Optimized 2'-Modified Ribose Derivatives and Methods of Use

Abstract

The present disclosure provides optimized isolated oligonucleotides comprising specific combinations of 2'-ribose derivatives or substitutions to enhance their stability and efficacy for delivery and use as therapeutic agents.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 359,969, filed on July 11, 2022, the entire content of which is incorporated herein by reference.

Background Art

[0002] Small interfering RNA (siRNA) can efficiently cause RNAi silencing of specific genes, but its therapeutic potential still faces challenges regarding efficacy, safety, and delivery. Completely chemically modified siRNA molecules with combinations of ribose modifications (e.g., 2'-O-methyl and 2'-deoxy-2'-fluoro), backbone modifications (e.g., phosphorothioate), and 5'-phosphate modifications (e.g., 5'-E-vinylphosphonate) have been widely investigated and have been shown to significantly improve metabolic stability and RNAi activity, leading to improved efficacy and duration in vivo.

[0003] Among these chemical modifications, substitution of the 2'-OH group of ribose with 2'-O-methyl (2'-OMe), 2'-deoxy-2'-fluoro (2'-F), and other 2'-O-alkyl groups (e.g., 2'-O-methoxyethyl) has been demonstrated to improve the metabolic stability and RNAi activity of siRNA. More specifically, 2'-O-methyl modification has been shown to be more sterically hindered than 2'-deoxy-2'-fluoro and have stronger metabolic stability against endogenous nucleases. Also, although 2'-fluoro-containing siRNAs have been well tested in preclinical trials and shown good tolerance in clinical trials, it may be desirable to reduce concerns regarding the tolerance of incorporating the unnatural 2'-fluoro modification into siRNA. Therefore, increasing the 2'-O-methyl content in siRNA while simultaneously decreasing the 2'-fluoro content is preferred from the perspective of improving metabolic stability and tolerance. However, the highly sterically hindered 2'-O-methyl group may cause steric clashes with the argonaute RISC catalytic component 2 (Ago2) residues, and thus, if not applied appropriately, loading of the guide strand into RISC is hindered and RNAi activity is significantly reduced. Therefore, an optimal chemical modification that comprehensively improves the metabolic stability, tolerance, and RNAi activity of siRNA continues to be needed. The present disclosure meets this need.

SUMMARY OF THE INVENTION

[0004] Provided herein is an isolated oligonucleotide comprising: (a) a sense strand comprising X1 nucleotides, wherein at least one nucleotide is modified with a first modification and each of the remaining nucleotides is independently modified with a second modification, X1 is an integer selected from 13 to 36, the first modification and the second modification are different, and (b) an antisense strand comprising X2 nucleotides, wherein at least one nucleotide is modified with a third modification and each of the remaining nucleotides is independently modified with a fourth modification, X2 is an integer selected from 18 to 31, the third modification and the fourth modification are different.

[0005] In some embodiments, the first modification is a modification of the sugar moiety of at least one nucleotide at the 2'-position selected from a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, a 2'-deoxy modification, and equivalents thereof, and combinations thereof. In some embodiments, the first modification is a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, or a 2'-deoxy modification, or stereoisomers thereof. In some embodiments, the first modification is a 2'-F modification, a 2'-CN modification, or a 2'-N3 modification, or stereoisomers thereof. In some embodiments, the first modification is a 2'-F modification or a stereoisomer thereof.

[0006] In some embodiments, the second modification is a modification of one or more sugar moieties of the remaining nucleotides at the 2'-position selected from 2'-C1-C6 alkyl, 2'-OR modification [wherein R is C1-C6 alkoxy, acetamide, phenyl, or heteroaryl containing a 5- or 6-membered ring and one or two heteroatoms selected from N, O, and S, and is optionally substituted C1-C6 alkyl], 2'-amino, and morpholino substitution, and equivalents thereof, and combinations thereof. In some embodiments, the second modification is a 2'-OR modification, or a morpholino substitution, or a combination thereof. In some embodiments, the second modification is a 2'-OR modification. In some embodiments, the second modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification. In some embodiments, the second modification is a 2'-O-methyl modification. In some embodiments, the second modification is a morpholino substitution.

[0007] In some embodiments, the first modification is a 2'-F modification or a stereoisomer thereof, and the second modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification. In some embodiments, the first modification is a 2'-F modification or a stereoisomer thereof, and the second modification is a 2'-O-methyl modification.

[0008] In some embodiments, the third modification is a modification of the sugar moiety of at least one nucleotide at the 2'-position selected from a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, a 2'-deoxy modification, and equivalents thereof, and combinations thereof. In some embodiments, the third modification is a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, or a 2'-deoxy modification, or a stereoisomer thereof. In some embodiments, the third modification is a 2'-F modification, a 2'-CN modification, or a 2'-N3 modification, or a stereoisomer thereof. In some embodiments, the third modification is a 2'-F modification or a stereoisomer thereof.

[0009] In some embodiments, the fourth modification is a modification of one or more sugar moieties of the remaining nucleotides at the 2'-position selected from 2'-C1-C6 alkyl, 2'-OR modification [wherein R is C1-C6 alkoxy, acetamide, phenyl, or heteroaryl containing a 5- or 6-membered ring and one or two heteroatoms selected from N, O, and S, optionally substituted C1-C6 alkyl], 2'-amino, and morpholino substitution, and equivalents thereof, and combinations thereof. In some embodiments, the fourth modification is a 2'-OR modification, or a morpholino substitution, or a combination thereof. In some embodiments, the fourth modification is a 2'-OR modification. In some embodiments, the fourth modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification. In some embodiments, the fourth modification is a 2'-O-methyl modification. In some embodiments, the fourth modification is a morpholino substitution.

[0010] In some embodiments, the third modification is a 2'-F modification or a stereoisomer thereof, and the fourth modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification. In some embodiments, the third modification is a 2'-F modification or a stereoisomer thereof, and the fourth modification is a 2'-O-methyl modification.

[0011] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 3 nucleotides are modified with a first modification. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 2 of the at least 3 nucleotides modified with the first modification are located consecutively. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 3 of the at least 3 nucleotides modified with the first modification are located consecutively.

[0012] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 4 nucleotides are modified with a first modification. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 3 of the at least 4 nucleotides modified with the first modification are located consecutively. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 4 of the at least 4 nucleotides modified with the first modification are located consecutively.

[0013] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 5 nucleotides are modified with a first modification. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 3 of the at least 5 nucleotides modified with the first modification are located consecutively, and at least 4 of the at least 5 nucleotides modified with the first modification are located consecutively.

[0014] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 3 nucleotides, at least 4 nucleotides, or at least 5 nucleotides modified with the first modification are located at positions 10 to 15 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand.

[0015] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, two of at least three nucleotides modified with a first modification are located at positions selected from the 10th, 11th, 12th, and 13th positions from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, three of at least three nucleotides modified with a first modification are located at positions selected from the 10th, 11th, 12th, and 13th positions from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand.

[0016] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, one of at least three nucleotides modified with a first modification is located at the 11th position from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, one of at least four nucleotides modified with a first modification is located at the 10th position from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, one of at least four nucleotides modified with a first modification is located at the 14th position from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand.

[0017] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, two of at least four nucleotides modified with a first modification are located at positions from the 10th to 14th positions from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand.

[0018] In some embodiments, not all of at least three nucleotides modified with a first modification are located consecutively in the sense strand of the isolated oligonucleotide of the present disclosure. In some embodiments, at least three nucleotides, at least four nucleotides, or at least five nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure are modified with a 2'-F modification.

[0019] In some embodiments, the sense strand of the isolated oligonucleotide of the present disclosure comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification, and has the formula: 5'(M) g (F) f (M) e (F) d (M) c (F) b (M) a 3' [wherein M is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and each of a, b, c, d, e, f, and g is any one of 0 to 16, indicating the number of consecutive nucleotides modified with the modification], and the sense strand is a) 5'(M)0(F)0(M)0(F)0(M)8(F)3(M) 10 3' (SEQ ID NO: 52); b) 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3' (SEQ ID NO: 53); c) 5'(M)0(F)0(M)0(F)0(M)7(F)3(M) 10 3' (SEQ ID NO: 54); d) 5'(M)0(F)0(M)5(F)1(M)1(F)2(M) 11 3' (SEQ ID NO: 55); e) 5'(M)0(F)0(M)6(F)1(M)1(F)2(M) 11 3' (SEQ ID NO: 56); f) 5'(M)6(F)1(M)1(F)2(M)1(F)1(M)9 3' (SEQ ID NO: 57); g) 5'(M)5(F)1(M)1(F)2(M)1(F)1(M)9 3' (SEQ ID NO: 58); h) 5’(M)0(F)0(M)0(F)0(M)7(F)4(M) 10 3’ (Sequence number: 59); i) 5’(M)0(F)0(M)6(F)1(M)1(F)4(M)93’ (Sequence number: 60); j) 5’(M)0(F)0(M)0(F)0(M)8(F)4(M)93’ (Sequence number: 61); k) 5’(M)0(F)0(M)0(F)0(M)6(F)4(M) 10 3’ (Sequence number: 62); l) 5’(M)0(F)0(M)5(F)1(M)1(F)4(M)93’ (Sequence number: 63); m) 5’(M)0(F)0(M)0(F)0(M)7(F)4(M)93’ (Sequence number: 64); n) 5’(M)0(F)0(M)0(F)0(M)7(F)5(M)93’ (Sequence number: 65); and o) any one of 5’(M)0(F)0(M)0(F)0(M)6(F)5(M)93’ (Sequence number: 66).

[0020] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 7 nucleotides are modified with a third modification.

[0021] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 4 of the at most 7 nucleotides modified with a third modification are located at positions 2 to 8 from the first nucleotide at the 5’ end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 2 of the at most 7 nucleotides modified with a third modification are located consecutively.

[0022] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 3 or 4 of at most 7 nucleotides modified with a third modification are located at positions selected from the 2nd, 3rd, 5th, 6th, 7th, and 8th positions from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at least 1 of at most 7 nucleotides modified with a third modification is located at the 14th position from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 2 or 3 of at most 7 nucleotides modified with a third modification are located at positions selected from the 2nd, 3rd, 5th, and 6th positions from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 3 of at most 7 nucleotides modified with a third modification are located at positions selected from the 2nd, 3rd, 5th, and 6th positions from the first nucleotide at the 5' end of the antisense strand.

[0023] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 2 of at most 7 nucleotides modified with a third modification are located at the 8th and 14th positions from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0024] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, one or two of at most seven nucleotides modified with a third modification are located at positions selected from positions 14 and 16 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, two of at most seven nucleotides modified with a third modification are located at positions 14 and 16 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most seven nucleotides are modified with a 2'-F modification.

[0025] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most six nucleotides are modified with a third modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most six nucleotides are modified with a 2'-F modification.

[0026] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most five nucleotides are modified with a third modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most five nucleotides are modified with a 2'-F modification.

[0027] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most four nucleotides are modified with a third modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most four nucleotides are modified with a 2'-F modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most three nucleotides are modified with a third modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at least three nucleotides are modified with a 2'-F modification.

[0028] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M) a (F) b (M) c (F) d (M) e (F) f (M) g (F) h (M) i (F) j (M) k (F) l (M) m (F) n (M) o 5' [wherein M is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and each of a, b, c, d, e, f, g, h, i, j, k, l, m, n, and o is any one of 0 to 16, indicating the number of consecutive nucleotides modified with the modification], and the antisense strand is 1) 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)7(F)2(M)2(F)1(M)15' (SEQ ID NO: 14); 2) 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)7(F)1(M)2(F)2(M)15' (SEQ ID NO: 15); 3) 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)6(F)2(M)3(F)1(M)15' (SEQ ID NO: 16); 4) 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)5(F)1(M)1(F)1(M)3(F)1(M)15' (SEQ ID NO: 17); 5) 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)1(M)2(F)1(M)3(F)1(M)15' (SEQ ID NO: 18); 6) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)8(F)1(M)2(F)1(M)15’(SEQ ID NO: 20); 7) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)0(F)0(M)9(F)1(M)7(F)1(M)3(F)1(M)15’(SEQ ID NO: 21); 8) 3’(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)2(F)2(M)15’(SEQ ID NO: 22); 9) 3’(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)2(M)2(F)1(M)1(F)2(M)15’(SEQ ID NO: 23); 10) 3’(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)1(M)1(F)1(M)1(F)2(M)15’(SEQ ID NO: 24); 11) 3’(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)3(F)1(M)2(F)1(M)1(F)1(M)1(F)2(M)15’(SEQ ID NO: 25); 12) 3’(M)0(F)0(M)4(F)1(M)1(F)1(M)1(F)1(M)3(F)1(M)1(F)1(M)1(F)3(M)1(F)1(M)15’(SEQ ID NO: 27); 13) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)3(F)1(M)15’(SEQ ID NO: 30); 14) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)2(M)2(F)1(M)15’(SEQ ID NO: 50); 15) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)2(F)2(M)15’(SEQ ID NO: 45); 16) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)6(F)2(M)3(F)1(M)15’(SEQ ID NO: 46); 17) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)5(F)1(M)1(F)1(M)3(F)1(M)15’(SEQ ID NO: 47); 18) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)1(M)2(F)1(M)3(F)1(M)15’(SEQ ID NO: 48); 19) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)3(F)1(M)15’(SEQ ID NO: 49); 20) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)8(F)1(M)2(F)1(M)15’(SEQ ID NO: 37); 21) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)0(F)0(M)8(F)1(M)7(F)1(M)3(F)1(M)15’(SEQ ID NO: 38); 22) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)2(F)2(M)15’(SEQ ID NO: 39); 23) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)2(F)1(M)1(F)2(M)15’(SEQ ID NO: 40); 24) 3’(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)1(M)1(F)1(M)1(F)1(M)1(F)2(M)15’(SEQ ID NO: 41); and 25) It is any one of 3’(M)0(F)0(M)6(F)1(M)1(F)1(M)3(F)1(M)2(F)1(M)1(F)1(M)1(F)2(M)15’(SEQ ID NO: 42).

[0029] In some embodiments, the X1 nucleotide of the sense strand of the isolated oligonucleotide of the present disclosure is from 18 to 21, and the X2 nucleotide of the antisense strand of the isolated oligonucleotide of the present disclosure is from 20 to 23. In some embodiments, the X1 nucleotide of the sense strand of the isolated oligonucleotide of the present disclosure is 20 or 21, and the X2 nucleotide of the antisense strand of the isolated oligonucleotide of the present disclosure is 22 or 23. In some embodiments, the X2 nucleotide of the antisense strand of the isolated oligonucleotide of the present disclosure is equal to the X1 nucleotide of the sense strand of the isolated oligonucleotide of the present disclosure plus 2.

[0030] In some embodiments, the sense strand of the isolated oligonucleotide of the present disclosure comprises at least one nucleotide having a modified phosphate backbone. In some embodiments, the antisense strand of the isolated oligonucleotide of the present disclosure comprises at least one nucleotide having a modified phosphate backbone. In some embodiments, in the sense strand or the antisense strand of the isolated oligonucleotide of the present disclosure, or both the sense strand and the antisense strand, the modified phosphate backbone comprises a modified phosphodiester bond. In some embodiments, the modified phosphodiester bond is modified by substituting one or more oxygen atoms in part, and the part is bonded to a phosphorus atom in a phosphodiester bond having a carbon, nitrogen, or sulfur atom in the part, or is modified by forming a 2'-5' linkage. In some embodiments, the modified phosphodiester bond comprises phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate diester, mesylphosphoramidate, or phosphonoacetate.

[0031] In some embodiments, the isolated oligonucleotide of the present disclosure comprises one or more nucleotides containing non-natural bases, locked nucleotides, or abasic nucleotides. In some embodiments, in the isolated oligonucleotide of the present disclosure, the terminal nucleotide at the 5' end comprises a phosphate mimetic. In some embodiments, the 5'-phosphate mimetic is ethylphosphonate, vinylphosphonate, or an analog thereof.

[0032] In some embodiments, the antisense strand of the isolated oligonucleotide of the present disclosure comprises an overhang having at least two single-stranded nucleotides at the 3' end.

[0033] In some embodiments, in the sense strand or the antisense strand, or both, of the isolated oligonucleotide of the present disclosure, the terminal nucleotide or internal nucleotide is linked to one or more targeting ligands. In some embodiments, the terminal nucleotide or internal nucleotide is directly linked to one or more targeting ligands. In some embodiments, the terminal nucleotide or internal nucleotide is indirectly linked to one or more targeting ligands by a linker. In some embodiments, one or more targeting ligands directly or indirectly linked to the terminal nucleotide or internal nucleotide may further comprise a PK modulator. In some embodiments, the PK modulator is a competitive modulator, a positive allosteric modulator, a negative allosteric modulator, or a neutral allosteric modulator. In some embodiments, the targeting ligand is selected from one or more of a carbohydrate, a peptide, a lipid, an antibody or a fragment thereof, an aptamer, albumin, fibrinogen, and folic acid.

[0034] In some embodiments, the antisense strand of the isolated oligonucleotide of the present disclosure is complementary to the mRNA, and sequence-specific hybridization of the mRNA to the antisense strand results in degradation of the mRNA.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification, the singular forms also include the plural forms unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described below. Publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting. In case of conflict between the chemical structure and the name of a compound disclosed herein, the chemical structure shall prevail.

[0036] Other features and advantages of the present disclosure will become apparent from the following detailed description of the invention and the claims.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0061] Provided herein is an isolated oligonucleotide comprising: (a) a sense strand comprising X1 nucleotides, wherein at least one nucleotide is modified with a first modification and each of the remaining nucleotides is independently modified with a second modification, X1 is an integer selected from 13 to 36, the first modification and the second modification are different; and (b) an antisense strand comprising X2 nucleotides, wherein at least one nucleotide is modified with a third modification and each of the remaining nucleotides is independently modified with a fourth modification, X2 is an integer selected from 18 to 31, the third modification and the fourth modification are different.

[0062] In some embodiments, the first modification is a modification of the sugar moiety of at least one nucleotide at the 2'-position selected from a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, a 2'-deoxy modification, and equivalents thereof, and combinations thereof. In some embodiments, the first modification is a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, or a 2'-deoxy modification, or stereoisomers thereof. In some embodiments, the first modification is a 2'-F modification, a 2'-CN modification, or a 2'-N3 modification, or stereoisomers thereof. In some embodiments, the first modification is a 2'-F modification or a stereoisomer thereof.

[0063] In some embodiments, the second modification is a modification of one or more sugar moieties of the remaining nucleotides at the 2'-position selected from a 2'-C1-C6 alkyl, a 2'-OR modification [wherein R is a C1-C6 alkoxy, an acetamide, a phenyl, or a heteroaryl containing a 5- or 6-membered ring and one or two heteroatoms selected from N, O, and S, optionally substituted C1-C6 alkyl], a 2'-amino, and a morpholino substitution, and equivalents thereof, and combinations thereof. In some embodiments, the second modification is a 2'-OR modification, or a morpholino substitution, or a combination thereof. In some embodiments, the second modification is a 2'-OR modification. In some embodiments, the second modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification. In some embodiments, the second modification is a 2'-O-methyl modification. In some embodiments, the second modification is a morpholino substitution.

[0064] In some embodiments, the first modification is a 2'-F modification or a stereoisomer thereof, and the second modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification. In some embodiments, the first modification is a 2'-F modification or a stereoisomer thereof, and the second modification is a 2'-O-methyl modification.

[0065] In some embodiments, the third modification is a modification of the sugar moiety of at least one nucleotide at the 2'-position selected from a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, a 2'-deoxy modification, and equivalents thereof, and combinations thereof. In some embodiments, the third modification is a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, or a 2'-deoxy modification, or stereoisomers thereof. In some embodiments, the third modification is a 2'-F modification, a 2'-CN modification, or a 2'-N3 modification, or stereoisomers thereof. In some embodiments, the third modification is a 2'-F modification or a stereoisomer thereof.

[0066] In some embodiments, the fourth modification is a modification of one or more sugar moieties of the remaining nucleotides at the 2'-position selected from 2'-C1-C6 alkyl, 2'-OR modification [wherein R is C1-C6 alkoxy, acetamide, phenyl, or heteroaryl containing a 5- or 6-membered ring and one or two heteroatoms selected from N, O, and S, optionally substituted C1-C6 alkyl], 2'-amino, and morpholino substitution, and equivalents thereof, and combinations thereof. In some embodiments, the fourth modification is a 2'-OR modification, or a morpholino substitution, or a combination thereof. In some embodiments, the fourth modification is a 2'-OR modification. In some embodiments, the fourth modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification. In some embodiments, the fourth modification is a 2'-O-methyl modification. In some embodiments, the fourth modification is a morpholino substitution.

[0067] In some embodiments, the third modification is a 2'-F modification or a stereoisomer thereof, and the fourth modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification. In some embodiments, the third modification is a 2'-F modification or a stereoisomer thereof, and the fourth modification is a 2'-O-methyl modification.

[0068] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 3 nucleotides are modified with a first modification. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 2 of the at least 3 nucleotides modified with the first modification are located consecutively. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 3 of the at least 3 nucleotides modified with the first modification are located consecutively.

[0069] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 4 nucleotides are modified with a first modification. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 3 of the at least 4 nucleotides modified with the first modification are located consecutively. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 4 of the at least 4 nucleotides modified with the first modification are located consecutively.

[0070] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 5 nucleotides are modified with a first modification. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 3 of the at least 5 nucleotides modified with the first modification are located consecutively, and at least 4 of the at least 5 nucleotides modified with the first modification are located consecutively.

[0071] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 3 nucleotides, at least 4 nucleotides, or at least 5 nucleotides modified with the first modification are located at positions 10 to 15 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0072] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, two of at least three nucleotides modified with the first modification are located at positions selected from the 10th, 11th, 12th, and 13th positions from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, three of at least three nucleotides modified with the first modification are located at positions selected from the 10th, 11th, 12th, and 13th positions from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, one of at least three nucleotides modified with the first modification is located at the 11th position from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand.

[0073] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, three of at least three nucleotides modified with the first modification are located at the 11th, 12th, and 13th positions from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, three of at least three nucleotides modified with the first modification are located at the 12th, 13th, and 14th positions from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, three of at least three nucleotides modified with the first modification are located at the 10th, 11th, and 12th positions from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand.

[0074] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, one of at least 4 nucleotides modified with a first modification is located at the 10th position from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, one of at least 4 nucleotides modified with a first modification is located at the 11th position from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, one of at least 4 nucleotides modified with a first modification is located at the 12th position from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, one of at least 4 nucleotides modified with a first modification is located at the 13th position from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, one of at least 4 nucleotides modified with a first modification is located at the 14th position from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, one of at least 4 nucleotides modified with a first modification is located at the 15th position from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0075] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 4 nucleotides modified with a first modification are located at positions 11, 12, 13, and 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 4 nucleotides modified with a first modification are located at positions 10, 11, 12, and 13 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 4 nucleotides modified with a first modification are located at positions 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0076] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, 2 of at least 4 nucleotides modified with a first modification are located at positions 10 to 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0077] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, not all of at least 3 nucleotides modified with a first modification are located continuously. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 3 nucleotides, at least 4 nucleotides, or at least 5 nucleotides are modified with a 2'-F modification.

[0078] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 3 nucleotides modified with a first modification are located at positions 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand (e.g., SEQ ID NO: 11, SEQ ID NO: 34). In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 4 nucleotides modified with a first modification are located at positions 10, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand (e.g., SEQ ID NO: 12, SEQ ID NO: 35). In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 4 nucleotides modified with a first modification are located at positions 11, 12, 13, and 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand (e.g., SEQ ID NO: 8, SEQ ID NO: 32). In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 4 nucleotides modified with a first modification are located at positions 10, 11, 12, and 13 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand (e.g., SEQ ID NO: 9, SEQ ID NO: 33). In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 5 nucleotides modified with a first modification are located at positions 10, 11, 12, 13, and 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand (e.g., SEQ ID NO: 10, SEQ ID NO: 36). In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least 5 nucleotides modified with a first modification are located at positions 10, 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand (e.g., SEQ ID NO: 13, SEQ ID NO: 44).

[0079] In some embodiments, the sense strand of the isolated oligonucleotide of the present disclosure comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification, and has the formula: 5'(M) g (F) f (M) e (F) d (M) c (F) b (M) a3’ [wherein M is a 2’-O-methyl modified nucleotide, F is a 2’-F modified nucleotide, and each of a, b, c, d, e, f, and g is any one of 0 to 16, indicating the number of consecutive nucleotides modified by the modification], and the sense strand is a) 5’(M)0(F)0(M)0(F)0(M)8(F)3(M) 10 3’ (SEQ ID NO: 52); b) 5’(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3’ (SEQ ID NO: 53); c) 5’(M)0(F)0(M)0(F)0(M)7(F)3(M) 10 3’ (SEQ ID NO: 54); d) 5’(M)0(F)0(M)5(F)1(M)1(F)2(M) 11 3’ (SEQ ID NO: 55); e) 5’(M)0(F)0(M)6(F)1(M)1(F)2(M) 11 3’ (SEQ ID NO: 56); f) 5’(M)6(F)1(M)1(F)2(M)1(F)1(M)9 3’ (SEQ ID NO: 57); g) 5’(M)5(F)1(M)1(F)2(M)1(F)1(M)9 3’ (SEQ ID NO: 58); h) 5’(M)0(F)0(M)0(F)0(M)7(F)4(M) 10 3’ (SEQ ID NO: 59); i) 5’(M)0(F)0(M)6(F)1(M)1(F)4(M)9 3’ (SEQ ID NO: 60); j) 5’(M)0(F)0(M)0(F)0(M)8(F)4(M)9 3’ (SEQ ID NO: 61); k) 5’(M)0(F)0(M)0(F)0(M)6(F)4(M) 10 3’ (SEQ ID NO: 62); l) 5’(M)0(F)0(M)5(F)1(M)1(F)4(M)9 3’ (SEQ ID NO: 63); m) 5’(M)0(F)0(M)0(F)0(M)7(F)4(M)9 3’ (SEQ ID NO: 64); n) 5’(M)0(F)0(M)0(F)0(M)7(F)5(M)9 3’ (SEQ ID NO: 65); and (o) Any one of 5’(M)0(F)0(M)0(F)0(M)6(F)5(M)93’ (SEQ ID NO: 66).

[0080] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2’-F modification and nucleotides modified with 2’-O-methyl modification, and has the formula: 5’(M)0(F)0(M)0(F)0(M)8(F)3(M) 10 3’ (SEQ ID NO: 52) [wherein M is a 2’-O-methyl modified nucleotide and F is a 2’-F modified nucleotide].

[0081] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2’-F modification and nucleotides modified with 2’-O-methyl modification, and has the formula: 5’(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3’ (SEQ ID NO: 53) [wherein M is a 2’-O-methyl modified nucleotide and F is a 2’-F modified nucleotide].

[0082] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2’-F modification and nucleotides modified with 2’-O-methyl modification, and has the formula: 5’(M)0(F)0(M)0(F)0(M)7(F)3(M) 10 3’ (SEQ ID NO: 54) [wherein M is a 2’-O-methyl modified nucleotide and F is a 2’-F modified nucleotide].

[0083] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2’-F modification and nucleotides modified with 2’-O-methyl modification, and has the formula: 5’(M)0(F)0(M)5(F)1(M)1(F)2(M) 11 3’ (SEQ ID NO: 55) [wherein M is a 2’-O-methyl modified nucleotide and F is a 2’-F modified nucleotide].

[0084] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)6(F)1(M)1(F)2(M) 11 3' (SEQ ID NO: 56) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0085] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)6(F)1(M)1(F)2(M)1(F)1(M)93' (SEQ ID NO: 57) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0086] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)5(F)1(M)1(F)2(M)1(F)1(M)93' (SEQ ID NO: 58) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0087] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)0(F)0(M)7(F)4(M) 10 3' (SEQ ID NO: 59) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0088] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)6(F)1(M)1(F)4(M)93' (SEQ ID NO: 60) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0089] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)0(F)0(M)8(F)4(M)93' (SEQ ID NO: 61) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0090] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)0(F)0(M)6(F)4(M) 10 3' (SEQ ID NO: 62) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0091] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)4(M)93' (SEQ ID NO: 63) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0092] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)0(F)0(M)7(F)4(M)93' (SEQ ID NO: 64) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0093] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)0(F)0(M)7(F)5(M)93' (SEQ ID NO: 65) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0094] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)0(F)0(M)6(F)5(M)93' (SEQ ID NO: 66) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0095] In some embodiments, in the antisense strand of the isolated oligonucleotides of the present disclosure, at most 7 nucleotides are modified with a third modification.

[0096] In some embodiments, in the antisense strand of the isolated oligonucleotides of the present disclosure, at most 4 of the at most 7 nucleotides modified with a third modification are located at positions 2 to 8 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotides of the present disclosure, at least 1 of the at most 7 nucleotides modified with a third modification is located at position 2 from the first nucleotide at the 5' end of the antisense strand.

[0097] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 2 out of at most 7 nucleotides modified with a third modification are located consecutively. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 2 out of at most 7 nucleotides modified with a third modification that are consecutively located are located at positions 2 and 3 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 2 out of at most 7 nucleotides modified with a third modification that are consecutively located are located at positions 5 and 6 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 2 out of at most 7 nucleotides modified with a third modification that are consecutively located are located at positions 6 and 7 from the first nucleotide at the 5' end of the antisense strand.

[0098] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 3 out of at most 7 nucleotides modified with a third modification are located consecutively. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 3 out of at most 7 nucleotides modified with a third modification that are consecutively located are located at positions 4, 5, and 6 from the first nucleotide at the 5' end of the antisense strand.

[0099] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 3 or 4 of at most 7 nucleotides modified with the third modification are located at positions selected from the 2nd, 3rd, 5th, 6th, 7th, and 8th positions from the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 3 of at most 7 nucleotides modified with the third modification are located at the 2nd, 5th, and 6th positions from the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 3 of at most 7 nucleotides modified with the third modification are located at the 2nd, 3rd, and 6th positions from the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 3 of at most 7 nucleotides modified with the third modification are located at the 2nd, 6th, and 7th positions from the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 3 of at most 7 nucleotides modified with the third modification are located at the 2nd, 6th, and 8th positions from the first nucleotide at the 5'-end of the antisense strand.

[0100] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at least one of at most 7 nucleotides modified with a third modification is located at the 14th position from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 2 or 3 of at most 7 nucleotides modified with a third modification are located at positions selected from the 2nd, 3rd, 5th, and 6th positions from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 3 of at most 7 nucleotides modified with a third modification are located at positions selected from the 2nd, 3rd, 5th, and 6th positions from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 2 of at most 7 nucleotides modified with a third modification are located at the 2nd and 6th positions from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 2 of at most 7 nucleotides modified with a third modification are located at the 2nd and 5th positions from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 2 of at most 7 nucleotides modified with a third modification are located at the 2nd and 3rd positions from the first nucleotide at the 5' end of the antisense strand.

[0101] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 3 out of at most 7 nucleotides modified with a third modification are located at the 2nd, 3rd, and 6th positions from the 1st nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 3 out of at most 7 nucleotides modified with a third modification are located at the 2nd, 3rd, and 5th positions from the 1st nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 3 out of at most 7 nucleotides modified with a third modification are located at the 2nd, 5th, and 6th positions from the 1st nucleotide at the 5' end of the antisense strand.

[0102] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, 2 out of at most 7 nucleotides modified with a third modification are located at the 8th and 14th positions from the nucleotide complementary to the 1st nucleotide at the 5' end of the antisense strand.

[0103] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, one or two of at most seven nucleotides modified with a third modification are located at positions selected from positions 14 and 16 from the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, two of at most seven nucleotides modified with a third modification are located at positions 14 and 16 from the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most seven nucleotides are modified with a 2'-F modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, one of at most seven nucleotides modified with a third modification is located at position 14 from the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, two of at most seven nucleotides modified with a third modification are located at positions 14 and 16 from the first nucleotide at the 5'-end of the antisense strand.

[0104] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 7 nucleotides modified with a third modification are located at positions selected from the 2nd, 3rd, 5th, 6th, 7th, 8th, 9th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 7 nucleotides modified with a third modification are located at the 2nd, 3rd, 6th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand (e.g., SEQ ID NO: 15, SEQ ID NO: 45). In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 7 nucleotides modified with a third modification are located at the 2nd, 6th, 7th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand (e.g., SEQ ID NO: 16, SEQ ID NO: 46). In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 7 nucleotides modified with a third modification are located at the 2nd, 3rd, 6th, 8th, 9th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand (e.g., SEQ ID NO: 22, SEQ ID NO: 39). In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 7 nucleotides modified with a third modification are located at the 2nd, 3rd, 5th, 8th, 9th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand (e.g., SEQ ID NO: 23, SEQ ID NO: 40). In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 7 nucleotides modified with a third modification are located at the 2nd, 3rd, 5th, 7th, 9th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand (e.g., SEQ ID NO: 24, SEQ ID NO: 41). In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 7 nucleotides modified with a third modification are located at the 2nd, 3rd, 5th, 7th, 10th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand (e.g., SEQ ID NO: 25, SEQ ID NO: 42).

[0105] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 6 nucleotides are modified with a third modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 6 nucleotides are modified with a 2'-F modification.

[0106] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 5 nucleotides are modified with a third modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 5 nucleotides are modified with a 2'-F modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 5 nucleotides modified with a third modification are located at positions selected from the 2nd, 3rd, 5th, 6th, 7th, 8th, 9th, 14th, and 16th positions from the first nucleotide at the 5'-end of the antisense strand.

[0107] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 5 nucleotides are modified with a third modification at positions 2, 3, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 5 nucleotides are modified with a third modification at positions 2, 6, 7, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 6 nucleotides are modified with a third modification at positions 2, 3, 6, 8, 9, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 6 nucleotides are modified with a third modification at positions 2, 3, 5, 8, 9, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 7 nucleotides are modified with a third modification at positions 2, 3, 5, 7, 9, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 7 nucleotides are modified with a third modification at positions 2, 3, 5, 7, 10, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0108] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 5 nucleotides modified with a third modification are located at the 2nd, 5th, 6th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 5 nucleotides modified with a third modification are located at the 2nd, 3rd, 6th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 5 nucleotides modified with a third modification are located at the 2nd, 6th, 8th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 5 nucleotides modified with a third modification are located at the 2nd, 6th, 9th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand.

[0109] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 4 nucleotides are modified with a third modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 4 nucleotides are modified with a 2'-F modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 4 nucleotides modified with a third modification are located at positions selected from the 2nd, 3rd, 5th, 6th, 7th, 8th, 9th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 4 nucleotides modified with a third modification are located at the 2nd, 6th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 4 nucleotides modified with a third modification are located at the 2nd, 5th, 14th, and 16th positions from the first nucleotide at the 5' end of the antisense strand.

[0110] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 3 nucleotides are modified with a third modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at least 3 nucleotides are modified with a 2'-F modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 3 nucleotides modified with a third modification are located at positions selected from the 2nd, 3rd, 5th, 6th, 7th, 8th, 9th, 14th, and 16th positions from the first nucleotide at the 5'-end of the antisense strand. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most 3 nucleotides modified with a third modification are located at the 2nd, 6th, and 14th positions from the first nucleotide at the 5'-end of the antisense strand.

[0111] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification, and has the formula: 3'(M) a (F) b (M) c (F) d (M) e (F) f (M) g (F) h (M) i (F) j (M) k (F) l (M) m (F) n (M) o 5' [wherein M is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and each of a, b, c, d, e, f, g, h, i, j, k, l, m, n, and o is any one of 0 to 16, indicating the number of consecutive nucleotides modified with a modification], and the antisense strand is 1) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)7(F)2(M)2(F)1(M)15’(SEQ ID NO: 14); 2) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)7(F)1(M)2(F)2(M)15’(SEQ ID NO: 15); 3) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)6(F)2(M)3(F)1(M)15’(SEQ ID NO: 16); 4) 3’(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)5(F)1(M)1(F)1(M)3(F)1(M)15’(SEQ ID NO: 17); 5) 3’(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)1(M)2(F)1(M)3(F)1(M)15’(SEQ ID NO: 18); 6) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)8(F)1(M)2(F)1(M)15’(SEQ ID NO: 20); 7) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)0(F)0(M)9(F)1(M)7(F)1(M)3(F)1(M)15’(SEQ ID NO: 21); 8) 3’(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)2(F)2(M)15’(SEQ ID NO: 22); 9) 3’(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)2(M)2(F)1(M)1(F)2(M)15’(SEQ ID NO: 23); 10) 3’(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)1(M)1(F)1(M)1(F)2(M)15’(SEQ ID NO: 24); 11) 3’(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)3(F)1(M)2(F)1(M)1(F)1(M)1(F)2(M)15’(SEQ ID NO: 25); 12) 3’(M)0(F)0(M)4(F)1(M)1(F)1(M)1(F)1(M)3(F)1(M)1(F)1(M)1(F)3(M)1(F)1(M)15’(SEQ ID NO: 27); 13) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)3(F)1(M)15’(SEQ ID NO: 30); 14) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)2(M)2(F)1(M)15’(SEQ ID NO: 50); 15) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)2(F)2(M)15’(SEQ ID NO: 45); 16) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)6(F)2(M)3(F)1(M)15’(SEQ ID NO: 46); 17) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)5(F)1(M)1(F)1(M)3(F)1(M)15’(SEQ ID NO: 47); 18) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)1(M)2(F)1(M)3(F)1(M)15’(SEQ ID NO: 48); 19) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)3(F)1(M)15’(SEQ ID NO: 49); 20) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)8(F)1(M)2(F)1(M)15’(SEQ ID NO: 37); 21) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)0(F)0(M)8(F)1(M)7(F)1(M)3(F)1(M)15’(SEQ ID NO: 38); 22) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)2(F)2(M)15’(SEQ ID NO: 39); 23) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)2(F)1(M)1(F)2(M)15’ (SEQ ID NO: 40); 24) 3’(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)1(M)1(F)1(M)1(F)1(M)1(F)2(M)15’ (SEQ ID NO: 41); and 25) Any one of 3’(M)0(F)0(M)6(F)1(M)1(F)1(M)3(F)1(M)2(F)1(M)1(F)1(M)1(F)2(M)15’ (SEQ ID NO: 42).

[0112] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2’-F modification and nucleotides modified with 2’-O-methyl modification, and is of the formula: 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)7(F)2(M)2(F)1(M)15’ (SEQ ID NO: 14) [wherein M is a 2’-O-methyl modified nucleotide and F is a 2’-F modified nucleotide].

[0113] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2’-F modification and nucleotides modified with 2’-O-methyl modification, and is of the formula: 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)7(F)1(M)2(F)2(M)15’ (SEQ ID NO: 15) [wherein M is a 2’-O-methyl modified nucleotide and F is a 2’-F modified nucleotide].

[0114] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)6(F)2(M)3(F)1(M)15'(SEQ ID NO: 16) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0115] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)5(F)1(M)1(F)1(M)3(F)1(M)15'(SEQ ID NO: 17) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0116] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)1(M)2(F)1(M)3(F)1(M)15'(SEQ ID NO: 18) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0117] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)8(F)1(M)2(F)1(M)15'(SEQ ID NO: 20) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0118] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)0(F)0(M)9(F)1(M)7(F)1(M)3(F)1(M)15' (SEQ ID NO: 21) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0119] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)2(F)2(M)15' (SEQ ID NO: 22) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0120] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)2(M)2(F)1(M)1(F)2(M)15' (SEQ ID NO: 23) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0121] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)1(M)1(F)1(M)1(F)2(M)15' (SEQ ID NO: 24) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0122] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)3(F)1(M)2(F)1(M)1(F)1(M)1(F)2(M)15' (SEQ ID NO: 25) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0123] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)4(F)1(M)1(F)1(M)1(F)1(M)3(F)1(M)1(F)1(M)1(F)3(M)1(F)1(M)15' (SEQ ID NO: 27) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0124] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)3(F)1(M)15' (SEQ ID NO: 30) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0125] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)2(M)2(F)1(M)15' (SEQ ID NO: 50) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0126] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)2(F)2(M)15' (SEQ ID NO: 45) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0127] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)6(F)2(M)3(F)1(M)15' (SEQ ID NO: 46) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0128] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)5(F)1(M)1(F)1(M)3(F)1(M)15' (SEQ ID NO: 47) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0129] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)1(M)2(F)1(M)3(F)1(M)15' (SEQ ID NO: 48) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0130] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)3(F)1(M)15' (SEQ ID NO: 49) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0131] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)8(F)1(M)2(F)1(M)15' (SEQ ID NO: 37) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0132] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)0(F)0(M)8(F)1(M)7(F)1(M)3(F)1(M)15' (SEQ ID NO: 38) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0133] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)2(F)2(M)15' (SEQ ID NO: 39) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0134] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)2(F)1(M)1(F)2(M)15' (SEQ ID NO: 40) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0135] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)1(M)1(F)1(M)1(F)1(M)1(F)2(M)15' (SEQ ID NO: 41) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0136] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)6(F)1(M)1(F)1(M)3(F)1(M)2(F)1(M)1(F)1(M)1(F)2(M)15' (SEQ ID NO: 42) [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0137] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a first modification located at positions 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5'-end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand.

[0138] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a first modification located at positions 11, 12, and 13 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0139] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a first modification located at positions 11, 12, 13, and 14 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0140] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a first modification located at positions 10, 11, 12, and 13 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0141] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a first modification located at positions 10, 11, 12, 13, and 14 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0142] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0143] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 10, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0144] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 10, 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0145] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 5, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0146] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 5, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0147] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 3, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0148] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 6, 7, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0149] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 6, 8, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0150] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 6, 9, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0151] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 6, 8, 9, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0152] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 5, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0153] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 6, and 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0154] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 10, 11, 12, 13 and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 3, 6, 8, 9, 14 and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0155] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 10, 11, 12 and 13 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 3, 6, 8, 9, 14 and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0156] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 10, 11, 12, 13 and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 3, 5, 8, 9, 14 and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0157] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 10, 11, 12, 13 and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 3, 5, 7, 9, 14 and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0158] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with modifications located at positions 10, 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with a first modification located at positions 2, 3, 5, 7, 10, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0159] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification, and is of the formula: 5'(M)0(F)0(M)6(F)1(M)1(F)4(M)93'; the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification, and is of the formula: 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)2(F)2(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0160] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification, and is of the formula: 5'(M)0(F)0(M)0(F)0(M)8(F)4(M)93'; the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification, and is of the formula: 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)2(F)2(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0161] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 5'(M)0(F)0(M)6(F)1(M)1(F)4(M)93'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)2(M)2(F)1(M)1(F)2(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0162] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 5'(M)0(F)0(M)6(F)1(M)1(F)4(M)93'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)1(M)1(F)1(M)1(F)2(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0163] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 5'(M)0(F)0(M)6(F)1(M)1(F)4(M)93'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)1(M)1(F)1(M)1(F)2(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0164] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, of the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 by 3'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, of the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)3(F)1(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0165] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, of the formula: 5'(M)0(F)0(M)0(F)0(M)7(F)3(M) 10 by 3'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, of the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)3(F)1(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0166] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, of the formula: 5'(M)0(F)0(M)0(F)0(M)6(F)4(M) 10It is due to 3'; the antisense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)3(F)1(M)15' [where M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0167] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 5'(M)0(F)0(M)0(F)0(M)7(F)4(M)93'; the antisense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)3(F)1(M)15' [where M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0168] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 5'(M)0(F)0(M)0(F)0(M)6(F)5(M)93'; the antisense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)3(F)1(M)15' [where M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0169] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)2(M) 11 at the 3'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)3(F)1(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0170] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)5(F)1(M)1(F)2(M)1(F)1(M)93' at the 3'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)3(F)1(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0171] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)4(M)93' at the 3'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)3(F)1(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0172] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 is by 3'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)3(F)1(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0173] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 is by 3'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)2(M)2(F)1(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0174] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10It is due to 3'; the antisense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)2(F)2(M)15' [where M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0175] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 It is due to 3'; the antisense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)6(F)2(M)3(F)1(M)15' [where M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0176] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 It is due to 3'; the antisense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 3'(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)5(F)1(M)1(F)1(M)3(F)1(M)15' [where M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0177] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, having the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 by 3'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, having the formula: 3'(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)1(M)2(F)1(M)3(F)1(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0178] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, having the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 by 3'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, having the formula: 3'(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)3(F)1(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0179] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, having the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10It is due to 3'; the antisense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)8(F)1(M)2(F)1(M)15' [where M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0180] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 It is due to 3'; the antisense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)0(F)0(M)8(F)1(M)7(F)1(M)3(F)1(M)15' [where M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0181] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)4(M)93' is due to; the antisense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is of the formula: 3'(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)2(F)2(M)15' [where M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0182] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 5'(M)0(F)0(M)0(F)0(M)7(F)4(M)93'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 3'(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)2(F)2(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0183] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)4(M)93'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 3'(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)2(F)1(M)1(F)2(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0184] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)4(M)93'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 3'(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)1(M)1(F)1(M)1(F)1(M)1(F)2(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0185] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)4(M)93'; the antisense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and is according to the formula: 3'(M)0(F)0(M)6(F)1(M)1(F)1(M)3(F)1(M)2(F)1(M)1(F)1(M)1(F)2(M)15' [wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide].

[0186] In some embodiments, the X1 nucleotide of the sense strand of the isolated oligonucleotide of the present disclosure is from 18 to 21, and the X2 nucleotide of the antisense strand of the isolated oligonucleotide of the present disclosure is from 20 to 23. In some embodiments, the X1 nucleotide of the sense strand of the isolated oligonucleotide of the present disclosure is 20 or 21, and the X2 nucleotide of the antisense strand of the isolated oligonucleotide of the present disclosure is 22 or 23. In some embodiments, the X2 nucleotide of the antisense strand of the isolated oligonucleotide of the present disclosure is equal to the X1 nucleotide of the sense strand of the isolated oligonucleotide of the present disclosure plus 2. In some embodiments, the X1 nucleotide of the sense strand of the isolated oligonucleotide of the present disclosure is 21, and the X2 nucleotide of the antisense strand of the isolated oligonucleotide of the present disclosure is 23. In some embodiments, the X1 nucleotide of the sense strand of the isolated oligonucleotide of the present disclosure is 20, and the X2 nucleotide of the antisense strand of the isolated oligonucleotide of the present disclosure is 22.

[0187] In some embodiments, the sense strand of the isolated oligonucleotide of the present disclosure comprises at least one nucleotide having a modified phosphate backbone. In some embodiments, the antisense strand of the isolated oligonucleotide of the present disclosure comprises at least one nucleotide having a modified phosphate backbone. In some embodiments, in the sense strand or the antisense strand, or both the sense strand and the antisense strand of the isolated oligonucleotide of the present disclosure, the modified phosphate backbone comprises a modified phosphodiester bond. In some embodiments, the modified phosphodiester bond is modified by substituting one or more oxygen atoms in part, and the part is bonded to the phosphorus atom in the phosphodiester bond having a carbon, nitrogen, or sulfur atom in the part, or is modified by forming a 2'-5' linkage. In some embodiments, the modified phosphodiester bond comprises phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate diester, mesylphosphoramidate, or phosphonoacetate.

[0188] In some embodiments, the isolated oligonucleotide of the present disclosure comprises one or more nucleotides containing non-natural bases, locked nucleotides, or abasic nucleotides. In some embodiments, in the isolated oligonucleotide of the present disclosure, the terminal nucleotide at the 5' end comprises a phosphate mimic. In some embodiments, the 5'-phosphate mimic is ethylphosphonate, vinylphosphonate, or an analog thereof.

[0189] In some embodiments, the antisense strand of the isolated oligonucleotide of the present disclosure comprises at least two single-stranded nucleotides at the 3' end. In some embodiments, the antisense strand of the isolated oligonucleotide of the present disclosure comprises two single-stranded nucleotides at the 3' end.

[0190] Targeting ligand In some embodiments, in the sense strand or the antisense strand, or both, of the isolated oligonucleotide of the present disclosure, the terminal nucleotide or internal nucleotide is linked to a targeting ligand. In some embodiments, the targeting ligand is attached to one or more nucleotides at the 5' end of the sense strand of the isolated oligonucleotide of the present disclosure. In some embodiments, the targeting ligand is attached to one or more nucleotides at the 3' end of the sense strand of the isolated oligonucleotide of the present disclosure. In some embodiments, the targeting ligand is attached to one or more nucleotides at the 5' end of the antisense strand of the isolated oligonucleotide of the present disclosure. In some embodiments, the targeting ligand is attached to one or more nucleotides at the 3' end of the antisense strand of the isolated oligonucleotide of the present disclosure. In some embodiments, the targeting ligand is attached to one or more nucleotides of at least two single-stranded nucleotides at the 3' end of the antisense strand of the isolated oligonucleotide of the present disclosure.

[0191] In some embodiments, the targeting ligand is selected from one or more of a carbohydrate, a peptide, a lipid, an antibody or fragment thereof, an aptamer, albumin, fibrinogen, and folic acid. In some embodiments, the targeting ligand binds to a surface protein on a cell that expresses the target mRNA of the isolated oligonucleotide of the present disclosure. In some embodiments, the targeting ligand mediates entry of the isolated oligonucleotide of the present disclosure into a cell that expresses the target mRNA of the isolated oligonucleotide of the present disclosure.

[0192] In some embodiments, the targeting ligand is a therapeutic ligand. In some embodiments, the targeting ligand is a therapeutic antibody.

[0193] In some embodiments, the targeting ligand is attached to the isolated oligonucleotide of the present disclosure by a linker. In some embodiments, the linker is any one of a protein, DNA, RNA, or chemical compound. In some embodiments, the isolated oligonucleotide, linker, and targeting ligand of the present disclosure form a scaffold. As used herein, the term "scaffold" refers to a compound or complex that includes the linker of the present disclosure, and the linker is covalently attached to either the ligand or the isolated oligonucleotide, or both.

[0194] In some embodiments, the isolated oligonucleotide, linker, and targeting ligand of the present disclosure form a conjugate. As used herein, the term "conjugate" refers to a compound or complex that includes an isolated oligonucleotide covalently attached to a ligand via the linker of the present disclosure.

[0195] As used herein, the term "targeting ligand" or "ligand" refers to a moiety that, when covalently attached to a nucleic acid agent (e.g., an oligonucleotide), can mediate its entry into a target site (e.g., a target cell or tissue), or facilitate or enable its delivery to the target site. In some embodiments, the targeting ligand includes a sugar ligand moiety (e.g., N-acetylgalactosamine (GalNAc)) that can direct the uptake of the oligonucleotide into the liver.

[0196] In some embodiments, the targeting ligand binds to the asialoglycoprotein receptor (ASGPR). In some embodiments, the targeting ligand binds to the liver (e.g., via ASGPR), such as to hepatocytes of the liver.

[0197] Suitable targeting ligands include, but are not limited to, the ligands disclosed in Winkler (Ther. Deliv., 2013, 4(7):791-809), International Publication Nos. 2016 / 100401, 2012 / 089352, and 2009 / 082607, and U.S. Patent Application Publication Nos. 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, and 2009 / 0247608 (each of which is incorporated by reference).

[0198] In some embodiments, the targeting ligand comprises a carbohydrate moiety.

[0199] As used herein, "carbohydrate moiety" refers to a moiety comprising one or more monosaccharide units, each having at least 6 carbon atoms (which can be linear, branched, or cyclic) and having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. In some embodiments, the carbohydrate moiety comprises a monosaccharide, disaccharide, trisaccharide, or tetrasaccharide. In some embodiments, the carbohydrate moiety comprises an oligosaccharide containing about 4 to 9 monosaccharide units. In some embodiments, the carbohydrate moiety comprises a polysaccharide (e.g., starch, glycogen, cellulose, or a polysaccharide gum).

[0200] In some embodiments, the carbohydrate moiety comprises a monosaccharide, disaccharide, trisaccharide, or tetrasaccharide. In some embodiments, the carbohydrate moiety comprises an oligosaccharide (e.g., containing about 4 to about 9 monosaccharide units). In some embodiments, the carbohydrate moiety comprises a polysaccharide (e.g., starch, glycogen, cellulose, or a polysaccharide gum).

[0201] In some embodiments, the ligand is capable of binding to the human asialoglycoprotein receptor (ASGPR), e.g., the human asialoglycoprotein receptor 2 (ASGPR2).

[0202] In some embodiments, the carbohydrate moiety comprises a sugar (e.g., 1, 2, or 3 sugars). In some embodiments, the carbohydrate moiety comprises galactose or a derivative thereof (e.g., 1, 2, or 3 galactoses or derivatives thereof). In some embodiments, the carbohydrate moiety comprises N-acetylgalactosamine or a derivative thereof (e.g., 1, 2, or 3 N-acetylgalactosamines or derivatives thereof). In some embodiments, the carbohydrate moiety comprises N-acetyl-D-galactosylamine or a derivative thereof (e.g., 1, 2, or 3 N-acetyl-D-galactosylamines or derivatives thereof).

[0203] In some embodiments, the carbohydrate moiety comprises N-acetylgalactosamine (e.g., 1, 2, or 3 N-acetylgalactosamines). In some embodiments, the carbohydrate moiety comprises N-acetyl-D-galactosylamine (e.g., 1, 2, or 3 N-acetyl-D-galactosylamines).

[0204] In some embodiments, the carbohydrate moiety comprises mannose or a derivative thereof (e.g., mannose-6-phosphate). In some embodiments, the carbohydrate moiety further comprises a linking moiety that connects one or more sugars (e.g., N-acetyl-D-galactosylamine) to the linker.

[0205] In some embodiments, the linker comprises a thioether (e.g., thiosuccinimide, or a hydrolytic analog thereof), disulfide, triazole, phosphorothioate, phosphodiester, ester, amide, or any combination thereof. In some embodiments, the linker is a trivalent linking moiety. Suitable targeting ligands include, but are not limited to, the ligands disclosed in International Publication Nos. WO 2015 / 006740, WO 2016 / 100401, WO 2017 / 214112, WO 2018 / 039364, and WO 2018 / 045317, each of which is incorporated herein by reference in its entirety.

[0206] In some embodiments, the targeting ligand comprises a lipid or lipid moiety (e.g., 1, 2, or 3 lipid moieties). In some embodiments, the lipid moiety comprises (e.g., 1, 2, or 3) C8-C24 fatty acids, cholesterol, vitamins, sterols, phospholipids, or any combination thereof.

[0207] In some embodiments, the targeting ligand comprises a peptide or peptide moiety (e.g., 1, 2, or 3 peptide moieties). In some embodiments, the peptide moiety comprises (e.g., 1, 2, or 3) integrins, insulin, glucagon-like peptide, or any combination thereof. In some embodiments, the targeting ligand comprises an antibody or antibody moiety (e.g., transferrin). In some embodiments, the targeting ligand comprises 1, 2, or 3 antibody moieties (e.g., transferrin).

[0208] In some embodiments, the targeting ligand comprises an oligonucleotide (e.g., an aptamer or CpG). In some embodiments, the targeting ligand comprises 1, 2, or 3 oligonucleotides (e.g., an aptamer or CpG).

[0209] In some embodiments, the ligand comprises 1, 2, or 3 sugars (e.g., N-acetyl-D-galactosylamine); 1, 2, or 3 lipid moieties, 1, 2, or 3 peptide moieties, 1, 2, or 3 antibody moieties, 1, 2, or 3 oligonucleotides, or any combination thereof.

[0210] In some embodiments, the linker is attached to the isolated oligonucleotide of the present disclosure via a phosphate group or an analog of a phosphate group in an isolated oligonucleotide.

[0211] Immunostimulatory oligonucleotide In some embodiments, the isolated oligonucleotides of the present disclosure are linked to a lipid, carbohydrate, or peptide that can be immunostimulatory, which includes immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded), and when administered to a subject that is a mammal or other patient, can induce an immune response. ISS includes, for example, specific palindromes that result in a hairpin secondary structure (see Yamamoto S., et al. (1992) J. Immunol. 148:4072-4076, which is incorporated by reference in its entirety), or CpG motifs, as well as other known ISS features (see, for example, multi-G domains, WO 96 / 11266, which is incorporated by reference in its entirety).

[0212] The immune response can be a innate immune response or an adaptive immune response. The immune system is divided into the more innate immune system and the acquired adaptive immune system of vertebrates, and the acquired adaptive immune system is further divided into humoral and cellular components. In some embodiments, the immune response can be mucosal.

[0213] In some embodiments, the immunostimulatory isolated oligonucleotide is immunostimulatory only when administered in combination with lipid particles and is not immunostimulatory when administered in its "free form". Such oligonucleotides are considered to be immunostimulatory.

[0214] An immunostimulatory isolated oligonucleotide is considered non-sequence specific if it is not required to specifically bind to a target polynucleotide to reduce its expression in order to elicit an immune response. Thus, certain immunostimulatory isolated oligonucleotides can contain sequences corresponding to regions of naturally occurring genes or mRNAs, but they can still be considered non-sequence specific immunostimulatory nucleic acids.

[0215] In some embodiments, the immunostimulatory isolated oligonucleotide or oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may or may not be methylated. In another embodiment, the immunostimulatory isolated oligonucleotide comprises at least one CpG dinucleotide having a methylated cytosine. In some embodiments, the nucleic acid comprises a single CpG dinucleotide, and the cytosine in the CpG dinucleotide is methylated. In an alternative embodiment, the isolated oligonucleotide comprises at least two CpG dinucleotides, and at least one cytosine in the CpG dinucleotide is methylated. In a further embodiment, each cytosine in the CpG dinucleotides present in the sequence is methylated. In another embodiment, the isolated oligonucleotide comprises a plurality of CpG dinucleotides, and at least one of the CpG dinucleotides comprises a methylated cytosine.

[0216] The bond between the linker unit, the isolated oligonucleotide, and the ligand

[0217] In some embodiments, the bond between the linker unit and the nucleic acid agent is a bond.

[0218] In some embodiments, the bond between the linker unit and the nucleic acid agent is a moiety (e.g., a moiety containing a cleavable group).

[0219] In some embodiments, the bond between the linker unit and the ligand is a bond.

[0220] In some embodiments, the bond between the linker unit and the ligand is a moiety (e.g., a moiety containing a cleavable group).

[0221] In some embodiments, the bond between the linker unit and the ligand comprises -C(=O)- connected to the linker unit.

[0222] This group may be cleavable or non-cleavable. Suitable groups include, for example, -NR-, -C(=O)-, -C(=O)NH-, -S(=O)-, -S(=O)2-, -S(=O)2NH, or, without limitation, alkylene, alkenylene, alkynylene, arylalkylene, arylalkenylene, arylalkynylene, heteroarylalkylene, heteroarylalkenylene, heteroarylalkynylene, heterocyclylalkylene, heterocyclylalkenylene, heterocyclylalkynylene, arylene, heteroarylene, heterocyclylene, cycloalkylene, cycloalkenylene, alkylarylalkylene, alkylarylalkenylene, alkylarylalkynylene, alkenylarylalkylene, alkenylarylalkenylene, alkenylarylalkynylene, alkynylarylalkylene, alkynylarylalkenylene, alkynylarylalkynylene, alkylheteroarylalkylene, alkylheteroarylalkenylene, alkylheteroarylalkynylene, alkenylheteroarylalkylene, alkenylheteroarylalkenylene, alkenylheteroarylalkynylene, alkynylheteroarylalkylene, alkynylheteroarylalkenylene, alkynylheteroarylalkynylene, alkylheterocyclylalkylene, alkylheterocyclylalkenylene, alkylheterocyclylalkynylene, alkenylheterocyclylalkylene, alkenylheterocyclylalkenylene, alkenylheterocyclylalkynylene, alkynylheterocyclylalkylene, alkynylheterocyclylalkenylene, alkynylheterocyclylalkynylene, alkylarylene, alkenylarylene, alkynylarylene, alkylheteroarylene, alkenylheteroarylene, alkynylheteroarylene, etc., and each of these may be substituted or unsubstituted, and one or more methylenes may be interrupted or terminated by -O-, -S-, -S(=O)-, -S(=O)2-, -NR-, -C(=O)-, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycle, and R is hydrogen, acyl, aliphatic or substituted aliphatic.

[0223] A cleavable group is a group that is sufficiently stable extracellularly but is cleaved upon entering the target cell, releasing two moieties that the group holds together. In a preferred embodiment, the cleavable group is cleaved at least 10-fold, preferably at least 100-fold faster in the target cell, or under a first reference condition (e.g., that can be selected to mimic or represent intracellular conditions) or a second reference condition (e.g., that can be selected to mimic or represent conditions found in blood or serum) than in the subject's blood.

[0224] A cleavable group is sensitive to a cleaving agent (e.g., pH, redox potential, or the presence of a degradable molecule). Generally, the cleaving agent is more prevalent or found at a higher level or activity intracellularly than in serum or blood. Examples of such degrading agents include: redox agents selected for or having no substrate specificity for a particular substrate (e.g., oxidases or reductases present intracellularly or reducing agents such as mercaptans that can degrade a redox-cleavable group by reduction), esterases; agents that can create an endosomal or acidic environment, e.g., an agent that brings about a pH of 5 or less, general acids, peptidases (which can be substrate-specific), and enzymes that can hydrolyze or degrade an acid-cleavable group by acting as phosphatases.

[0225] A cleavable group, e.g., a disulfide bond, can be sensitive to pH. The pH of human serum is 7.4, but the average intracellular pH is slightly lower, in the range of about 7.1 - 7.3. Endosomes have a more acidic pH in the range of 5.5 - 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have cleavable groups that are cleaved at a preferred pH, thereby releasing a cationic lipid from an intracellular ligand or within a desired compartment of the cell.

[0226] The conjugate can include a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the conjugate can vary depending on the cell to be targeted. For example, a liver-targeting ligand can be conjugated to a cationic lipid via a chemical moiety containing an ester group. Hepatocytes are rich in esterases, and thus this group is cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include cells of the lung, renal cortex, and testis.

[0227] Coupling groups containing peptide bonds can be used when targeting cell types rich in peptidases, such as hepatocytes and synoviocytes.

[0228] Generally, the suitability of a candidate cleavable group can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the candidate group. It is also desirable to test the candidate cleavable group for its ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative sensitivity to cleavage between a first condition and a second condition can be determined, where the first condition is selected to show cleavage in the target cell and the second condition is selected to show cleavage in other tissues or biological fluids, such as blood or serum. The evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to perform an initial evaluation under cell-free or culture conditions and confirm it by further evaluation in whole animals. In a preferred embodiment, a useful candidate compound is cleaved at least 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0229] Redox-cleavable group. One class of cleavable groups are redox-cleavable groups that are cleaved upon reduction or oxidation. An example of a reductively cleavable group is a disulfide linking group (-S-S-). The methods described herein can be considered to determine whether a candidate cleavable group is a suitable "reductively cleavable linking group" or, for example, suitable for use with a particular iRNA moiety and a particular targeting agent. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that reproduce the cleavage rate observed in cells, e.g., target cells. A candidate can also be evaluated under conditions selected to reproduce blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved by no more than 10% in blood. In a preferred embodiment, a useful candidate compound is degraded at least 2, 4, 10, or 100 times faster in cells (or in vitro conditions selected to reproduce intracellular conditions) compared to blood (or in vitro conditions selected to reproduce extracellular conditions). The cleavage rate of a candidate compound can be determined using standard enzyme kinetics assays under conditions selected to reproduce the intracellular medium and compared to conditions selected to reproduce the extracellular medium.

[0230] Phosphate-based cleavable groups. Phosphate-based cleavable groups are cleaved by an agent that decomposes or hydrolyzes the phosphate group. Examples of agents that cleave phosphate groups (phosphoric acid groups) within cells are enzymes such as phosphatases within cells. In some embodiments, the phosphate-based linking group is -O-P(=O)(ORk)-O-, -O-P(=S)(ORk)-O-, -O-P(=S)(SRk)-O-, -S-P(=O)(ORk)-O-, -O-P(=O)(ORk)-S-, -S-P(=O)(ORk)-S-, -O-P(=S)(ORk)-S-, -S-P(=S)(ORk)-O-, -O-P(=O)(Rk)-O-, -O-P(=S)(Rk)-O-, -S-P(=O)(Rk)-O-, -S-P(=S)(Rk)-O-, -S-P(=O)(Rk)-S-, or -O-P(=S)(Rk)-S-. In some embodiments, the phosphate-based linking group is -O-P(=O)(OH)-O-, -O-P(=S)(OH)-O-, -O-P(=S)(SH)-O-, -S-P(=O)(OH)-O-, -O-P(=O)(OH)-S-, -S-P(=O)(OH)-S-, -O-P(=S)(OH)-S-, -S-P(=S)(OH)-O-, -O-P(=O)(H)-O-, -O-P(=S)(H)-O-, -S-P(=O)(H)-O-, -S-P(=S)(H)-O-, -S-P(=O)(H)-S-, or -O-P(=S)(H)-S-. In some embodiments, the phosphate-based linking group is -O-P(=O)(OH)-O-.

[0231] Acid-cleavable group. An acid-cleavable group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by an agent such as an enzyme that can act as a general acid. In cells, certain low pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable groups include, but are not limited to, hydrazones, esters, and esters of amino acids. The acid-cleavable group can have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using a method similar to the method described above.

[0232] Ester-based cleavable group. An ester-based cleavable group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. The ester-cleavable linking group has the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using a method similar to the method described above.

[0233] Peptide-based cleavable groups. Peptide-based cleavable groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not contain an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a specific type of amide bond formed between amino acids to yield peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that yield peptides and proteins and do not include the entire amide functional group. The peptide-based cleavable linking group has the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using a method similar to the above method. As used herein, "carbohydrate" refers to a compound that is essentially composed of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched, or cyclic) and having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom, or a compound having as part of it a carbohydrate moiety composed of one or more monosaccharide units each having at least 6 carbon atoms (which can be linear, branched, or cyclic) and having an oxygen atom, nitrogen atom, or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4 to 9 monosaccharide units), as well as polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars having C5 or more (preferably C5 - C8), and disaccharides and trisaccharides include sugars having 2 or 3 monosaccharide units (preferably C5 - C8).

[0234] In some embodiments, the antisense strand of the isolated oligonucleotide of the present disclosure is complementary to the mRNA, and sequence-specific hybridization of the mRNA to the antisense strand results in degradation of the mRNA.

[0235] Method for preparing the isolated oligonucleotides of the present disclosure In some embodiments, the present disclosure provides a pharmaceutical composition comprising the isolated oligonucleotides described herein, or a compound, scaffold, or conjugate comprising the isolated oligonucleotides.

[0236] In some embodiments, the present disclosure provides a method for regulating the expression of a target gene in a subject, the method comprising administering to the subject the isolated oligonucleotides described herein, or a compound, scaffold, or conjugate comprising the isolated oligonucleotides.

[0237] In some embodiments, the present disclosure provides a method for delivering a nucleic acid agent to a subject, the method comprising administering to the subject the isolated oligonucleotides described herein, or a compound, scaffold, or conjugate comprising the isolated oligonucleotides.

[0238] In some embodiments, the present disclosure provides a method for performing it in a subject in need of treatment or prevention of a disease, the method comprising administering to the subject a therapeutically effective amount of the isolated oligonucleotides described herein, or a compound, scaffold, or conjugate comprising the isolated oligonucleotides.

[0239] In some embodiments, the present disclosure provides the use of the isolated oligonucleotides described herein, or a compound, scaffold, or conjugate comprising the isolated oligonucleotides, in the manufacture of a medicament for regulating the expression of a target gene in a subject.

[0240] In some embodiments, the present disclosure provides the use of the isolated oligonucleotides described herein, or a compound, scaffold, or conjugate comprising the isolated oligonucleotides, in the manufacture of a medicament for delivering a nucleic acid agent to a subject.

[0241] In some aspects, the present disclosure provides the use of an isolated oligonucleotide described herein, or a compound, scaffold, or conjugate comprising an isolated oligonucleotide, in the manufacture of a medicament therefor in a subject in need of treating or preventing a disease.

[0242] In some embodiments, the isolated oligonucleotide of the present disclosure comprises one or more phosphate groups or one or more analogs of a phosphate group.

[0243] In some embodiments, the isolated oligonucleotide is siRNA (e.g., single-stranded siRNA (e.g., hairpin single-stranded siRNA) or double-stranded siRNA), microRNA, anti-microRNA, microRNA mimic, anti-miR, antagomir, dsRNA, aptamer, immunostimulatory oligonucleotide, decoy oligonucleotide, splice-modifying oligonucleotide, triple-stranded-forming oligonucleotide, G-quadruplex, or antisense oligonucleotide.

[0244] In some embodiments, the isolated oligonucleotide is double-stranded RNA (dsRNA).

[0245] The sense strand of the isolated oligonucleotide of the present disclosure is also known as the passenger strand, and it is understood that the terms "sense strand" and "passenger strand" are used interchangeably herein.

[0246] The antisense strand of the isolated oligonucleotide of the present disclosure is also known as the guide strand, and it is understood that the terms "antisense strand" and "guide strand" are used interchangeably herein.

[0247] In some embodiments, the isolated oligonucleotide is iRNA.

[0248] The term "iRNA" refers to an RNA agent that can down-regulate the expression of a target gene (e.g., siRNA), e.g., an endogenous or pathogen target RNA. Without being bound by theory, iRNA can act by one or more of several mechanisms, including post-transcriptional cleavage of the target mRNA (referred to in the art as RNAi), or pre-transcriptional or pre-translational mechanisms. An iRNA can comprise a single strand or can comprise two or more strands, e.g., it can be a double-stranded iRNA. When the iRNA is single-stranded, it can comprise a 5' modification that includes one or more phosphate groups or one or more analogs of a phosphate group. In some embodiments, the iRNA is double-stranded. In some embodiments, one or both strands of the double-stranded iRNA can be modified, e.g., 5' modified.

[0249] An iRNA typically comprises a region of sufficient homology to the target gene and is of sufficient length with respect to nucleotides such that the iRNA or a fragment thereof can mediate down-regulation of the target gene. An iRNA comprises or is a region that is at least partially, and in some embodiments completely, complementary to the target RNA. While perfect complementarity between the iRNA and the target is not necessarily required, the pairing can be sufficient to enable the iRNA or a cleavage product thereof to direct sequence-specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA.

[0250] The nucleotides of the isolated oligonucleotide can be further modified. The single-stranded or double-stranded regions of the isolated oligonucleotide may be further modified or may further contain nucleotide surrogates. For example, the unpaired regions of the hairpin structure, such as the regions connecting two complementary regions, may have modifications or nucleotide surrogates. For example, modifications for stabilizing one or more 3' or 5' ends of the isolated oligonucleotide against exonuclease. Modifications include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotide spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), phosphoramidites, and may include specific biotin or fluorescein reagents having another DMT-protected hydroxyl group, enabling multiple couplings during RNA synthesis. Further modifications may also include, for example, the use of modifications at the 2'OH group of the ribose sugar, such as the use of deoxyribonucleotides instead of ribonucleotides, such as the use of deoxythymidine, and modifications at the phosphate group, such as phosphorothioate modifications. In some embodiments, different strands contain different further modifications.

[0251] In some embodiments, the length of the double-stranded region between the strands of the isolated oligonucleotide is from 6 to 30 nucleotides in length. In some embodiments, the double-stranded region is from 15 to 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length. In some embodiments, the double-stranded region is from 6 to 20 nucleotides, most preferably 6, 7, 8, 9, 10, 11 and 12 nucleotides in length.

[0252] The isolated oligonucleotide may be one described in U.S. Patent Application Publication Nos. 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, or 2009 / 0247608, each of which is incorporated herein by reference.

[0253] In some embodiments, the isolated oligonucleotide is siRNA.

[0254] In some embodiments, the isolated oligonucleotide is a double-stranded siRNA, e.g., a double-stranded siRNA as described herein.

[0255] As used herein, "double-stranded siRNA" is an siRNA comprising two or more, optionally two, strands capable of forming a double-stranded region by intermolecular hybridization. In some embodiments, the sense strand of the double-stranded siRNA may correspond to or be at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides in length. In some embodiments, the antisense strand of the double-stranded siRNA may correspond to or be at least 18, 19, 20, 21, 22, 23, 24, 25, 29, 30, or 31 nucleotides in length.

[0256] In some embodiments, the isolated oligonucleotide of the disclosure can be converted or dissociated into a single-stranded siRNA to form a single-stranded siRNA.

[0257] As used herein, "single-stranded siRNA" is an siRNA composed of a single strand that contains a double-stranded region formed by intramolecular pairing, e.g., it may be or contain a hairpin or panhandle structure. The single-stranded siRNA can be antisense with respect to the target molecule. The single-stranded siRNA may be long enough to enter RISC and participate in RISC-mediated cleavage of the target mRNA. The single-stranded siRNA is at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides in length. The single-stranded siRNA is at least 18, 19, 20, 21, 22, 23, 24, 25, 29, 30, or 31 nucleotides in length.

[0258] In some embodiments, the isolated oligonucleotide is a hairpin siRNA.

[0259] Hairpin siRNA may have a double-stranded region corresponding to 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs or a double-stranded region of at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs. In some embodiments, the range of the double-stranded region is 15 - 30, 17 - 23, 19 - 23, 18 - 21, and 19 - 21 nucleotide pairs in length. The hairpin may have a single-stranded overhang or a terminal unpaired region. In some embodiments, the length of the overhang is at least two. In some embodiments, the overhang is on the antisense side of the hairpin.

[0260] In some embodiments, the siRNA is large enough to be cleaved by an endogenous molecule, for example, Dicer, to produce a smaller siRNA, for example, an siRNA agent.

[0261] The sense strand and the antisense strand can be selected such that the double-stranded siRNA contains a single-stranded or unpaired region at one end of the molecule. Thus, the double-stranded siRNA can contain a sense strand and an antisense strand paired such that it contains a 3' overhang of two or more nucleotides. In some embodiments, the overhang is two nucleotides.

[0262] The siRNAs described herein, including double-stranded siRNAs and single-stranded siRNAs generated from double-stranded siRNAs, can mediate the silencing of a target RNA, for example, an mRNA, for example, a transcript of a gene encoding a protein. For convenience, such an mRNA is also referred to herein as the mRNA to be silenced. Such a gene is also referred to as the target gene. Generally, the RNA to be silenced is an endogenous gene or a pathogen gene. Further, RNAs other than mRNA, for example, tRNA and viral RNA, can also be targeted.

[0263] As used herein, the phrases “mediated by RNAi” or “mRNA silencing” or “targeting mRNA”, “inhibiting mRNA” or “degrading mRNA” refer to the ability to silence a target RNA in a sequence-specific manner. Without being bound by theory, silencing is thought to utilize the RNAi machinery or process and a guide RNA, such as a 21-23 nucleotide ssRNA.

[0264] In some embodiments, the isolated oligonucleotide is a siRNA that is “sufficiently complementary” to a target RNA, such as a target mRNA, such that the siRNA silences the production of the protein encoded by the target mRNA. In another embodiment, the siRNA is “exactly complementary” to the target RNA, such that, for example, the target RNA and the siRNA anneal to form a hybrid made up of only Watson-Crick base pairs in the region of exact complementarity. A “sufficiently complementary” target RNA may include an internal region that is exactly complementary to the target RNA (e.g., at least 10 nucleotides). Further, in some embodiments, the siRNA specifically discriminates a single nucleotide difference. In this case, the siRNA mediates RNAi only if exact complementarity is found in the region of the single nucleotide difference (e.g., within 7 nucleotides thereof).

[0265] In some embodiments, the isolated oligonucleotide is an antisense oligonucleotide targeted to a target polynucleotide. The term "antisense oligonucleotide" or simply "antisense" means including an oligonucleotide that is complementary to a target polynucleotide sequence. An antisense oligonucleotide is a single strand of DNA or RNA that is complementary to a selected sequence, for example, the mRNA of a target gene. An antisense oligonucleotide is thought to inhibit gene expression by binding to the complementary mRNA. Binding to the target mRNA can result in inhibition of gene expression by preventing translation of the complementary mRNA strand by binding to it, or by causing degradation of the target mRNA. In some embodiments, the antisense oligonucleotide comprises from about 13 to about 36 nucleotides, more preferably from about 20 to about 25 nucleotides. The term also encompasses antisense oligonucleotides that may not be exactly complementary to the desired target gene. Thus, it is contemplated that if non-target-specific activity is found with the antisense, or if an antisense sequence containing one or more mismatches with the target sequence is most preferred for a particular use.

[0266] Antisense oligonucleotides have been demonstrated to be effective inhibitors of protein synthesis and can therefore be used to specifically inhibit protein synthesis by a target gene. The effectiveness of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of polygalacturonase and the muscarinic 2-type acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U.S. Patent Nos. 5,739,119 and 5,759,829, each of which is incorporated by reference). Further, examples of antisense inhibition have been demonstrated with nuclear protein cyclin, multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAA receptor, and human EGF (Jaskulski et al., Science. 1988 Jun. 10;240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989, 1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998 Jun. 15;57(2):310-20; U.S. Patent No. 5,801,154; 5,789,573, 5,718,709, and 5,610,288, each of which is incorporated by reference). Further, antisense constructs that can be used to inhibit and treat various abnormal cell proliferations (e.g., cancer) have also been described (U.S. Patent Nos. 5,747,470, 5,591,317, and 5,783,683, each of which is incorporated by reference).

[0267] Methods for producing antisense oligonucleotides are known in the art and can be readily adapted to produce antisense oligonucleotides that target any polynucleotide sequence. The selection of an antisense oligonucleotide sequence specific for a given target sequence is based on the analysis of the selected target sequence and the determination of secondary structure, Tm, binding energy, and relative stability. Antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prevent specific binding to the target mRNA in the host cell. Highly preferred target regions of the mRNA include the region around or at the AUG translation initiation codon and sequences substantially complementary to the 5' region of the mRNA. Consideration of these secondary structure analyses and target site selection can be accomplished, for example, using OLIGO Primer Analysis Software, v.4 (Molecular Biology Insights) and / or the BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

[0268] In some embodiments, the isolated oligonucleotide can be an antagomir. An antagomir is an RNA-like oligonucleotide having various modifications for pharmacological properties such as RNase protection and enhanced tissue and cell uptake. Antagomirs differ from normal RNA, for example, by complete 2'-O-methylation of the sugars, a phosphorothioate backbone, and, for example, a cholesterol moiety at the 3' end. Antagomirs can be used to efficiently silence endogenous miRNAs by forming a duplex containing the antagomir and the endogenous miRNA, thereby preventing miRNA-induced gene silencing. An example of antagomir-mediated miRNA silencing is the silencing of miR-122 described in Krutzfeldt et al., Nature, 2005, 438:685-689, which is hereby incorporated by reference in its entirety. Antagomir RNAs can be synthesized using standard solid-phase oligonucleotide synthesis protocols. See U.S. Patent Application Publication Nos. 2007 / 0123482 and 2007 / 0213292, each of which is incorporated herein by reference.

[0269] An antagomir can comprise a ligand conjugate monomer subunit and a monomer for oligonucleotide synthesis. Exemplary monomers are described in U.S. Patent Application Publication No. 2005 / 0107325, which is incorporated herein by reference in its entirety. An antagomir can have a ZXY structure such as that described in International Publication No. 2004 / 080406, which is incorporated herein by reference in its entirety. An antagomir can complex with an amphiphilic moiety. Exemplary amphiphilic moieties for use with oligonucleotide agents are described in International Publication No. 2004 / 080406, which is incorporated herein by reference in its entirety.

[0270] In some embodiments, the isolated oligonucleotides of the disclosure linked to a lipid are associated with a ribozyme. A ribozyme is an RNA molecule complex that has a specific catalytic domain with endonuclease activity (Kim and Cech, Proc Natl Acad Sci USA. 1987 December; 84(24):8788-92; Forster and Symons, Cell. 1987 Apr. 24; 49(2):211-20). For example, many ribozymes accelerate phosphoester transfer reactions with a high degree of specificity and often cleave only one of several phosphoesters in an oligonucleotide substrate (Cech et al., Cell. 1981 December; 27(3 Pt 2):487-96; Michel and Westhof, J Mol Biol. 1990 Dec. 5; 216(3):585-610; Reinhold-Hurek and Shub, Nature. 1992 May 14; 357(6374):173-6). This specificity is due to the requirement that the substrate bind to the internal guide sequence ("IGS") of the ribozyme via specific base pairing interactions prior to the chemical reaction.

[0271] At least six basic types of naturally occurring enzymatic RNAs are currently known. Each can catalyze the hydrolysis of RNA phosphodiester bonds in transit under physiological conditions (and thus can cleave other RNA molecules). Generally, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through a target binding portion of the enzymatic nucleic acid that is held in close proximity to the enzymatic portion of the molecule that acts to cleave the target RNA. Thus, an enzymatic nucleic acid first recognizes a target RNA, then binds to the target RNA via complementary base pairing, and upon binding to the correct site, acts enzymatically to cleave the target RNA. Such strategic cleavage of the target RNA disrupts its ability to direct the synthesis of the encoded protein. After binding to and cleaving its RNA target, the enzymatic nucleic acid can be released from that RNA to search for another target and repeatedly bind to and cleave new targets.

[0272] The enzymatic nucleic acids can be formed, for example, in a hammerhead, hairpin, hepatitis delta virus, group I intron or RNaseP RNA (associated with an RNA guide sequence) or Neurospora VS RNA motif. Specific examples of the hammerhead motif are described in Rossi et al., Nucleic Acids Res. 1992 Sep. 11;20(17):4559-65. Examples of the hairpin motif are described in Hampel et al. (European Patent Application Publication No. 0360257), Hampel and Tritz, Biochemistry 1989 Jun. 13;28(12):4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan. 25;18(2):299-304 and U.S. Patent No. 5,631,359. Examples of the hepatitis delta virus motif are described in Perrotta and Been, Biochemistry. 1992 Dec. 1;31(47):11843-52; examples of the RNaseP motif are described in Guerrier-Takada et al., Cell. 1983 December;35(3 Pt 2):849-57; the Neurospora VS RNA ribozyme motif is described in Collins (Saville and Collins, Cell. 1990 May 18;61(4):685-96; Saville and Collins, Proc Natl Acad Sci USA. 1991 Oct. 1;88(19):8826-30; Collins and Olive, Biochemistry. 1993 Mar. 23;32(11):2795-9), and examples of group I introns are described in U.S. Patent No. 4,987,071. An important feature of the enzymatic nucleic acids used is that they have specific substrate binding sites that are complementary to one or more of the target gene DNA or RNA regions, and that they have nucleotide sequences within or surrounding the substrate binding sites that confer RNA cleavage activity to the molecule. Thus, ribozyme constructs need not be limited to the specific motifs referred to herein.

[0273] Methods for producing ribozymes that target any polynucleotide sequence are known in the art. Ribozymes can be designed as described in International Publication Nos. 93 / 23569 and 94 / 02595 (each specifically incorporated herein by reference) and synthesized as described therein to be tested in vitro and in vivo.

[0274] Ribozyme activity can be optimized by changing the length of the ribozyme binding arms, or by chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see, e.g., International Publication Nos. 92 / 07065, 93 / 15187, and 91 / 03162, European Patent Application Publication No. 92110298.4, U.S. Patent No. 5334711, and International Publication No. 94 / 13688, which describe various chemical modifications that can be added to the sugar moiety of enzymatic RNA molecules), modifications that enhance efficacy in cells, and removal of stem II bases to shorten RNA synthesis time and reduce the chemicals required.

[0275] Synthesis methods In some aspects, the disclosure provides methods for preparing the isolated oligonucleotides of the disclosure, or compounds, conjugates or scaffolds comprising the isolated oligonucleotides.

[0276] In some aspects, the disclosure provides intermediates described herein that are suitable for use in methods for preparing the isolated oligonucleotides of the disclosure, or compounds, conjugates or scaffolds comprising the isolated oligonucleotides.

[0277] The isolated oligonucleotides of the present disclosure, or compounds, conjugates or scaffolds comprising the isolated oligonucleotides, can be prepared by any suitable technique known in the art. Specific processes for the preparation of the isolated oligonucleotides of the present disclosure, or compounds, conjugates or scaffolds comprising the isolated oligonucleotides, are further described in the appended examples.

[0278] In the description of the synthetic methods described herein, and in any reference synthetic methods used to prepare starting materials, it should be understood that all proposed reaction conditions, including solvent selection, reaction atmosphere, reaction temperature, experimental duration and work-up procedures, can be selected by those skilled in the art. It is understood by those skilled in organic synthesis that the functional groups present in the various parts of the molecule need to be compatible with the reagents and reaction conditions utilized.

[0279] It will be understood that it may be desirable to protect certain substituents during the synthesis of the isolated oligonucleotides of the present disclosure, or compounds, conjugates or scaffolds comprising the isolated oligonucleotides, to prevent their unwanted reactions. Skilled chemists will understand when such protection is required and how such protecting groups can be placed in place and later removed. For examples of protecting groups, see one of the many general documents on the subject, for example, ‘Protective Groups in Organic Synthesis’ by Theodora Green (publisher: John Wiley & Sons). The protecting groups can be removed by any convenient method described in the literature as being appropriate for the removal of the protecting group in question or known to the skilled chemist, and such methods are selected to effect the removal of the protecting group with minimal interference to groups elsewhere in the molecule. Thus, when the reactants contain groups such as, for example, amino, carboxy or hydroxy, it may be desirable to protect this group in some of the reactions referred to herein.

[0280] As an example, protecting groups suitable for amino or alkylamino groups are, for example, acyl groups such as alkanoyl groups like acetyl, alkoxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl or t-butoxycarbonyl groups, arylmethoxycarbonyl groups such as benzyloxycarbonyl, or aroyl groups such as benzoyl. Protecting groups suitable for hydroxy or alkylhydroxy groups are, for example, acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl (DMT), methoxymethyl ether (MOM), methoxytrityl (MMT), p-methoxybenzyl ether (PMB), p-methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ethers (e.g., trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers), methyl ether, or ethoxyethyl ether (EE). Protecting groups suitable for 1,2-diols can be, for example, acetals. Protecting groups suitable for 1,3-diols can be, for example, tetraisopropyldisiloxanilidene (TIPDS).

[0281] The deprotection conditions of the above protecting groups will necessarily vary depending on the choice of the protecting group. Thus, for example, an acyl group such as an alkanoyl or alkoxycarbonyl group or an aroyl group can be removed by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium hydroxide or sodium hydroxide. Alternatively, an acyl group such as a tert-butoxycarbonyl group can be removed by treatment with a suitable acid such as hydrochloric acid, sulfuric acid or phosphoric acid or trifluoroacetic acid, and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group can be removed by hydrogenation on a catalyst such as palladium on carbon or by treatment with a Lewis acid such as tris(trifluoroacetate) borate. A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group, which can be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or hydrazine.

[0282] Suitable protecting groups for a hydroxy group are, for example, an acyl group such as an alkanoyl group such as acetyl, an aroyl group such as benzoyl, or an arylmethyl group such as benzyl. The deprotection conditions of the above protecting groups will necessarily vary depending on the choice of the protecting group. Thus, for example, an acyl group such as an alkanoyl or aroyl group can be removed by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium hydroxide, sodium hydroxide or ammonia. Alternatively, an arylmethyl group such as a benzyl group can be removed by hydrogenation on a catalyst such as palladium on carbon.

[0283] Suitable protecting groups for a carboxy group are, for example, an esterifying group such as a methyl or ethyl group which can be removed by hydrolysis with a base such as sodium hydroxide, or a tert-butyl group which can be removed by treatment with an organic acid such as trifluoroacetic acid, or a benzyl group which can be removed by hydrogenation on a catalyst such as palladium on carbon.

[0284] For convenience, the reactions of the compounds are preferably carried out in the presence of a suitable solvent that is inert under the respective reaction conditions. Examples of suitable solvents include hydrocarbons such as hexane, petroleum ether, benzene, toluene, or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, carbon tetrachloride, chloroform, or dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol, or tert-butanol; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 2-methyltetrahydrofuran, cyclopentyl methyl ether (CPME), methyl tert-butyl ether (MTBE), or dioxane; glycol ethers such as ethylene glycol monomethyl ether or ethylene glycol monoethyl ether or ethylene glycol dimethyl ether (diglyme); ketones such as acetone, methyl isobutyl ketone (MIBK), or butanone; amides such as acetamide, dimethylacetamide, dimethylformamide (DMF), N-methylpyrrolidinone (NMP); nitriles such as acetonitrile; sulfoxides such as dimethyl sulfoxide (DMSO); nitro compounds such as nitromethane or nitrobenzene; esters such as ethyl acetate or methyl acetate; or mixtures of these solvents or mixtures with water, but are not limited thereto.

[0285] The reaction temperature is preferably about -100°C to 300°C, depending on the reaction step and conditions used.

[0286] The reaction time generally ranges from a fraction of a minute to several days, depending on the reactivity of the respective compounds and the respective reaction conditions. Suitable reaction times can be readily determined by methods known in the art (e.g., reaction monitoring). Based on the above reaction temperature, suitable reaction times are generally in the range of 10 minutes to 48 hours.

[0287] Furthermore, additional compounds of the present disclosure can be readily prepared by utilizing the procedures described herein in conjunction with ordinary techniques in the art. One of ordinary skill in the art will readily appreciate that the conditions of the following preparative procedures and known variations of the processes can be used to prepare these compounds.

[0288] As will be appreciated by one of ordinary skill in organic synthesis, the isolated oligonucleotides of the present disclosure, or compounds, conjugates or scaffolds comprising isolated oligonucleotides, are readily available by a variety of synthetic routes, some of which are exemplified in the accompanying examples. One of ordinary skill in the art will readily recognize which types of reagents and reaction conditions should be used to obtain the compounds of the present disclosure, and how they should be applied and adapted in any given case (whenever necessary or useful). Furthermore, some of the isolated oligonucleotides of the present disclosure, or compounds, conjugates or scaffolds comprising isolated oligonucleotides, can be readily synthesized by reacting other compounds of the present disclosure under suitable conditions, for example, by applying standard synthetic methods such as reduction, oxidation, addition or substitution reactions, or by converting one particular functional group present in a compound of the present disclosure or a suitable precursor molecule thereof into another, and these methods are well known to those of ordinary skill in the art. Similarly, one of ordinary skill in the art can apply synthetic protecting (or protective) groups, suitable protecting groups, whenever necessary or useful, and the methods for introducing and removing them are well known to those of ordinary skill in chemical synthesis, and more particularly, are described, for example, in P.G.M. Wuts, T.W. Greene, “Greene’s Protective Groups in Organic Synthesis”, 4th edition (2006) (John Wiley & Sons).

[0289] Biological assay The isolated oligonucleotides, or compounds, conjugates or scaffolds of the present disclosure that have been designed, selected, prepared and / or optimized by the above-described method, once generated, can be characterized using various assays known to those skilled in the art to determine whether the compound, scaffold or conjugate has biological activity. For example, the isolated oligonucleotides of the present disclosure, or compounds, conjugates or scaffolds comprising an isolated oligonucleotide, can be characterized by conventional assays including, but not limited to, the assays described below to determine whether they have the desired activity, such as target binding activity and / or specificity and / or stability.

[0290] Furthermore, high-throughput screening can be used to accelerate the analysis using such assays. As a result, it may be possible to rapidly screen the molecules described herein for activity using techniques known in the art. General methodologies for performing high-throughput screening are described, for example, in Devlin (1998) High Throughput Screening, Marcel Dekker, and U.S. Patent No. 5,763,263. High-throughput assays can use one or more different assay techniques including, but not limited to, those described below.

[0291] A variety of in vitro or in vivo biological assays may be suitable for detecting the effects of the compounds, scaffolds, or conjugates of the present disclosure. These in vitro or in vivo biological assays can include, but are not limited to, enzyme activity assays, electrophoretic mobility shift assays, reporter gene assays, in vitro cell viability assays, and the assays described herein.

[0292] In some embodiments, the biological assay is described in the examples herein.

[0293] Pharmaceutical composition In some aspects, the present disclosure provides a pharmaceutical composition comprising an isolated oligonucleotide of the present disclosure, or a compound, conjugate or scaffold comprising the isolated oligonucleotide as an active ingredient.

[0294] As used herein, the term "composition" is intended to encompass a product comprising specific ingredients in specific amounts, as well as any product that results directly or indirectly from the combination of specific amounts of the specific ingredients.

[0295] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water-soluble), dispersions, and sterile powders for the extemporaneous preparation of sterile injection solutions or dispersions. In the case of intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include in the composition isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride. Prolonged absorption of the injectable composition can be achieved by including in the composition agents that delay absorption, for example, aluminum monostearate and gelatin.

[0296] A sterile injectable solution can be prepared by incorporating the required amount of the active compound in a suitable solvent, optionally with one or a combination of the ingredients listed above, followed by sterile filtration. Generally, a dispersion is prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and the necessary other ingredients from those listed above. In the case of a sterile powder for the preparation of a sterile injectable solution, the preparation methods include vacuum drying and lyophilization, and powders of the active ingredient and any further desired ingredients are obtained from a previously sterile-filtered solution.

[0297] The formulations of the present disclosure may be in the form of an aqueous solution containing an aqueous vehicle. The aqueous vehicle component may include water and at least one pharmaceutically acceptable excipient. Suitable acceptable excipients include those selected from the group consisting of solubilizers, chelating agents, preservatives, isotonic agents, viscosity / suspending agents, buffers, and pH adjusters, and mixtures thereof.

[0298] Any suitable solubilizer can be used. Examples of solubilizers include hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, randomly methylated-β-cyclodextrin, ethylated-β-cyclodextrin, triacetyl-β-cyclodextrin, fully acetylated-β-cyclodextrin, carboxymethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 2-hydroxy-3-(trimethylammonio)propyl-β-cyclodextrin, glucosyl-β-cyclodextrin, sulfated β-cyclodextrin (S-β-CD), maltosyl-β-cyclodextrin, β-cyclodextrin sulfobutyl ether, branched-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, randomly methylated-γ-cyclodextrin, and trimethyl-γ-cyclodextrin, and cyclodextrins such as those selected from the group consisting of mixtures thereof.

[0299] Any suitable chelating agent can be used. Examples of suitable chelating agents include those selected from the group consisting of ethylenediaminetetraacetic acid and its metal salts, disodium edetate, trisodium edetate, and tetrasodium edetate, and mixtures thereof.

[0300] Any suitable preservative can be used. Examples of preservatives include quaternary ammonium salts such as benzalkonium halide (preferably benzalkonium chloride), chlorhexidine gluconate, benzethonium chloride, cetylpyridinium chloride, benzyl bromide, phenylmercuric nitrate, phenylmercuric acetate, phenylmercuric neodecanoate, mercthiolate, methylparaben, propylparaben, sorbic acid, potassium sorbate, sodium benzoate, sodium propionate, ethyl p-hydroxybenzoate, propylaminopropylbiguanide, and butyl p-hydroxybenzoate, and sorbic acid, and mixtures thereof.

[0301] The aqueous vehicle may also contain an isotonic agent to adjust the tonicity (osmotic pressure). The isotonic agent can be selected from the group consisting of glycols (such as propylene glycol, diethylene glycol, triethylene glycol, etc.), glycerol, dextrose, glycerin, mannitol, potassium chloride, and sodium chloride, and mixtures thereof.

[0302] To adjust the formulation to an acceptable pH (typically a pH range of about 5.0 to about 9.0, more preferably about 5.5 to about 8.5, particularly about 6.0 to about 8.5, about 7.0 to about 8.5, about 7.2 to about 7.7, about 7.1 to about 7.9, or about 7.5 to about 8.0), the formulation may contain a pH adjuster. The pH adjuster is typically a mineral acid or metal hydroxide base selected from the group consisting of potassium hydroxide, sodium hydroxide, and hydrochloric acid, and mixtures thereof, preferably sodium hydroxide and / or hydrochloric acid. These acidic and / or basic pH regulators are added to adjust the formulation to the target acceptable pH range. Thus, it may not be necessary to use both an acid and a base, and depending on the formulation, adding only one of the acid or base may be sufficient to bring the mixture to the desired pH range.

[0303] The aqueous vehicle may also contain a buffer to stabilize the pH. When used, the buffer is selected from the group consisting of phosphate buffers (such as sodium dihydrogen phosphate and disodium hydrogen phosphate), borate buffers (such as boric acid or its salts containing disodium tetraborate), citrate buffers (such as citric acid or its salts containing sodium citrate), and ε-aminocaproic acid, and mixtures thereof.

[0304] According to a further aspect of the disclosure, there is provided a pharmaceutical composition comprising a compound of the disclosure as defined herein, or a pharmaceutically acceptable salt, hydrate or solvate thereof, together with a pharmaceutically acceptable diluent or carrier.

[0305] The compositions of the present disclosure may be in a form suitable for oral use (for example, as tablets, troches, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), topical use (for example, as creams, ointments, gels, or aqueous or oily solutions or suspensions), administration by inhalation (for example, as fine powders or liquid aerosols), administration by insufflation (for example, as fine powders), or parenteral administration (for example, as sterile aqueous or oily solutions for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular administration, or as suppositories for rectal administration).

[0306] The compositions of the present disclosure can be obtained by conventional procedures using conventional pharmaceutical excipients well known in the art. Thus, compositions intended for oral use may, for example, contain one or more coloring agents, sweetening agents, flavoring agents and / or preservatives.

[0307] An effective amount of the compounds of the present disclosure for use in therapy is an amount sufficient to treat or prevent the inflammasome-related conditions referred to herein, slow their progression, and / or reduce the symptoms associated with such conditions.

[0308] An effective amount of the compounds of the present disclosure for use in therapy is an amount sufficient to treat the inflammasome-related conditions referred to herein, slow their progression, and / or reduce the symptoms associated with such conditions.

[0309] The magnitude of the dose of the compounds of formula (I) or (II) for therapeutic or prophylactic purposes will, of course, vary according to the nature and severity of the condition, the age and sex of the animal or patient, and the route of administration, in accordance with well-known medical principles. It should also be understood that the present disclosure provides pharmaceutical compositions comprising any of the compounds, scaffolds, or conjugates described herein in combination with at least one pharmaceutically acceptable excipient or carrier.

[0310] As used herein, the term "pharmaceutical composition" refers to a formulation containing a compound, scaffold, or conjugate of the present disclosure in a form suitable for administration to a subject. In some embodiments, the pharmaceutical composition is in bulk or unit dosage form. The unit dosage form can be in any of a variety of forms, including, for example, capsules, IV bags, tablets, a single pump on an aerosol inhaler, or vials. The amount of the active ingredient (e.g., a formulation of the disclosed compound or a salt, hydrate, solvate, or isomer thereof) in a unit dose of the composition is an effective amount and varies according to the particular treatment involved. One of ordinary skill in the art will understand that it may be necessary to routinely vary the dosage according to the age and condition of the patient. The dosage also varies depending on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intrathecal, intranasal, etc. Dosage forms for topical or transdermal administration of the compounds of the present disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. In some embodiments, the active compound is mixed, under sterile conditions, with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants as required.

[0311] As used herein, the term "pharmaceutically acceptable" refers to compounds, scaffolds, conjugates, anions, cations, materials, compositions, carriers, and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0312] As used herein, the term "pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and not otherwise undesirable biologically and that is useful in the preparation of pharmaceutical compositions, and includes excipients that are acceptable for veterinary use and human pharmaceutical use. The "pharmaceutically acceptable excipient" as used herein and in the claims includes both one excipient and two or more such excipients.

[0313] It should be understood that the pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., ingestion), inhalation, transdermal (topical), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben, an antioxidant such as ascorbic acid or sodium bisulfite, a chelating agent such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate, and an isotonicity agent, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0314] It should be understood that the compounds or pharmaceutical compositions of the present disclosure can be administered to a subject in many of the well-known methods currently used for chemotherapy treatment. For example, the compounds of the present disclosure can be injected into the bloodstream or body cavity, or can be orally ingested, or can be applied through the skin using a patch. The dose selected needs to be sufficient to constitute an effective treatment, but not so high as to cause unacceptable side effects. The condition of the disease state (e.g., the diseases or disorders disclosed herein) and the health status of the patient should preferably be closely monitored over a reasonable period during and after treatment.

[0315] It should be understood that the compounds, scaffolds, and conjugates of the present disclosure can be prepared in various ways using commercially available starting materials, compounds known in the literature, or intermediates that are readily prepared, by using standard synthetic methods and procedures known to those of skill in the art or that are apparent to those of skill in the art in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and for functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field. Representative texts, without limitation to any one or several sources, such as Smith, M.B., March, J., March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition, John Wiley & Sons: New York, 2001; Greene, T.W., Wuts, P.G.M., Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999; R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser, Fieser and Fieser’s Reagents forganic Synthesis, John Wiley and Sons (1994), and L. Paquette, ed., Encyclopedia of Reagents forganic Synthesis, John Wiley and Sons (1995) are incorporated herein by reference and are useful and recognized reference textbooks of organic synthesis known to those of skill in the art.

[0316] One of ordinary skill in the art will note that in the reaction sequences and synthetic schemes described herein, the order of certain steps, such as the introduction and removal of protecting groups, can be changed. One of ordinary skill in the art will recognize that certain groups may require protection from reaction conditions by use of a protecting group. Protecting groups can also be used to distinguish similar functional groups in a molecule. A list of protecting groups and methods for introducing and removing these groups can be found in Greene, T.W., Wuts, P.G.M., Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999.

[0317] Techniques for the formulation and administration of the disclosed compounds of the present disclosure can be found in Remington: the Science and Practice of Pharmacy, 19th edition, Mack Publishing Co., Easton, PA (1995). In one embodiment, the compounds described herein and their pharmaceutically acceptable salts are used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous solutions of organic solvents. The compounds are present in such pharmaceutical compositions in an amount sufficient to provide the desired dosage within the ranges described herein.

[0318] Pharmaceutical compositions containing the active compounds of the present disclosure can be manufactured in a generally known manner, for example, by conventional mixing, dissolving, granulating, tablet coating, levigating, emulsifying, encapsulating, entrapping, or lyophilization processes. The pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers including excipients and / or auxiliaries that facilitate processing of the active compounds into preparations that can be pharmaceutically used. Of course, the appropriate formulation will vary depending on the chosen route of administration.

[0319] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble), dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In the case of intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycols, etc.), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include in the composition isotonic agents such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride. Sustained absorption of the injectable composition can be achieved by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin.

[0320] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in an appropriate solvent, with one or a combination of the ingredients enumerated above as required, followed by sterile filtration. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying, whereby powders of the active ingredient and any additional desired ingredients are obtained from a previously sterile-filtered solution.

[0321] Oral compositions generally contain an inert diluent or a pharmaceutically acceptable carrier for food use. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, and the compound in the fluid carrier is applied orally, rinsed, spat out, or swallowed. Pharmaceutically compatible binders and / or auxiliary materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. can contain any of the following components or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0322] For administration by inhalation, the compound is delivered in the form of an aerosol spray from a suitable pressurized container or dispenser containing a gas such as carbon dioxide as a propellant, or a nebulizer.

[0323] For nasal administration, the compound is delivered in a solution or a solid formulation. In some embodiments, the compound is delivered in a solution as a mist, drop, or swab. In some embodiments, the compound is delivered as a powder. In some embodiments, the compound is included in a kit that further includes a nasal applicator.

[0324] Systemic administration may be by transmucosal or transdermal means. In the case of transmucosal or transdermal administration, suitable penetration enhancers are used in the formulation for the barrier to be penetrated. Such penetration enhancers are generally known in the art and include, for example, surfactants, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, plasters, gels, or creams as generally known in the art.

[0325] The active compound can be prepared with a pharmaceutically acceptable carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, as described, for example, in U.S. Patent No. 4,522,811.

[0326] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in unit dosage form. As used herein, unit dosage form refers to physically discrete units suitable as unit doses for the subject to be treated, each unit containing a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present disclosure are determined by the unique characteristics of the active compound and the particular therapeutic effect to be achieved and vary directly in accordance therewith.

[0327] In therapeutic use, the dosage of the pharmaceutical composition used in accordance with the present disclosure will vary, inter alia, according to factors that influence the selected dosage, such as the drug, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the treatment. Generally, the dosage should be sufficient to retard, preferably regress, and more preferably cause complete regression of the symptoms of the disease or disorder disclosed herein. The dosage can range from about 0.01 mg / kg / day to about 5000 mg / kg / day. The effective amount of the pharmaceutical is the amount that provides an objectively identifiable improvement as recognized by a clinician or other qualified observer. Improvement in survival and growth indicates regression. As used herein, the term "dosage effective format" refers to the amount of the active compound that produces the desired biological effect in a subject or cell.

[0328] It should be understood that the pharmaceutical composition can be contained in a container, pack, or dispenser together with instructions for administration.

[0329] It should be understood that for the compounds, scaffolds, or conjugates of the present disclosure that can further form salts, all of these forms are also contemplated within the scope of the claimed disclosure.

[0330] As used herein, the term "pharmaceutically acceptable salt" refers to a derivative of a compound of the present disclosure in which the parent compound is modified by making its acid or base salt. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral acid organic acid salts of basic residues such as amines, and alkali organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic organic acids. For example, such conventional non-toxic salts include 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, 1,2-ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, glycollyarsanilic acid, hexylresorcinic acid, hydrabamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxymaleic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, laurylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, napsylic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannic acid, tartaric acid, toluenesulfonic acid, and those derived from inorganic and organic acids selected from commonly occurring amino acids such as glycine, alanine, phenylalanine, arginine, etc., but are not limited thereto.

[0331] In some embodiments, the pharmaceutically acceptable salt is a sodium salt, potassium salt, calcium salt, magnesium salt, diethylamine salt, choline salt, meglumine salt, benzathine salt, tromethamine salt, ammonia salt, arginine salt, or lysine salt.

[0332] Other examples of pharmaceutically acceptable salts include hexanoic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, muconic acid, and the like. The present disclosure also includes salts formed when the acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion, or when it coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine. It is understood that in the salt form, the ratio of the compound to the cation or anion of the salt can be 1:1 or any ratio other than 1:1, e.g., 3:1, 2:1, 1:2, or 1:3.

[0333] It should be understood that all references to pharmaceutically acceptable salts include the solvate (solvent addition form) or crystalline form (polymorph) of the same salt as defined herein.

[0334] The compound or its pharmaceutically acceptable salt is administered orally, nasally, transdermally, by the pulmonary route, by inhalation, buccally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally, and parenterally. In some embodiments, the compound is administered orally. One of ordinary skill in the art will recognize the advantages of a particular route of administration.

[0335] The dosing regimen using the compound is selected according to various factors including the type, species, age, weight, sex, and medical condition of the patient, the severity of the condition to be treated, the route of administration, the renal and hepatic function of the patient, and the particular compound or its salt being used. A typical skilled physician or veterinarian can readily determine and prescribe an effective amount of the drug necessary to prevent, counteract, or stop the progression of the condition. A typical skilled physician or veterinarian can readily determine and prescribe an effective amount of the drug necessary to counteract or stop the progression of the condition.

[0336] How to use In some aspects, the present disclosure provides a method of modulating (e.g., reducing or eliminating) expression of a target gene in a subject, the method comprising administering to the subject an isolated oligonucleotide of the present disclosure, or a compound, conjugate, or scaffold comprising the isolated oligonucleotide.

[0337] In some aspects, the disclosure provides a method of modulating (e.g., reducing or eliminating) expression of a target gene in a cell or tissue of a subject, the method comprising administering to a subject an isolated oligonucleotide of the disclosure, or a compound, conjugate, or scaffold comprising the isolated oligonucleotide.

[0338] In some aspects, the present disclosure provides a method of delivering an isolated oligonucleotide of the present disclosure, or a compound, conjugate, or scaffold comprising the isolated oligonucleotide, to a subject, comprising administering to the subject an isolated oligonucleotide of the present disclosure, or a compound, conjugate, or scaffold comprising the isolated oligonucleotide.

[0339] In some aspects, the disclosure provides a method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated oligonucleotide of the disclosure, or a compound, conjugate, or scaffold comprising the isolated oligonucleotide.

[0340] In some aspects, the present disclosure provides an isolated oligonucleotide of the present disclosure, or a compound, conjugate, or scaffold comprising the isolated oligonucleotide, for modulating (e.g., reducing or eliminating) expression of a target gene in a subject.

[0341] In some embodiments, the present disclosure provides an isolated oligonucleotide of the present disclosure, or a compound, conjugate or scaffold comprising the isolated oligonucleotide, for regulating (e.g., reducing or eliminating) the expression of a target gene in a subject cell or tissue.

[0342] In some embodiments, the present disclosure provides a compound, conjugate or scaffold comprising the isolated oligonucleotide of the present disclosure for delivering the isolated oligonucleotide of the present disclosure.

[0343] In some embodiments, the present disclosure provides an isolated oligonucleotide of the present disclosure, or a compound, conjugate or scaffold comprising the isolated oligonucleotide, for the treatment or prevention of a disease in a subject in need thereof.

[0344] In some embodiments, the present disclosure provides the use of an isolated oligonucleotide of the present disclosure, or a compound, conjugate or scaffold comprising the isolated oligonucleotide, in the manufacture of a medicament for regulating (e.g., reducing or eliminating) the expression of a target gene in a subject.

[0345] In some embodiments, the present disclosure provides the use of an isolated oligonucleotide of the present disclosure, or a compound, conjugate or scaffold comprising the isolated oligonucleotide, in the manufacture of a medicament for regulating (e.g., reducing or eliminating) the expression of a target gene in a subject cell or tissue.

[0346] In some embodiments, the present disclosure provides the use of a compound, conjugate or scaffold comprising the isolated oligonucleotide of the present disclosure in the manufacture of a medicament for delivering the isolated oligonucleotide of the present disclosure to a subject.

[0347] In some embodiments, the present disclosure provides the use of an isolated oligonucleotide of the present disclosure, or a compound, conjugate or scaffold comprising the isolated oligonucleotide, in the manufacture of a medicament for the treatment or prevention of a disease in a subject in need thereof.

[0348] In some embodiments, the subject is a cell. In some embodiments, the subject is a tissue. In some embodiments, the subject is a human.

[0349] In some embodiments, the modified siRNA molecule having the chemically modified sense and antisense strands of the present disclosure can be directed against any target gene, such as PCSK9, ANGPTL-3, AGT, HSD17b3, C3, CfB, APOC3, C5, SOD1, GPAM, LPA, F11, Factor VII, Eg5, TPX2, apoB, SAA, TTR, HBV, HCV, RSV, the PDGF beta gene, the Erb-B gene, the Src gene, the CRK gene, the GRB2 gene, the RAS gene, the MEKK gene, the JNK gene, the RAF gene, the Erk1 / 2 gene, the PCNA (p21) gene, the MYB gene, the JUN gene, the FOS gene, the BCL-2 gene, the Cyclin D gene, the VEGF gene, the EGFR gene, the Cyclin A gene, the Cyclin E gene, the WNT-1 gene, the beta-catenin gene, the c-MET gene, the PKC gene, the NFKB gene, the STAT3 gene, the survivin gene, the Her2 / Neu gene, the topoisomerase I gene, the topoisomerase II alpha gene, the p73 gene, the p21 (WAF1 / CIP1) gene, the p27 (KIP1) gene, the PPM1D gene, the RAS gene, the caveolin I gene, the MIB I gene, the MTAI gene, the M68 gene, mutations in tumor suppressor genes, the p53 tumor suppressor gene, LDHA, or any combination thereof.

[0350] In some embodiments, the disease is characterized by unwanted or abnormal expression of a target gene. In some embodiments, administration results in a reduction or elimination of the expression of the target gene in the subject.

[0351] In some embodiments, the disease is a viral infection, such as HCV, HBV, HPV, HSV or HIV infection.

[0352] In some embodiments, the disease is cancer. In some embodiments, the cancer is biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, glioma, astrocytoma, breast cancer, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal cancer, colon cancer, hereditary nonpolyposis colorectal cancer, colorectal adenocarcinoma, gastrointestinal stromal tumor (GIST), endometrial cancer, endometrial stromal sarcoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, uveal melanoma, choroidal melanoma, gallbladder cancer, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinoma, Wilms tumor, leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic (CLL), chronic myelogenous (CML), chronic myelomonocytic leukemia (CMML), liver cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, hepatic adenoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B-cell lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, T-cell lymphoma, non-Hodgkin lymphoma, precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary neoplasm, acinar cell carcinoma. Prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, gastric cancer, gastric cancer, gastrointestinal stromal tumor (GIST), uterine cancer, or uterine sarcoma.

[0353] In some embodiments, the cancer is liver cancer, hepatocellular carcinoma, cholangiocarcinoma, or hepatic adenoma.

[0354] In some embodiments, the disease is a proliferative, inflammatory, autoimmune, neurological, ocular, respiratory, metabolic, cutaneous, auditory, hepatic, renal, or infectious disease. In some embodiments, the disease is a hepatic disease. In some embodiments, the hepatic disease is cirrhosis, steatosis or a combination thereof.

[0355] In some embodiments, the disease is a metabolic disorder. In some embodiments, the metabolic disorder is hypercholesterolemia, hypo-beta-lipoproteinemia, coronary heart disease, peripheral arterial disease, stroke, type 2 diabetes, obesity, or hypertension, or a combination thereof.

[0356] Unless otherwise specified, any description of a method of treatment or prevention is to be understood to include the use of a compound, scaffold, and conjugate for providing treatment or prevention as described herein. Unless otherwise specified, any description of a method of treatment or prevention is to be further understood to include the use of a compound, scaffold, and conjugate for preparing a medicament for treating or preventing such a condition. Treatment or prevention includes treatment or prevention of non-human animals, including humans or rodents, and other disease models.

[0357] Unless otherwise specified, any description of a method of treatment is to be understood to include the use of a compound, scaffold, and conjugate for providing treatment as described herein. Unless otherwise specified, any description of a method of treatment is to be further understood to include the use of a compound, scaffold, and conjugate for preparing a medicament for treating such a condition. Treatment includes treatment of non-human animals, including humans or rodents, and other disease models.

[0358] As used herein, the term "subject" is interchangeable with the term "subject in need thereof", and both refer to a subject having a disease or at high risk of developing a disease. "Subject" includes mammals. Mammals can be, for example, humans or suitable non-human mammals such as primates, mice, rats, dogs, cats, cows, horses, goats, camels, sheep, or pigs. The subject may be a bird or poultry. In some embodiments, the mammal is a human. The subject in need thereof can be a subject previously diagnosed or identified as having a disease or disorder disclosed herein. The subject in need thereof can also be a subject suffering from a disease or disorder disclosed herein. Alternatively, the subject in need thereof can be a subject at high risk of developing such a disease or disorder compared to the population as a whole (i.e., a subject likely to develop such a disorder compared to the population as a whole). The subject in need thereof can be refractory or resistant to a disease or disorder disclosed herein (i.e., a disease or disorder disclosed herein that does not respond or has not yet responded to treatment). The subject may be resistant at the start of treatment or may become resistant during treatment. In some embodiments, the subject in need thereof has received and failed all known effective therapies for a disease or disorder disclosed herein. In some embodiments, the subject in need thereof has received at least one prior treatment.

[0359] As used herein, the terms "treating" or "treatment" describe the management and care of a patient for the purpose of combating a disease, condition, or disorder, and includes the administration of a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph, or solvate thereof, to alleviate the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term "treatment" can also include the treatment of cells or animal models in vitro. It should be understood that reference to "treating" or "treatment" includes the alleviation of established symptoms of a condition. Thus, "treating" a condition, disorder, or state, or "treatment" of a condition, disorder, or state, includes (1) preventing or delaying the onset of clinical symptoms of a condition, disorder, or state in a human who has or is at risk of having, but has not yet experienced or exhibited, clinical or preclinical symptoms of the condition, disorder, or state, (2) inhibiting the condition, disorder, or state, i.e., arresting, reducing, or delaying the development or recurrence (in the case of maintenance therapy) of the disease, or at least one of its clinical or preclinical symptoms, or (3) alleviating or reducing the disease, i.e., causing regression of the condition, disorder, or state, or at least one of its clinical or preclinical symptoms.

[0360] It should be understood that the compounds, scaffolds, and conjugates of the present disclosure, or pharmaceutically acceptable salts, polymorphs, or solvates thereof, can also be used to prevent a related disease, condition, or disorder, or to identify suitable candidates for such purposes.

[0361] As used herein, the terms "preventing", "prevention", or "protecting against" describe reducing or eliminating the onset of symptoms or complications of such a disease, condition, or disorder.

[0362] As used herein, the term "therapeutically effective amount" refers to an amount of an agent that is used to treat, ameliorate, or prevent a specified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The exact effective amount for a subject will depend upon the subject's weight, size, and general health, the nature and extent of the condition, and the particular therapeutic agent or combination of therapeutic agents selected for administration. The therapeutically effective amount for a given situation can be determined by routine experimentation within the skill and judgment of the clinician.

[0363] As used herein, the term "therapeutically effective amount" refers to an amount of an agent that is used to treat or ameliorate a specified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The exact effective amount for a subject will depend upon the subject's weight, size, and general health, the nature and extent of the condition, and the particular therapeutic agent or combination of therapeutic agents selected for administration. The therapeutically effective amount for a given situation can be determined by routine experimentation within the skill and judgment of the clinician.

[0364] It should be understood that for any compound, a therapeutically effective amount can first be estimated, for example, in cell culture assays of neoplastic cells or in animal models, usually rats, mice, rabbits, dogs, or pigs. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful dosages and routes of administration in humans. Therapeutic / preventive efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, ED 50 (the dose therapeutically effective in 50% of the population), and LD 50 (the dose lethal to 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, LD 50 / ED 50It can be expressed as a ratio. Pharmaceutical compositions showing a large therapeutic index are preferred. The dosage can vary within this range depending on the dosage form used, the sensitivity of the patient, and the route of administration.

[0365] Dosage and administration are adjusted to provide a sufficient level of the active agent or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the general health of the subject, the age, weight, and sex of the subject, diet, time and frequency of administration, drug combinations, response sensitivity, and tolerance / response to treatment. Long-acting pharmaceutical compositions can be administered once every 3 - 4 days, weekly, or every two weeks depending on the half-life and clearance rate of the particular formulation.

[0366] All publications and patent documents are incorporated herein by reference to the same extent as if each such publication or patent document was specifically and individually indicated to be incorporated herein by reference. The citation of publications and patent documents is not intended to admit that any of them are relevant prior art, nor does it constitute any admission as to their content or date. Although the present invention has been described by way of written explanation, those skilled in the art will recognize that the present invention can be implemented in various embodiments, and the foregoing description and the following examples are for illustrative purposes and do not limit the scope of the following claims.

Example

[0367] Example 1: Optimization of chemical modification patterns to improve the stability and efficacy of siRNA targeting Gene 1.

[0368] Tests are described herein that show further improvement in the stability and tolerability of siRNA by decreasing the 2'-fluoro nucleotide content and simultaneously increasing the 2'-O-methyl content. The 2'-fluoro and 2'-O-methyl content across both the sense and antisense strands of siRNA were systematically tested to identify highly modified designs with improved in vitro activity and metabolic stability, which may lead to improved in vivo potency, duration, and tolerability. In the tests described herein, the positional effects of 2'-O-methyl modification and 2'-fluoro modification on both the sense and antisense strands were systematically tested, and several highly advanced designs with significantly improved in vitro activity were identified for both the 21 / 23-mer platform and the 20 / 22-mer platform.

[0369] In the tests described herein, a published prototype compound (21 / 23-mer), which is a clinically approved drug, was used. Based on the chemical modification of the prototype compound, the positional effects of 2'-O-methyl modification and 2'-fluoro modification on both the sense and antisense strands were systematically tested for both the 21 / 23-mer platform and the 20 / 22-mer platform. Several advanced designs are described herein that have in vitro activity improved up to 60-fold compared to that of the prototype compound (4 pm vs 245 pm IC 50 ).

[0370] Materials and Methods

[0371] Compounds (e.g., nucleic acid agents, nucleic acid agents including nucleotide-based enhancers, and conjugates) were prepared by solid-phase synthesis according to standard synthetic protocols.

[0372] Briefly, oligonucleotide synthesis was carried out on a solid support, incorporating each nucleoside phosphoramidite from the 3'-end to the 5'-end to prepare a single-stranded oligo. ETT or BTT was used as the activating substance for the coupling reaction. Phosphorous triester (P(III)) was oxidized using iodine in water / pyridine / THF to obtain a phosphate backbone, and phosphorothioate linkages were prepared using DDTT. Aqueous ammonium was used to cleave the oligo from the solid support and remove the protecting groups globally. The crude oligo was then concentrated and purified by strong anion exchange or reverse phase HPLC. The purified fractions were combined and concentrated.

[0373] In some cases, the single-stranded oligo was then conjugated with a targeting ligand (e.g., sugar, peptide, antibody) via conjugation after synthesis to obtain a conjugate. The conjugation reaction was carried out using standard conjugation methods. The crude conjugate was further purified by strong anion exchange or reverse phase HPLC. The purified fractions were combined and concentrated. The synthesized single-strand was then dialyzed against water, concentrated, and the OD amount was measured. The sense strand and the antisense strand were annealed based on a 1:1 molar ratio. The solution of the annealed duplex was lyophilized to obtain the desired siRNA molecule.

[0374] To measure in vitro activity, all test substances in the optimization series were transfected into Huh7 cells using Lipofectamine RNAiMax. In 96-well plates, 20,000 cells / well were transfected with each compound at concentrations ranging from 5000 pM to 2 pM according to the manufacturer's protocol (Invitrogen - 13778 - 150). All transfections were repeated three times. Before RNA isolation, the cells were incubated at 37 °C for 24 hours. Then, intracellular RNA was isolated using the RNeasy 96 kit according to the manufacturer's protocol (Qiagen - 74182). Target gene 1 cDNA was detected by qPCR, and GAPDH cDNA was used as an internal control and detected in parallel. GraphPad Prism was used to plot the graph and calculate the IC 50 value.

[0375] In the tests described herein, the position effects of 2'-F and 2'-OMe modifications of the sense and antisense strands of siRNA molecules against target gene 1 on their stability and tolerance were tested.

[0376] Testing of chemical modifications of 21 / 23-mer prototype siRNA molecules against target gene 1

[0377] In the first set of optimizations described herein (Figure 1), the modification pattern of an exemplary siRNA prototype compound against target gene 1 was utilized as the parental design [SEQ ID NO: 1: SEQ ID NO: 2], which inhibited target gene 1 with an IC 50 of 245 pm. The further modified siRNA molecule [SEQ ID NO: 3: SEQ ID NO: 4] showed an equivalent IC 50 of 243 pm, and [SEQ ID NO: 5: SEQ ID NO: 6] showed an approximately 10-fold increase in RNAi activity compared to the parental design (25 pm vs. 245 pm IC 50 )(Figures 2A - 2C).

[0378] Subsequent optimization of the prototype siRNA molecule: position effects of 2'-OMe and 2'-F modifications of the sense strand

[0379] In the subsequent sets of optimizations described herein (Figures 3 and 5), the improvement in potency (IC 50 ) due to the improved modification content (2'-OMe and 2'-F) on the sense strand was determined by focusing on positions 10 - 15. As shown in Figures 4A - 4E, the in vitro activities of the siRNA molecules [SEQ ID NO:7:SEQ ID NO:6], [SEQ ID NO:8:SEQ ID NO:6], and [SEQ ID NO:9:SEQ ID NO:6] were well maintained at IC 50 of 33 pM, 30 pM, and 31 pM, respectively. However, [SEQ ID NO:10:SEQ ID NO:6] showed an IC 50 of 14 pM, which is an approximately 2-fold improvement in potency compared to that of the siRNA molecule [SEQ ID NO:7 and SEQ ID NO:6], and also an approximately 2-fold improvement compared to the siRNA molecule [SEQ ID NO:5:SEQ ID NO:6]. The results described herein showed that 2'-F modifications at positions 10 and 14, in combination with adjacent modifications, can additively improve in vitro activity.

[0380] Furthermore, when the 2'-OMe modification was moved to position 11 of the siRNA molecule [SEQ ID NO:11:SEQ ID NO:6] (Figure 5), as shown in Figures 6A - 6C, the in vitro potency decreased significantly, and an IC 50 of 110 pM was obtained. However, further modification to the siRNA molecule [SEQ ID NO:12:SEQ ID NO:6] was shown to partially rescue the potency at an IC 50 of 67 pM. Furthermore, the siRNA molecule [SEQ ID NO:13:SEQ ID NO:6] showed an excellent IC 50 of 14 pM, showing an approximately 2-fold improvement in in vitro activity compared to that of [SEQ ID NO:5 and SEQ ID NO:6].

[0381] Further optimization of the 2'-F content of the antisense strand of the siRNA molecule

[0382] The results described herein demonstrate the optimization of the 2'-F content on the antisense strand to generate a third set of siRNAs (Figures 7 and 9). The design of siRNAs containing sense and antisense strands with siRNA molecules [SEQ ID NO:5:SEQ ID NO:6] was used as the parental design, and the effect of 2'-F modification on the seed region (positions 2 to 8) was mainly tested. 2'-F modifications were added at positions 3, 5, 7, 8, and 9, respectively. Among these individual modifications, siRNA molecules [SEQ ID NO:5:SEQ ID NO:15] and [SEQ ID NO:5:SEQ ID NO:14] showed IC 50 as shown in Figures 8A - 8F, which was a 2 - 3 - fold improvement in activity compared to the parental design. The remaining modified siRNA molecules [SEQ ID NO:5:SEQ ID NO:16], [SEQ ID NO:5:SEQ ID NO:17], and [SEQ ID NO:5:SEQ ID NO:18] also showed a slight effect on in vitro activity.

[0383] As shown in Figure 9, the combined effect of 2'-F modifications on some of these important positions was further tested. [SEQ ID NO:5:SEQ ID NO:19], [SEQ ID NO:5:SEQ ID NO:20], and [SEQ ID NO:5:SEQ ID NO:21] did not further improve in vitro activity compared to the parental design [SEQ ID NO:5:SEQ ID NO:6] of the siRNA molecule. See Figures 10A - 10C.

[0384] Fourth Optimization of Composite Modifications on Sense and Antisense Strands

[0385] Herein, a subsequent set of optimizations, namely, antisense with a further optimized 2'-F content, is described to obtain antisense strands according to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25. The antisense strands were then paired with the optimized sense strand according to the identified SEQ ID NO:13 or the sub - optimized sense strand according to SEQ ID NO:9 (Figures 7 and 8A - 8F), and their overall effect on potency was investigated (Figure 11). As shown in Figures 12A - 12F, the siRNA molecule [SEQ ID NO:9 and SEQ ID NO:22] showed IC 50is shown, which is an approximately 3-fold increase in in vitro activity compared to that of the siRNA molecule [SEQ ID NO: 9 and SEQ ID NO: 6] (IC of 31 pm 50 ), and is an approximately 3-fold improvement compared to that of the parental design [SEQ ID NO: 5:SEQ ID NO: 6] (IC of 25 pm 50 ). The pairing of the optimal sense strand according to SEQ ID NO: 13 and the further optimized antisense strands according to SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25 all showed excellent potency with an IC 50 of 12 - 14 pm.

[0386] Optimization of chemical modifications on 20 / 22mers to improve the stability and potency of siRNAs targeting Gene 1

[0387] Referring to the results of 2’-F and 2’-OMe modifications in the 21 / 23mer platform above, the positional effects in the 20 / 22mer platform after the same siRNA modification design were tested. To generate a 20 / 22mer duplex based on the 21 / 23mer, both nucleosides were clipped at the 21st position of the sense strand according to SEQ ID NO: 1 and the 23rd position of the antisense strand according to SEQ ID NO: 2, resulting in a design containing the sense and antisense strands of the siRNA molecule [SEQ ID NO: 26:SEQ ID NO: 27]. Thus, [SEQ ID NO: 26:SEQ ID NO: 27] was designed as the 20 / 22mer counterpart of the 21 / 23mer [SEQ ID NO: 1:SEQ ID NO: 2] and was used as the parental design in the following optimizations of the 20 / 22mer platform. The number of nucleoside positions on the 20mer sense strand is one less than the corresponding positions on the 21mer sense strand, but due to the substantial siRNA modification trends and positional effects observed in the 21 / 23mer platform, it could be converted to the 20 / 22mer platform.

[0388] In the tests described herein, the first set of siRNA modifications of the 20 / 22mer platform, [SEQ ID NO: 26:SEQ ID NO: 27], was used as the parental design, with an IC 50is shown (Figure 13). The modified siRNA molecule [SEQ ID NO: 28: SEQ ID NO: 27] showed equivalent potency as shown in Figures 14A - 14C. The further chemically modified siRNA molecule [SEQ ID NO: 29: SEQ ID NO: 30] showed an IC 50 of 15 pm, which is an approximately 4-fold improvement in RNAi activity compared to that of [SEQ ID NO: 26: SEQ ID NO: 27]. The substantial improvement in in vitro activity was consistent with the trend observed for [SEQ ID NO: 5: SEQ ID NO: 6] when the same chemical modification was applied to the 21 / 23-mer sequences.

[0389] Second modification of 20 / 22-mer: positional effects of 2'-OMe and 2'-F modifications on the sense strand

[0390] In the tests described herein, the 2'-OMe content on the second SAR set of the sense strand was improved (Figures 15 and 17). The tests described herein confirmed that the 2'-OMe modification at the 15th position can sufficiently maintain the in vitro potency regardless of the modification at the 14th position [SEQ ID NO: 31: SEQ ID NO: 30] or [SEQ ID NO: 32: SEQ ID NO: 30]. The siRNA molecules [SEQ ID NO: 33: SEQ ID NO: 30] and [SEQ ID NO: 36: SEQ ID NO: 30] showed excellent IC 50 of 4 - 5 pm (Figures 16A - 16E). The combined effects of these modifications at the 10th, 14th, and 15th positions on the 20-mer sense strand were consistent with the effects on the 21-mer sense strand.

[0391] Furthermore, the siRNA molecules [SEQ ID NO: 34: SEQ ID NO: 30], [SEQ ID NO: 35: SEQ ID NO: 30], and [SEQ ID NO: 44: SEQ ID NO: 30] all showed excellent IC 50 of 5 - 10 pm as shown in Figures 18A - 18C.

[0392] Third modification of 20 / 22-mer: positional effects of 2'-OMe and 2'-F modifications on the antisense strand

[0393] This specification describes the generation of a third set of siRNA modifications to improve the 2'-F content on the antisense strand and enhance efficacy (Figure 19). In the results described herein, the 3-position of the siRNA molecule [SEQ ID NO: 29:SEQ ID NO: 45] was shown to exhibit an IC 50 of 5 pm. 2'-F modifications at other positions in the seed region, including the 3-position [SEQ ID NO: 29:SEQ ID NO: 50], 5-position [SEQ ID NO: 29:SEQ ID NO: 46], 7-position [SEQ ID NO: 29:SEQ ID NO: 47], 8-position [SEQ ID NO: 29:SEQ ID NO: 47], and 9-position [SEQ ID NO: 29:SEQ ID NO: 48] of the siRNA molecule, also showed a positive effect on in vitro activity in the range of 7 - 11 pm of IC 50 (Figures 20B and 20D - 20F).

[0394] Furthermore, the combinatorial effects of 2'-F modifications on some of these important positions in the seed region were tested (Figure 21). As shown in Figures 22A - 22C, the siRNA molecule [SEQ ID NO: 29:SEQ ID NO: 37] showed a two-fold improvement in efficacy compared to that of the parental design [SEQ ID NO: 29:SEQ ID NO: 30]. Also, the original efficacy of the siRNA molecule [SEQ ID NO: 29:SEQ ID NO: 38] was well maintained.

[0395] Fourth modification of the 20 / 22-mer: Combinatorial effect of an optimized sense strand and an optimized (or further optimized) antisense strand

[0396] To examine the combinatorial effect of an optimized sense strand and an optimized (or further optimized) antisense strand, a fourth set of siRNA modifications is described herein (Figure 23). In the results described herein, all double-optimized duplexes were shown to exhibit an IC 50 ≤ 7 pm and a two-fold improvement in efficacy compared to the parental design (Figures 24A - 24F). Among these molecules, the design containing the sense and antisense strands of the siRNA molecule [SEQ ID NO: 44:SEQ ID NO: 42] showed the lowest IC 50 of 4 pm, which was an IC of 245 pm50 Compared with those of the parental siRNA molecule having [SEQ ID NO: 1: SEQ ID NO: 2], it is an improvement of about 60 times the in vitro activity.

[0397] Summary

[0398] In the results described herein, on both the 21 / 23-mer and 20 / 22-mer platforms, the 2'-O-methyl and 2'-fluoro contents across the sense and antisense strands of the siRNA molecule were systematically improved, and advanced modification designs related to the improvement of in vitro activity and stability were identified. The improved in vitro activity and stability disclosed herein may improve in vivo efficacy, duration, and tolerability.

[0399] In the tests described herein, the optimized pairing of the sense and antisense strands resulted in a combined improvement in activity, and several advanced designs, such as [SEQ ID NO: 44: SEQ ID NO: 42], were also identified with a maximum 60-fold improvement in in vitro activity compared to that of the prototype benchmark.

[0400] Additional Embodiments Additional embodiments of the present disclosure include the following: Embodiment 1. (a) A sense strand comprising X1 nucleotides, wherein at least one nucleotide is modified with a first modification, each of the remaining nucleotides is independently modified with a second modification, X1 is an integer selected from 13 to 36, the first modification and the second modification are different, and the sense strand, (b) An antisense strand comprising X2 nucleotides, wherein at least one nucleotide is modified with a third modification, each of the remaining nucleotides is independently modified with a fourth modification, X2 is an integer selected from 18 to 31, the third modification and the fourth modification are different, and the antisense strand, and an isolated oligonucleotide comprising the same. Embodiment 2. The isolated oligonucleotide according to Embodiment 1, wherein the first modification is a modification of the sugar moiety of at least one nucleotide at the 2'-position selected from a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, a 2'-deoxy modification, and equivalents thereof, and combinations thereof. Embodiment 3. The isolated oligonucleotide according to Embodiment 1 or 2, wherein the first modification is a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, or a 2'-deoxy modification, or stereoisomers thereof. Embodiment 4. The isolated oligonucleotide according to any one of Embodiments 1 to 3, wherein the first modification is a 2'-F modification, a 2'-CN modification, or a 2'-N3 modification, or stereoisomers thereof. Embodiment 5. The isolated oligonucleotide according to any one of Embodiments 1 to 4, wherein the first modification is a 2'-F modification or a stereoisomer thereof. Embodiment 6. The isolated oligonucleotide according to any one of Embodiments 1 to 5, wherein the second modification is a modification of one or more sugar moieties of the remaining nucleotides at the 2'-position selected from a 2'-C1-C6 alkyl, a 2'-OR modification [wherein R is a C1-C6 alkoxy, an acetamide, a phenyl, or a heteroaryl containing a 5-membered or 6-membered ring and one or two heteroatoms selected from N, O, and S, and is an optionally substituted C1-C6 alkyl], a 2'-amino, and a morpholino substitution, and equivalents thereof, and combinations thereof. Embodiment 7. The isolated oligonucleotide according to any one of Embodiments 1 to 6, wherein the second modification is a 2'-OR modification. Embodiment 8. The isolated oligonucleotide according to any one of Embodiments 1 to 7, wherein the second modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification. Embodiment 9. The isolated oligonucleotide according to any one of Embodiments 1 to 8, wherein the second modification is a 2'-O-methyl modification. Embodiment 10. The isolated oligonucleotide according to any one of Embodiments 1 to 8, wherein the first modification is a 2'-F modification or a stereoisomer thereof, and the second modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification. Embodiment 11. The isolated oligonucleotide according to any one of Embodiments 1 to 10, wherein the first modification is a 2'-F modification or a stereoisomer thereof, and the second modification is a 2'-O-methyl modification. Embodiment 12. The isolated oligonucleotide according to any one of Embodiments 1 to 11, wherein the third modification is a modification of the sugar moiety of at least one nucleotide at the 2'-position selected from a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, a 2'-deoxy modification, and equivalents thereof, and combinations thereof. Embodiment 13. The isolated oligonucleotide according to any one of Embodiments 1 to 12, wherein the third modification is a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, or a 2'-deoxy modification, or a stereoisomer thereof. Embodiment 14. The isolated oligonucleotide according to any one of Embodiments 1 to 13, wherein the third modification is a 2'-F modification, a 2'-CN modification, or a 2'-N3 modification, or a stereoisomer thereof. Embodiment 15. The isolated oligonucleotide according to any one of Embodiments 1 to 14, wherein the third modification is a 2'-F modification or a stereoisomer thereof. Embodiment 16. The isolated oligonucleotide according to any one of Embodiments 1 to 15, wherein the fourth modification is a modification of one or more sugar moieties of the remaining nucleotides at the 2'-position selected from 2'-C1-C6 alkyl, 2'-OR modification [wherein R is C1-C6 alkoxy, acetamide, phenyl, or heteroaryl containing one or two heteroatoms selected from N, O, and S and optionally substituted C1-C6 alkyl], 2'-amino, and morpholino substitution, and equivalents thereof, and combinations thereof. Embodiment 17. The isolated oligonucleotide according to any one of Embodiments 1 to 16, wherein the fourth modification is a 2'-OR modification. Embodiment 18. The isolated oligonucleotide according to any one of Embodiments 1 to 17, wherein the fourth modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification. Embodiment 19. The isolated oligonucleotide according to any one of Embodiments 1 to 18, wherein the fourth modification is a 2'-O-methyl modification. Embodiment 20. The isolated oligonucleotide according to any one of Embodiments 1 to 18, wherein the third modification is a 2'-F modification or a stereoisomer thereof, and the fourth modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification. Embodiment 21. The isolated oligonucleotide according to any one of Embodiments 1 to 20, wherein the third modification is a 2'-F modification or a stereoisomer thereof, and the fourth modification is a 2'-O-methyl modification. Embodiment 22. The isolated oligonucleotide according to any one of Embodiments 1 to 21, wherein at least 3 nucleotides in the sense strand are modified with the first modification. Embodiment 23. The isolated oligonucleotide according to Embodiment 22, wherein at least 2 of the at least 3 nucleotides modified with the first modification are located continuously. Embodiment 24. The isolated oligonucleotide according to Embodiment 22 or 23, wherein at least 3 of the at least 3 nucleotides modified with the first modification are located continuously. Embodiment 25. The isolated oligonucleotide according to any one of Embodiments 1 to 24, wherein at least 4 nucleotides in the sense strand are modified with the first modification. Embodiment 26. The isolated oligonucleotide according to Embodiment 25, wherein at least 3 of the at least 4 nucleotides modified with the first modification are located continuously. Embodiment 27. The isolated oligonucleotide according to Embodiment 25 or 26, wherein at least 4 of the at least 4 nucleotides modified with the first modification are located continuously. Embodiment 28. The isolated oligonucleotide according to any one of Embodiments 1 to 27, wherein at least 5 nucleotides in the sense strand are modified with a first modification. Embodiment 29. The isolated oligonucleotide according to Embodiment 28, wherein at least 3 of the at least 5 nucleotides modified with the first modification are located consecutively. Embodiment 30. The isolated oligonucleotide according to Embodiment 28 or 29, wherein at least 4 of the at least 5 nucleotides modified with the first modification are located consecutively. Embodiment 31. The isolated oligonucleotide according to any one of Embodiments 22 to 30, wherein at least 3 nucleotides, at least 4 nucleotides, or at least 5 nucleotides modified with the first modification are located at positions 10 to 15 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 32. The isolated oligonucleotide according to any one of Embodiments 22 to 31, wherein 2 of the at least 3 nucleotides modified with the first modification are located at positions selected from positions 10, 11, 12, and 13 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 33. The isolated oligonucleotide according to any one of Embodiments 22 to 32, wherein 3 of the at least 3 nucleotides modified with the first modification are located at positions selected from positions 10, 11, 12, and 13 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 34. The isolated oligonucleotide according to any one of Embodiments 22 to 33, wherein 1 of the at least 3 nucleotides modified with the first modification is located at position 11 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 35. The isolated oligonucleotide according to any one of Embodiments 22 to 34, wherein 1 of the at least 4 nucleotides modified with the first modification is located at position 10 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 36. The isolated oligonucleotide according to any one of Embodiments 22 to 34, wherein one of at least 4 nucleotides modified with the first modification is located at the 14th position from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 37. The isolated oligonucleotide according to any one of Embodiments 22 to 34, wherein two of at least 4 nucleotides modified with the first modification are located at the 10th to 14th positions from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 38. The isolated oligonucleotide according to any one of Embodiments 22 to 37, wherein all of at least 3 nucleotides modified with the first modification are not located continuously. Embodiment 39. The isolated oligonucleotide according to any one of Embodiments 22 to 38, wherein at least 3 nucleotides, at least 4 nucleotides, or at least 5 nucleotides are modified with a 2'-F modification. Embodiment 40. The isolated oligonucleotide according to Embodiment 31, wherein in the sense strand, at most 3 nucleotides are modified with the first modification at the 12th, 13th, and 15th positions from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 41. The isolated oligonucleotide according to Embodiment 31, wherein in the sense strand, at most 4 nucleotides are modified with the first modification at the 11th, 12th, 13th, and 14th positions from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 42. The isolated oligonucleotide according to Embodiment 31, wherein in the sense strand, at most 4 nucleotides are modified with the first modification at the 10th, 11th, 12th, and 13th positions from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 43. The isolated oligonucleotide according to Embodiment 31, wherein at most 4 nucleotides in the sense strand are modified with the first modification at positions 10, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 44. The isolated oligonucleotide according to Embodiment 31, wherein at most 5 nucleotides in the sense strand are modified with the first modification at positions 10, 11, 12, 13, and 15 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 45. The isolated oligonucleotide according to Embodiment 31, wherein at most 5 nucleotides in the sense strand are modified with the first modification at positions 10, 11, 12, 13, and 14 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 46. The sense strand comprises nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 5’(M) g (F) f (M) e (F) d (M) c (F) b (M) a 3’ [wherein M is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and each of a, b, c, d, e, f, and g is any one of 0 to 16, indicating the number of consecutive nucleotides modified with the modification], and the sense strand is a) 5’(M)0(F)0(M)0(F)0(M)8(F)3(M) 10 3’ (SEQ ID NO: 52); b) 5’(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3’ (SEQ ID NO: 53); c) 5’(M)0(F)0(M)0(F)0(M)7(F)3(M) 10 3’ (SEQ ID NO: 54); d) 5’(M)0(F)0(M)5(F)1(M)1(F)2(M) 113’ (SEQ ID NO: 55); e) 5’ (M) 0 (F) 0 (M) 6 (F) 1 (M) 1 (F) 2 (M) 11 3’ (SEQ ID NO: 56); f) 5’ (M) 6 (F) 1 (M) 1 (F) 2 (M) 1 (F) 1 (M) 9 3’ (SEQ ID NO: 57); g) 5’ (M) 5 (F) 1 (M) 1 (F) 2 (M) 1 (F) 1 (M) 9 3’ (SEQ ID NO: 58); h) 5’ (M) 0 (F) 0 (M) 0 (F) 0 (M) 7 (F) 4 (M) 10 3’ (SEQ ID NO: 59); i) 5’ (M) 0 (F) 0 (M) 6 (F) 1 (M) 1 (F) 4 (M) 9 3’ (SEQ ID NO: 60); j) 5’ (M) 0 (F) 0 (M) 0 (F) 0 (M) 8 (F) 4 (M) 9 3’ (SEQ ID NO: 61); k) 5’ (M) 0 (F) 0 (M) 0 (F) 0 (M) 6 (F) 4 (M) 10 3’ (SEQ ID NO: 62); l) 5’ (M) 0 (F) 0 (M) 5 (F) 1 (M) 1 (F) 4 (M) 9 3’ (SEQ ID NO: 63); m) 5’ (M) 0 (F) 0 (M) 0 (F) 0 (M) 7 (F) 4 (M) 9 3’ (SEQ ID NO: 64); n) 5’ (M) 0 (F) 0 (M) 0 (F) 0 (M) 7 (F) 5 (M) 9 3’ (SEQ ID NO: 65); and o) an isolated oligonucleotide according to any one of embodiments 1 to 45, which is any one of 5’ (M) 0 (F) 0 (M) 0 (F) 0 (M) 6 (F) 5 (M) 9 3’ (SEQ ID NO: 66). Embodiment 47. An isolated oligonucleotide according to embodiments 1 to 46, wherein at most 7 nucleotides in the antisense strand are modified with a third modification. Embodiment 48. An isolated oligonucleotide according to embodiment 47, wherein at most 4 of the at most 7 nucleotides modified with the third modification are located at positions 2 to 8 from the first nucleotide at the 5’ end of the antisense strand. Embodiment 49. The isolated oligonucleotide according to Embodiment 47 or 48, wherein at most 2 out of at most 7 nucleotides modified with the third modification are located continuously. Embodiment 50. The isolated oligonucleotide according to any one of Embodiments 47 to 49, wherein 3 or 4 out of at most 7 nucleotides modified with the third modification are located at positions selected from the 2nd, 3rd, 5th, 6th, 7th, and 8th positions from the first nucleotide at the 5'-end of the antisense strand. Embodiment 51. The isolated oligonucleotide according to any one of Embodiments 47 to 50, wherein at least 1 out of at most 7 nucleotides modified with the third modification is located at the 14th position from the first nucleotide at the 5'-end of the antisense strand. Embodiment 52. The isolated oligonucleotide according to any one of Embodiments 47 to 51, wherein 2 or 3 out of at most 7 nucleotides modified with the third modification are located at positions selected from the 2nd, 3rd, 5th, and 6th positions from the first nucleotide at the 5'-end of the antisense strand. Embodiment 53. The isolated oligonucleotide according to any one of Embodiments 47 to 52, wherein 3 out of at most 7 nucleotides modified with the third modification are located at positions selected from the 2nd, 3rd, 5th, and 6th positions from the first nucleotide at the 5'-end of the antisense strand. Embodiment 54. The isolated oligonucleotide according to any one of Embodiments 47 to 53, wherein 2 out of at most 7 nucleotides modified with the third modification are located at the 8th and 14th positions from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 55. The isolated oligonucleotide according to any one of Embodiments 47 to 54, wherein 1 or 2 out of at most 7 nucleotides modified with the third modification are located at positions selected from the 14th and 16th positions from the first nucleotide at the 5'-end of the antisense strand. Embodiment 56. The isolated oligonucleotide according to any one of Embodiments 47 to 55, wherein two of at most seven nucleotides modified with a third modification are located at positions 14 and 16 from the first nucleotide at the 5' end of the antisense strand. Embodiment 57. The isolated oligonucleotide according to any one of Embodiments 47 to 56, wherein at most seven nucleotides are modified with a 2'-F modification. Embodiment 58. The isolated oligonucleotide according to any one of Embodiments 47 to 57, wherein at most six nucleotides are modified with a third modification. Embodiment 59. The isolated oligonucleotide according to any one of Embodiments 47 to 58, wherein at most six nucleotides are modified with a 2'-F modification. Embodiment 60. The isolated oligonucleotide according to any one of Embodiments 47 to 59, wherein at most five nucleotides are modified with a third modification. Embodiment 61. The isolated oligonucleotide according to any one of Embodiments 47 to 60, wherein at most five nucleotides are modified with a 2'-F modification. Embodiment 62. The isolated oligonucleotide according to any one of Embodiments 47 to 61, wherein at most four nucleotides are modified with a third modification. Embodiment 63. The isolated oligonucleotide according to any one of Embodiments 47 to 62, wherein at most four nucleotides are modified with a 2'-F modification. Embodiment 64. The isolated oligonucleotide according to any one of Embodiments 47 to 63, wherein at most three nucleotides are modified with a third modification. Embodiment 65. The isolated oligonucleotide according to any one of Embodiments 47 to 64, wherein at least three nucleotides are modified with a 2'-F modification. Embodiment 66. The isolated oligonucleotide according to any one of Embodiments 60 to 61, wherein in the antisense strand, at most five nucleotides are modified with a third modification at positions 2, 3, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. Embodiment 67. The isolated oligonucleotide according to any one of Embodiments 60 to 61, wherein in the antisense strand, at most 5 nucleotides are modified with a third modification at positions 2, 6, 7, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 68. The isolated oligonucleotide according to any one of Embodiments 58 to 59, wherein in the antisense strand, at most 6 nucleotides are modified with a third modification at positions 2, 3, 6, 8, 9, and 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 69. The isolated oligonucleotide according to any one of Embodiments 58 to 59, wherein in the antisense strand, at most 6 nucleotides are modified with a third modification at positions 2, 3, 5, 8, 9, and 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 70. The isolated oligonucleotide according to any one of Embodiments 47 to 57, wherein in the antisense strand, at most 7 nucleotides are modified with a third modification at positions 2, 3, 5, 7, 9, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 71. The isolated oligonucleotide according to any one of Embodiments 47 to 57, wherein in the antisense strand, at most 7 nucleotides are modified with a third modification at positions 2, 3, 5, 7, 10, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand. Embodiment 72. The antisense strand contains nucleotides modified with 2'-F modification and nucleotides modified with 2'-O-methyl modification, and has the formula: 3’(M) a (F) b (M) c (F) d (M) e (F) f (M) g (F) h (M) i (F)j (M) k (F) l (M) m (F) n (M) o 5', [wherein, M is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and each of a, b, c, d, e, f, g, h, i, j, k, l, m, n, and o is any one of 0 to 16, indicating the number of consecutive nucleotides modified by the modification], and the antisense strand is 1) 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)7(F)2(M)2(F)1(M)15'(SEQ ID NO: 14); 2) 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)7(F)1(M)2(F)2(M)15'(SEQ ID NO: 15); 3) 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)6(F)2(M)3(F)1(M)15'(SEQ ID NO: 16); 4) 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)5(F)1(M)1(F)1(M)3(F)1(M)15'(SEQ ID NO: 17); 5) 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)1(M)2(F)1(M)3(F)1(M)15'(SEQ ID NO: 18); 6) 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)8(F)1(M)2(F)1(M)15'(SEQ ID NO: 20); 7) 3'(M)0(F)0(M)0(F)0(M)0(F)0(M)0(F)0(M)9(F)1(M)7(F)1(M)3(F)1(M)15'(SEQ ID NO: 21); 8) 3'(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)2(F)2(M)15'(SEQ ID NO: 22); 9) 3’(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)2(M)2(F)1(M)1(F)2(M)15’(SEQ ID NO: 23); 10) 3’(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)4(F)1(M)1(F)1(M)1(F)2(M)15’(SEQ ID NO: 24); 11) 3’(M)0(F)0(M)0(F)0(M)7(F)1(M)1(F)1(M)3(F)1(M)2(F)1(M)1(F)1(M)1(F)2(M)15’(SEQ ID NO: 25); 12) 3’(M)0(F)0(M)4(F)1(M)1(F)1(M)1(F)1(M)3(F)1(M)1(F)1(M)1(F)3(M)1(F)1(M)15’(SEQ ID NO: 27); 13) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)3(F)1(M)15’(SEQ ID NO: 30); 14) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)2(M)2(F)1(M)15’(SEQ ID NO: 50); 15) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)7(F)1(M)2(F)2(M)15’(SEQ ID NO: 45); 16) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)6(F)2(M)3(F)1(M)15’(SEQ ID NO: 46); 17) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)5(F)1(M)1(F)1(M)3(F)1(M)15’(SEQ ID NO: 47); 18) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)1(M)2(F)1(M)3(F)1(M)15’(SEQ ID NO: 48); 19) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)3(F)1(M)15’(SEQ ID NO: 49); 20) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)8(F)1(M)2(F)1(M)15’ (SEQ ID NO: 37); 21) 3’(M)0(F)0(M)0(F)0(M)0(F)0(M)0(F)0(M)8(F)1(M)7(F)1(M)3(F)1(M)15’ (SEQ ID NO: 38); 22) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)1(F)1(M)2(F)2(M)15’ (SEQ ID NO: 39); 23) 3’(M)0(F)0(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)2(M)2(F)1(M)1(F)2(M)15’ (SEQ ID NO: 40); 24) 3’(M)0(F)0(M)6(F)1(M)1(F)1(M)4(F)1(M)1(F)1(M)1(F)1(M)1(F)2(M)15’ (SEQ ID NO: 41); and 25) any one of 3’(M)0(F)0(M)6(F)1(M)1(F)1(M)3(F)1(M)2(F)1(M)1(F)1(M)1(F)2(M)15’ (SEQ ID NO: 42), an isolated oligonucleotide according to any one of Embodiments 1 to 71. Embodiment 73. The sense strand is nucleotides modified with 2’-F modification and nucleotides modified with 2’-O-methyl modification, of the formula: 5’(M) g (F) f (M) e (F) d (M) c (F) b (M) a 3’, [wherein M is a 2’-O-methyl modified nucleotide, F is a 2’-F modified nucleotide, and each of a, b, c, d, e, f, and g is any one of 0 to 16, indicating the number of consecutive nucleotides modified with the modification], and the sense strand is a) 5’(M)0(F)0(M)0(F)0(M)8(F)3(M) 10 3’ (SEQ ID NO: 52); b) 5’(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3’(SEQ ID NO: 53); c) 5’(M)0(F)0(M)0(F)0(M)7(F)3(M) 10 3’(SEQ ID NO: 54); d) 5’(M)0(F)0(M)5(F)1(M)1(F)2(M) 11 3’(SEQ ID NO: 55); e) 5’(M)0(F)0(M)6(F)1(M)1(F)2(M) 11 3’(SEQ ID NO: 56); f) 5’(M)6(F)1(M)1(F)2(M)1(F)1(M)93’(SEQ ID NO: 57); g) 5’(M)5(F)1(M)1(F)2(M)1(F)1(M)93’(SEQ ID NO: 58); h) 5’(M)0(F)0(M)0(F)0(M)7(F)4(M) 10 3’(SEQ ID NO: 59); i) 5’(M)0(F)0(M)6(F)1(M)1(F)4(M)93’(SEQ ID NO: 60); j) 5’(M)0(F)0(M)0(F)0(M)8(F)4(M)93’(SEQ ID NO: 61); k) 5’(M)0(F)0(M)0(F)0(M)6(F)4(M) 10 3’(SEQ ID NO: 62); l) 5’(M)0(F)0(M)5(F)1(M)1(F)4(M)93’(SEQ ID NO: 63); m) 5’(M)0(F)0(M)0(F)0(M)7(F)4(M)93’(SEQ ID NO: 64); n) 5’(M)0(F)0(M)0(F)0(M)7(F)5(M)93’(SEQ ID NO: 65); and o) an isolated oligonucleotide according to embodiment 72, which is any one of 5’(M)0(F)0(M)0(F)0(M)6(F)5(M)93’(SEQ ID NO: 66). Embodiment 74. The isolated oligonucleotide according to any one of Embodiments 40 to 45, wherein at most 5 nucleotides in the antisense strand are modified with a third modification at positions 2, 3, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. Embodiment 75. The isolated oligonucleotide according to any one of Embodiments 40 to 45, wherein at most 5 nucleotides in the antisense strand are modified with a third modification at positions 2, 6, 7, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. Embodiment 76. The isolated oligonucleotide according to any one of Embodiments 40 to 45, wherein at most 6 nucleotides in the antisense strand are modified with a third modification at positions 2, 3, 6, 8, 9, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. Embodiment 77. The isolated oligonucleotide according to any one of Embodiments 40 to 45, wherein at most 6 nucleotides in the antisense strand are modified with a third modification at positions 2, 3, 5, 8, 9, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. Embodiment 78. The isolated oligonucleotide according to any one of Embodiments 40 to 45, wherein at most 7 nucleotides in the antisense strand are modified with a third modification at positions 2, 3, 5, 7, 9, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. Embodiment 79. The isolated oligonucleotide according to any one of Embodiments 40 to 45, wherein at most 7 nucleotides in the antisense strand are modified with a third modification at positions 2, 3, 5, 7, 10, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand. Embodiment 80. The isolated oligonucleotide according to any one of Embodiments 1 to 79, wherein X1 is 18 to 21 and X2 is 20 to 23. Embodiment 81. The isolated oligonucleotide according to Embodiment 80, wherein X1 is 20 or 21 and X2 is 22 or 23. Embodiment 82. The isolated oligonucleotide according to any one of Embodiments 1 to 80, wherein X2 is equal to X1 plus 2. Embodiment 83. The isolated oligonucleotide according to any one of Embodiments 1 to 82, wherein the sense strand contains at least one nucleotide having a modified phosphate backbone. Embodiment 84. The isolated oligonucleotide according to any one of Embodiments 1 to 83, wherein the antisense strand contains at least one nucleotide having a modified phosphate backbone. Embodiment 85. The isolated oligonucleotide according to Embodiment 83 or 84, wherein the modified phosphate backbone contains a modified phosphodiester bond. Embodiment 86. The isolated oligonucleotide according to Embodiment 85, wherein the modified phosphodiester bond is modified by substituting one or more oxygen atoms in part, and the part is bonded to the phosphorus atom in the phosphodiester bond having a carbon, nitrogen, or sulfur atom in the part, or is modified by forming a 2'-5' linkage. Embodiment 87. The isolated oligonucleotide according to Embodiment 85, wherein the modified phosphodiester bond contains phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate diester, mesylphosphoramidate, or phosphonoacetate. Embodiment 88. The isolated oligonucleotide according to any one of Embodiments 1 to 84, containing one or more nucleotides containing non-natural bases, locked nucleotides, or abasic nucleotides. Embodiment 89. The isolated oligonucleotide according to any one of Embodiments 1 to 88, wherein the terminal nucleotide at the 5' end contains a phosphate mimic. Embodiment 90. The isolated oligonucleotide according to Embodiment 89, wherein the 5'-phosphate mimic is ethylphosphonate, vinylphosphonate, or an analog thereof. Embodiment 91. The isolated oligonucleotide according to any one of Embodiments 1 to 90, wherein the antisense strand contains at least two single-stranded nucleotides at the 3'-end. Embodiment 92. The isolated oligonucleotide according to any one of Embodiments 1 to 91, wherein a terminal nucleotide or an internal nucleotide is linked to a targeting ligand in the sense strand, the antisense strand, or both. Embodiment 93. The isolated oligonucleotide according to Embodiment 92, wherein the targeting ligand is selected from one or more of carbohydrates, peptides, lipids, antibodies or fragments thereof, aptamers, albumin, fibrinogen, and folic acid. Embodiment 94. The isolated oligonucleotide according to Embodiment 93, wherein the targeting ligand is GalNAc. Embodiment 95. The isolated oligonucleotide according to any one of Embodiments 1 to 94, wherein the antisense strand is complementary to mRNA, and sequence-specific hybridization of the antisense strand to mRNA results in degradation of the mRNA. Embodiment 96. The isolated oligonucleotide according to any one of Embodiments 1 to 95, which can target any specific gene target including but not limited to PCSK9, ANGPTL-3, AGT, HSD17b3, C3, CFB, and HBV.

[0401] Equivalents Details of one or more embodiments of the present disclosure are described in the accompanying specification above. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, but the preferred methods and materials are described herein. Other features, objects, and advantages of the present disclosure will become apparent from this specification and the claims. In this specification and the appended claims, the singular form includes plural objects unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications cited herein are incorporated by reference.

[0402] The foregoing description is presented for purposes of illustration only and is not intended to limit the disclosure to the precise form disclosed, but rather is intended to be limited by the scope of the claims appended hereto.

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