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 present disclosure is based in part on chemically modified inhibitory nucleic acids that enable target RNA knock-down with high specificity and low off-target effects.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 386,153, filed on December 5, 2022, under 35 U.S.C. 119(e), the entire content of which is incorporated herein by reference.

[0003] Reference to Electronic Sequence Listing

[0004] The content of the electronic sequence listing (E058570012WO00-SEQ-KZM.xml; size: 2,820,627 bytes; created on November 30, 2023) is incorporated herein by reference in its entirety. Background Art

[0005] Targeted knockdown of proteins can be achieved by targeting mRNA with inhibitory nucleic acids. Chemically modified nucleic acids have unique functional properties and can specifically target mRNA. Therapeutic agents containing chemically modified oligonucleotides with a low frequency of off-target knockdown effects have clinical application value. Summary of the Invention

[0006] Aspects of the present disclosure relate to chemically modified nucleic acids that bind to the mRNA transcripts of target genes. In some embodiments, the compositions of the present disclosure can be used to treat diseases or disorders associated with dysregulation of the expression of mRNAs and / or their encoded protein products. The present disclosure is in part based 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 the translation of the target mRNA in a cell or a subject.

[0007] Thus, in some aspects, 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 comprise no more than five 2'-fluoro (2'F)-modified nucleotides, and wherein the 2'F-modified nucleotides are located at the 2nd, 5th, 7th, 14th, and 16th positions from the 5' end of the antisense strand.

[0008] In some aspects, 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 comprise no more than five 2'F-modified nucleotides, and wherein the 2'F-modified nucleotides are at the 2nd, 3rd, 7th, 14th, and 16th positions from the 5' end of the antisense strand.

[0009] In some aspects, 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 comprise no more than five 2'F-modified nucleotides, and wherein the 2'F-modified nucleotides are located at the 2nd, 5th, 8th, 14th, and 16th positions counting from the 5' end of the antisense strand.

[0010] In some aspects, 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 comprise no more than six 2'F-modified nucleotides, and wherein the 2'F-modified nucleotides are located at the 2nd, 4th, 6th, 8th, 14th, and 16th positions counting from the 5' end of the antisense strand.

[0011] In some aspects, 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 comprise no more than five 2'F-modified nucleotides, and wherein the 2'F-modified nucleotides are located at the 2nd, 6th, 8th, 14th, and 16th positions counting from the 5' end of the antisense strand.

[0012] In some embodiments, the sense strand comprises a 5' end and a 3' end, and at the 9th, 10th, and 11th positions counting from the 5' end of the sense strand; at the 7th, 9th, and 11th positions counting from the 5' end of the sense strand; at the 7th, 9th, and 10th positions counting from the 5' end of the sense strand; or at the 7th, 10th, and 11th positions counting from the 5' end of the sense strand comprise 2'F-modified nucleotides. In some embodiments, the sense strand does not comprise any other 2'F-modified nucleotides.

[0013] In some aspects, 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, wherein the antisense strand comprises a sequence complementary to a portion of a target mRNA, and wherein the antisense strand comprises five 2'F-modified nucleotides at one of the following sets of positions counting from its 5' end and does not comprise other 2'F-modified nucleotides: (a) the 2nd, 5th, 7th, 14th, and 16th positions; (b) the 2nd, 3rd, 7th, 14th, and 16th positions; (c) the 2nd, 5th, 8th, 14th, and 16th positions.

[0014] In some embodiments, the sense strand comprises three 2'F-modified nucleotides at one of the following sets of positions counting from its 5' end: (a) the 9th, 10th, and 11th positions; (b) the 7th, 9th, and 11th positions; (c) the 7th, 9th, and 10th positions; or (d) the 7th, 10th, and 11th positions.

[0015] In some embodiments, the antisense strand contains 2'-O-methyl modified nucleotides at sites other than the 2'F-modified sites.

[0016] In some embodiments, the sense strand contains 2'-O-methyl modified nucleotides or 2'-O-alkyl modified nucleotides (e.g., 2'-O-C 12-16 alkyl modified nucleotides) at sites other than the 2'F-modified sites. In some embodiments, the sense strand contains one or more abasic moieties at sites other than the 2'F-modified sites.

[0017] In some embodiments, the first position counting from the 5' end of the sense strand or the first position counting from the 5' end of the antisense strand contains a 5' phosphate analogue. In some embodiments, the 5' phosphate analogue contains a 5'-vinyl phosphonate group. In some embodiments, the antisense strand contains a 5' phosphate analogue.

[0018] In some embodiments, the sense strand has a length of 18 to 24 nucleotides. In some embodiments, the sense strand has a length of 21 nucleotides.

[0019] In some embodiments, the antisense strand has a length of 18 to 24 nucleotides. In some embodiments, the antisense strand has a length of 23 nucleotides.

[0020] In some embodiments, the sense strand and the antisense strand have different lengths. In some embodiments, the antisense strand is longer than the sense strand. In some embodiments, the antisense strand is 2 to 10 nucleotides longer than the sense strand.

[0021] In some embodiments, the modified nucleotides are modified ribonucleotides. In some embodiments, the modified nucleotides of the sense strand contain one or more 2'-O-methyl (2'OMe)-modified nucleotides or one or more 2'-O-alkyl modified nucleotides (e.g., 2'-O-C 12-16 alkyl modified nucleotides).

[0022] In some embodiments, the double-stranded nucleic acid contains one or more abasic moieties.

[0023] In some embodiments, the modified nucleotides of the sense strand contain only 2'OMe-modified nucleotides except for 2'F-modified nucleotides at the listed sites.

[0024] In some embodiments, the modified nucleotides of the sense strand contain one or more 2'-O-methyl (2'OMe)-modified nucleotides, one or more 2'-O-alkyl modified nucleotides (e.g., 2'-O-C 12-16 alkyl modified nucleotides) or one or more abasic moieties.

[0025] In some embodiments, the modified nucleotides of the antisense strand contain only 2’OMe-modified nucleotides except for the 2’F-modified nucleotides at the listed sites.

[0026] In some embodiments, the sense strand contains one or more modified internucleotide linkages. In some embodiments, the sense strand contains four modified internucleotide linkages. In some embodiments, the modified internucleotide linkages contain one or more phosphorothioate (PS) internucleotide linkages. In some embodiments, each of the modified internucleotide linkages of the sense strand of the double-stranded nucleic acid is a PS internucleotide linkage.

[0027] In some embodiments, the antisense strand contains one or more modified internucleotide linkages. In some embodiments, the antisense strand contains four modified internucleotide linkages. In some embodiments, the modified internucleotide linkages contain one or more phosphorothioate (PS) internucleotide linkages. In some embodiments, each of the modified internucleotide linkages of the antisense strand of the double-stranded nucleic acid is a PS internucleotide linkage.

[0028] In some embodiments, the 1st and 2nd positions (e.g., starting from the 5’ end) of the sense strand are connected by a modified internucleotide linkage. In some embodiments, the 2nd and 3rd positions (e.g., starting from the 5’ end) of the sense strand are connected by a modified internucleotide linkage. In some embodiments, the 1st, 2nd, 3rd, and 4th positions (e.g., starting from the 5’ end) of the sense strand are connected by modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is a phosphorothioate (PS) internucleotide linkage.

[0029] In some embodiments, the 1st and 2nd positions (e.g., starting from the 5’ end) of the antisense strand are connected by a modified internucleotide linkage. In some embodiments, the 2nd and 3rd positions (e.g., starting from the 5’ end) of the antisense strand are connected by a modified internucleotide linkage. In some embodiments, the 1st, 2nd, 3rd, and 4th positions (e.g., starting from the 5’ end) of the antisense strand are connected by modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is a phosphorothioate (PS) internucleotide linkage.

[0030] In some embodiments, at least two of the 1st, 2nd, and 3rd positions starting from the 3’ end of the sense strand are connected by a modified internucleotide linkage, optionally by a phosphorothioate (PS) internucleotide linkage.

[0031] In some embodiments, at least two of the first, second, and third positions from the 3'-end of the antisense strand are linked by a modified internucleotide linkage, optionally by a phosphorothioate (PS) internucleotide linkage.

[0032] In some embodiments, each of the first, second, and third positions from the 3'-end of the sense strand is linked by a modified internucleotide linkage, optionally by a phosphorothioate (PS) internucleotide linkage.

[0033] In some embodiments, each of the first, second, and third positions from the 3'-end of the antisense strand is linked by a modified internucleotide linkage, optionally by a phosphorothioate (PS) internucleotide linkage.

[0034] In some aspects, the present disclosure provides a double-stranded nucleic acid comprising a sense strand and / or an antisense strand, the strands comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 1-199 (e.g., as listed in Tables 1-5). In some aspects, the present disclosure provides a double-stranded nucleic acid comprising a sense strand and / or an antisense strand, the strands comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 1-199 (e.g., as listed in Tables 1-5) and a modification pattern. In some aspects, the present disclosure provides a double-stranded nucleic acid comprising a sense strand and an antisense strand, the strands comprising a SEQ ID NO selected from any one of Tables 1-5 (e.g., a sense strand comprising any one of SEQ ID NO: 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, 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 an antisense strand comprising any one of SEQ ID NO: 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 complementary to the human α-synuclein (SNCA) mRNA transcript.In some embodiments, the double-stranded nucleic acid comprises a region complementary to the human apolipoprotein E (APOE) mRNA transcript.

[0035] In some aspects, the present disclosure provides conjugates comprising the double-stranded nucleic acids described herein.

[0036] In some embodiments, the conjugate comprises a structure of Formula I, wherein Formula I comprises: A - B - C Formula I, wherein "A" of Formula I comprises the double-stranded nucleic acid described herein, "B" of Formula I comprises a bond or linker, and "C" of Formula I comprises a delivery molecule.

[0037] In some embodiments, "B" of Formula I is attached to the 5' end or the 3' end of the sense strand of the double-stranded nucleic acid. In some embodiments, "B" of Formula I is attached to the 3' end of the sense strand of the double-stranded nucleic acid. In some embodiments, "B" of Formula I is attached to the 5' end or the 3' end of the antisense strand of the double-stranded nucleic acid. In some embodiments, "B" of Formula I comprises a triethylene glycol (TEG) linker. In some embodiments, "B" of Formula I comprises a linker containing a C6-NH2 group. In some embodiments, "B" of Formula I does not comprise: a maleimide-methyl-tetrazine-trans-cyclooctene (mal-tet-TCO) linker; a N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker; a linker comprising a tertiary amide bonded to a geminal dimethyl (GDM) group; or a linker containing a C6-NH2 group.

[0038] 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.

[0039] In some embodiments, the double-stranded RNA described in the present disclosure does not comprise a nucleotide linked to: a maleimide group; a tertiary amide bonded to a geminal dimethyl (GDM) group; or a C6-NH2 group.

[0040] In some aspects, the present disclosure provides a pharmaceutical composition comprising the double-stranded nucleic acid described herein or the conjugate described herein, and a pharmaceutically acceptable carrier.

[0041] In some aspects, the present disclosure provides a method of inhibiting or reducing intracellular target mRNA, the method comprising contacting a cell comprising the target mRNA with the double-stranded nucleic acid, conjugate, or pharmaceutical composition described herein.

[0042] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is located within a subject. In some embodiments, the subject is a human subject. Detailed Description

[0043] Aspects of the present disclosure relate to compositions and methods for modulating the levels and / or translation of one or more RNA transcripts (e.g., mRNA transcripts) in a cell or a subject. The present disclosure is in part based on chemically modified double-stranded inhibitory nucleic acids having certain 2'-fluoro (2'F) modification patterns that bind to a target mRNA transcript, alter the target mRNA transcript levels, and alter the activity and / or levels of the protein expressed from the target mRNA transcript. In some embodiments, the present disclosure provides a double-stranded nucleic acid having an antisense strand that comprises 2'F-modified nucleotides at the following positions starting from its 5'-end: the 2nd, 5th, 7th, 14th, and 16th positions; the 2nd, 3rd, 7th, 14th, and 16th positions; the 2nd, 5th, 8th, 14th, and 16th positions; the 2nd, 4th, 6th, 8th, 14th, and 16th positions; or the 2nd, 6th, 8th, 14th, and 16th positions. In some embodiments, the antisense strand does not comprise any other 2'F-modified nucleotides. In some embodiments, the double-stranded nucleic acids described herein are more persistent in vivo, maintain potency, and / or have fewer off-target effects than unmodified or double-stranded nucleic acids having different chemical modification patterns.

[0044] In some embodiments, the double-stranded nucleic acids described in 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 the double-stranded nucleic acids described herein and one or more delivery molecules.

[0045] In some embodiments, the compositions of the present disclosure can be used to inhibit the expression or activity of a target mRNA, and / or to treat a disease or disorder associated with an altered expression of a target mRNA and / or protein associated with a particular disease.

[0046] Double-stranded nucleic acid

[0047] Aspects of the present disclosure relate to double-stranded nucleic acids. Nucleic acids comprise two or more nucleotides. As used herein, "nucleotide" refers to an organic compound having a nucleoside (a nucleobase, such as adenine, cytosine, guanine, thymine or uracil, and a pentose, such as ribose or 2'-deoxyribose) linked to a phosphate group. "Nucleotides" can serve as monomeric units of nucleic acid polymers, such as oligonucleotides, for example 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 moieties), each nucleotide of which can be modified or unmodified. The length of an oligonucleotide is typically less than about 100 nucleotides. Oligonucleotides can comprise unmodified DNA nucleotides, chemically modified DNA nucleotides, unmodified RNA nucleotides, chemically modified RNA nucleotides, unnatural nucleotides (e.g., nucleotides that do not occur naturally), abasic moieties, or any combination thereof.

[0048] Double-stranded nucleic acids typically form duplexes. As used herein, "duplex" refers to a structure formed by complementary base pairing of two antiparallel (i.e., opposite direction) nucleotide sequences, whether formed by two separate nucleic acid strands or by a folded nucleic acid strand (e.g., a hairpin structure). As used herein, "strand" refers to a single continuous nucleotide sequence linked together by internucleotide linkages, such as phosphodiester linkages or phosphorothioate linkages. A strand can 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. The sense and antisense strands of the nucleotides are further described in the section herein entitled "Sense and Antisense Strands".

[0049] The length of each strand of the double-stranded nucleic acid can vary. In some embodiments, the length of each strand of the double-stranded nucleic acid ranges from about 10 nucleotides to about 50 nucleotides (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, the length of each strand of the double-stranded nucleic acid is from about 18 to 24 nucleotides. 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 moieties within such ranges.

[0050] In some embodiments, the sense and antisense strands of the double-stranded nucleic acid are of 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 and antisense strands have different lengths and the double-stranded nucleic acid contains one or two overhangs. As used herein, an "overhang" refers to one or more unpaired nucleotides that protrude from the duplex structure of a double-stranded oligonucleotide. An overhang can include one or more unpaired nucleotides that extend from the duplex region at the 5' or 3' end of the double-stranded oligonucleotide. An overhang can be a 3' overhang or a 5' overhang on the antisense or sense strand of the double-stranded oligonucleotide. In some embodiments, the sense strand is 1-10 nucleotides longer than the antisense strand (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides). In some embodiments, the antisense strand is 1-10 nucleotides longer than the sense strand (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides). In some embodiments, the double-stranded nucleic acid contains an overhang on the sense strand (e.g., a sequence at the 5' end of the sense strand that extends beyond the 5' most terminal nucleotide of the antisense strand). In some embodiments, the double-stranded nucleic acid contains an overhang on the antisense strand (e.g., a sequence at the 5' end of the antisense strand that extends beyond the 5' most terminal nucleotide of the sense strand). In some embodiments, the double-stranded nucleic acid contains two overhangs on the sense strand. In some embodiments, the double-stranded nucleic acid contains two overhangs on the antisense strand. In some embodiments, the double-stranded nucleic acid contains one overhang on the sense strand and one overhang on the antisense strand. In some embodiments, the length of the overhang sequence is about 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0051] In some embodiments, the double-stranded nucleic acid is an inhibitory nucleic acid. As used herein, an "inhibitory nucleic acid" refers to an oligonucleotide that is capable of reducing the level, function, and / or activity of an intracellular mRNA transcript (e.g., a target mRNA transcript) or a protein encoded by said mRNA. As used herein, a "target mRNA" is an mRNA that encodes a protein associated with a disease or disorder, including but not limited to an mRNA that encodes a mutant protein, a pathogenic protein isoform, or an mRNA that is not normally expressed in healthy cells. In some embodiments, the target mRNA is a wild-type mRNA (e.g., an mRNA that encodes a wild-type protein). In some embodiments, the inhibitory nucleic acid can be used to cleave a target mRNA or inhibit the translation of a target mRNA that contains one or more mutations (e.g., substitutions, deletions, and / or insertions) relative to the wild-type sequence found in nature. In some embodiments, the inhibitory nucleic acid can be used to inhibit the translation of a target mRNA that encodes a commonly occurring gene-shortened version in nature (e.g., a truncated mutant or a mutant that lacks catalytic and / or regulatory domains).

[0052] In some embodiments, the antisense strand of the double-stranded nucleic acid hybridizes to a target mRNA molecule. Nucleic acid hybridization generally involves the binding of one nucleic acid strand to a complementary region on another nucleic acid strand. Unless otherwise specified, the term "binding" as used herein refers to the ability of a molecule to form a chemical bond or attractive interaction with another molecule, resulting in the proximity of the two molecules, as determined by conventional methods known in the art. "Complementary" as used herein refers to the structural relationship between two nucleotides (e.g., on two opposing nucleic acids, or on opposing regions of a single nucleic acid strand such as a hairpin structure) that allows the two nucleotides to form base pairs with each other. For example, a purine nucleotide of one nucleic acid and a pyrimidine nucleotide of the complementary 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 for the formation of a stable duplex. Similarly, two nucleic acids can have regions of multiple nucleotides that are complementary to each other to form complementary regions, as described herein.

[0053] In some embodiments, the target mRNA has a degree of sequence identity with the sense strand sufficient to promote specific binding (e.g., hybridization) of the antisense strand of a double-stranded nucleic acid (e.g., an inhibitory nucleic acid) thereto. The definition of the term “% sequence identity” or “sequence identity percentage” with respect to a reference nucleic acid sequence is: 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 the sequences have been optimally aligned and gaps or overhangs have been introduced, if necessary, to achieve the maximum percentage of sequence identity. The alignment for determining the nucleic acid sequence identity percentage can be accomplished in a variety of ways known to those of skill in the art, e.g., using publicly available computer software programs such as those described in Current Protocols in Molecular Biology (edited by Ausubel et al., 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 of skill in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. The “sequence identity” percentage can be determined by comparing two optimally aligned sequences over a window of alignment, wherein the nucleic acid sequence segment in the window of alignment may include additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (not including additions or deletions) to achieve the optimal alignment of the two sequences. The percentage can be calculated as follows: determine the number of positions at which the identical nucleotides, nucleosides or nucleobases occur in the two sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the window of alignment, and then multiply the result by 100 to obtain the sequence identity percentage. The output is the percentage of identity of the target sequence relative to the query sequence.

[0054] In some embodiments, a strand of the double-stranded nucleic acid (e.g., the sense strand) has about 80% to 100% (e.g., about 80%, 85%, 90%, 95%, 99%, 99.9% or 100%) sequence identity with the target mRNA. In some embodiments, a strand of the double-stranded nucleic acid (e.g., the sense strand) 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 the target mRNA. In some embodiments, the inhibitory nucleic acid comprises an antisense strand that comprises a complementary region 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 the ribonucleotides of the target mRNA). In some embodiments, the inhibitory nucleic acid comprises an antisense strand that comprises a complementary region that is fully complementary to a portion of the target mRNA (e.g., 100% of the nucleotides of the antisense strand hybridize to the ribonucleotides of the target mRNA).

[0055] In some embodiments, the double-stranded inhibitory nucleic acid comprises a single oligonucleotide that comprises a sense strand and an antisense strand linked by a linker, a hairpin loop, and / or a common backbone and that undergoes intramolecular hybridization via nucleotides in the sense strand and the antisense strand. Such double-stranded inhibitory nucleic acids will comprise a single 5' and 3' end and a loop region located between the two sense and antisense sequences that does not base pair with any other nucleotides within the inhibitory nucleic acid. In some embodiments, the double-stranded inhibitory nucleic acid comprises two complementary oligonucleotides that have their respective 5' and 3' ends and their respective backbones. In some embodiments, the inhibitory nucleic acid is double-stranded RNA (dsRNA). In some embodiments, the inhibitory nucleic acid that is dsRNA functions like an RNA interference (RNAi) molecule and thus can be referred to as an RNAi agent. In some embodiments, the inhibitory nucleic acid that is dsRNA functions like a small interfering RNA molecule and thus can be referred to as siRNA. The term "knockdown" or "expression knockdown" refers to a decrease in the mRNA or protein expression of a gene following treatment with an inhibitory nucleic acid (e.g., an RNAi agent).

[0056] In some embodiments, the double-stranded nucleic acid is an RNAi agent. As used herein, "RNAi agent", "iRNA", "iRNA agent", "RNAi", and "RNA interference agent" refer to an agent containing ribonucleotides or RNA that mediates the targeted cleavage of an RNA transcript through an RNA interference mechanism (e.g., via 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 the antisense strand form a duplex. In some embodiments, the sense strand and the antisense strand of the RNAi agent are 21-23 nucleotides in length.

[0057] To provide desired properties, such as minimizing the likelihood of off-target effects, increasing potency, and / or increasing persistence, the double-stranded nucleic acid can be designed to ensure that it does not have a sequence (e.g., 5 or more consecutive nucleotides) complementary to an off-target nucleic acid (e.g., an mRNA that does not contain the target mRNA sequence), or the double-stranded nucleic acid can be engineered to contain 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 having certain 2'F-modification patterns, which, compared to unmodified or non-identical 2'F-modification patterns as described herein, reduce the promiscuity (e.g., off-target binding) of the nucleic acid.

[0058] Aspects of the present disclosure relate to double-stranded nucleic acids containing certain chemical modification patterns (e.g., modified nucleotides, abasic moieties, modified internucleotide linkages, etc.). As used herein, "modified nucleotide" refers to a nucleotide having one or more chemical modifications compared to the corresponding reference nucleotide, which is selected from: adenosine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide, uridine ribonucleotide, deoxyadenosine nucleotide, deoxyguanosine nucleotide, deoxycytidine nucleotide, and thymidine deoxynucleotide.

[0059] Chemical modifications can be used to provide specific functional properties to the double-stranded nucleic acid that are not present in the unmodified nucleic acid. The double-stranded nucleic acids of the present disclosure can be modified to achieve one or more desired properties, such as improved cellular uptake, improved stability, reduced immunogenicity, improved potency, improved target hybridization, sensitivity to RNase cleavage, etc. In some embodiments, the inhibitory nucleic acid is modified such that when present in a cell containing the mRNA target, it is capable of hybridizing to the mRNA transcribed from the DNA sequence and inducing cleavage of the mRNA.

[0060] Modified nucleotides can have, for example, one or more chemical modifications in their sugar, nucleobase, and / or phosphate group. Additionally or alternatively, the modified nucleotides can have one or more chemical moieties conjugated to the corresponding reference nucleotides. In some embodiments, the modified nucleotides are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or 2'-O-alkyl-modified nucleotides such as 2'-O-C 12-16 alkyl-modified nucleotides. As used herein, the term "alkyl" refers to a saturated straight-chain or branched-chain monovalent hydrocarbon group containing the specified number of carbon atoms. For example, "C 12-16 alkyl" refers to a group having a straight-chain or branched-chain arrangement of 12-16 carbon atoms. Other examples of nucleotides containing a modification at the 2'-carbon of the sugar moiety 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 (OCH2CH=CH2), 2'-propargyl, 2'-propyl, ethynyl, vinyl, propenyl, and cyano, etc. In some embodiments, the 2'-modified nucleotides include a 2'-O-4'-C methylene bridge, such as a locked nucleic acid (LNA) nucleotide. In some embodiments, the 2'-hydroxy group is linked to the 3'- or 4'-carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. Other 2'-modifications are also found in the art.

[0061] In some embodiments, the modified nucleotides have a phosphate analogue. As used herein, "phosphate analogue" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analogue is located at the 5'-terminal nucleotide of the oligonucleotide, replacing the 5'-phosphate ester that is typically susceptible to enzymatic removal. The 5'-phosphate analogue can include a phosphatase-resistant linkage. Examples of phosphate analogues include 5'-methylphosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). In some embodiments, the phosphate analogue is located at the 1st position of the sense or antisense strand of a double-stranded RNA. In some embodiments, the phosphate analogue is 5'-VP. In some embodiments, the antisense strand contains a 5'-phosphate analogue.

[0062] Double-stranded nucleic acids can contain one or more abasic moieties or inverted abasic moieties. As used herein, "abasic moiety" or "apurinic / apyrimidinic site" refers to a molecule derived from a nucleotide that contains a ribose (or deoxyribose) and a phosphate bond, wherein the nitrogenous base (e.g., nucleobase) has been removed by cleavage of the glycosidic bond between the ribose (or deoxyribose) and the base.

[0063] In some embodiments, the double-stranded nucleic acid comprises one or more (e.g., 1, 2, 3, 4, 5 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 can 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, boranophosphates, alkylphosphonate inhibitory nucleic acids, peptide inhibitory nucleic acids, and morpholino backbones. Morpholino backbones have been described, for example, in Corey and Abrams, Genome Biol. 2001;2(5): reviews1015.1–reviews1015.3.

[0064] The number of modified internucleotide linkages in each strand of the double-stranded nucleic acid can vary. In some embodiments, each strand of the double-stranded nucleic acid comprises from 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 the double-stranded nucleic acid comprises from 1 to 5 (e.g., 1, 2, 3, 4 or 5) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are arranged 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., at one or more of positions 1, 2, and 3 counting from the 3' end of the sense strand). In some embodiments, the antisense strand of the double-stranded nucleic acid comprises from 1 to 5 (e.g., 1, 2, 3, 4 or 5) modified internucleotide linkages. In some embodiments, the modified internucleotide linkages are arranged 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., at one or more of positions 1, 2, and 3 counting from the 3' end of the antisense strand). In some embodiments, each of the modified internucleotide linkages is a phosphorothioate (PS) linkage.

[0065] In some embodiments, the antisense strand of the double-stranded nucleic acid comprises no more than four phosphorothioate internucleotide linkages. In some embodiments, the first and second positions of the sense strand (e.g., starting from the 5' or 3' end) are linked by a modified internucleotide linkage. In some embodiments, the first and second positions of the antisense strand (e.g., starting from the 5' or 3' end) are linked by a phosphorothioate internucleotide linkage. In some embodiments, the second and third positions of the sense strand (e.g., starting from the 5' or 3' end) are linked by a modified internucleotide linkage. In some embodiments, the second and third positions of the antisense strand (e.g., starting from the 5' or 3' end) are linked by a phosphorothioate internucleotide linkage.

[0066] In some embodiments, the target mRNA of the inhibitory nucleic acid corresponds to a gene sequence encoding a wild-type protein or any of its variants (e.g., mutants, disease-related alleles, subtypes, etc.). However, these examples should be considered non-limiting as the chemical modifications described in the present disclosure can be applied to almost any inhibitory nucleic acid sequence for targeted knockdown of the target mRNA.

[0067] In some embodiments, the double-stranded nucleic acid downregulates the expression or activity of the target mRNA. The amount of downregulation mediated by the double-stranded nucleic acid may vary. In some embodiments, the double-stranded nucleic acid reduces the expression level or activity of the target mRNA transcript by 1-fold to 100-fold, 2-fold to 10-fold, 5-fold to 20-fold, 10-fold to 30-fold, 20-fold to 50-fold, or 25-fold to 100-fold, or any value therebetween (e.g., compared to the expression or activity of the target mRNA that has not been 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 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., compared to the expression or activity of the target mRNA that has not been 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, such as at least 200-fold, 400-fold, 500-fold, or 1000-fold (e.g., compared to the expression or activity of the target mRNA that has not been contacted with the double-stranded nucleic acid).

[0068] In some embodiments, the double-stranded nucleic acid is a separated nucleic acid. Those skilled in the art should understand that a "separated" nucleic acid is an artificially produced nucleic acid. The artificial production of separated nucleic acids can be achieved, for example, by in vitro amplification through polymerase chain reaction (PCR), in vitro transcription, in vitro reverse transcription, recombinant cloning, or chemical synthesis. Methods for synthesizing separated nucleic acids (such as RNA) are known in the art, for example, as described by Soukchareun et al. in "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 synthesis of the sense and antisense strands of the inhibitory nucleic acid can be carried out, for example, using solid-phase synthesis methods, by using phosphoramidite chemistry (for example, "Current Protocols in Inhibitory nucleic acid Chemistry", Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphotriester chemistry, or enzymatic synthesis. Automated commercial synthesizers can be used, such as the MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems. Sulfurizing reagents such as phenylacetyl disulfide or DDTT (((dimethylaminomethylene)amino)-3H-1,2,4-dithiazoline-3-thione) can be used to introduce phosphorothioate linkages. It is well known that similar techniques and commercially available modified amidites and controlled pore glass (CPG) products can be utilized to synthesize modified oligonucleotides or conjugated oligonucleotides.

[0069] Purification methods can be used to exclude unwanted impurities from the final oligonucleotide product. Commonly used single-stranded oligonucleotide purification techniques 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 oligonucleotide can be analyzed by mass spectrometry and quantified spectrophotometrically at a wavelength of 260 nm. The sense strand and the antisense strand can then be annealed to form double-stranded RNA.

[0070] Sense strand and antisense strand

[0071] Aspects of the present disclosure relate to double-stranded nucleic acids having a sense strand and an antisense strand, each strand comprising certain chemical modification patterns (e.g., a pattern of 2′F-modified nucleotides). As used herein, an "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, a "sense strand" refers to a single-stranded oligonucleotide that is complementary to a region of the antisense strand.

[0072] Aspects of the present disclosure are based in part on the antisense strand having 2′F-modified nucleotides at the combination of sites starting from the 5′ end of the antisense strand: the 2nd, 5th, 7th, 14th, and 16th positions; the 2nd, 3rd, 7th, 14th, and 16th positions; the 2nd, 5th, 8th, 14th, and 16th positions; the 2nd, 4th, 6th, 8th, 14th, and 16th positions; or the 2nd, 6th, 8th, 14th, and 16th positions. In some embodiments, the 2′F-modified antisense strand contains 2′F-modified nucleotides only at the listed sites and no such modification at other sites. In some embodiments, the antisense strand (and the double-stranded nucleic acid comprising such an antisense strand) has improved potency, persistence, and / or reduced off-target effects compared to previously described chemically modified double-stranded nucleic acids.

[0073] In some embodiments, the antisense strand contains 2′F-modified nucleotides at the 2nd, 5th, 7th, 14th, and 16th positions starting from its 5′ end and contains 2′F-modified nucleotides only at the listed sites and no such modification at other sites. 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, one or more of the additional modified nucleotides each comprise a 2′O-alkyl modification, e.g., a 2′O-methyl (2′O-Me) modification or a 2′O-C 12-16Alkyl modification. In some embodiments, each of 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 linkage is a phosphorothioate (PS) linkage. In some embodiments, the antisense strand comprises a PS linkage between the 1st and 2nd positions counting from the 5′-end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between the 1st and 2nd positions counting from the 3′-end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between the 1st and 2nd positions counting from the 5′-end of the antisense strand and a PS linkage between the 1st and 2nd positions counting from the 3′-end of the antisense strand. In some embodiments, the antisense strand comprises PS linkages between the 1st and 2nd positions and between the 2nd and 3rd positions counting from the 3′-end of the antisense strand. In some embodiments, the antisense strand comprises PS linkages between the 1st and 2nd positions and between the 2nd and 3rd positions counting from the 5′-end of the antisense strand, and PS linkages between the 1st and 2nd positions and between the 2nd and 3rd positions counting from the 3′-end of the antisense strand.

[0074] In some embodiments, the antisense strand hybridizes with 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 comprises 2′F-modified nucleotides only at the listed positions and does not contain any such modification at other positions. 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, 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 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 linkage is a phosphorothioate (PS) linkage. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 3′ end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the sense strand and a PS linkage between positions 1 and 2 counting from the 3′ end of the sense strand. In some embodiments, the sense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the sense strand. In some embodiments, the sense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 5′ end of the sense strand, and PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the sense strand.

[0075] 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 composed of modified nucleotides, wherein the modified nucleotides are composed of 2'F-modified nucleotides located at the 2nd, 3rd, 7th, 14th, and 16th positions from the 5' end of the antisense strand and 2'O-Me modified nucleotides located at other positions of the antisense strand, and wherein the sense strand comprises 2'F-modified nucleotides only at the following positions: (a) the 9th, 10th, and 11th positions; (b) the 7th, 9th, and 11th positions; (c) the 7th, 9th, and 10th positions; or (d) the 7th, 10th, and 11th positions, and comprises 2'O-Me modified nucleotides, 2'O-C 12-16 alkyl modified nucleotides or abasic moieties at other positions of the sense strand; and wherein each sense strand and antisense strand comprises a PS linkage between the 1st and 2nd positions and between the 2nd and 3rd positions from their respective 5' and 3' ends.

[0076] In some embodiments, the antisense strand comprises 2′F-modified nucleotides at positions 2, 3, 7, 14, and 16 counting from the 5′ end thereof, and comprises 2′F-modified nucleotides only at the listed positions, with no such modification at other 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 linkage is a phosphorothioate (PS) linkage. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 3′ end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the antisense strand and a PS linkage between positions 1 and 2 counting from the 3′ end of the antisense strand. In some embodiments, the antisense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the antisense strand. In some embodiments, the antisense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 5′ end of the antisense strand, and PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the antisense strand.

[0077] In some embodiments, the antisense strand hybridizes with 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 comprises 2′F-modified nucleotides only at the listed positions and does not contain any such modification at other positions. 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, 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 sense strand comprises one or more 2′O-C 12-16 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 linkage is a phosphorothioate (PS) linkage. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counted from the 5′ end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counted from the 3′ end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counted from the 5′ end of the sense strand and a PS linkage between positions 1 and 2 counted from the 3′ end of the sense strand. In some embodiments, the sense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counted from the 3′ end of the sense strand. In some embodiments, the sense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counted from the 5′ end of the sense strand and PS linkages between positions 1 and 2 and between positions 2 and 3 counted from the 3′ end of the sense strand.

[0078] 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 composed of modified nucleotides, wherein the modified nucleotides are composed of 2'-F-modified nucleotides located at the 2nd, 5th, 7th, 14th, and 16th positions from the 5'-end of the antisense strand and 2'-O-Me modified nucleotides at other positions of the antisense strand, and wherein the sense strand comprises 2'-F-modified nucleotides only at the following positions: (a) the 9th, 10th, and 11th positions; (b) the 7th, 9th, and 11th positions; (c) the 7th, 9th, and 10th positions; or (d) the 7th, 10th, and 11th positions, and comprises 2'-O-Me modified nucleotides, 2'-O-C 12-16 alkyl modified nucleotides or abasic moieties at other positions of the sense strand; and wherein each sense strand and antisense strand comprises a PS linkage between the 1st and 2nd positions and between the 2nd and 3rd positions from their respective 5'-ends and 3'-ends.

[0079] In some embodiments, the antisense strand comprises 2′F-modified nucleotides at positions 2, 5, 7, 14, and 16 counting from the 5′ end of the antisense strand, and comprises 2′F-modified nucleotides only at the listed positions and no such modification at other 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 linkage is a phosphorothioate (PS) linkage. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 3′ end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the antisense strand and a PS linkage between positions 1 and 2 counting from the 3′ end of the antisense strand. In some embodiments, the antisense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the antisense strand. In some embodiments, the antisense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 5′ end of the antisense strand, and PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the antisense strand.

[0080] In some embodiments, the antisense strand hybridizes with 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 comprises 2′F-modified nucleotides only at the listed positions and does not contain any such modification at other positions. 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, 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 sense strand comprises one or more 2′O-C 12-16 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 linkage is a phosphorothioate (PS) linkage. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 3′ end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the sense strand and a PS linkage between positions 1 and 2 counting from 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 counting from 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 counting from the 5′ end of the sense strand, and a PS linkage between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the sense strand.

[0081] 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 consist of 2'F-modified nucleotides located at the 2nd, 5th, 8th, 14th, and 16th positions from the 5' end of the antisense strand and 2'O-Me modified nucleotides at other positions of the antisense strand, and wherein the sense strand comprises 2'F-modified nucleotides at and only at the following positions: (a) the 9th, 10th, and 11th positions; (b) the 7th, 9th, and 11th positions; (c) the 7th, 9th, and 10th positions; or (d) the 7th, 10th, and 11th positions, and comprises 2'O-Me modified nucleotides, 2'O-C 12-16 alkyl modified nucleotides or abasic moieties at other positions of the sense strand; and wherein each sense strand and antisense strand comprises a PS linkage between the 1st and 2nd positions and between the 2nd and 3rd positions from their respective 5' and 3' ends.

[0082] In some embodiments, the antisense strand comprises 2′F-modified nucleotides at positions 2, 5, 8, 14, and 16 counting from the 5′-end of the antisense strand, and comprises 2′F-modified nucleotides only at the listed positions and no such modification at other 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 linkage is a phosphorothioate (PS) linkage. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 5′-end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 3′-end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 5′-end of the antisense strand and a PS linkage between positions 1 and 2 counting from the 3′-end of the antisense strand. In some embodiments, the antisense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′-end of the antisense strand. In some embodiments, the antisense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 5′-end of the antisense strand, and PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′-end of the antisense strand.

[0083] In some embodiments, the antisense strand hybridizes with 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 comprises 2′F-modified nucleotides only at the listed positions and does not contain any such modification at other positions. 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, 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 sense strand comprises one or more 2′O-C 12-16 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 linkage is a phosphorothioate (PS) linkage. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 3′ end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the sense strand and a PS linkage between positions 1 and 2 counting from the 3′ end of the sense strand. In some embodiments, the sense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the sense strand. In some embodiments, the sense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 5′ end of the sense strand, and PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the sense strand.

[0084] 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 consist of 2'-F-modified nucleotides located at the 2nd, 4th, 6th, 8th, 14th, and 16th positions counting from the 5'-end of the antisense strand and 2'-O-Me modified nucleotides located at other positions of the antisense strand, and wherein the sense strand comprises 2'-F-modified nucleotides at and only at the following positions: (a) the 9th, 10th, and 11th positions; (b) the 7th, 9th, and 11th positions; (c) the 7th, 9th, and 10th positions; or (d) the 7th, 10th, and 11th positions, and comprises 2'-O-Me modified nucleotides, 2'-O-C 12-16 alkyl modified nucleotides or abasic moieties at other positions of the sense strand; and wherein each sense strand and antisense strand comprises PS linkages between the 1st and 2nd positions and between the 2nd and 3rd positions counting from their respective 5'-ends and 3'-ends.

[0085] In some embodiments, the antisense strand comprises 2′F-modified nucleotides at positions 2, 4, 6, 8, 14, and 16 counting from the 5′ end of the antisense strand, and comprises 2′F-modified nucleotides only at the listed positions and no such modification at other 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 linkage is a phosphorothioate (PS) linkage. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 3′ end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the antisense strand and a PS linkage between positions 1 and 2 counting from the 3′ end of the antisense strand. In some embodiments, the antisense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the antisense strand. In some embodiments, the antisense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 5′ end of the antisense strand, and PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the antisense strand.

[0086] In some embodiments, the antisense strand hybridizes with 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 comprises 2′F-modified nucleotides only at the listed positions and does not contain any such modification at other positions. 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, 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 sense strand comprises one or more 2′O-C 12-16 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 linkage is a phosphorothioate (PS) linkage. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 3′ end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the sense strand and a PS linkage between positions 1 and 2 counting from the 3′ end of the sense strand. In some embodiments, the sense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the sense strand. In some embodiments, the sense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 5′ end of the sense strand, and PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the sense strand.

[0087] 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 composed of modified nucleotides, wherein the modified nucleotides are composed of 2'F-modified nucleotides located at the 2nd, 6th, 8th, 14th, and 16th positions from the 5' end of the antisense strand and 2'O-Me modified nucleotides located at other positions of the antisense strand, and wherein the sense strand comprises 2'F-modified nucleotides only at the following positions: (a) the 9th, 10th, and 11th positions; (b) the 7th, 9th, and 11th positions; (c) the 7th, 9th, and 10th positions; or (d) the 7th, 10th, and 11th positions, and comprises 2'O-Me modified nucleotides, 2'O-C 12-16 alkyl modified nucleotides or abasic moieties at other positions of the sense strand; and wherein the sense strand and the antisense strand each comprise a PS linkage between the 1st and 2nd positions and between the 2nd and 3rd positions from their respective 5' and 3' ends.

[0088] In some embodiments, the antisense strand comprises 2′F-modified nucleotides at positions 2, 6, 8, 14, and 16 counting from the 5′ end of the antisense strand, and comprises 2′F-modified nucleotides only at the listed positions and no such modification at other 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 linkage is a phosphorothioate (PS) linkage. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 3′ end of the antisense strand. In some embodiments, the antisense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the antisense strand and a PS linkage between positions 1 and 2 counting from the 3′ end of the antisense strand. In some embodiments, the antisense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the antisense strand. In some embodiments, the antisense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 5′ end of the antisense strand, and PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the antisense strand.

[0089] In some embodiments, the antisense strand hybridizes with 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 comprises 2′F-modified nucleotides only at the listed positions and no such modification at other positions. 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, 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 sense strand comprises one or more 2′O-C 12-16 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 linkage is a phosphorothioate (PS) linkage. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 3′ end of the sense strand. In some embodiments, the sense strand comprises a PS linkage between positions 1 and 2 counting from the 5′ end of the sense strand and a PS linkage between positions 1 and 2 counting from the 3′ end of the sense strand. In some embodiments, the sense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the sense strand. In some embodiments, the sense strand comprises PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 5′ end of the sense strand and PS linkages between positions 1 and 2 and between positions 2 and 3 counting from the 3′ end of the sense strand.

[0090] In some embodiments, the sense strand comprises a nucleotide sequence having about 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the sequence shown in any of SEQ ID NOs: 1-199. In some embodiments, the sense strand comprises a nucleotide sequence having 100% identity to any of SEQ ID NOs: 1-199. In some embodiments, the antisense strand comprises a nucleotide sequence having about 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the sequence shown in any of SEQ ID NOs: 1-199. In some embodiments, the antisense strand comprises a nucleotide sequence having 100% identity to any of SEQ ID NOs: 1-199.

[0091] In some embodiments, the double-stranded nucleic acid comprises a sense strand and an antisense strand having about 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sense-antisense strand pair shown in any of SEQ ID NOs: 1-199. In some embodiments, the double-stranded nucleic acid comprises a sense strand and an antisense strand having 100% identity to the sense-antisense strand pair shown in any of SEQ ID NOs: 1-199. In some embodiments, the double-stranded nucleic acid comprises a sense strand having the modification pattern described in any of SEQ ID NOs: 1-199. In some embodiments, the double-stranded nucleic acid comprises an antisense strand having the modification pattern shown in any of SEQ ID NOs: 1-199.

[0092] Aspects of the present disclosure relate to double-stranded nucleic acids that are not conjugated to any other molecule (e.g., a delivery molecule such as an antibody, lipid, sugar, 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 (e.g., 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 (e.g., N-acetylgalactosamine (GalNAc)). However, those skilled in the art will recognize that in some embodiments, the double-stranded nucleic acids described herein can be linked to one or more delivery molecules to form conjugates, as described in further detail below.

[0093] Conjugate

[0094] Aspects of the present disclosure relate to compositions comprising the double-stranded nucleic acids described herein, which are optionally linked to a delivery molecule. As used herein, a "delivery molecule" refers to a reagent that aids in delivering the double-stranded nucleic acid into a target cell, directs tissue or cell specificity of the double-stranded nucleic acid, and / or confers additional functionality to the double-stranded nucleic acid. In some embodiments, the delivery molecule includes a lipid, a peptide, a protein, an antibody, or a small molecule. Examples of delivery molecules include, but are not limited to, lipids (e.g., fatty acids, cholesterol, tocopherols, etc.), antibodies (e.g., cell receptor-specific antibodies, therapeutic antibodies, etc.), antigen-binding fragments (e.g., Fab, single-chain variable fragments (scFv)), radioligands, 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 an 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 (e.g., GalNAc).

[0095] In some embodiments, the delivery molecule is linked (e.g., conjugated or bound) to the double-stranded nucleic acid. The delivery molecule can be directly linked to the double-stranded nucleic acid (e.g., by forming one or more interactions or bonds with the nucleotides or internucleotide linkages of the double-stranded nucleic acid), or indirectly linked (e.g., via one or more linker molecules). The delivery molecule can be linked (e.g., conjugated or bound) to any suitable site of the double-stranded nucleic acid. For example, the delivery molecule can be conjugated to the end of the double-stranded nucleic acid (e.g., the 5' end or the 3' end), or conjugated to an internal nucleotide or internucleotide linkage of the double-stranded nucleic acid.

[0096] 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, e.g., via a linker. In some embodiments, the linker comprises an amino acid linker (e.g., glycine-rich linker, glycine-serine linker, proline linker, etc.) or a small molecule (e.g., polyethylene 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, a TEG linker, and cholesterol or tocopherol, and the double-stranded nucleic acid comprises a sequence shown in any one of SEQ ID NO: 1-199.

[0097] Aspects of the present disclosure relate to conjugates comprising a double-stranded nucleic acid as described herein and a GalNAc delivery molecule. Delivery molecules comprising N-acetylgalactosamine (GalNAc) are known to target the asialoglycoprotein receptor on hepatocytes and are a means of delivering double-stranded nucleic acids to a desired tissue. In some embodiments, the double-stranded nucleic acid is conjugated to a GalNAc delivery molecule comprising a structure of Formula II: Formula II.

[0098] 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, suitably 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 Linker 1 having attachment points A and B as shown below (Formula III). In another embodiment, the linker comprises piperidine. In another suitable embodiment, the linker is Linker 2 having attachment points C and D as shown below (Formula IV). Formula III Formula IV.

[0099] In one embodiment, attachment point A of Linker 1 (Formula III) or attachment point C of Linker 2 (Formula IV) is conjugated to Formula II. In one embodiment, attachment point A of Linker 1 is conjugated to Formula II and attachment point B of Linker 1 is conjugated to the double-stranded nucleic acid. In one embodiment, attachment point C of Linker 2 is conjugated to Formula II and attachment point D of Linker 2 is conjugated to the double-stranded nucleic acid. In one embodiment, attachment point A of Linker 1 is conjugated to Formula II and attachment point B of Linker 1 is conjugated to a phosphate group that is conjugated to the double-stranded nucleic acid. In one embodiment, attachment point C of Linker 2 is conjugated to Formula II and attachment point D of Linker 2 is conjugated to a phosphate group that is conjugated to the double-stranded nucleic acid.

[0100] Those skilled in the art will recognize that the linking group can be located at the 5' or 3' end of the double-stranded nucleic acid, or attached to one of the internal nucleotides or nucleobases. Those skilled in the art will also recognize that the linking group can be linked or conjugated to the 5' or 3' end of the double-stranded nucleic acid. Those skilled in the art will further recognize that when a delivery molecule (such as a delivery molecule comprising Formula II) is placed at the 5' end of the double-stranded nucleic acid (whether or not through a linking group), it may be necessary to overcome potential inefficient loading of Ago2, or other hindrances to the activity of the RISC complex. For example, for a delivery molecule comprising Formula II, which is linked or directly conjugated to an siRNA comprising a sense strand and an antisense strand, placing the delivery moiety at the 5' end of the antisense strand may pose difficulties for Ago2 loading and prevent effective knockdown. In suitable embodiments, the one or more double-stranded nucleic acids comprise an siRNA comprising a sense strand and an antisense strand, and the delivery moiety of Formula II is present at the 3' end of the sense strand. In further embodiments, the delivery moiety of Formula II is conjugated to the 3' end of the sense strand through a linking group. In yet another embodiment, the linking group comprises a ring structure, suitably a piperidine ring. In yet another embodiment, the linking group comprises Linker 2.

[0101] Pharmaceutical composition

[0102] The compositions described herein may also comprise pharmaceutically acceptable excipients, buffers or diluents, and may be formulated for ex vivo or in situ administration to host cells of an animal (especially a human). Such compositions may also optionally comprise liposomes, lipids, lipid complexes, lipid nanoparticles (LNPs), microspheres, microparticles, nanospheres or nanoparticles, or may otherwise be formulated for administration to the cells, tissues, organs or body of a subject in need thereof. Such compositions may be formulated for a variety of therapies, for example, for ameliorating, preventing and / or treating conditions associated with the expression of mutant proteins, polypeptides or peptides or the overexpression of proteins, polypeptides or peptides.

[0103] Preparations comprising pharmaceutically acceptable excipients and / or carrier solutions are well known to those skilled in the art, and similarly, developing suitable dosage regimens and treatment protocols for using the specific compositions described herein in various therapeutic regimens is also well known to those skilled in the art, including, for example, oral, parenteral, intravenous, intrathecal, subcutaneous or intracisternal, intranasal, intra-articular and intramuscular administrations and formulations.

[0104] Typically, these formulations may contain at least about 0.1% or more of a therapeutic agent (e.g., a nucleic acid or a liposome containing the nucleic acid), although the percentage of the active ingredient can of course vary and can conveniently range from about 1% or 2% to about 70% or 90% or more of the total formulation weight or volume. The amount of the therapeutic agent in each therapeutically effective composition can be prepared to allow for a suitable dosage of the compound in any given unit dose. When preparing such pharmaceutical formulations, those skilled in the art will consider factors such as solubility, stability, bioavailability, biological half-life, route of administration, product shelf life, etc. as well as other pharmacological considerations. In addition, multiple dosages and treatment regimens may be required.

[0105] In certain cases, it is desirable to deliver the nucleic acid or its preparation in a suitably formulated pharmaceutical composition in the following ways: subcutaneously, intraocularly, intravitreally, parenterally, intravenously, intracerebroventricularly, intrathecal, intramuscularly, intrathecally, orally, intraperitoneally, or by inhalation through the mouth or nose, or by direct injection into one or more cells, tissues, or organs.

[0106] The pharmaceutical formulations of compositions suitable for injection comprise sterile aqueous solutions or dispersions. In some embodiments, the formulation is sterile and has a fluidity that ensures easy injection with a syringe. In some embodiments, the form is stable under the manufacturing and storage conditions and can prevent the contaminating action of microorganisms (e.g., bacteria and fungi). The carrier can be a solvent or a dispersion medium, including, for example, water, saline, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, vegetable oils, or other pharmaceutically acceptable carriers, such as carriers generally recognized as safe (GRAS) by the U.S. Food and Drug Administration. For example, by using coatings (e.g., lecithin), by maintaining the desired particle size in the case of a dispersion, and by using surfactants, the appropriate fluidity can be maintained. Thus, the nucleic acid can be delivered together with various other pharmaceutically acceptable reagents as needed for the specific situation. Such compositions can be purified from host cells or other biological sources, or can be chemically synthesized as described herein.

[0107] The amount of the nucleic acid and / or nucleic acid composition and the time of administration of such composition will be within the knowledge of those skilled in the art who benefit from this teaching. In some embodiments, the administration is a single administration. In some embodiments, the administration is more than one administration. In certain cases, the dosing schedule can be determined by a physician overseeing the administration of such composition.

[0108] The toxicity and efficacy of the compositions used in the methods of the present disclosure can be determined by standard pharmaceutical procedures, using cultured cells or experimental animals to determine the LD50 (the dose lethal to 50% of the population) and / or the IC50 (the half-maximal inhibitory concentration). The dose of the composition for therapeutic purposes can be selected based on the ratio between toxicity and efficacy (corresponding to the therapeutic index), and thus can be expressed as the ratio LD50 / ED50 or LD50 / IC50 (where "ED50" represents the dose effective for 50% of the population, and "IC50" represents the dose effective to inhibit the translation of 50% of the target mRNA). Preferred are those compositions that exhibit a large therapeutic index. Although compositions that exhibit toxic side effects can be used, the delivery system should be carefully designed to minimize the potential damage of such side effects. The dose of the compositions described herein generally falls within the range including the ED50 or IC50 and has little or no toxicity. The dose may vary within this range depending on the dosage form employed and the route of administration employed.

[0109] Other aspects of the present disclosure relate to methods and formulations for a subject (e.g., a human or non-human subject), in situ host cells in a subject, ex vivo host cells, or host cells derived from a subject. Non-limiting examples of subjects include dogs and cats; livestock such as horses, cows, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject is a human subject.

[0110] 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 to form a pharmaceutical formulation suitable for in vivo delivery to a subject (e.g., a human).

[0111] A pharmaceutical composition or a drug comprises a pharmacologically effective amount of at least one therapeutic agent and optionally one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are substances intentionally included in a drug delivery system in addition to the active pharmaceutical ingredient (API, therapeutic product). Excipients do not exert or are not expected to exert a therapeutic effect at the intended dose. Excipients can serve the following functions: a) assist 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) contribute to product identification, and / or d) enhance any other properties of the overall safety and effectiveness of API delivery during storage or use. Pharmaceutically acceptable excipients can be inert substances or not inert substances.

[0112] Excipients include, but are not limited to: absorption promoters, anti-adhesives, antifoaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery promoters, delivery polymers, dextrans, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, glidants, humectants, lubricants, oils, polymers, preservatives, saline solutions, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water repellents, and wetting agents.

[0113] The pharmaceutical composition may contain other additional components common in pharmaceutical compositions. These additional components may include, but are not limited to: antipruritics, astringents, local anesthetics or anti-inflammatory agents (such as antihistamines, diphenhydramine).

[0114] The carrier may be, but is not limited to, a solvent or a dispersion medium, such as one containing water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof. Adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents may also be present in the carrier. The carrier may also incorporate into the composition isotonic agents (such as sugars, polyols, sodium chloride, etc.).

[0115] "Pharmaceutically acceptable" refers to those properties and / or substances that are acceptable to a subject from a pharmacological / toxicological perspective. The phrase "pharmaceutically acceptable" refers to molecular entities, compositions, and properties that are physiologically tolerable and generally do not produce allergic or other adverse or toxic reactions when administered to a subject. In some embodiments, a pharmaceutically acceptable compound is approved by a regulatory agency of the federal or state government of the United States or listed in the United States Pharmacopeia or other recognized pharmacopeias for use in animals, and more particularly in humans.

[0116] Method

[0117] Aspects of the present disclosure relate to methods of inhibiting or reducing target mRNA in a cell using a composition as described herein (e.g., double-stranded nucleic acid, conjugate, pharmaceutical composition, etc.). In some embodiments, the method includes contacting a cell containing the target mRNA with a double-stranded nucleic acid (or a conjugate containing a double-stranded nucleic acid). In some embodiments, the method includes delivering the double-stranded nucleic acid to a cell, such as a mammalian cell or a human cell (e.g., contacting the cell with the double-stranded nucleic acid). In some embodiments, the cell is located within a subject (e.g., a human subject).

[0118] The amount of target mRNA inhibited or reduced may vary. In some embodiments, the target mRNA is reduced by about 50% to about 100% compared 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% compared 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 compared 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 compared 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.

[0119] Aspects of the present disclosure relate to double-stranded nucleic acids having certain 2'-F-modification patterns (e.g., having 2'-F-modified nucleotides at positions 2, 5, 7, 14, and 16 from the 5' end of the antisense strand; positions 2, 3, 7, 14, and 16; positions 2, 5, 8, 14, and 16; positions 2, 4, 6, 8, 14, and 16; or positions 2, 6, 8, 14, and 16 of the antisense strand), which have increased persistence in vivo compared to double-stranded nucleic acids having other modification patterns. In some embodiments, the increased persistence refers to the persistence of knockdown or gene silencing mediated by the double-stranded nucleic acid. In some embodiments, the increased persistence refers to resistance to degradation of the double-stranded nucleic acid in vivo (e.g., enzymatic degradation). In some embodiments, the double-stranded nucleic acids described herein have a duration in vivo that is about 10% to about 100% longer than that of double-stranded nucleic acids having different modification patterns (or unmodified double-stranded nucleic acids). In some embodiments, the double-stranded nucleic acids described herein have a duration in vivo that is about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% longer than that of double-stranded nucleic acids having different modification patterns (or unmodified double-stranded nucleic acids). In some embodiments, the double-stranded nucleic acids described herein have a duration in vivo that is about 5-fold to about 100-fold longer than that of double-stranded nucleic acids having different modification patterns (or unmodified double-stranded nucleic acids). In some embodiments, the double-stranded nucleic acids described herein have a duration in vivo that is about 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold longer than that of double-stranded nucleic acids having different modification patterns (or unmodified double-stranded nucleic acids).

[0120] Aspects of the present disclosure relate to double-stranded nucleic acids having reduced (or decreased) off-target effects compared to double-stranded nucleic acids having different modification patterns (or unmodified double-stranded nucleic acids). In some embodiments, the off-target effect is reduced by about 50% to about 100% compared to double-stranded nucleic acids having different modification patterns (or unmodified double-stranded nucleic acids). In some embodiments, the off-target effect is reduced by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% compared to double-stranded nucleic acids having different modification patterns (or unmodified double-stranded nucleic acids). In some embodiments, the off-target effect is reduced by about 2-fold to about 100-fold compared to double-stranded nucleic acids having different modification patterns (or unmodified double-stranded nucleic acids). In some embodiments, the off-target effect is reduced by about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold compared to double-stranded nucleic acids having different modification patterns (or unmodified double-stranded nucleic acids).

[0121] 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 (also referred to as a "patient"). Treatment of the subject comprises administering to the subject a composition as described herein (e.g., a double-stranded nucleic acid, a conjugate, a pharmaceutical composition, etc.). As used herein, "treatment" refers to all processes that slow, control, delay or stop the development or progression of a disorder or disease disclosed herein, or improve the symptoms of a disorder or disease, but does not necessarily mean complete elimination of all disorder or disease symptoms. Treatment includes administering a composition as described herein (e.g., a double-stranded nucleic acid, a conjugate, a pharmaceutical composition, etc.) to treat a disease or disorder in a patient (especially a human). In some embodiments, administering the double-stranded nucleic acid as described herein is a prophylactic treatment for a subject who is not currently ill or has never been ill but is at risk of developing the disease, or who has previously had a disease and is currently not ill but is at risk of disease recurrence. In certain embodiments, the subject has a higher risk of developing a disease or a higher risk of disease recurrence compared to the average healthy member of the population.

[0122] "Development" or "progression" of a disease, disorder or condition refers to the initial manifestation and / or subsequent progression of the disease, disorder or condition. The development of a disease, disorder or condition can be detected and evaluated by standard clinical techniques well known in the art. However, progression also refers to progression that may not be detectable. For the purposes of the present disclosure, development or progression refers to the biological process of symptoms.

[0123] The optimal process for the administration or delivery of the compositions of the present disclosure (e.g., double-stranded nucleic acids, conjugates, pharmaceutical compositions, etc.) can vary depending on the desired outcome and / or the subject to be treated. As used herein, "administration" refers to contacting a cell with a double-stranded nucleic acid and its preparation, which can be carried out in vitro (including ex vivo) or in vivo. The compositions provided herein (e.g., double-stranded nucleic acids, conjugates, pharmaceutical compositions, etc.) can be administered by a variety of 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.

[0124] Generally, the most suitable route of administration depends on a variety of factors, including the nature of the agent (e.g., its stability in the patient environment) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration, injection, etc.). In some embodiments, the composition is administered to the subject by only one route of administration. In some embodiments, multiple routes of administration (e.g., sequentially or simultaneously) can be employed to administer the composition to the subject.

[0125] In some embodiments, an effective amount (e.g., an amount sufficient to reduce the expression or activity of the target mRNA) is administered to the subject. "Effective amount" refers to the amount necessary to achieve the desired therapeutic outcome (taking into account the duration of administration and the route of administration). The effective amount can vary depending on factors such as the individual's disease state, age, gender, and weight, as well as the ability of the double-stranded nucleic acid to elicit the desired response in the individual. The effective amount is also the amount at which any toxic or harmful effects produced during administration are offset by the therapeutic beneficial effects. The effective amount depends primarily on factors such as the species, age, weight, health status of the subject, and the target tissue, and thus may vary from animal to animal and tissue to tissue. In some embodiments, the effective amount can be a combination of an effective dose, frequency, and duration of administration.

[0126] During the course of treatment, the administration of the composition (e.g., double-stranded nucleic acids, conjugates, pharmaceutical compositions, etc.) can be changed or adjusted accordingly. For example, the expression of the target mRNA and / or the protein product encoded by the target mRNA can be monitored to guide the method of using the composition. For example, expression information can be obtained by measuring changes in the levels of the protein or RNA products of the target mRNA.

[0127] Examples

[0128] Example 1: Synthesis of RNAi agent

[0129] Single strands (sense and antisense strands) of RNA duplexes were synthesized on solid supports by MerMade™ 12 (LGC Biosearch Technologies). The sequences of the unmodified and modified sense and antisense strands are shown in Tables 1-4. The mRNA targets 1 (APOE) and 2 (SNCA) molecules are not encoded by the same gene. Oligonucleotides were synthesized by phosphoramidite chemistry. In some embodiments, the sense strands described in Table 2 or Table 4 contain delivery molecules, such as lipids (e.g., cholesterol esters, cholesteryl). In some embodiments, the delivery molecule (e.g., lipid) is linked to the sense strand via a linker (e.g., a TEG linker). Table 1: Unmodified nucleic acid sequences of dsRNA targeting APOE. Table 2: Modified nucleic acid sequences of dsRNA targeting APOE. Abbreviations - “m” represents 2’-OMe; “f” represents 2’-fluoro; “ ” represents a modified linkage (e.g., phosphorothioate linkage); “VP” represents 5’-vinylphosphonate. Table 3: Unmodified nucleic acid sequences of dsRNA targeting SNCA. Table 4: Modified nucleic acid sequences of dsRNA targeting SNCA. Abbreviations - “m” represents 2’-OMe; “f” represents 2’-fluoro; “ ” represents a modified linkage (e.g., phosphorothioate linkage); “VP” represents 5’-vinylphosphonate; “(Ab)” represents an abasic moiety, “r” represents a site containing ribose; “d” represents a site containing deoxyribose. Table 5: Conjugates containing modified nucleic acid sequences of dsRNA targeting SNCA. Abbreviations - “m” represents 2’-OMe; “f” represents 2’-fluoro; “ ” represents a modified linkage (e.g., phosphorothioate linkage); “VP” represents 5’-vinylphosphonate; “(Ab)” represents an abasic moiety, “r” represents a site containing ribose; “d” represents a site containing deoxyribose.

[0130] In some embodiments, the conjugates described herein comprise the sequences listed in Table 2 or Table 4 or Table 5. In some embodiments, the conjugates described herein comprise double-stranded RNA shown in Table 2 or Table 4 or Table 5 (e.g., conjugates comprising the sense and antisense strands of the double-stranded RNA shown in Table 2 or Table 4 or Table 5, wherein the sense strand is conjugated to tocopherol or cholesterol). In some embodiments, the conjugates described herein comprise the sequences (e.g., sense strand) listed in Table 2 or Table 4 or Table 5, which are bonded (e.g., covalently bonded) to the linker groups described herein (e.g., TEG linker groups, such as linker groups that link the sense strand of double-stranded RNA to tocopherol or cholesterol).

[0131] For the sense strand, the types of solid-phase carriers are 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), all of which are commercially available. For all antisense strands, commercially available standard carriers are used and selected according to the sequences. Standard reagents (Table 6) are used in oligonucleotide synthesis, in which pyridine containing 0.1M xanthane hydride is used as a sulfurization reagent, and ACN containing 20% DEA is used as an auxiliary detergent after synthesis. All monomers (Table 7) are dissolved in acetonitrile (ACN) at a concentration of 0.1M and are equipped with molecular sieve trapping bags.

[0132] Cleavage and deprotection (C / D) of the oligonucleotides was carried out at 45 °C for 20 h. The sense strand was C / D from the CPG using ammonium hydroxide (28 - 30%, cold), while the antisense strand was C / D using ammonium hydroxide (28 - 30%, cold) containing 3% DEA. After confirming that the resulting mass data was consistent with the sequence by IP-RP LCMS, the C / D was determined to be complete. Depending on the scale, the CPG was filtered through a 0.45 µm PVDF syringe-free filter, a 0.22 µm PVDF Steriflip® vacuum filter, or a 0.22 µm PVDF Stericup® quick-release filter. The CPG was backwashed / rinsed with 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. Then the material was evenly divided into 50 mL falcon tubes to remove organics by Genevac™. After concentration, the crude oligonucleotides were diluted back to the scale at synthesis with RNase-free water and filtered through a 0.45 µm PVDF syringe-free filter, a 0.22 µm PVDF Steriflip® vacuum filter, or a 0.22 µm PVDF Stericup® quick-release filter.

[0133] The crude oligonucleotides were purified by an AKTA™ Pure purification system using an anion exchange (AEX) or reverse phase (RP) Source™ 15Q-RP column. For AEX, an ES Industry Source™ 15Q column was used, maintaining the column temperature at 65 °C, mobile phase A: 20 mM NaH2PO4, 15% ACN, pH 7.4; mobile phase B: 20 mM NaH2PO4, 1 M NaBr, 15% ACN, pH 7.4. For RP, a Source™ 15Q-RP chromatographic column was used, mobile phase A: 50 mM NaOAc and 10% ACN; mobile phase B: 50 mM NaOAc and 80% ACN. In all cases, the integrations containing materials with a purity greater than 85% and impurity content ≤5% were combined.

[0134] The purified oligonucleotides were desalted using a 15 mL 3K MWCO centrifuge tube at 3500 x g for approximately 30 minutes. The oligonucleotides were rinsed with RNAse-free water until the conductivity of the eluate reached <100 usemi / cm. After desalting was complete, 2 - 3 mL of RNAse-free water was added, and then aspirated 10 times. The retention was transferred to a 50 mL falcon tube, and this operation was repeated until the transfer of the oligonucleotides was confirmed by measuring the concentration of the compound on the filter using a Nanodrop spectrophotometer. Then, the final oligonucleotides were nanofiltered twice through a 15 mL 100K MWCO centrifuge tube at 3500 x g for 2 minutes. The concentration of the oligonucleotides after final desalting was analyzed (detected at A260 using a Nanodrop spectrophotometer), the mass purity was characterized by IP-RPLCMS, and the UV purity was characterized by UPLC.

[0135] The duplexes were prepared as follows: equimolar amounts of the sense and antisense strands were mixed and heated at 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 using IP-RPLCMS. All duplexes were nanofiltered and then the endotoxin level was measured using a Charles River Endosafe® endotoxin detection card to obtain the final conjugated RNAi compounds. For in vivo analysis, an appropriate amount of the duplex was lyophilized, and then, for rodent studies, it was reconstituted with 1X PBS, and for non-human primate studies, it was reconstituted with CSF. Table 6 - Oligonucleotide Synthesis Reagents Table 7 - Phosphoramidite

[0136] Example 2. In Vitro Characterization of RNAi Agents

[0137] The selected RNAi agents were tested for their inhibition of mRNA target 1 (APOE) or mRNA target 2 (SNCA) in cultured cells including HEP3B, SH-SY5Y, iPSC astrocytes, or mouse primary cortical neurons.

[0138] Materials and Methods

[0139] HEP3B, SH-SY5Y, and Astrocyte Cultures with RNAi Treatment and Analysis: SH-SY5Y cells (ATCC CRL-2266) were derived from the SK-N-SH neuroblastoma cell line (Ross, R. A. et al., 1983. J Natl Cancer Inst 71, 741-747). The basal medium was a 1:1 mixture of Eagle's Minimum Essential Medium (catalog number 30-2003) formulated by ATCC and F12 medium. The complete growth medium was supplemented with 10% fetal bovine serum, 1X amino acids, 1X sodium bicarbonate, and 1X penicillin-streptomycin (Gibco), and the cells were incubated in a humidified environment at 37°C and 5% CO2. On the first day, SH-SY5Y cells were seeded into fibronectin-coated 96-well tissue culture plates and allowed to adhere overnight. On the second day, the complete medium was removed and replaced with serum-free medium containing the RNAi agent. The cells were incubated with the RNAi agent for 72 hours, and then gene expression was analyzed. Changes in gene expression in RNAi-treated SH-SY5Y cells were analyzed using the Cells-to-C T Kit (ThermoFisher A35377). Predesigned gene expression assays (provided as 20X mixes) were selected from Applied Bio-systems (Foster City, CA, USA). The efficiency of these assays (mRNA targets 1 or 2 and ThermoFisher Hs99999905_m1 GAPDH) was characterized by a cDNA dilution series. RT-QPCR was performed in a MicroAmp Optical 384-well reaction plate using the QuantStudio7 Flex system. The ΔΔCT method was used to normalize to the housekeeping gene GAPDH to calculate the relative amount of gene expression. The IC50 was determined by four-parameter logistic fitting using GraphPad Prism v9.0.

[0140] HEP3B cells (ATCC HB-8064) were cultured in a medium containing Eagle's Minimum Essential Medium (Catalog No. 30-2003) formulated by ATCC, and the medium contained fetal bovine serum at a final concentration of 10%. On the first day, HEP3B cells were seeded in a 96-well tissue culture plate and allowed to adhere overnight. On the second day, the complete medium was removed and replaced with serum-free medium containing the RNAi agent. The cells were incubated with the RNAi agent for 72 hours, and then gene expression was analyzed. Using the Perkin Elmer AlphaLISA assay, according to the protocol provided by the manufacturer, the changes in protein expression in HEP3B cells treated with RNAi were analyzed to evaluate the protein level corresponding to the expression of mRNA target 1 (AL395). The IC50 was determined by four-parameter logistic fitting using GraphPad Prism v9.0.

[0141] Astrocytes derived from iPSC (Cellular Dynamics 01434) were cultured according to the protocol provided by the manufacturer. On the first day, astrocytes were seeded in a 96-well tissue culture plate and allowed to adhere overnight. On the second day, the complete medium was removed and replaced with serum-free medium containing the RNAi agent. The cells were incubated with the RNAi agent for 72 hours, and then gene expression was analyzed using the above method (Thermofisher Assay ID Hs00171168_ml).

[0142] Mouse primary cortical neuron (MCN) culture, RNAi treatment, and analysis: Mouse primary cortical neurons were isolated from E18 wild-type C57BL6 mouse embryos. Cells were seeded at a density of 40,000 cells / well in poly-D-lysine-coated 96-well plates and cultured at 37 °C for 7 days in NbActiv1 (BrainBits, LLC) containing 1% antibiotic / antifungal (Corning) in a humidified incubator with 5% CO2. On day 7, half of the medium was removed from each well and replaced with 2% FBS medium containing 2-fold concentrated RNAi (as CRC treatment), and the cells were incubated for an additional 7, 14, or 21 days. Half of the medium was replaced with fresh medium every 7 days. At the end of the RNAi treatment, RT-qPCR was performed using the TaqMan Fast Advanced Cell-to-CT kit to quantify SNCA levels. Specifically, the cells were lysed, cDNA was generated on a Mastercycler X50a (Eppendorf), and qPCR was performed on a QuantStudio 7 Flex real-time PCR system (Applied Biosystems). The APO gene expression level was normalized to β-actin (ThermoFisher, Mm02619580_gl) using the corresponding probe.

[0143] 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, 5% CO2. The next day, the cells were transfected with siRNA using RNAiMAX (Invitrogen, Waltham, MA) according to the manufacturer's protocol. The cells were incubated at 37 °C, 5% CO2 for 48 hours. The culture plates were cooled to room temperature, and an equal volume of Bio-Glo (Promega, Madison, WI) was added to each well. The plates were incubated in the dark at room temperature and then read on a BioTek Neo s2 plate reader (Agilent, Santa Clara, CA).

[0144] Representative results of these analyses are shown in Table 11.

[0145] Results

[0146] Tables 8, 9, 10, and 11 show the in vitro activities of the RNAi agents against mRNA target 1 (APOE) and mRNA target 2 (SNCA) in SH-SY5Y, HEP3B, astrocytes, and mouse primary cortical neurons (MCN). Table 8: In vitro activity of double-stranded RNA against mRNA target 1 (APOE) in HEP3B and astrocytes. Table 9: In vitro activity of double-stranded RNA against mRNA target 2 (SNCA) in Sy5y neurons. Table 10: In vitro activity of double-stranded RNA against mRNA target 2 (SNCA) in mouse cortical neurons (MCN). Table 11: Luciferase reporter gene assay of in vitro activity of double-stranded RNA against mRNA target 2 (SNCA).

[0147] Example 3. Characterization of selected RNAi agents in mice

[0148] The efficacy of RNAi agents against mRNA target 1 (APOE) was studied in the brains and livers of mRNA target 1 knock-in (KI) mice. Six mice received intracerebroventricular (ICV) injection of 100 μg of the RNAi agent (e.g., the dsRNA described in Table 2) or PBS (phosphate-buffered solution) and were sacrificed on day 15 (D15) or day 57 (D57) after injection. The expression levels of human mRNA target 1 (APOE) in the spinal cord and brain were detected and analyzed by quantitative PCR (qPCR). Another group of mRNA target 1 (APOE) KI mice received subcutaneous injection of the RNAi agent or PBS and had blood samples collected continuously for up to 12 weeks after injection, and the protein levels of mRNA target 1 (APOE) in the serum were analyzed as described above.

[0149] Representative results are shown in Table 12. The data in Table 12 show that dsRNA constructs containing 2′F-modifications at positions 2, 5, 7, 14, and 16 of the antisense strand are more persistent in vivo than dsRNA constructs containing 2′F-modifications at other positions. In addition, 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 decrease in the serum concentration of the mRNA target 1 (APOE) protein in vivo for at least 8 weeks and a decrease in the expression level of the mRNA target 1 (APOE) in several brain tissues including the brain stem (BS), hippocampus (H), hippocampal cortex (HCTX), and frontal cortex (FCTX).

[0150] The efficacy of the RNAi agent against mRNA target 2 (SNCA) was studied in wild-type C56BL / 6N mice. Six mice received an intracerebroventricular (ICV) injection of 30 μg of the RNAi agent (e.g., the dsRNA described in Table 4) or PBS (phosphate-buffered solution) and were sacrificed at D15 or D57 after injection. The mRNA expression levels of mRNA target 2 (SNCA) in the spinal cord and brain of the mice were detected and analyzed by quantitative PCR (qPCR). The data are shown in Table 13, indicating a higher level of knockdown of mRNA target 2 (SNCA) 2 months after ICV injection. The highest levels of knockdown of mRNA target 2 (SNCA) were observed in the brain stem, frontal cortex, striatum (STR), and lumbar spinal cord tissues (e.g., LDRG cells) of the mouse subjects (see Table 13). Table 12: In vivo activity of mRNA target 1 (APOE). Table 13: In vivo activity of double-stranded RNA against mRNA target 2 (SNCA).

[0151] Example 4. siRNA Conjugates

[0152] This example describes the preparation of double-stranded nucleic acid-GalNAc conjugates. To synthesize the sense strand conjugated with GalNAc, first, the sense strand with a 3’ C6-NH2 functional group was synthesized using standard phosphoramidite chemistry. A stock solution of GalNAc ligand-NHS ester (in acetonitrile, 10 mmol / L; 1 equivalent) was prepared. Borate buffer (10% v / v; 20x) was added to the oligonucleotide C6-NH2 sense strand in an Eppendorf tube, and then the GalNAc ligand (5 equivalents) was added. The mixture was shaken at ambient temperature for 16 hours. Thereafter, the mixture was transferred to a 15 mL falcon tube, ammonium hydroxide (28 mass%) was added, and the mixture was shaken at ambient temperature for 2 hours. Then ammonia was removed in vacuo. The residue was purified by ion-exchange chromatography. Conditions: Solvent A: 15% MeCN / 20 mM NaH2PO4, Solvent B: 15% MeCN / 20 mM NaH2PO4, 1 M NaBr; the concentration of Solvent B was gradually changed from 35% to 55% during 5 column volumes, the flow rate was 8 mL / min, and the column temperature was 60 °C. The desired fractions were pooled and desalted using a spin filter or a desalting column with an Eppendorf centrifuge. After desalting, the material was recovered and the OD and volume were measured to obtain the concentration.

[0153] Alternatively, for 5’-position conjugation of the sense strand, the GalNAc ligand was immobilized on microporous polystyrene resin or controlled pore glass, and synthesis was carried out using the established solid-phase oligonucleotide synthesis method with 5’-CE (β-cyanoethyl) phosphoramidite.

[0154] Alternatively, the GalNAc ligand was converted into a suitable phosphoramidite and delivered to the 5’-position of the sense strand using standard phosphoramidite chemistry.

[0155] To generate the siRNA duplex of the sense and antisense strands, the following steps were performed. The corresponding antisense oligonucleotide (1 equivalent) was added to a Falcon tube containing the oligonucleotide sense strand-GalNAc conjugate, vortexed for 10 seconds, and then spin-filtered through a 100K MWCO Amicon filtration device to remove particles. The filtrate was recovered and concentrated in vacuo on a Genevac evaporator. The residue was reconstituted in 1x PBS, filtered through a 0.2 µm filter, and the OD and volume were measured to obtain the concentration.

[0156] The endotoxin test was performed using Limulus amebocyte lysate on an Endosafe® NexGen PTS instrument.

[0157] Equivalent protocols

[0158] Although several embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of these variations and / or modifications is to be regarded as being within the scope of the embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are exemplary, and the actual parameters, dimensions, materials, and / or configurations will depend upon the particular application for which the teachings of the invention are directed. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Accordingly, it is to be understood that the foregoing embodiments are presented by way of example only, and that the embodiments of the invention may be practiced otherwise than as specifically described and claimed within the scope of the claims and their equivalents. The embodiments of the present disclosure are directed to each and every separate feature, system, article, material, kit, and / or method described herein. Additionally, any combination of two or more such features, systems, articles, materials, kits, and / or methods (where not mutually inconsistent) is included within the inventive scope of the present disclosure.

[0159] All definitions defined and used herein shall be understood to be controlling over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0160] All references, patents, and patent applications disclosed herein are hereby incorporated by reference in their entirety for each cited subject matter, in some cases covering the entire document.

[0161] References in the specification and claims to an indefinite number of an item, unless otherwise expressly stated, shall be understood to mean "at least one".

[0162] The phrase "and / or" as used in the specification and claims shall be understood to mean "either or both" of the connected elements, i.e., the elements may co-occur in some cases and occur separately in other cases. Multiple elements listed in "and / or" shall be construed in the same manner, i.e., "one or more" of the connected elements. In addition to the elements expressly referred to by "and / or", there may optionally be other elements different from those expressly referred to, whether or not related to the expressly referred to elements. Thus, as a non-limiting example, when referring to "A and / or B" in conjunction with an open-ended statement such as "comprising", in one embodiment it may refer only to A (optionally including elements other than B); in another embodiment it may refer only to B (optionally including elements other than A); in yet another embodiment it refers to both A and B (optionally including other elements); and so on.

[0163] As used in this specification and the claims, "or" shall 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 interpreted inclusively, i.e., including at least one of the listed elements, but also including more than one, and optionally other unlisted items. Only terms that explicitly indicate the contrary meaning, such as "only one" or "exactly one", or in the case of using "consisting of..." in the claims, refer to exactly one of the listed elements. Generally, the term "or" as used herein shall be interpreted as representing an exclusive alternative (i.e., "one or the other, but not both") only when preceded by an exclusive term (such as "either", "one of", "only one of", or "exactly one of"). "Consisting essentially of..." when used in the claims shall have its ordinary meaning in the field of patent law.

[0164] As used in this specification and the claims, when referring to a list of one or more elements, the phrase "at least one" shall 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 element specifically listed in the list of elements, nor excluding any combination of the elements in the list of elements. The definition also allows for the optional presence of elements other than those explicitly referred to in the list of elements to which the phrase "at least one" refers, whether or not they are related to the explicitly referred to 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"), in one embodiment may refer to at least one A, optionally including more than one A, no B (and optionally including elements other than B); in another embodiment may refer to at least one B, optionally including more than one B, no A (and optionally including elements other than A); in yet another embodiment may refer to at least one A, optionally including more than one A, and at least one B, optionally including more than one B (and optionally including other elements); and so on.

[0165] It should also be understood that, unless explicitly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are described.

Claims

1. A double-stranded nucleic acid, comprising: (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 comprise no more than five 2'-fluoro (2'F)-modified nucleotides, and wherein the 2'F-modified nucleotides are located at the 2nd, 5th, 7th, 14th, and 16th positions from the 5' end of the antisense strand.

2. A double-stranded nucleic acid, comprising: (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 comprise no more than five 2'F-modified nucleotides, and wherein the 2'F-modified nucleotides are located at the 2nd, 3rd, 7th, 14th, and 16th positions from the 5' end of the antisense strand.

3. A double-stranded nucleic acid, comprising: (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 comprise no more than five 2'F-modified nucleotides, and wherein the 2'F-modified nucleotides are located at the 2nd, 5th, 8th, 14th, and 16th positions from the 5' end of the antisense strand.

4. A double-stranded nucleic acid, comprising: (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 comprise no more than six 2'F-modified nucleotides, and wherein the 2'F-modified nucleotides are located at the 2nd, 4th, 6th, 8th, 14th, and 16th positions from the 5' end of the antisense strand.

5. A double-stranded nucleic acid, comprising: (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 comprise no more than five 2'F-modified nucleotides, and wherein the 2'F-modified nucleotides are located at the 2nd, 6th, 8th, 14th, and 16th positions from the 5' end of the antisense strand.

6. The double-stranded nucleic acid according to any one of claims 1 to 5, wherein the sense strand comprises a 5' end and a 3' end, and comprises 2'F-modified nucleotides at the following positions: (i) the 9th, 10th, and 11th positions from the 5' end of the sense strand; (ii) the 7th, 9th, and 11th positions from the 5' end of the sense strand; (iii) the 7th, 9th, and 10th positions from the 5' end of the sense strand; or (iv) the 7th, 10th, and 11th positions from the 5' end of the sense strand.

7. The double-stranded nucleic acid according to claim 6, wherein the sense strand does not comprise 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, wherein the antisense strand comprises a sequence complementary to a portion of a target mRNA, and wherein the antisense strand comprises five 2'F-modified nucleotides at one of the following groups of positions from its 5' end and does not comprise any other 2'F-modified nucleotides: (a) the 2nd, 5th, 7th, 14th, and 16th positions; (b) the 2nd, 3rd, 7th, 14th, and 16th positions; (c) the 2nd, 5th, 8th, 14th, and 16th positions.

9. The double-stranded nucleic acid according to claim 8, wherein the sense strand contains three 2′F-modified nucleotides at one of the following site groups starting from its 5′ end: (a) the 9th, 10th, and 11th positions; (b) the 7th, 9th, and 11th positions; (c) the 7th, 9th, and 10th positions; or (d) the 7th, 10th, and 11th positions.

10. The double-stranded nucleic acid according to claim 8 or 9, wherein the antisense strand contains 2′-O-methyl modified nucleotides at sites that are not 2′F-modified sites.

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

12. The double-stranded nucleic acid according to any one of claims 1 to 11, wherein the 1st position starting from the 5′ end of the sense strand or the 1st position starting from the 5′ end of the antisense strand contains a 5′ phosphate analogue.

13. The double-stranded nucleic acid according to claim 12, wherein the 5′ phosphate analogue contains a 5′ vinyl phosphonate group.

14. The double-stranded nucleic acid according to claim 12 or 13, wherein the antisense strand contains a 5′ phosphate analogue.

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

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

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

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

19. The double-stranded nucleic acid according to any one of claims 1 to 18, wherein the sense strand and the antisense strand have different lengths.

20. The double-stranded nucleic acid according to any one of claims 1 to 19, wherein the antisense strand is longer than the sense strand, optionally wherein 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 double-stranded nucleic acid according to any one of claims 1 to 21, wherein the modified nucleotides of the sense strand comprise one or more 2'-O-methyl (2'OMe)-modified nucleotides or one or more 2'-O-C 12-16 alkyl-modified nucleotides.

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

24. The double-stranded nucleic acid according to any one of claims 1 to 23, wherein the modified nucleotides of the sense strand contain only 2′OMe-modified nucleotides except for containing 2′-fluoro (2′F)-modified nucleotides at the listed sites.

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

26. The double-stranded nucleic acid according to any one of claims 1 to 25, wherein the modified nucleotides of the antisense strand contain only 2′OMe-modified nucleotides except for containing 2′-fluoro (2′F)-modified nucleotides at the listed sites.

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

28. The double-stranded nucleic acid according to claim 27, wherein the sense strand contains four modified internucleotide linkages.

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

30. The double-stranded nucleic acid according to claim 29, wherein each of the modified internucleotide linkages is a phosphorothioate (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 linkages.

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

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

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

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

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

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

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

39. The double-stranded nucleic acid according to any one of claims 27 to 38, wherein at least two of the first, second, and third positions from the 3'-end of the sense strand are linked by a modified internucleotide linkage, optionally by a phosphorothioate (PS) internucleotide linkage.

40. The double-stranded nucleic acid according to any one of claims 27 to 39, wherein at least two of the first, second, and third positions from the 3'-end of the antisense strand are linked by a modified internucleotide linkage, optionally by a phosphorothioate (PS) internucleotide linkage.

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

42. The double-stranded nucleic acid according to any one of claims 27 to 41, wherein each of the first, second, and third positions from 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 according to claim 43, wherein the conjugate comprises a structure of Formula I, wherein Formula I comprises: A-B-C Formula I, wherein "A" of Formula I comprises a double-stranded nucleic acid, "B" of Formula I comprises a bond or a linker, and "C" of Formula I comprises a delivery molecule.

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

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

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

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

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

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

51. The conjugate according to any one of claims 43 to 50, wherein the double-stranded RNA does not comprise a nucleotide linked to the following groups: a maleimide group, a tertiary amide bonded to a gem-dimethyl (GDM) group, or a C6-NH2 group.

52. The conjugate according to any one of claims 43 to 51, wherein "B" of Formula I does not comprise: a maleimide-methyl-tetrazine-trans-cyclooctene (mal-tet-TCO) linker, a N-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.

53. The conjugate according to any one of claims 43 to 47, wherein "B" of Formula I comprises a linker containing a C6-NH2 group.

54. The conjugate according to 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. A method for inhibiting or reducing a target mRNA in a cell, the method comprising contacting a cell containing the target mRNA with 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 or the pharmaceutical composition according to claim 55.

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

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

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

60. The double-stranded nucleic acid according to claim 59, wherein (i) The sense strand contains the sequence shown by any one of SEQ ID NO: 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, 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 contains the sequence shown by any one of SEQ ID NO: 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.

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