Modified Oligonucleotides

Abstract

Aspects of the present disclosure relate to compositions and methods for modulating the level, function, and / or activity of one or more RNA transcripts (e.g., mRNA transcripts) or proteins in a cell or subject. The disclosure is based, in part, on chemically modified inhibitory nucleic acids capable of target RNA knockdown with high specificity and low off-target effects.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 386,153, filed December 5, 2022, which is incorporated herein by reference in its entirety.

[0002] Reference to Electronic Sequence Listing The contents of the Electronic Sequence Listing (E058570012WO00-SEQ-KZM.xml, size: 2,820,627 bytes, and creation date: November 30, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] background Targeted knockdown of protein can be achieved by targeting mRNA with inhibitory nucleic acid.Chemically modified nucleic acid provides unique functional properties that enable specific targeting of mRNA.Therapeutic agents comprising chemically modified oligonucleotides associated with low frequency of off-target knockdown effects are clinically meaningful. Summary of the Invention

[0004] overview Aspects of the present disclosure relate to chemically modified nucleic acids that bind to mRNA transcripts of target genes. In some embodiments, the compositions of the present disclosure are useful for treating diseases or disorders associated with dysregulated expression of mRNA and / or the protein product encoded by the mRNA. The present disclosure is based, in part, on compositions and methods for modulating the function, activity, and / or level of the protein product encoded by a target mRNA by reducing the level of the target mRNA and / or translation of the target mRNA in a cell or subject.

[0005] Thus, in some embodiments, the present disclosure provides a double-stranded nucleic acid comprising a sense strand and an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides, wherein the modified nucleotides do not comprise more than five 2'-fluoro (2'F) modified nucleotides, and the 2'F modified nucleotides are present at positions 2, 5, 7, 14, and 16 relative to the 5' end of the antisense strand.

[0006] In some embodiments, the disclosure provides a double-stranded nucleic acid comprising a sense strand and an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides, wherein the modified nucleotides do not include more than five 2'F-modified nucleotides, and the 2'F-modified nucleotides are present at positions 2, 3, 7, 14, and 16 relative to the 5' end of the antisense strand.

[0007] In some embodiments, the disclosure provides a double-stranded nucleic acid comprising a sense strand and an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides, wherein the modified nucleotides do not include more than five 2'F-modified nucleotides, and the 2'F-modified nucleotides are present at positions 2, 5, 8, 14, and 16 relative to the 5' end of the antisense strand.

[0008] In some embodiments, the disclosure provides a double-stranded nucleic acid comprising a sense strand and an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides, wherein the modified nucleotides do not include more than six 2'F-modified nucleotides, and the 2'F-modified nucleotides are present at positions 2, 4, 6, 8, 14, and 16 relative to the 5' end of the antisense strand.

[0009] In some embodiments, the disclosure provides a double-stranded nucleic acid comprising a sense strand and an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides, wherein the modified nucleotides do not include more than five 2'F-modified nucleotides, and the 2'F-modified nucleotides are present at positions 2, 6, 8, 14, and 16 relative to the 5' end of the antisense strand.

[0010] In some embodiments, the sense strand comprises a 5'-end and a 3'-end, and comprises 2'F-modified nucleotides at positions 9, 10, and 11 relative to the 5'-end of the sense strand, positions 7, 9, and 11 relative to the 5'-end of the sense strand, positions 7, 9, and 10 relative to the 5'-end of the sense strand, or positions 7, 10, and 11 relative to the 5'-end of the sense strand. In some embodiments, the sense strand does not comprise any other 2'F-modified nucleotides.

[0011] In some embodiments, the present disclosure provides a double-stranded nucleic acid comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex, the antisense strand comprises a sequence complementary to a portion of a target mRNA, and the antisense strand comprises five 2'F-modified nucleotides at one set of positions from the 5' end of the antisense strand: (a) positions 2, 5, 7, 14, and 16; (b) positions 2, 3, 7, 14, and 16; or (c) positions 2, 5, 8, 14, and 16, but no other 2'F-modified nucleotides.

[0012] In some embodiments, the sense strand comprises three 2'F-modified nucleotides at one of the following sets of positions from the 5' end of the sense strand: (a) positions 9, 10, and 11; (b) positions 7, 9, and 11; (c) positions 7, 9, and 10; or (d) positions 7, 10, and 11.

[0013] In some embodiments, the antisense strand comprises 2'-O-methyl modified nucleotides at positions other than the 2'F modified positions.

[0014] In some embodiments, the sense strand contains 2'-O-methyl modified nucleotides or 2'-O-alkyl modified nucleotides (e.g., 2'-OC) at positions other than the 2'F modified positions. 12~16 In some embodiments, the sense strand comprises one or more abasic moieties at a position other than the 2'F modified position.

[0015] In some embodiments, position 1 relative to the 5' end of the sense strand or the 5' end of the antisense strand comprises a 5' phosphate analog. In some embodiments, the 5' phosphate analog comprises a 5' vinylphosphonate group. In some embodiments, the antisense strand comprises a 5' phosphate analog.

[0016] In some embodiments, the sense strand is 18 to 24 nucleotides in length, hi some embodiments, the sense strand is 21 nucleotides in length.

[0017] In some embodiments, the antisense strand is 18 to 24 nucleotides in length, hi some embodiments, the antisense strand is 23 nucleotides in length.

[0018] In some embodiments, the sense strand and the antisense strand are not the same length. In some embodiments, the antisense strand is longer than the sense strand. In some embodiments, the antisense strand is 2-10 nucleotides longer than the sense strand.

[0019] In some embodiments, the modified nucleotides are modified ribonucleotides. In some embodiments, the modified nucleotides of the sense strand are one or more 2'-O-methyl (2'OMe) modified nucleotides or one or more 2'-O-alkyl modified nucleotides (e.g., 2'-OC 12~16 alkyl-modified nucleotides).

[0020] In some embodiments, the double-stranded nucleic acid comprises one or more abasic moieties.

[0021] In some embodiments, the modified nucleotides in the sense strand include only 2'OMe-modified nucleotides, excluding 2'F-modified nucleotides at the recited positions.

[0022] In some embodiments, the modified nucleotides in the sense strand include one or more 2'-O-methyl (2'OMe) modified nucleotides, one or more 2'-O-alkyl modified nucleotides (e.g., 2'-OC 12~16 alkyl-modified nucleotides), or contain one or more abasic moieties.

[0023] In some embodiments, the modified nucleotides in the antisense strand include only 2'OMe-modified nucleotides, excluding 2'F-modified nucleotides at the recited positions.

[0024] In some embodiments, the sense strand comprises one or more modified internucleotide linkages. In some embodiments, the sense strand comprises four modified internucleotide linkages. In some embodiments, the modified internucleotide linkages comprise one or more phosphorothioate (PS) internucleotide linkages. In some embodiments, each modified internucleotide linkage in the double-stranded nucleic acid sense strand is a PS internucleotide linkage.

[0025] In some embodiments, the antisense strand comprises one or more modified internucleotide linkages. In some embodiments, the antisense strand comprises four modified internucleotide linkages. In some embodiments, the modified internucleotide linkages comprise one or more phosphorothioate (PS) internucleotide linkages. In some embodiments, each modified internucleotide linkage of the double-stranded nucleic acid antisense strand is a PS internucleotide linkage.

[0026] In some embodiments, positions 1 and 2 (e.g., relative to the 5' end) of the sense strand are linked by a modified internucleotide linkage. In some embodiments, positions 2 and 3 (e.g., relative to the 5' end) of the sense strand are linked by a modified internucleotide linkage. In some embodiments, positions 1, 2, 3, and 4 (e.g., relative to the 5' end) of the sense strand are linked by a modified internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate (PS) internucleotide linkage.

[0027] In some embodiments, positions 1 and 2 (e.g., relative to the 5' end) of the antisense strand are linked by a modified internucleotide linkage. In some embodiments, positions 2 and 3 (e.g., relative to the 5' end) of the antisense strand are linked by a modified internucleotide linkage. In some embodiments, positions 1, 2, 3, and 4 (e.g., relative to the 5' end) of the antisense strand are linked by a modified internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate (PS) internucleotide linkage.

[0028] In some embodiments, at least two of positions 1, 2, and 3 at the 3' end of the sense strand are linked by modified internucleotide linkages, optionally by phosphorothioate (PS) internucleotide linkages.

[0029] In some embodiments, at least two of positions 1, 2, and 3 at the 3' end of the antisense strand are linked by modified internucleotide linkages, optionally by phosphorothioate (PS) internucleotide linkages.

[0030] In some embodiments, each of positions 1, 2, and 3 relative to the 3' end of the sense strand are linked by a modified internucleotide linkage, optionally a phosphorothioate (PS) internucleotide linkage.

[0031] In some embodiments, each of positions 1, 2, and 3 relative to the 3' end of the antisense strand are linked by a modified internucleotide linkage, optionally a phosphorothioate (PS) internucleotide linkage.

[0032] In some embodiments, the present disclosure provides double-stranded nucleic acids comprising a sense strand and / or an antisense strand comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1-199 (e.g., listed in Tables 1-5). In some embodiments, the present disclosure provides double-stranded nucleic acids comprising a sense strand and / or an antisense strand comprising a nucleotide sequence and modification pattern set forth in any one of SEQ ID NOs: 1-199 (e.g., listed in Tables 1-5).In some embodiments, the present disclosure provides sense and antisense strands comprising a SEQ ID NO: selected from any one of Tables 1-5 (e.g., SEQ ID NOs: 1, 2, 3, 4, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 77, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 40, 41, 42, 77, 79, 80, 81, 82, 7, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 145, 146, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, or 175 Sense strand and SEQ ID NOs: 5, 6, 7, 8, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 147, 148, 149, 150, 151, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199). In some embodiments, the double-stranded nucleic acid comprises a region of complementarity to a human alpha synuclein (SNCA) mRNA transcript. In some embodiments, the double-stranded nucleic acid comprises a region of complementarity to a human apolipoprotein E (APOE) mRNA transcript.

[0033] In some embodiments, the present disclosure provides a conjugate comprising a double-stranded nucleic acid described herein.

[0034] In some embodiments, the conjugate comprises a structure of Formula I, wherein Formula I is: ABC Formula I wherein "A" of Formula I comprises a double-stranded nucleic acid described herein, "B" of Formula I comprises a bond or linker, and "C" of Formula I comprises a delivery molecule.

[0035] In some embodiments, "B" in Formula I is attached to the 5'-end or 3'-end of the sense strand of a double-stranded nucleic acid. In some embodiments, "B" in Formula I is attached to the 3'-end of the sense strand of a double-stranded nucleic acid. In some embodiments, "B" in Formula I is attached to the 5'-end or 3'-end of the antisense strand of a double-stranded nucleic acid. In some embodiments, "B" in Formula I comprises a triethylene glycol (TEG) linker. In some embodiments, "B" in Formula I comprises a linker comprising a C6-NH2 group. In some embodiments, "B" in Formula I does not comprise a maleimido-methyl-tetrazine-trans-cyclo-octene (mal-tet-TCO) linker, a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, a linker comprising a tertiary amide bonded to a gem-dimethyl (GDM) group, or a linker comprising a C6-NH2 group.

[0036] In some embodiments, "C" of Formula I comprises one or more N-acetylgalactosamine (GalNAc) moieties. In some embodiments, "C" of Formula I comprises cholesterol. In some embodiments, "C" of Formula I comprises tocopherol.

[0037] In some embodiments, the double-stranded RNA described by the present disclosure does not contain a nucleotide attached to a maleimide group, a tertiary amide bonded to a gem-dimethyl (GDM) group, or a C6-NH2 group.

[0038] In some aspects, the present disclosure provides a pharmaceutical composition comprising a double-stranded nucleic acid described herein, or a conjugate described herein, and a pharmaceutically acceptable carrier.

[0039] In some aspects, the present disclosure provides a method for inhibiting or reducing a target mRNA in a cell, the method comprising contacting a cell containing the target mRNA with a double-stranded nucleic acid, conjugate, or pharmaceutical composition described herein.

[0040] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is in a subject. In some embodiments, the subject is a human subject. DETAILED DESCRIPTION OF THE INVENTION

[0041] Detailed Description Aspects of the present disclosure relate to compositions and methods for modulating the level and / or translation of one or more RNA transcripts (e.g., mRNA transcripts) in a cell or subject. The disclosure is based, in part, on chemically modified double-stranded inhibitory nucleic acids having a specific pattern of 2' fluoro (2'F) modifications that bind to a target mRNA transcript, alter the level of the target mRNA transcript, and alter the activity and / or level of a protein expressed by the target mRNA transcript. In some embodiments, the disclosure provides double-stranded nucleic acids having an antisense strand that includes 2'F-modified nucleotides at positions 2, 5, 7, 14, and 16, or positions 2, 3, 7, 14, and 16, or positions 2, 5, 8, 14, and 16, or positions 2, 4, 6, 8, 14, and 16, or positions 2, 6, 8, 14, and 16, relative to the 5' end of the antisense strand. In some embodiments, the antisense strand does not contain any other 2'F-modified nucleotides. In some embodiments, the double-stranded nucleic acids described herein are more durable in vivo, retain potency, and / or have fewer off-target effects than double-stranded nucleic acids that are unmodified or have a different pattern of chemical modifications.

[0042] In some embodiments, the double-stranded nucleic acids described by the present disclosure are not conjugated to any other moiety (e.g., a delivery molecule). However, some aspects of the present disclosure relate to conjugates comprising a double-stranded nucleic acid described herein and one or more delivery molecules.

[0043] In some embodiments, the compositions of the present disclosure are useful for inhibiting the expression or activity of a target mRNA and / or treating a disease or disorder associated with altered expression of a target mRNA and / or protein associated with a particular disease.

[0044] [Double-stranded nucleic acid] Aspects of the present disclosure relate to double-stranded nucleic acids. Nucleic acids contain two or more nucleotides. As used herein, "nucleotide" refers to an organic compound in which a nucleoside (a nucleic acid base, e.g., adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar, e.g., ribose or 2'-deoxyribose) is linked to a phosphate group. A "nucleotide" can function as a monomer unit of nucleic acid polymers (e.g., oligonucleotides), such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In some embodiments, the nucleic acid is an oligonucleotide. As used herein, "oligonucleotide" refers to a polymer of linked nucleotides (including abasic portions), each of which may be modified or unmodified. Oligonucleotides are typically less than about 100 nucleotides in length. Oligonucleotides may contain unmodified DNA nucleotides, chemically modified DNA nucleotides, unmodified RNA nucleotides, chemically modified RNA nucleotides, non-natural nucleotides (e.g., non-naturally occurring nucleotides), abasic portions, or any combination thereof.

[0045] Double-stranded nucleic acids typically form duplexes. As used herein, "duplex" refers to a structure formed through complementary base pairing of two antiparallel sequences (i.e., in opposite orientation) of nucleotides, whether formed by two separate nucleic acid strands or by a single folded strand (e.g., via a hairpin). As used herein, "strand" refers to a single, continuous sequence of nucleotides linked together through internucleotide bonds (e.g., phosphodiester or phosphorothioate bonds). A strand may have two free ends (e.g., a 5' end and a 3' end). In some embodiments, the double-stranded nucleic acids described herein form duplexes comprising a sense strand and an antisense strand. Sense and antisense strands of nucleotides are further described herein in the section entitled "Sense and Antisense Strands."

[0046] The length of each strand of the double-stranded nucleic acid can vary. In some embodiments, each strand of the double-stranded nucleic acid ranges from about 10 nucleotides to about 50 nucleotides in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length). In some embodiments, each strand of the double-stranded nucleic acid ranges from about 18 to 24 nucleotides in length. In some embodiments, the sense strand comprises or consists of 21 nucleotides. In some embodiments, the antisense strand comprises or consists of 23 nucleotides. It should be understood that although the foregoing numerical ranges refer to "nucleotides," the present disclosure also contemplates including abasic portions within the ranges.

[0047] In some embodiments, the sense strand and antisense strand of a double-stranded nucleic acid are the same length (e.g., the double-stranded nucleic acid is "blunt-ended"). In some embodiments, the sense strand is longer than the antisense strand. In some embodiments, the antisense strand is longer than the sense strand. In some embodiments, the sense strand and the antisense strand are different lengths, and the double-stranded nucleic acid comprises one or two overhangs. As used herein, "overhang" refers to an unpaired nucleotide or a nucleotide that protrudes from the duplex structure of a double-stranded oligonucleotide. An overhang can comprise one or more unpaired nucleotides extending from the duplex region at the 5'-end or 3'-end of a double-stranded oligonucleotide. An overhang can be a 3'-overhang or a 5'-overhang in the antisense strand or the sense strand of a double-stranded oligonucleotide. In some embodiments, the sense strand is 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) longer than the antisense strand. In some embodiments, the antisense strand is 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) longer than the sense strand. In some embodiments, the double-stranded nucleic acid comprises one overhang in the sense strand (e.g., the 5' sequence of the sense strand extending beyond the 5'-most nucleotide of the antisense strand). In some embodiments, the double-stranded nucleic acid comprises one overhang in the antisense strand (e.g., the 5' sequence of the antisense strand extending beyond the 5'-most nucleotide of the sense strand). In some embodiments, the double-stranded nucleic acid comprises two overhangs in the sense strand. In some embodiments, the double-stranded nucleic acid comprises two overhangs in the antisense strand. In some embodiments, the double-stranded nucleic acid comprises one overhang in the sense strand and one overhang in the antisense strand. In some embodiments, the overhang sequence is about 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.

[0048] In some embodiments, the double-stranded nucleic acid is an inhibitory nucleic acid. As used herein, "inhibitory nucleic acid" refers to an oligonucleotide capable of reducing the level, function, and / or activity of an mRNA transcript (e.g., a target mRNA transcript) or a protein encoded by the mRNA in a cell. As used herein, a "target mRNA" is an mRNA encoding a protein associated with a disease or disorder, including, but not limited to, an mRNA encoding a mutant protein, a pathogenic protein isoform, or an mRNA not normally expressed in healthy cells. In some embodiments, the target mRNA is a wild-type mRNA (e.g., an mRNA encoding a wild-type protein). In some embodiments, an inhibitory nucleic acid can be used to cleave a target mRNA or inhibit the translation of a target mRNA containing one or more mutations (e.g., substitutions, deletions, and / or insertions) relative to the wild-type version of the sequence found in nature. In some embodiments, an inhibitory nucleic acid can be used to inhibit the translation of a target mRNA encoding a shortened version of a gene commonly found in nature (e.g., a truncation mutant or a mutant lacking a catalytic domain and / or a regulatory domain).

[0049] In some embodiments, the antisense strand of a double-stranded nucleic acid hybridizes with a target mRNA molecule. Nucleic acid hybridization typically involves one nucleic acid strand binding to a complementary region of another nucleic acid strand. As used herein, the terms "bind" and "binds," unless otherwise specified, are intended to mean that the proximity of two molecules, as determined by common methods known in the art, occurs as a result of the ability of a molecule to form a chemical bond or attractive interaction with another molecule. As used herein, "complementary" refers to a structural relationship between two nucleotides (e.g., in two opposing nucleic acids or in opposing regions of a single nucleic acid strand, e.g., a hairpin) that allows the two nucleotides to base pair with each other. For example, purine nucleotides in one nucleic acid that are complementary to pyrimidine nucleotides in an opposing nucleic acid can base pair together by forming hydrogen bonds with each other. Complementary nucleotides can base pair in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. Likewise, two nucleic acids can have a region of multiple nucleotides that are complementary to one another to form a region of complementarity, as described herein.

[0050] In some embodiments, the target mRNA comprises a degree of sequence identity to the sense strand that is sufficient to promote specific binding (e.g., hybridization) by the antisense strand of a double-stranded nucleic acid (e.g., an inhibitory nucleic acid). The term "% sequence identity" or "percentage sequence identity" with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleobases in a candidate sequence that are identical to the nucleotides, nucleosides, or nucleobases in the reference nucleic acid sequence after optimally aligning the candidate and reference nucleic acid sequences and introducing gaps or overhangs, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be accomplished by a variety of means within the skill of one in the art, including publicly available computer software programs such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supplement 30, Section 7.7.18, Table 7.7.1), including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), Clustal W2.0, or Clustal X2.0 software. Those skilled in the art can determine appropriate parameters for measuring alignment, including the algorithms necessary to achieve maximal alignment over the entire length of the sequences being compared. The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences in a comparison window, where a fragment of a nucleic acid sequence within the comparison window may contain additions or deletions (e.g., gaps or overhangs) relative to a reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. This percentage can be calculated by determining the number of positions in both sequences where the same nucleotide, nucleoside, or nucleobase occurs to determine the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to determine the percentage sequence identity. The output is the percent identity of the subject sequence relative to the query sequence.

[0051] In some embodiments, a strand (e.g., the sense strand) of a double-stranded nucleic acid comprises about 80% to 100% (e.g., about 80%, 85%, 90%, 95%, 99%, 99.9%, or 100%) sequence identity with a target mRNA. In some embodiments, a strand (e.g., the sense strand) of a double-stranded nucleic acid has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a target mRNA. In some embodiments, the inhibitory nucleic acid comprises an antisense strand that includes a region of complementarity that is at least 80% complementary to a portion of the target mRNA (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleotides of the antisense strand hybridize to ribonucleotides of the target mRNA). In some embodiments, the inhibitory nucleic acid comprises an antisense strand that includes a region of complementarity that is completely complementary to a portion of the target mRNA (e.g., 100% of the nucleotides of the antisense strand hybridize to ribonucleotides of the target mRNA).

[0052] In some embodiments, a double-stranded inhibitory nucleic acid comprises a single oligonucleotide comprising a sense strand and an antisense strand linked by a linker, hairpin loop, and / or a common backbone that hybridizes intramolecularly via nucleotides in the sense and antisense strands. Such a double-stranded inhibitory nucleic acid will comprise a single 5'-end and a 3'-end, and a loop region between the two sense and antisense sequences that does not base pair with any other nucleotides in the inhibitory nucleic acid. In some embodiments, a double-stranded inhibitory nucleic acid comprises two complementary oligonucleotides with respective 5'-ends and 3'-ends and respective backbones. In some embodiments, an inhibitory nucleic acid that is a double-stranded RNA (dsRNA) functions like an RNA interference (RNAi) molecule and therefore may be referred to as an RNAi agent. In some embodiments, an inhibitory nucleic acid that is a dsRNA functions like a small interfering RNA molecule and therefore may be referred to as an siRNA. The term "knockdown" or "expression knockdown" refers to the reduction of mRNA or protein expression of a gene following treatment with an inhibitory nucleic acid (eg, an RNAi agent).

[0053] In some embodiments, the double-stranded nucleic acid is an RNAi agent. As used herein, "RNAi agent," "iRNA," "iRNA agent," "RNAi," and "RNA interfering agent" refer to an agent that contains ribonucleotides or RNA and mediates targeted cleavage of an RNA transcript via RNA interference, e.g., through the RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi agent comprises a sense strand and an antisense strand, and the sense strand and antisense strand form a duplex. In some embodiments, the sense strand and antisense strand of the RNAi agent are 21-23 nucleotides in length.

[0054] To provide desirable characteristics, such as minimizing the possibility of off-target effects, increasing potency, and / or increasing durability, double-stranded nucleic acids can be designed to ensure that they do not have sequences complementary to off-target nucleic acids (e.g., sequences of five or more consecutive nucleotides) with off-target nucleic acids (e.g., mRNAs that do not contain the sequence of the target mRNA), or can be engineered to include one or more modified nucleotides that reduce or prevent off-target interactions or degradation (e.g., enzymatic cleavage). Aspects of the present disclosure relate to double-stranded nucleic acids with certain patterns of 2'F modifications that reduce nucleic acid promiscuity (e.g., off-target binding) relative to double-stranded nucleic acids that do not have the modifications or the same patterns of 2'F modifications described herein.

[0055] Aspects of the present disclosure relate to double-stranded nucleic acids that contain a particular pattern of chemical modifications (e.g., modified nucleotides, abasic moieties, modified internucleotide linkages, etc.). As used herein, "modified nucleotide" refers to a nucleotide that has one or more chemical modifications when compared to a corresponding reference nucleotide selected from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymidine deoxyribonucleotides.

[0056] Chemical modifications can be used to confer specific functional characteristics to double-stranded nucleic acids that are not exhibited by unmodified nucleic acids. The double-stranded nucleic acids of the present disclosure can be modified to achieve one or more desired properties, such as, for example, improved cellular uptake, improved stability, reduced immunogenicity, improved efficacy, improved target hybridization, susceptibility to RNAse cleavage, etc. In some embodiments, the inhibitory nucleic acid is modified so that when the inhibitory nucleic acid is present in a cell containing the mRNA target, it can hybridize with mRNA transcribed from the DNA sequence and induce cleavage of the mRNA.

[0057] A modified nucleotide can have, for example, one or more chemical modifications on its sugar, nucleobase, and / or phosphate group. Additionally or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to the corresponding reference nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, or a 2'-O-alkyl-modified nucleotide, e.g., 2'OC 12~16 As used herein, the term "alkyl" refers to a saturated, straight- or branched-chain monovalent hydrocarbon radical containing the indicated number of carbon atoms. For example, "C 12 ~ 16"Alkyl" refers to a radical having 12 to 16 carbon atoms in a linear or branched arrangement. Further examples of nucleotides containing modifications on the 2' carbon of the sugar group include, but are not limited to, D-ribose, 2'-deoxy, 2'-O-alkyl (including 2'-O-methyl and 2'-O-ethyl), 2'-alkoxy, 2'-amino, 2'-aminoalkoxy, 2'-S-alkyl, 2'-2-O-methoxyethoxy, 2'-O-methoxyethyl, 2'-allyloxy (OCHCH=CH), 2'-propargyl, 2'-propyl, ethynyl, ethenyl, propenyl, and cyano. In some embodiments, a 2'-modified nucleotide contains a 2'-O-4'-C methylene bridge, such as in locked inhibitory nucleic acid (LNA) nucleotides. In some embodiments, a 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring to form a bicyclic sugar moiety. Other 2' modifications are found in the art.

[0058] In some embodiments, the modified nucleotide has a phosphate analog. As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is positioned at the 5'-terminal nucleotide of the oligonucleotide in place of the 5'-phosphate, which is often susceptible to enzymatic removal. The 5' phosphate analog may contain a phosphatase-resistant bond. Examples of phosphate analogs include 5' methylene phosphonic acid (5'-MP) and 5'-(E)-vinyl phosphonic acid (5'-VP). In some embodiments, the phosphate analog is positioned at the 1-position relative to the sense or antisense strand of a double-stranded RNA. In some embodiments, the phosphate analog is 5'-VP. In some embodiments, the antisense strand comprises a 5' phosphate analog.

[0059] A double-stranded nucleic acid may contain one or more abasic moieties or inverted abasic moieties. As used herein, "abasic moiety" or "apurinating / apyrimidinating site" refers to a molecule derived from a nucleotide containing a ribose (or deoxyribose) sugar and a phosphate bond, in which a nitrogenous base (e.g., a nucleobase) has been removed by cleaving the glycosidic bond between the ribose (or deoxyribose) sugar and the base.

[0060] In some embodiments, a double-stranded nucleic acid contains one or more (e.g., one, two, three, four, five, or more) modified internucleotide linkages. As used herein, a "modified internucleotide linkage" refers to an internucleotide linkage having one or more chemical modifications compared to a reference internucleotide linkage having a phosphodiester bond. The modified internucleotide linkage may be a non-naturally occurring linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate (PS) linkage. Examples of other modified nucleic acids and modified internucleotide linkages include, but are not limited to, boranophosphate, alkylphosphonate-inhibitory nucleic acids, peptide-inhibitory nucleic acids, and morpholino. Morpholino backbones are described, for example, by Corey and Abrams Genome Biol. 2001;2(5):reviews1015.1-reviews1015.3.

[0061] The number of modified internucleotide linkages in each strand of a double-stranded nucleic acid can vary. In some embodiments, each strand of a double-stranded nucleic acid comprises 1 to 23 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23) modified internucleotide linkages. In some embodiments, the sense strand of a double-stranded nucleic acid comprises 1 to 5 (e.g., 1, 2, 3, 4, or 5) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are positioned starting from the first nucleotide at the 5' end of the sense strand. In some embodiments, the modified internucleotide linkage is located between the last two nucleotides at the 3' end of the sense strand (e.g., one or more of positions 1, 2, and 3 relative to the 3' end of the sense strand). In some embodiments, the antisense strand of the double-stranded nucleic acid comprises 1 to 5 (e.g., 1, 2, 3, 4, or 5) modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is located starting from the first nucleotide at the 5' end of the antisense strand. In some embodiments, the modified internucleotide linkage is located between the last two nucleotides at the 3' end of the antisense strand (e.g., one or more of positions 1, 2, and 3 relative to the 3' end of the antisense strand). In some embodiments, each of the modified internucleotide linkages is a phosphorothioate (PS) linkage.

[0062] In some embodiments, the antisense strand of the double-stranded nucleic acid comprises four or fewer phosphorothioate internucleotide linkages. In some embodiments, positions 1 and 2 (e.g., relative to the 5' or 3' end) of the sense strand are linked by a modified internucleotide linkage. In some embodiments, positions 1 and 2 (e.g., relative to the 5' or 3' end) of the antisense strand are linked by a phosphorothioate internucleotide linkage. In some embodiments, positions 2 and 3 (e.g., relative to the 5' or 3' end) of the sense strand are linked by a modified internucleotide linkage. In some embodiments, positions 2 and 3 (e.g., relative to the 5' or 3' end) of the antisense strand are linked by a phosphorothioate internucleotide linkage.

[0063] In some embodiments, the target mRNA of an inhibitory nucleic acid corresponds to a gene sequence encoding a wild-type protein, or any variant thereof (e.g., a mutant, a disease-associated allele, an isoform, etc.), however, these examples should be considered non-limiting, as the chemical modifications described in this disclosure can be applied to virtually any inhibitory nucleic acid sequence to effect targeted knockdown of the target mRNA.

[0064] In some embodiments, the double-stranded nucleic acid down-regulates the expression or activity of a target mRNA. The amount of down-regulation mediated by the double-stranded nucleic acid can vary. In some embodiments, the double-stranded nucleic acid reduces the expression level or activity of a target mRNA transcript by 1-100-fold, 2-10-fold, 5-20-fold, 10-30-fold, 20-50-fold, or 25-100-fold, or any value therebetween (e.g., relative to the expression or activity of a target mRNA not contacted with the double-stranded nucleic acid). In some embodiments, the double-stranded nucleic acid reduces the expression level or activity of a target mRNA transcript by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold (e.g., relative to the expression or activity of a target mRNA not contacted with the double-stranded nucleic acid). In some embodiments, the double-stranded nucleic acid reduces the expression level or activity of the target mRNA transcript by more than 100-fold, e.g., at least 200-fold, 400-fold, 500-fold, or 1000-fold (e.g., relative to the expression or activity of the target mRNA not contacted with the double-stranded nucleic acid).

[0065] In some embodiments, the double-stranded nucleic acid is an isolated nucleic acid. Those skilled in the art will understand that an "isolated" nucleic acid is one that has been artificially produced. Artificial production of an isolated nucleic acid can be achieved, for example, through in vitro amplification via polymerase chain reaction (PCR), in vitro transcription, in vitro reverse transcription, recombinant cloning, or chemical synthesis. Methods for synthesizing isolated nucleic acids, such as RNA, are known in the art, as described, for example, by Soukchareun et al., "Preparation and characterization of antisense oligonucleotide-peptide hybrids containing viral fusion peptides." Bioconjug Chem. 1995 Jan-Feb;6(1):43-53. doi:10.1021 / bc00031a004. PMID:7711103. In some embodiments, the sense and antisense strands of inhibitory nucleic acids can be synthesized using solid-phase synthesis, for example, by using phosphoramidite chemistry (e.g., Current Protocols in Inhibitory Nucleic Acid Chemistry, Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphortriester chemistry, or enzymatic synthesis. Automated commercial synthesizers, such as the MerMade by LGC Biosearch Technologies, are also available. TM 12, or other synthesizers from BioAutomation or Applied Biosystems, can be used. Phosphorothioate linkages can be introduced using sulfurizing reagents such as phenylacetyl disulfide or DDTT ((dimethylaminomethylidene)amino)-3H-1,2,4-dithiazaolin-3-thione). The use of similar techniques, as well as commercially available modified amidites and controlled-pore glass (CPG) products, to synthesize modified or conjugated oligonucleotides is well known.

[0066] Purification methods can be used to remove unwanted impurities from the final oligonucleotide product. Commonly used purification techniques for single-stranded oligonucleotides include reverse-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC), capillary gel electrophoresis (CGE), anion-exchange HPLC (AX-HPLC), and size-exclusion chromatography (SEC). After purification, the oligonucleotides can be analyzed by mass spectrometry and quantified by spectrophotometry at a wavelength of 260 nm. The sense and antisense strands can then be annealed to form double-stranded RNA.

[0067] [Sense and antisense strands] Aspects of the present disclosure relate to double-stranded nucleic acids having a sense strand and an antisense strand, each of which comprises a specific pattern of chemical modification (e.g., a pattern of 2'F-modified nucleotides). As used herein, "antisense strand" refers to a single-stranded oligonucleotide that is complementary to a region of a target sequence (e.g., the sequence of a target mRNA). Similarly, as used herein, "sense strand" refers to a single-stranded oligonucleotide that is complementary to a region of the antisense strand.

[0068] Aspects of the present disclosure are based, in part, on antisense strands having 2'F-modified nucleotides at positions 2, 5, 7, 14, and 16, or 2, 3, 7, 14, and 16, or 2, 5, 8, 14, and 16, or 2, 4, 6, 8, 14, and 16, or any combination of positions 2, 6, 8, 14, and 16, relative to the 5' end of the antisense strand. In some embodiments, the 2'F-modified antisense strand does not contain 2'F-modified nucleotides other than those at the recited positions. In some embodiments, the antisense strand (and double-stranded nucleic acids comprising the antisense strand) have improved potency, durability, and / or reduced off-target effects relative to previously described chemically modified double-stranded nucleic acids.

[0069] In some embodiments, the antisense strand comprises 2'F-modified nucleotides at positions 2, 5, 7, 14, and 16 relative to the 5' end of the antisense strand, and does not comprise 2'F-modified nucleotides other than those at the recited positions. In some embodiments, the antisense strand further comprises one or more additional modified nucleotides. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 additional modified nucleotides. In some embodiments, the one or more additional modified nucleotides each have a 2'O-alkyl modification, e.g., a 2'O-methyl (2'O-Me) modification or a 2'OC 12~16 In some embodiments, the antisense strand comprises an alkyl modification. In some embodiments, each of the one or more additional modified nucleotides comprises a 2'O-methyl (2'O-Me) modification. In some embodiments, the antisense strand comprises one or more abasic moieties. In some embodiments, the antisense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate (PS) linkages. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the antisense strand and between positions 1 and 2 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' end of the antisense strand, and between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the antisense strand.

[0070] In some embodiments, the antisense strand is hybridized to the sense strand. In some embodiments, the sense strand comprises one or more 2'F-modified nucleotides. In some embodiments, the sense strand comprises 2'F-modified nucleotides at the following positions: (a) positions 9, 10, and 11; (b) positions 7, 9, and 11; (c) positions 7, 9, and 10; or (d) positions 7, 10, and 11. In some embodiments, the sense strand does not comprise 2'F-modified nucleotides at positions other than those listed. In some embodiments, the sense strand further comprises one or more additional modified nucleotides. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 additional modified nucleotides. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-alkyl modification. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-methyl (2'O-Me) modification. In some embodiments, the sense strand comprises one or more abasic moieties. In some embodiments, the sense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate (PS) linkages. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 3' end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the sense strand and between positions 1 and 2 relative to the 3' end of the sense strand. In some embodiments, the sense strand comprises PS bond between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the sense strand.In some embodiments, the sense strand contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' end of the sense strand, and between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the sense strand.

[0071] In some embodiments, the disclosure provides a double-stranded nucleic acid comprising a sense strand and an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides, wherein the modified nucleotides consist of 2'F-modified nucleotides present at positions 2, 3, 7, 14, and 16 relative to the 5' end of the antisense strand, and 2'O-Me-modified nucleotides at other positions in the antisense strand; and wherein the sense strand consists of 2'F-modified nucleotides present at, and only at, the following positions: (a) positions 9, 10, and 11; (b) positions 7, 9, and 11; (c) positions 7, 9, and 10; or (d) positions 7, 10, and 11, and 2'O-Me-modified nucleotides at other positions in the sense strand, 2'OC 12~16 A double-stranded nucleic acid is provided, which contains alkyl-modified nucleotides or abasic portions, and each of the sense and antisense strands contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' and 3' ends, respectively.

[0072] In some embodiments, the antisense strand comprises 2'F-modified nucleotides at positions 2, 3, 7, 14, and 16 relative to the 5' end of the antisense strand, and does not comprise 2'F-modified nucleotides other than those at the recited positions. In some embodiments, the antisense strand further comprises one or more additional modified nucleotides. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 additional modified nucleotides. In some embodiments, each of the one or more additional modified nucleotides comprises a 2'O-alkyl modification. In some embodiments, each of the one or more additional modified nucleotides comprises a 2'O-methyl (2'O-Me) modification. In some embodiments, the antisense strand comprises one or more abasic moieties. In some embodiments, the antisense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate (PS) linkages. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the antisense strand and between positions 1 and 2 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' end of the antisense strand, and between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the antisense strand.

[0073] In some embodiments, the antisense strand is hybridized to the sense strand. In some embodiments, the sense strand comprises one or more 2'F-modified nucleotides. In some embodiments, the sense strand comprises 2'F-modified nucleotides at the following positions: (a) positions 9, 10, and 11; (b) positions 7, 9, and 11; (c) positions 7, 9, and 10; or (d) positions 7, 10, and 11. In some embodiments, the sense strand does not comprise 2'F-modified nucleotides at positions other than those listed. In some embodiments, the sense strand further comprises one or more additional modified nucleotides. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 additional modified nucleotides. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-alkyl modification. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-methyl (2'O-Me) modification. In some embodiments, the sense strand comprises one or more 2'OC 12~16The sense strand comprises alkyl-modified nucleotides. In some embodiments, the sense strand comprises one or more abasic moieties. In some embodiments, the sense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate (PS) linkages. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 5'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 3'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 5'-end of the sense strand and between positions 1 and 2 relative to the 3'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 and between positions 2 and 3 relative to the 3'-end of the sense strand. In some embodiments, the sense strand contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' end of the sense strand, and between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the sense strand.

[0074] In some embodiments, the disclosure provides a double-stranded nucleic acid comprising a sense strand and an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides, wherein the modified nucleotides consist of 2'F-modified nucleotides present at positions 2, 5, 7, 14, and 16 relative to the 5' end of the antisense strand, and 2'O-Me-modified nucleotides at other positions in the antisense strand; and wherein the sense strand consists of 2'F-modified nucleotides present at, and only at, the following positions: (a) positions 9, 10, and 11; (b) positions 7, 9, and 11; (c) positions 7, 9, and 10; or (d) positions 7, 10, and 11, and 2'O-Me-modified nucleotides at other positions in the sense strand, 2'OC 12~16 A double-stranded nucleic acid is provided, which contains alkyl-modified nucleotides or abasic portions, and each of the sense and antisense strands contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' and 3' ends, respectively.

[0075] In some embodiments, the antisense strand comprises 2'F-modified nucleotides at positions 2, 5, 7, 14, and 16 relative to the 5' end of the antisense strand, and does not comprise 2'F-modified nucleotides other than those at the recited positions. In some embodiments, the antisense strand further comprises one or more additional modified nucleotides. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 additional modified nucleotides. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-alkyl modification. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-methyl (2'O-Me) modification. In some embodiments, the antisense strand comprises one or more abasic moieties. In some embodiments, the antisense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate (PS) linkages. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the antisense strand and between positions 1 and 2 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' end of the antisense strand, and between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the antisense strand.

[0076] In some embodiments, the antisense strand is hybridized to the sense strand. In some embodiments, the sense strand comprises one or more 2'F-modified nucleotides. In some embodiments, the sense strand comprises 2'F-modified nucleotides at the following positions: (a) positions 9, 10, and 11; (b) positions 7, 9, and 11; (c) positions 7, 9, and 10; or (d) positions 7, 10, and 11. In some embodiments, the sense strand does not comprise 2'F-modified nucleotides at positions other than those listed. In some embodiments, the sense strand further comprises one or more additional modified nucleotides. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 additional modified nucleotides. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-alkyl modification. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-methyl (2'O-Me) modification. In some embodiments, the sense strand comprises one or more 2'OC 12~16The sense strand comprises alkyl-modified nucleotides. In some embodiments, the sense strand comprises one or more abasic moieties. In some embodiments, the sense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate (PS) linkages. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 5'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 3'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 5'-end of the sense strand and between positions 1 and 2 relative to the 3'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 and between positions 2 and 3 relative to the 3'-end of the sense strand. In some embodiments, the sense strand contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' end of the sense strand, and between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the sense strand.

[0077] In some embodiments, the disclosure provides a double-stranded nucleic acid comprising a sense strand and an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides, wherein the modified nucleotides consist of 2'F-modified nucleotides present at positions 2, 5, 8, 14, and 16 relative to the 5' end of the antisense strand, and 2'O-Me-modified nucleotides at other positions in the antisense strand; and wherein the sense strand consists of 2'F-modified nucleotides present at, and only at, the following positions: (a) positions 9, 10, and 11; (b) positions 7, 9, and 11; (c) positions 7, 9, and 10; or (d) positions 7, 10, and 11, and 2'O-Me-modified nucleotides at other positions in the sense strand, 2'OC 12~16 A double-stranded nucleic acid is provided, which contains alkyl-modified nucleotides or abasic portions, and each of the sense and antisense strands contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' and 3' ends, respectively.

[0078] In some embodiments, the antisense strand comprises 2'F-modified nucleotides at positions 2, 5, 8, 14, and 16 relative to the 5' end of the antisense strand, and does not comprise 2'F-modified nucleotides other than those at the recited positions. In some embodiments, the antisense strand further comprises one or more additional modified nucleotides. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 additional modified nucleotides. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-alkyl modification. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-methyl (2'O-Me) modification. In some embodiments, the antisense strand comprises one or more abasic moieties. In some embodiments, the antisense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate (PS) linkages. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the antisense strand and between positions 1 and 2 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' end of the antisense strand, and between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the antisense strand.

[0079] In some embodiments, the antisense strand is hybridized to the sense strand. In some embodiments, the sense strand comprises one or more 2'F-modified nucleotides. In some embodiments, the sense strand comprises 2'F-modified nucleotides at the following positions: (a) positions 9, 10, and 11; (b) positions 7, 9, and 11; (c) positions 7, 9, and 10; or (d) positions 7, 10, and 11. In some embodiments, the sense strand does not comprise 2'F-modified nucleotides at positions other than those listed. In some embodiments, the sense strand further comprises one or more additional modified nucleotides. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 additional modified nucleotides. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-alkyl modification. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-methyl (2'O-Me) modification. In some embodiments, the sense strand comprises one or more 2'OC 12~16The sense strand comprises alkyl-modified nucleotides. In some embodiments, the sense strand comprises one or more abasic moieties. In some embodiments, the sense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate (PS) linkages. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 5'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 3'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 5'-end of the sense strand and between positions 1 and 2 relative to the 3'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 and between positions 2 and 3 relative to the 3'-end of the sense strand. In some embodiments, the sense strand contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' end of the sense strand, and between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the sense strand.

[0080] In some embodiments, the disclosure provides a double-stranded nucleic acid comprising a sense strand and an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides, wherein the modified nucleotides consist of 2'F-modified nucleotides present at positions 2, 4, 6, 8, 14, and 16 relative to the 5' end of the antisense strand, and 2'O-Me-modified nucleotides at other positions in the antisense strand; and wherein the sense strand consists of 2'F-modified nucleotides present at, and only at, the following positions: (a) positions 9, 10, and 11; (b) positions 7, 9, and 11; (c) positions 7, 9, and 10; or (d) positions 7, 10, and 11, and 2'O-Me-modified nucleotides at other positions in the sense strand, 2'OC 12~16 A double-stranded nucleic acid is provided, which contains alkyl-modified nucleotides or abasic portions, and each of the sense and antisense strands contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' and 3' ends, respectively.

[0081] In some embodiments, the antisense strand comprises 2'F-modified nucleotides at positions 2, 4, 6, 8, 14, and 16 relative to the 5' end of the antisense strand, and does not comprise 2'F-modified nucleotides other than those at the recited positions. In some embodiments, the antisense strand further comprises one or more additional modified nucleotides. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 additional modified nucleotides. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-alkyl modification. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-methyl (2'O-Me) modification. In some embodiments, the antisense strand comprises one or more abasic moieties. In some embodiments, the antisense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate (PS) linkages. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the antisense strand and between positions 1 and 2 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' end of the antisense strand, and between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the antisense strand.

[0082] In some embodiments, the antisense strand is hybridized to the sense strand. In some embodiments, the sense strand comprises one or more 2'F-modified nucleotides. In some embodiments, the sense strand comprises 2'F-modified nucleotides at the following positions: (a) positions 9, 10, and 11; (b) positions 7, 9, and 11; (c) positions 7, 9, and 10; or (d) positions 7, 10, and 11. In some embodiments, the sense strand does not comprise 2'F-modified nucleotides at positions other than those listed. In some embodiments, the sense strand further comprises one or more additional modified nucleotides. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 additional modified nucleotides. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-alkyl modification. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-methyl (2'O-Me) modification. In some embodiments, the sense strand comprises one or more 2'OC 12~16The sense strand comprises alkyl-modified nucleotides. In some embodiments, the sense strand comprises one or more abasic moieties. In some embodiments, the sense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate (PS) linkages. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 5'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 3'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 5'-end of the sense strand and between positions 1 and 2 relative to the 3'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 and between positions 2 and 3 relative to the 3'-end of the sense strand. In some embodiments, the sense strand contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' end of the sense strand, and between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the sense strand.

[0083] In some embodiments, the disclosure provides a double-stranded nucleic acid comprising a sense strand and an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides, wherein the modified nucleotides consist of 2'F-modified nucleotides present at positions 2, 6, 8, 14, and 16 relative to the 5' end of the antisense strand, and 2'O-Me-modified nucleotides at other positions in the antisense strand; and wherein the sense strand consists of 2'F-modified nucleotides present at, and only at, the following positions: (a) positions 9, 10, and 11; (b) positions 7, 9, and 11; (c) positions 7, 9, and 10; or (d) positions 7, 10, and 11, and 2'O-Me-modified nucleotides at other positions in the sense strand, 2'OC 12~16 A double-stranded nucleic acid is provided, which contains alkyl-modified nucleotides or abasic portions, and each of the sense and antisense strands contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' and 3' ends, respectively.

[0084] In some embodiments, the antisense strand comprises 2'F-modified nucleotides at positions 2, 6, 8, 14, and 16 relative to the 5' end of the antisense strand, and does not comprise 2'F-modified nucleotides other than those at the recited positions. In some embodiments, the antisense strand further comprises one or more additional modified nucleotides. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 additional modified nucleotides. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-alkyl modification. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-methyl (2'O-Me) modification. In some embodiments, the antisense strand comprises one or more abasic moieties. In some embodiments, the antisense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate (PS) linkages. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 relative to the 5' end of the antisense strand and between positions 1 and 2 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the antisense strand. In some embodiments, the antisense strand contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' end of the antisense strand, and between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the antisense strand.

[0085] In some embodiments, the antisense strand is hybridized to the sense strand. In some embodiments, the sense strand comprises one or more 2'F-modified nucleotides. In some embodiments, the sense strand comprises 2'F-modified nucleotides at the following positions: (a) positions 9, 10, and 11; (b) positions 7, 9, and 11; (c) positions 7, 9, and 10; or (d) positions 7, 10, and 11. In some embodiments, the sense strand does not comprise 2'F-modified nucleotides at positions other than those listed. In some embodiments, the sense strand further comprises one or more additional modified nucleotides. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 additional modified nucleotides. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-alkyl modification. In some embodiments, the one or more additional modified nucleotides each comprise a 2'O-methyl (2'O-Me) modification. In some embodiments, the sense strand comprises one or more 2'OC 12~16The sense strand comprises alkyl-modified nucleotides. In some embodiments, the sense strand comprises one or more abasic moieties. In some embodiments, the sense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate (PS) linkages. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 5'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 3'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 relative to the 5'-end of the sense strand and between positions 1 and 2 relative to the 3'-end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 and between positions 2 and 3 relative to the 3'-end of the sense strand. In some embodiments, the sense strand contains PS bonds between positions 1 and 2 and between positions 2 and 3 relative to the 5' end of the sense strand, and between positions 1 and 2 and between positions 2 and 3 relative to the 3' end of the sense strand.

[0086] In some embodiments, the sense strand comprises a nucleotide sequence that is about 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence set forth in any one of SEQ ID NOs: 1-199. In some embodiments, the sense strand comprises a nucleotide sequence that is 100% identical to a sequence set forth in any one of SEQ ID NOs: 1-199. In some embodiments, the antisense strand comprises a nucleotide sequence that is about 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any of the sequences set forth in any one of SEQ ID NOs: 1-199. In some embodiments, the antisense strand comprises a nucleotide sequence that is 100% identical to a sequence set forth in any one of SEQ ID NOs: 1-199.

[0087] In some embodiments, the double-stranded nucleic acid comprises a sense strand and an antisense strand that have about 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a pair of sense and antisense strands set forth in any of SEQ ID NOs: 1-199. In some embodiments, the double-stranded nucleic acid comprises a sense strand and an antisense strand that are 100% identical to a pair of sense and antisense strands set forth in any of SEQ ID NOs: 1-199. In some embodiments, the double-stranded nucleic acid comprises a sense strand having a modification pattern set forth in any one of SEQ ID NOs: 1-199. In some embodiments, the double-stranded nucleic acid comprises an antisense strand having a modification pattern set forth in any one of SEQ ID NOs: 1-199.

[0088] Aspects of the present disclosure relate to double-stranded nucleic acids that are not conjugated to any other molecule(s) (e.g., delivery molecules such as antibodies, lipids, sugars, etc.). In some embodiments, the double-stranded nucleic acid is not conjugated to a lipid, e.g., cholesterol, tocopherol, etc. In some embodiments, the double-stranded nucleic acid is not conjugated to an antibody, such as a transferrin receptor antibody, a low-density lipoprotein receptor (LDLR) antibody, etc. In some embodiments, the double-stranded nucleic acid is not conjugated to a sugar, such as N-acetylgalactosamine (GalNAc). However, one of skill in the art will appreciate that in some embodiments, the double-stranded nucleic acids described herein may be attached to one or more delivery molecules to form a conjugate, as described in more detail below.

[0089] [Conjugate] Aspects of the present disclosure relate to compositions comprising a double-stranded nucleic acid described herein, optionally attached to a delivery molecule. As used herein, "delivery molecule" refers to an agent that helps deliver the double-stranded nucleic acid to a target cell, directs tissue- or cell-specificity of the double-stranded nucleic acid, and / or adds additional functionality to the double-stranded nucleic acid. In some embodiments, the delivery molecule comprises a lipid, peptide, protein, antibody, or small molecule. Examples of delivery molecules include, but are not limited to, lipids (e.g., fatty acids, cholesterol, tocopherol, etc.), antibodies (e.g., cell receptor-specific antibodies, therapeutic antibodies, etc.), antigen-binding fragments (e.g., Fabs, single-chain variable fragments (scFvs)), radioligands, and sugars (e.g., GalNAc, etc.). In some embodiments, the double-stranded nucleic acid is conjugated to a lipid (e.g., cholesterol, tocopherol). In some embodiments, the double-stranded nucleic acid is conjugated to a peptide or antibody (e.g., protamine, cell receptor-specific antibody, etc.). In some embodiments, the antibody is not a transferrin receptor antibody. In some embodiments, the double-stranded nucleic acid is conjugated to a sugar (eg, GalNAc).

[0090] In some embodiments, a delivery molecule is connected (e.g., conjugated or bound) to a double-stranded nucleic acid. The delivery molecule may be directly connected to the double-stranded nucleic acid (e.g., by forming one or more interactions or bonds with nucleotides or internucleotide bonds of the double-stranded nucleic acid) or indirectly connected (e.g., via one or more linker molecules). The delivery molecule may be connected (e.g., conjugated or bound) to the double-stranded nucleic acid at any suitable position. For example, the delivery molecule may be conjugated to an end (e.g., the 5' end or 3' end) of the double-stranded nucleic acid or to an internal nucleotide or internucleotide bond of the double-stranded nucleic acid.

[0091] In some embodiments, the delivery molecule is directly conjugated to the double-stranded nucleic acid. In some embodiments, the delivery molecule is indirectly conjugated to the double-stranded nucleic acid, for example, via a linker. In some embodiments, the linker comprises an amino acid linker (e.g., a glycine-rich linker, a glycine-serine linker, a proline linker, etc.) or a small molecule (e.g., a poly-ethylene glycol linker, etc.). In some embodiments, the linker is a Mal-Tet-TCO linker, an SMCC linker, or a GDM linker, and the delivery molecule is an antibody that does not bind to the transferrin receptor. In some embodiments, the linker is not a Mal-Tet-TCO linker, an SMCC linker, or a GDM linker, and the antibody is an antibody that does not bind to the transferrin receptor. In some embodiments, the linker is a triethylene glycol (TEG) linker. In some embodiments, the conjugate comprises a double-stranded nucleic acid comprising a sequence set forth in any one of SEQ ID NOs: 1-199, a TEG linker, and cholesterol or tocopherol.

[0092] Aspects of the present disclosure relate to a conjugate comprising a double-stranded nucleic acid described herein and a GalNAc delivery molecule. Delivery molecules comprising N-acetylgalactosamine (GalNAc) are known to target the asialoglycoprotein receptor of hepatocytes, and are one modality for delivering double-stranded nucleic acids to desired tissues. In some embodiments, the double-stranded nucleic acid is represented by Formula II: [ka] It is conjugated to a GalNAc delivery molecule comprising the structure:

[0093] In some embodiments, the double-stranded nucleic acid is conjugated to Formula II via a linker. Suitable linkers are known in the art. In one embodiment, the linker comprises an alkyl chain, preferably a C1-10 (e.g., a C6 chain). In some embodiments, the linker comprises a C6 amide (C6-NH2). In a further embodiment, the linker is shown below as Linker 1 with connection points A and B (Formula III). In another embodiment, the linker comprises piperidine. In a further preferred embodiment, the linker is shown below as Linker 2 with connection points C and D (Formula IV). [ka]

[0094] In some embodiments, Linker 1 (Formula III), connection point A, or Linker 2 (Formula IV), connection point C, is conjugated to Formula II. In some embodiments, connection point A of Linker 1 is conjugated to Formula II, and connection point B of Linker 1 is conjugated to a double-stranded nucleic acid. In some embodiments, connection point C of Linker 2 is conjugated to Formula II, and connection point D of Linker 2 is conjugated to a double-stranded nucleic acid. In some embodiments, connection point A of Linker 1 is conjugated to Formula II, and connection point B of Linker 1 is conjugated to a phosphate group conjugated to a double-stranded nucleic acid. In some embodiments, connection point C of Linker 2 is conjugated to Formula II, and connection point D of Linker 2 is conjugated to a phosphate group conjugated to a double-stranded nucleic acid.

[0095] Those skilled in the art will understand that the linker may be at the 5' or 3' end of the double-stranded nucleic acid, or may be attached to one of the internal nucleotide or nucleoside bases. Those skilled in the art will also understand that the linker may be linked or conjugated to the 5' or 3' end of the double-stranded nucleic acid. Those skilled in the art will also understand that placement of a delivery molecule, such as a delivery molecule comprising Formula II, at the 5' end of the double-stranded nucleic acid, whether via a linker or not, may be necessary to overcome potentially inefficient loading of Ago2 or other interference with RISC complex activity. For example, in the case of a delivery molecule comprising Formula II linked to or directly conjugated to an siRNA comprising a sense strand and an antisense strand, placement of the delivery moiety at the 5' end of the antisense strand may create difficulties with Ago2 loading and prevent efficient knockdown. In a preferred embodiment, one or more double-stranded nucleic acids comprise an siRNA comprising a sense strand and an antisense strand, and the delivery moiety comprising Formula II is present at the 3' end of the sense strand. In a further embodiment, the delivery moiety comprising Formula II is conjugated to the 3'-end of the sense strand via a linker. In yet a further embodiment, the linker comprises a ring structure, preferably a piperidine ring. In yet a further embodiment, the linker comprises Linker 2.

[0096] [Pharmaceutical composition] The compositions described herein may further comprise pharmaceutical excipients, buffers, or diluents and may be formulated for administration to host cells ex vivo or for administration in animals, particularly humans, in situ. Such compositions may optionally further comprise liposomes, lipids, lipid complexes, lipid nanoparticles (LNPs), microspheres, microparticles, nanospheres, or nanoparticles, or may be otherwise formulated for administration to cells, tissues, organs, or the body of a subject in need thereof. Such compositions may be formulated for use in a variety of therapies, such as ameliorating, preventing, and / or treating conditions associated with expression of mutant proteins, polypeptides, or peptides, or overexpression of proteins, polypeptides, or peptides.

[0097] Formulations, including pharmaceutically acceptable excipient and / or carrier solutions, are well known to those skilled in the art, as is the development of suitable dosing and treatment regimens for use with the particular compositions described herein in a variety of treatment regimens, including, for example, oral, parenteral, intravenous, intrathecal, subcutaneous, or intracisternal, intranasal, intraarticular, and intramuscular administration and formulations.

[0098] Typically, these formulations will contain at least about 0.1% or more of a therapeutic agent (e.g., nucleic acid or liposomes containing nucleic acid), although the percentage of active ingredient(s) may, of course, vary and may conveniently be from about 1% or 2% to about 70% or 90% or more by weight or volume of the total formulation. The amount of therapeutic agent(s) in each therapeutically useful composition may be prepared in such a way that a suitable dosage is obtained in any unit dose of the compound. Factors such as solubility, stability, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be considered by those skilled in the art when preparing such pharmaceutical formulations. Furthermore, various dosages and treatment regimens may be desirable.

[0099] In certain circumstances, it may be desirable to deliver a nucleic acid or preparation thereof in a suitably formulated pharmaceutical composition disclosed herein to one or more cells, tissues, or organs either subcutaneously, intraocularly, intravitreally, parenterally, intravenously, intraventricularly, intracisternally, intramuscularly, intrathecally, orally, intraperitoneally, or by oral or nasal inhalation, or by direct injection.

[0100] Pharmaceutical formulations of compositions suitable for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the formulations are sterile and fluid to the extent that easy syringability exists. In some embodiments, the form is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, vegetable oils, or other pharmaceutically acceptable carriers generally recognized as safe (GRAS) by the U.S. Food and Drug Administration. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Thus, nucleic acids can be delivered in conjunction with various other pharmaceutically acceptable agents as required in specific cases. Such compositions may be purified from host cells or other biological sources, or alternatively, chemically synthesized as described herein.

[0101] The amount of nucleic acid and / or nucleic acid composition and the time of administration of the composition will be within the purview of one of ordinary skill in the art having the benefit of the teachings herein. In some embodiments, the administration is a single administration. In some embodiments, the administration is more than one administration. In some situations, the administration schedule can be determined by a physician overseeing the administration of the composition.

[0102] The toxicity and efficacy of the compositions utilized in the methods of the present disclosure can be determined by standard pharmaceutical procedures using either cells in culture or experimental animals to determine the LD50 (the dose lethal to 50% of a population) and / or IC50 (the half maximal inhibitory concentration). The dose of a composition used for therapeutic purposes can be selected based on its therapeutic index, which is the ratio of toxicity to efficacy and can therefore be expressed as the ratio LD50 / ED50 or LD50 / IC50 (where "ED50" means the dose effective in 50% of a population, and "IC50" means the dose effective for 50% inhibition of target mRNA translation). Compositions that exhibit large therapeutic indices are preferred. Compositions that exhibit toxic side effects may be used, although care should be taken to design a delivery system that minimizes the potential for damage from such side effects. The dosage of the compositions described herein generally lies within a range that includes the ED50 or IC50 with little or no toxicity. Dosages within this range may vary depending on the dosage form employed and the route of administration utilized.

[0103] Other aspects of the present disclosure relate to methods and preparations for use with subjects, such as human or non-human subjects, host cells in situ in a subject, host cells ex vivo, or host cells derived from a subject. Non-limiting examples of subjects include domestic animals such as dogs and cats, horses, cows, pigs, sheep, goats, and chickens, as well as other animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject is a human subject.

[0104] In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) are added to a pharmaceutical composition comprising a therapeutic agent, thereby forming a pharmaceutical formulation suitable for in vivo delivery to a subject, such as a human.

[0105] A pharmaceutical composition or drug product contains at least one therapeutic agent in a pharmacologically effective amount and, optionally, one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (vehicle) is a substance other than the active pharmaceutical ingredient (API, therapeutic product) that is intentionally included in a drug delivery system. An excipient does not, and is not intended to, exert a therapeutic effect at the intended dosage. Excipients may a) function to aid in the processing of the drug delivery system during manufacturing; b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API; c) aid in product identification; and / or d) enhance other aspects of the overall safety, efficacy, or delivery of the API during storage or use. Pharmaceutically acceptable excipients may or may not be inert substances.

[0106] Excipients include, but are not limited to, absorption enhancers, anti-adherents, anti-foaming agents, antioxidants, binders, buffers, carriers, coatings, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavorings, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, water repellents, and wetting agents.

[0107] The pharmaceutical composition may contain other additional ingredients commonly found in pharmaceutical compositions, such as, but not limited to, antipruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine).

[0108] The carrier can be a solvent or dispersion medium containing, for example, but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The carrier may also contain auxiliary substances such as preservatives, wetting agents, emulsifying agents, and dispersing agents. The carrier may also contain isotonic agents, such as sugars, polyalcohols, sodium chloride, etc., in the composition.

[0109] Pharmaceutically acceptable refers to properties and / or substances that are acceptable to a subject from a pharmacological / toxicological standpoint. The phrase pharmaceutically acceptable refers to molecular entities, compositions, and properties that are physiologically tolerable and do not typically produce allergic or other harmful or toxic reactions when administered to a subject. In some embodiments, a pharmaceutically acceptable compound is approved by a federal or state regulatory agency or is listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, more particularly in humans.

[0110] [method] Aspects of the present disclosure relate to methods for inhibiting or reducing target mRNA in a cell using the compositions (e.g., double-stranded nucleic acids, conjugates, pharmaceutical compositions, etc.) described herein. In some embodiments, the method includes contacting a cell containing the target mRNA with the double-stranded nucleic acid (or a conjugate containing the double-stranded nucleic acid). In some embodiments, the method includes delivering the double-stranded nucleic acid to a cell, e.g., a mammalian cell or a human cell (e.g., contacting the cell with the double-stranded nucleic acid). In some embodiments, the cell is in a subject (e.g., a human subject).

[0111] The amount of target mRNA inhibition or reduction can vary. In some embodiments, the target mRNA is reduced by about 50% to about 100% relative to the amount of target mRNA prior to administration. In some embodiments, the target mRNA is reduced by about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% relative to the amount of target mRNA prior to administration. In some embodiments, the target mRNA is reduced by about 5-fold to about 100-fold relative to the amount of target mRNA prior to administration. In some embodiments, the target mRNA is reduced by about 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold relative to the amount of target mRNA prior to administration. In some embodiments, the target mRNA is SNCA mRNA. In some embodiments, the target mRNA is APOE mRNA.

[0112] Aspects of the present disclosure relate to double-stranded nucleic acids having a particular pattern of 2'-F modifications (e.g., an antisense strand having 2'F-modified nucleotides at positions 2, 5, 7, 14, and 16, or positions 2, 3, 7, 14, and 16, or positions 2, 5, 8, 14, and 16, or positions 2, 6, 8, 14, and 16, relative to the 5' end of the antisense strand), which have increased durability in vivo relative to double-stranded nucleic acids having other modification patterns. In some embodiments, increased durability refers to the persistence of knockdown or gene silencing mediated by the double-stranded nucleic acid. In some embodiments, increased durability refers to the resistance of the double-stranded nucleic acid to degradation (e.g., enzymatic degradation) in vivo. In some embodiments, the double-stranded nucleic acids described herein persist in vivo about 10% to about 100% longer relative to double-stranded nucleic acids with a different pattern of modifications (or unmodified double-stranded nucleic acids). In some embodiments, the double-stranded nucleic acids described herein persist in vivo about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% longer than double-stranded nucleic acids with a different pattern of modifications (or unmodified double-stranded nucleic acids). In some embodiments, the double-stranded nucleic acids described herein persist in vivo about 5-fold and about 100-fold longer relative to double-stranded nucleic acids with a different pattern of modifications (or unmodified double-stranded nucleic acids). In some embodiments, the double-stranded nucleic acids described herein persist in vivo about 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold longer relative to double-stranded nucleic acids with a different pattern of modifications (or unmodified double-stranded nucleic acids).

[0113] Aspects of the present disclosure relate to double-stranded nucleic acids that have reduced (or decreased) off-target effects relative to double-stranded nucleic acids with a different pattern of modification (or unmodified double-stranded nucleic acids). In some embodiments, off-targeting is reduced by about 50% to about 100% relative to double-stranded nucleic acids with a different pattern of modification (or unmodified double-stranded nucleic acids). In some embodiments, off-targeting is reduced by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% relative to double-stranded nucleic acids with a different pattern of modification (or unmodified double-stranded nucleic acids). In some embodiments, off-targeting is reduced by about 2-fold to about 100-fold relative to double-stranded nucleic acids with a different pattern of modification (or unmodified double-stranded nucleic acids). In some embodiments, off-targeting is reduced by about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold relative to a double-stranded nucleic acid having a different pattern of modification (or an unmodified double-stranded nucleic acid).

[0114] In some aspects, the present disclosure provides methods for treating a subject having or suspected of having a disease, disorder, or condition. In some embodiments, the subject is a human (sometimes referred to as a "patient"). Treating a subject includes administering a composition (e.g., a double-stranded nucleic acid, a conjugate, a pharmaceutical composition, etc.) as described herein to the subject. As used herein, "treatment" or "treating" refers to any process that may slow, control, delay, or halt the onset or progression of a disorder or disease disclosed herein, or ameliorate the symptoms of the disorder or disease, but does not necessarily indicate the complete disappearance of all disorder or disease symptoms. Treatment includes administering a composition (e.g., a double-stranded nucleic acid, a conjugate, a pharmaceutical composition, etc.) described herein for the treatment of a disease or condition in a patient, particularly a human. In some embodiments, administering a double-stranded nucleic acid described herein is a prophylactic treatment of a subject who does not or has never had the disease but is at risk of developing the disease, or a subject who has had the disease and is no longer disease-free but is at risk of disease relapse. In certain embodiments, the subject is at increased risk of developing the disease or at increased risk of recurrence of the disease relative to the average healthy person in the population.

[0115] "Onset" or "progression" of a disease, disorder, or condition refers to the initial manifestation and / or subsequent progression of the disease, disorder, or condition. Onset of a disease, disorder, or condition is detectable and assessable using standard clinical techniques well known in the art. However, onset also refers to progression, which may be undetectable. In this disclosure, onset or progression refers to the biological process of the condition.

[0116] The optimal course of administration or delivery of the compositions (e.g., double-stranded nucleic acids, conjugates, pharmaceutical compositions, etc.) of the present disclosure can vary depending on the desired outcome and / or the subject being treated. As used herein, "administration" refers to contacting a cell with double-stranded nucleic acids and preparations thereof, and can be performed in vitro (including ex vivo) or in vivo. The compositions (e.g., double-stranded nucleic acids, conjugates, pharmaceutical compositions, etc.) provided herein can be administered by several routes, including, but not limited to, oral administration, intravenous administration (e.g., systemic intravenous injection / administration), intrathecal administration, subcutaneous administration, or intracisternal administration to the brain.

[0117] Generally, the most appropriate route of administration will depend on a variety of factors, including the properties of the agent (e.g., the stability of the agent in the patient's environment) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration, injection, etc.). In some embodiments, a composition is administered to a subject via only one route of administration. In some embodiments, multiple routes of administration may be utilized to administer a composition to a subject (e.g., sequentially or simultaneously).

[0118] In some embodiments, an effective amount (e.g., an amount sufficient to reduce the expression or activity of a target mRNA) is administered to a subject. An "effective amount" refers to the amount (for the duration and means of administration) necessary to achieve the desired therapeutic result. An effective amount may vary according to factors such as the individual's condition, age, sex, and weight, and the ability of the double-stranded nucleic acid to elicit a desired response in the individual. An effective amount is also one in which any toxic or harmful effects of administration are outweighed by the therapeutically beneficial effects. An effective amount depends primarily on factors such as the subject's species, age, weight, health condition, and tissue being targeted, and therefore may vary between animals and tissues. In some embodiments, an effective amount may be a combination of an effective dosage, frequency of administration, and duration of administration.

[0119] During the course of treatment, the administration of the composition (e.g., double-stranded nucleic acid, conjugate, pharmaceutical composition, etc.) can be changed or adjusted as appropriate. For example, the expression of the target mRNA and / or the protein product encoded by the target mRNA can be monitored to obtain information for how to use the composition. Expression information can be obtained, for example, by measuring changes in the levels of the protein product or RNA product of the target mRNA. [Example]

[0120] Example 1: Synthesis of RNAi Agents Single strands of the RNA duplex (sense and antisense) are transfected onto a solid support using MerMade TM The oligonucleotides were synthesized by 12 (LGC Biosearch Technologies). The sequences of the unmodified and modified sense and antisense strands are shown in Tables 1-4. The mRNA target 1 (APOE) molecule and the mRNA target 2 (SNCA) molecule are not encoded by the same gene. These oligonucleotides were synthesized using phosphoramidite chemistry. In some embodiments, the sense strand described in Table 2 or Table 4 includes a delivery molecule such as a lipid (e.g., cholesterol ester, cholesteryl). In some embodiments, the delivery molecule (e.g., lipid) is attached to the sense strand via a linker, such as a TEG linker.

[0121] [Table 1]

[0122] [Table 2] TIFF2026504639000005.tif201149

[0123] [Table 3]

[0124] [Table 4] TIFF2026504639000008.tif213149 TIFF2026504639000009.tif249149

[0125] [Table 5] TIFF2026504639000011.tif211149 TIFF2026504639000012.tif186149

[0126] In some embodiments, a conjugate described herein comprises a sequence defined in Table 2 or Table 4 or Table 5. In some embodiments, a conjugate described herein comprises a double-stranded RNA defined in Table 2 or Table 4 or Table 5 (e.g., a conjugate comprising a sense strand and an antisense strand of a double-stranded RNA defined in Table 2 or Table 4 or Table 5, wherein the sense strand is conjugated to tocopherol or cholesterol). In some embodiments, a conjugate described herein comprises a sequence defined in Table 2 or Table 4 or Table 5 (e.g., a sense strand) linked (e.g., covalently linked) to a linker described herein (e.g., a TEG linker such as one that connects the sense strand of the double-stranded RNA to a tocopherol or cholesterol).

[0127] For the sense strand, the solid support type was Universal CPG: Universal UnyLinker (Chemgenes, catalog number AT273-27), 3'Teg-tocopherol (LGC Biosearch Technologies, catalog number BG7-1190), and 3'Teg-cholesterol (Chemgenes, catalog number N-9166-05) were purchased commercially. For all antisense strands, commercially available standard supports were utilized and varied depending on the sequence. Standard reagents were used in oligo synthesis (Table 6), with 0.1 M xanthan hydride-pyridine used as the sulfurizing reagent and 20% DEA-ACN used as a post-synthesis auxiliary detergent. All monomers (Table 7) were made up at 0.1 M in ACN and included molecular sieve collection bags.

[0128] Oligonucleotide cleavage and deprotection (C / D) were carried out at 45°C for 20 hours. C / D of the sense strand from the CPG was performed using ammonium hydroxide (28-30%, cold), while that of the antisense strand was performed using 3% DEA-ammonium hydroxide (28-30%, cold). C / D was deemed complete when the resulting mass data confirmed sequence identity by IP-RP LCMS. Depending on the scale, the CPG was filtered through a 0.45 μm PVDF syringeless filter, a 0.22 μm PVDF Steriflip® vacuum filtration, or a 0.22 μm PVDF Stericup® Quick Release. The CPG was backwashed / rinsed with either 30% ACN / RNAse-free water or 30% EtOH / RNAse-free water, then filtered through the same filtration device and combined with the first filtrate. This was repeated twice. This material was then equally divided into 50 mL Falcon tubes and placed in a Genevac. TMAfter concentration, the crude oligonucleotides were diluted back to the synthesis scale with RNAse-free water and filtered through either a 0.45 μm PVDF syringeless filter, a 0.22 μm PVDF Steriflip® vacuum filtration, or a 0.22 μm PVDF Stericup® Quick release.

[0129] AKTA using source 15Q-RP columns for either anion exchange (AEX) or reversed phase (RP) TM Crude oligonucleotides were purified using a Pure purification system. AEX was performed using ES Industry's Source HPLC system, maintained at a column temperature of 65°C. TM 15Q column, MPA: 20 mM NaH2PO4, 15% ACN, pH 7.4, and MPB: 20 ​​mM NaH2PO4, 1 M NaBr, 15% ACN, pH 7.4. TM 15Q-RP column with MPA: 50 mM NaOAc with 10% ACN and MPB: 50 mM NaOAc with 80% ACN. In all cases, fractions contained greater than 85% mass purity and contained no more than 5% combined impurities.

[0130] The purified oligonucleotides were desalted using a 15 mL 3K MWCO centrifuge spin tube at 3500 x g for approximately 30 minutes. The oligonucleotides were rinsed with RNAse-free water until the conductivity of the eluate reached less than 100 μg / cm. After desalting was complete, 2-3 mL of RNAse-free water was added, followed by 10 cycles of aspirating, and the retentate was transferred to a 50 mL Falcon tube. This process was repeated until the oligos were completely transferred, as measured by nanodrop filtration of the compound concentration on the filter. The final oligonucleotides were then nanofiltered twice using a 15 mL 100K MWCO centrifuge spin tube at 3500 x g for 2 minutes. The final desalted oligonucleotides were analyzed for concentration (nanodrop at A260) and characterized for mass purity by IP-RP LCMS and UV purity by UPLC.

[0131] To prepare the duplexes, equimolar amounts of the sense and antisense strands were combined and heated to 65°C for 10 minutes, then slowly cooled to ambient temperature over 40 minutes. The integrity of the duplexes was confirmed by UPLC analysis and characterized by LCMS using IP-RP. All duplexes were nanofiltered, and endotoxin levels were measured using a Charles River Endosafe® cartridge device to obtain the final conjugated RNAi compounds. For in vivo analysis, the appropriate amount of duplex was lyophilized and then reconstituted with 1X PBS for rodent studies and CSF for non-human primate studies.

[0132] [Table 6]

[0133] [Table 7]

[0134] Example 2. In vitro characterization of RNAi agents Selected RNAi agents were tested for inhibition of mRNA target 1 (APOE) or mRNA target 2 (SNCA) in cultured cells, including HEP3B, SH-SY5Y, iPSC astrocyte cells, or mouse primary cortical neurons.

[0135] Materials and Methods Culture, RNAi Treatment, and Analysis of HEP3B, SH-SY5Y, and Astrocyte Cells: SH-SY5Y cells (ATCC CRL-2266) were derived from the SK-N-SH neuroblastoma cell line (Ross, RA et al., 1983. J Natl Cancer Inst 71, 741-747). Basal medium consisted of a 1:1 mixture of ATCC-formulated Eagle's Minimum Essential Medium (Cat. No. 30-2003) and F12 medium. Complete growth medium was supplemented with 10% fetal bovine serum, 1X amino acids, 1X sodium bicarbonate, and 1X penicillin-streptomycin (Gibco), and cells were incubated at 37°C in a humidified atmosphere with 5% CO2. On day 1, SH-SY5Y cells were plated onto 96-well fibronectin-coated tissue culture plates and allowed to adhere overnight. On day 2, the complete medium was removed and replaced with RNAi agent in serum-free medium. Cells were incubated with RNAi agents for 72 hours, after which gene expression was analyzed. Analysis of changes in gene expression in RNAi-treated SH-SY5Y cells was performed using Cells-to-C T Measurements were performed using a kit according to the manufacturer's protocol (ThermoFisher A35377). Predesigned gene expression assays (supplied as a 20X mixture) were selected from Applied Biosystems (Foster City, CA, USA). The efficiency of these assays (mRNA target 1 or mRNA target 2 and ThermoFisher Hs99999905_m1 GAPDH) was characterized using a dilution series of cDNA. RT-QPCR was performed using a QuantStudio 7 Flex system in a MicroAmp Optical 384-well reaction plate. Relative amounts of gene expression were determined using the delta-delta CTC method with normalization to the housekeeping gene GAPDH. IC50 values ​​were determined using a four-parameter logistic fit using GraphPad Prism v9.0.

[0136] HEP3B cells (ATCC HB-8064) were cultured in medium containing ATCC-formulated Eagle's Minimum Essential Medium (Cat. No. 30-2003) containing fetal bovine serum at a final concentration of 10%. On day 1, HEP3B cells were plated into 96-well tissue culture plates and allowed to adhere overnight. On day 2, the complete medium was removed and replaced with RNAi agent in serum-free medium. Cells were incubated with RNAi agent for 72 hours before protein expression analysis. Analysis of changes in protein expression in RNAi-treated HEP3B cells was measured using a Perkin-Elmer AlphaLISA assay (AL395) to assess protein levels corresponding to mRNA target 1 expression, according to the manufacturer's protocol. IC50 values ​​were determined using a four-parameter logistic fit using GraphPad Prism v9.0.

[0137] iPSC-derived astrocytes (Cellular Dynamics 01434) were grown according to the manufacturer's protocol. On day 1, astrocytes were plated into 96-well tissue culture plates and allowed to adhere overnight. On day 2, the complete medium was removed and replaced with RNAi agents in serum-free medium. After incubating the cells with the RNAi agents for 72 hours, gene expression was analyzed using the method described above (Thermofisher assay ID Hs00171168_m1).

[0138] Culture, RNAi Treatment, and Analysis of Mouse Primary Cortical Neurons (MCNs): Mouse primary cortical neurons were isolated from wild-type C57BL6 mouse embryos at E18. Cells were plated at a density of 40,000 (40k) cells / well in poly-D-lysine-coated 96-well plates and cultured in NbActiv1 (BrainBits, LLC) containing 1% antibiotic / antimycotic (Corning) for 7 days at 37°C in a humidified chamber containing 5% CO2 in a tissue culture incubator. On day 7, half of the medium was removed from each well, and RNAi was added at a 2x concentration in culture medium containing 2% FBS for treatment as CRCs. The cells were incubated for an additional 7, 14, or 21 days. Half the medium was replaced with fresh culture medium every 7 days. At the end of RNAi treatment, SNCA levels were quantified by RT-qPCR using the TaqMan Fast Advanced Cell-to-CT kit. Specifically, cells were lysed, cDNA was generated using a Mastercycler X50a (Eppendorf), and qPCR was performed using a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). APOE gene expression levels were normalized to β-actin (ThermoFisher, Mm02619580_g1) using the respective probes.

[0139] 293T luciferase transfection, RNAi treatment, and analysis: 293T cells transfected with a pMIR-luciferase construct (Invitrogen, Waltham, MA) containing the target SNCA sequence were plated overnight at 37°C and 5% CO2. On day 2, cells were transfected with siRNA using RNAiMAX (Invitrogen, Waltham, MA) according to the manufacturer's protocol. Cells were incubated for 48 hours at 37°C and 5% CO2. After the plates were cooled to room temperature, an equal volume of Bio-Glo (Promega, Madison, WI) was added to each well. Plates were incubated in the dark at room temperature and read on a BioTek Neos2 plate reader (Agilent, Santa Clara, CA).

[0140] Representative results from these analyses are shown in Table 11.

[0141] result Tables 8, 9, 10, and 11 show the in vitro activity of RNAi agents against mRNA target 1 (APOE) and mRNA target 2 (SNCA) in SH-SY5Y, HEP3B, astrocyte cells, and mouse primary cortical neurons (MCN).

[0142] [Table 8]

[0143] [Table 9] TIFF2026504639000017.tif115149

[0144] [Table 10] TIFF2026504639000019.tif69149

[0145] [Table 11]

[0146] Example 3. In vivo characterization of selected RNAi agents in mice The efficacy of RNAi agents against mRNA target 1 (APOE) was studied in the brain and liver of mRNA target 1 knock-in (KI) mice. Six mice received intracerebroventricular (ICV) injections of 100 μg of RNAi agent (e.g., dsRNAs described in Table 2) or PBS (phosphate-buffered saline) and were sacrificed either on Day 15 or Day 57 after injection. Human mRNA target 1 (APOE) expression in the spinal cord and brain was measured and analyzed by quantitative PCR (qPCR). Another cohort of mRNA target 1 (APOE) KI mice received SC injections of RNAi agent or PBS. Blood was collected serially for up to 12 weeks after injection, and serum was analyzed for mRNA target 1 (APOE) protein levels as described above.

[0147] Representative results are shown in Table 12. The data in Table 12 indicate that dsRNA constructs containing 2'F modifications at positions 2, 5, 7, 14, and 16 of the antisense strand were more durable in vivo than dsRNA constructs containing 2'F modifications at other positions. Furthermore, injection of two different concentrations of dsRNA constructs containing 2'F modifications at positions 2, 5, 7, 14, and 16 of the antisense strand resulted in a reduction in serum levels of mRNA target 1 (APOE) protein in vivo for at least 8 weeks and in reduced mRNA target 1 (APOE) expression in several brain tissues, including the brainstem (BS), hippocampus (H), hippocampal cortex (HCTX), and frontal cortex (FCTX).

[0148] The efficacy of RNAi agents against mRNA target 2 (SNCA) was studied in wild-type C56BL / 6N mice. Six mice received an intracerebroventricular (ICV) injection of 30 μg of RNAi agent (e.g., dsRNA described in Table 4) or PBS (phosphate-buffered saline) and were sacrificed either on D15 or D57 post-injection. Mouse mRNA target 2 (SNCA) mRNA expression in the spinal cord and brain was measured and analyzed by quantitative PCR (qPCR). The data are presented in Table 13 and demonstrate high levels of mRNA target 2 (SNCA) knockdown two months after ICV injection. The highest levels of mRNA target 2 (SNCA) knockdown were observed in the brainstem, frontal cortex, striatum (STR), and lumbar spinal cord tissue (e.g., LDRG cells) of mouse subjects (see Table 13).

[0149] [Table 12]

[0150] [Table 13]

[0151] Example 4. siRNA conjugates This example describes the production of double-stranded nucleic acid-GalNAc conjugates. To synthesize GalNAc-conjugated sense strands, the sense strand bearing a 3'C6-NH2 functional group is first synthesized using standard phosphoramidite chemistry. A stock solution of GalNAc ligand-NHS ester (10 mmol / L in acetonitrile, 1 equivalent) is prepared. In an Eppendorf tube, borate buffer (10% v / v; 20x) is added to the oligonucleotide C6-NH2 sense strand, followed by the addition of GalNAc ligand (5 equivalents). The mixture is then shaken at ambient temperature for 16 hours. The mixture is then transferred to a 15 mL Falcon tube, ammonium hydroxide (28% by weight) is added, and the mixture is shaken at ambient temperature for 2 hours. The ammonia is then removed in vacuo. The residue is purified by ion exchange chromatography. Conditions: Solvent A: 15% MeCN / 20 mM NaH2PO4, Solvent B: 15% MeCN / 20 mM NaH2PO4, 1 M NaBr; 35-55% B over 5 CV at 8 mL / min, column temperature 60 °C. Desired fractions are pooled and desalted by spin filtration using an Eppendorf centrifuge column or desalting column. After desalting, the material is collected and the OD and volume are measured to determine the concentration.

[0152] Alternatively, conjugation is performed at the 5' position of the sense strand by immobilizing the GalNAc ligand on a microporous polystyrene resin or controlled pore glass and synthesizing it using established solid-phase oligonucleotide synthesis methods using 5'-CEβ-cyanoethyl) phosphoramidites.

[0153] Alternatively, the GalNAc ligand is converted to a suitable phosphoramidite and delivered to the 5' position of the sense strand using standard phosphoramidite chemistry.

[0154] To generate sense and antisense siRNA duplexes, perform the following procedure: Add the corresponding antisense oligonucleotide (1 equivalent) to the Falcon tube containing the oligonucleotide sense-GalNAc conjugate, vortex for 10 seconds, and then spin-filter through a 100K MWCO Amicon filter unit to remove particulate matter. The filtrate is collected and concentrated in vacuo in a Genevac evaporator. The residue is reconstituted in 1x PBS, filtered through a 0.2μ filter, and the concentration is determined by measuring the OD and volume.

[0155] Endotoxin testing is performed using Limulus amebocyte lysate on an Endosafe® NexGen PTS instrument.

[0156] equivalent While several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision a wide variety of other means and / or structures for performing the functions described herein and / or achieving one or more of the results and / or advantages described herein, and each of such modifications and / or variations is deemed to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application or applications in which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, it is to be understood that the foregoing embodiments are presented by way of example only, and that, within the scope of the appended claims and their equivalents, embodiments of the invention may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0157] All definitions and definitions used herein should be understood to supersede any dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0158] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and incorporation by reference may include the entire document in some cases.

[0159] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0160] The term "and / or," as used herein in the specification and claims, should be understood to mean "either or both" of the elements conjuncted by "and / or," i.e., elements that are present conjunctively in some cases and disjunctively in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements conjuncted by "and / or." Other elements, whether related or unrelated to the elements specifically identified, may optionally be present other than the elements specifically identified by the "and / or" phrase. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may, in one embodiment, refer to A only (optionally including elements other than B); in another embodiment, it may refer to B only (optionally including elements other than A); in yet another embodiment, it may refer to both A and B (optionally including other elements); and so on.

[0161] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be construed as inclusive, i.e., the including of at least one, but also more than one, of a plurality of elements or list of elements, optionally including additional unlisted items. Only terms clearly indicated to the contrary, e.g., "only one of" or "only one of," or, when used in the claims, "consisting of," shall refer to the inclusion of only one element of a plurality of elements or list of elements. Generally, the term "or" as used herein shall be construed to indicate exclusive alternatives (i.e., "one or the other, but not both") only when preceded by terms of exclusivity such as "either," "one of," "only one of," or "only one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0162] As used herein in the specification and claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also provides that elements other than those specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether related or unrelated to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, optionally including more than one, A, and no B (and optionally including elements other than B); in another embodiment to at least one, optionally including more than one, B, and no A (and optionally including elements other than A); in yet another embodiment to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); and so on.

[0163] It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

Claims

1. (i) a sense strand; and (ii) an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides; wherein the modified nucleotides do not include more than five 2'-fluoro (2'F) modified nucleotides, and the 2'F modified nucleotides are present at positions 2, 5, 7, 14, and 16 relative to the 5' end of the antisense strand.

2. (i) a sense strand; and (ii) an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides; wherein the modified nucleotides do not include more than five 2'F modified nucleotides, and the 2'F modified nucleotides are present at positions 2, 3, 7, 14, and 16 relative to the 5' end of the antisense strand.

3. (i) a sense strand; and (ii) an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides; wherein the modified nucleotides do not include more than five 2'F modified nucleotides, and the 2'F modified nucleotides are present at positions 2, 5, 8, 14, and 16 relative to the 5' end of the antisense strand.

4. (i) a sense strand; and (ii) an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides; wherein the modified nucleotides do not include more than six 2'F modified nucleotides, and the 2'F modified nucleotides are present at positions 2, 4, 6, 8, 14, and 16 relative to the 5' end of the antisense strand.

5. (i) a sense strand; and (ii) an antisense strand having a 5' end and a 3' end and consisting of modified nucleotides; wherein the modified nucleotides do not include more than five 2'F modified nucleotides, and the 2'F modified nucleotides are present at positions 2, 6, 8, 14, and 16 relative to the 5' end of the antisense strand.

6. the sense strand comprises a 5' end and a 3' end; (i) positions 9, 10, and 11 relative to the 5′ end of the sense strand; (ii) positions 7, 9, and 11 relative to the 5′ end of the sense strand; (iii) positions 7, 9, and 10 relative to the 5′ end of the sense strand; or (iv) positions 7, 10, and 11 relative to the 5′ end of the sense strand The double-stranded nucleic acid according to any one of claims 1 to 5, comprising a 2'F modified nucleotide at position 1.

7. The double-stranded nucleic acid of claim 6, wherein the sense strand does not contain any other 2'F-modified nucleotides.

8. A double-stranded nucleic acid comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex, the antisense strand comprises a sequence complementary to a portion of a target mRNA, and the antisense strand comprises five 2'F-modified nucleotides at one set of positions from the 5' end of the antisense strand: (a) positions 2, 5, 7, 14, and 16; (b) positions 2, 3, 7, 14, and 16; or (c) positions 2, 5, 8, 14, and 16, but does not comprise any other 2'F-modified nucleotides.

9. 9. The double-stranded nucleic acid of claim 8, wherein the sense strand comprises three 2′F-modified nucleotides at one set of positions from the 5′ end of the sense strand: (a) positions 9, 10, and 11; (b) positions 7, 9, and 11; (c) positions 7, 9, and 10; or (d) positions 7, 10, and 11.

10. The double-stranded nucleic acid according to claim 8 or 9, wherein the antisense strand comprises 2'-O-methyl modified nucleotides at positions other than the 2'F modified positions.

11. The sense strand contains 2'-O-methyl modified nucleotides at positions other than the 2'F modified positions, or 2'-O-C １２～１６ The double-stranded nucleic acid according to any one of claims 8 to 10, comprising alkyl-modified nucleotides or optionally comprising one or more abasic moieties.

12. The double-stranded nucleic acid of any one of claims 1 to 11, wherein position 1 relative to the 5' end of the sense strand or the 5' end of the antisense strand comprises a 5' phosphate analog.

13. 13. The double-stranded nucleic acid of claim 12, wherein the 5' phosphate analog comprises a 5' vinylphosphonate group.

14. The double-stranded nucleic acid of claim 12 or 13, wherein the antisense strand comprises the 5' phosphate analog.

15. The double-stranded nucleic acid according to any one of claims 1 to 14, wherein the sense strand is 18 to 24 nucleotides in length.

16. The double-stranded nucleic acid according to any one of claims 1 to 15, wherein the sense strand is 21 nucleotides in length.

17. The double-stranded nucleic acid according to any one of claims 1 to 16, wherein the antisense strand is 18 to 24 nucleotides in length.

18. The double-stranded nucleic acid according to any one of claims 1 to 17, wherein the antisense strand is 23 nucleotides in length.

19. The double-stranded nucleic acid according to any one of claims 1 to 18, wherein the sense strand and the antisense strand do not have the same length.

20. 20. The double-stranded nucleic acid of any one of claims 1 to 19, wherein the antisense strand is longer than the sense strand, optionally, the antisense strand is 2 to 10 nucleotides longer than the sense strand.

21. The double-stranded nucleic acid according to any one of claims 1 to 20, wherein the modified nucleotide is a modified ribonucleotide.

22. The modified nucleotides of the sense strand are one or more 2'-O-methyl (2'OMe) modified nucleotides or one or more 2'-O-C １２～１６ The double-stranded nucleic acid according to any one of claims 1 to 21, comprising alkyl-modified nucleotides.

23. The double-stranded nucleic acid according to any one of claims 1 to 22, comprising one or more abasic portions.

24. 24. The double-stranded nucleic acid of any one of claims 1 to 23, wherein the modified nucleotides of the sense strand comprise only 2'OMe modified nucleotides, excluding the 2'-fluoro (2'F) modified nucleotides at the recited positions.

25. The modified nucleotides of the sense strand may be one or more 2'-O-methyl (2'OMe) modified nucleotides, one or more 2'-O-C １２～１６ The double-stranded nucleic acid according to any one of claims 1 to 23, comprising alkyl-modified nucleotides or one or more abasic moieties.

26. 26. The double-stranded nucleic acid of any one of claims 1 to 25, wherein the modified nucleotides of the antisense strand comprise only 2'OMe modified nucleotides, excluding the 2'-fluoro (2'F) modified nucleotides at the recited positions.

27. The double-stranded nucleic acid of any one of claims 1 to 26, wherein the sense strand comprises one or more modified internucleotide bonds.

28. 28. The double-stranded nucleic acid of claim 27, wherein the sense strand comprises four modified internucleotide linkages.

29. 29. The double-stranded nucleic acid of claim 27 or 28, wherein the modified internucleotide linkages comprise one or more phosphorothioate (PS) internucleotide linkages.

30. 30. The double-stranded nucleic acid of claim 29, wherein each of the modified internucleotide linkages is a PS internucleotide linkage.

31. The double-stranded nucleic acid according to any one of claims 1 to 30, wherein the antisense strand comprises one or more modified internucleotide bonds.

32. 32. The double-stranded nucleic acid of claim 31 , wherein the antisense strand comprises four modified internucleotide linkages.

33. 33. The double-stranded nucleic acid of claim 31 or 32, wherein the modified internucleotide linkages comprise one or more phosphorothioate (PS) internucleotide linkages.

34. 34. The double-stranded nucleic acid of claim 33, wherein each of the modified internucleotide linkages is a PS internucleotide linkage.

35. 35. The double-stranded nucleic acid of any one of claims 27 to 34, wherein positions 1 and 2 relative to the 5' end of the sense strand are linked by a modified internucleotide bond, optionally by a phosphorothioate (PS) internucleotide bond.

36. 36. The double-stranded nucleic acid of any one of claims 27 to 35, wherein positions 1 and 2 relative to the 5' end of the antisense strand are linked by a modified internucleotide bond, optionally by a phosphorothioate (PS) internucleotide bond.

37. 37. The double-stranded nucleic acid of any one of claims 27 to 36, wherein positions 2 and 3 relative to the 5' end of the sense strand are linked by a modified internucleotide bond, optionally by a phosphorothioate (PS) internucleotide bond.

38. 38. The double-stranded nucleic acid of any one of claims 27 to 37, wherein positions 2 and 3 relative to the 5' end of the antisense strand are linked by a modified internucleotide bond, optionally by a phosphorothioate (PS) internucleotide bond.

39. 39. The double-stranded nucleic acid of any one of claims 27 to 38, wherein at least two of positions 1, 2, and 3 of the 3' end of the sense strand are linked by modified internucleotide linkages, optionally by phosphorothioate (PS) internucleotide linkages.

40. 40. The double-stranded nucleic acid of any one of claims 27 to 39, wherein at least two of positions 1, 2, and 3 at the 3' end of the antisense strand are linked by modified internucleotide linkages, optionally by phosphorothioate (PS) internucleotide linkages.

41. 41. The double-stranded nucleic acid of any one of claims 27 to 40, wherein each of positions 1, 2, and 3 relative to the 3' end of the sense strand is linked by a modified internucleotide linkage, optionally by a phosphorothioate (PS) internucleotide linkage.

42. 42. The double-stranded nucleic acid of any one of claims 27 to 41, wherein each of positions 1, 2, and 3 relative to the 3' end of the antisense strand is linked by a modified internucleotide linkage, optionally by a phosphorothioate (PS) internucleotide linkage.

43. A conjugate comprising the double-stranded nucleic acid according to any one of claims 1 to 42.

44. The conjugate comprises the structure of Formula I, wherein Formula I is A-B-C Formula I wherein "A" of Formula I comprises the double-stranded nucleic acid, "B" of Formula I comprises a bond or linker, and "C" of Formula I comprises a delivery molecule.

45. 45. The conjugate of claim 44, wherein "B" of formula I is attached to the 5' end or the 3' end of the sense strand of the double-stranded nucleic acid.

46. 46. ​​The conjugate of claim 44 or 45, wherein "B" of formula I is attached to the 3' end of the sense strand of the double-stranded nucleic acid.

47. 45. The conjugate of claim 44, wherein "B" of formula I is attached to the 5' end or the 3' end of the antisense strand of the double-stranded nucleic acid.

48. The conjugate of any one of claims 44 to 47, wherein "B" of formula I comprises a triethylene glycol (TEG) linker.

49. The conjugate of any one of claims 44 to 48, wherein "C" in formula I comprises cholesterol.

50. The conjugate of any one of claims 44 to 48, wherein "C" of formula I comprises tocopherol.

51. The double-stranded RNA may be a tertiary amide linked to a maleimide group; a gem-dimethyl (GDM) group; or a C ６ -NH ２ 51. The conjugate of any one of claims 43 to 50, which does not comprise a nucleotide connected to a group.

52. "B" in Formula I is a maleimido-methyl-tetrazine-trans-cyclo-octene (mal-tet-TCO) linker; a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker; a linker containing a tertiary amide bonded to a gem-dimethyl (GDM) group; or C ６ -NH ２ 52. The conjugate of any one of claims 43 to 51, which does not include a linker comprising a group.

53. "B" in Formula I is C ６ -NH ２ 48. The conjugate of any one of claims 43 to 47, comprising a linker comprising a group.

54. 48. The conjugate of any one of claims 43 to 47, wherein "C" of formula I comprises one or more N-acetylgalactosamine (GalNAc) moieties.

55. A pharmaceutical composition comprising the double-stranded nucleic acid according to any one of claims 1 to 42 or the conjugate according to any one of claims 43 to 54, and a pharmaceutically acceptable carrier.

56. 56. A method for inhibiting or reducing a target mRNA in a cell, the method comprising contacting the cell containing the target mRNA with the double-stranded nucleic acid of any one of claims 1 to 42, or the conjugate of any one of claims 43 to 54, or the pharmaceutical composition of claim 55.

57. 57. The method of claim 56, wherein the cell is a mammalian cell, optionally a human cell.

58. 58. The method of claim 56 or 57, wherein the cell is in a subject, optionally a human subject.

59. A double-stranded nucleic acid comprising a sense strand comprising a sequence set forth in any one of Tables 1-5 and an antisense strand comprising a sequence set forth in any one of Tables 1-5.

60. (i) the sense strand is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 77, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 98 , 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 145, 146, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, or 175; and / or (ii) the antisense strand is selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 70, 71, 72, 73, 74, 75, 76, 78, 112, 113, 114, 1 9, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 147, 148, 149, 150, 151, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199, 60. The double-stranded nucleic acid of claim 59.

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