, 13, 14 or -13, 14, 15, numbers starting from the first nucleotide from the 5' end, or numbers starting from the 5' end of the duplex as appropriate It may start at the first paired nucleotide within the region. Preferably, the Y'Y'Y' motif occurs at positions 11,12,13.

[0336] In one embodiment, the Y'Y'Y' motifs are all 2'-OMe modified nucleotides.

[0337] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

[0338] Thus, the antisense strand has the formula: 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a ’-n p’ 3' (IIb), 5'n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3’ (IIc), or 5'n q’ -N a ’-Z’Z’Z’-N b '-Y'Y'Y'-N b ’-X’X’X’-N a ’-n p’ 3' (IId) can be represented by

[0339] When the antisense strand is represented by formula (IIb), N b ’ represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0340] When the antisense strand is represented as formula (IIc), N b ' represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0341] When the antisense strand is represented as formula (IId), each N b ' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides. preferably N b is 0, 1, 2, 3, 4, 5 or 6.

[0342] In other embodiments, k is 0, l is 0, and the antisense strand has the formula: 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3' (Ia) can be represented by

[0343] When the antisense strand is represented as formula (IIa), each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0344] Each of X', Y' and Z' may be the same or different from each other.

[0345] Each nucleotide in the sense and antisense strands can be LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl or 2 '-Fluoro may be independently modified. For example, each nucleotide of the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y' and Z' may represent a 2'-O-methyl or 2'-fluoro modification, among others.

[0346] In one embodiment, the sense strand of the RNAi agent may contain YYY motifs occurring at positions 9, 10 and 11 of the strand when the duplex region is 21 nt, numbered from the first nucleotide from the 5' end. Starting, or optionally the number may start at the first paired nucleotide within the duplex region from the 5' end, Y represents a 2'-F modification. The sense strand may further contain XXX or ZZZ motifs as wing modifications at opposite ends of the duplex region, with XXX and ZZZ each independently representing a 2'-OMe or 2'-F modification.

[0347] In one embodiment, the antisense strand may contain a Y'Y'Y' motif that occurs at positions 11, 12, 13 of the strand, the number starting at the first nucleotide from the 5' end, or optionally the number Starting at the first paired nucleotide in the duplex region from the 5' end, Y' represents a 2'-O-methyl modification. The antisense strand may further contain X'X'X' or Z'Z'Z' motifs as wing modifications at opposite ends of the duplex region, with X'X'X' and Z'Z' Each Z' independently represents a 2'-OMe modification or a 2'-F modification.

[0348] The sense strand represented by any one of formulas (Ia), (Ib), (Ic) and (Id) above is represented by any one of formulas (IIa), (IIb), (IIc) and (IId) respectively. or forms a duplex with the antisense strand represented by one.

[0349] Thus, an RNAi agent for use in the disclosed methods can include a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the RNAi duplex having the formula (III): Sense: 5'n p -N a -(X X X) i -N b -YYY-Nb -(Z Z Z) j -N a -n q 3' Antisense: 3'n p ’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) [In the formula, i, j, k and l are each independently 0 or 1, p, p', q and q' are each independently 0 to 6, each N a and N a ’ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least 2 differently modified nucleotides, each N b and N b ’ independently represents an oligonucleotide sequence containing 0-10 modified nucleotides, each n p ’, n p , n q ’ and n q independently represent overhanging nucleotides, each of which may or may not be present, XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides] represented by

[0350] In one embodiment, i is 0 and j is 0, or i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 0, or i and j are both 1. In another embodiment, k is 0 and l is 0, or k is 1, l is 0, k is 0, l is 1, or k and l are Either both are 0 or k and l are both 1.

[0351] An exemplary combination of sense and antisense strands to form an RNAi duplex has the following formula: 5'n p -N a -Y Y Y -N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y' -N a ’ n q ’ 5' (IIIa) 5'n p -N a -Y Y Y -N b -Z Z Z -N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ Five' (IIIb) 5'n p -N a - X X X -N b -YYYY-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a ’ -n q ’ 5’ (IIIc) 5'n p -N a -X X X -N b -Y Y Y -N b - Z Z Z -N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ Five' (IIId) including.

[0352] When the RNAi agent is represented by formula (IIIa), each N a independently represent oligonucleotide sequences containing 2-20, 2-15 or 2-10 modified nucleotides.

[0353] When the RNAi agent is represented by formula (IIIb), each N b independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5 or 1-4 modified nucleotides. each N a independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0354] When the RNAi agent is represented by Formula (IIIc), each N b , N b ' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. each N a independently represent oligonucleotide sequences containing 2-20, 2-15 or 2-10 modified nucleotides.

[0355] When the RNAi agent is represented by formula (IIId), each N b , N b ' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. each N a , N a ’ independently represent oligonucleotide sequences containing 2-20, 2-15 or 2-10 modified nucleotides. N. a , N a ’, N b and N b ’ independently contain an alternating pattern of modifications.

[0356] In one embodiment, when the RNAi agent is represented by formula (IIId), N a Modifications are 2'-O-methyl or 2'-fluoro modifications. In another embodiment, when the RNAi agent is represented by formula (IIId), N a Modifications are 2'-O-methyl or 2'-fluoro modifications, n p ’>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages. In yet another embodiment, when the RNAi agent is represented by formula (IIId), N a Modifications are 2'-O-methyl or 2'-fluoro modifications, n p ’>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages and the sense strand is linked to one or more C16 (or related) moieties attached by bivalent or trivalent branched linkers (described below). be conjugated. In another embodiment, when the RNAi agent is represented by formula (IIId), N a Modifications are 2'-O-methyl or 2'-fluoro modifications, n p ’>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, the sense strand may be attached by a bivalent or trivalent branched linker, one or more is conjugated to a lipophilic, eg, C16 (or related) moiety.

[0357] In one embodiment, when the RNAi agent is represented by Formula (IIIa), N a Modifications are 2'-O-methyl or 2'-fluoro modifications, n p ’>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, and the sense strand comprises one or more lipophilic molecules attached by bivalent or trivalent branched linkers. is conjugated to, eg, a C16 (or related) portion of.

[0358] In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by Formulas (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the duplex are connected by a linker. A linker may or may not be cleavable. A multimer may further comprise a ligand. Each of the duplexes may target the same gene, two different genes, or each of the duplexes may target the same gene at two different target sites. There is also

[0359] In one embodiment, the RNAi agent contains 3, 4, 5, 6 or more duplexes represented by Formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) The duplexes are connected by linkers. A linker may or may not be cleavable. A multimer may further comprise a ligand. Each of the duplexes may target the same gene, two different genes, or each of the duplexes may target the same gene at two different target sites. There is also

[0360] In one embodiment, two RNAi agents represented by Formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) are linked to each other at their 5' ends and one or both at their 3' ends. may be conjugated to a ligand. Each of the agents may target the same gene, two different genes, or each of the agents may target the same gene at two different target sites.

[0361] Various publications describe multimeric RNAi agents that can be used in the methods of the present disclosure. Such publications include WO2007 / 091269, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520 and US7858769, the entire contents of each of which are incorporated herein by reference.

[0362] In certain embodiments, the compositions and methods of this disclosure comprise vinylphosphonate (VP) modifications of RNAi agents as described herein. In an exemplary embodiment, the 5'-vinylphosphonate of the present disclosure has the structure:

[0363] [formation] [In the formula, X is O or S; R is hydrogen, hydroxy, fluoro, or C 1~20 is alkoxy (e.g. methoxy or n-hexadecyloxy); R. 5’ =C(H)-P(O)(OH) 2 , and the C5′ carbon and R 5’ the double bond between is in the E or Z configuration (e.g., the E configuration); B is a nucleobase or modified nucleobase, where B may be adenine, guanine, cytosine, thymine, or uracil]

[0364] Vinyl phosphonates of the disclosure can be attached to either the antisense or sense strands of the dsRNAs of the disclosure. In certain embodiments, a vinyl phosphonate of the disclosure is attached to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

[0365] Vinyl phosphate modifications are also contemplated for the compositions and methods of this disclosure. Exemplary vinyl phosphate structures include the structures above, where R 5’ =C(H)-OP(O)(OH) 2 , and the C5′ carbon and R 5’ The double bond between is in the E or Z configuration (eg, E configuration).

[0366] E. Thermal destabilizing modifications In certain embodiments, thermal destabilizing modifications are incorporated into the seed region of the antisense strand (i.e., positions 2-9 at the 5' end of the antisense strand) to reduce or inhibit off-target gene silencing. dsRNA molecules can be optimized for RNA interference by that dsRNAs with antisense strands containing at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end of the antisense strand had reduced off-target gene silencing activity has been discovered. Thus, in some embodiments, the antisense strand has at least one (eg, 1, 2, 3, 4, 5 or more) two nucleotide positions within the first 9 nucleotide positions of the 5' region of the antisense strand. Includes thermally destabilizing modifications of the heavy chain. In some embodiments, the one or more duplex thermally destabilizing modifications are located in positions 2-9, or preferably in positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the duplex thermally destabilizing modification(s) is located at position 6, 7 or 8 from the 5' end of the antisense strand. In still some further embodiments, the thermal destabilizing modification of the duplex is located at position 7 from the 5' end of the antisense strand. The term "thermally destabilizing modification(s)" includes modification(s) that would result in a dsRNA having a lower overall melting temperature (Tm) (preferably such modification(s) 1, 2, 3, or 4 degrees lower than the Tm of the dsRNA without the dsRNA.In some embodiments, the thermally destabilizing modification of the duplex is at position 2 from the 5' end of the antisense strand. , 3, 4, 5 or 9.

[0367] Thermally destabilizing modifications include, but are not limited to, abasic modifications, mismatches with opposing nucleotides on opposite strands, and sugar modifications such as 2'-deoxy modifications or acyclic nucleotides such as non-locked nucleotides. Nucleic acids (UNA) or glycolic nucleic acids (GNA) may be mentioned.

[0368] Exemplary abasic modifications include, but are not limited to:

[0369] [formation] [wherein R = H, Me, Et or OMe; R' = H, Me, Et or OMe; R" = H, Me, Et or OMe]

[0370] [formation] [wherein B is a modified or unmodified nucleobase] is mentioned.

[0371] Exemplary sugar modifications include, but are not limited to:

[0372] [formation] [wherein B is a modified or unmodified nucleobase] is mentioned.

[0373] In some embodiments, the thermally destabilizing modifications of the duplex are:

[0374] [formation] [wherein B is a modified or unmodified nucleobase and each structural asterisk represents either R, S or racemic] selected from the group consisting of

[0375] The term "acyclic nucleotide" refers to, for example, any of the bonds between the ribose carbons (e.g. C1'-C2', C2'-C3', C3'-C4', C4'-O4' or C1'- O4') is absent, or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4' or O4') is absent, either independently or in combination, in the nucleotide. It refers to any nucleotide having the formula ribose sugar. In some embodiments, the acyclic nucleotide is

[0376] [formation] [Wherein, B is a modified or unmodified nucleobase; 1 and R 2 independently H, halogen, OR 3 or is alkyl; and R 3is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar]. The term "UNA" refers to an unlocked acyclic nucleic acid in which any sugar linkages have been removed, forming an unlocked "sugar" residue. In one example, UNA also includes monomers in which the bond between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) has been removed [herein incorporated by reference in their entirety. Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009)]. Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0377] The term "GNA" refers to glycol nucleic acid, a polymer similar to DNA or RNA, but differing in the composition of its "backbone" in that it consists of repeating glycerol units linked by phosphodiester bonds:

[0378] [formation]

[0379] A thermally destabilizing modification of a duplex can be a mismatch (ie, a non-complementary base pair) between a thermally destabilizing nucleotide and the opposing nucleotide in the opposing strand within the dsRNA duplex. Exemplary mismatched base pairs are G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T , U:T or combinations thereof. Other mismatch base pairings known in the art are also suitable for the present invention. Mismatches can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., mismatched base pairing originates from each nucleotide independently of the modification on the nucleotide's ribose sugar. can occur between two nucleobases. In certain embodiments, the dsRNA molecule contains at least one nucleobase in mismatch pairing that is a 2'-deoxy nucleobase, e.g., the 2'-deoxy nucleobase is in the sense strand.

[0380] In some embodiments, the duplex thermally destabilizing modification in the seed region of the antisense strand comprises nucleotides with impaired W-C H bonding with complementary bases on the target mRNA, e.g.:

[0381] [formation] including.

[0382] More examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in WO2011 / 133876, which is incorporated herein by reference in its entirety.

[0383] Thermally destabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0384] In some embodiments, the thermally destabilizing modification of the duplex includes nucleotides with non-canonical bases, such as, but not limited to, impairing the ability to form hydrogen bonds with bases in the opposite strand. nucleobase modifications that have been added or completely eliminated. These nucleobase modifications have been evaluated for destabilization of the central region of dsRNA duplexes, as described in WO2010 / 0011895, which is hereby incorporated by reference in its entirety. Exemplary nucleobase modifications include the following:

[0385] [formation]

[0386] In some embodiments, the duplex thermally destabilizing modification in the seed region of the antisense strand includes one or more α-nucleotides that are complementary to bases on the target mRNA, such as the following:

[0387] [formation] [In the formula, R is H, OH, OCH 3 , F, NH 2 , NHMe, NMe 2 or is O-alkyl] is included.

[0388] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to native phosphodiester linkages include:

[0389] [formation]

[0390] The alkyl of the R group is C 1 ~C 6 can be alkyl. Specific alkyl for R groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.

[0391] As those skilled in the art will appreciate, given that the functional role of nucleobases defines the specificity of the RNAi agents of the present disclosure, nucleobase modifications may be used, e.g., on versus off-target effects. For the purpose of enhancing the targeting effect, e.g., to introduce destabilizing modifications into the RNAi agents of the present disclosure, which can be performed in a variety of ways as described herein, available In general, the range of modifications present on the RNAi agents of the present disclosure tends to be greater for non-nucleobase modifications, such as modifications to the sugar groups or phosphate backbone of polyribonucleotides. Such modifications are described in more detail in other sections of this disclosure, either naturally occurring nucleobases or modified nucleobases, as described above or elsewhere herein. is expressly contemplated for RNAi agents of the present disclosure having

[0392] In addition to the antisense strand containing thermally destabilizing modifications, the dsRNA may also contain one or more stabilizing modifications. For example, the dsRNA can include at least two (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications. Without limitation, all stabilizing modifications may be present in one strand. In some embodiments, both the sense and antisense strands contain at least two stabilizing modifications. Stabilizing modifications can occur at any nucleotide of the sense or antisense strand. For example, a stabilizing modification can occur at any nucleotide on the sense or antisense strand; each stabilizing modification can occur in an alternating pattern on the sense or antisense strand; Both sense strands contain stabilizing modifications in an alternating pattern. The alternating pattern of stabilizing modifications on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of stabilizing modifications on the sense strand is equal to the alternating pattern of stabilizing modifications on the antisense strand. It may have a shift compared to the pattern.

[0393] In some embodiments, the antisense strand comprises at least two (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications. Without limitation, stabilizing modifications in the antisense strand can be at any position. In some embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 8, 9, 14 and 16 from the 5'end. In some other embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 14 and 16 from the 5'end. In still some other embodiments, the antisense comprises stabilizing modifications at positions 2, 14 and 16 from the 5'end.

[0394] In some embodiments, the antisense strand comprises at least one stabilizing modification flanked by destabilizing modifications. For example, the stabilizing modification can be a nucleotide at the 5' or 3' end of the destabilizing modification, i.e., position -1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5' and 3' ends of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification.

[0395] In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3' end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

[0396] In some embodiments, the sense strand comprises at least two (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications. Without limitation, stabilizing modifications in the sense strand can be at any position. In some embodiments, the sense strand contains stabilizing modifications at positions 7, 10 and 11 from the 5' end. In some other embodiments, the sense strand contains stabilizing modifications at positions 7, 9, 10 and 11 from the 5' end. In some embodiments, the sense strand contains stabilizing modifications at positions opposite or complementary to positions 11, 12 and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand has stabilizing modifications at positions opposite or complementary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5' end of the antisense strand. include. In some embodiments, the sense strand comprises blocks of 2, 3 or 4 stabilizing modifications.

[0397] In some embodiments, the sense strand does not contain stabilizing modifications at positions opposite or complementary to thermally destabilizing modifications of the duplex in the antisense strand.

[0398] Exemplary thermal stabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermal stabilizing modifications include, but are not limited to, LNA.

[0399] In some embodiments, a dsRNA of the present disclosure comprises at least four (eg, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. Without limitation, all 2'-fluoronucleotides may be present in one strand. In some embodiments both the sense and antisense strands comprise at least two 2'-fluoronucleotides. A 2'-fluoro modification can occur at any nucleotide in the sense or antisense strand. For example, a 2'-fluoro modification can occur at any nucleotide on the sense or antisense strand, each 2'-fluoro modification can occur in an alternating pattern on the sense or antisense strand, or Both the sense or antisense strands contain 2'-fluoro modifications in alternating patterns. The alternating pattern of 2'-fluoro modifications on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand can be two-fold on the antisense strand. It may have a shift compared to the alternating pattern of '-fluoro modifications.

[0400] In some embodiments, the antisense strand comprises at least two (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. Without limitation, the 2'-fluoro modification in the antisense strand can be at any position. In some embodiments, the antisense comprises 2'-fluoronucleotides at positions 2, 6, 8, 9, 14 and 16 from the 5' end. In some other embodiments, the antisense comprises 2'-fluoronucleotides at positions 2, 6, 14 and 16 from the 5' end. In still some other embodiments, the antisense comprises 2'-fluoronucleotides at positions 2, 14 and 16 from the 5' end.

[0401] In some embodiments, the antisense strand comprises at least one 2'-fluoronucleotide flanked by destabilizing modifications. For example, a 2'-fluoro nucleotide can be the nucleotide at the 5' or 3' end of the destabilizing modification, i.e., at position -1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a 2'-fluoronucleotide at each of the 5' and 3' ends of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification. .

[0402] In some embodiments, the antisense strand comprises at least two 2'-fluoronucleotides at the 3' end of the destabilizing modification, i.e., positions +1 and +2 from the position of the destabilizing modification.

[0403] In some embodiments, the sense strand comprises at least two (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. Without limitation, the 2'-fluoro modification in the sense strand can be at any position. In some embodiments, the antisense comprises 2'-fluoronucleotides at positions 7, 10 and 11 from the 5' end. In some other embodiments, the sense strand comprises 2'-fluoronucleotides at positions 7, 9, 10 and 11 from the 5' end. In some embodiments, the sense strand comprises 2'-fluoronucleotides at positions opposite or complementary to positions 11, 12 and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand has a 2'-fluoro moiety at positions opposite or complementary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5' end of the antisense strand. Contains nucleotides. In some embodiments, the sense strand comprises blocks of 2, 3 or 4 2'-fluoronucleotides.

[0404] In some embodiments, the sense strand does not contain a 2'-fluoronucleotide at the position opposite or complementary to the thermally destabilizing modification of the duplex in the antisense strand.

[0405] In some embodiments, a dsRNA molecule of the present disclosure comprises a 21 nucleotide (nt) sense strand and a 23 nucleotide (nt) antisense strand, wherein the antisense strand contains at least one thermally labile nucleotide. at least one thermally destabilizing nucleotide occurs in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand) and one end of the dsRNA is blunt; The other end comprises a 2nt overhang, and the dsRNA may further have at least one (eg, 1, 2, 3, 4, 5, 6 or all 7) of the following characteristics: (i) the antisense contains 2, 3, 4, 5 or 6 2'-fluoro modifications, (ii) the antisense contains 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages, ( iii) the sense strand is conjugated to the ligand, (iv) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications, (v) the sense strand contains 1, 2, 3, 4 or five phosphorothioate internucleotide linkages, (vi) the dsRNA contains at least four 2'-fluoro modifications, and (vii) the dsRNA contains a blunt end at the 5' end of the antisense strand. Preferably, a 2nt overhang is at the 3' end of the antisense.

[0406] In some embodiments, the dsRNA molecules of this disclosure comprise a sense and an antisense strand, the sense strand being 25-30 nucleotide residues in length and starting at the 5' terminal nucleotide (position 1). , positions 1-23 of the sense strand contain at least 8 ribonucleotides, the antisense strand is 36-66 nucleotide residues long, and starting from the 3′ terminal nucleotide, positions 1-23 of the sense strand At least 8 ribonucleotides in positions paired with ~23 form a duplex, at least the 3' terminal nucleotides of the antisense strand are not paired with the sense strand, and up to 6 consecutive 3' ends The nucleotides do not pair with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides, and the 5' end of the antisense strand is a stretch of 10-30 that does not pair with the sense strand. nucleotides thereby forming a single-stranded 5' overhang of 10-30 nucleotides, at least the 5' and 3' terminal nucleotides of the sense strand so that the sense and antisense strands are aligned for maximum complementarity. are bases paired with nucleotides of the antisense strand, thereby forming a substantially double-stranded region between the sense and antisense strands, the antisense strand being the base of the antisense strand. sufficiently complementary to the target RNA along at least 19 ribonucleotides in length to reduce target gene expression when the double-stranded nucleic acid is introduced into mammalian cells; It contains thermally labile nucleotides, and at least one thermally labile nucleotide is in the seed region of the antisense strand (ie, at positions 2-9 of the 5' end of the antisense strand). For example, the thermally labile nucleotide occurs between positions opposite or complementary to positions 14-17 on the 5' end of the sense strand, and the dsRNA has at least one of the following characteristics (e.g., 1, 2, 3, 4, 5, 6 or all 7): (i) the antisense comprises 2, 3, 4, 5 or 6 2'-fluoro modifications, (ii) The antisense comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages, (iii) the sense strand is conjugated to the ligand, (iv) the sense strand comprises 2, 3, 4 or 5 (v) the sense strand contains 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages, (vi) the dsRNA contains at least 4 2'-fluoro modifications, and (vii) the dsRNA contains a double-stranded region 12-30 nucleotide pairs in length;

[0407] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense and an antisense strand, said dsRNA molecule being a sense strand having a length that is at least 25 and at most 29 nucleotides and at most 30 nucleotides. and the sense strand comprises a modified nucleotide susceptible to enzymatic degradation from the 5' end to position 11, the 3' end of said sense strand and the 5' end of said antisense strand. The ends form blunt ends, the antisense strand is 1-4 nucleotides longer at its 3' end than the sense strand, the double-stranded region is at least 25 nucleotides long, and the antisense strand is at least 19 nucleotides. is sufficiently complementary to a target mRNA along the length of said antisense strand of to reduce target gene expression when said dsRNA molecule is introduced into a mammalian cell, and Dicer cleavage of said dsRNA results in said Preferentially resulting in an siRNA comprising the 3' end of the antisense strand, thereby reducing target gene expression in a mammal, the antisense strand containing at least one heat-labile nucleotide and at least one The two thermally labile nucleotides are in the seed region of the antisense strand (i.e., positions 2-9 at the 5' end of the antisense strand), and the dsRNA has at least one of the following characteristics (e.g., 1 , 2, 3, 4, 5, 6 or all 7): (i) the antisense comprises 2, 3, 4, 5 or 6 2'-fluoro modifications; ) the antisense contains 2, 3, 4, 5 or 6 2'-fluoro modifications, (ii) the antisense contains 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages, (iii) The sense strand is conjugated to the ligand; (iv) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4 or 5 (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA has a duplex region 12-29 nucleotide pairs in length.

[0408] In some embodiments, any nucleotide in the sense and antisense strands of the dsRNA molecule can be modified. Each nucleotide has one or more alterations of one or more of the non-linking phosphate oxygens, or one or more of the linking phosphate oxygens, or both, a component of the ribose sugar, e.g., 2' on the ribose sugar Can be modified with the same or different modifications, which can include hydroxyl changes, massive replacement of the phosphate moiety with a "dephospho" linker, modification or replacement of naturally occurring bases, and replacement or modification of the ribose-phosphate backbone. .

[0409] Since nucleic acids are polymers of subunits, many of the modifications occur at repeated positions within the nucleic acid, eg, modifications of bases or phosphate moieties or non-linking Os of phosphate moieties. In some cases, modifications occur at all positions of interest in the nucleic acid, but in many cases, they do not. By way of example, modifications may occur only at the 3' or 5' terminal positions, only in the terminal regions, e.g. may occur. Modifications may occur in double-stranded regions, single-stranded regions or both. Modifications may occur only in double-stranded regions of RNA, or may occur only in single-stranded regions of RNA. For example, phosphorothioate modifications at non-linking O-positions may occur only at one or both termini, and only in the terminal regions, e.g. It may occur at 5 or 10 nucleotides, or in double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends can be phosphorylated.

[0410] For example, to enhance stability, inclusion of specific bases in overhangs, or modified nucleotides or nucleotide substitutions in single-stranded overhangs, such as in 5′ or 3′ overhangs, or both. It may be possible to include objects. For example, it may be desirable to include purine nucleotides in overhangs. In some embodiments, all or some of the bases in the 3' or 5' overhangs can be modified, eg, with modifications described herein. Modifications include, for example, the use of modifications at the 2′ position of the ribose sugar in modifications known in the art, e.g., 2′-deoxy-2′-fluoro(2′ -F) or 2'-O-methyl modified, use of deoxyribonucleotides, and modifications in the phosphate group, such as phosphorothioate modifications. Overhangs need not be homologous to the target sequence.

[0411] In some embodiments, each residue of the sense and antisense strands is LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C- independently modified with allyl, 2'-deoxy, or 2'-fluoro. A strand may contain more than one modification. In some embodiments, each residue of the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro. It should be understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0412] At least two different modifications are usually present on the sense and antisense strands. Those two modifications can be 2'-deoxy, 2'-O-methyl or 2'-fluoro modifications, acyclic nucleotides, and the like. In some embodiments, the sense and antisense strands each comprise two differently modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue of the sense and antisense strands is a 2'-O-methyl nucleotide, 2'-deoxy nucleotide, 2'-deoxy-2'-fluoro nucleotide, 2'-O-N-methyl Acetamide (2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides or 2'- independently modified with ara-F nucleotides. Again, it should be understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0413] In some embodiments, the dsRNA molecules of the present disclosure comprise alternating patterns of modifications, particularly in the B1, B2, B3, B1', B2', B3', B4' regions. The terms "alternating motif" or "alternating pattern" as used herein refer to a motif having one or more modifications, each modification occurring at alternating nucleotides of one strand. Alternating nucleotides may refer to one every other nucleotide or one every third nucleotide or similar patterns. For example, where A, B and C each represent one type of modification to a nucleotide, the alternating motifs are "ABABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB …” or “ABCABCABCABC …” and so on.

[0414] The types of modifications contained in alternating motifs may be the same or different. For example, if A, B, C, D each represent one type of modification on a nucleotide, then the alternating pattern, ie, the modifications on every other nucleotide, may be identical, but the sense Each of the strands or antisense strands can be selected from several possibilities of modifications within alternating motifs such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0415] In some embodiments, the dsRNA molecules of this disclosure comprise a modification pattern of alternating motifs on the sense strand that is shifted relative to the modification pattern of alternating motifs on the antisense strand. The shift can be such that a modified group of nucleotides in the sense strand corresponds to a differently modified group of nucleotides in the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with "ABABAB" from 5'-3' of the strand, Alternating motifs in the antisense strand may start with "BABABA" from 3'-5' of the strands within the duplex region. As another example, the alternating motif in the sense strand may start from the 5'-3' of the strand with "AABBAABB" and the alternating motif in the antisense strand may start at the 3' of the strand within the duplex region. -5' may begin with 'BBAABBAA', resulting in a complete or partial shift in the modification pattern between the sense and antisense strands.

[0416] The dsRNA molecules of this disclosure can further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. A phosphorothioate or methylphosphonate internucleotide linkage modification can occur at any nucleotide in the sense or antisense strand or both at any position on the strand. For example, an internucleotide linkage modification can occur at any nucleotide on the sense or antisense strand, each internucleotide linkage modification can occur in an alternating pattern on the sense or antisense strand, or The antisense strand contains both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same as or different from the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may be the same as the internucleotide linkage modifications on the antisense strand. It may have a shift to the alternating pattern of linking modifications.

[0417] In some embodiments, the dsRNA molecules include phosphorothioate or methylphosphonate internucleotide linkage modifications in the overhang regions. For example, the overhang region comprises two nucleotides with a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications can also be made to link overhanging nucleotides with terminal paired nucleotides within the duplex region. For example, at least 2, 3, 4 or all overhanging nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkages, and additional phosphorothioate or methyl There may be phosphonate internucleotide linkages. For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhanging nucleotides and the third is the paired nucleotide adjacent to the overhanging nucleotide. Preferably, these terminal 3 nucleotides can be the 3' end of the antisense strand.

[0418] In some embodiments, the sense strand of the dsRNA molecule has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate internucleotide comprising 1-10 blocks of 2-10 phosphorothioate or methylphosphonate internucleotide linkages separated by linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being placed at any position in the oligonucleotide sequence, said The sense strand is paired with an antisense strand containing any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages, or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0419] In some embodiments, the antisense strand of the dsRNA molecule is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being placed at any position in the oligonucleotide sequence , the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages, and an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0420] In some embodiments, the antisense strand of the dsRNA molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 nucleotide phosphates comprising two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by interlinkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being placed at any position in the oligonucleotide sequence; The strands are paired with a sense strand containing any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages, or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0421] In some embodiments, the antisense strand of the dsRNA molecule is separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages. comprising two blocks of four phosphorothioate or methylphosphonate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being placed at any position in the oligonucleotide sequence, the antisense strand comprising phosphorothioate , with a sense strand containing any combination of methylphosphonate and phosphate internucleotide linkages, or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0422] In some embodiments, the antisense strand of the dsRNA molecule is separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphate internucleotide linkages of 5 comprising two blocks of phosphorothioate or methylphosphonate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being placed at any position in the oligonucleotide sequence; Paired with a sense strand containing any combination of phosphate internucleotide linkages, or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0423] In some embodiments, the antisense strand of the dsRNA molecule comprises six phosphorothioate or methylphosphonates separated by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate internucleotide linkages. comprising two blocks of internucleotide linkages, one of which is a phosphorothioate or methylphosphonate internucleotide linkage located anywhere in the oligonucleotide sequence; Paired with a sense strand containing any combination of linkages, or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0424] In some embodiments, the antisense strand of the dsRNA molecule is composed of 7 phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages. comprising two blocks, one of phosphorothioate or methylphosphonate internucleotide linkages positioned at any position in the oligonucleotide sequence; Paired with a sense strand containing the combination, or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0425] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages. wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the antisense strand comprises any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages in a sense strands, or antisense strands containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0426] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3 or 4 phosphate internucleotide linkages, phosphorothioate or one of the methylphosphonate internucleotide linkages is positioned anywhere in the oligonucleotide sequence, the antisense strand with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages; or It is paired with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0427] In some embodiments, the dsRNA molecules of this disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within one to ten terminal positions of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkages at either or both ends of the sense or antisense strand.

[0428] In some embodiments, the dsRNA molecules of this disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1-10 of the internal region of each duplex of the sense or antisense strand. . For example, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides are linked by phosphorothioate methylphosphonate internucleotide linkages at positions 8-16 of the duplex region counting from the 5' end of the sense strand. can be The dsRNA molecule may further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within one to ten terminal positions.

[0429] In some embodiments, the dsRNA molecules of this disclosure have 1-5 phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 of the sense strand and 1-5 phosphorothioate or methylphosphonate modifications within positions 18-23 of the sense strand. Internucleotide linkage modifications (counting from the 5' end) and 1-5 phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and 1-5 within positions 18-23 (counting from the 5' end). counting).

[0430] In some embodiments, the dsRNA molecules of this disclosure have one phosphorothioate internucleotide linkage modification within positions 1-5 of the sense strand and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 18-23 (5' counting from the end) and one phosphorothioate internucleotide linkage modification at positions 1 and 2 of the antisense strand and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 (counting from the 5' end). .

[0431] In some embodiments, the dsRNA molecules of this disclosure have two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand. ) and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0432] In some embodiments, the dsRNA molecules of this disclosure have two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the sense strand. ) and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0433] In some embodiments, the dsRNA molecules of this disclosure have two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the sense strand. ) and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0434] In some embodiments, the dsRNA molecules of this disclosure have one phosphorothioate internucleotide linkage modification within positions 1-5 of the sense strand and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand. ) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0435] In some embodiments, the dsRNA molecules of this disclosure have one phosphorothioate internucleotide linkage modification within positions 1-5 and one within positions 18-23 (counting from the 5' end) of the sense strand and the antisense strand. It further contains two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the strand and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the strand.

[0436] In some embodiments, the dsRNA molecules of this disclosure have one phosphorothioate internucleotide linkage modification within positions 1-5 of the sense strand (counting from the 5' end) and two modifications at positions 1 and 2 of the antisense strand. Further includes a phosphorothioate internucleotide linkage modification and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end).

[0437] In some embodiments, the dsRNA molecules of this disclosure have two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end) and one at positions 1 and 2 of the antisense strand. Further includes a phosphorothioate internucleotide linkage modification and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end).

[0438] In some embodiments, the dsRNA molecules of this disclosure have two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand and one within positions 18-23 (counting from the 5' end) and the antisense strand. It further contains two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the strand and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the strand.

[0439] In some embodiments, the dsRNA molecules of this disclosure have two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand. ) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0440] In some embodiments, the dsRNA molecules of this disclosure have two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand. ) and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0441] In some embodiments, the dsRNA molecules of this disclosure have two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the sense strand. and one phosphorothioate internucleotide linkage modification at position 1 and one at position 21 (counting from the 5' end) of the antisense strand.

[0442] In some embodiments, the dsRNA molecules of this disclosure have one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand and the antisense strand. further includes two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end).

[0443] In some embodiments, the dsRNA molecules of this disclosure have two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the sense strand. and further comprising one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0444] In some embodiments, the dsRNA molecules of this disclosure have one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand and the antisense strand. further includes two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end).

[0445] In some embodiments, the dsRNA molecules of this disclosure have two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 (counting from the 5' end) of the sense strand. and further comprising one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0446] In some embodiments, the dsRNA molecules of this disclosure have one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand and the antisense strand. further includes two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 (counting from the 5' end).

[0447] In some embodiments, the compounds of this disclosure contain a pattern of backbone chiral centers. In some embodiments, the general pattern of backbone chiral centers comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers includes at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers includes at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers includes at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers includes at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers includes at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers includes at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers includes at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers includes at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises no more than 8 internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises no more than 7 internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers includes no more than 6 internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises no more than 5 internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers includes no more than 4 internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises no more than 3 internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers includes no more than two internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers includes no more than one internucleotide linkage in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises 8 or fewer non-chiral internucleotide linkages (phosphodiesters as a non-limiting example). In some embodiments, the general pattern of backbone chiral centers includes no more than 7 non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers includes no more than 6 non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers includes no more than 5 non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers includes no more than 4 non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers includes no more than three non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers includes no more than two non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers includes no more than one non-chiral internucleotide linkage. In some embodiments, the general pattern of backbone chiral centers includes at least 10 internucleotide linkages and no more than 8 non-chiral internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers includes at least 11 internucleotide linkages and no more than 7 non-chiral internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers includes at least 12 internucleotide linkages and no more than 6 non-chiral internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers includes at least 13 internucleotide linkages and no more than 6 non-chiral internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers includes at least 14 internucleotide linkages and no more than 5 non-chiral internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers includes at least 15 internucleotide linkages and no more than 4 non-chiral internucleotide linkages in the Sp configuration. In some embodiments, the internucleotide linkages in the Sp configuration may or may not be contiguous. In some embodiments, the internucleotide linkages in the Rp configuration may or may not be continuous. In some embodiments, non-chiral internucleotide linkages may or may not be continuous.

[0448] In some embodiments, the compounds of the present disclosure contain blocks that are stereochemical blocks. In some embodiments, the block is an Rp block in that each internucleotide linkage of the block is Rp. In some embodiments, the 5'-block is the Rp block. In some embodiments, the 3'-block is the Rp block. In some embodiments, the block is an Sp block in that each internucleotide linkage of the block is Sp. In some embodiments, the 5'-block is the Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, provided oligonucleotides include both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp, but no Sp block. In some embodiments, provided oligonucleotides contain one or more Sp but do not contain an Rp block. In some embodiments, provided oligonucleotides comprise one or more PO blocks where each internucleotide linkage is a natural phosphate linkage.

[0449] In some embodiments, the compounds of the present disclosure contain a 5'-block where each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety contains a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a phosphorothioate linkage and each sugar moiety contains a 2'-F modification. In some embodiments, the 5'-block comprises 4 or more nucleoside units. In some embodiments, the 5'-block comprises 5 or more nucleoside units. In some embodiments, the 5'-block comprises 6 or more nucleoside units. In some embodiments, the 5'-block comprises 7 or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety contains a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety contains a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each internucleotide linkage is a phosphorothioate linkage and each sugar moiety contains a 2'-F modification. In some embodiments, the 3'-block comprises 4 or more nucleoside units. In some embodiments, the 3'-block comprises 5 or more nucleoside units. In some embodiments, the 3'-block comprises 6 or more nucleoside units. In some embodiments, the 3'-block comprises 7 or more nucleoside units.

[0450] In some embodiments, the compounds of the present disclosure contain certain types of nucleosides in the region or have specific types of internucleotide linkages in the oligonucleotide, e.g., natural phosphate linkages, modified internucleotide linkages. , Rp chiral internucleotide linkages, Sp chiral internucleotide linkages, and so on. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0451] In some embodiments, the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, and the antisense strand is attached to the seed region of the antisense strand (i.e., containing at least one thermally destabilizing modification of the duplex located at positions 2-9 of the 5' end of the antisense strand, the dsRNA exhibits at least one (e.g., 1, 2) of the following characteristics: , 3, 4, 5, 6, 7 or all 8): (i) the antisense comprises 2, 3, 4, 5 or 6 2'-fluoro modifications; (ii ) the antisense contains 3, 4 or 5 phosphorothioate internucleotide linkages, (iii) the sense strand is conjugated to the ligand, (iv) the sense strand has 2, 3, 4 or 5 2'- (v) the sense strand contains 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages, (vi) the dsRNA contains at least 4 2'-fluoro modifications, (vii) the dsRNA contains a double-stranded region 12-40 nucleotide pairs in length, and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0452] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23. , the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one of the following characteristics (e.g. 1, 2, 3, 4, 5, 6, 7 or all 8): (i) the antisense is 2, 3, contains 4, 5 or 6 2'-fluoro modifications, (ii) the sense strand is conjugated to the ligand, (iii) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications (iv) the sense strand contains 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages, (v) the dsRNA contains at least four 2'-fluoro modifications, (vi) the dsRNA contains 12- (vii) the dsRNA contains a double-stranded region 12-40 nucleotide pairs long; and (viii) the dsRNA contains the 5' end of the antisense strand. have blunt ends.

[0453] In some embodiments, the sense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and the antisense strand is in the seed region of the antisense strand (i.e., anti containing at least one thermally destabilizing modification of the duplex located at positions 2-9 of the 5' end of the sense strand, the dsRNA exhibits at least one of the following characteristics (e.g., 1, 2, 3, 4, 5, 6, 7 or all 8): (i) the antisense comprises 2, 3, 4, 5 or 6 2'-fluoro modifications, (ii) The antisense comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages, (iii) the sense strand is conjugated to the ligand, (iv) the sense strand comprises 2, 3, 4 or 5 (v) the sense strand contains 3, 4 or 5 phosphorothioate internucleotide linkages, (vi) the dsRNA contains at least 4 2'-fluoro modifications, (vii) the dsRNA contains , contains a double-stranded region 12-40 nucleotide pairs in length, and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0454] In some embodiments, the sense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand comprises between nucleotide positions 1 and 2 and between nucleotide positions 2 and 2. and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, the antisense strand is attached to the seed region of the antisense strand (i.e., the 5' end of the antisense strand). containing at least one thermally destabilizing modification of the duplex located at positions 2-9 of the dsRNA, and the dsRNA has at least one of the following characteristics (e.g., 1, 2, 3, 4, 5, 6 or all 7): (i) the antisense contains 2, 3, 4, 5 or 6 2'-fluoro modifications, (ii) the sense strand is conjugated to the ligand (iii) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications, (iv) the sense strand contains 3, 4 or 5 phosphorothioate internucleotide linkages, (v) the dsRNA contains at least four 2'-fluoro modifications, (vi) the dsRNA contains a duplex region 12-40 nucleotide pairs in length, and (vii) the dsRNA contains at the 5' end of the antisense strand It has blunt ends.

[0455] In some embodiments, the dsRNA molecules of this disclosure contain mismatch(es) or a combination thereof within the duplex with the target. Mismatches can occur in overhang regions or duplex regions. Base pairs can be ranked based on their propensity to promote dissociation or melting (e.g., the free energy of association or dissociation for a particular pairing; the simplest approach is to examine the pairs on the basis of individual pairs). but the next neighbor analysis or similar analysis could also be used). In terms of promotion of dissociation: A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical pairings or anything other than canonical pairings (as described elsewhere herein), canonical (A:T, A:U, G:C) Preferred over formation, pairing involving universal bases is preferred over canonical pairing.

[0456] In some embodiments, the dsRNA molecules of the present disclosure comprise A:U, G:U, I:C and mismatched pairs, e.g. , non-canonical pairing or non-canonical pairing, or the first 1, 2 in the duplex region from the 5' end of the antisense strand, which may be independently selected from the pairing group comprising universal bases, Contains at least one of 3, 4 or 5 base pairs.

[0457] In some embodiments, the nucleotide at position 1 within the duplex region from the 5' end in the antisense strand is selected from the group consisting of A, dA, dU, U and dT. Alternatively, at least one of the first 1, 2 or 3 base pairs in the duplex region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the duplex region from the 5' end of the antisense strand is an AU base pair.

[0458] 4'-modified or It has been found that introducing 5'-modified nucleotides can exert steric effects on the internucleotide linkage, thus protecting it from nucleases and stabilizing it.

[0459] In some embodiments, a 5'-modified nucleoside is introduced at the 3' end of the dinucleotide at any position in the single- or double-stranded siRNA. For example, a 5'-alkylated nucleoside can be introduced at the 3' end of a dinucleotide at any position in a single- or double-stranded siRNA. The alkyl group at the 5' position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 5'-alkylated nucleoside is a 5'-methyl nucleoside. The 5'-methyl can be either racemic or chirally pure R or S isomer.

[0460] In some embodiments, a 4'-modified nucleoside is introduced at the 3' end of the dinucleotide at any position in the single- or double-stranded siRNA. For example, a 4'-alkylated nucleoside can be introduced at the 3' end of a dinucleotide at any position in a single- or double-stranded siRNA. The alkyl group at the 4' position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside. The 4'-methyl can be either racemic or chirally pure R or S isomer. Alternatively, a 4'-O-alkylated nucleoside can be introduced at the 3' end of the dinucleotide at any position in the single- or double-stranded siRNA. The 4'-O-alkyl of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is a 4'-O-methyl nucleoside. 4'-O-methyl can be either racemic or chirally pure R or S isomer.

[0461] In some embodiments, 5'-alkylated nucleosides are introduced at any position of the sense or antisense strand of the dsRNA, and such modifications maintain or improve the potency of the dsRNA. The 5'-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 5'-alkylated nucleoside is a 5'-methyl nucleoside. The 5'-methyl can be either racemic or chirally pure R or S isomer.

[0462] In some embodiments, 4'-alkylated nucleosides are introduced at any position of the sense or antisense strand of the dsRNA, and such modifications maintain or improve the potency of the dsRNA. The 4'-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is a 4'-methyl nucleoside. The 4'-methyl can be either racemic or chirally pure R or S isomer.

[0463] In some embodiments, 4'-O-alkylated nucleosides are introduced at any position of the sense or antisense strand of the dsRNA and such modifications maintain or improve the potency of the dsRNA. The 5'-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is a 4'-O-methyl nucleoside. 4'-O-methyl can be either racemic or chirally pure R or S isomer.

[0464] In some embodiments, the dsRNA molecules of the present disclosure have 2'-5' linkages (having 2'-H, 2'-OH and 2'-OMe with P=O or P=S). can contain. For example, 2'-5' linkage modifications can be used to promote nuclease resistance, or to inhibit sense binding to the antisense strand, or to avoid sense strand activation by RISC. Available at the 5' end.

[0465] In another embodiment, the dsRNA molecules of the present disclosure may include L-sugars (eg, L-ribose, L-arabinose with 2'-H, 2'-OH and 2'-OMe). For example, these L sugar modifications can be used to promote nuclease resistance, or to inhibit sense binding to the antisense strand, or to avoid sense strand activation by RISC. available on the edge.

[0466] Various publications describe multimeric siRNAs, all of which can be used with the dsRNAs of the present disclosure. Such publications include WO2007 / 091269, US7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, which are incorporated herein in their entirety.

[0467] As described in more detail below, RNAi agents containing conjugation of one or more carbohydrate moieties to the RNAi agent can optimize one or more properties of the RNAi agent. Carbohydrate moieties are often attached to the modified subunits of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of the dsRNA agent can be replaced with another moiety, eg, a non-carbohydrate (preferably cyclic) carrier to which the carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit is so replaced is referred to herein as a ribose replacement modified subunit (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e. all ring atoms are carbon atoms, or a heterocyclic ring structure, i.e. one or more ring atoms are heteroatoms such as nitrogen , oxygen, sulfur. Cyclic carriers can be monocyclic ring structures or can contain more than one ring, eg, fused rings. Cyclic carriers can be fully saturated ring structures or can contain one or more double bonds.

[0468] A ligand can be attached to a polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point", preferably two "backbone attachment points" and (ii) at least one "tethering point". A "backbone attachment point", as used herein, is a functional group, such as a hydroxyl group, or, in general, a backbone, such as a phosphate or a modified phosphate, such as sulfur, of a ribonucleic acid. Refers to linkages available and suitable for incorporation of the carrier into the containing scaffold. A “tethered point of attachment” (TAP) is, in some embodiments, a constituent ring atom of a cyclic carrier that connects selected moieties, e.g. separate). Moieties can be, for example, carbohydrates such as monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides and polysaccharides. The selected moieties may be connected to the cyclic carrier by intervening tethers. Thus, the cyclic carrier may contain a functional group, such as an amino group, or generally provide a bond suitable for incorporation or tethering of another chemical entity, such as a ligand, to the constituent ring. There will be many.

[0469] RNAi agents may be conjugated to ligands via carriers, which may be cyclic or acyclic groups, preferably cyclic groups are pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl , piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin, preferably the acyclic group is It is selected from a serinol skeleton or a diethanolamine skeleton.

[0470] In certain specific embodiments, an RNAi agent for use in the methods of the disclosure is an agent selected from the group of agents listed in any one of Tables 2-5, and 7-10. . These agents may further comprise a ligand, eg, one or more lipophilic moieties, one or more GalNAc derivatives, or both one or more lipophilic moieties and one or more GalNAc derivatives.

[0471] IV. iRNA Conjugated to Ligand Another modification of the RNA of the iRNA of the present invention is to chemically link the iRNA with one or more ligands, moieties or conjugates that enhance the iRNA's activity, cellular distribution or, for example, cellular uptake into cells. include. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309). Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), fatty chains, For example, dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie 1993, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783); 969-973) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237) or octadecyl Amine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0472] In certain embodiments, the ligand alters the distribution, targeting or longevity of the iRNA agent into which it is incorporated. In some embodiments, the ligand is a selected target, e.g., a molecule, cell or cell type, compartment, e.g., of a cell or of an organ, e.g., compared to a species in which such ligand does not exist. Provides enhanced affinity for a compartment, tissue, organ or region of the body. Ordinary ligands do not participate in duplex pairing in double-stranded nucleic acids.

[0473] As ligands, naturally occurring substances such as proteins (e.g. human serum albumin (HSA), low density lipoprotein (LDL) or globulin), carbohydrates (e.g. dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid) or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer. Coalescing, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid) (ethylacryllic acid)), N-isopropylacrylamide polymer or polyphosphazine. Examples of polyamines include polyethylenimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, polyamine quaternaries. grade salts or alpha-helical peptides.

[0474] Ligands can also include targeting groups, such as cell or tissue targeting agents such as lectins, glycoproteins, lipids or proteins, such as antibodies that bind to specific cell types, such as glial cells. Targeting groups include thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose , polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol steroids, bile acids, folic acid, vitamin B12, biotin or RGD peptides or RGD peptide mimetics. . In certain embodiments, the ligand is a polyvalent galactose, such as N-acetyl-galactosamine.

[0475] Other examples of ligands include dyes, intercalating agents (e.g. acridine), cross-linking agents (e.g. psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sappphyrin), polycyclic aromatic hydrocarbons ( phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol , geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g. antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g. PEG-40K), MPEG, [MPEG] 2 , polyaminos, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g. biotin), transport / absorption enhancers (e.g. aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g. imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.

[0476] The ligand can be a protein, eg, a glycoprotein or peptide, eg, a molecule or an antibody, eg, an antibody that has a specific affinity for the co-ligand, eg, an antibody that binds to a particular cell type such as brain cells or glial cells. Ligands can also include hormones and hormone receptors. They also include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polymannose or polyvalent fucose. can be A ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase or an activator of NF-κB.

[0477] The ligand is a substance, e.g., a drug, that can increase cellular uptake of an iRNA agent, e.g., by disrupting the cytoskeleton of the cell, e.g., by disrupting the microtubules, microfilaments or intermediate fibers of the cell. could be. The drug can be, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanosine or myocerbin.

[0478] In some embodiments, ligands attached to iRNAs as described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogues, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins and thus short oligonucleotides, e.g. Oligonucleotides of 20 bases are also suitable for the present invention as ligands (eg, as PK modulating ligands). Additionally, aptamers that bind serum components (eg, serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

[0479] Ligand-conjugated iRNAs of the invention can be synthesized through the use of oligonucleotides (described below) derived from, for example, the attachment of linking molecules onto oligonucleotides having pendant reactive functionalities. This reactive oligonucleotide can be reacted directly with commercially available ligands, synthesized ligands with any of a variety of protecting groups, or ligands with linking moieties attached thereto.

[0480] Oligonucleotides used in the conjugates of the invention can be conveniently and routinely made by the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems® (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be used. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioate and alkylated derivatives.

[0481] For ligand-conjugated oligonucleotides and molecules having ligand-sequence specific linked nucleosides of the invention, oligonucleotides and oligonucleosides can be either standard nucleotide or nucleoside precursors or nucleotide or nucleoside conjugates already having a linking moiety. Gate precursors, ligand-nucleotide or nucleoside conjugate precursors already having ligand molecules or non-nucleoside ligands having building blocks can be utilized and assembled in a suitable DNA synthesizer.

[0482] When using a nucleotide-conjugate precursor that already has a linking moiety, the synthesis of the sequence-specific linked nucleosides is usually completed, then the ligand molecule is reacted with the linking moiety to conjugate the ligand. An oligonucleotide is formed. In some embodiments, the oligonucleotides or linked nucleosides of the invention are commercially available and can be used in ligand-nucleoside conjugates in addition to standard and non-standard phosphoramidites routinely used in oligonucleotide synthesis. Synthesized by an automated synthesizer using the phosphoramidites from which it was derived.

[0483] A. Lipid conjugate In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such lipids or lipid-based molecules are typically capable of binding serum proteins, such as human serum albumin (HSA). HSA-binding ligands allow distribution of the conjugate to target tissues of the body, eg, non-kidney target tissues. For example, the target tissue can be the liver, which contains liver parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipids or lipid-based ligands can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into target cells or cell membranes, or (c) to serum proteins such as HSA. can adjust the binding of

[0484] Lipid-based ligands can be used to modulate, eg, control (eg, inhibit) binding of the conjugate to the target tissue. For example, a lipid or lipid-based ligand that binds HSA more strongly is less likely to be targeted to the kidney and thus less likely to be cleared from the body. A lipid or lipid-based ligand that binds HSA less strongly can be used to target the conjugate to the kidney.

[0485] In certain embodiments, the lipid-based ligand binds HSA. For example, the ligand can bind to HSA with sufficient affinity, resulting in enhanced distribution of the conjugate to non-kidney tissues. However, the affinity is usually not so strong that HSA-ligand binding cannot be reversed.

[0486] In certain embodiments, the lipid-based ligand binds HSA weakly or not at all, resulting in enhanced distribution of the conjugate to the kidney. Other moieties that target kidney cells can also be used in place of, or in addition to, lipid-based ligands.

[0487] In certain embodiments, the lipid-based ligand binds HSA weakly or not at all, resulting in enhanced distribution of the conjugate to the kidney. Other moieties that target kidney cells can also be used in place of, or in addition to, lipid-based ligands.

[0488] In another aspect, the ligand is a moiety, eg, a vitamin, that is taken up by target cells, eg, proliferating cells. They are particularly useful, for example, for the treatment of disorders characterized by unwanted cell proliferation of malignant or non-malignant species, such as cancer cells. Exemplary vitamins include vitamins A, E and K. Other exemplary vitamins include B vitamins such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

[0489] B. Cell permeabilization agent In another aspect, the ligand is a cell permeation agent, eg, a helical cell permeation agent. In certain embodiments, these cell-penetrating agents are amphipathic. Exemplary cell penetrating agents include peptides such as tat or antennopedia. If the agent is a peptide, it may be modified, including the use of peptidyl mimetics, reverse isomers, non-peptide or pseudopeptide linkages and D-amino acids. A helical agent is usually an α-helical agent and can have a lipophilic and a lipophobic phase.

[0490] A ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptide mimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and uptake. A peptide or peptidomimetic portion can be about 5-50 amino acids long, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids long.

[0491] A peptide or peptidomimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphiphilic peptide or a hydrophobic peptide (eg consisting mainly of Tyr, Trp or Phe). Peptide moieties can be dendrimer peptides, constrained peptides or bridging peptides. In another alternative, the peptide portion may contain a hydrophobic membrane translocating sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 11). RFGF analogues containing a hydrophobic MTS [eg amino acid sequence AALLPVLLAAP (SEQ ID NO: 12)] can also be targeting moieties. The peptide moiety can be a "delivery" peptide capable of carrying large polar molecules including peptides, oligonucleotides and proteins across cell membranes. For example, a sequence from the HIV Tat protein [GRKKRRQRRRPPQ (SEQ ID NO: 13)] and a sequence from the Drosophila Antennapedia protein [RQIKIWFQNRRMKWKK (SEQ ID NO: 14)] have been found to be functionalizable as delivery peptides. . Peptides or peptidomimetics are DNA, such as peptides identified from phage display libraries or 1-bead-1-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). can be encoded by a random sequence of Peptides or peptidomimetics that are commonly tethered to dsRNA agents via incorporated monomeric units include cell-targeting peptides, such as arginine-glycine-aspartic acid (RGD)-peptides or RGD mimetics. Peptide portions can range from about 5 amino acids to about 40 amino acids in length. The peptide portion may have structural modifications that, for example, increase stability or direct conformational properties. Any of the structural modifications described below can be used.

[0492] RGD peptides for use in the compositions and methods of the invention may be linear or cyclic, modified to facilitate targeting to a particular tissue(s). For example, it may be glycosylated or methylated. RGD-containing peptides and peptidiomimemtics can include D-amino acids as well as synthetic RGD mimetics. In addition to RGD, other moieties that target integrin ligands can be used. Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0493] RGD peptide moieties can be used to target specific cell types, such as tumor cells, such as endothelial or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). . RGD peptides can facilitate targeting of dsRNA agents to tumors in a variety of other tissues, including lung, kidney, spleen or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). In general, RGD peptides facilitate targeting of iRNA agents to the kidney. RGD peptides may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to specific tissue(s). . For example, a glycosylated RGD peptide can convert an iRNA agent to α V β 3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0494] A "cell penetrating peptide" is capable of penetrating cells, eg, microbial cells, eg, bacterial or fungal cells, or mammalian cells, eg, human cells. Microbial cell-penetrating peptides are, for example, α-helical linear peptides (eg LL-37 or Seropin P1), disulfide bond-containing peptides (eg α-defensins, β-defensins or bactenesins) or one or two dominant It can be a peptide containing only amino acids (eg, PR-39 or indolicidin). A cell penetrating peptide may also contain a nuclear localization signal (NLS). For example, the cell penetrating peptide can be a bisected amphiphilic peptide such as MPG derived from, for example, the fusion peptide domain of the NLS of HIV-1 gp41 and SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31 : 2717-2724, 2003).

[0495] C. Carbohydrate Conjugates In some embodiments of the compositions and methods of this invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo delivery of nucleic acids as described herein as well as compositions suitable for in vivo therapeutic use. As used herein, a "carbohydrate" is one having at least 6 carbon atoms (which can be linear, branched or cyclic) with oxygen, nitrogen or sulfur atoms attached to each carbon atom. or as a carbohydrate itself or part thereof, which is composed of multiple monosaccharide units, each of at least 6 carbon atoms (which can be linear, branched or cyclic), with oxygen, nitrogen or sulfur atoms attached to each carbon atom. ), which is any compound having a carbohydrate moiety made up of one or more monosaccharide units. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides and oligosaccharides containing about 4, 5, 6, 7, 8 or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose and polysaccharide gums. Particular monosaccharides include C5 and the above (e.g. C5, C6, C7 or C8) sugars, disaccharides and trisaccharides are sugars with two or three monosaccharide units (e.g. C5, C6, C7 or C8).

[0496] In certain embodiments the carbohydrate conjugate comprises a monosaccharide.

[0497] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in US 8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, GalNAc conjugates act as ligands to target iRNAs to specific cells. In some embodiments, GalNAc conjugates target iRNAs to liver cells, eg, by serving as ligands for asialoglycoprotein receptors on liver cells (eg, hepatocytes).

[0498] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. A GalNAc derivative can be attached via a linker, eg, a bivalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (eg, at the 3' end of the sense strand) via a linker, eg, a linker as described herein. In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (eg, at the 5' end of the sense strand) via a linker, eg, a linker as described herein.

[0499] In certain embodiments of the invention, a GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, a GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In still other embodiments of the invention, a GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker. In other embodiments of the invention, a GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a tetravalent linker.

[0500] In certain embodiments, double-stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent. In certain embodiments, a double-stranded RNAi agent of the invention has a plurality (e.g., 2, 3) each independently attached to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers. , 4, 5 or 6) GalNAc or GalNAc derivatives.

[0501] In some embodiments, for example, two strands of an iRNA agent of the invention form a hairpin loop comprising multiple unpaired nucleotides, the 3′ end of one strand and the The unpaired nucleotides in the hairpin loop are each attached via a monovalent linker, GalNAc or A GalNAc derivative may be included independently. A hairpin loop may also be formed by an extended overhang on one strand of the duplex.

[0502] In some embodiments, for example, two strands of an iRNA agent of the invention form a hairpin loop comprising multiple unpaired nucleotides, the 3′ end of one strand and the The unpaired nucleotides in the hairpin loop are each attached via a monovalent linker, GalNAc or A GalNAc derivative may be included independently. A hairpin loop may also be formed by an extended overhang on one strand of the duplex.

[0503] In some embodiments, the GalNAc conjugate is

[0504] [formation] is.

[0505] In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the schematic below, where X is O or S.

[0506] [formation]

[0507] In some embodiments, the RNAi agent is defined in Table 1 and conjugated to L96 as shown below:

[0508] [formation]

[0509] In certain embodiments, carbohydrate conjugates for use in the compositions and methods of the invention are selected from the group consisting of:

[0510] [formation] [formation] [formation] [formation] [formation] [formation] [formation] [formation]

[0511] In certain embodiments, carbohydrate conjugates for use in the compositions and methods of the invention are monosaccharides. In certain embodiments, the monosaccharide is N-acetylgalactosamine,

[0512] [formation] is.

[0513] Other exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to:

[0514] [formation] [wherein one of X or Y is an oligonucleotide and the other is hydrogen] is mentioned.

[0515] In some embodiments, suitable ligands are those disclosed in WO2019 / 055633, the entire contents of which are incorporated herein by reference. In one embodiment, the ligand has the following structure:

[0516] [formation] including.

[0517] In certain embodiments, the RNAi agents of the present disclosure are GalNAc ligands such GalNAc ligands are of limited value for the presently preferred intrathecal / CNS delivery route(s) of the present disclosure. It can be included even if it is predicted that

[0518] In certain embodiments of the invention, a GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, a GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In still other embodiments of the invention, a GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker. In other embodiments of the invention, a GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a tetravalent linker.

[0519] In certain embodiments, the double-stranded RNAi agent of the invention is the 5' end of the sense strand of an iRNA agent, e.g., a dsRNA agent, or one of the dual-targeting RNAi agents described herein or It contains one GalNAc or GalNAc derivative attached to the 5' ends of both sense strands. In certain embodiments, a double-stranded RNAi agent of the invention comprises a plurality of (e.g., 2, 3, 4, 5 or 6) GalNAc or GalNAc derivatives.

[0520] In some embodiments, for example, two strands of an iRNA agent of the invention form a hairpin loop comprising multiple unpaired nucleotides, the 3′ end of one strand and the The unpaired nucleotides in the hairpin loop are each attached via a monovalent linker, GalNAc or A GalNAc derivative may be included independently.

[0521] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, PK modulators or cell penetrating peptides.

[0522] Additional carbohydrate conjugates and linkers suitable for use in the present invention include those described in WO2014 / 179620 and WO2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0523] D. Linker In some embodiments, the conjugates or ligands described herein can be attached to iRNA oligonucleotides using a variety of linkers that may or may not be cleavable.

[0524] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, eg, covalently attaches two parts of a compound. Linkers are usually direct bonds or atoms such as oxygen or sulfur, NR8, C(O), C(O)NH, SO, SO 2 , SO 2 Units such as NH or, but not limited to, one or more methylenes are O, S, S(O), SO 2 , N(R8), C(O), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkyl, which may be interrupted or terminated by C(O) arylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl , alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, substituted or unsubstituted aryl, substituted or unsubstituted, wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic Including chains of atoms such as heteroaryl, substituted or unsubstituted heterocyclic. In certain embodiments, the linker is about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16 or 8-16 atoms.

[0525] A cleavable linking group is one that is sufficiently stable outside the cell, but once inside the target cell it is cleaved to release the two moieties held together by the linker. In preferred embodiments, the cleavable linking group is in the subject's blood or in target cells rather than under a second reference condition (which may be selected to mimic or represent conditions found in blood or serum), or at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, under a first reference condition (e.g., can be selected to mimic or represent intracellular conditions); Cuts 90 times or more, or at least about 100 times faster.

[0526] Cleavable linking groups are susceptible to the presence of cleaving agents such as pH, redox potential or degradable molecules. In general, cleaving agents are more prevalent or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents that are selected for a particular substrate or have no substrate specificity, e.g. Reducing agents such as mercaptans, esterases, endosomes, or agents that can create an acidic environment, such as those that result in a pH of 5 or less, common acids, peptidases (which can be substrate-specific), present in cells that can degrade groups, such as and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as phosphatases.

[0527] Cleavable linking groups, such as disulfide bonds, can be pH sensitive. Human serum has a pH of 7.4, but the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5-6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell into the desired compartment of the cell.

[0528] A linker may comprise a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into the linker can vary depending on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid via a linker containing an ester group. Liver cells are rich in esterases, therefore linkers are cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortical and testicular cells.

[0529] Linkers containing peptide bonds can be used when targeting peptidase-rich cell types such as liver cells and synoviocytes.

[0530] In general, the suitability of a candidate cleavable linking group can be assessed by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test candidate cleavable linking groups for their ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, one can determine the relative susceptibility to cleavage between a first and second condition, the first selected to exhibit cleavage in the target cell and the second selected for other tissues or biology. are selected to exhibit cleavage in biological fluids such as blood or serum. Assessment can be performed in cell-free systems, in cells, in cell culture, in organs or in tissue culture, or in whole animals. It may be helpful to perform an initial evaluation in cell-free or culture conditions and confirm by further evaluation in whole animals. In preferred embodiments, useful candidate compounds are tested in cells (or selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extra...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of APOE, or a pharma- ceutically acceptable salt thereof, comprising: a) wherein said dsRNA agent or a pharma- ceutically acceptable salt thereof comprises a sense strand and an antisense strand which form a double-stranded region; the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the complement of nucleotides 57-84 of SEQ ID NO:1; and the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties that are conjugated to one or more internal positions in at least one strand; b) wherein said dsRNA agent or a pharma- ceutically acceptable salt thereof comprises a sense strand and an antisense strand which form a double-stranded region; The antisense strand is 5'-AAACCUUCAUCUUCCUGCCUGUG-3' of SEQ ID NO:151; 5'-UAACCUUCAUCUUCCUGCCUGUG-3' of SEQ ID NO:700; 5'-ACACAGAACCUUCAUCUUCCUGC-3' of SEQ ID NO:152; and 5'-UCACAGAACCUUCAUCUUCCUGC-3' of SEQ ID NO:701; and wherein the antisense strand nucleotide sequence comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from an antisense strand nucleotide sequence selected from the group consisting of: the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties that are conjugated to one or more internal positions in at least one strand; c) wherein said dsRNA agent or a pharma- ceutically acceptable salt thereof comprises a sense strand and an antisense strand which form a double-stranded region; the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the complement of nucleotides 1128-1155 of SEQ ID NO:1; and the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties that are conjugated to one or more internal positions in at least one strand; d) wherein said dsRNA agent or a pharma- ceutically acceptable salt thereof comprises a sense strand and an antisense strand which form a double-stranded region; The antisense strand is 5'-AAUCUUUAUUAAACUAGGGUCCA-3' of SEQ ID NO:281; 5'-AGGUGAAUCUUUAUUAAACUAGG-3' of SEQ ID NO:282; and 5'-UAUCUUUAUUAAACUAGGGUCCA-3' of SEQ ID NO:708; and wherein the antisense strand nucleotide sequence comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from an antisense strand nucleotide sequence selected from the group consisting of: the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties that are conjugated to one or more internal positions in at least one strand; e) wherein said dsRNA agent or a pharma- ceutically acceptable salt thereof comprises a sense strand and an antisense strand which form a double-stranded region; the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides with 0 or 1 mismatch of a portion of the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having at least 90% nucleotide sequence identity with the entire nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides with 0 or 1 mismatch of a corresponding portion of the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least 90% nucleotide sequence identity with the entire nucleotide sequence of SEQ ID NO:2, such that the sense strand is complementary to at least 15 consecutive nucleotides in the antisense strand; or f) wherein said dsRNA agent or a pharma- ceutically acceptable salt thereof comprises a sense strand and an antisense strand which form a double-stranded region; the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-5 and 7-10; A dsRNA agent, or a pharma- ceutically acceptable salt thereof.

2. dsRNA agent of claim 1 e) or f), or a pharma- ceutically acceptable salt thereof, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences 57-79, 62-84, 75-97, 86-108, 207-229, 213-235, 218-240, 898-920, 1128-1150, 637-659 of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:

2.

3. 3. The dsRNA agent, or pharma- ceutically acceptable salt thereof, of claim 1 or 2, wherein said lipophilic moiety is conjugated to one or more positions in the double-stranded region of said dsRNA agent, or pharma- ceutically acceptable salt thereof.

4. 4. The dsRNA agent of any one of claims 1 to 3, or a pharma- ceutically acceptable salt thereof, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a nucleotide modification.

5. At least one of the nucleotide modifications is a deoxynucleotide modification, a 3'-terminal deoxythymidine (dT) nucleotide modification, a 2'-O-methyl nucleotide modification, a 2'-fluoro nucleotide modification, a 2'-deoxynucleotide modification, a locked nucleotide modification, a non-locked nucleotide modification, a conformationally restricted nucleotide modification, a constrained ethyl nucleotide modification, an abasic nucleotide modification, a 2'-amino nucleotide modification, a 2'-O-allyl nucleotide modification, a 2'-C-alkyl nucleotide modification, a 2'-hydroxyl nucleotide modification, a 2'-methoxyethyl nucleotide modification, a 2'-O-alkyl nucleotide modification, a morpholino nucleotide modification, a phosphoramidate modification, a nucleotide modification containing a non-natural base, a tetrahydropyran nucleotide modification, a 1,5-anhydrohexitol nucleotide modification, a cyclohexyl ...

5. The dsRNA agent of claim 4, or a pharma- ceutically acceptable salt thereof, selected from the group consisting of xenyl nucleotide modifications, nucleotide modifications comprising a 5'-phosphorothioate group, nucleotide modifications comprising a 5'-methylphosphonate group, nucleotide modifications comprising a 5' phosphate or a 5' phosphate mimetic, nucleotide modifications comprising a vinyl phosphonate, nucleotide modifications comprising adenosine-glycol nucleic acid (GNA), nucleotide modifications comprising thymidine-glycol nucleic acid (GNA) S-isomers, nucleotide modifications comprising 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotide modifications comprising 2'-deoxythymidine-3' phosphate, nucleotide modifications comprising 2'-deoxyguanosine-3'-phosphate, and terminal nucleotide modifications linked to cholesteryl derivatives and dodecanoic acid bisdecylamide groups; and combinations thereof.

6. 6. The dsRNA agent of any one of claims 1 to 5, or a pharma- ceutically acceptable salt thereof, further comprising at least one phosphorothioate internucleotide linkage.

7. The dsRNA agent of any one of claims 1-6, or a pharma- ceutically acceptable salt thereof, wherein each strand is 30 or fewer nucleotides in length.

8. 8. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein said at least one strand comprises a 3' overhang of at least one nucleotide.

9. 9. The dsRNA agent of any one of claims 1 to 8, or a pharma- ceutically acceptable salt thereof, wherein said double-stranded region is 15 to 30 nucleotide pairs in length.

10. 10. The dsRNA agent of any one of claims 1-9, or a pharma- ceutically acceptable salt thereof, wherein said one or more lipophilic moieties are conjugated to one or more internal positions in at least one strand via a linker or carrier.

11. 11. The dsRNA agent of any one of claims 1-10, or a pharma- ceutically acceptable salt thereof, wherein said one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 4-8 and 13-18 in the sense strand, and positions 6-10 and 15-18 in the antisense strand, counting from the 5' end of each strand.

12. 12. The dsRNA agent of any one of claims 1-11, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

13. 13. The dsRNA agent of claim 12, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

14. 14. The dsRNA agent of any one of claims 1-13, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides at an internal position or in a double-stranded region.

15. The double-stranded iRNA agent of any one of claims 1-14, wherein the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

16. The dsRNA agent of any one of claims 1-15, or a pharma- ceutically acceptable salt thereof, further comprising a phosphate or a phosphate mimetic at the 5'-end of the antisense strand.

17. 17. An isolated cell comprising the dsRNA agent of any one of claims 1 to 16, or a pharma- ceutically acceptable salt thereof.

18. A pharmaceutical composition for inhibiting the expression of a gene encoding APOE, comprising the dsRNA agent according to any one of claims 1 to 16, or a pharma- ceutically acceptable salt thereof.

19. 1. An in vitro method for inhibiting expression of APOE in a cell, comprising: (a) contacting said cell with a dsRNA agent according to any one of claims 1 to 16 or a pharma- ceutically acceptable salt thereof, or with a pharmaceutical composition according to claim 18; and (b) maintaining the cells produced in step (a) for a period of time sufficient to result in degradation of the mRNA transcripts of the APOE gene, thereby inhibiting expression of the APOE gene in the cells. A method comprising:

20. Use of a dsRNA agent according to any one of claims 1 to 16 or a pharma- ceutically acceptable salt thereof or a pharmaceutical composition according to claim 18 in the manufacture of a medicament for treating a subject diagnosed with an APOE-associated neurodegenerative disease or for preventing the onset of an APOE-associated neurodegenerative disease in a subject who meets at least one diagnostic criterion for an APOE-associated neurodegenerative disease.

21. 21. The use according to claim 20, wherein the subject is a human.

22. 21. The use of claim 20, wherein the treating comprises amelioration of at least one sign or symptom of the disease.

23. 22. The use of claim 21, wherein the use of the medicament comprises preventing the progression of an APOE-associated neurodegenerative disease.

24. The use according to claim 20, wherein the subject has been diagnosed with an APOE-associated neurodegenerative disease.

25. The use according to claim 23, wherein the APOE-associated neurodegenerative disease is an amyloid-β mediated disease.

26. 26. The use of claim 25, wherein the amyloid-β mediated disease is selected from the group consisting of Alzheimer's disease, Down's syndrome, and cerebral amyloid angiopathy.

27. 24. The use according to claim 23, wherein the APOE-associated neurodegenerative disease is a tau-mediated disease.

28. 28. The use according to claim 27, wherein the tau-mediated disease is a primary tauopathy or a secondary tauopathy.

29. 29. The use according to claim 28, wherein the primary tauopathy is selected from the group consisting of frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), bulbar glial tauopathy (GGT), frontotemporal dementia with parkinsonism (FTDP, FTDP-17), chronic traumatic encephalopathy (CTE), dementia pugilistica, frontotemporal lobar degeneration (FTLD), argyrophilic grain disease (AGD), and primary age-related tauopathy (PART).

30. 29. The use of claim 28, wherein the secondary tauopathy is selected from the group consisting of AD, Creutzfeldt-Jakob disease, Down's syndrome, and Familial British Dementia.

31. The use of any one of claims 20 to 30, wherein the dsRNA agent is for administration at a dose of from about 0.01 mg / kg to about 50 mg / kg.

32. The use according to any one of claims 20 to 31, wherein the dsRNA agent is for intrathecal administration.

33. The use according to any one of claims 20 to 32, further comprising administering to the subject an additional agent suitable for treating or preventing an APOE-associated neurodegenerative disorder.

34. 1. An in vitro method for inhibiting expression of the APOE gene in astrocytes, comprising: (a) contacting the astrocyte with a dsRNA agent according to any one of claims 1 to 16 or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition according to claim 18; and (b) maintaining the astrocytes produced in step (a) for a time sufficient to result in degradation of the mRNA transcripts of the APOE gene, thereby inhibiting expression of the APOE gene in the astrocytes. A method comprising:

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