Apolipoprotein E (ApoE)iRNA preparation composition and method 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 applications This application claims priority to U.S. Provisional Patent Application No. 63 / 015,867, filed on 27 April 2020, the entire contents of which are incorporated herein by reference.

[0002] Sequence List This application includes an electronically submitted sequence listing in ASCII format, which is incorporated herein by reference in its entirety. The 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 an 18-amino acid signal peptide is cleaved. There are three common isoforms of APOE: APOE2, APOE3, and APOE4, each encoded by a corresponding allele. The three APOE isoforms, ApoE ε2 (APOE2), ApoE ε3 (APOE3), and ApoE ε4 (APOE4), differ from each other only at amino acid positions 112 and 158: APOE2 has Cys112 and Cys158, APOE3 has Cys112 and Arg158, and APOE4 has Arg112 and Arg158. APOE is widely expressed but is mainly peripherally expressed in hepatocytes of the liver and glial cells of 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 microglial cells are the main source of apoE in the brain [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. 47(3):566-581.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 cerebral vascular integrity. For example, when APOE is secreted from cells, several transporters (e.g., ATP-binding cassette (cssestte) transporters) transport cholesterol and phospholipids to the newly synthesized APOE to form lipoprotein particles, which APOE then distributes to neurons through binding to APOE receptors, such as members of the LDL receptor (LDLR) family. Furthermore, it has been observed that after liver transplantation, the recipient's serum APOE phenotype is completely converted to the donor's phenotype, but the cerebrospinal fluid (CSF) ApoE phenotype is not. In addition, astrocytes produce APOE in the form of high-density lipoprotein (HDL)-like particles, which have properties distinct from APOE derived from other sources [see, for example, Morikawa, et al., Neurobiol Dis.. Jun-Jul 2005;19(1-2): 66-76]. Therefore, APOE in CSF cannot be obtained from the plasma pool 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 several proteinopathy. The most well-established association between APOE polymorphism and disease is between APOE genotype and Alzheimer's disease (AD), which has been shown to be a risk determinant for late-onset Alzheimer's disease, where symptoms begin after age 65. In addition, a recent study from the Haltzman lab reported that having an ε4 allele significantly accelerates disease progression (p=0.02), with one ε4 allele increasing the rate of progression by 14% compared to non-carriers, and two ε4 alleles increasing it by 23% [Holtzman, et al. (2017) Nature 549:523]. AD is the leading cause of dementia in the elderly, and its pathological features include the deposition of extracellular amyloid-β (Aβ) aggregates as amyloid plaques, intracellular hyperphosphorylated tau aggregates as neurofibrillary tangles, as well as neuronal loss and glial activation. Since individuals with late-onset Alzheimer's disease (AD) account for more than 95% of the entire 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 more than three times higher risk of developing AD, subjects with two copies of APOE4 have a more than twelve times increased risk of developing AD, while subjects with two copies of APOE2 have been shown to be protective against AD development in subjects [Reiman EM, et al. (2020) Nature Communications 11 (1); 667]. In addition, there are three reported cases of human APOE knockout, and none of these subjects experienced dementia at the time of hospital visits (ages 40-60) [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 male), MRI and cerebrospinal fluid (CSF) biomarker tests demonstrated no signs of neurodegeneration, no impairment of brain structure, and normal levels of tau and p-tau. Furthermore, recent studies have shown that the presence of a Christchurch mutation in ApoE3 may be protective against pyresenilin 1-driven dementia, as evidenced by preserved cognitive function and limited tauopathy on PET. In the presence of the Christchurch mutation, 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 Medicien 25:1680].

[0007] In ApoE-induced amyloid mouse models, increased ApoE4 expression accelerates amyloid accumulation and neuritis dystrophy [Liu, et al. (2017) Neuron 96:1024] and Huynh, et al. [Neuron (2017) 96:1013]. Furthermore, antisense inhibition of APOE4 has been shown to be protective in mice transgenic to the amyloid precursor protein (APP) / presenilin 1 (PS1-21). In addition, it has been shown that deletion of ApoE4 in a tauopathy mouse model provided protection against neurodegeneration [Holtzman, et al. (2017) Nature 549:523], and that reintroduction of APOE4 expression in human neurons derived from induced pluripotent stem cells that express APOE4 but have nullified APOE, resulted in toxic effects from APOE4 [wang, et al. (2018) Nat Medicine 24:647]. Furthermore, it has been shown that restricting APOE4 expression in the mouse liver may affect cognitive ability, impair the blood-brain barrier, and increase neuroinflammation (alzforum.org / news / research-news / apoe-has-hand-Alzheimer'ss-beyond-av-beyond-brain).

[0008] Currently, there are no therapeutic or prophylactic treatments for individuals with APOE-related neurodegenerative diseases such as Alzheimer's disease (AD), and supportive and symptomatic treatments are the mainstays of care. Therefore, there is a need in the art for compositions and methods for treating individuals with neurodegenerative diseases or at risk of developing them. [Overview of the project]

[0009] This disclosure provides RNAi compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the apolipoprotein E (APOE) gene. The APOE gene may be intracellular, for example, in cells within a subject, such as a human. This disclosure also provides methods of using the RNAi compositions of this disclosure for inhibiting the expression of the APOE gene, or for treating subjects, such as those suffering from or prone to suffering from APOE-related neurodegenerative diseases, such as amyloid-beta-mediated diseases or tau-mediated diseases, for inhibiting or reducing the expression of the APOE gene, for example, the pathogenic APOE allele, i.e., APOE4. In particular, the RNAi compositions of this specification can affect specific APOE expression by astrocytes in the CNS to treat APOE-related neurodegenerative diseases.

[0010] Accordingly, in one embodiment, the present disclosure provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of the apolipoprotein E (APOE) gene, wherein the RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides) from any one of the antisense sequences listed in any one of Tables 2-5 and 7-10. In a particular embodiment, the antisense strand comprises a complementary region comprising at least 15 consecutive nucleotides from any one of the antisense sequences listed in any one of Tables 2-5 and 7-10. In a particular embodiment, the antisense strand comprises a complementary region comprising at least 15 consecutive nucleotides from any one of the antisense sequences listed in any one of Tables 7 and 8. In a particular embodiment, the antisense strand comprises a complementary region comprising at least 15 consecutive nucleotides from any one of the antisense sequences listed in any one of Tables 9 and 10. In certain embodiments, the antisense strand includes a complementary region containing at least 19 consecutive nucleotides that differ by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides) from any one of the antisense sequences listed in any one of Tables 2-5 and 7-10. In certain embodiments, the antisense strand includes a complementary region containing at least 19 consecutive nucleotides (i.e., three, two, one, or zero nucleotides) from any one of the antisense sequences listed in any one of Tables 7 and 8. In certain embodiments, the antisense strand includes a complementary region containing at least 19 consecutive nucleotides (i.e., three, two, one, or zero nucleotides) from any one of the antisense sequences listed in any one of Tables 9 and 10. In certain embodiments, the antisense strand includes a complementary region containing at least 19 consecutive nucleotides from any one of the antisense sequences listed in any one of Tables 2-5 and 7-10.In certain embodiments, the antisense strand includes a complementary region containing at least 19 consecutive nucleotides of any one of the antisense sequences listed in either Table 7 or 8. In certain embodiments, the antisense strand includes a complementary region containing at least 19 consecutive nucleotides of any one of the antisense sequences listed in either Table 9 or 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 drug comprises one or more lipophilic moieties conjugated to one or more internal nucleotide positions via a linker or carrier, as appropriate.

[0012] In other embodiments, the agent further comprises a targeted ligand that targets liver tissue, such as one or more GalNAc derivatives, which is appropriately conjugated to the double-stranded RNAi agent via a linker or carrier.

[0013] In yet another embodiment, the agent further comprises one or more lipophilic moieties conjugated to one or more internal nucleotide positions via a linker or carrier as appropriate, and a targeted ligand for liver tissue, such as one or more GalNAc derivatives, which is conjugated to a double-stranded RNAi agent via a linker or carrier as appropriate.

[0014] In one particular embodiment, the double-stranded RNAi agent inhibits the expression of the APOE2, APOE3, and APOE4 alleles. In another embodiment, the double-stranded RNAi agent inhibits the expression of APOE4, but does not substantially inhibit the expression of APOE2 and APOE3; for example, the expression of APOE2 and APOE3 is inhibited by about 10% or less.

[0015] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting the expression of the apolipoprotein E (APOE) gene, wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides) from any one of the sense strand sequences presented in Tables 2-5 and 7-10, and the antisense strand comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense strand nucleotide sequences presented in Tables 2-5 and 7-10. In a particular embodiment, the sense strand comprises at least 15 consecutive nucleotides from any one of the sense strand sequences presented in Tables 2-5 and 7-10, and the antisense strand comprises at least 15 consecutive nucleotides from any one of the antisense strand nucleotide sequences presented in Tables 2-5 and 7-10. In certain embodiments, the sense strand comprises at least 15 consecutive nucleotides from any one of the sense strand sequences presented in Tables 7 and 8, and the antisense strand comprises at least 15 consecutive nucleotides from any one of the antisense strand nucleotide sequences presented in Tables 7 and 8. In certain embodiments, the sense strand comprises at least 15 consecutive nucleotides from any one of the sense strand sequences presented in Tables 9 and 10, and the antisense strand comprises at least 15 consecutive nucleotides from any one of the antisense strand nucleotide sequences presented in Tables 9 and 10. In certain embodiments, the sense strand comprises at least 19 consecutive nucleotides from any one of the sense strand sequences presented in Tables 2-5 and 7-10 (i.e., 3, 2, 1, or 0 nucleotides differing), and the antisense strand comprises at least 19 consecutive nucleotides from any one of the antisense strand nucleotide sequences presented in Tables 2-5 and 7-10 (i.e., 3, 2, 1, or 0 nucleotides differing).In a particular embodiment, the sense strand comprises at least 19 consecutive nucleotides from any one of the sense strand sequences presented in Tables 7 and 8 (i.e., differing by 3, 2, 1, or 0 nucleotides), and the antisense strand comprises at least 19 consecutive nucleotides from any one of the antisense strand nucleotide sequences presented in Tables 7 and 8 (i.e., differing by 3, 2, 1, or 0 nucleotides). In a particular embodiment, the sense strand comprises at least 19 consecutive nucleotides from any one of the sense strand sequences presented in Tables 9 and 10 (i.e., differing by 3, 2, 1, or 0 nucleotides), and the antisense strand comprises at least 19 consecutive nucleotides from any one of the antisense strand nucleotide sequences presented in Tables 9 and 10 (i.e., differing by 3, 2, 1, or 0 nucleotides).

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

[0017] In other embodiments, the agent further comprises a targeted ligand that targets liver tissue, such as one or more GalNAc derivatives, which is appropriately conjugated to the double-stranded RNAi agent via a linker or carrier.

[0018] In yet another embodiment, the agent further comprises one or more lipophilic moieties conjugated to one or more internal nucleotide positions via a linker or carrier as appropriate, and a targeted ligand for liver tissue, such as one or more GalNAc derivatives, which is conjugated to a double-stranded RNAi agent via a linker or carrier as appropriate.

[0019] In one particular embodiment, the double-stranded RNAi agent inhibits the expression of the APOE2, APOE3, and APOE4 alleles. In another embodiment, the double-stranded RNAi agent inhibits the expression of APOE4, but does not substantially inhibit the expression of APOE2 and APOE3; for example, the expression of APOE2 and APOE3 is inhibited by about 10% or less.

[0020] A further aspect of the present disclosure relates to a double-stranded RNAi agent for inhibiting the expression of the apolipoprotein E (APOE) gene, wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides) from any one of the nucleotide sequences of SEQ ID NOs. 1, 3, 5, 7, or 9, or 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, and the thymine of any of SEQ ID NOs. 1, 3, 5, 7, and 9 in uracil A substitution (when comparing aligned sequences) is not counted as a difference that contributes to at least 90% nucleotide sequence identity between any one of the nucleotide sequences of SEQ ID NOs. 1, 3, 5, 7, and 9, or any one of the entire nucleotide sequences of SEQ ID NOs. 1, 3, 5, 7, or 9, and a nucleotide sequence having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, and an antisense strand is not counted as a difference that contributes to at least 90% nucleotide sequence identity between any one of the nucleotide sequences of SEQ ID NOs. 2, 4, 6, 8, or 10, or any one of the entire nucleotide sequences of SEQ ID NOs. 2, 4, 6, 8, or 10,For example, a nucleotide sequence having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity contains at least 15 consecutive nucleotides that differ by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides), and the substitution of thymine to uracil in any of sequence numbers 2, 4, 6, 8, and 10 (when comparing aligned sequences) is equivalent to any one of sequence numbers 2, 4, 6, 8, and 10, or any one of sequence numbers 2, 4, 6, 8, or 10. The present invention provides a double-stranded RNAi agent comprising a nucleotide sequence having at least 90% nucleotide sequence identity with the ocide sequence, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, with no differences of three or fewer nucleotides (i.e., differences of three, two, one, or zero nucleotides), and at least one of the sense strand and antisense strand contains one or more lipophilic moieties conjugated to one or more internal nucleotide positions via a linker or carrier as appropriate.

[0021] In one embodiment, a double-stranded RNAi agent targeting APOE includes a sense strand containing at least 15 consecutive nucleotides that differ from the nucleotide sequence of the double-stranded sense strand nucleotide sequence described in Tables 2-5 and 7-10 by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides). In another embodiment, a double-stranded RNAi agent targeting APOE includes a sense strand containing at least 15 consecutive nucleotides that differ from the nucleotide sequence of the double-stranded sense strand nucleotide sequence described in Tables 7 and 8 by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides). A double-stranded RNAi agent targeting APOE includes a sense strand containing at least 15 consecutive nucleotides that differ from the nucleotide sequence of the double-stranded sense strand nucleotide sequence described in Tables 9 and 10 by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides).

[0022] In one embodiment, a double-stranded RNAi agent targeting APOE includes an antisense strand containing at least 15 consecutive nucleotides that differ by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides) from any one of the double-stranded antisense nucleotide sequences described in Tables 2-5 and 7-10. In one embodiment, a double-stranded RNAi agent targeting APOE includes an antisense strand containing at least 15 consecutive nucleotides that differ by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides) from the double-stranded antisense nucleotide sequence described in Tables 7 and 8. In one embodiment, a double-stranded RNAi agent targeting APOE includes an antisense strand containing at least 15 consecutive nucleotides that differ by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides) from the double-stranded antisense nucleotide sequence described in Tables 9 and 10.

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

[0024] In one embodiment, a double-stranded RNAi agent targeting APOE includes a sense strand containing at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the nucleotide sequences 59-90, 330-357, 568-594, 1019-1044, and 1130-1155 of SEQ ID NO: 1 (i.e., differing by three, two, one, or zero nucleotides), and an antisense strand containing at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2.

[0025] In one embodiment, a double-stranded RNAi agent targeting APOE includes a sense strand containing at least 15 consecutive nucleotides that differ by three or fewer 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, and 637-659 of SEQ ID NO: 1 (i.e., differing by three, two, one, or zero nucleotides), and an antisense strand containing at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2.

[0026] In one embodiment, a double-stranded RNAi agent targeting APOE includes a sense strand containing at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the nucleotide sequences 57-79, 62-84, 207-229, and 1128-1150 of SEQ ID NO: 1 (i.e., differing by three, two, one, or zero nucleotides), and an antisense strand containing at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2.

[0027] In one embodiment, a double-stranded RNAi agent targeting APOE includes an antisense strand containing at least 15 consecutive nucleotides that differ from one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1204704, AD-1204705, AD-1204705, AD-1204706, AD-1204707, AD-1204708, AD-1204709, AD-1204710, AD-1204711, AD-1204712, and AD-1204713 by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides).

[0028] In one embodiment, a double-stranded RNAi agent targeting APOE includes an antisense strand containing at least 15 consecutive nucleotides that differ from one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1204704, AD-1204705, AD-1204708, and AD-1204712 by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides).

[0029] In some embodiments, the agent further comprises a targeted ligand that targets liver tissue, such as one or more GalNAc derivatives, which is appropriately conjugated to the double-stranded RNAi agent via a linker or carrier.

[0030] In one specific embodiment of the present invention, the double-stranded RNAi agent inhibits the expression of the APOE2, APOE3, and APOE4 alleles. In another embodiment, the double-stranded RNAi agent inhibits the expression of APOE4, but does not substantially inhibit the expression of APOE2 and APOE3, for example, inhibiting the expression of APOE2 and APOE3 by about 10% or less.

[0031] The double-stranded RNAi agent may contain at least one modified nucleotide.

[0032] In a particular embodiment, logK owThe lipophilicity of the lipophilic portion, as measured by [the method], is greater than 0.

[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 related embodiments, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0034] In a particular embodiment, substantially all of the nucleotides in the sense strand are modified nucleotides. All of the nucleotides in the sense strand may be modified nucleotides.

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

[0036] All nucleotides in the sense strand and all nucleotides in 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 nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a restricted ethyl nucleotide, a debasalized nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramide, a nucleotide containing a non-natural base, or a tetrahydropyran modified nucleotide. These are decorated nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5'-phosphate or 5'-phosphate mimetic, nucleotides containing vinylphosphonate, nucleotides containing adenosine glycol nucleic acid (GNA), nucleotides containing thymidine glycol nucleic acid (GNA) S-isomers, nucleotides containing 2-hydroxymethyltetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, or terminal nucleotides linked to a cholesteryl derivative or a dodecanoate bisdecylamide group.

[0038] In related embodiments, the modified nucleotides are nucleotides containing a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a 3'-terminal deoxythymidine nucleotide (dT), a locked nucleotide, a debasalized nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramide, or a non-natural base.

[0039] In one embodiment, the modified nucleotide includes a short sequence of a 3'-terminal deoxythymidine nucleotide (dT).

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

[0041] In further embodiments, the double-stranded RNAi agent comprises at least one phosphorothioate nucleotide linkage. The double-stranded RNAi agent may also comprise 6 to 8 (e.g., 6, 7, or 8) phosphorothioate nucleotide linkages.

[0042] In certain embodiments, the complementary region is at least 17 nucleotides long. The complementary region may be 19 to 23 nucleotides long. The complementary region may be 19 nucleotides long.

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

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

[0045] In certain embodiments, the double-stranded RNAi agent further comprises a lipophilic ligand, such as a C16 ligand, conjugated at the 3' end of the sense strand by a monovalent or branched divalent or trivalent linker.

[0046] In one embodiment, the ligand is

[0047] [ka] (In the formula, B is a nucleotide base or a nucleotide base analogue, and B may be adenine, guanine, cytosine, thymine, or uracil.) That is the case.

[0048] In other embodiments, the agent further comprises a targeted ligand that targets liver tissue, such as one or more GalNAc derivatives, which is appropriately conjugated to the double-stranded RNAi agent via a linker or carrier.

[0049] In yet another embodiment, the agent further comprises a lipophilic ligand, e.g., a C16 ligand, conjugated at an internal nucleotide position by, for example, a monovalent or branched divalent or trivalent linker, and a targeted ligand that targets liver tissue, e.g., one or more GalNAc derivatives, conjugated at the 3' end of the sense strand by a monovalent or branched divalent or trivalent linker.

[0050] In another embodiment, the region complementary to APOE includes one of the antisense sequences listed in any one of Tables 2-5 and 7-10. In a particular embodiment, the region complementary to APOE includes one of the antisense sequences listed in any one of Tables 7 and 8. In a particular embodiment, the region complementary to APOE includes one of the antisense sequences listed in any one of Tables 9 and 10.

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

[0052] In the relevant embodiments, the internal position includes all positions except the three terminal positions from each end of the chain. The internal position may also exclude the cleavage region of the sense chain.

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

[0054] In other embodiments, the internal positions exclude positions 11-13, counting from the 3' end of the sense strand. The internal positions may also exclude the cleavage region of the antisense strand. In a particular embodiment, the sense strand is 21 nucleotides long.

[0055] In some embodiments, the internal positions exclude positions 12–14, counting from the 5' end of the antisense strand. In a particular embodiment, the antisense strand is 23 nucleotides long.

[0056] In another embodiment, the internal positions are excluding positions 11-13 counting from the 3' end in the sense strand and positions 12-14 counting from the 5' end in the antisense strand. In a particular embodiment, the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

[0057] In a further embodiment, one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 4–8 and 13–18 on the sense strand, and positions 6–10 and 15–18 on the antisense strand, counting from the 5' end of each strand. One or more lipophilic moieties may also be conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand. In a particular embodiment, the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

[0058] In certain embodiments, the lipophilic portion is an aliphatic compound, an alicyclic compound, or a polyalicyclic compound. The lipophilic portion may also be a lipid, cholesterol, retinoic acid, cholic acid, adamantane acetate, 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 portion is saturated or unsaturated C4-C 30 It contains a hydrocarbon chain and a suitable functional group selected from hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, or alkyne.

[0060] In a particular embodiment, the lipophilic portion is saturated or unsaturated C6-C 18 Contains hydrocarbon chains. The lipophilic portion is saturated or unsaturated carbon. 16The hydrocarbon chain may be included. In the relevant embodiments, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides at an internal position. In certain embodiments, the carrier is a cyclic group that is pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranil, or dekalinyl, or an acyclic moiety based on a serinol skeleton or a diethanolamine skeleton.

[0061] In one embodiment, the lipophilic portion is conjugated to a double-stranded RNAi agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphate diester, sulfonamide linkage, click reaction product, or carbamate.

[0062] In one embodiment, the lipophilic portion is conjugated to a nucleic acid base, a sugar portion, or an 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. The phosphate mimetic may be a 5'-vinyl phosphonate (VP).

[0064] In certain embodiments, the double-stranded RNAi agent further comprises a targeted ligand, such as a hydrophilic ligand, that targets a receptor that mediates delivery to CNS tissue. In certain embodiments, the targeted ligand is a C16 ligand.

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

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

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

[0068] In related embodiments, the 3' end of the sense chain is protected via an end cap which is a cyclic group having an amine, and the cyclic group is pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranil, or dekalinyl.

[0069] In one embodiment, the RNAi agent comprises at least one modified nucleotide, which 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. The RNAi agent may also comprise at least one of the following modifications: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a glycol nucleic acid (GNA)-containing nucleotide, and a vinyl phosphonate-containing nucleotide.

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

[0071] Another aspect of the disclosure is a double-stranded RNAi agent for inhibiting the expression of the APOE gene, the double-stranded RNAi agent comprising a sense strand complementary to the antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding APOE, each strand being from about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent being of formula (III): Sense: 5’ n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) 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) (wherein, i, j, k, and l are each independently 0 or 1, p, p', q, and q' are each independently between 0 and 6. each N a and N a ' independently represents oligonucleotide sequences containing 0 to 25 nucleotides, which may be modified, unmodified, or a combination thereof, and each sequence contains at least two differently modified nucleotides. each N b and N b The ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified, unmodified, or a combination thereof. each n p , n p ',n q , and n q Each of these may or may not be present, and independently represents an overhang nucleotide. 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 above modifier is different from the above modifier, N b (The above modifier is different from the above modifier of Y) Represented by, The sense strand is conjugated to at least one ligand. We provide double-stranded RNAi agents.

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

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

[0074] In a particular embodiment, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

[0075] In one particular embodiment, the double-stranded RNAi agent inhibits the expression of the APOE2, APOE3, and APOE4 alleles. In another embodiment, the double-stranded RNAi agent inhibits the expression of APOE4, but does not substantially inhibit the expression of APOE2 and APOE3; for example, the expression of APOE2 and APOE3 is inhibited by about 10% or less.

[0076] In another embodiment, the YYY motif occurs at or near the break point of the sense chain.

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

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

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

[0080] In a further embodiment, equation (III) is equation (IIIc): Sense: 5' n p -N a -XX XN b -YY YN a -n q 3' Antisense: 3' n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N a’ -n q’ 5' (IIIc) (In the formula, each N b and N b (' represents an oligonucleotide sequence containing 1 to 5 modified nucleotides independently.) It is represented by [this].

[0081] In a particular embodiment, equation (III) is equation (IIId): Sense: 5' n p -N a -XX XN b -YY YN b -ZZ ZN 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) (In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, Na and N a (Each nucleotide independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides.) It is represented by [this].

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

[0083] In a particular embodiment, the double-stranded region is 17 to 25 nucleotide pairs long. The double-stranded region may also be 23 to 27 nucleotide pairs long.

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

[0085] In a particular embodiment, each chain has 15 to 30 nucleotides. Each chain may have 19 to 30 nucleotides. Each chain may have 19 to 23 nucleotides.

[0086] In a particular embodiment, the double-stranded region is 19 to 21 nucleotide pairs long, and each strand has 19 to 23 nucleotides.

[0087] In another embodiment, the nucleotide modifications of the RNAi agent include 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. Nucleotide modifications include 2'-O-methyl, 2'-fluoro, or GNA, and combinations thereof. In related embodiments, the nucleotide modifications are 2'-O-methyl or 2'-fluoro modifications.

[0088] In one embodiment, the RNAi agent comprises one or more lipophilic ligands, such as a C16 moiety, attached by a divalent or trivalent branched linker, or a ligand containing the same.

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

[0090] In yet another embodiment, the agent further comprises a lipophilic ligand, such as a C16 ligand, conjugated at the 3' end of a sense chain by a monovalent or branched divalent or trivalent linker, and a targeted ligand that targets liver tissue, such as one or more GalNAc derivatives, conjugated at the 3' end of a sense chain by a monovalent or branched divalent or trivalent linker.

[0091] In a particular embodiment, the ligand is attached to the 3' end of the sense chain.

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

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

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

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

[0096] In some embodiments, the Y' nucleotide contains a 2'-O-methyl modification.

[0097] In a particular embodiment, p'>0. p'=2 is also acceptable.

[0098] In some embodiments, q'=0, p=0, q=0, and the overhang nucleotide of p' is complementary to the target mRNA.

[0099] In a particular embodiment, q'=0, p=0, q=0, and the overhang nucleotide of p' is 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 ' is linked to adjacent nucleotides via phosphorothioate linkage. All n p ' may be linked to an adjacent nucleotide via phosphorothioate linkage.

[0102] In certain embodiments, the APOE RNAi agent of the present disclosure is one of those listed in Tables 2-5 and 7-10. In certain embodiments, the APOE RNAi agent of the present disclosure is 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 include modifications. In certain embodiments, the APOE RNAi agent of the present disclosure is 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 include modifications.

[0103] Another aspect of the present disclosure is a double-stranded RNAi agent for inhibiting the expression of the APOE gene in a cell, wherein the double-stranded RNAi agent includes a sense strand complementary to the antisense strand, and the antisense strand includes 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 is of formula (III): Sense: 5’ n p -N a -(XXX) i -N b -YYY-N [[ENDID=14]] b -(ZZZ) 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) (wherein, i, j, k, and l are each independently 0 or 1, p, p’, q, and q’ are each independently 0-6, each N a and N a' independently represents oligonucleotide sequences containing 0 to 25 nucleotides, which may be modified, unmodified, or a combination thereof, and each sequence contains at least two differently modified nucleotides. each N b and N b The ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified, unmodified, or a combination thereof. each n p , n p ',n q , and n q Each of these may or may not be present, and independently represents an overhang nucleotide. 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 modifications are 2'-O-methyl or 2'-fluoro modifications. N b The above modifier is different from the above modifier, N b (The above modifier is different from the above modifier of Y) Represented by, The sense chain is conjugated to at least one ligand, which may be one or more lipophilic ligands, for example, a C16 ligand, and / or one or more GalNAc derivatives. We provide double-stranded RNAi agents.

[0104] In one particular embodiment, the double-stranded RNAi agent inhibits the expression of the APOE2, APOE3, and APOE4 alleles. In another embodiment, the double-stranded RNAi agent inhibits the expression of APOE4, but does not substantially inhibit the expression of APOE2 and APOE3; for example, the 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 the expression of the APOE gene in cells, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding APOE, each strand being approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent is given by formula (III): Sense: 5' n p -N a -(XXX) i -N b -YY YN b -(ZZZ) 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 either 0 or 1. each n p , n p ',n q , and n q Each of these may or may not be present, and independently represents an overhang nucleotide. p, q, and q' are each independently between 0 and 6. n p '>0 and at least one n p ' is linked to an adjacent nucleotide via phosphorothioate linkage, each N a and N a ' independently represents oligonucleotide sequences containing 0 to 25 nucleotides, which may be modified, unmodified, or a combination thereof, and each sequence contains at least two differently modified nucleotides. each N b and N bThe ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified, 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, where the modifications are 2'-O-methyl, glycol nucleic acid (GNA), or 2'-fluoro modifications. N b The above modifier is different from the above modifier, N b [The above modifier is different from the above modifier of Y] Represented by, The sense chain is conjugated to at least one ligand, which may be one or more lipophilic ligands, for example, a C16 ligand, and / or one or more GalNAc derivatives. We provide double-stranded RNAi agents.

[0106] In one particular embodiment, the double-stranded RNAi agent inhibits the expression of the APOE2, APOE3, and APOE4 alleles. In another embodiment, the double-stranded RNAi agent inhibits the expression of APOE4, but does not substantially inhibit the expression of APOE2 and APOE3; for example, the 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 the expression of the APOE gene in cells, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding APOE (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, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity), each strand being approximately 14 to approximately 30 nucleotides long, the double-stranded RNAi agent having formula (III): Sense: 5' n p -N a-(XXX) i -N b -YY YN b -(ZZZ) 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 either 0 or 1. each n p , n p ',n q , and n q Each of these may or may not be present, and independently represents an overhang nucleotide. p, q, and q' are each independently between 0 and 6. n p '>0 and at least one n p ' is linked to an adjacent nucleotide via phosphorothioate linkage, each N a and N a ' independently represents oligonucleotide sequences containing 0 to 25 nucleotides, which may be modified, unmodified, or a combination thereof, and each sequence contains at least two differently modified nucleotides. each N b and N b The ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified, 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 modifications are 2'-O-methyl or 2'-fluoro modifications. N bThe modification above is different from the modification on Y, N b (The modification above is different from the modification on Y') is represented by The sense strand is conjugated to at least one ligand, and the ligand may be one or more lipophilic ligands, for example, C16 ligands, and / or one or more GalNAc derivatives Provide a double-stranded RNAi agent

[0108] In certain embodiments, the double-stranded RNAi agent inhibits the expression of the APOE2 allele, the APOE3 allele, and the APOE4 allele. In other embodiments, the double-stranded RNAi agent inhibits the expression of APOE4 but does not substantially inhibit the expression of APOE2 and APOE3. For example, the 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 the expression of the APOE gene in cells. The double-stranded RNAi agent includes a sense strand complementary to the antisense strand. The antisense strand includes a region complementary to a portion of the mRNA encoding APOE (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, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity). 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 -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3’ Antisense: 3’ n p ’-N a ’-(X’X’X’) k -N b ’-Y’Y’Y’-N b ​l -N a '-n q ' 5' (III) (In the formula, i, j, k, and l are each independently either 0 or 1. each n p , n p ',n q , and n q Each of these may or may not be present, and independently represents an overhang nucleotide. p, q, and q' are each independently between 0 and 6. n p '>0 and at least one n p ' is linked to an adjacent nucleotide via phosphorothioate linkage, each N a and N a ' independently represents oligonucleotide sequences containing 0 to 25 nucleotides, which may be modified, unmodified, or a combination thereof, and each sequence contains at least two differently modified nucleotides. each N b and N b The ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified, 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 modifications are 2'-O-methyl or 2'-fluoro modifications. N b The above modifier is different from the above modifier, N b (The above modifier is different from the above modifier of Y) Represented by, The sense strand contains at least one phosphorothioate linkage, The sense chain is conjugated to at least one ligand, which may be one or more lipophilic ligands, for example, a C16 ligand, and / or one or more GalNAc derivatives. We provide double-stranded RNAi agents.

[0110] In one particular embodiment, the double-stranded RNAi agent inhibits the expression of the APOE2, APOE3, and APOE4 alleles. In another embodiment, the double-stranded RNAi agent inhibits the expression of APOE4, but does not substantially inhibit the expression of APOE2 and APOE3; for example, the 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 the expression of the APOE gene in cells, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding APOE (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, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity), each strand being approximately 14 to approximately 30 nucleotides long, the double-stranded RNAi agent having formula (III): Sense: 5' n p -N a -YY YN 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 of these may or may not be present, and independently represents an overhang nucleotide. p, q, and q' are each independently between 0 and 6. n p '>0 and at least one n p ' is linked to an adjacent nucleotide via phosphorothioate linkage, each N aand N a ' independently represents oligonucleotide sequences containing 0 to 25 nucleotides, which may be modified, unmodified, or a combination thereof, and each sequence contains at least two differently modified nucleotides. YYY and Y'Y'Y' each independently represent one motif of three identical modifications on a triple nucleotide sequence, where the modifications are 2'-O-methyl or 2'-fluoro modifications. Represented by, The sense strand contains at least one phosphorothioate linkage, The sense chain is conjugated to at least one ligand, which may be one or more lipophilic ligands, for example, a C16 ligand, and / or one or more GalNAc derivatives. We provide double-stranded RNAi agents.

[0112] In one particular embodiment, the double-stranded RNAi agent inhibits the expression of the APOE2, APOE3, and APOE4 alleles. In another embodiment, the double-stranded RNAi agent inhibits the expression of APOE4, but does not substantially inhibit the expression of APOE2 and APOE3; for example, the 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 the 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, the sense strand having at least 90% nucleotide sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, or 9 The antisense strand contains at least 15 consecutive nucleotides that differ by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides), and the antisense strand contains at least 90% nucleotide sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, with any one of the nucleotide sequences of SEQ ID NOs. 2, 4, 6, 8, and 10, or any one of the nucleotide sequences of SEQ ID NOs. 3 or fewer nucleotides The sense strand contains at least 15 consecutive nucleotides that differ in rheotides (i.e., differ by 3, 2, 1, or 0 nucleotides), and any substitution of thymine to uracil in any of the sequences shown in SEQ ID NOs. 1-10 (when comparing aligned sequences) is not counted as a difference that contributes to a difference of 3 or fewer nucleotides from any one of the nucleotide sequences shown in SEQ ID NOs. 1-10, substantially all of the nucleotides in the sense strand contain modifications that are 2'-O-methyl modifications, GNAs, or 2'-fluoro modifications, and the sense strand has 2 at its 5' end. The sense strand comprises a phosphorothioate nucleotide linkage, substantially all of the nucleotides of the antisense strand include modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications, the antisense strand comprises two phosphorothioate nucleotide linkages at the 5' end and two phosphorothioate nucleotide linkages at the 3' end, and the sense strand is conjugated to one or more lipophilic ligands, for example, C16, and may further include ligands that target the liver, for example, ligands comprising one or more GalNAc derivatives.We provide double-stranded RNAi agents.

[0114] In one particular embodiment, the double-stranded RNAi agent inhibits the expression of the APOE2, APOE3, and APOE4 alleles. In another embodiment, the double-stranded RNAi agent inhibits the expression of APOE4, but does not substantially inhibit the expression of APOE2 and APOE3; for example, the 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 the expression of the APOE gene, wherein the APOE-targeting double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides (i.e., three, two, one, or zero 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 with any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, and 9, or any of the entire nucleotide sequence of SEQ ID NOs: 1, 3, 5, 7, or 9, and the antisense strand comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides), the antisense strand comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, and 10, or any of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, and 10 The present invention provides a double-stranded RNAi agent comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides (i.e., three, two, one, or zero nucleotides) from a nucleotide sequence having at least 90% nucleotide sequence identity with any one of the total nucleotide sequences of a creotide sequence, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with any one of the nucleotide sequences of the creotide sequence, wherein any substitution of thymine to uracil in any of the sequences shown in SEQ ID NOs. 1 to 10 (when comparing aligned sequences) is not counted as a difference that contributes to the difference of three or fewer nucleotides from any one of the nucleotide sequences shown in SEQ ID NOs. The sense strand comprises at least one 3'-terminal deoxythymidine nucleotide (dT), and the antisense strand comprises a 3'-terminal deoxythymidine nucleotide (dT).

[0116] In one particular embodiment, the double-stranded RNAi agent inhibits the expression of the APOE2, APOE3, and APOE4 alleles. In another embodiment, the double-stranded RNAi agent inhibits the expression of APOE4, but does not substantially inhibit the expression of APOE2 and APOE3; for example, the expression of APOE2 and APOE3 is inhibited by about 10% or less.

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

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

[0119] In certain embodiments, the antisense strand of the RNAi agent comprises at least one thermally destabilizing modification (or a precursor thereof) of the duplex within the first 9 nucleotide positions of the 5' region. The thermally destabilizing modification of the duplex may be one or more of the following

[0120]

Chemical formula

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

[0122] <​​​​​​​​​​​​​​​​In one embodiment, the lipid formulation includes lipid nanoparticles (LNPs).

[0127] Further aspects of the present disclosure provide a method for inhibiting the expression of the APOE gene in cells, comprising: (a) contacting cells with a double-stranded RNAi agent of the present disclosure or a pharmaceutical composition of the present disclosure; and (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the APOE gene, thereby inhibiting the expression of the APOE gene in cells.

[0128] In one embodiment, the cells are located within the subject. The subject may be a human.

[0129] In certain embodiments, the subject may be a rhesus macaque, a cynomolgus macaque, a mouse, or a rat.

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

[0131] In certain embodiments, the method further comprises administering additional therapeutic agents, such as cholinesterase inhibitors and / or memantine, to the target.

[0132] In certain embodiments, the double-stranded RNAi agent is administered in doses ranging from approximately 0.01 mg / kg to approximately 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 the expression of the APOE gene in brain tissue (e.g., striatum) or spinal tissue. The brain or spinal tissue may be the striatum, cortex, cerebellum, cervical vertebrae, lumbar vertebrae, or thoracic vertebrae.

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

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

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

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

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

[0140] In certain embodiments, APOE-related neurodegenerative diseases are amyloid-beta-mediated diseases, selected from the group consisting of, for example, Alzheimer's disease, Down syndrome, and cerebral amyloid angiopathy.

[0141] In certain embodiments, APOE-associated neurodegenerative diseases are tau-mediated diseases such as primary or secondary tauopathy.

[0142] In a particular embodiment, primary tauopathy is selected from the group consisting of 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's dementia, frontotemporal lobar degeneration (FTLD), argyrophilic granulopathy (AGD), and primary age-related tauopathy (PART).

[0143] In a particular embodiment, secondary tauopathy is selected from the group consisting of AD, Creutzfeldt-Jakob disease, Down syndrome, and familial British dementia.

[0144] Another aspect of the present disclosure provides a kit for carrying out the method of the present disclosure, comprising: a) a double-stranded RNAi agent of the present disclosure; b) instructions for use; and c) a device for administering the double-stranded RNAi agent to a target, as appropriate.

[0145] Further aspects of the present disclosure provide a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of the APOE gene, wherein the RNAi agent has a sense strand and an antisense strand, the antisense strand comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides (i.e., 3, 2, 1, or 0 nucleotides) from any one of the antisense strand nucleic acid base sequences in Tables 2-5 and 7-10, for example, at least 15 nucleotides (i.e., 3, 2, 1, or 0 nucleotides differ), and at least 19 nucleotides (i.e., 3, 2, 1, or 0 nucleotides differ). In one embodiment, the RNAi agent comprises one or more of the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C-alkyl modified nucleotides, glycol nucleic acid (GNA), phosphorothioate (PS), and vinyl phosphonate (VP) nucleotides. The RNAi agent may contain at least one of the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C-alkyl modified nucleotides, glycol nucleic acids (GNAs), phosphorothioates (PSs), and vinyl phosphonates (VPs).

[0146] In one particular embodiment, the double-stranded RNAi agent inhibits the expression of the APOE2, APOE3, and APOE4 alleles. In another embodiment, the double-stranded RNAi agent inhibits the expression of APOE4, but does not substantially inhibit the expression of APOE2 and APOE3; for example, the expression of APOE2 and APOE3 is inhibited by about 10% or less.

[0147] In another embodiment, the RNAi agent may contain four or more PS modifications, six to ten PS modifications, or eight 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 contains eight PS modifications located at the second-to-last and last nucleotide junctions, respectively, of the 3' and 5' ends of each of the sense and antisense strands of the RNAi agent.

[0149] In another embodiment, each of the sense and antisense strands of the RNAi agent includes a 5' end and a 3' end, and the RNAi agent contains a single nucleotide containing a GNA. The nucleotide containing the GNA may be located on the antisense strand as the seventh nucleic acid base residue from the 5' end of the antisense strand.

[0150] In further embodiments, each of the sense and antisense strands of the RNAi agent includes a 5' end and a 3' end, and the RNAi agent contains 1 to 4 2'-C-alkyl-modified nucleotides. The 2'-C-alkyl-modified nucleotides may also be 2'-C16-modified nucleotides. The RNAi agent may contain a single 2'-C-alkyl, e.g., a C16-modified nucleotide. The single 2'-C-alkyl, e.g., a C16-modified nucleotide, may be located on the sense strand at the 6th position from the 5' end of the sense strand.

[0151] In another embodiment, each of the sense and antisense strands of the RNAi agent includes a 5' end and a 3' end, and the RNAi agent contains two or more 2'-fluoromodified nucleotides. Each of the sense and antisense strands of the RNAi agent may contain two or more 2'-fluoromodified nucleotides. The 2'-fluoromodified nucleotides may be located on the sense strand at nucleic acid base positions 7, 9, 10, and 11 from the 5' end of the sense strand, and on the antisense strand at nucleic acid base positions 2, 14, and 16 from the 5' end of the antisense strand.

[0152] In further embodiments, each of the sense and antisense strands of the RNAi agent includes a 5' end and a 3' end, and the RNAi agent includes one or more VP modifications. The RNAi agent may also include 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 includes a 5' end and a 3' end, and the RNAi agent contains two or more 2'-O-methyl modified nucleotides. The RNAi agent may also contain 2'-O-methyl modified nucleotides at all nucleic acid base locations that are not modified by 2'-fluoro, 2'-alkyl, or glycol nucleic acid (GNA). The two or more 2'-O-methyl modified nucleotides may be located on the sense strand at positions 1, 2, 3, 4, 5, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 from the 5' end of the sense strand, and on the antisense strand at positions 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 from the 5' end of the antisense strand.

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

[0155] In a particular embodiment, the cells are within a subject, for example, a human subject.

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

[0157] In a particular embodiment, the antisense strand of the dsRNA agent includes at least 15 consecutive nucleotides that differ by three or fewer nucleotides from one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1204704, AD-1204705, AD-1204708, and AD-1204712. [Brief explanation of the drawing]

[0158] [Figure 1-1] Figure 1A is a graph showing the percentage of APOE mRNA remaining in the right hemisphere (BRH) of the brain of homozygous humanized APOE knock-in mice 14 days after administration, administered by intracerebroventricular injection (ICV) with a single 300 μg dose of a double-stranded or artificial CSF (aCSF) control. Figure 1B is a graph showing the percentage of APOE mRNA remaining in the liver of homozygous humanized APOE knock-in mice 14 days after administration, administered by intracerebroventricular injection (ICV) with a single 300 μg dose of a double-stranded or artificial CSF (aCSF) control. [Figure 1-2] Figure 1A is a graph showing the percentage of APOE mRNA remaining in the right hemisphere (BRH) of the brain of homozygous humanized APOE knock-in mice 14 days after administration, administered by intracerebroventricular injection (ICV) with a single 300 μg dose of a double-stranded or artificial CSF (aCSF) control. Figure 1B is a graph showing the percentage of APOE mRNA remaining in the liver of homozygous humanized APOE knock-in mice 14 days after administration, administered by intracerebroventricular injection (ICV) with a single 300 μg dose of a double-stranded or artificial CSF (aCSF) control. [Figure 2] Figure 2 is a graph showing the correlation between the in vitro activity of drugs AD-1204704, AD-1204705, AD-1204706, AD-1204707, AD-1204708, AD-1204709, AD-1204710, AD-1204711, AD-1204712, and AD-1204713 and their in vivo activity. [Modes for carrying out the invention]

[0159] The present invention provides RNAi compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of genes. APOE genes may be located within cells, for example, within cells in subjects such as humans. The disclosure also provides methods of using the RNAi compositions of the disclosure to inhibit the expression of APOE genes, or to treat subjects having disorders in which inhibition or reduction of the expression of APOE genes, for example, pathogenic APOE alleles, i.e., APOE4, would be beneficial, such as APOE-related neurodegenerative diseases, such as amyloid-beta-mediated diseases or tau-mediated diseases.

[0160] The RNAi agents of this disclosure are approximately 30 nucleotides or less in length, for example, 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, 1 The RNAi agent comprises an RNA strand (antisense strand) having a region of length 9-25, 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, wherein the region is at least a portion of the mRNA transcript of the APOE gene. In certain embodiments, the RNAi agent of the present disclosure comprises an RNA strand (antisense strand) having a region of about 21-23 nucleotides in length, wherein the region is substantially complementary to at least a portion of the mRNA transcript of the APOE gene.

[0161] In certain embodiments, the RNAi agents of the present disclosure include an RNA chain (antisense chain) having a region of at least 19 consecutive nucleotides that is substantially complementary to at least a portion of the mRNA transcript of the APOE gene, and which can be longer in length, for example, up to 66 nucleotides, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides. These RNAi agents having longer antisense chains preferably include a second RNA chain (sense chain) of 20-60 nucleotides in length, in which case the sense and antisense chains form a double helix of 18-30 consecutive nucleotides.

[0162] The use of these RNAi agents enables targeted degradation of APOE gene mRNA in mammals. Therefore, methods and compositions containing these RNAi agents are useful for treating subjects in which a reduction in the level or activity of APOE protein would be beneficial, such as subjects with APOE-related neurodegenerative diseases, such as amyloid-beta-mediated or tau-mediated diseases.

[0163] The following detailed description discloses methods for preparing and using compositions containing RNAi agents to inhibit the expression of the APOE gene, as well as compositions or methods for treating subjects with a disease or disorder in which inhibition or reduction of gene expression would be beneficial.

[0164] I. Definition To make this disclosure more easily understandable, certain terms are defined first. In addition, whenever parameter values ​​or ranges of values ​​are listed, intermediate values ​​and ranges of the listed values ​​are also intended to be part of this disclosure.

[0165] The articles "a" and "an" are used herein to mean one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one or more elements, e.g., multiple elements.

[0166] The term "including" is used herein to mean "including but not limited to" and is interchangeable with the phrase. The term "or" is used herein to mean "and / or" unless explicitly indicated in the context and is interchangeable with the phrase.

[0167] The term “approximately” is used herein to mean within a typical range of crossover in the art. For example, “approximately” can be understood as approximately 2 standard deviations from the mean. In a particular embodiment, approximately means ±10%. In a particular embodiment, approximately means ±5%. It will be understood that when “approximately” precedes a series of numbers or ranges, it can modify each of the numbers or ranges in that series.

[0168] The terms “at least,” “greater than,” or “more than” preceding a number or sequence of numbers are understood to include the number adjacent to the term “at least,” and all subsequent numbers or integers that may logically be included, as is evident from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 18 nucleotides in a 21-nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the stated characteristic. It will be understood that when the term “at least” precedes a sequence of numbers or ranges, “at least” can modify each of the numbers and ranges in the sequence.

[0169] Where used herein, “less than” or “less than” is understood to mean from the value adjacent to the phrase and any value or integer logically smaller than that value, down to zero, where logical in context. For example, a double helix with an overhang of “2 nucleotides or less” has an overhang of 2, 1, or 0 nucleotides. Where “less than” precedes a series of numbers or ranges, it will be understood that “less than” can modify each of the numbers or ranges in that series.

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

[0171] If the indicated target site does not match the nucleotide sequence on the sense or antisense strand, the indicated sequence takes precedence.

[0172] If the chemical structure and chemical name do not match, the chemical structure takes precedence.

[0173] The term "APOE," also known as "apolipoprotein E," "Alzheimer's disease 2," "LPG," and "LDLCQ5," refers to the well-known gene that codes for the protein APOE. APOE is synthesized throughout the body, primarily in the liver, and functions as a lipid transporter protein and is the primary 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, it has been identified as a major risk factor for Alzheimer's disease and has been associated with the pathology of other neurodegenerative diseases.

[0174] The nucleotide and amino acid sequences of APOE are, for example, GenBank accession number NM_000041.4 [Human (Homo sapiens) APOE, SEQ ID NO: 1, reverse complement, SEQ ID NO: 2]; GenBank accession number NM_001270681.1 [Rat (Rattus norvegicus) APOE, SEQ ID NO: 3; reverse complement, SEQ ID NO: 4]; GenBank accession number NM_001305843.1 [House mouse (Mus musculus) APOE, SEQ ID NO: 5, reverse complement, SEQ ID NO: 6]; GenBank accession number XM_028839202.1 [Rhesus macaque (Macaca mulatta) APOE, SEQ ID NO: 7, reverse complement, SEQ ID NO: 8]; and GenBank accession number XM_005589554.2 [Cynomolgus macaque (Macaca This can be found in [fascicularis)APOE, SEQ ID NO: 9; reverse complement, SEQ ID NO: 10].

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

[0176] When used herein, the term APOE also refers to variations of the APOE gene, including human APOE variants, provided in SNP databases, for example, at ncbi.nlm.nih.gov / clinvar / ?term=APOE[gene].

[0177] The human APOE gene contains two single nucleotide polymorphisms, which result in the three most common variants: APOE2 (also referred to as APOE*ε2 or ε2; Cys112, Cys158), APOE3 (also referred to as APOE*ε3 or ε3; Cys112, Arg158), and APOE4 (also referred to as APOE*ε4 or ε4; Arg112, Arg158). GenBank accession number NM_000041.4 (Human APOE, Sequence ID 1, Reverse Complement, Sequence ID 2) is the nucleotide sequence of the APOE*ε3 (APOE3) variant; the APOE*ε2 (APOE2) variant has a single nucleotide change at nucleotide 595C>T in Sequence ID 1, and the APOE*ε4 (APOE4) variant has a single nucleotide change at nucleotide 457T>C in Sequence ID 1.

[0178] Unless otherwise 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 the APOE2 allele, the APOE3 allele, and / or the APOE4 allele, while the term “APOE4 allele,” etc., refers to the APOE4 allele only.

[0179] As used herein, “target sequence” means a contiguous portion of a nucleotide sequence in an mRNA molecule formed during the transcription of the APOE gene, such as mRNA which is the product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence will be at least sufficiently long to function as a substrate for RNAi-dependent cleavage in or near a portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the APOE gene.

[0180] The target sequence is approximately 15-30 nucleotides long. For example, the target sequence is approximately 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, 1 The target sequence may be 9-28, 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. In certain embodiments, the target sequence is 19-23 nucleotides long and may optionally be 21-23 nucleotides long. It is conceivable that intermediate ranges and lengths between those listed above are also part of this disclosure.

[0181] As used herein, the term “sequence-containing chain” means an oligonucleotide containing a chain of nucleotides described by a sequence as referred to using the standard nucleotide terminology.

[0182] "G," "C," "A," "T," and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively, in relation to modified or unmodified nucleotides. However, it will be understood that the terms "ribonucleotide" or "nucleotide" can also mean modified nucleotides or substituted parts (see, for example, Table 1), as will be described in more detail below. Those skilled in the art are well aware that guanine, cytosine, adenine, thymidine, and uracil can be replaced by other parts without substantially altering the base-pairing properties of oligonucleotides containing such substituted parts. For example, but not limited to, nucleotides containing inosine as a base can base-pair with nucleotides containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine can be substituted, for example, with nucleotides containing 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 substituted with guanine and uracil, respectively, to form G-UWobble base pairs with the target mRNA. Sequences containing such substitutions 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, mean agents containing RNA as defined herein, which mediate targeted cleavage in RNA transcription via the RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs sequence-specific degradation of mRNA. RNAi modulates, for example, inhibits, APOE expression in cells, for example, in cells within a target, for example, a mammalian target.

[0184] In one embodiment, the RNAi agent of the present disclosure comprises a single-stranded RNAi that interacts with a target RNA sequence, e.g., an APOE target mRNA sequence, to direct the cleavage of the target RNA. While we do not wish to be bound by theory, it is thought that long double-stranded RNA introduced into a cell is degraded into double-stranded small interfering RNA (siRNA) containing sense and antisense strands by a type III endonuclease known as Dicer [Sharp et al. (2001) Genes Dev. 15:485]. Dicer, a ribonuclease III-like enzyme, processes these dsRNAs into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs [Bernstein, et al., (2001) Nature 409:363]. These siRNAs are then introduced into an RNA-induced silencing complex (RISC), in which one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to induce target recognition [Nykanen, et al., (2001) Cell 107:309]. Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing [Elbashir, et al., (2001) Genes Dev. 15:188]. Thus, in one embodiment, this disclosure relates to single-stranded RNA (ssRNA) (the antisense strand of the siRNA double helix) that is generated in a cell and facilitates the formation of the RISC complex, thereby silencing a target gene, namely the APOE gene. Accordingly, the term "siRNA" is used herein to also mean the RNAi described above.

[0185] In another embodiment, the RNAi agent may be a single-stranded RNA introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaut-2 and then cleaves the target mRNA. Single-stranded siRNAs are generally 15–30 nucleotides long and are chemically modified. Designs and tests of single-stranded RNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883–894, the entire contents of which are incorporated herein by reference. Any antisense nucleotide sequences described herein may be used as single-stranded siRNAs described herein or as single-stranded siRNAs chemically modified by the methods described in Lima et al., (2012) Cell 150:883–894.

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

[0187] Generally, dsRNA molecules may contain ribonucleotides, but as will be described in detail herein, each or both strands may also contain one or more ribonucleotides, such as deoxyribonucleotides, modified nucleotides, etc. In addition, as used herein, “RNAi agent” may include ribonucleotides having chemical modifications; an RNAi agent may include substantial modifications in multiple nucleotides. As used herein, the term “modified nucleotide” means a nucleotide having independently modified sugar moieties, modified nucleotide linkages, or modified nucleic acid bases. Thus, the term modified nucleotide includes substitution, addition, or removal of, for example, functional groups or atoms, to nucleoside linkages, sugar moieties, or nucleic acid bases. Modifications suitable for use in the agents of this disclosure include all types of modifications disclosed herein or known in the art. Any such modifications used in siRNA-type molecules are encompassed by “RNAi agent” for the purposes of this specification and the claims.

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

[0189] The double-stranded region can be of any length that allows for the specific degradation of the desired target RNA by the RISC pathway, as well as lengths of approximately 15–36 base pairs, e.g., approximately 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 The base pair lengths can range from ~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. In certain embodiments, the double-stranded region is 19 to 21 base pairs long, for example, 21 base pairs long. It is conceivable that intermediate ranges and lengths between those listed above are also part of this disclosure.

[0190] The two strands forming a double helix structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. If the two strands are part of one larger molecule and are therefore connected by an unpaired nucleotide chain between the 3' end of one strand and the 5' end of the other strand forming the double helix structure, then the connecting RNA strands are called a “hairpin loop”. A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides or nucleotides not targeting the dsRNA site. In some embodiments, a hairpin loop may contain 10 or fewer nucleotides. In some embodiments, a hairpin loop may contain 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 8 unpaired nucleotides.

[0191] The two substantially complementary strands of dsRNA are contained within separate RNA molecules, which can, though not necessarily, be covalently linked. In certain embodiments, where the two strands are covalently linked between the 3' end of one strand and the 5' end of the other strand forming a double-stranded structure by means other than an uninterrupted chain of nucleotides, the connecting structure is called a “linker” (although certain other structures defined elsewhere in this specification may also be called “linkers”). RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus all the overhangs present in the double-stranded structure. In addition to the double-stranded structure, RNAi may contain one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent includes a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand includes a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent include an overhang of at least one nucleotide.

[0192] In one embodiment, the RNAi agent of the present disclosure is a dsRNA, each strand comprising 19 to 23 nucleotides that independently interact with a target RNA sequence, such as an APOE target mRNA sequence, to induce cleavage of the target RNA.

[0193] As used herein, the term “nucleotide overhang” means at least one unpaired nucleotide protruding from the double-stranded structure of an RNAi agent, such as a dsRNA. For example, a nucleotide overhang exists if the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; or the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may contain or consist of nucleotide / nucleoside analogs such as deoxynucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located at the 5' end, the 3' end, or both of either the antisense strand or the sense strand of the dsRNA.

[0194] In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at its 3' or 5' end, for example, an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are replaced with a nucleoside thiophosphate.

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

[0196] In certain embodiments, the overhang in the sense or antisense strand may include an extended length longer than 10 nucleotides, for example, 1–30 nucleotides, 2–30 nucleotides, 10–30 nucleotides, or 10–15 nucleotides. In certain embodiments, the extended overhang is located in the sense strand of the double helix. In certain embodiments, the extended overhang is located at the 3' end of the sense strand of the double helix. In certain embodiments, the extended overhang is located at the 5' end of the sense strand of the double helix. In certain embodiments, the extended overhang is located in the antisense strand of the double helix. In certain embodiments, the extended overhang is located at the 3' end of the antisense strand of the double helix. In certain embodiments, the extended overhang is located at the 5' end of the antisense strand of the double helix. In certain embodiments, one or more nucleotides in the overhang are replaced with a nucleoside thiophosphate. In a particular embodiment, the overhang includes a self-complementary portion such that the overhang can form a stable hairpin structure under physiological conditions.

[0197] The terms “blunt” or “blunt-ended,” as used herein in relation to dsRNA, mean that there are no unpaired nucleotides or nucleotide analogs at any given end of the dsRNA; that is, there are no nucleotide overhangs. One or both ends of a dsRNA can be blunt. If both ends of a dsRNA are blunt, it is said to be blunt-ended. For clarity, a “blunt-ended” dsRNA is a dsRNA that is blunt at both ends, i.e., a dsRNA in which there are no nucleotide overhangs at either end of the molecule. In most cases, such a molecule will be double-stranded along its entire length.

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

[0199] As used herein, the term “complementary region” means a region on an antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., an APOE nucleotide sequence, as defined herein. If the complementary region is not fully complementary to the target sequence, the mismatch may be in an internal or terminal region of the molecule. Generally, the most acceptable mismatch is in a terminal region, e.g., within 5, 4, 3, or 2 nucleotides of the 5' or 3' end of an RNAi agent.

[0200] When used herein, the terms “sense strand” or “passenger strand” mean a strand of an RNAi agent that contains a region substantially complementary to the antisense strand region as defined herein.

[0201] As used herein, the term “cleavage region” means a region located directly adjacent to a cleavage site. A cleavage site is a site on the target where a cleavage occurs. In some embodiments, a cleavage region includes three bases directly adjacent to either end of a cleavage site. In some embodiments, a cleavage region includes two bases directly adjacent to either end of a cleavage site. In some embodiments, in detail, a cleavage site occurs at a site bound 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” means, as understood by those skilled in the art, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence to form a double helix under certain conditions.

[0203] In RNAi agents, for example, in dsRNA as described herein, complementary sequences include base pairings of an oligonucleotide or polynucleotide containing a first nucleotide sequence with an oligonucleotide or polynucleotide containing a second nucleotide sequence over the full length of one or both nucleotide sequences. Such sequences may be referred to herein as “fully complementary” with respect to each other. However, where herein the first sequence is considered “substantially complementary” to the second sequence, the two sequences may be fully complementary, or they may form one or more, but generally 5, 4, 3, or 2 or fewer, mismatched base pairs during hybridization in the case of a double helix of up to 30 base pairs, while retaining their ability to hybridize under conditions most relevant to their final application, e.g., inhibition of gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs are not considered mismatches with respect to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides and another oligonucleotide of 23 nucleotides, wherein the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be considered "perfectly complementary" for the purposes described herein.

[0204] When used herein, “complementary” sequences may include, or may be entirely formed from, non-Watson-Crick base pairs or non-naturally modified nucleotides, provided that the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogsteen base pairings.

[0205] The terms “complementary,” “fully complementary,” and “substantially complementary” can be used herein, as understood from the context in which they are used, in relation to base matching between two oligonucleotides or polynucleotides, for example, between the sense strand and antisense strand of a dsRNA, or between the antisense strand and target sequence of an RNAi agent.

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

[0207] Accordingly, 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 the target APOE sequence and comprise a sequence of nucleotides that are at least about 80% complementary over their entire length to the equivalent region of the nucleotide sequence of APOE SEQ ID NO: 1, 3, 5, 7, or 9, or a fragment of APOE SEQ ID NO: 1, 3, 5, 7, or 9, 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% complementary.

[0208] In other embodiments, the antisense polynucleotides disclosed herein include a sequence of nucleotides that is substantially complementary to a target APOE sequence and is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2-5 and 7-10 of the APOE, for example, 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% complementary.

[0209] In one embodiment, the RNAi agent of the present disclosure comprises a sense strand substantially complementary to an antisense polynucleotide which is identical to the target APOE sequence, wherein the sense strand polynucleotide comprises a sequence of nucleotides which is at least about 80% complementary over its entire length to an equivalent region of SEQ ID NOs. 2, 4, 6, 8, and 10, or any one of the SEQ ID NOs. 2, 4, 6, 8, and 10, 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% complementary.

[0210] In one embodiment, at least partial suppression of APOE gene expression is assessed by a reduction in the amount of APOE mRNA, which can be isolated from or detected in a first cell or cell population in which the APOE gene is transcribed and has been treated to inhibit APOE gene expression, or has been treated in such a first cell or cell population, and compared to a second cell or cell population (control cell) that is substantially identical to the first cell or cell population but has not been treated in such a way. The degree of inhibition can be expressed in the following units:

[0211]

number

[0212] The phrase "contacting cells with an RNAi agent," as used herein, includes contacting cells by any possible means. Contacting cells with an RNAi agent includes contacting cells with an RNAi agent in vitro or in vivo. Contact may be direct or indirect. For example, an RNAi agent may be brought into physical contact with cells by performing the method individually, or an RNAi agent may be placed in a situation that allows or causes subsequent contact with cells.

[0213] Cell contact in vitro can be achieved, for example, by incubating cells with an RNAi agent. Cell contact in vivo can be achieved, for example, by injecting an RNAi agent into or near the tissue in which the cells are located, or by injecting an RNAi agent into another region, such as the central nervous system (CNS), by intrathecal, intravitreous, or other injection, as appropriate, or by injecting an RNAi agent into the bloodstream or subcutaneous space so that the agent subsequently reaches the tissue in which the cells to be contacted are located. For example, an RNAi agent may include or be coupled to a ligand that directs or stabilizes the RNAi agent to a site of interest, such as the CNS, such as a lipophilic moiety, as described below and further detailed, for example, in PCT / US2019 / 031170, which is incorporated herein by reference. In some embodiments, the RNAi agent may contain or be coupled to a ligand, for example, one or more GalNAc derivatives, which are described below, and which direct the RNAi agent to a site of interest, for example, the liver, or otherwise stabilize it. In other embodiments, the RNAi agent may contain or be coupled to a lipophilic moiety and one or more GalNAc derivatives. Combinations of in vitro and in vivo methods for contact are also possible. For example, cells may be contacted with the RNAi agent in vitro and then transferred to a target.

[0214] In one embodiment, contacting cells with an RNAi agent includes “introducing” or “delivering the RNAi agent into cells” by promoting or carrying out cellular uptake or absorption. Absorption or uptake of the RNAi agent may occur by spontaneous diffusive or active cellular processes, or by adjuvants or devices. Introducing the RNAi agent into cells may be in vitro or in vivo. For example, in the case of in vivo introduction, the RNAi agent may 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. Further approaches are described below herein or are 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 by the octanol-water partition coefficient logK. ow This is by which, in this case, K ow The octanol-water partition coefficient is the ratio of the concentration of a chemical in the octanol phase to the concentration of a chemical in the aqueous phase in a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using a coefficient derived from the structural components of the chemical, calculated using first-principles or empirical methods [see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), whose entirety is incorporated herein by reference]. It provides a thermodynamic measure of a substance's tendency to prefer non-aqueous or oily environments rather than water (i.e., the hydrophilic / lipophilic balance). In principle, a chemical is logK ow If logK is greater than 0, it is lipophilic. Typically, the lipophilic portion is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. owIt has, for example, the logK of 6-aminohexanol. ow It is expected to be approximately 0.7. Using the same method, the logK of cholesteryl N-(hexane-6-ol) carbamate can be obtained. ow It 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 a hydroxyl group or an amine group to the end of the lipophilic moiety, the partition coefficient (e.g., logK) of the lipophilic moiety can be changed. ow The 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 a particular embodiment, the unbound fraction of a double-stranded RNAi agent in a plasma protein-binding assay can be determined to be positively correlated with the relative hydrophobicity of the double-stranded RNAi agent, which may be positively correlated with the silencing activity of the double-stranded RNAi agent.

[0218] In one embodiment, the plasma protein binding assay to be 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, 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, greater than 0.4, greater than 0.45, or greater than 0.5 in the case of in vivo delivery of enhanced dsRNA.

[0219] Therefore, by conjugating the lipophilic portion to the internal position of the double-stranded RNAi agent, optimal hydrophobicity for enhanced in vivo delivery in siRNA is provided.

[0220] The term “lipid nanoparticle” or “LNP” refers to a vesicle containing 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 U.S. Patents 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, “Subject” means an animal, e.g., a mammal, e.g., a primate (e.g., human, non-human primates, e.g., monkeys and chimpanzees), or a non-primate (e.g., a rat or mouse). In a preferred embodiment, the subject is a human, e.g., a human treated or evaluated for a disease, disorder, or condition for which a reduction in APOE expression would be beneficial; a human at risk of a disease, disorder, or condition for which a reduction in APOE expression would be beneficial; a human having a disease, disorder, or condition for which a reduction in APOE expression would be beneficial; or a human treated for a disease, disorder, or condition for which a reduction in APOE expression would be beneficial as described herein.

[0222] As used herein, the terms “to treat” or “treatment” refer to beneficial or desired outcomes, such as one or more signs or symptoms associated with APOE gene expression or APOE protein production, e.g., APOE-related neurodegenerative diseases, e.g., amyloid-beta mediated diseases, e.g., Alzheimer's disease, Down syndrome, and cerebral amyloid angiopathy, or tau mediated diseases, e.g., primary tauopathy, e.g., frontotemporal dementia, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), chronic traumatic encephalopathy (CBT). Treatment may include, but is not limited to, the alleviation or improvement of encelopathy (CTE), frontotemporal dementia (FTD, FTDP-17), frontotemporal lobar degeneration (FTLD), argyrophilic granulopathy (AGD), primary age-related tauopathy (PART), and glial tauopathy (GGT), or secondary tauopathy such as AD, Creutzfeldt-Jakob disease, Down syndrome, familial British dementia, and boxer dementia. "Treatment" may also mean extending survival compared to the survival expected without treatment.

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

[0224] Where used herein, “prevention” or “preventing” means, when used in relation to a disease, disorder, or condition in which a reduction in APOE gene expression or APOE protein production would be beneficial, a reduction in the likelihood of developing symptoms associated with such disease, disorder, or condition, e.g., symptoms of APOE-associated neurodegenerative disease. The absence of developing a disease, disorder, or condition, or a reduction in the development of symptoms associated with such disease, disorder, or condition (e.g., a reduction of at least about 10% of a clinically acceptable scale for that disease or disorder), or a delay in the onset of delayed symptoms (e.g., a delay of days, weeks, months, or years) is considered effective prevention.

[0225] As used herein, the terms “APOE-related neurodegenerative disease” or “APOE-related neurodegenerative disorder” are understood to mean 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-beta-mediated diseases such as Alzheimer's disease, Down syndrome, and cerebral amyloid angiopathy, as well as tau-mediated diseases such as primary tauopathy, such as 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's dementia, frontotemporal lobar degeneration (FTLD), argyrophilic granulopathy (AGD), and primary age-related tauopathy (PART), as well as secondary tauopathy, such as Alzheimer's disease (AD), Creutzfeldt-Jakob disease, Down syndrome, and familial British dementia.

[0226] As used herein, the term “amyloid-beta-mediated disease” refers to disorders resulting from the extracellular accumulation of amyloid-beta, which leads to the formation of amyloid plaques in brain tissue. Exemplary amyloid-beta-mediated diseases include Alzheimer's disease, Down syndrome, and cerebral amyloid angiopathy (CAA).

[0227] As used herein, the term “tau-mediated disease” refers to disorders resulting from the aggregation of tau protein and leading to neurofibrillary tangles (tangles). Tangles are formed by the hyperphosphorylation of tau, causing the protein to dissociate from microtubules and aggregates. Tauopathies can be divided into “primary tauopathies,” in which the pathology is primarily driven by tau aggregation, and “secondary tauopathies” (e.g., amyloid-beta plaques in Alzheimer’s disease), in which other factors drive the disease, and the presence of tauopathies exacerbates disease progression. Examples of primary tauopathy 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's dementia, argyrophilic granulopathy (AGD), and primary age-related tauopathy (PART). Examples of secondary tauopathy include Alzheimer's disease (AD), Creutzfeldt-Jakob disease, Down syndrome, and familial British dementia.

[0228] APOE polymorphisms are associated with numerous tauopathies. The APOE4 allele has been 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 correlated with more advanced chronic traumatic encephalopathy (CTE) in the brains of football players with low frequency of repetitive head impacts [Verscaj, C. et al. (2017) Neurology 88 (16) Supplement S9.001] and in the brains of boxers [Jordan, BD et al. (1997) JAMA 278(2): 136-140]. The presence of the APOE4 allele is associated with an 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. described a significant increase in tau levels, brain atrophy, and neuroinflammation in the P301S mouse model of FTD when APOE4 was present compared to when APOE2, APOE3, or APOE knockout was present [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 worsened compared to 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, suggesting that APOE2 may be protective in the presence of amyloid pathology and 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 disease that usually begins slowly and gradually worsens over time. The most common initial symptom is difficulty recalling recent events. As the disease progresses, symptoms may include language problems, disorientation (including easily getting lost), mood swings, loss of motivation, inability to manage oneself, and behavioral problems. As the person's condition deteriorates, they often withdraw from family and society. They gradually lose physical function and eventually die.

[0231] Neuropathologically, Alzheimer's disease (AD) is characterized by the 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, as well as in parts of the frontal cortex and cingulate gyrus. Degeneration is also present in brainstem nuclei such as the locus coeruleus. Studies using MRI and PET have documented a reduction in the size of specific brain regions in individuals with AD as they progress from mild cognitive impairment to Alzheimer's disease, compared to similar images from healthy older adults.

[0232] Both amyloid plaques and neurofibrillary tangles are clearly visible under a microscope in the brains of people with Alzheimer's disease (AD). Plaques are dense, mostly insoluble deposits of beta-amyloid peptides and cytomaterial outside and around neurons. Tangles (neurofibrillary tangles) are aggregates of microtubule-associated protein tau that are hyperphosphorylated and accumulate within cells themselves. Many older adults develop some plaques and tangles as a result of aging, but 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 commonly known as the Alzheimer's Disease Association) established the NINCDS-ADRDA Alzheimer's disease criteria, the most commonly used diagnostic criteria, in 1984 and extensively updated them in 2007. These criteria require confirmation by neuropsychological testing of the presence of cognitive impairment and suspected cognitive syndromes for a clinical diagnosis of possible or certain AD. Definitive diagnosis requires histopathological confirmation, including microscopic examination of brain tissue. Excellent statistical reliability and justification have been demonstrated between the diagnostic criteria and definitive histopathological confirmation. Most commonly, eight intellectual domains—memory, language, perceptual skills, attention, motor skills, orientation, problem-solving, and executive function—are impaired in AD. 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, drugs available to treat patients with Alzheimer's disease (AD) include cholinesterase inhibitors and memantine. These drugs can improve a patient's quality of life by addressing symptoms related to memory, thinking, and language, for example, but they do not alter the rate of disease progression or decline.

[0235] Although the causes of AD are not fully understood, as discussed above, the presence of APOE4 has been shown to be a major risk determinant for late-onset Alzheimer's disease (AD) that develops after the age of 65, and numerous studies in non-human animal models of amyloid-beta-mediated disease (AD) and tau-mediated disease have demonstrated that inhibiting APOE, such as APOE4, has beneficial effects on amyloid plaque formation and cognitive function.

[0236] Down syndrome ("DS"), also known as trisomy 21, is a genetic disorder caused by the presence of all or part of a third copy of chromosome 21. DS is a lifelong condition characterized by intellectual disability, distinctive facial features, and hypotonia in early childhood. Individuals with DS often experience a progressive decline in cognitive function. The third copy of chromosome 21 contains an extra amyloid precursor protein (APP) gene, and the excess amyloid production leads to the accumulation of amyloid-beta plaques and, consequently, a more than 50% increased risk of early-onset Alzheimer's disease (AD). Another gene that is tripled in DS is DYRK1A, which affects alternative splicing of tau, stimulating tau to abnormal hyperphosphorylation and promoting neurofibrillary degeneration [Hartley D. et al. (2016) Alzheimers Dement 11(6): 700-709]. Individuals with AD who are also DS exhibit neuropathological changes similar to those seen in typical 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, NC et al. (2017) Neurology and Therapy 6: 69-81].

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

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

[0239] Pick's disease is characterized by prominent, well-defined atrophy of the frontal, temporal, and cingulate regions, with the parietal lobe being better preserved.

[0240] Corticobasal degeneration ("CBD") is characterized by the predominant loss of cells in the dorsal prefrontal cortex, supplementary motor area, perirowlandic cortex, and subcortical nuclei.

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

[0242] Spheroidal glial tauopathy ("GGT") is a rare form of frontotemporal lobar degeneration (FLD) characterized by widespread spherical inclusions in astrocytes and oligodendrocytes containing four-repeat tau isoforms. These cases are associated with a range of clinical features 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 motor function. Chromosome 17 has been found to be linked to FTDP (FTDP-17), and mutations in microtubule-associated protein tau (MAPT) on chromosome 17 have been found in many families with familial FTDP-17. FTDP-17 mutations in MAPT begin between the ages of 25 and 65, with penetrance close to 100%. Symptoms include executive dysfunction and personality and behavioral changes, often accompanied by aphasia and parkinsonism [Boeve, BF et al. (2008) Arch Neurol 65(4): 460-464].

[0244] Chronic traumatic encephalopathy ("CTE") is a debilitating neurodegenerative disease resulting from recurrent mild traumatic brain injury, commonly found in many athletes, particularly football players. The neuropathological signature of CTE includes accumulation of phosphorylated tau in the sulci and perivascular zones, microgliosis, and astrocytosis; starting with some tau deposits in the early stages, the disease can progress to whole-brain atrophy in the later stages. CTE can progress from mild symptoms, such as short-term memory impairment and mild aggression, to advanced language impairment, as well as psychotic symptoms including paranoia and parkinsonism, over many years [Fesharaki-Zadeh, A.(2019) Front Neurol 10:713].

[0245] "Boxer's dementia" is a form of CTE that includes severe cognitive and motor impairment due to repetitive blows to the head in boxing [Castellani. RJ et al. (2017) J Alzheimers Dis 60(4): 1209-1221].

[0246] "Argyrophilic granulopathy" ("AGD") is a very common sporadic tauopathy and, in some studies, is the second most common neurodegenerative disease after Alzheimer's disease. AGD is a late-onset neurodegenerative disease characterized by small, spindle-shaped or oval-shaped silver-stained lesions in neural processes called argyrophilic granules (AGs). Phosphorylated tau is the main component of AGs. The most common AGD findings are slowly progressive amnesia and mild cognitive impairment, as well as associated high-prevalence neuropsychiatric symptoms. Due to the lack of prominent clinical features, AGD is often diagnosed only post-mortemly, based on three pathological features: AGs, oligodendrocyte coil bodies, and neural tangles [Rodriguez, RD 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 mildly impaired cognitive function. Brains with PART have tau neurofibrillary tangles indistinguishable from those of Alzheimer's disease, but lack amyloid-beta plaques [Crary, JF 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 encephalopathy (TSE) or prion disease. Prions, a term derived from "protein" and "infectious," cause CJD in humans and TSE in animals. Spongy refers to the characteristic appearance of the brain, which, when viewed under a microscope, appears filled with holes until it resembles a sponge. CJD is a rare, degenerative, and fatal brain disorder that usually develops late and follows a rapid course. Typical symptom onset occurs around age 60, and about 70 percent of individuals die within one year. In the early stages of the disease, people may have memory loss, behavioral changes, lack of coordination, and visual impairment. As the disease progresses, cognitive decline becomes more pronounced, and involuntary movements, blindness, limb weakness, and coma may 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 high levels of total tau protein [Kovacs, GG et al. (2017) Brain Pathol 3: 332-344].

[0249] Familial British dementia ("FBD") is a type of cerebral amyloid angiopathy first reported in affected members of a large British family, presenting with a clinical picture including dementia, spastic tetreparesis, and cerebellar ataxia. 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 the affected areas of amyloid-Bri lesions. Tau immunoblotting in FBD is similar to the tau pattern in Alzheimer's disease [Holton JL et al. (2001) Am J Patho 2: 515-526].

[0250] When used herein, “therapeutic dose” is intended to include an amount of RNAi agent sufficient to treat the disease (e.g., by reducing, improving, or maintaining one or more symptoms of the existing disease or disease) when administered to a subject with APOE-associated neurodegenerative disease. “Therapeutic dose” may vary depending on the RNAi agent, how the drug is administered, the disease and its severity, as well as the patient’s medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, and any other individual characteristics of the subject being treated.

[0251] When used herein, “Prophylactic effective dose” is intended to include an amount of RNAi agent sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to a subject with APOE-associated neurodegenerative disorder. Improvement of the disease includes slowing the course of the disease or reducing the severity of the disease if it develops later. The “Prophylactic effective dose” may vary depending on the RNAi agent, how the drug is administered, the degree of the disease risk, and the patient’s medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, and any other individual characteristics of the treated patient.

[0252] The “therapeutic dose” or “preventive dose” also includes the amount of RNAi agent that produces several desired local or systemic effects in a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the method of this disclosure may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0253] The term "pharmaceutically acceptable" is used herein to mean a compound, material, composition, or dosage form that is suitable for use in contact with the tissues of human and animal subjects within the bounds of sound medical judgment, at a reasonable benefit / risk ratio, without excessive toxicity, irritation, allergic reaction, or other problems or complications.

[0254] When used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in the transport or delivery of the compound of interest from one organ or part of the body to another organ, e.g., another part of the body. Each carrier must be “acceptable” in the sense that it is compatible with the other raw materials of the formulation and must not be harmful to the subject being treated. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants, e.g., magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, e.g., cocoa butter and suppository waxes; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil. (10) Glycols, e.g., propylene glycol; (11) Polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; (12) Esters, e.g., ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, e.g., magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Phenothermally hydrated; (17) Isotonic saline; (18) Ringer's solution; (19) Ethyl alcohol; (20) pH buffer solution; (21) Polyesters, polycarbonates, or polyanhydrides; (22) Bulking agents, e.g., polypeptides and amino acids; (23) Serum components, e.g., serum albumin, HDL, and LDL; and (22) other non-toxic affinity substances used in pharmaceutical formulations.

[0255] The term “sample,” as used herein, encompasses similar bodily fluids, cells, or tissues isolated from a subject, as well as collections of bodily fluids, cells, or tissues present within the subject. Examples of bodily fluids include blood, serum, and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs, or local areas. For example, a sample may be obtained from a specific organ, a part of an organ, or bodily fluids or cells within those organs. In certain embodiments, a sample may be obtained from the brain (e.g., the whole brain or a segment of the brain, e.g., striatum, or certain types of cells in the brain, e.g., neurons and glial cells (astrocytes, oligodendrocytes, small glial cells)). In other embodiments, “sample obtained from subject” means liver tissue (or its components) obtained from the subject. In some embodiments, “sample obtained from subject” means blood obtained from the subject or plasma or serum obtained therefrom. In a further embodiment, “sample obtained from subject” means brain tissue (or its minor components) or retinal tissue (or its minor components) obtained from subject.

[0256] II. RNAi agents of the present disclosure RNAi agents that inhibit the expression of APOE genes are described herein. In some embodiments, the RNAi agents provided herein inhibit the expression of the APOE2 allele, the APOE3 allele, and the APOE4 allele. In other embodiments, the RNAi agents provided herein inhibit the expression of the APOE4 allele, and for example, the RNAi agent does not substantially inhibit the expression of the APOE2 or APOE3 allele, for example, the inhibition of APOE2 and / or APOE3 expression is about 10% or less. In one embodiment, RNAi agents are used to treat cells, for example, target mammals, for example, APOE-related neurodegenerative diseases, for example, amyloid-beta-mediated diseases, for example, Alzheimer's disease, Down syndrome, and cerebral amyloid angiopathy, and tau-mediated diseases, for example, primary tauopathy, for example, frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), glial tauopathy (GGT), and Parkinson's disease. This invention includes a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the APOE gene in cells within humans with sonism-associated frontotemporal dementia (FTDP, FTDP-17), chronic traumatic encephalopathy (CTE), boxer's dementia, frontotemporal lobar degeneration (FTLD), argyrophilic granulopathy (AGD), and primary age-related tauopathy (PART), or secondary tauopathy, such as AD, Creutzfeldt-Jakob disease, Down syndrome, and familial British dementia. The dsRNA contains an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during APOE gene expression. The complementary region is approximately 15-30 nucleotides or less in length. When RNAi agents come into contact with cells expressing the APOE gene, they inhibit the expression of the APOE gene (e.g., human gene, primate gene, non-primate gene) by at least 50%, as assayed by, for example, PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence analysis using Western blotting or flow cytometry techniques.In one embodiment, the level of knockdown is assayed with a 10 nM concentration of siRNA in human neuroblastoma BE(2)-C cells using the dual luciferase assay method provided in Example 1 below.

[0257] dsRNA comprises two RNA strands, which are complementary and hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially complementary and generally perfectly complementary to the target sequence. The target sequence can be obtained from the sequence of mRNA formed during the expression of the APOE gene. The other strand (the sense strand) contains a region complementary to the antisense strand, thereby the two strands hybridize to form a double-stranded structure when combined under favorable conditions. As described elsewhere in this specification and known in the art, the complementary sequence of the dsRNA can also be included as a self-complementary region of a single nucleic acid molecule, so as to be relative on separate oligonucleotides.

[0258] Generally, double-stranded structures are 15 to 30 base pairs long, for example, 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, 19-2 The lengths are 9, 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 certain preferred embodiments, the double-stranded structure is 18 to 25 base pairs long, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, or 24-25 base pairs long, e.g., 19-21 base pairs long. It is conceivable that intermediate ranges and lengths between those listed above are also part of this disclosure.

[0259] Similarly, the complementary region to the target sequence is 15 to 30 nucleotides long, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19- 27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotide lengths, for example, 19-23 nucleotide lengths or 21-23 nucleotide lengths. It is conceivable that intermediate ranges and lengths between the above-listed ranges and lengths are also part of this disclosure.

[0260] In some embodiments, dsRNAs are 15 to 23 nucleotides long, or 25 to 30 nucleotides long. Generally, dsRNAs are long enough to function as substrates for Dicer enzymes. 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 recognize, the RNA region targeted for cleavage is in most cases a longer RNA molecule, often a portion of an mRNA molecule. Where applicable, the “portion” of the mRNA target is a sequence of mRNA targets long enough to allow it to be a substrate for RNAi-dependent cleavage (i.e., cleavage via the RISC pathway).

[0261] Those skilled in the art will know that the double-stranded region is the primary functional portion of dsRNA, for example, 15 to 36 base pairs, for example, 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 1 You will also recognize that these are 8-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, for example, a double-stranded region of 19-21 base pairs. Therefore, in one embodiment, an RNA molecule or complex of RNA molecules having a double-stranded region of more than 30 base pairs is a dsRNA, insofar as it is processed into a functional double helix of, for example, 15-30 base pairs, which targets the desired RNA for cleavage. Thus, those skilled in the art will recognize that, in one embodiment, miRNA is a dsRNA. In another embodiment, a 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 dsRNAs.

[0262] The dsRNAs described herein may further include one or more single-stranded nucleotide overhangs, for example, 1, 2, 3, or 4 nucleotides. The nucleotide overhangs may include or consist of nucleotide / nucleoside analogs such as deoxynucleotides / nucleosides. The overhangs may be on the sense strand, on the antisense strand, or in any combination thereof. Furthermore, the nucleotides of the overhangs may be located on the 5' end, the 3' end, or both of either the antisense strand or the sense strand of the dsRNA.

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

[0264] In one embodiment, the dsRNA of the present disclosure comprises at least two nucleotide sequences, namely a sense strand and an antisense strand. The sense strand sequence for APOE may 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 and antisense strand may be selected from the group of sequences in any one of Tables 2-5 and 7-10. In this embodiment, one of the two sequences is complementary to the other of the two sequences, in which case one of the sequences is substantially complementary to the mRNA sequence produced during the expression of the APOE gene. Thus, in this embodiment, the dsRNA would comprise two oligonucleotides, one of which is described as the sense strand (passenger strand) in any one of Tables 2-5 and 7-10 for APOE, and the second oligonucleotide being described as the corresponding antisense strand (guide strand) to the sense strand in any one of Tables 2-5 and 7-10.

[0265] In one embodiment, a substantially complementary sequence to the dsRNA is contained in separate oligonucleotides. In another embodiment, a substantially complementary sequence to the dsRNA is 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. However, it will be understood that the RNA of the RNAi agent of this disclosure, e.g., the dsRNA of this disclosure, may include any one of the sequences described in any one of Tables 2-5 and 7-10, which may be unmodified, unconjugated, or modified or conjugated in a manner different from those described herein. One or more lipophilic ligands and / or one or more GalNAc ligands may be included in any of the positions of the RNAi agent provided in this application.

[0267] Those skilled in the art are well aware that dsRNAs having double-stranded structures of about 20 to 23 base pairs, for example, 21 base pairs, have been welcomed as particularly effective in introducing RNA interference [Elbashir et al., (2001) EMBO J., 20:6877-6888]. However, others have found that shorter or longer RNA double-stranded 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 may include at least one strand of a minimum length of 21 nucleotides. It can be reasonably expected that shorter double-stranded structures, with some nucleotides subtracted from one or both ends, may be equally effective compared to the dsRNAs described above. Therefore, dsRNAs having sequences of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides derived from one of the sequences provided herein, which differ from dsRNAs containing full-length sequences in their ability to inhibit APOE gene expression by only 10, 15, 20, 25, or 30% or less when using in vitro and PCR assays with Cos7 and 10 nM RNA agents, as provided in the examples herein.

[0268] In addition, the RNAs described herein identify sites in the APOE transcript that are susceptible to RISC-mediated cleavage. Therefore, this disclosure further features RNAi agents that target these sites. As used herein, an RNAi agent is said to target a specific site in an RNA transcript if it promotes cleavage of the transcript at any of these specific sites. Such an RNAi agent would generally consist of at least about 15 nucleotides, preferably at least 19 nucleotides, from one of the sequences provided herein coupled to an additional nucleotide sequence taken from a region adjacent to a selected sequence in the APOE gene.

[0269] Therefore, the RNAi agents described herein may contain one or more mismatches with respect to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In a particular embodiment, if the antisense strand of the RNAi agent contains a mismatch with respect to the target sequence, the mismatch may, as appropriate, be limited to within the last five nucleotides from the 5' or 3' end of the complementary region. For example, in such an embodiment, for a 23-nucleotide RNAi agent, the strand complementary to the APOE gene region generally does not contain any mismatches within the central 13 nucleotides. By using the methods described herein or methods known in the art, it is possible to determine whether an RNAi agent containing a mismatch with respect to the target sequence is effective in inhibiting the expression of the APOE gene. In particular, when specific complementary regions in the APOE gene are known to exhibit polymorphic sequence variations within a population, it is important to consider the effectiveness of mismatched RNAi agents in inhibiting APOE gene expression.

[0270] III. Modified RNAi agents of this disclosure In one embodiment, the RNA of the RNAi agent of the Disclosure, e.g., dsRNA, is unmodified and does not contain, for example, chemical modifications or conjugations known in the Art and described herein. In a preferred embodiment, the RNA of the RNAi agent of the Disclosure, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the Disclosure, substantially all of the nucleotides of the RNAi agent of the Disclosure are modified. In other embodiments of the Disclosure, all of the nucleotides of the RNAi agent of the Disclosure are modified. The RNAi agent of the Disclosure that is "substantially all of its nucleotides modified" may be mostly modified, but not entirely, and may contain 5, 4, 3, 2, 1 or unmodified nucleotides. In yet another embodiment of the Disclosure, the RNAi agent of the Disclosure may contain 5, 4, 3, 2 or 1 modified nucleotides.

[0271] The nucleic acids featured in this disclosure can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse linking) or 3'-end modifications (conjugation, DNA nucleotides, reverse linking, etc.), base modifications, such as replacement of stabilizing bases, destabilizing bases or bases that form base pairs with partners in an expanded repertoire, base removal (debasing nucleotides) or conjugated bases, sugar modifications (e.g., at the 2' or 4' position) or sugar replacement, or skeletal modifications including modification or replacement of phosphodiester bonds. Specific examples of RNAi agents useful in the embodiments described herein, but not limited to, RNA containing a modified skeleton or lacking natural nucleoside linkages, include RNA containing a modified skeleton or lacking natural nucleoside linkages. Among RNAs having a modified skeleton, those lacking a phosphorus atom in their skeleton are particularly noteworthy. For the purposes of this specification, as sometimes mentioned in the art, modified RNAs lacking a phosphorus atom in their internucleoside skeleton can also be considered oligonucleosides. In some embodiments, the modified RNAi agent has a phosphorus atom in its internucleoside skeleton.

[0272] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methylphosphonates, and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates having the usual 3'-5' linkage, their analogues with 2'-5' linkages, and those with reverse polarity where adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or 2'-5' to 5'-2'. 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. Patents 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, and 5,286, No. 717, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5, No. 476,925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5,587,361, No. 5,625,050, No. 6,028,188, No. 6,124,445, No. 6,160,109, No. 6,169,170, No. 6,172,2 No. 09, No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,53 Examples include Nos. 4,639, 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 these are incorporated herein by reference.

[0274] Modified RNA skeletons that do not contain phosphorus atoms have skeletons formed by short alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside linkages, or one or more short heteroatoms or heterocyclic nucleoside linkages. These include morpholino linkages (some formed from the sugar moiety of nucleosides), siloxane skeletons, sulfide, sulfoxide and sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamate skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and others having mixed N, O, S and CH2 component moieties.

[0275] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides include, but are not limited to, U.S. Patents 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, and the same. Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439 are examples, the entire contents of each of these are incorporated herein by reference.

[0276] In other embodiments, RNA mimetic compounds suitable for use in RNAi agents are envisioned, in which both the sugar and nucleoside linkages of the nucleotide units, i.e., the backbone, are replaced with novel groups. The base units are maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an RNA mimetic compound 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, in particular an aminoethylglycine backbone. The nucleic acid bases are retained and directly or indirectly bonded to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds, but not limited to, include U.S. Patents 5,539,082, 5,714,331, and 5,719,262, the entire contents of each of which are incorporated herein by reference. Further PNA compounds suitable for use in the RNAi agents of this disclosure are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0277] Some embodiments featured in this disclosure include RNAs and heteroatom skeletons having a phosphorothioate backbone, in particular the --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as the methylene(methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- and oligonucleosides having an amide backbone as referenced in U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure as referenced in U.S. Patent No. 5,034,506. Natural phosphodiester backbones may be represented as OP(O)(OH)-OCH2-.

[0278] Modified RNA may also contain one or more substituted sugar moieties. RNAi agents characterized herein, such as dsRNA, may contain one of the following at the 2' position: OH;F;O-, S- or N-alkyl;O-, S- or N-alkenyl;O-, S- or N-alkynyl or O-alkyl-O-alkyl, where alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 It can be an alkenyl or alkinyl. An exemplary suitable modification is O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)2 is an example, where n and m are approximately 1 to 10. In other embodiments, the dsRNA is located at the 2' position as follows: C1~C 10The modifications include one of the following: lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, interfering substance, group for improving the pharmacokinetic properties of RNAi agents or group for improving the pharmacokinetic properties of RNAi agents, and other substituents having similar properties. In some embodiments, the modifications include 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O--CH2CH2OCH3) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Other exemplary modifications include 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group also known as 2'-DMAOE, as described below in the examples herein, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2. Further exemplary modifications include 5'-Me-2'-F nucleotide, 5'-Me-2'-OMe nucleotide, 5'-Me-2'-deoxynucleotide (both R and S isomers in these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0279] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-O-hexadecyl, and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the RNAi agent, particularly on the 3' terminal nucleotide or at the 3' position and 5' position of the sugar in the 2'-5' ligated dsRNA. The RNAi agent may also have sugar mimetic moieties, such as a cyclobutyl moiety instead of a pentofuranosyl sugar. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patents 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, and 5,56 Examples include patents 7,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, some of which are jointly owned with this application. The entire content of each of the aforementioned is incorporated herein by reference.

[0280] The RNAi agents of this disclosure may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), the pyrimidine base thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include other synthetic and natural nucleic acid bases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and This includes thymine, 5-uracil (pseudolacil), 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, as well as 3-deazaguanine and 3-deazaadenine.Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, 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, those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleic acid bases 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 substitution has been shown to increase nucleic acid double-strand stability by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278), and more specifically, is an exemplary base substitution when combined with 2'-O-methoxyethyl sugar modification.

[0281] Representative U.S. patents teaching the preparation of the above-mentioned modified nucleic acid bases and certain other modified nucleic acid bases include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, and 5,587,469. This includes, for example, Nos. 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088, the entire contents of each of these are incorporated herein by reference.

[0282] In some embodiments, the 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”) is a nucleoside having a sugar moiety that includes a ring formed by bridging two carbons of a sugar ring, whether adjacent or not, thereby forming a bicyclic ring structure. In certain embodiments, the bridging connects the 4'-carbon and 2'-carbon of the sugar ring, optionally via a 2'-acyclic oxygen atom. Thus, in some embodiments, the agents of the present invention may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, wherein the ribose moiety includes an additional bridging that connects the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridging. This structure efficiently “locks” the ribose into a 3'-end 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, Mook, OR. 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 present invention include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, one or more bicyclic nucleosides containing a 4'-to-2' bridge may be used as the antisense polynucleotide agent of the present invention.

[0283] A locked nucleoside has the following structure (stereochemistry is omitted):

[0284] [ka] It can be represented by (wherein B is a nucleic acid base or modified nucleic acid base, and L is a linking group that connects 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'-(CH2)--O-2'(LNA), 4'-(CH2)--S-2', 4'-(CH2)2--O-2'(ENA), 4'-CH(CH3)--O-2' (also called "restricted ethyl" or "cEt") and 4'-CH(CH2OCH3)--O-2' (and its analogues, see, for example, U.S. Patent No. 7,399,845), 4'-C(CH3)(CH3)--O-2' (and its analogues, see, for example, U.S. Patent No. 8, See Patent Nos. 278,283), 4'-CH2-N(OCH3)-2' (and its analogues, e.g., see U.S. Patent No. 8,278,425), 4'-CH2--O--N(CH3)-2' (e.g., see U.S. Patent Publication No. 2004 / 0171570), 4'-CH2-N(R)--O-2' (wherein R is H, C1-C12 alkyl or nitrogen protecting group) (e.g., see U.S. Patent No. 7,427,672), 4'-CH2-C(H)(CH3)-2' (e.g., Chattopadhyaya Examples include 4'-CH2--C(=CH2)-2' (and its analogues, see, for example, U.S. Patent No. 8,278,426), and 4'-CH2--C(=CH2)-2' (see, et al., J. Org. Chem., 2009, 74, 118-134). 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, the following: U.S. Patent Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, and the same. Examples include patent numbers 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 these are incorporated herein by reference.

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

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

[0288] The RNAi agents of this disclosure may also comprise one or more “conformation-restricted nucleotides” (“CRNs”). CRNs are nucleotide analogs having a linker connecting the C2' and C4' carbons of ribose, or the C3 and C5' carbons of ribose. CRNs lock the ribose ring into a stable conformation and increase its hybridization affinity to mRNA. The linker is long enough to position the oxygen optimally for stability and affinity, resulting in less ribose ring puckering.

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

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

[0291] Representative U.S. publications teaching the preparation of UNAs include, but are not limited to, U.S. 8,314,227 and U.S. Patent Publications 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0292] Potentially 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'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyluridine-3"-phosphate, reverse base dT (idT), and others. The disclosure of these modifications can be found in WO2011 / 005861.

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

[0294] A. Modified RNAi agents containing motifs of the present disclosure In certain embodiments of this disclosure, the double-stranded RNAi agents of this disclosure include agents having chemical modifications such as those disclosed in WO2013 / 075035, the entirety of which is incorporated herein by reference. Excellent results can be obtained by introducing one or more motifs of three identical modifications on a triple nucleotide into the sense or antisense strand of the RNAi agent at or near the cleavage site, as shown herein and in WO2013 / 075035. In some embodiments, the sense and antisense strands of the RNAi agent may otherwise be fully modified. The introduction of these motifs disrupts the modification pattern of the sense or antisense strand, if present. The RNAi agent may be conjugated with a lipophilic ligand, for example, a C16 ligand on the sense strand. The RNAi agent may be modified, for example, with (S)-glycol nucleic acid (GNA) modification at one or more residues on the antisense strand. The resulting RNAi agent exhibits excellent gene silencing activity.

[0295] Accordingly, this disclosure provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., the APOE gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be 15 to 30 nucleotides long. For example, each strand may be 16 to 30 nucleotides long, 17 to 30 nucleotides long, 25 to 30 nucleotides long, 27 to 30 nucleotides long, 17 to 23 nucleotides long, 17 to 21 nucleotides long, 17 to 19 nucleotides long, 19 to 25 nucleotides long, 19 to 23 nucleotides long, 19 to 21 nucleotides long, 21 to 25 nucleotides long, or 21 to 23 nucleotides long. In a particular embodiment, each strand is 19 to 23 nucleotides long.

[0296] The sense strand and antisense strand typically form a double-stranded RNA ("dsRNA"), also referred herein as the "RNAi agent." The double-stranded region of the RNAi agent may be 15–30 nucleotide pairs long. For example, the double-stranded 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, 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 21–23 nucleotide pairs long. In another example, the double-stranded region is selected from lengths of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides. In a preferred embodiment, the double-stranded region is 19–21 nucleotide pairs long.

[0297] In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhangs may be 1 to 6 nucleotides long, for example, 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides. In a preferred embodiment, the nucleotide overhang region is 2 nucleotides long. The overhangs may result from one strand being longer than the other or from two strands of equal length being twisted. The overhangs may form a mismatch with the target mRNA, or may be complementary to the gene sequence being targeted, or may be a different sequence. The first and second strands may also be joined, for example, by additional bases forming a hairpin, or by other non-base linkers.

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

[0299] For example, TT could be an overhang sequence at any end of either strand. The overhang could form a mismatch with the target mRNA, or it could be complementary to the targeted gene sequence, or it could be a different sequence altogether.

[0300] The sense strand, antisense strand, or 5'- or 3'-overhangs of both strands of an RNAi agent can be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between them, and the two nucleotides may be identical or different. In one embodiment, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3'-overhang is located in the antisense strand. In one embodiment, this 3'-overhang is located in the sense strand.

[0301] RNAi agents may contain only a single overhang that can enhance RNAi interference activity without affecting their overall stability. For example, a single-stranded overhang may be located at the 3' end of the sense strand or the 3' end of the antisense strand. RNAi may 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 a blunt end at the 5' end. Without getting bogged down in theory, the blunt end at the 5' end of an asymmetric antisense strand and the 3' end overhang of the antisense strand are advantageous for guide strand loading into RISC processes.

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

[0303] In another embodiment, the RNAi agent is blunt-terminated at both ends and 20 nucleotides long, with the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

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

[0305] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, with one end of the RNAi agent being blunt and the other end containing a 2-nucleotide overhang. Preferably, the 2-nucleotide overhang is at the 3' end of the antisense strand. If the 2-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate nucleotide linkages between the three terminal nucleotides, where two of the three nucleotides are the overhang nucleotide and the third nucleotide is the nucleotide that follows the overhang nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate nucleotide linkages between the three terminal 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 strand and antisense strand of the RNAi agent, including a nucleotide that is part of a motif, is a modified nucleotide. In one embodiment, each residue is independently modified, for example, with 2'-O-methyl or 2'-fluoro in an alternating motif. The RNAi agent may further contain a ligand (e.g., a lipophilic ligand, optionally a C16 ligand).

[0306] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the sense strand being 25-30 nucleotides long and starting from the 5' terminal nucleotide (position 1), positions 1-23 of the first strand containing at least 8 ribonucleotides; the antisense strand being 36-66 nucleotides long and starting from the 3' terminal nucleotide, containing at least 8 ribonucleotides at positions 1-23 of the sense strand to form a double helix, at least 3' terminal nucleotides of the antisense strand not pairing with the sense strand, up to 6 consecutive 3' terminal nucleotides not pairing with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides, and the 5' end of the antisense strand containing 10-30 consecutive nucleotides not pairing with the sense strand The sense strand contains a conjugate nucleotide, thereby forming a single-stranded 5' overhang of 10–30 nucleotides, and at least the 5' and 3' terminal nucleotides of the sense strand are bases that pair with the nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, the antisense strand being sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand, and reducing target gene expression when the double-stranded nucleic acid is introduced into mammalian cells, the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of which occurs at or near the cleavage site, and 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 sense and antisense strands, the RNAi agent comprising a first strand having a length of at least 25 and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides and having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand form blunt ends, the second strand is 1 to 4 nucleotides longer at its 3' end than the first strand, the double-stranded region is at least 25 nucleotides long, the second strand is sufficiently complementary to the target mRNA along the length of the second strand of at least 19 nucleotides, and the RNAi agent reduces the expression of the target gene when introduced into mammalian cells, the dicer cleavage of the RNAi agent preferentially yields the siRNA including the 3' end of the second strand, thereby reducing the expression of the target gene in mammals. 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 a triple nucleotide sequence, one of which occurs at a cleavage site in the sense strand.

[0309] In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif of three identical modifications on a triple nucleotide, one of which occurs at or near a cleavage site in the antisense strand.

[0310] For RNAi agents having a double-stranded region of 17–23 nucleotides in length, the cleavage sites on the antisense strand are typically at positions 10, 11, and 12 from the 5' end. Therefore, three identical modification motifs may occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, with the number starting from the first nucleotide from the 5' end of the antisense strand, or the number starting from the first pair-formed nucleotide within the double-stranded region from the 5' end of the antisense strand. The cleavage sites in the antisense strand may also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0311] The sense strand of an RNAi agent may contain at least one motif of three identical modifications on a triple nucleotide at the cleavage site of the strand, and the antisense strand may have at least one motif of three identical modifications on a triple nucleotide at or near the cleavage site of the strand. When the sense strand and antisense strand form a dsRNA double helix, the sense strand and antisense strand can be sequenced such that one motif of three nucleotides on the sense strand and one motif of three nucleotides on the antisense strand have at least one nucleotide duplication, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may be duplicated, or all three nucleotides may be duplicated.

[0312] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications on a triple nucleotide sequence. The first motif may occur at or near a cleavage site on the strand, and the other motifs may be wing modifications. In this specification, the term “wing modification” refers to a motif occurring on a different part of the strand, away from the motif at or near the cleavage site on the same strand. Wing modifications are either adjacent to the first motif or at least one or more nucleotides away. If the motifs are immediately adjacent to each other, their chemistry is distinct from each other; if the motifs are one or more nucleotides away, their chemistry may be identical or different. There may be two or more wing modifications. For example, if there are two wing modifications, each wing modification may occur at one end relative to the first motif at or near the cleavage site, or on either side of the read motif.

[0313] Similar to the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications on a triple nucleotide sequence, with at least one motif occurring at or near a cleavage site on the strand. This antisense strand may also contain one or more wing modifications in a sequence similar to those 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, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two pairs of nucleotides in the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0316] If the sense strand and antisense strand of an RNAi agent each contain at least one wing modification, the wing modification may be located at the same end of the double-stranded region and may have one, two, or three nucleotide duplicates.

[0317] If the sense strand or antisense strand of the RNAi agent each contains at least two wing modifications, the sense strand and antisense strand can be arranged such that two modifications from one strand each enter one end of the double-stranded region with 1, 2, or 3 nucleotide duplicates, and two modifications from one strand each enter the other end of the double-stranded region with 1, 2, or 3 nucleotide duplicates, and one strand of the two modifications enters each side of the read motif with 1, 2, or 3 nucleotide duplicates in the double-stranded region.

[0318] In one embodiment, the RNAi agent comprises a double-stranded mismatch(s) or combination thereof with the target. Mismatches may occur in overhang regions or double-stranded regions. Base pairs can be ranked based on their tendency to promote dissociation or dissolution (e.g., by the free energy of association or dissociation of a particular pairing, the simplest approach being to examine pairs on a basis of individual pairs, although the following adjacent analysis or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical pairing or non-canonical pairing (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairing, and pairings involving universal bases are preferred over canonical pairing.

[0319] In one embodiment, the RNAi agent includes at least one of the first 1, 2, 3, 4, or 5 base pairs in the double-stranded region from the 5' end of the antisense strand, independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical pairing or pairing other than canonical pairing or pairing including universal bases, in order to promote the dissociation of the antisense strand at the 5' end of the double helix.

[0320] In one embodiment, the nucleotide at position 1 in the double-strand region from the 5' end of 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 double-strand region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-strand 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 deoxythymidine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, e.g., two dT nucleotides at the 3' end of the sense or antisense strand.

[0322] In one embodiment, the sense strand sequence is given by formula (I): 5'n p -N a -(XXX) i -N b -YY -N b -(ZZ )Z j -N a -n q 3' (I) [In the formula, i and j are independently either 0 or 1. p and q are each independently between 0 and 6. each N a Each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two differently modified nucleotides. each N b Each independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides. each n p and n q These independently represent overhang nucleotides, Nb and Y do not have the same modifications, and XXX, YYY, and ZZZ each independently represent one motif of three identical modifications on a sequence of three nucleotides. It can be represented by the following. Preferably, all YYY nucleotides are 2'-F modified nucleotides.

[0323] In one embodiment, N a or N b This includes alternating modification patterns.

[0324] In one embodiment, the YYY motif occurs at or near the sense strand cleavage site. For example, if the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the YYY motif may occur at or near the sense strand cleavage site (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13), the number may start from the first nucleotide from the 5' end, or, as appropriate, the number may start from the first pair-formed nucleotide in the double-stranded region from the 5' end.

[0325] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Therefore, the sense chain is given by the following equation: 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) It can be represented by [this].

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

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

[0328] If the sense chain is expressed as equation (Ic), then N b This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. a These may also independently represent oligonucleotide sequences containing 2–20, 2–15, or 2–10 modified nucleotides.

[0329] When the sense chain is expressed as formula (Id), each N b Each independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6. a These can also independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

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

[0331] In another embodiment, i is 0, j is 0, and the sense chain is given by: 5' n p -N a -YYY-N a -n q 3' (Ia) It can be represented by [this].

[0332] If the sense chain is represented by equation (Ia), then each N a These may independently contain oligonucleotide sequences comprising 2-20, 2-15, or 2-10 modified nucleotides.

[0333] In one embodiment, the antisense strand sequence of RNAi is given by formula (II): 5' n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p ' 3' (II) [In the formula, k and l are independently either 0 or 1. p' and q' are each independently between 0 and 6. each N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two differently modified nucleotides. each N b 'Independently, this represents an oligonucleotide sequence containing 0 to 10 modified nucleotides, each n p 'and n q ' independently represents an overhang nucleotide, N b 'and Y' do not have the same modifier, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on a triple nucleotide chain. It can be represented by [this].

[0334] In one embodiment, N a'or N b ' includes alternating modification patterns.

[0335] The 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 of 17-23 nucleotides in length, the Y'Y'Y' motif may occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, with the number starting from the first nucleotide from the 5' end, or optionally starting from the first pair-formed nucleotide in the double-stranded region from the 5' end. Preferably, the Y'Y'Y' motif occurs at positions 11, 12, and 13.

[0336] In one embodiment, the Y'Y'Y' motif consists entirely of nucleotides modified with 2'-OMe.

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

[0338] Therefore, the antisense chain is given by the following equation: 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) It can be represented by [this].

[0339] If the antisense chain is represented by equation (IIb), then N b ’ This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0340] If the antisense chain is expressed as equation (IIc), then N b ' represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0341] When the antisense chain is expressed as equation (IId), each N b ' independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6.

[0342] In another embodiment, k is 0, l is 0, and the antisense chain is given by: 5' n p’ -N a’ -Y'Y'Y'- N a’ -n q’ 3' (Ia) It can be represented by [this].

[0343] If the antisense chain is expressed as equation (IIa), then each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0344] Each of X', Y', and Z' may be identical or different from the others.

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

[0346] In one embodiment, the sense strand of the RNAi agent may contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the double-stranded region is 21nt, the number starting from the first nucleotide from the 5' end, or optionally starting from the first pair-formed nucleotide in the double-stranded region from the 5' end, where Y represents a 2'-F modification. The sense strand may further contain an XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region, where XXX and ZZZ independently represent a 2'-OMe modification or a 2'-F modification.

[0347] In one embodiment, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, and 13 of the strand, the number starting from the first nucleotide from the 5' end, or optionally starting from the first pair-formed nucleotide in the double-stranded region from the 5' end, where Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region, where X'X'X' and Z'Z'Z' independently represent a 2'-OMe modification or a 2'-F modification.

[0348] A sense strand represented by any one of the above equations (Ia), (Ib), (Ic), and (Id) forms a double helix with an antisense strand represented by any one of the above equations (IIa), (IIb), (IIc), and (IId).

[0349] Therefore, the RNAi agent for use in the method of this disclosure may include a sense strand and an antisense strand, each having 14 to 30 nucleotides, and the RNAi double helix is ​​given by formula (III): Sense: 5' n p -N a -(XXX) i -N b - YYY -N b -(ZZZ) 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 either 0 or 1. p, p', q, and q' are each independently between 0 and 6. each N a and N a ’ Each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two differently modified nucleotides. each N b and N b ’ Each independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides. each n p ',np , n q 'and n q Each of these may or may not be present, but they independently represent an overhang nucleotide. XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on three consecutive nucleotides. It is represented by [this].

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

[0351] An exemplary combination of sense and antisense strands that form an RNAi double helix is ​​given by the following formula: 5' n p - N a -YYY -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y' -N a ’ n q ’ 5' (IIIa) 5' n p -N a -YYY -N b -ZZZ -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q’ 5' (IIIb) 5' n p -N a - XXX -N b -YYY - 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 -XXX -N b -YYY -N b - ZZZ -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 ’ 5' (IIId) Includes.

[0352] When an RNAi agent is represented by formula (IIIa), each N a Each of these independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

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

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

[0355] When an RNAi agent is represented by formula (IIId), each N b , N b ' independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a , N a ’ N independently represents oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides. a , N a ', N b and N b ’ Each of these independently includes alternating modification patterns.

[0356] In one embodiment, if the RNAi agent is represented by formula (IIId), then N a The modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, if the RNAi agent is represented by formula (IIId), then N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is linked to an adjacent nucleotide via phosphorothioate linkage. In yet another embodiment, if the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, np '>0 and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more C16 (or related) portions attached by a divalent or trivalent branched linker (described below). In another embodiment, if the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The sense strand is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, for example, C16 (or related) moieties, which may be attached by a divalent or trivalent branched linker.

[0357] In one embodiment, when the RNAi agent is represented by formula (IIIa), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The sense strand is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand contains at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties attached by a divalent or trivalent branched linker.

[0358] In one embodiment, the RNAi agent is a multimer containing at least two double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the double helixes being linked by a linker. The linker may or may not be cleavable. The multimer may further contain ligands. Each double helix may target the same gene, or two different genes, or each double helix may target the same gene at two different target sites.

[0359] In one embodiment, the RNAi agent is a multimer containing 3, 4, 5, 6 or more double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), where the double helixes are linked by linkers. The linkers may or may not be cleavable. The multimer may further contain ligands. Each double helix may target the same gene, or two different genes, or each double helix may target the same gene at two different target sites.

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

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

[0362] In certain embodiments, the compositions and methods of the Disclosure include vinyl phosphonate (VP) modification of an RNAi agent as described herein. In exemplary embodiments, the 5'-vinyl phosphonate of the Disclosure has the following structure:

[0363] [ka] [In the formula, X is either O or S; R is hydrogen, hydroxyl, fluoro, or C1~20 It is an alkoxy (e.g., methoxy or n-hexadecyloxy); R 5’ This is =C(H)-P(O)(OH)2, with C5' carbon and R 5’ The double bond between them is in an E or Z configuration (for example, an E configuration); B is a nucleic acid base or a modified nucleic acid base, and here B may be adenine, guanine, cytosine, thymine, or uracil.

[0364] The vinyl phosphonates of the Disclosure may be attached to either the antisense or sense strand of the dsRNA of the Disclosure. In certain embodiments, the vinyl phosphonates of the Disclosure may be attached to the antisense strand of the dsRNA at its 5' end, as appropriate.

[0365] Vinyl phosphate modifications are also intended for the compositions and methods of this disclosure. The above structure is an example of a vinyl phosphate structure, where R 5’ This is =C(H)-OP(O)(OH)2, with C5' carbon and R 5’ The double bond between them is in an E or Z configuration (for example, an E configuration).

[0366] E. Thermal destabilization modification In certain embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating thermal destabilization modifications within 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. dsRNAs having an antisense strand containing at least one double-strand thermal destabilization modification within the first nine nucleotide positions counting from the 5' end of the antisense strand have been found to exhibit reduced off-target gene silencing activity. Therefore, in some embodiments, the antisense strand contains at least one (e.g., 1, 2, 3, 4, 5 or more) double-strand thermal destabilization modification within the first nine nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more double-strand thermal destabilization modifications are located within positions 2–9, or preferably 4–8, from the 5' end of the antisense strand. In some further embodiments, the double-strand thermal destabilization modifications are located at positions 6, 7, or 8 from the 5' end of the antisense strand. In some further embodiments, the double-strand thermal destabilization modification is located at position 7 from the 5' end of the antisense strand. The term “thermal destabilization modification” includes modifications(or modifications) that would result in a dsRNA having a lower overall melting temperature (Tm) (preferably 1, 2, 3, or 4 degrees lower than the Tm of a dsRNA without such modifications(or modifications). In some embodiments, the double-strand thermal destabilization modification is located at positions 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0367] Examples of thermal destabilization modifications, though not limited to these, include debasing modifications, mismatches with opposing nucleotides on opposing chains, and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs).

[0368] Examples of debase modification include, but are not limited to, the following:

[0369] [ka] [In the formula, R = H, Me, Et or OMe; R' = H, Me, Et or OMe; R” = H, Me, Et or OMe]

[0370] [ka] [In the formula, B is a modified or unmodified nucleic acid base.] These are some examples.

[0371] Examples of sugar modifications include, but are not limited to, the following:

[0372] [ka] [In the formula, B is a modified or unmodified nucleic acid base.] These are some examples.

[0373] In some embodiments, the thermal destabilization modification of the double chain is as follows:

[0374] [ka] [In the formula, B is a modified or unmodified nucleic acid base, and each asterisk in the structure represents either R, S, or racemic.] It is selected from the group consisting of the following.

[0375] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar in which, for example, one of the bonds between 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 oxygen atoms (e.g., C1', C2', C3', C4', or O4') is absent independently or in combination in the nucleotide. In some embodiments, acyclic nucleotides are,

[0376] [ka] [In the formula, B is a modified or unmodified nucleic acid base, and R 1 and R 2 R3 is independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which any sugar bond has been removed, forming an unlocked "sugar" residue. In one example, UNA also includes monomers in which the bond between C1'-C4' (i.e., the carbon-oxygen-carbon bond of the covalent bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon bond of the covalent bond between the C2' and C3' carbons) has been removed [see Mikhailov et al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), the whole of which is incorporated herein by reference]. Acyclic derivatives offer greater skeletal flexibility without affecting Watson-Crick pair formation. Acyclic nucleotides can be linked via 2'-5' or 3'-5' ligatures.

[0377] The term "GNA" refers to glycol nucleic acids, which are polymers similar to DNA or RNA, but differ in the composition of their "backbone" in that it consists of repeating glycerol units linked by phosphodiester bonds.

[0378] [ka]

[0379] Double-strand thermal destabilization modifications can be a mismatch (i.e., a non-complementary base pair) between a thermally destabilized nucleotide and an opposing nucleotide in the opposing strand within the dsRNA double-strand. Exemplary mismatch base pairs include 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 pair formations known in the art are also suitable for the present invention. Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides; that is, mismatch base pair formation can occur between nucleic acid bases derived from each nucleotide independently of modifications on the ribose sugar of the nucleotides. In certain embodiments, the dsRNA molecule contains at least one nucleic acid base in the mismatch pair formation, for example, the 2'-deoxynucleotide is in the sense strand.

[0380] In some embodiments, thermal destabilization modification of the double helix in the seed region of the antisense strand results in a nucleotide whose WHC bond with the complementary base on the target mRNA is impaired, for example:

[0381] [ka] Includes.

[0382] More examples of debasalized 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] Thermal destabilization modifications may also include universal base and phosphate modifications in which the ability to form hydrogen bonds with opposing bases is reduced or lost.

[0384] In some embodiments, thermal destabilization modifications of the double helix include nucleotides with non-canonical bases, for example, but not limited to, nucleic acid base modifications in which the ability to form hydrogen bonds with bases in the opposing strand is impaired or completely lost. These nucleic acid base modifications have been evaluated for destabilization of the central region of the dsRNA double helix, as described in WO2010 / 0011895, which is incorporated in its entirety herein by reference. Exemplary nucleic acid base modifications include:

[0385] [ka]

[0386] In some embodiments, the thermal destabilization modification of the double helix in the seed region of the antisense strand involves one or more α-nucleotides complementary to the base on the target mRNA, for example:

[0387] [ka] [In the formula, R is H, OH, OCH3, F, NH2, NHMe, NMe2, or O-alkyl] It includes.

[0388] As an example of phosphate modifications known to reduce the thermal stability of dsRNA double helix compared to natural phosphodiester bonds:

[0389] [ka]

[0390] The alkyl group of the R group can be C1-C6 alkyl. Specific examples of alkyl groups of the R group, though not limited to these, include methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0391] As those skilled in the art will recognize, given that the functional roles of nucleic acid bases define the specificity of the RNAi agents of this disclosure, nucleic acid base modifications can be carried out in various ways as described herein, for example, to enhance on-target effects against off-target effects, or to introduce destabilizing modifications into the RNAi agents of this disclosure. However, the range of modifications available and generally present on the RNAi agents of this disclosure tends to be greater with respect to non-nucleonucleotide 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 and are explicitly intended for the RNAi agents of this disclosure having either natural nucleic acid bases or modified nucleic acid bases, as described above or elsewhere herein.

[0392] In addition to the antisense strand containing thermal destabilization modifications, the dsRNA may also contain one or more stabilization modifications. For example, the dsRNA may contain at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilization modifications. While not limiting, all stabilization modifications may be present on one of the strands. In some embodiments, both the sense and antisense strands contain at least two stabilization modifications. Stabilization modifications can occur on any nucleotide of the sense or antisense strand. For example, a stabilization modification may occur on any nucleotide on the sense or antisense strand, each stabilization modification may occur in an alternating pattern on the sense or antisense strand, or both the sense or antisense strand may contain stabilization modifications in an alternating pattern. The alternating pattern of stabilization modifications on the sense strand may be identical or different to that on the antisense strand, and the alternating pattern of stabilization modifications on the sense strand may have a shift compared to the alternating pattern of stabilization modifications on the antisense strand.

[0393] In some embodiments, the antisense chain includes at least two stabilizing modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). Stabilizing modifications in the antisense chain may be located at any position, but are not limited. In some embodiments, the antisense includes stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense includes stabilizing modifications at positions 2, 6, 14, and 16 from the 5' end. In some further embodiments, the antisense includes stabilizing modifications at positions 2, 14, and 16 from the 5' end.

[0394] In some embodiments, the antisense strand includes at least one stabilizing modification adjacent to the destabilizing modification. For example, the stabilizing modification may be a 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 includes stabilizing modifications at each of the 5' and 3' ends of the destabilizing modification, i.e., at positions -1 and +1 from the position of the destabilizing modification.

[0395] In some embodiments, the antisense chain includes 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 chain includes at least two stabilization modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). Stabilization modifications in the sense chain may be located at any position, but are not limited to these. In some embodiments, the sense chain includes stabilization modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense chain includes stabilization modifications at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense chain includes stabilization modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense chain, counting from the 5' end of the antisense chain. In some other embodiments, the sense chain includes stabilization modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense chain, counting from the 5' end of the antisense chain. In some embodiments, the sense chain includes blocks of two, three, or four stabilization modifications.

[0397] In some embodiments, the sense chain does not contain stabilizing modifications in positions that counteract or complement the thermal destabilizing modifications of the double chain in the antisense chain.

[0398] Examples of thermal stabilization modifications include, but are not limited to, 2'-fluoro modifications. Other examples of thermal stabilization modifications include, but are not limited to, LNA.

[0399] In some embodiments, the dsRNA of this disclosure contains at least four (e.g., 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. Not limited to, all 2'-fluoronucleotides may be present in one of the strands. In some embodiments, both the sense and antisense strands contain at least two 2'-fluoronucleotides. 2'-fluoro modifications may occur on any nucleotide of the sense or antisense strand. For example, a 2'-fluoro modification may occur on any nucleotide on the sense or antisense strand, each 2'-fluoro modification may occur in an alternating pattern on the sense or antisense strand, or both the sense or antisense strand may contain 2'-fluoro modifications in an alternating pattern. The alternating pattern of 2'-fluoro modifications on the sense strand may be identical or different from that on the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand may have a shift compared to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0400] In some embodiments, the antisense chain contains at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. While not limiting, 2'-fluoro modifications in the antisense chain can be located at any position. In some embodiments, the antisense contains 2'-fluoronucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense contains 2'-fluoronucleotides at positions 2, 6, 14, and 16 from the 5' end. In yet another embodiment, the antisense contains 2'-fluoronucleotides at positions 2, 14, and 16 from the 5' end.

[0401] In some embodiments, the antisense strand includes at least one 2'-fluoronucleotide adjacent to the destabilization modification. For example, the 2'-fluoronucleotide may be at the 5' or 3' end of the destabilization modification, i.e., at position -1 or +1 from the position of the destabilization modification. In some embodiments, the antisense strand includes 2'-fluoronucleotides at each of the 5' and 3' ends of the destabilization modification, i.e., at positions -1 and +1 from the position of the destabilization modification.

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

[0403] In some embodiments, the sense strand contains at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. While not limiting, 2'-fluoro modifications in the sense strand can be present at any position. In some embodiments, the antisense strand contains 2'-fluoronucleotides at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand contains 2'-fluoronucleotides at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand contains 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 contains 2'-fluoronucleotides at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand contains blocks of 2, 3, or 4 2'-fluoronucleotides.

[0404] In some embodiments, the sense strand does not contain a 2'-fluoronucleotide in a position opposite or complementary to the thermal destabilization modification of the double helix in the antisense strand.

[0405] In some embodiments, the dsRNA molecule of the present disclosure comprises a sense strand of 21 nucleotides (nt) and an antisense strand of 23 nucleotides (nt), wherein the antisense strand contains at least one thermally destabilized nucleotide, the at least one thermally destabilized nucleotide occurring in the seed region of the antisense strand (i.e., at position 2-9 at the 5' end of the antisense strand), one end of the dsRNA is blunt, the other end contains a 2nt overhang, and the dsRNA further has at least one of the following features (e.g., 1, 2, 3, 4, 5, 6, or all of 7): (i) The antisense strand contains 2, 3, 4, 5 or 6 2'-fluoro modifications; (ii) The antisense strand contains 1, 2, 3, 4 or 5 phosphorothioate nucleotide linkages; (iii) The sense strand is conjugated with a 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 phosphorothioate nucleotide linkages; (vi) The dsRNA contains at least 4 2'-fluoro modifications; and (vii) The dsRNA has a blunt end at the 5' end of the antisense strand. Preferably, the 2nt overhang is at the 3' end of the antisense.

[0406] In some embodiments, the dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, the sense strand being 25–30 nucleotides long and starting from the 5' terminal nucleotide (position 1), positions 1–23 of the sense strand contain at least 8 ribonucleotides; the antisense strand being 36–66 nucleotides long and starting from the 3' terminal nucleotide, at least 8 ribonucleotides in positions that pair with positions 1–23 of the sense strand form a double helix; at least 3' terminal nucleotides of the antisense strand do not pair with the sense strand, up to 6 consecutive 3' terminal 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 contains 10–30 consecutive nucleotides that do not pair with the sense strand. The antisense strand contains a single-stranded 5' overhang of 10–30 nucleotides, the sense strand's 5' and 3' terminal nucleotides being bases that pair with the antisense strand's nucleotides when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, the antisense strand being sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand's length, and reducing target gene expression when the double-stranded nucleic acid is introduced into mammalian cells, the antisense strand containing at least one thermally destabilized nucleotide, the at least one of which is located in the seed region of the antisense strand (i.e., at positions 2–9 at the 5' end of the antisense strand).For example, thermally destabilized nucleotides occur between positions opposite or complementary to positions 14-17 at the 5' end of the sense strand, and the dsRNA may further have at least one of the following features (e.g., all 1, 2, 3, 4, 5, 6, or 7): (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate nucleotide linkages; (iii) the sense strand is conjugated with a 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 phosphorothioate nucleotide linkages; (vi) the dsRNA contains at least 4 2'-fluoro modifications; and (vii) the dsRNA contains a double-stranded region 12-30 nucleotide pairs long.

[0407] In some embodiments, the dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, the dsRNA molecule comprising a sense strand having a length of at least 25 and at most 29 nucleotides, and an antisense strand having a length of at most 30 nucleotides, wherein the sense strand comprises a modified nucleotide sensitive to enzymatic degradation at position 11 from its 5' end, the 3' end of the sense strand and the 5' end of the antisense strand form a blunt end, the antisense strand is 1 to 4 nucleotides longer at its 3' end than the sense strand, the double-stranded region is at least 25 nucleotides long, the antisense strand is sufficiently complementary to the target mRNA along the length of the antisense strand by at least 19 nucleotides, the dsRNA molecule reduces target gene expression when introduced into mammalian cells, the dicer cleavage of the dsRNA preferentially yields siRNA including the 3' end of the antisense strand, thereby reducing target gene expression in mammals, the antisense strand is less Each contains one thermally destabilized nucleotide, and at least one thermally destabilized nucleotide is 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 features (e.g., all of 1, 2, 3, 4, 5, 6, or 7): (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications, (ii) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications, (ii) (iii) the antisense strand contains 1, 2, 3, 4 or 5 phosphorothioate nucleotide interlinks, (iv) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications, (v) the sense strand contains 1, 2, 3, 4 or 5 phosphorothioate nucleotide interlinks, (vi) the dsRNA contains at least 4 2'-fluoro modifications, and (vii) the dsRNA has a double-stranded region of 12–29 nucleotide pairs in length.

[0408] In some embodiments, any nucleotide in the sense and antisense strands of a dsRNA molecule may be modified. Each nucleotide may be modified by the same or different modifications, which may include alterations of one or more unbound phosphate oxygens, or one or more bound phosphate oxygens, alterations of the 2' hydroxyl group on the ribose sugar components, large-scale substitution of the phosphate moiety with a "dephospho" linker, modifications or substitutions of naturally occurring bases, and substitutions or modifications of the ribose-phosphate backbone.

[0409] Since nucleic acids are polymers of subunits, many modifications occur at repeating positions within the nucleic acid, for example, modifications of bases or phosphate moieties or unbound oxygen atoms of phosphate moieties. In some cases, modifications occur at all target positions in the nucleic acid, but often they do not. For example, modifications may occur only at the 3' or 5' end, or only in the terminal region, for example, at the terminal nucleotides of the strand, or at the last 2, 3, 4, 5, or 10 nucleotides. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in the double-stranded regions of RNA, or only in the single-stranded regions of RNA. For example, phosphorothioate modifications at unbound oxygen atoms may occur only at one or both ends, or only in the terminal region, for example, at the terminal nucleotides of the strand, or at the last 2, 3, 4, 5, or 10 nucleotides, or in both double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends may be phosphorylated.

[0410] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in single-stranded overhangs, e.g., in the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, with modifications described herein. Modifications may include, for example, the use of 2'-position modification of ribose sugars in modifications known in the art, e.g., the use of 2'-deoxy-2'-fluoro(2'-F) or 2'-O-methyl-modified deoxyribonucleotides instead of ribosaccharides in nucleic acid bases, and modifications at phosphate groups, e.g., phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0411] In some embodiments, each residue in the sense and antisense chains is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. The chains may contain two or more modifications. In some embodiments, each residue in the sense and antisense chains is independently modified with 2'-O-methyl or 2'-fluoro. It should be understood that these modifications are in addition to at least one thermal destabilization modification of the double helix present in the antisense chain.

[0412] At least two distinct modifications are typically present on the sense and antisense strands. These two modifications may include 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense and antisense strands each contain two distinctly modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue in the sense and antisense strands is independently modified with 2'-O-methyl nucleotide, 2'-deoxy nucleotide, 2'-deoxy-2'-fluoro nucleotide, 2'-ON-methylacetamide (2'-O-NMA) nucleotide, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, 2'-O-aminopropyl (2'-O-AP) nucleotide, or 2'-ala-F nucleotide. Again, it should be understood that these modifications are in addition to at least one thermal destabilization modification of the double helix present in the antisense strand.

[0413] In some embodiments, the dsRNA molecules of this disclosure include alternating pattern modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions. The terms “alternating motif” or “alternating pattern,” as used herein, refer to a motif having one or more modifications, each modification occurring in alternating nucleotides on a single strand. Alternating nucleotides may refer to one every other nucleotide, one every three nucleotides, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif may be “ABABABABABAB…”, “AABBAABBAABB…”, “AABAABAABAAB…”, “AAABAAABAAAB…”, “AAABBBAAABBB…”, or “ABCABCABCABC…”.

[0414] The types of modifications contained within an alternating motif may be identical or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating pattern, i.e., the modifications on every other nucleotide, may be identical, but each of the sense strand or antisense strand may be selected from several possible modifications within the alternating motif, such as "ABABAB…", "ACACAC…", "BDBDBD…", or "CDCDCD…".

[0415] In some embodiments, the dsRNA molecules of this disclosure include 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 may be such that modified groups of nucleotides on the sense strand correspond to differently modified groups of nucleotides on the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA double helix, the alternating motifs on the sense strand may begin with "ABABAB" from 5'-3' of the strand, and the alternating motifs on the antisense strand may begin with "BABABA" from 3'-5' of the strand in the double helix region. As another example, the alternating motifs on the sense strand may begin with "AABBAABB" from 5'-3' of the strand, and the alternating motifs on the antisense strand may begin with "BBAABBAA" at 3'-5' of the strand in the double helix region, resulting in a complete or partial shift of the modification patterns between the sense and antisense strands.

[0416] The dsRNA molecules of this disclosure may further include at least one phosphorothioate or methylphosphonate internucleotide ligation. Phosphothioate or methylphosphonate internucleotide ligation modifications may occur at any position on the chain, on the sense strand, the antisense strand, or on any nucleotide of both. For example, an internucleotide ligation modification may occur on any nucleotide on the sense strand or the antisense strand, each internucleotide ligation modification may occur in an alternating pattern on the sense strand or the antisense strand, or the sense strand or the antisense strand may contain both internucleotide ligation modifications in an alternating pattern. The alternating pattern of internucleotide ligation modifications on the sense strand may be identical or different to that on the antisense strand, and the alternating pattern of internucleotide ligation modifications on the sense strand may have a shift relative to the alternating pattern of internucleotide ligation modifications on the antisense strand.

[0417] In some embodiments, the dsRNA molecule includes phosphorothioate or methylphosphonate internucleotide ligation modifications within the overhang region. For example, the overhang region includes two nucleotides having a phosphorothioate or methylphosphonate internucleotide ligation between them. The internucleotide ligation modifications may also be made to ligate the overhang nucleotides to the terminal pair-forming nucleotides in the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be ligated by phosphorothioate or methylphosphonate internucleotide ligations, and there may be further phosphorothioate or methylphosphonate internucleotide ligations ligating the overhang nucleotides to the pair-forming nucleotides adjacent to the overhang nucleotides. For example, there may be at least two phosphorothioate internucleotide ligations between three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third is the pair-forming nucleotide adjacent to the overhang nucleotide. Preferably, these terminal 3 nucleotides may be the 3' end of the antisense strand.

[0418] In some embodiments, the sense strand of a dsRNA molecule comprises 1 to 10 blocks of 2 to 10 phosphorothioate or methylphosphonate nucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate nucleotide links, one of which is positioned at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide links, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

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

[0420] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate nucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate nucleotide links, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide links, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

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

[0422] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkage.

[0423] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkage.

[0424] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide links, separated by one, two, three, four, five, six, seven, or eight phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

[0425] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide links, separated by one, two, three, four, five, or six phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

[0426] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate nucleotide links, separated by one, two, three, or four phosphate nucleotide links, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide links, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

[0427] In some embodiments, the dsRNA molecules of this disclosure further include one or more phosphorothioate or methylphosphonate internucleotide ligation modifications within 1 to 10 terminal positions of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be ligated by phosphorothioate or methylphosphonate internucleotide ligations at one or both ends of the sense or antisense strand.

[0428] In some embodiments, the dsRNA molecules of this disclosure further include one or more phosphorothioate or methylphosphonate nucleotide ligation modifications within 1 to 10 of the internal regions of each of the sense or antisense strands of the double helix. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be ligated by phosphorothioate-methylphosphonate ligations at positions 8 to 16 of the double helix region, counting from the 5' end of the sense strand. The dsRNA molecules may further include one or more phosphorothioate or methylphosphonate nucleotide ligation modifications within terminal positions 1 to 10.

[0429] In some embodiments, the dsRNA molecule of the present disclosure further comprises 1 to 5 phosphorothioate or methylphosphonate internucleotide ligation modifications within positions 1 to 5 of the sense strand and 1 to 5 phosphorothioate or methylphosphonate internucleotide ligation modifications within positions 18 to 23 (counting from the 5' end), as well as 1 to 5 phosphorothioate or methylphosphonate internucleotide ligation modifications within positions 1 and 2 of the antisense strand and 1 to 5 within positions 18 to 23 (counting from the 5' end).

[0430] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification within positions 1-5 of the sense strand and one phosphorothioate or methylphosphonate nucleotide ligation modification within positions 18-23 (counting from the 5' end), and one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and two phosphorothioate or methylphosphonate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0431] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1-5 of the sense strand and one phosphorothioate nucleotide ligation modification within positions 18-23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0432] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1-5 of the sense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0433] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1-5 of the sense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and one phosphorothioate nucleotide ligation modification within positions 18-23 (counting from the 5' end).

[0434] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification within positions 1-5 of the sense strand and one phosphorothioate nucleotide ligation modification within positions 18-23 (counting from the 5' end), as well as two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0435] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification within positions 1-5 of the sense strand and one within positions 18-23 (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and one phosphorothioate nucleotide ligation modification within positions 18-23 (counting from the 5' end).

[0436] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification (counting from the 5' end) within positions 1-5 of the sense strand, and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and one phosphorothioate nucleotide ligation modification (counting from the 5' end) within positions 18-23.

[0437] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications (counting from the 5' end) within positions 1-5 of the sense strand, one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand, and two phosphorothioate nucleotide ligation modifications (counting from the 5' end) within positions 18-23.

[0438] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1-5 of the sense strand and one within positions 18-23 (counting from the 5' end), as well as two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and one within positions 18-23 (counting from the 5' end).

[0439] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1-5 of the sense strand and one phosphorothioate nucleotide ligation modification within positions 18-23 (counting from the 5' end), as well as two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0440] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1-5 of the sense strand and one phosphorothioate nucleotide ligation modification within positions 18-23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0441] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the sense strand and two phosphorothioate nucleotide ligation modifications at positions 20 and 21 (counting from the 5' end), and one phosphorothioate nucleotide ligation modification at position 1 of the antisense strand and one at position 21 (counting from the 5' end).

[0442] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification at position 1 of the sense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications at positions 20 and 21 (counting from the 5' end).

[0443] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the sense strand and two phosphorothioate nucleotide ligation modifications at positions 21 and 22 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at position 1 of the antisense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end).

[0444] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification at position 1 of the sense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications at positions 21 and 22 (counting from the 5' end).

[0445] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the sense strand and two phosphorothioate nucleotide ligation modifications at positions 22 and 23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at position 1 of the antisense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end).

[0446] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification at position 1 of the sense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications at positions 23 and 23 (counting from the 5' end).

[0447] In some embodiments, the compounds of the Disclosure include a pattern of skeletal chiral centers. In some embodiments, the general pattern of skeletal chiral centers includes at least five nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least six nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least seven nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least eight nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least nine nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least ten nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least eleven nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least twelve nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least thirteen nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 14 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 15 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 16 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 17 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 18 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 19 nucleotide linkages in the Sp configuration.In some embodiments, the general pattern of the skeletal chiral center includes eight or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes seven or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes six or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes five or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes four or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes three or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes two or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes one or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes eight or fewer non-chiral nucleotide linkages (phosphodiesters are an example, though not limited to them). In some embodiments, the general pattern of the skeletal chiral center includes seven or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes six or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes five or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes four or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes three or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes two or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes one or fewer non-chiral nucleotide linkages.In some embodiments, the general pattern of the skeletal chiral center includes at least 10 internucleotide links and 8 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 11 internucleotide links and 7 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 12 internucleotide links and 6 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 13 internucleotide links and 6 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 14 internucleotide links and 5 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 15 internucleotide links and 4 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the internucleotide links in the Sp configuration may be continuous or not. In some embodiments, the nucleotide linkages in the Rp configuration may be continuous or not. In some embodiments, the non-chiral nucleotide linkages may be continuous or not.

[0448] In some embodiments, the compounds of the Disclosure include blocks that are stereochemical blocks. In some embodiments, a block is an Rp block in that each nucleotide linkage in the block is Rp. In some embodiments, a 5'-block is an Rp block. In some embodiments, a 3'-block is an Rp block. In some embodiments, a block is an Sp block in that each nucleotide linkage in the block is Sp. In some embodiments, a 5'-block is an Sp block. In some embodiments, a 3'-block is an Sp block. In some embodiments, the oligonucleotides provided include both Rp and Sp blocks. In some embodiments, the oligonucleotides provided include one or more Rp but do not include Sp blocks. In some embodiments, the oligonucleotides provided include one or more Sp but do not include Rp blocks. In some embodiments, the oligonucleotides provided include one or more PO blocks in which each nucleotide linkage is a native phosphate linkage.

[0449] In some embodiments, the compounds of the present disclosure include a 5'-block in which 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 nucleotide linkage is a modified nucleotide linkage and 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 nucleotide linkage is a phosphorothioate linkage and each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 5'-block contains four or more nucleoside units. In some embodiments, the 5'-block contains five or more nucleoside units. In some embodiments, the 5'-block contains six or more nucleoside units. In some embodiments, the 5'-block contains seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each nucleotide linkage is a modified nucleotide linkage and each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each nucleotide linkage is a phosphorothioate linkage and each sugar moiety contains a 2'-F modification. In some embodiments, the 3'-block contains four or more nucleoside units. In some embodiments, the 3'-block contains five or more nucleoside units. In some embodiments, the 3'-block contains six or more nucleoside units. In some embodiments, the 3'-block contains seven or more nucleoside units.

[0450] In some embodiments, the compounds of the Disclosure comprise a nucleoside of a certain type in the region, or an oligonucleotide followed by a specific type of internucleotide linkage, such as a native phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, and the like. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a native 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 native 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 native 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 native 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 includes phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, the antisense strand contains at least one double-stranded thermal destabilization modification 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 features (e.g., all of 1, 2, 3, 4, 5, 6, 7, or 8): (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, (i i) the antisense strand contains 3, 4, or 5 phosphorothioate nucleotide interlinks, (iii) the sense strand is conjugated with a 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 phosphorothioate nucleotide interlinks, (vi) the dsRNA contains at least 4 2'-fluoro modifications, (vii) the dsRNA contains a double-stranded region of 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 includes phosphorothioate internucleotide links 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, and the antisense strand contains at least one double-stranded thermal destabilization modification 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 features (e.g., 1, 2, 3, 4, 5, 6, 7, or all 8): (i) the antisense strand is 2, 3, 4, 5 or (ii) the sense strand contains six 2'-fluoro modifications, is conjugated with a ligand, (iii) the sense strand contains two, three, four or five 2'-fluoro modifications, (iv) the sense strand contains one, two, three, four or five phosphorothioate nucleotide interlinks, (v) the dsRNA contains at least four 2'-fluoro modifications, (vi) the dsRNA contains a double-stranded region of 12-40 nucleotide pairs in length, (vii) the dsRNA contains a double-stranded region of 12-40 nucleotide pairs in length, and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0453] In some embodiments, the sense strand includes phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand includes at least one double-stranded thermal destabilization modification 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 features (e.g., all of 1, 2, 3, 4, 5, 6, 7, or 8): (i) the antisense includes 2, 3, 4, 5, or 6 2'-fluoro modifications, (ii) an (iii) the sense strand contains 1, 2, 3, 4 or 5 phosphorothioate nucleotide interlinks, (iv) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications, (v) the sense strand contains 3, 4 or 5 phosphorothioate nucleotide interlinks, (vi) the dsRNA contains at least 4 2'-fluoro modifications, (vii) the dsRNA contains a double-stranded region of 12 to 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 includes phosphorothioate internucleotide links between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand includes phosphorothioate internucleotide links 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 double helix thermal destabilization modification 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 has at least one of the following features (e.g., 1, The dsRNA may further have 2, 3, 4, 5, 6 or 7 of the following: (i) the antisense strand contains 2, 3, 4, 5 or 6 2'-fluoro modifications; (ii) the sense strand is conjugated with a 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 4 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region 12–40 nucleotide pairs long; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0455] In some embodiments, the dsRNA molecules of this disclosure include double-stranded mismatches(s) or combinations thereof with respect to the target. Mismatches may occur in overhang regions or double-stranded regions. Base pairs can be ranked based on their tendency to promote dissociation or fusion (e.g., by the free energy of association or dissociation of a particular pairing, the simplest approach being to examine pairs on a basis of individual pairs, although the following adjacency analysis or similar analysis may also be used). In terms of promoting dissociation: A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical pairing or non-canonical pairing (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairing, and pairings involving universal bases are preferred over canonical pairing.

[0456] In some embodiments, the dsRNA molecule of the present disclosure includes at least one of the first 1, 2, 3, 4, or 5 base pairs in the double-stranded region from the 5' end of the antisense strand, which can be independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical pairing or pairing other than canonical pairing or pairing including universal bases, in order to facilitate the dissociation of the antisense strand at the 5' end of the double helix.

[0457] In some embodiments, the nucleotide at position 1 in the double-strand region from the 5' end of 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 double-strand region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-strand region from the 5' end of the antisense strand is an AU base pair.

[0458] It has been found that introducing a 4'-modified or 5'-modified nucleotide to the 3' end of a phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage of a dinucleotide at any position on a single-stranded or double-stranded oligonucleotide exerts a steric effect on the nucleotide linkage, thereby protecting and stabilizing it from nucleases.

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

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

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

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

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

[0464] In some embodiments, the dsRNA molecules of this disclosure may include a 2'-5' ligation (having 2'-H, 2'-OH, and 2'-OMe, and being P=O or P=S). For example, the 2'-5' ligation modification can be used to promote nuclease resistance, to inhibit the binding of sense to the antisense strand, or to avoid sense strand activation by RISC at the 5' end of the sense strand.

[0465] In another embodiment, the dsRNA molecule of this disclosure may contain L-sugars (e.g., L-ribose having 2'-H, 2'-OH and 2'-OMe, L-arabinose). For example, these L-sugar modifications can be used to promote nuclease resistance, to inhibit the binding of sense to the antisense strand, or to avoid sense strand activation by RISC at the 5' end of the sense strand.

[0466] Multimeric siRNAs have been described in various publications, all of which can be used in conjunction with the dsRNAs of this disclosure. Such publications include WO2007 / 091269, US7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, which are incorporated in their entirety herein.

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

[0468] Ligands can be attached to polynucleotides via a carrier. The carrier comprises (i) at least one “skeleton attachment site,” preferably two “skeleton attachment sites,” and (ii) at least one “tethering attachment site.” “Skeleton attachment site,” as used herein, refers to a functional group, e.g., a hydroxyl group, or generally, a bond available and suitable for the incorporation of the carrier into a skeleton, e.g., a phosphate or modified phosphate of ribonucleic acid, e.g., a sulfur-containing skeleton. “Tethering attachment site” (TAP) refers, in some embodiments, to a constituent ring atom of the cyclic carrier connecting a selected moiety, e.g., a carbon atom or heteroatom (separate from the atom providing the skeleton attachment sites). The moiety may be, for example, a carbohydrate, e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides. The selected moiety may be connected to the cyclic carrier by an intervening tether. Thus, the cyclic carrier will often provide a bond suitable for the incorporation or tethering of another chemical entity, e.g., a ligand, into a constituent ring, e.g., containing a functional group, e.g., an amino group.

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

[0470] In a particular embodiment, the RNAi agent for use in the method of the present 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, for example, 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 a ligand Another modification of the iRNA of the present invention involves chemically linking the iRNA with one or more ligands, a moiety or conjugate that enhances the activity, cell distribution, or, for example, cellular uptake into cells of the iRNA. These include, but are not limited to, lipid parts such as cholesterol (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, for example, beryl-S-tritylthiol (Manoharan et al., Ann. NY 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 acid chains, e.g., 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, e.g., di-hexadecyl-rac-glycerol or triethylammonium 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), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetate (Manoharan et al., Tetrahedron Lett.Examples include the palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237) or the octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0472] In certain embodiments, ligands alter the distribution, targeting, or lifespan of the iRNA agent into which they are incorporated. In some embodiments, ligands provide enhanced affinity to selected targets, such as molecules, cells or cell types, compartments, such as cellular or organ compartments, tissues, organs, or regions of the body, compared to species in which such ligands are absent. Conventional ligands do not participate in double-strand pairing in double-stranded nucleic acids.

[0473] Ligands can be 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-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helix peptides.

[0474] Ligands may 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 may 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, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol steroids, bile acids, folic acid, vitamin B12, biotin, or RGD peptides or RGD peptide mimetic. In certain embodiments, the ligand is polyvalent galactose, such as N-acetyl-galactosamine.

[0475] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyllin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantane acetate, 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) lithoglycerol Examples include oenic acid, O3-(oleoyl)colenic acid, dimethoxytrityl or phenoxazine, and peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphoric acid, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, 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 tetraaza macrocyclic molecules), dinitrophenyl, HRP, or AP.

[0476] Ligands can be proteins, such as glycoproteins or peptides, molecules or antibodies that have a specific affinity for a co-ligand, such as antibodies that bind to specific cell types, such as brain cells or glial cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. Ligands can also be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0477] A ligand can be a substance, such as a drug, that can increase the uptake of an iRNA agent into a cell by, for example, disrupting the cytoskeleton of a cell, for example, by disrupting the microtubules, microfibrils, or intermediate fibers of a cell. A drug may be, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latruncrine A, phalloidin, swinford A, indanosine, or myoserbine.

[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 analogs, peptides, protein binders, PEGs, vitamins, and the like. Exemplary PK modulators, but not limited to, include cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins; therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in their backbone, are also suitable as ligands (e.g., as PK-modulating ligands) in the present invention. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0479] iRNAs conjugated with the ligand of the present invention can be synthesized using oligonucleotides having pendant-reactive functionality, for example, those derived from the attachment of a linking molecule to an oligonucleotide (as described below). These reactive oligonucleotides can be directly reacted with commercially available ligands, synthetic ligands having any of the various protecting groups, or ligands having a linking portion attached thereto.

[0480] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared by known solid-phase synthesis techniques. Equipment for such synthesis is available from several vendors, including, for example, Applied Biosystems® (Foster City, California). Any other means for such synthesis known in the art may be used further or instead. It is also known that similar techniques can be used to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0481] In the ligand-conjugated oligonucleotide and ligand-sequence-specific linked nucleosides of the present invention, the oligonucleotide and oligonucleosides can be assembled in a suitable DNA synthesizer using a standard nucleotide or nucleoside precursor, a nucleotide or nucleoside conjugate precursor already having a linking portion, a ligand-nucleotide or nucleoside conjugate precursor already having a ligand molecule, or a non-nucleoside ligand having a building block.

[0482] When using nucleotide-conjugate precursors that already have a linking region, the synthesis of a sequence-specific linked nucleoside is usually completed, and then the ligand molecule reacts with the linking region to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates, in addition to commercially available and standard and non-standard phosphoramidites routinely used in oligonucleotide synthesis.

[0483] A. Lipid conjugates In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecules can typically bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the conjugate to target tissues in the body, such as non-renal target tissues. For example, the target tissue could be the liver, including the parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can (a) increase the resistance of the conjugate to degradation, (b) increase the targeting or transport into target cells or cell membranes, or (c) modulate binding to serum proteins, such as HSA.

[0484] Lipid-based ligands can be used to modulate, for example, control (e.g., inhibit) the binding of conjugates to target tissues. For instance, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidneys and therefore less likely to be eliminated from the body. Lipids or lipid-based ligands that do not bind less strongly to HSA can be used to target conjugates to the kidneys.

[0485] In certain embodiments, lipid-based ligands bind to HSA. For example, a ligand can bind to HSA with sufficient affinity, resulting in enhanced distribution of the conjugate to non-renal tissue. However, the affinity is usually not strong enough to reverse the HSA-ligand binding.

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

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

[0488] In another embodiment, the ligand is a portion taken up by target cells, e.g., proliferating cells, e.g., a vitamin. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant species, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

[0489] B. Cell permeability agents In another embodiment, the ligand is a cell permeabilizer, such as a helix cell permeabilizer. In certain embodiments, these cell permeabilizers are amphiphilic. Exemplary cell permeabilizers include peptides, such as tat or antennopedia. If the agent is a peptide, it may be modified, including peptidyl mimetic, inverted isomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. Helix agents are typically α-helix agents and may have lipophilic and oleophobic phases.

[0490] Ligands can be peptides or peptidomimetic molecules. Peptidomimetic molecules (also referred to herein as oligopeptidomimetic molecules) are molecules that can fold into a defined three-dimensional structure similar to that of natural peptides. The attachment of peptides and peptidomimetic molecules to iRNA agents can affect the pharmacokinetic distribution of the iRNA, for example, by enhancing cell recognition and absorption. The peptide or peptidomimetic moiety may be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0491] Peptides or peptidomimetic molecules can be, for example, cell-permeable peptides, cationic peptides, amphiphilic peptides, or hydrophobic peptides (e.g., mainly composed of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, a restrictive peptide, or a cross-linked peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane-transfer sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF with the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 11). RFGF analogs containing hydrophobic MTS [e.g., 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 the cell membrane. For example, sequences derived from the HIV Tat protein [GRKKRRQRRRPPQ (SEQ ID NO: 13)] and sequences derived from the Drosophila Antennapedia protein [RQIKIWFQNRRMKWKK (SEQ ID NO: 14)] have been found to be functional as delivery peptides. Peptides or peptidomimetic molecules can be encoded by random sequences of 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). Typically, peptides or peptidomimetic molecules tethered to dsRNA agents via incorporated monomer units include cell-targeting peptides, such as arginine-glycine-aspartate (RGD) peptides or RGD mimics. The peptide moiety can range in length from approximately 5 to 40 amino acids. The peptide moiety may have structural modifications that increase, for example, stability or direct conformational properties. Any of the structural modifications described below are available.

[0492] The RGD peptides for use in the compositions and methods of the present invention may be linear or cyclic, and may be modified to facilitate targeting of specific tissues, for example, by glycosylation or methylation. RGD-containing peptides and peptidiomimemtics may include D-amino acids and synthetic RGD mimics. In addition to RGD, other parts that target integrin ligands may be used. Preferred conjugates of these ligands target PECAM-1 or VEGF.

[0493] The RGD peptide portion can be used to target specific cell types, such as tumor cells, endothelial tumor cells, or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can facilitate the targeting of dsRNA agents to tumors in various other tissues, including the lungs, kidneys, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, RGD peptides facilitate the targeting of iRNA agents to the kidneys. RGD peptides can be linear or cyclic and can be modified to facilitate targeting to specific tissues, for example, by glycosylation or methylation. For example, glycosylated RGD peptides can α V It can be delivered to tumor cells that express β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0494] A "cell-permeable peptide" is capable of permeating cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-permeable peptides may be, for example, α-helix linear peptides (e.g., LL-37 or seropin P1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bactenesin), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidine). Cell-permeable peptides may also contain nuclear localization signals (NLS). For example, a cell-permeable peptide may be a bifid amphiphilic peptide such as MPG derived from 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 Conjugate In some embodiments of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNA is advantageous for the in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” means a compound that is either a carbohydrate itself, or a compound having a carbohydrate portion composed of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic), along with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom, and each having at least six monosaccharide units (which may be linear, branched, or cyclic), along with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Typical 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 gum. C5 is an example of a specific monosaccharide, and the above-mentioned (e.g., C5, C6, C7, or C8) sugars, disaccharides, and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0496] In certain embodiments, the carbohydrate conjugate includes 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 US8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate acts as a ligand that targets iRNA to specific cells. In some embodiments, the GalNAc conjugate targets iRNA to liver cells, for example, by acting as a ligand for the asialocrycoprotein receptor in liver cells (e.g., hepatocytes).

[0498] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be attached via a linker, for example, a divalent 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 (for example, to the 3' end of the sense strand) via a linker, for example, 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 (for example, to the 5' end of the sense strand) via a linker, for example, a linker as described herein.

[0499] In a particular embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a trivalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a tetravalent linker.

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

[0501] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each of the other strands, forming a hairpin loop containing a plurality of unpaired nucleotides, each unpaired nucleotide in the hairpin loop may independently contain GalNAc or a GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one of the two strands.

[0502] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each of the other strands, forming a hairpin loop containing a plurality of unpaired nucleotides, each unpaired nucleotide in the hairpin loop may independently contain GalNAc or a GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one of the two strands.

[0503] In some embodiments, the GalNAc conjugate is

[0504] [ka] That is the case.

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

[0506] [ka]

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

[0508] [ka]

[0509] In a particular embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is selected from the group consisting of:

[0510] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0511] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the present invention is a monosaccharide. In certain embodiments, the monosaccharide is N-acetylgalactosamine.

[0512] [ka] That is the case.

[0513] Other representative carbohydrate conjugates for use in the embodiments described herein, but not limited to,

[0514] [ka] [In the formula, one of X or Y is an oligonucleotide, and the other is hydrogen.] These are some examples.

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

[0516] [ka] Includes.

[0517] In certain embodiments, the RNAi agents of the Disclosure may include a GalNAc ligand, even if such a GalNAc ligand is currently expected to have limited value for the preferred intrathecal / CNS delivery pathway(s) of the Disclosure.

[0518] In a particular embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a trivalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a tetravalent linker.

[0519] In certain embodiments, the double-stranded RNAi agent of the present invention comprises one GalNAc or GalNAc derivative attached to the 5' end of the sense strand of an iRNA agent, e.g., a dsRNA agent, or to the 5' end of one or both sense strands of a dual-targeting RNAi agent as described herein. In certain embodiments, the double-stranded RNAi agent of the present invention comprises a plurality of (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives attached independently to each of a plurality of nucleotides of the double-stranded RNAi agent via a plurality of linkers, e.g., monovalent linkers.

[0520] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of the other strand, forming a hairpin loop containing a plurality of unpaired nucleotides, each of the unpaired nucleotides in the hairpin loop may independently contain GalNAc or a GalNAc derivative attached via a monovalent linker.

[0521] In some embodiments, the carbohydrate conjugate further comprises one or more of the above-mentioned ligands, but is not limited to a PK modulator or a cell-permeable peptide.

[0522] Further carbohydrate conjugates and linkers suitable for use in the present invention include those described in WO2014 / 179620 and WO2014 / 179627, the entire contents 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 various linkers, which may or may not be cleavable.

[0524] The term "linker" or "linking group" refers to an organic part that connects two parts of a compound, for example, by covalent bonding.Linkers are typically directly bonded or composed of atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or not, but one or more methylene groups may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylal Quinnyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylal Kenyl, alkyl heteroarylalkynyl, alkenyl heteroarylalkyl, alkenyl heteroarylalkenyl, alkenyl heteroarylalkynyl, alkenyl heteroarylalkynyl, alkynyl heteroarylalkyl, alkynyl heteroarylalkenyl, alkynyl heteroarylalkynyl, alkyl heterocyclylalkyl, alkyl heterocyclylalkenyl, alkyl heterocyclylalkynyl (alkylhererocyclylalkynyl), alkenyl heterocyclylalkyl, alke...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting APOE expression, or a pharmaceutically acceptable salt thereof, Here, the dsRNA agent or a pharmaceutically acceptable salt thereof comprises a sense strand and an antisense strand that form a double-stranded region. The sense strand is 17 to 23 nucleotides long, and the antisense strand is 19 to 25 nucleotides long. The antisense strand comprises at least 19 consecutive nucleotides having zero or one mismatch from the nucleotide sequence 5'-UAACCUUCAUCUUCCUGCCUGUGU-3' of SEQ ID NO: 700, or the nucleotide sequence 5'-UCACAGAACCUUCAUCUUCCUGC-3' of SEQ ID NO: 701, All of the nucleotides of the sense strand and all of the nucleotides of the antisense strand independently contain nucleotide modifications selected from the group consisting of deoxynucleotide modifications, 2'-O-methylnucleotide modifications, 2'-fluoronucleotide modifications, and thermal destabilization modifications, and if the thermal destabilization modifications are present, only the antisense strand of the dsRNA agent or a pharmaceutically acceptable salt thereof contains the thermal destabilization modifications. The sense strand and the antisense strand independently comprise at least one phosphorothioate nucleotide linkage. One or more lipophilic portions containing saturated or unsaturated C6-C18 hydrocarbon chains are conjugated at one or more internal positions selected from the group consisting of positions 4-8 and 13-18, counting from the 5' end of the sense chain. dsRNA agents, or pharmaceutically acceptable salts thereof.

2. The dsRNA agent or a pharmaceutically acceptable salt thereof according to Claim 1, wherein the sense strand comprises at least 17 consecutive nucleotides from the nucleotide sequence 5'-CAGGCAGGGAAGAUGAAGGUUA-3' of SEQ ID NO: 690 or the nucleotide sequence 5'-AGGAAGAUGAAGGUUCUGUGA-3' of SEQ ID NO:

691.

3. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the sense strand comprises at least 17 consecutive nucleotides from the nucleotide sequence 5'-CAGGCAGGGAAGAUGAAGGUUA-3' of SEQ ID NO: 690, and the antisense strand comprises at least 19 consecutive nucleotides having 0 or 1 mismatch from the nucleotide sequence 5'-UAACCUUCAUCUUCCUGCCUGUGUG-3' of SEQ ID NO:

700.

4. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the sense strand comprises at least 17 consecutive nucleotides from the nucleotide sequence 5'-AGGAAGAUGAAGGUUCUGUGA-3' of SEQ ID NO: 691, and the antisense strand comprises at least 19 consecutive nucleotides having 0 or 1 mismatch from the nucleotide sequence 5'-UCACAGAACCUUCAUCUCUCUGC-3' of SEQ ID NO:

701.

5. A dsRNA agent according to any one of claims 1 to 4, comprising 6 to 8 phosphorothioate nucleotide linkages, or a pharmaceutically acceptable salt thereof.

6. A dsRNA agent or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the at least one strand comprises a 3' overhang of at least one nucleotide.

7. A dsRNA agent or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein the at least one strand comprises a 3' overhang of at least two nucleotides.

8. The dsRNA agent or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein the double-stranded region is 19 to 23 nucleotide pairs long.

9. The dsRNA agent according to claim 8 or a pharmaceutically acceptable salt thereof, wherein the double-stranded region is 21 to 23 nucleotide pairs long.

10. A dsRNA agent according to any one of claims 1 to 9, wherein each strand is independently 19 to 23 nucleotides long, or a pharmaceutically acceptable salt thereof.

11. The dsRNA agent according to claim 10 or a pharmaceutically acceptable salt thereof, wherein each chain is independently 21 to 23 nucleotides long.

12. The dsRNA agent or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein the one or more lipophilic moieties are conjugated to one or more internal positions in at least one strand via a linker or carrier.

13. The dsRNA agent according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, wherein the lipophilic portion contains a saturated or unsaturated C16 hydrocarbon chain.

14. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 13, wherein the saturated or unsaturated C16 hydrocarbon chain is conjugated at position 6, counting from the 5' end of the chain.

15. The dsRNA agent or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, wherein the lipophilic portion is conjugated via a carrier that replaces one or more nucleotides in an internal position or double-stranded region.

16. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 15, wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinil, pyrazolinil, pyrazolidinil, imidazolinil, imidazolidinil, piperidinil, piperazinil, [1,3]dioxolanil, oxazolidinil, isoxazolidinil, morpholinil, thiazolidinil, isothiazolidinil, quinoxalinil, pyridadinil, tetrahydrofuranil, and dekalinil, or an acyclic portion based on a serinol skeleton or a diethanolamine skeleton.

17. The dsRNA agent or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 16, wherein the lipophilic portion is conjugated to the dsRNA agent or a pharmaceutically acceptable salt thereof via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfamide linkage, click reaction product, or carbamate.

18. The dsRNA agent or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 17, wherein the lipophilic portion is conjugated to a nucleic acid base, a sugar portion, or an internucleoside linkage.

19. A dsRNA agent according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof, wherein the 3' end of the sense strand is protected via an end cap which is a cyclic group having an amine, and the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinil, pyrazolidinyl, imidazolinil, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinil, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranil, and dekalinil.

20. A dsRNA agent according to any one of claims 1 to 19, further comprising a phosphate or phosphate mimetic at the 5' end of the antisense strand, or a pharmaceutically acceptable salt thereof.

21. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 20, wherein the phosphate mimetic is 5'-vinylphosphonate (VP).

22. Isolated cells containing a dsRNA agent according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof.

23. A pharmaceutical composition for inhibiting the expression of a gene encoding APOE, comprising a dsRNA agent according to any one of claims 1 to 22 or a pharmaceutically acceptable salt thereof.

24. An in vitro method for inhibiting the expression of the APOE gene in cells, (a) Contacting the cells with the dsRNA agent described in any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in claim 23; and (b) Maintain the cells produced in step (a) for a sufficient time to obtain degradation of the mRNA transcript of the APOE gene, thereby inhibiting the expression of the APOE gene in the cells. Methods that include...

25. Use of a dsRNA agent according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 23 in the manufacture of a pharmaceutical for treating a subject diagnosed with APOE-associated neurodegenerative disease, or a pharmaceutical for preventing the onset of APOE-associated neurodegenerative disease in a subject that meets at least one diagnostic criterion for APOE-associated neurodegenerative disease.

26. The use according to claim 25, wherein the subject is a human.

27. The use according to claim 25, wherein the treatment includes improvement of at least one sign or symptom of the disease.

28. The use of the pharmaceutical product according to claim 26, wherein the use of the pharmaceutical product includes preventing the progression of APOE-related neurodegenerative disease.

29. The use according to claim 25, wherein the subject is diagnosed with APOE-associated neurodegenerative disease.

30. The use according to claim 28, wherein the APOE-related neurodegenerative disease is an amyloid-beta-mediated disease.

31. The use according to claim 30, wherein the amyloid-beta-mediated disease is selected from the group consisting of Alzheimer's disease, Down syndrome, and cerebral amyloid angiopathy.

32. The use according to claim 28, wherein the APOE-related neurodegenerative disease is a tau-mediated disease.

33. The use according to claim 32, wherein the tau-mediated disease is primary tauopathy or secondary tauopathy.

34. The use according to claim 33, 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), glial tauopathy (GGT), frontotemporal dementia with parkinsonism (FTDP, FTDP-17), chronic traumatic encephalopathy (CTE), boxer's dementia, frontotemporal lobar degeneration (FTLD), argyrophilic granulopathy (AGD), and primary age-related tauopathy (PART).

35. The use according to claim 33, wherein the secondary tauopathy is selected from the group consisting of AD, Creutzfeldt-Jakob disease, Down syndrome, and familial British dementia.

36. The use according to any one of claims 25 to 35, wherein the dsRNA agent is for administration in a dose of about 0.01 mg / kg to about 50 mg / kg.

37. The use according to any one of claims 25 to 36, wherein the dsRNA agent is for intrathecal administration.

38. The use according to any one of claims 25 to 37, further comprising the use of an additional agent suitable for the treatment or prevention of APOE-related neurodegenerative disorders.

39. An in vitro method for inhibiting the expression of the APOE gene in astrocytes, (a) Contacting the astrocytes with a dsRNA agent according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 23; and (b) Maintain the astrocytes produced in step (a) for a sufficient time to obtain degradation of the mRNA transcript of the APOE gene, thereby inhibiting the expression of the APOE gene in the astrocytes. Methods that include...

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