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 potency for delivery and use as therapeutic agents.

Description

[0001] Related Applications

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 359,969, filed on July 11, 2022, the entire contents of which are incorporated herein by reference.

[0003] background

[0004] Small interfering RNA (siRNA) can effectively trigger RNAi silencing of specific genes, but its therapeutic potential still faces challenges involving efficacy, safety and delivery. Fully 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 extensively studied and shown to significantly improve metabolic stability and RNAi activity, leading to enhanced in vivo efficacy and duration.

[0005] Among these chemical modifications, the 2'-OH group of ribose is substituted with 2'-O-methyl (2'-OMe), 2'-deoxy-2'-fluoro (2'-F) and other 2'-O-alkyl groups (e.g., 2'-O-methoxyethyl) and has been shown to enhance the metabolic stability and RNAi activity of siRNA. More specifically, 2'-O-methyl modification is more spatially hindered than 2'-deoxy-2'-fluoro, and shows a stronger metabolic stability against endogenous nucleases. In addition, it can be expected to alleviate the concern about the tolerance of non-natural 2'-fluoro modification incorporated into siRNA, although siRNA containing 2'-fluoro has been thoroughly examined in preclinical studies and well tolerated in clinical trials. Therefore, in terms of improving metabolic stability and tolerance, the increase of 2'-O-methyl content in siRNA is preferred with the reduction of 2'-fluoro content. However, the more sterically hindered 2'-O-methyl group can cause steric clashes with Argonaute RISC catalytic component 2 (Ago2) residues, thereby preventing the guide strand from loading into RISC and significantly reducing RNAi activity if not applied judiciously. Therefore, there remains a continuing need for optimal chemical modifications that comprehensively enhance the metabolic stability, tolerance, and RNAi activity of siRNAs. The present disclosure addresses this need.

[0006] Overview

[0007] 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, each of the remaining nucleotides is independently modified with a second modification, and X1 is an integer selected from 13-36, wherein 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, each of the remaining nucleotides is independently modified with a fourth modification, and X2 is an integer selected from 18-31, wherein the third modification and the fourth modification are different.

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

[0009] In some embodiments, the second modification is a modification selected from 2'-C1-C6 alkyl (alkyl), 2'-OR modification, 2'-amino and morpholino replacement (morpholinoreplacement), and its equivalents and combinations thereof at the 2'-position of the sugar moiety in one or more of the remaining nucleotides, in which R is a C1-C6 alkyl optionally substituted with: C1-C6 alkoxy, acetamide, phenyl or a heteroaryl comprising a 5- or 6-ring and 1 or 2 heteroatoms selected from N, O and S. In some embodiments, the second modification is a 2'-OR modification or a morpholino replacement, 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 replacement.

[0010] 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.

[0011] In some embodiments, the third modification is a modification selected from 2'-F modification, 2'-CN modification, 2'-N3 modification, 2'-deoxy modification, and equivalents thereof, and combinations thereof at the 2'-position of the sugar portion of at least one nucleotide. In some embodiments, the third modification is 2'-F modification, 2'-CN modification, 2'-N3 modification, or 2'-deoxy modification, or a stereoisomer thereof. In some embodiments, the third modification is 2'-F modification, 2'-CN modification, or 2'-N3 modification, or a stereoisomer thereof. In some embodiments, the third modification is 2'-F modification, 2'-CN modification, or 2'-N3 modification, or a stereoisomer thereof. In some embodiments, the third modification is 2'-F modification or a stereoisomer thereof.

[0012] In some embodiments, the fourth modification is a modification selected from 2'-C1-C6 alkyl, 2'-OR modification, 2'-amino and morpholino substitution, and its equivalents and combinations thereof at the 2'-position of the sugar moiety in one or more of the remaining nucleotides, in which R is a C1-C6 alkyl optionally substituted with: C1-C6 alkoxy, acetamide, phenyl, or a heteroaryl comprising a 5- or 6-membered ring and 1 or 2 heteroatoms selected from N, O, and S. 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.

[0013] In some embodiments, the third modification is a 2'-F modification or a stereoisomer thereof, and the fourth modification is a 2'-O-methyl or 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.

[0014] In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least three nucleotides are modified with a first modification. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least two of the at least three nucleotides modified with a first modification are positioned consecutively. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least three of the at least three nucleotides modified with a first modification are positioned consecutively.

[0015] In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least four nucleotides are modified with a first modification. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least three of the at least four nucleotides modified with a first modification are positioned consecutively. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least four of the at least four nucleotides modified with a first modification are positioned consecutively.

[0016] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least five nucleotides are modified with the first modification. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least three of the at least five nucleotides modified with the first modification are positioned consecutively. At least four of the at least five nucleotides modified with the first modification are positioned consecutively.

[0017] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least three nucleotides, at least four nucleotides, or at least five nucleotides modified with the first modification are positioned at position 10 to position 15 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0018] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, two of the at least three nucleotides modified with the first modification are positioned at a position selected from position 10, position 11, position 12, and position 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, three of the at least three nucleotides modified with the first modification are positioned at a position selected from position 10, position 11, position 12, and position 13 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0019] In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, one of the at least three nucleotides modified with the first modification is positioned at position 11 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, one of the at least four nucleotides modified with the first modification is positioned at position 10 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, one of the at least four nucleotides modified with the first modification is positioned at position 14 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand.

[0020] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, two of the at least four nucleotides modified with the first modification are located at position 10 to position 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0021] In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least three nucleotides modified with a first modification are not all positioned consecutively. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least three nucleotides, at least four nucleotides, or at least five nucleotides are modified with a 2'-F modification.

[0022] In some embodiments, the sense strand of an isolated oligonucleotide of the present disclosure comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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 0-16 and indicates the number of consecutive nucleotides modified with the modification, and wherein the sense strand is any one of:

[0023] a)5'(M)0(F)0(M)0(F)0(M)8(F)3(M) 10 3' (SEQ ID NO:52);

[0024] b)5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3' (SEQ ID NO:53);

[0025] c)5'(M)0(F)0(M)0(F)0(M)7(F)3(M) 10 3' (SEQ ID NO:54);

[0026] d)5'(M)0(F)0(M)5(F)1(M)1(F)2(M) 11 3' (SEQ ID NO:55);

[0027] e)5'(M)0(F)0(M)6(F)1(M)1(F)2(M) 11 3' (SEQ ID NO:56);

[0028] f)5'(M)6(F)1(M)1(F)2(M)1(F)1(M)9 3' (SEQ ID NO:57);

[0029] g)5'(M)5(F)1(M)1(F)2(M)1(F)1(M)9 3' (SEQ ID NO:58);

[0030] h)5'(M)0(F)0(M)0(F)0(M)7(F)4(M) 10 3' (SEQ ID NO:59);

[0031] i)5'(M)0(F)0(M)6(F)1(M)1(F)4(M)9 3'(SEQ ID NO:60);

[0032] j)5'(M)0(F)0(M)0(F)0(M)8(F)4(M)9 3'(SEQ ID NO:61);

[0033] k)5'(M)0(F)0(M)0(F)0(M)6(F)4(M) 10 3' (SEQ ID NO: 62);

[0034] l)5'(M)0(F)0(M)5(F)1(M)1(F)4(M)9 3'(SEQ ID NO:63);

[0035] m)5'(M)0(F)0(M)0(F)0(M)7(F)4(M)9 3'(SEQ ID NO:64);

[0036] n)5'(M)0(F)0(M)0(F)0(M)7(F)5(M)9 3' (SEQ ID NO:65); and

[0037] o)5'(M)0(F)0(M)0(F)0(M)6(F)5(M)9 3' (SEQ ID NO: 66).

[0038] In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, up to seven nucleotides are modified with the third modification.

[0039] In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, up to four of the up to seven nucleotides modified with the third modification are positioned at position 2 to position 8 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, up to two of the up to seven nucleotides modified with the third modification are positioned consecutively.

[0040] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, three or four of the up to seven nucleotides modified with the third modification are positioned at a position selected from position 2, position 3, position 5, position 6, position 7, and position 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 least one of the up to seven nucleotides modified with the third modification is positioned 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 or three of the up to seven nucleotides modified with the third modification are positioned at a position selected from position 2, position 3, position 5, and position 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, three of the up to seven nucleotides modified with the third modification are positioned at a position selected from position 2, position 3, position 5, and position 6 from the first nucleotide at the 5' end of the antisense strand.

[0041] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, two of the up to seven nucleotides modified with the third modification are located at position 8 and position 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0042] In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, one or two of the up to seven nucleotides modified with the third modification are positioned at a position selected from position 14 and position 16 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, two of the up to seven nucleotides modified with the third modification are positioned at position 14 and position 16 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, up to seven nucleotides are modified with a 2'-F modification.

[0043] In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, up to six nucleotides are modified with the third modification. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, up to six nucleotides are modified with the 2'-F modification.

[0044] In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, up to five nucleotides are modified with the third modification. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, up to five nucleotides are modified with the 2'-F modification.

[0045] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most four nucleotides are modified with the third modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most four nucleotides are modified with the 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 the third modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at least three nucleotides are modified with the 2'-F modification.

[0046] In some embodiments, in the antisense strand of an 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 according to the following 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 0-16 and indicates the number of consecutive nucleotides modified with the modification, and wherein the antisense strand is any one of the following:

[0047] 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)1 5'(SEQ IDNO:14);

[0048] 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)1 5'(SEQ IDNO:15);

[0049] 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)1 5'(SEQ IDNO:16);

[0050] 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)1 5’(SEQ IDNO:17);

[0051] 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)1 5’(SEQ IDNO:18);

[0052] 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)1 5’(SEQ IDNO:20);

[0053] 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)1 5’(SEQ IDNO:21);

[0054] 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)1 5’(SEQ IDNO:22);

[0055] 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)1 5’(SEQ IDNO:23);

[0056] 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)1 5’(SEQID NO:24);

[0057] 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);

[0058] 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);

[0059] 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)1 5’(SEQID NO:30);

[0060] 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)1 5’(SEQID NO:50);

[0061] 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)1 5’(SEQID NO:45);

[0062] 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)1 5’(SEQID NO:46);

[0063] 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)1 5’(SEQID NO:47);

[0064] 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)1 5’(SEQID NO:48);

[0065] 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)1 5’(SEQID NO:49);

[0066] 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)1 5’(SEQID NO:37);

[0067] 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)1 5’(SEQID NO:38);

[0068] 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)1 5'(SEQID NO:39);

[0069] 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)1 5'(SEQID NO:40);

[0070] and

[0071] 25) 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)1 5' (SEQ ID NO: 42).

[0072] In some embodiments, the X1 nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure is 18-21, and the X2 nucleotides of the antisense strand of the isolated oligonucleotide of the present disclosure is 20-23. In some embodiments, the X1 nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure is 20 or 21, and the X2 nucleotides of the antisense strand of the isolated oligonucleotide of the present disclosure is 22 or 23. In some embodiments, the X2 nucleotides of the antisense strand of the isolated oligonucleotide of the present disclosure is equal to the X1 nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure plus 2.

[0073] In some embodiments, the sense strand of the oligonucleotide of the separation of the present disclosure comprises at least one nucleotide with a modified phosphate backbone. In some embodiments, the antisense strand of the oligonucleotide of the separation of the present disclosure comprises at least one nucleotide with a modified phosphate backbone. In some embodiments, in the sense strand or antisense strand of the oligonucleotide of the separation 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 replacing one or more oxygen atoms with a part or by forming a 2'-5' bond, wherein the part is bonded to the phosphorus atom in the phosphodiester bond by a carbon, nitrogen or sulfur atom in the part. In some embodiments, the modified phosphodiester bond comprises a thiophosphate, a dithiophosphate, a methylphosphonate, a phosphoramidate diester, a methylsulfonyl phosphoramidate or a phosphonoacetate.

[0074] In some embodiments, the isolated oligonucleotides of the present disclosure comprise one or more nucleotides containing non-natural bases, locked nucleotides or abasic nucleotides. In some embodiments, in the isolated oligonucleotides 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 its analog.

[0075] In some embodiments, the antisense strand of an isolated oligonucleotide of the present disclosure comprises an overhang at the 3' end having at least two single-stranded nucleotides.

[0076] In some embodiments, in the sense strand or antisense strand or both of the isolated oligonucleotides of the present disclosure, the terminal or internal nucleotide is connected to one or more targeting ligands. In some embodiments, the terminal or internal nucleotide is directly connected to one or more targeting ligands. In some embodiments, the terminal or internal nucleotide is indirectly connected to one or more targeting ligands through a joint. In some embodiments, one or more targeting ligands directly or indirectly connected to the terminal or internal nucleotide can also include a PK regulator. In some embodiments, the PK regulator is a competitive regulator, a positive allosteric regulator, a negative allosteric regulator or a neutral allosteric regulator. In some embodiments, the targeting ligand is selected from one or more of carbohydrates, peptides, lipids, antibodies or fragments thereof, aptamers, albumin, fibrinogen and folic acid.

[0077] In some embodiments, the antisense strand of an isolated oligonucleotide of the present disclosure is complementary to an mRNA, wherein sequence-specific hybridization of the antisense strand to the mRNA results in degradation of the mRNA.

[0078] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the field to which the present disclosure belongs. In this specification, unless the context clearly specifies otherwise, the singular form also includes the plural. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference. It is not recognized that the references cited herein are prior art of the claimed invention. In the case of a conflict, the present specification including the definition will prevail. In addition, materials, methods and examples are only illustrative and are not intended to be restrictive. In the case of a conflict between the chemical structure and the name of the compound disclosed herein, the chemical structure will prevail.

[0079] Other features and advantages of the disclosure will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 is a diagram depicting a set of siRNA 21 / 23mer molecules directed against target gene 1 with different chemical modifications as indicated.

[0082] Figure 2A-2C It is a depiction Figure 1 Figure 2 is a graph of the efficacy of siRNA molecules. The X-axis indicates increasing concentrations of siRNA and the Y-axis indicates the remaining % IC of human mRNA of target gene 1. 50 Values ​​are as indicated.

[0083] Figure 3 is a diagram depicting a set of siRNA 21 / 23mer molecules optimized with the indicated chemical modifications on the sense strand (eg, at position 7, position 8, and position 12).

[0084] Figure 4A-4E It is a depiction Figure 3 Figure 2 is a graph of the efficacy of siRNA molecules. The X-axis indicates increasing concentrations of siRNA and the Y-axis indicates the remaining % IC of human mRNA of target gene 1. 50 Values ​​are as indicated.

[0085] Figure 5 is a diagram depicting a set of siRNA 21 / 23mer molecules optimized with the indicated chemical modifications on the sense strand (eg, at positions 11 and 12).

[0086] Figure 6A-6C It is a depiction Figure 5 Figure 2 is a graph of the efficacy of siRNA molecules. The X-axis indicates increasing concentrations of siRNA and the Y-axis indicates the remaining % IC of human mRNA of target gene 1. 50 Values ​​are as indicated.

[0087] Figure 7 is a diagram depicting a set of siRNA 21 / 23mer molecules optimized with the indicated chemical modifications on the antisense strand (eg, at position 3, position 5, and positions 7-9).

[0088] Figure 8A-8F It is a depiction Figure 7 Figure 2 is a graph of the efficacy of siRNA molecules. The X-axis indicates increasing concentrations of siRNA and the Y-axis indicates the remaining % IC of human mRNA of target gene 1. 50 Values ​​are as indicated.

[0089] Fig. 9 is a diagram depicting a set of siRNA 21 / 23mer molecules optimized with chemical modifications on the antisense strand (eg, at the seed region and position 16) as indicated.

[0090] Figure 10A-10C It is a depiction Fig. 9 Figure 2 is a graph of the efficacy of siRNA molecules. The X-axis indicates increasing concentrations of siRNA and the Y-axis indicates the remaining % IC of human mRNA of target gene 1. 50 Values ​​are as indicated.

[0091] Fig.11 is a diagram depicting a set of siRNA 21 / 23mer molecules optimized with chemical modifications as indicated on both the sense and antisense strands.

[0092] Figures 12A-12F It is a depiction Fig.11 Figure 2 is a graph of the efficacy of siRNA molecules. The X-axis indicates increasing concentrations of siRNA and the Y-axis indicates the remaining % IC of human mRNA of target gene 1. 50 Values ​​are as indicated.

[0093] Fig.13 is a graph depicting a set of siRNA 20 / 22 molecules optimized with the indicated chemical modifications.

[0094] Figure 14A-Figure 14C It is a depiction Fig.13 Figure 2 is a graph of the efficacy of siRNA molecules. The X-axis indicates increasing concentrations of siRNA and the Y-axis indicates the remaining % IC of human mRNA of target gene 1. 50 Values ​​are as indicated.

[0095] Fig.15 is a diagram depicting a set of siRNA 20 / 22mer molecules optimized with the indicated chemical modifications on the sense strand (eg, at position 6, position 7, and position 11).

[0096] Figure 16A-16E It is a depiction Fig.15 Figure 2 is a graph of the efficacy of siRNA molecules. The X-axis indicates increasing concentrations of siRNA and the Y-axis indicates the remaining % IC of human mRNA of target gene 1. 50 Values ​​are as indicated.

[0097] Fig.17 is a diagram depicting a set of siRNA 20 / 22mer molecules optimized with the indicated chemical modifications on the sense strand (eg, at positions 10 and 11).

[0098] Figures 18A-18C It is a depiction Fig.17 Figure 2 is a graph of the efficacy of siRNA molecules. The X-axis indicates increasing concentrations of siRNA and the Y-axis indicates the remaining % IC of human mRNA of target gene 1. 50Values ​​are as indicated.

[0099] Fig.19 is a diagram depicting a set of siRNA 20 / 22mer molecules optimized with the indicated chemical modifications on the antisense strand (eg, at position 3, position 5, and positions 7-9).

[0100] Figures 20A-20F It is a depiction Fig.19 Figure 2 is a graph of the efficacy of siRNA molecules. The X-axis indicates increasing concentrations of siRNA and the Y-axis indicates the remaining % IC of human mRNA of target gene 1. 50 Values ​​are as indicated.

[0101] Fig.21 is a diagram depicting a set of siRNA 20 / 22mer molecules optimized with chemical modifications on the antisense strand as indicated.

[0102] Figure 22A-22C It is a depiction Fig.21 Figure 2 is a graph of the efficacy of siRNA molecules. The X-axis indicates increasing concentrations of siRNA and the Y-axis indicates the remaining % IC of human mRNA of target gene 1. 50 Values ​​are as indicated.

[0103] Fig.23 is a diagram depicting a set of siRNA 20 / 22mer molecules optimized with chemical modifications as indicated on both the sense and antisense strands.

[0104] Figure 24A-24F It is a depiction Fig.23 Figure 2 is a graph of the efficacy of siRNA molecules. The X-axis indicates increasing concentrations of siRNA and the Y-axis indicates the remaining % IC of human mRNA of target gene 1. 50 Values ​​are as indicated.

[0105] Details

[0106] 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, each of the remaining nucleotides is independently modified with a second modification, and X1 is an integer selected from 13-36, wherein 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, each of the remaining nucleotides is independently modified with a fourth modification, and X2 is an integer selected from 18-31, wherein the third modification and the fourth modification are different.

[0107] In some embodiments, the first modification is a modification selected from 2'-F modification, 2'-CN modification, 2'-N3 modification, 2'-deoxy modification, and equivalents thereof, and combinations thereof at the 2'-position of the sugar portion of at least one nucleotide. In some embodiments, the first modification is 2'-F modification, 2'-CN modification, 2'-N3 modification, or 2'-deoxy modification, or a stereoisomer thereof. In some embodiments, the first modification is 2'-F modification, 2'-CN modification, or 2'-N3 modification, or a stereoisomer thereof. In some embodiments, the first modification is 2'-F modification, 2'-CN modification, or 2'-N3 modification, or a stereoisomer thereof. In some embodiments, the first modification is 2'-F modification or a stereoisomer thereof.

[0108] In some embodiments, the second modification is a modification selected from 2'-C1-C6 alkyl, 2'-OR modification, 2'-amino and morpholino substitution, and equivalents and combinations thereof at the 2'-position of the sugar moiety of one or more of the remaining nucleotides, in which R is a C1-C6 alkyl optionally substituted with: C1-C6 alkoxy, acetamide, phenyl, or a heteroaryl comprising a 5- or 6-membered ring and 1 or 2 heteroatoms selected from N, O, and S. 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.

[0109] 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.

[0110] In some embodiments, the third modification is a modification selected from 2'-F modification, 2'-CN modification, 2'-N3 modification, 2'-deoxy modification, and equivalents thereof, and combinations thereof at the 2'-position of the sugar portion of at least one nucleotide. In some embodiments, the third modification is 2'-F modification, 2'-CN modification, 2'-N3 modification, or 2'-deoxy modification, or a stereoisomer thereof. In some embodiments, the third modification is 2'-F modification, 2'-CN modification, or 2'-N3 modification, or a stereoisomer thereof. In some embodiments, the third modification is 2'-F modification, 2'-CN modification, or 2'-N3 modification, or a stereoisomer thereof. In some embodiments, the third modification is 2'-F modification or a stereoisomer thereof.

[0111] In some embodiments, the fourth modification is a modification selected from 2'-C1-C6 alkyl, 2'-OR modification, 2'-amino and morpholino substitution, and equivalents thereof and combinations thereof at the 2'-position of the sugar moiety of one or more of the remaining nucleotides, in which R is a C1-C6 alkyl optionally substituted with: C1-C6 alkoxy, acetamide, phenyl, or a heteroaryl comprising a 5- or 6-membered ring and 1 or 2 heteroatoms selected from N, O, and S. 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.

[0112] In some embodiments, the third modification is a 2'-F modification or a stereoisomer thereof, and the fourth modification is a 2'-O-methyl or 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.

[0113] In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least three nucleotides are modified with a first modification. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least two of the at least three nucleotides modified with a first modification are positioned consecutively. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least three of the at least three nucleotides modified with a first modification are positioned consecutively.

[0114] In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least four nucleotides are modified with a first modification. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least three of the at least four nucleotides modified with a first modification are positioned consecutively. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least four of the at least four nucleotides modified with a first modification are positioned consecutively.

[0115] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least five nucleotides are modified with the first modification. In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least three of the at least five nucleotides modified with the first modification are positioned consecutively. At least four of the at least five nucleotides modified with the first modification are positioned consecutively.

[0116] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least three nucleotides, at least four nucleotides, or at least five nucleotides modified with the first modification are positioned at position 10 to position 15 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0117] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, two of the at least three nucleotides modified with the first modification are positioned at a position selected from position 10, position 11, position 12, and position 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, three of the at least three nucleotides modified with the first modification are positioned at a position selected from position 10, position 11, position 12, and position 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, one of the at least three nucleotides modified with the first modification is positioned at position 11 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0118] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, three of the at least three nucleotides modified with the first modification are positioned at position 11, position 12, and position 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, three of the at least three nucleotides modified with the first modification are positioned at position 12, position 13, and position 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, three of the at least three nucleotides modified with the first modification are positioned at position 10, position 11, and position 12 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0119] In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, one of the at least four nucleotides modified with the first modification is positioned at position 10 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, one of the at least four nucleotides modified with the first modification is positioned at position 11 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, one of the at least four nucleotides modified with the first modification is positioned at position 12 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, one of the at least four nucleotides modified with the first modification is positioned at position 13 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, one of the at least four nucleotides modified with the first modification is positioned at position 14 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, one of the at least four nucleotides modified with the first modification is positioned at position 15 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand.

[0120] In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least four nucleotides modified with the first modification are positioned at position 11, position 12, position 13, and position 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 an isolated oligonucleotide of the present disclosure, at least four nucleotides modified with the first modification are positioned at position 10, position 11, position 12, and position 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 an isolated oligonucleotide of the present disclosure, at least four nucleotides modified with the first modification are positioned at position 11, position 12, position 13, and position 15 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0121] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, two of the at least four nucleotides modified with the first modification are located at position 10 to position 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0122] In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least three nucleotides modified with a first modification are not all positioned consecutively. In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least three nucleotides, at least four nucleotides, or at least five nucleotides are modified with a 2'-F modification.

[0123] In some embodiments, in the sense strand of the isolated oligonucleotide of the present disclosure, at least three nucleotides modified with the first modification are positioned at position 11, position 12, position 13, and position 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).

[0124] In some embodiments, in the sense strand of an isolated oligonucleotide of the present disclosure, at least four nucleotides modified with a first modification are positioned at position 10, position 12, position 13, and position 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 an isolated oligonucleotide of the present disclosure, at least four nucleotides modified with a first modification are positioned at position 11, position 12, position 13, and position 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 an isolated oligonucleotide of the present disclosure, at least four nucleotides modified with a first modification are positioned at position 11, position 10, position 11, position 12, and position 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 an isolated oligonucleotide of the present disclosure, at least five nucleotides modified with the first modification are positioned at position 10, position 11, position 12, position 13, and position 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 an isolated oligonucleotide of the present disclosure, at least five nucleotides modified with the first modification are positioned at position 10, position 11, position 12, position 13, and position 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).

[0125] In some embodiments, the sense strand of an isolated oligonucleotide of the present disclosure comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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 0-16 and indicates the number of consecutive nucleotides modified with the modification, and wherein the sense strand is any one of:

[0126] a)5'(M)0(F)0(M)0(F)0(M)8(F)3(M) 10 3' (SEQ ID NO:52);

[0127] b)5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3' (SEQ ID NO:53);

[0128] c)5'(M)0(F)0(M)0(F)0(M)7(F)3(M) 10 3' (SEQ ID NO:54);

[0129] d)5'(M)0(F)0(M)5(F)1(M)1(F)2(M) 11 3' (SEQ ID NO:55);

[0130] e)5'(M)0(F)0(M)6(F)1(M)1(F)2(M) 11 3' (SEQ ID NO:56);

[0131] f)5'(M)6(F)1(M)1(F)2(M)1(F)1(M)9 3' (SEQ ID NO:57);

[0132] g)5'(M)5(F)1(M)1(F)2(M)1(F)1(M)9 3' (SEQ ID NO:58);

[0133] h)5'(M)0(F)0(M)0(F)0(M)7(F)4(M) 10 3' (SEQ ID NO:59);

[0134] i)5'(M)0(F)0(M)6(F)1(M)1(F)4(M)9 3'(SEQ ID NO:60);

[0135] j)5'(M)0(F)0(M)0(F)0(M)8(F)4(M)9 3'(SEQ ID NO:61);

[0136] k)5'(M)0(F)0(M)0(F)0(M)6(F)4(M) 10 3' (SEQ ID NO: 62);

[0137] l)5'(M)0(F)0(M)5(F)1(M)1(F)4(M)9 3'(SEQ ID NO:63);

[0138] m)5'(M)0(F)0(M)0(F)0(M)7(F)4(M)9 3'(SEQ ID NO:64);

[0139] n)5'(M)0(F)0(M)0(F)0(M)7(F)5(M)9 3' (SEQ ID NO:65); and

[0140] o)5'(M)0(F)0(M)0(F)0(M)6(F)5(M)9 3' (SEQ ID NO: 66).

[0141] 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 according to 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.

[0142] 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 according to 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.

[0143] 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 according to 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.

[0144] 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 according to 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.

[0145] 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 according to 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.

[0146] 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 according to the following formula: 5'(M)6(F)1(M)1(F)2(M)1(F)1(M)9 3' (SEQ ID NO:57), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0147] 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 according to the following formula: 5'(M)5(F)1(M)1(F)2(M)1(F)1(M)9 3' (SEQ ID NO:58), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0148] 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 according to 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.

[0149] 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 according to the formula: 5'(M)0(F)0(M)6(F)1(M)1(F)4(M)9 3' (SEQ ID NO:60), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0150] 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 according to the following formula: 5'(M)0(F)0(M)0(F)0(M)8(F)4(M)9 3' (SEQ ID NO:61), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0151] 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 according to 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.

[0152] 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 according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)4(M)9 3' (SEQ ID NO:63), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0153] 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 according to the formula: 5'(M)0(F)0(M)0(F)0(M)7(F)4(M)9 3' (SEQ ID NO:64), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0154] 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 according to the formula: 5'(M)0(F)0(M)0(F)0(M)7(F)5(M)9 3' (SEQ ID NO:65), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0155] 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 according to the formula: 5'(M)0(F)0(M)0(F)0(M)6(F)5(M)9 3' (SEQ ID NO:66), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0156] In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, up to seven nucleotides are modified with the third modification.

[0157] In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, up to four of up to seven nucleotides modified with the third modification are positioned at position 2 to position 8 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, at least one of up to seven nucleotides is modified, wherein the third modification is positioned at position 2 from the first nucleotide at the 5' end of the antisense strand.

[0158] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most two of the at most seven nucleotides modified with the third modification are positioned continuously. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most two of the at most seven nucleotides modified with the third modification are positioned continuously at position 2 and position 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 two of the at most seven nucleotides modified with the third modification are positioned continuously at position 5 and position 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 two of the at most seven nucleotides modified with the third modification are positioned continuously at position 6 and position 7 from the first nucleotide at the 5' end of the antisense strand.

[0159] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, up to three of the up to seven nucleotides modified with the third modification are positioned consecutively. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, up to three of the up to seven nucleotides modified with the third modification are positioned consecutively at position 4, position 5, and position 6 from the first nucleotide at the 5' end of the antisense strand.

[0160] In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, three or four of the up to seven nucleotides modified with the third modification are positioned at a position selected from position 2, 3, 5, 6, 7, and 8 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, three of the up to seven nucleotides modified with the third modification are positioned at position 2, position 5, and position 6 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, three of the up to seven nucleotides modified with the third modification are positioned at position 2, position 3, and position 6 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, three of the up to seven nucleotides modified with the third modification are positioned at position 2, position 6, and position 7 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, three of the up to seven nucleotides modified with the third modification are located at position 2, position 6, and position 8 from the first nucleotide at the 5' end of the antisense strand.

[0161] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at least one of the up to seven nucleotides modified with the third modification is positioned 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 or three of the up to seven nucleotides modified with the third modification are positioned at positions selected from position 2, position 3, position 5, and position 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, three of the up to seven nucleotides modified with the third modification are positioned at positions selected from position 2, position 3, position 5, and position 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, two of the up to seven nucleotides modified with the third modification are positioned at positions 2 and 6 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, two of the up to seven nucleotides modified with the third modification are positioned at position 2 and position 5 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, two of the up to seven nucleotides modified with the third modification are positioned at position 2 and position 3 from the first nucleotide at the 5' end of the antisense strand.

[0162] In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, three of up to seven nucleotides modified with the third modification are positioned at position 2, position 3, and position 6 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, three of up to seven nucleotides modified with the third modification are positioned at position 2, position 3, and position 5 from the first nucleotide at the 5' end of the antisense strand. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, three of up to seven nucleotides modified with the third modification are positioned at position 2, position 5, and position 6 from the first nucleotide at the 5' end of the antisense strand.

[0163] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, two of the up to seven nucleotides modified with the third modification are located at position 8 and position 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0164] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, one or two of the up to seven nucleotides modified with the third modification are positioned at a position selected from position 14 and position 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 the up to seven nucleotides modified with the third modification are positioned at position 14 and position 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, up to 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 the up to seven nucleotides modified with the third modification is positioned 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 the up to seven nucleotides modified with the third modification are positioned at position 14 and position 16 from the first nucleotide at the 5' end of the antisense strand.

[0165] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, up to seven nucleotides modified with the third modification are positioned at a position selected from position 2, position 3, position 5, position 6, position 7, position 8, position 9, position 14, and position 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, up to seven nucleotides modified with the third modification are positioned at position 2, position 3, position 6, position 14, and position 16 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, up to seven nucleotides modified with the third modification are positioned at position 2, position 6, position 7, position 14, and position 16 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, up to seven nucleotides modified with the third modification are positioned at position 2, position 3, position 6, position 8, position 9, position 14, and position 16 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, up to seven nucleotides modified with the third modification are positioned at position 2, position 3, position 5, position 8, position 9, position 14, and position 16 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, up to seven nucleotides modified with the third modification are positioned at position 2, position 3, position 5, position 7, position 9, position 14, and position 16 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, up to seven nucleotides modified with the third modification are positioned at position 2, position 3, position 5, position 7, position 10, position 14 and position 16 from the first nucleotide at the 5' end of the antisense strand (e.g., SEQ ID NO: 25, SEQ ID NO: 42).

[0166] In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, up to six nucleotides are modified with the third modification. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, up to six nucleotides are modified with the 2'-F modification.

[0167] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, up to five nucleotides are modified with the third modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, up to five nucleotides are modified with the 2'-F modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, up to five nucleotides modified with the third modification are positioned at a position selected from position 2, position 3, position 5, position 6, position 7, position 8, position 9, position 14, and position 16 from the first nucleotide at the 5' end of the antisense strand.

[0168] In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, at position 2, position 3, position 6, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to five nucleotides are modified with the third modification. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, at position 2, position 6, position 7, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to five nucleotides are modified with the third modification. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, at position 2, position 3, position 6, position 8, position 9, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to six nucleotides are modified with the third modification. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, at position 2, position 3, position 5, position 8, position 9, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to six nucleotides are modified with the third modification. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, at position 2, position 3, position 5, position 7, position 9, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to seven nucleotides are modified with the third modification. In some embodiments, in the antisense strand of an isolated oligonucleotide of the present disclosure, at position 2, position 3, position 5, position 7, position 10, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to seven nucleotides are modified with the third modification.

[0169] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, up to five nucleotides modified with the third modification are positioned at position 2, position 5, position 6, position 14, and position 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, up to five nucleotides modified with the third modification are positioned at position 2, position 3, position 6, position 14, and position 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, up to five nucleotides modified with the third modification are positioned at position 2, position 6, position 8, position 14, and position 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, up to five nucleotides modified with the third modification are positioned at position 2, position 6, position 9, position 14, and position 16 from the first nucleotide at the 5' end of the antisense strand.

[0170] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, up to four nucleotides are modified with the third modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, up to four nucleotides are modified with 2'-F modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, up to four nucleotides modified with the third modification are positioned at positions selected from the first nucleotide at the 5' end of the antisense strand, position 2, position 3, position 5, position 6, position 7, position 8, position 9, position 14 and position 16. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, up to four nucleotides modified with the third modification are positioned at positions 2, position 6, position 14 and position 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, up to four nucleotides modified with the third modification are positioned at positions 2, position 5, position 14 and position 16 from the first nucleotide at the 5' end of the antisense strand.

[0171] In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most three nucleotides are modified with the third modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at least three nucleotides are modified with the 2'-F modification. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most three nucleotides modified with the third modification are positioned at positions selected from the first nucleotide at the 5' end of the antisense strand, position 2, position 3, position 5, position 6, position 7, position 8, position 9, position 14, and position 16. In some embodiments, in the antisense strand of the isolated oligonucleotide of the present disclosure, at most three nucleotides modified with the third modification are positioned at positions 2, position 6, and position 14 from the first nucleotide at the 5' end of the antisense strand.

[0172] 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 according to the following formula: 3'

[0173] (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 0-16 and indicates the number of consecutive nucleotides modified with the modification, and wherein the antisense strand is any one of the following:

[0174] 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)1 5'(SEQ IDNO:14);

[0175] 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)1 5'(SEQ IDNO:15);

[0176] 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)1 5’(SEQ IDNO:16);

[0177] 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)1 5’(SEQ IDNO:17);

[0178] 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)1 5’(SEQ IDNO:18);

[0179] 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)1 5’(SEQ IDNO:20);

[0180] 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)1 5’(SEQ IDNO:21);

[0181] 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)1 5’(SEQ IDNO:22);

[0182] 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)1 5’(SEQ IDNO:23);

[0183] 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)1 5’(SEQID NO:24);

[0184] 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);

[0185] 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);

[0186] 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)1 5’(SEQID NO:30);

[0187] 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)1 5’(SEQID NO:50);

[0188] 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)1 5’(SEQID NO:45);

[0189] 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)1 5’(SEQID NO:46);

[0190] 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)1 5’(SEQID NO:47);

[0191] 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)1 5’(SEQID NO:48);

[0192] 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)1 5’(SEQID NO:49);

[0193] 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)1 5’(SEQID NO:37);

[0194] 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)1 5'(SEQID NO:38);

[0195] 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)1 5'(SEQID NO:39);

[0196] 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)1 5'(SEQID NO:40);

[0197] and

[0198] 25) 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)1 5' (SEQ ID NO: 42).

[0199] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO: 14), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0200] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:15), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0201] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following formula: 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)1 5' (SEQ ID NO:16), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0202] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO: 17), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0203] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO: 18), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0204] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:20), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0205] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:21), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0206] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the formula: 3'(M)0(F)0

[0207] (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)1 5' (SEQ ID NO: 22), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0208] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:23), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0209] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:24), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0210] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:25), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0211] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to 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)1 5' (SEQ ID NO:27), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0212] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:30), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0213] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to 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)1 5' (SEQ ID NO:50), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0214] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:45), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0215] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to 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)1 5' (SEQ ID NO:46), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0216] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:47), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0217] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:48), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0218] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:49), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0219] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:37), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0220] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:38), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0221] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:39), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0222] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification 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)1 5' (SEQ ID NO:40), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0223] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:41), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0224] In some embodiments of the isolated oligonucleotides of the present disclosure, the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5' (SEQ ID NO:42), wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0225] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with the first modification positioned at position 11, position 12, position 13, and position 15 from the nucleotide that is complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with the first modification positioned at position 2, position 6, position 14, and position 16 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand.

[0226] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with the first modification positioned at position 11, position 12, and position 13 from the nucleotide that is complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with the first modification positioned at position 2, position 6, position 14, and position 16 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand.

[0227] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with the first modification positioned at position 11, position 12, position 13, and position 14 from the nucleotide that is complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with the first modification positioned at position 2, position 6, position 14, and position 16 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand.

[0228] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with the first modification positioned at position 10, position 11, position 12, and position 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 the first modification positioned at position 2, position 6, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0229] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with the first modification positioned at position 10, position 11, position 12, position 13, and position 14 from the nucleotide that is complementary to the first nucleotide at the 5' end of the sense strand, and the antisense strand comprises nucleotides modified with the first modification positioned at position 2, position 6, position 14, and position 16 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand.

[0230] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with the first modification positioned 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 the first modification positioned at positions 2, 6, 14, and 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0231] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 10, position 12, position 13, and position 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 the first modification positioned at position 2, position 6, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0232] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 10, position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 6, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0233] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 5, position 6, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0234] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 5, position 6, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0235] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 3, position 6, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0236] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 6, position 7, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0237] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 6, position 8, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0238] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 6, position 9, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0239] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 6, position 8, position 9, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0240] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 5, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0241] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 6, and position 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0242] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 10, position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 3, position 6, position 8, position 9, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0243] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 10, position 11, position 12, and position 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 the first modification positioned at position 2, position 3, position 6, position 8, position 9, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0244] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 10, position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 3, position 5, position 8, position 9, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0245] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 10, position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 3, position 5, position 7, position 9, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0246] In some embodiments of the isolated oligonucleotides of the present disclosure, the sense strand comprises nucleotides modified with a modification positioned at position 10, position 11, position 12, position 13, and position 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 the first modification positioned at position 2, position 3, position 5, position 7, position 10, position 14, and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0247] 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 according to the following formula: 5'(M)0(F)0(M)6(F)1(M)1(F)4(M)93'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0248] 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 according to the following formula: 5'(M)0(F)0(M)0(F)0(M)8(F)4(M)93'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0249] 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 according to the following formula: 5'(M)0(F)0(M)6(F)1(M)1(F)4(M)93'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0250] 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: 5'(M)0(F)0(M)6(F)1(M)1(F)4(M)9 3'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the formula:

[0251] 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0252] 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 according to the following formula: 5'(M)0(F)0(M)6(F)1(M)1(F)4(M)93'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0253] 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 according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0254] 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 according to the formula: 5'(M)0(F)0(M)0(F)0(M)7(F)3(M) 103'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0255] 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 according to the formula: 5'(M)0(F)0(M)0(F)0(M)6(F)4(M) 10 3'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0256] 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 according to the following formula: 5'(M)0(F)0(M)0(F)0(M)7(F)4(M)93'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0257] 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 according to the formula: 5'(M)0(F)0(M)0(F)0(M)6(F)5(M)93'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0258] 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 according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)2(M) 11 3'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0259] 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 according to the following formula: 5'(M)5(F)1(M)1(F)2(M)1(F)1(M)93'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0260] 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 according to the following formula: 5'(M)0(F)0(M)5(F)1(M)1(F)4(M)93'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0261] 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 according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 103'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0262] 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 according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0263] 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 according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0264] 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 according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0265] 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 according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0266] 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 according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0267] 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 according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0268] 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 according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 103'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0269] 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 according to the formula: 5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0270] 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 according to the following formula: 5'(M)0(F)0(M)5(F)1(M)1(F)4(M)93'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0271] 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 according to the formula: 5'(M)0(F)0(M)0(F)0(M)7(F)4(M)93'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0272] 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 according to the following formula: 5'(M)0(F)0(M)5(F)1(M)1(F)4(M)93'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0273] 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 according to the following formula: 5'(M)0(F)0(M)5(F)1(M)1(F)4(M)93'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0274] 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 according to the following formula: 5'(M)0(F)0(M)5(F)1(M)1(F)4(M)93'; and the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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)1 5', wherein M is a 2'-O-methyl modified nucleotide and F is a 2'-F modified nucleotide.

[0275] In some embodiments, the X1 nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure are 18-21, and the X2 nucleotides of the antisense strand of the isolated oligonucleotide of the present disclosure are 20-23. In some embodiments, the X1 nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure are 20 or 21, and the X2 nucleotides of the antisense strand of the isolated oligonucleotide of the present disclosure are 22 or 23. In some embodiments, the X2 nucleotides of the antisense strand of the isolated oligonucleotide of the present disclosure is equal to the X1 nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure plus 2. In some embodiments, the X1 nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure is 21, and the X2 nucleotides of the antisense strand of the isolated oligonucleotide of the present disclosure is 23. In some embodiments, the X1 nucleotides of the sense strand of the isolated oligonucleotide of the present disclosure is 20, and the X2 nucleotides of the antisense strand of the isolated oligonucleotide of the present disclosure is 22.

[0276] In some embodiments, the sense strand of the oligonucleotide of the separation of the present disclosure comprises at least one nucleotide with a modified phosphate backbone. In some embodiments, the antisense strand of the oligonucleotide of the separation of the present disclosure comprises at least one nucleotide with a modified phosphate backbone. In some embodiments, in the sense strand or antisense strand of the oligonucleotide of the separation 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 replacing one or more oxygen atoms with a part or by forming a 2'-5' bond, wherein the part is bonded to the phosphorus atom in the phosphodiester bond by a carbon, nitrogen or sulfur atom in the part. In some embodiments, the modified phosphodiester bond comprises phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate diester, methylsulfonylphosphoramidate or phosphonoacetate.

[0277] In some embodiments, the isolated oligonucleotides of the present disclosure comprise one or more nucleotides containing non-natural bases, locked nucleotides or abasic nucleotides. In some embodiments, in the isolated oligonucleotides 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 its analog.

[0278] In some embodiments, the antisense strand of an isolated oligonucleotide of the present disclosure comprises at least two single-stranded nucleotides at the 3' terminus. In some embodiments, the antisense strand of an isolated oligonucleotide of the present disclosure comprises two single-stranded nucleotides at the 3' terminus.

[0279] Targeting ligands

[0280] In some embodiments, in the sense strand or antisense strand or both of the isolated oligonucleotide of the present disclosure, the terminal or internal nucleotide is connected to the 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 in at least two single-stranded nucleotides at the 3' end of the antisense strand of the isolated oligonucleotide of the present disclosure.

[0281] 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, an albumin, a fibrinogen, and a folic acid. In some embodiments, the targeting ligand binds to a surface protein on a cell expressing a target mRNA of an isolated oligonucleotide of the present disclosure. In some embodiments, the targeting ligand mediates entry of an isolated oligonucleotide of the present disclosure into a cell expressing a target mRNA of an isolated oligonucleotide of the present disclosure.

[0282] In some embodiments, the targeting ligand is a therapeutic ligand. In some embodiments, the targeting ligand is a therapeutic antibody.

[0283] In some embodiments, the targeting ligand is attached to the isolated oligonucleotide of the present disclosure via a linker. In some embodiments, the linker is any one of a protein, DNA, RNA, or chemical compound. In some embodiments, the isolated oligonucleotide of the present disclosure, the linker, and the targeting ligand form a scaffold. As used herein, the term "scaffold" refers to a compound or complex comprising a linker of the present disclosure, wherein the linker is covalently attached to the ligand or the isolated oligonucleotide or both.

[0284] In some embodiments, the isolated oligonucleotide of the present disclosure, the linker and the targeting ligand form a conjugate. As used herein, the term "conjugate" refers to a compound or complex comprising an isolated oligonucleotide covalently attached to a ligand via a linker of the present disclosure.

[0285] 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), is capable of mediating its entry into a target site (e.g., a target cell or target tissue) or promoting or allowing its delivery to a target site (e.g., a target cell or target tissue). In some embodiments, the targeting ligand comprises a sugar ligand moiety (e.g., N-acetylgalactosamine (GalNAc)) that can direct the uptake of the oligonucleotide into the liver.

[0286] In some embodiments, the targeting ligand binds to the asialoglycoprotein receptor (ASGPR).In some embodiments, the targeting ligand binds to (eg, via the ASGPR) the liver, such as a parenchymal cell of the liver.

[0287] Suitable targeting ligands include, but are not limited to, those disclosed in Winkler (Ther. Deliv., 2013, 4(7):791-809), PCT Patent Application Publication Nos. WO / 2016 / 100401, WO / 2012 / 089352, and WO / 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.

[0288] In some embodiments, the targeting ligand comprises a carbohydrate moiety.

[0289] As used herein, "carbohydrate moiety" refers to a moiety comprising one or more monosaccharide units, each of which has at least six carbon atoms (which may be linear, branched or cyclic), each of which is bonded to an oxygen, nitrogen or sulfur atom. In some embodiments, the carbohydrate moiety comprises a monosaccharide, a disaccharide, a trisaccharide or a tetrasaccharide. In some embodiments, the carbohydrate moiety comprises an oligosaccharide containing from about 4-9 monosaccharide units. In some embodiments, the carbohydrate moiety comprises a polysaccharide (e.g., starch, glycogen, cellulose or polysaccharide gum).

[0290] In some embodiments, the carbohydrate moiety comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide. In some embodiments, the carbohydrate moiety comprises an oligosaccharide (e.g., comprising from about four to about nine monosaccharide units). In some embodiments, the carbohydrate moiety comprises a polysaccharide (e.g., starch, glycogen, cellulose, or a polysaccharide gum).

[0291] In some embodiments, the ligand is capable of binding to a human asialoglycoprotein receptor (ASGPR), such as human asialoglycoprotein receptor 2 (ASGPR2).

[0292] In some embodiments, the carbohydrate moiety comprises a sugar (e.g., one, two, or three sugars). In some embodiments, the carbohydrate moiety comprises galactose or a derivative thereof (e.g., one, two, or three galactoses or derivatives thereof). In some embodiments, the carbohydrate moiety comprises N-acetylgalactosamine or a derivative thereof (e.g., one, two, or three N-acetylgalactosamine or a derivative thereof). In some embodiments, the carbohydrate moiety comprises N-acetyl-D-galactosamine or a derivative thereof (e.g., one, two, or three N-acetyl-D-galactosamine or a derivative thereof).

[0293] In some embodiments, the carbohydrate moiety comprises N-acetylgalactosamine (e.g., one, two, or three N-acetylgalactosamines). In some embodiments, the carbohydrate moiety comprises N-acetyl-D-galactosamine (e.g., one, two, or three N-acetyl-D-galactosamines).

[0294] 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-galactosamine) to a linker.

[0295] In some embodiments, the linker comprises a thioether (e.g., thiosuccinimide or a hydrolyzed analog thereof), a disulfide, a triazole, a thiophosphate, a phosphodiester, an ester, an amide, or any combination thereof. In some embodiments, the linker is a three-branched connecting portion. Suitable targeting ligands include, but are not limited to, those disclosed in PCT Application 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.

[0296] In some embodiments, the targeting ligand comprises a lipid or lipid moiety (e.g., one, two, or three lipid moieties). In some embodiments, the lipid moiety comprises C8-C24 fatty acids, cholesterol, vitamins, sterols, phospholipids, or any combination thereof (e.g., one, two, or three of them).

[0297] In some embodiments, the targeting ligand comprises a peptide or peptide portion (e.g., one, two, or three peptide portions). In some embodiments, the peptide portion comprises an integrin, insulin, a glucagon-like peptide, or any combination thereof (e.g., one, two, or three thereof). In some embodiments, the targeting ligand comprises an antibody or antibody portion (e.g., transferrin). In some embodiments, the targeting ligand comprises one, two, or three antibody portions (e.g., transferrin).

[0298] In some embodiments, the targeting ligand comprises an oligonucleotide (e.g., an aptamer or a CpG). In some embodiments, the targeting ligand comprises one, two, or three oligonucleotides (e.g., an aptamer or a CpG).

[0299] In some embodiments, the ligand comprises: one, two, or three sugars (e.g., N-acetyl-D-galactosamine); one, two, or three lipid moieties; one, two, or three peptide moieties; one, two, or three antibody moieties; one, two, or three oligonucleotides; or any combination thereof.

[0300] In some embodiments, a linker is attached to an isolated oligonucleotide of the disclosure via a phosphate group or an analog of a phosphate group in the isolated oligonucleotide.

[0301] Immunostimulatory oligonucleotides

[0302] In some embodiments, the isolated oligonucleotides of the present disclosure linked to lipids, carbohydrates or peptides that may be immunostimulatory include immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded) that are capable of inducing an immune response when administered to a subject (which may be a mammal or other patient). ISSs include, for example, certain palindromes that result in hairpin secondary structures (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 (such as poly-G domains, see WO 96 / 11266, which is incorporated by reference in its entirety).

[0303] The immune response may be an innate immune response or an adaptive immune response. The vertebrate immune system is further divided into an innate immune system and an acquired adaptive immune system, which is further divided into humoral cellular components. In some embodiments, the immune response may be mucosal.

[0304] In some embodiments, the immunostimulatory isolated oligonucleotide is immunostimulatory only when administered in combination with a lipid particle, but not when administered in its "free form." Such oligonucleotides are considered immunostimulatory.

[0305] When it is not necessary for the immunostimulatory isolated oligonucleotide to specifically bind to the target polynucleotide and reduce the expression of the target polynucleotide so as to stimulate an immune response, the immunostimulatory isolated oligonucleotide is considered to be non-sequence specific. Therefore, some immunostimulatory isolated oligonucleotides may contain sequences corresponding to regions of naturally occurring genes or mRNAs, but they can still be considered to be non-sequence specific immunostimulatory nucleic acids.

[0306] In some embodiments, the oligonucleotide of immunostimulatory separation comprises at least one CpG dinucleotide.Oligonucleotide or CpG dinucleotide can be non-methylated or methylated.In another embodiment, the oligonucleotide of immunostimulatory separation comprises at least one CpG dinucleotide with methylated cytosine.In some embodiments, nucleic acid comprises single CpG dinucleotide, and the cytosine in wherein said CpG dinucleotide is methylated.In selectable embodiment, the oligonucleotide of separation comprises at least two CpG dinucleotide, and at least one cytosine in wherein said CpG dinucleotide is methylated.In another embodiment, each cytosine in the CpG dinucleotide present in the sequence is methylated.In another embodiment, the oligonucleotide of separation comprises more than one CpG dinucleotide, and at least one in wherein said CpG dinucleotide comprises methylated cytosine.

[0307] Attachments between linker units, isolated oligonucleotides and ligands

[0308] In some embodiments, the attachment between the Linker unit and the nucleic acid agent is a bond.

[0309] In some embodiments, the attachment between the Linker unit and the nucleic acid agent is a moiety (eg, a moiety comprising a cleavable group).

[0310] In some embodiments, the attachment between the Linker unit and the Ligand is a bond.

[0311] In some embodiments, the attachment between the Linker unit and the Ligand is a moiety (eg, a moiety comprising a cleavable group).

[0312] In some embodiments, the attachment between the Linker Unit and the Ligand comprises a -C(=O)- linked to the Linker Unit.

[0313] The 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 a chain of atoms such as, but not limited to, alkylene, alkenylene, alkynylene, arylalkylene, arylalkenylene, arylalkynylene, heteroarylalkylene, heteroarylalkenylene, heteroarylalkynylene, heterocyclylalkyl ... cyclylalkylene), heterocyclylalkenylene, heterocyclylalkynylene, arylene, heteroarylene, heterocyclylene, cycloalkylene, cycloalkenylene, alkylarylalkylene, alkylarylalkenylene, alkylarylalkynylene, alkenylarylalkylene, alkenylarylalkenylene ), alkenylarylalkynylene, alkynylarylalkylene, alkynylarylalkenylene, alkynylarylalkynylene, alkylheteroarylalkylene, alkylheteroarylalkenylene, alkylheteroarylalkynylene, alkenylheteroarylalkylene, lheteroarylalkylene), alkenylheteroarylalkenylene, alkenylheteroarylalkynylene, alkynylheteroarylalkylene, alkynylheteroarylalkenylene, alkynylheteroarylalkynylene, alkylheterocyclylalkylene,Alkylheterocyclylalkenylene, Alkylheterocyclylalkynylene, Alkenylheterocyclylalkylene, Alkenylheterocyclylalkenylene, Alkenylheterocyclylalkynylene, Alkynylheterocyclylalkylene, Alkynylheterocyclylalkylene, Alkynylheterocyclylalkenylene, Alkynylheterocyclylalkynylene lkynylene), alkylarylene, alkenylarylene, alkynylarylene, alkylheteroarylene, alkenylheteroarylene, alkynylhereroarylene, each of which may be substituted or unsubstituted and one or more of the methylene groups 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, wherein R is hydrogen, acyl, aliphatic or substituted aliphatic.

[0314] A cleavable group is a group that is sufficiently stable outside the cell, but is cleaved after entering the target cell to release the two parts of the group held together. In a preferred embodiment, the cleavable group is cleaved at least 10 times or more faster, preferably at least 100 times, in the target cell or under a first reference condition (which can, for example, be selected to simulate or represent intracellular conditions) than in the blood of the subject or under a second reference condition (which can, for example, be selected to simulate or represent conditions present in blood or serum).

[0315] Cleavable groups are susceptible to cleavage agents, such as the presence of pH, redox potential or degradation molecules. Typically, cleavage agents are more prevalent or present at higher levels or activities in cells than in serum or blood. Examples of such degradation agents include: redox agents, which are selected for specific substrates or which do not have substrate specificity, including, for example, oxidases or reductases or reducing agents such as thiols present in cells, which can degrade redox cleavable groups by reduction; esterases; endosomes or agents that can produce an acidic environment, such as those that cause a pH of 5 or less; enzymes, which can hydrolyze or degrade acid cleavable groups by acting as general acids, peptidases (which can be substrate specific) and phosphatases.

[0316] Cleavable groups, such as disulfide bonds, can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from 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 at about 5.0. Some linkers will have cleavable groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand within the cell, or into a desired compartment of the cell.

[0317] The conjugate may include a cleavable group that is cleavable by a specific enzyme. The type of cleavable group incorporated into the conjugate may depend on the cell to be targeted. For example, a liver targeting ligand may be attached to a cationic lipid by a chemical moiety comprising an ester group. Hepatocytes are rich in esterases, and therefore the group is more effectively cleaved in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include lung cells, renal cortical cells, and testicular cells.

[0318] When targeting peptidase-rich cell types such as hepatocytes and synoviocytes, coupling groups containing peptide bonds can be used.

[0319] In general, the suitability of candidate cleavable groups can be assessed by testing the ability of degradation agents (or conditions) to crack candidate groups. It will also be desirable to test the ability of candidate cleavable groups to resist cracking in blood or when contacting with other non-target tissues. Therefore, the relative susceptibility to cracking between the first condition and the second condition can be determined, wherein the first condition is selected to indicate the cracking in the target cell, and the second condition is selected to indicate other tissues or biological fluids, such as the cracking in blood or serum. Assessment can be carried out in a cell-free system, in cells, in cell culture, in organ or tissue culture or in whole animals. Preliminary assessment is carried out in cell-free or culture conditions and further assessment is carried out in whole animals to confirm that it may be useful. In a preferred embodiment, useful candidate compounds in cells (or under the in vitro conditions selected as the simulated intracellular conditions) are faster than cracking in blood or serum (or under the in vitro conditions selected as the simulated extracellular conditions) by at least 2 times, 4 times, 10 times or 100 times.

[0320] Redox cleavable groups. A class of cleavable groups is a redox cleavable group that is cleaved when reduced or oxidized. An example of a reduction cleavable group is a disulfide linking group (-SS-). In order to determine whether a candidate cleavable group is a suitable "reduction cleavable linking group", or whether it is suitable for use with a specific iRNA portion and a specific targeting agent, the technician can refer to the method described herein. For example, the candidate can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art, and the reagent simulates the cleavage rate observed in cells such as target cells. Candidates can also be evaluated under conditions selected to simulate blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved up to 10% in blood. In a preferred embodiment, useful candidate compounds are degraded at least 2 times, 4 times, 10 times or 100 times faster in cells (or under in vitro conditions selected to simulate intracellular conditions) than in blood (or under in vitro conditions selected to simulate extracellular conditions). The rate of cleavage of a candidate compound can be determined using standard enzyme kinetic assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

[0321] Phosphate-based cleavable groups. Phosphate-based cleavable groups are cleaved by agents that degrade or hydrolyze phosphate groups. Examples of agents that cleave phosphate groups in cells are enzymes in cells, such as phosphatases. In some embodiments, the phosphate-based linking group is -OP(=O)(ORk)-O-, -OP(=S)(ORk)-O-, -OP(=S)(SRk)-O-, -SP(=O)(ORk)-O-, -OP(=O)(ORk)-S-, -SP(=O)(ORk)-S-, -OP(=S)(ORk)-S-, -SP(=S)(ORk)-O-, -OP(=O)(Rk)-O-, -OP(=S)(Rk)-O-, -SP(=O)(Rk)-O-, -SP(=S)(Rk)-O-, -SP(=O)(Rk)-O-, -SP(=S)(Rk)-O-, -SP(=O)(Rk)-S- or -OP(=S)(Rk)-S-. In some embodiments, the phosphate-based linking group is -OP(=O)(OH)-O-, -OP(=S)(OH)-O-, -OP(=S)(SH)-O-, -SP(=O)(OH)-O-, -OP(=O)(OH)-S-, -SP(=O)(OH)-S-, -OP(=S)(OH)-S-, -SP(=S)(OH)-O-, -OP(=O)(H)-O-, -OP(=S)(H)-O-, -SP(=O)(H)-O-, -SP(=S)(H)-O-, -SP(=O)(H)-S-, or -OP(=S)(H)-S-. In some embodiments, the phosphate-based linking group is -OP(=O)(OH)-O-.

[0322] Acid cleavable group. 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 with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower), or is cleaved by an agent that can act as a general acid, such as an enzyme. In cells, specific low pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid cleavable linking group. Examples of acid cleavable groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid cleavable group can have the general formula -C=NN-, C(O)O or -OC(O). A preferred embodiment is when the carbon attached to the oxygen (alkoxy group) of the ester is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or tert-butyl. These candidates can be evaluated using a method similar to the method described above.

[0323] Based on ester cleavable groups. Based on ester cleavable groups are cleaved by enzymes in cells such as esterases and amidases. Based on the example of ester cleavable groups include but are not limited to esters of alkylene groups, alkenylene groups and alkynylene groups. The ester cleavable linking group has the general formula-C (O) O- or-OC (O)-. Can use methods similar to the method described above to evaluate these candidates.

[0324] Cleavable groups based on peptides. Cleavable groups based on peptides are cleaved by enzymes such as peptidases and proteases in cells. Cleavable groups based on peptides are peptide bonds formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Cleavable groups based on peptides do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene or alkynylene groups. Peptide bonds are a special type of amide bonds formed between amino acids to produce peptides and proteins. Cleavable groups based on peptides are usually limited to the peptide bonds (i.e., amide bonds) formed between amino acids to produce peptides and proteins, and do not include the entire amide functional group. Cleavable linking groups based on peptides have 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 methods similar to those described above. As used herein, "sugar" refers to a compound that is a sugar that itself is composed of one or more monosaccharide units, each of which has at least 6 carbon atoms (which may be linear, branched or cyclic), each of which has an oxygen, nitrogen or sulfur atom bonded to it; or a compound that has as a part of it a sugar moiety composed of one or more monosaccharide units, each of which has at least six carbon atoms (which may be linear, branched or cyclic), each of which has an oxygen, nitrogen or sulfur atom bonded to it. Representative sugars include sugars (monosaccharides, disaccharides, trisaccharides and oligosaccharides containing from about 4-9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (preferably C5-C8) sugars; disaccharides and trisaccharides include sugars with two or three monosaccharide units (preferably C5-C8).

[0325] In some embodiments, the antisense strand of an isolated oligonucleotide of the present disclosure is complementary to an mRNA, wherein sequence-specific hybridization of the antisense strand to the mRNA results in degradation of the mRNA.

[0326] Methods of preparing isolated oligonucleotides of the present disclosure

[0327] In some aspects, the present disclosure provides a pharmaceutical composition comprising an isolated oligonucleotide described herein or a compound, scaffold, or conjugate comprising an isolated oligonucleotide described herein.

[0328] In some aspects, the disclosure provides a method of modulating expression of a target gene in a subject, the method comprising administering to the subject an isolated oligonucleotide described herein, or a compound, scaffold, or conjugate described herein comprising the isolated oligonucleotide.

[0329] In some aspects, the disclosure provides a method of delivering a nucleic acid agent to a subject, the method comprising administering to the subject an isolated oligonucleotide described herein, or a compound, scaffold, or conjugate comprising an isolated oligonucleotide described herein.

[0330] In some aspects, the disclosure provides a method of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated oligonucleotide described herein, or a compound, scaffold, or conjugate comprising the isolated oligonucleotide described herein.

[0331] In some aspects, the disclosure provides use of an isolated oligonucleotide described herein, or a compound, scaffold, or conjugate comprising an isolated oligonucleotide described herein, in the preparation of a medicament for modulating expression of a target gene in a subject.

[0332] In some aspects, the disclosure provides use of an isolated oligonucleotide described herein, or a compound, scaffold, or conjugate comprising an isolated oligonucleotide described herein, in the preparation of a medicament for delivering a nucleic acid agent to a subject.

[0333] In some aspects, the disclosure provides use of an isolated oligonucleotide described herein, or a compound, scaffold, or conjugate comprising an isolated oligonucleotide described herein, in the preparation of a medicament for treating or preventing a disease in a subject in need thereof.

[0334] In some embodiments, the isolated oligonucleotides of the present disclosure comprise one or more phosphate groups or one or more analogs of a phosphate group.

[0335] In some embodiments, the isolated oligonucleotide is a siRNA (e.g., a single-stranded siRNA (e.g., a hairpin single-stranded siRNA) or a double-stranded siRNA), a microRNA, an antisense microRNA, a microRNA mimic, an antisense miR, an antagomir, a dsRNA, an aptamer, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a splice altering oligonucleotide, a riplex forming oligonucleotide, a G-quadruplex, or an antisense oligonucleotide.

[0336] In some embodiments, the isolated oligonucleotide is a double-stranded RNA (dsRNA), wherein the double-stranded RNA.

[0337] It will be understood that the sense strand of the isolated oligonucleotide of the present disclosure is also referred to as the passenger strand, and the terms "sense strand" and "passenger strand" are used interchangeably herein.

[0338] It will be understood that the antisense strand of the isolated oligonucleotide of the present disclosure is also referred to as the guide strand, and the terms "antisense strand" and "guide strand" are used interchangeably herein.

[0339] In some embodiments, the isolated oligonucleotide is an iRNA.

[0340] The term "iRNA" refers to an RNA agent that can down-regulate the expression of a target gene (e.g., siRNA), such as an endogenous or pathogen target RNA. Although not wishing to be bound by theory, iRNA can work by one or more of a variety of mechanisms, including post-transcriptional cleavage of target mRNA (referred to as RNAi in the art), or pre-transcriptional or pre-translation mechanisms. iRNA can include a single strand or can include more than one strand, for example, it can be a double-stranded iRNA. If the iRNA is a single strand, it can include a 5' modification, which includes one or more phosphate groups or one or more phosphate group analogs. In some embodiments, the iRNA is double-stranded. In some embodiments, one or both strands of the double-stranded iRNA can be modified, such as a 5' modification.

[0341] iRNA generally includes a zone with enough homology to the target gene, and has enough length in nucleotides so that iRNA or its fragment can mediate the downward regulation of the target gene. iRNA is or includes a zone that is at least partially complementary to the target RNA and fully complementary to the target RNA in some embodiments. There may not necessarily be complete complementarity between iRNA and the target, but this corresponding relationship may be enough to enable iRNA or its cleavage product to guide sequence-specific silencing, for example, by the RNAi cleavage of the target RNA (e.g., mRNA).

[0342] The nucleotide of the oligonucleotide of separation can be modified in addition.The single-stranded or double-stranded region of the oligonucleotide of separation can be modified in addition or also include nucleotide substitutes, such as an unpaired region or more unpaired regions of hairpin structure, such as connecting the region of two complementary regions, can have modification or nucleotide substitutes.One or more 3'-or 5'-ends of the oligonucleotide of stable separation are for example modified for exonuclease.Modification can include C3 (or C6, C7, C12) amino joint, thiol joint, carboxyl joint, non-nucleotide spacer (C3, C6, C9, C12, abasic (abasic), triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagent, they appear as phosphoramidite (phosphoramidite) and have the hydroxyl group of another DMT protection, allow repeatedly coupling during RNA synthesis.Modification can also include for example use at the modification of 2'OH group of ribose in addition, for example use deoxyribonucleotide such as deoxythymidine to replace ribonucleotide, and use the modification of phosphate group, for example phosphorothioate modification. In some embodiments, different chains will include different additional modifications.

[0343] In some embodiments, the length of the double-stranded region between the strands of the isolated oligonucleotide is between 6 nucleotides and 30 nucleotides in length. In some embodiments, the length of the double-stranded region is between 15 nucleotides and 30 nucleotides, most preferably 18, 19, 20, 21, 22, and 23 nucleotides. In some embodiments, the length of the double-stranded region is between 6 nucleotides and 20 nucleotides, most preferably 6, 7, 8, 9, 10, 11, and 12 nucleotides.

[0344] The isolated oligonucleotide can be an oligonucleotide described in U.S. Patent Publication Nos. 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, or 2009 / 0247608, each of which is hereby incorporated by reference.

[0345] In some embodiments, the isolated oligonucleotide is siRNA.

[0346] In some embodiments, the isolated oligonucleotide is a double-stranded siRNA, such as a double-stranded siRNA described herein.

[0347] As used herein, "double-stranded siRNA" is an siRNA that includes more than one strand and in some cases two strands, wherein the interstrand hybridization can form a region of a duplex structure. In some embodiments, the length of the sense strand of the double-stranded siRNA can be equal to or 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 some embodiments, the length of the antisense strand of the double-stranded siRNA can be equal to or at least 18, 19, 20, 21, 22, 23, 24, 25, 29, 30, or 31 nucleotides.

[0348] In some embodiments, the isolated oligonucleotides of the present disclosure can be converted or dissociated to form single-stranded siRNA.

[0349] " Single-stranded siRNA " as used herein is the siRNA consisting of a single strand, which includes a double-stranded region formed by intrachain pairing, for example, it can be a hairpin structure or a coiled handle structure or includes a hairpin structure or a coiled handle structure. Single-stranded siRNA can be antisense relative to the target molecule. Single-stranded siRNA can be long enough so that it can enter RISC and participate in the RISC-mediated cracking of target mRNA. The length of 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. The length of single-stranded siRNA is at least 18, 19, 20, 21, 22, 23, 24, 25, 29, 30 or 31 nucleotides.

[0350] In some embodiments, the isolated oligonucleotide is a hairpin siRNA.

[0351] The hairpin siRNA can have a duplex region equal to or at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs. In some embodiments, the length of the duplex region ranges from 15 to 30, 17 to 23, 19 to 23, 18 to 21, and 19 to 21 nucleotide pairs. The hairpin can have a single-stranded overhang or an unpaired terminal region. In some embodiments, the overhang is at least two in length. In some embodiments, the overhang is positioned on the antisense side of the hairpin.

[0352] In some embodiments, the siRNA is large enough that it can be cleaved by endogenous molecules, such as by Dicer, to produce smaller siRNAs, such as siRNA agents.

[0353] The sense strand and the antisense strand can be selected so that the double-stranded siRNA includes a single-stranded region or an unpaired region at one end of the molecule. Therefore, the double-stranded siRNA can include a sense strand and an antisense strand, and the sense strand and the antisense strand are paired to include a 3' overhang of 2 or more nucleotides. In some embodiments, the overhang is 2 nucleotides.

[0354] The siRNA described herein (including double-stranded siRNA and single-stranded siRNA produced by double-stranded siRNA) can mediate the silencing of target RNA (e.g., mRNA) (e.g., transcripts of genes encoding proteins). For convenience, such mRNA is also referred to as mRNA to be silenced herein. Such genes are also referred to as target genes. In general, RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA other than mRNA, such as tRNA and viral RNA, can also be targeted.

[0355] As used herein, the phrases "mediate 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. While not wishing to be bound by theory, it is believed that silencing uses the RNAi mechanism or process and a guide RNA, such as a 21 to 23 nucleotide ssRNA.

[0356] In some embodiments, the isolated oligonucleotide is "fully complementary" to the target RNA (e.g., target mRNA), so that the siRNA silences the production of a protein encoded by the target mRNA. In another embodiment, the siRNA is "precisely complementary" to the target RNA, such as the target RNA and the siRNA anneal, for example to form a hybrid made exclusively of Watson-Crick base pairs in a precisely complementary region. The "fully complementary" target RNA can include an internal region (e.g., an internal region of at least 10 nucleotides) that is precisely complementary to the target RNA. In addition, in some embodiments, the siRNA specifically distinguishes between single nucleotide differences. In this case, the siRNA mediates RNAi only when there is precise complementarity in the region of single nucleotide differences (e.g., within 7 nucleotides).

[0357] In some embodiments, the isolated oligonucleotide is an antisense oligonucleotide for a target polynucleotide. The term "antisense oligonucleotide" or "antisense" for short means an oligonucleotide complementary to a targeted polynucleotide sequence. Antisense oligonucleotides are single strands of DNA or RNA complementary to a selected sequence (e.g., target gene mRNA). Antisense oligonucleotides are considered to inhibit gene expression by binding to complementary mRNA. Binding to target mRNA can inhibit gene expression by preventing the translation of complementary mRNA chains via binding to complementary mRNA chains or by causing degradation of target mRNA. In some embodiments, antisense oligonucleotides include about 13 nucleotides to about 36 nucleotides, more preferably about 20 nucleotides to about 25 nucleotides. The term also encompasses antisense oligonucleotides that may not be precisely complementary to the desired target gene. Therefore, it is envisioned that a situation in which non-target specific activity is found with antisense, or an antisense sequence comprising one or more mismatches with a target sequence is the most preferred situation for a specific purpose.

[0358] Antisense oligonucleotides have been shown to be effective and targeted inhibitors of protein synthesis, and therefore, can be used to specifically inhibit protein synthesis of targeted genes. Antisense oligonucleotides are well confirmed for the effectiveness of inhibiting protein synthesis. For example, the synthesis of polygalacturonase and muscarinic type 2 acetylcholine receptors is 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). In addition, examples of antisense inhibition have been demonstrated with the nuclear proteins cyclins, multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAA receptors, 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. Pat. Nos. 5,801,154; 5,789,573; 5,718,709 and 5,610,288, each of which is incorporated by reference). Additionally, antisense constructs have been described that inhibit and can be used to treat a variety of abnormal cell proliferations, such as cancer (U.S. Pat. Nos. 5,747,470; 5,591,317; and 5,783,683, each of which is incorporated by reference).

[0359] The method for producing antisense oligonucleotide is known in the art, and can be easily applied to produce antisense oligonucleotide targeting any polynucleotide sequence. The selection of antisense oligonucleotide sequence specific to 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 oligonucleotide can be selected based on their relative inability to form dimer, hairpin or other secondary structures, which will reduce or forbid specific binding with target mRNA in host cells. The highly preferred target region of mRNA includes those regions at or near the AUG translation initiation codon and those sequences substantially complementary to the 5' region of mRNA. These secondary structure analyses and target site selection considerations can be carried out, for example, using the 4th edition and / or BLASTN2.0.5 algorithm software of OLIGO primer analysis software (Molecular Biology Insights) (Altschul et al., Nucleic Acids Res.1997, 25 (17): 3389-402).

[0360] In some embodiments, the isolated oligonucleotide can be an antagomir.Antagomir is an RNA-like oligonucleotide with a variety of modifications for RNA enzyme protection and pharmacological properties, such as enhanced tissue and cell uptake. They differ from normal RNA in that, for example, sugars, phosphorothioate backbones are completely 2'-O-methylated, and for example, a cholesterol moiety at the 3'-end.Antagomir can be used to effectively silence endogenous miRNA by forming a duplex comprising antagomir and endogenous miRNA, thereby preventing gene silencing induced by miRNA.An example of miRNA silencing mediated by antagomir is the silencing of miR-122, described in Krutzfeldt et al., Nature, 2005, 438:685-689, which is expressly incorporated herein by reference in its entirety.Antagomir RNA can be synthesized using a standard solid phase oligonucleotide synthesis protocol. See U.S. Patent Application Publication Nos. 2007 / 0123482 and 2007 / 0213292 (each of which is incorporated herein by reference).

[0361] Antagomir can include monomeric subunits and monomers for ligand conjugation of oligonucleotide synthesis. Exemplary monomers are described in U.S. Patent Application Publication No. 2005 / 0107325, which is incorporated by reference in its entirety. Antagomir can have a ZXY structure, such as the ZXY structure described in WO 2004 / 080406, which is incorporated by reference in its entirety. Antagomir can be complexed with an amphipathic part. Exemplary amphipathic parts for use with oligonucleotide agents are described in WO 2004 / 080406, which is incorporated by reference in its entirety.

[0362] In some embodiments, the isolated oligonucleotide of the present disclosure linked to a lipid is associated with a ribozyme. Ribozymes are RNA molecule complexes with specific catalytic domains that possess endonuclease activity (Kim and Cech, Proc Natl Acad Sci USA. 1987 December; 84(24):8788-92; Forster and Symons, Cell. 1987 April 24; 49(2):211-20). For example, a large number of ribozymes accelerate phosphate transfer reactions with high specificity, often cleaving only one of several phosphates in an oligonucleotide substrate (Cech et al., Cell. 1981 Dec; 27(3Pt 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 has been attributed 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.

[0363] At least six kinds of naturally occurring enzymatic RNAs are known to exist. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans under physiological conditions (and therefore can cleave other RNA molecules). In general, enzymatic nucleic acids first act by binding to target RNA. Such binding occurs through the target binding portion of the enzymatic nucleic acid, which is adjacent to the enzymatic portion in the molecule that plays the role of cleaving the target RNA. Therefore, the enzymatic nucleic acid first recognizes the target RNA, and then binds to the target RNA through complementary base pairing, and once bound to the correct site, it enzymatically plays the role of cleaving the target RNA. The strategic cleavage of such a target RNA will destroy its ability to guide the synthesis of the encoded protein. After the enzymatic nucleic acid binds and cleaves its RNA target, it is released from the RNA to find another target, and can repeatedly bind and cleave new targets.

[0364] For example, the enzymatic nucleic acid molecule can be formed as a hammerhead, hairpin, hepatitis delta virus, group I intron or RNase P RNA (associated with an RNA guide sequence) or Neurospora VS RNA motif. Specific examples of hammerhead motifs are described by Rossi et al. Nucleic Acids Res. 1992 Sep 11; 20(17): 4559-65. Examples of hairpin motifs are described by Hampel et al. (European Patent Application Publication No. EP 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. Pat. No. 5,631,359; examples of hepatitis delta virus motifs are described by Perrotta and Been, Biochemistry. 1992 Dec 1;31(47):11843-52; examples of RNase P motifs are described by Guerrier-Takada et al., Cell. 1983 Dec;35(3Pt 2):849-57; Neurospora VS RNA ribozyme motifs are described by 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. Pat. No. 4,987,071. The important features of the enzymatic nucleic acid molecules used are that they have a specific substrate binding site that is complementary to one or more target gene DNA or RNA regions, and that they have a nucleotide sequence within or around the substrate binding site that confers RNA cleavage activity to the molecule. Thus, ribozyme constructs need not be limited to the specific motifs mentioned herein.

[0365] Methods for producing ribozymes targeting any polynucleotide sequence are known in the art.Ribozymes can be designed as described in International Patent Application Publication Nos. WO 93 / 23569 and WO 94 / 02595, each expressly incorporated herein by reference, and synthesized as described therein for in vitro and in vivo testing.

[0366] Ribozyme activity can be optimized by varying the length of the ribozyme binding arms or chemically synthesizing ribozymes with modifications that protect them from degradation by serum ribonucleases (see, e.g., International Patent Application Publication Nos. WO 92 / 07065, WO 93 / 15187, and WO 91 / 03162; European Patent Application Publication No. 92110298.4; U.S. Pat. No. 5,334,711; and International Patent Application Publication No. WO 94 / 13688, which describe a variety of chemical modifications that can be made to the sugar portion of the enzymatic RNA molecule), modifications that enhance their potency in cells, and removal of the Stem II base to shorten RNA synthesis time and reduce chemical requirements.

[0367] Synthesis method

[0368] In some aspects, the disclosure provides a method of preparing an isolated oligonucleotide or a compound, conjugate or scaffold comprising an isolated oligonucleotide of the disclosure.

[0369] In some aspects, the disclosure provides an intermediate as described herein suitable for use in a method for preparing an isolated oligonucleotide or a compound, conjugate or scaffold comprising an isolated oligonucleotide of the disclosure.

[0370] The isolated oligonucleotide of the present disclosure or the compound, conjugate or scaffold comprising the isolated oligonucleotide of the present disclosure can be prepared by any suitable technique known in the art. The specific method for preparing the isolated oligonucleotide of the present disclosure or the compound, conjugate or scaffold comprising the isolated oligonucleotide of the disclosure is further described in the attached examples.

[0371] In the descriptions of the synthetic methods described herein, as well as in any referenced synthetic methods used to prepare starting materials, it is understood that all proposed reaction conditions, including the choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment, and work-up procedures, can be selected by one skilled in the art. Those skilled in the art of organic synthesis understand that the functionality present on various portions of the molecule must be compatible with the reagents and reaction conditions being utilized.

[0372] It should be understood that during the synthesis of the oligonucleotide of the separation of the present disclosure or the compound, conjugate or support comprising the oligonucleotide of the separation of the present disclosure, it may be desirable to protect certain substituent groups to prevent their undesirable reactions. A skilled chemist will understand when such protection is needed and how such blocking groups can be placed in place and removed later. For the example of blocking groups, see one of many general textbooks on this theme, for example, by Theodora Green (publisher: John Wiley & Sons) 'Protective Groups in Organic Synthesis '. Blocking groups can be removed by any convenient method described in the literature or known to a skilled chemist as being suitable for removing the blocking groups under discussion, selecting such a method to achieve removal of blocking groups in the case of a group elsewhere in the minimum interference molecule. Therefore, if reactant comprises a group such as, for example, an amino, carboxyl or hydroxyl group, it may be desirable to protect the group in some of the reactions mentioned herein.

[0373] For example, a suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyl group such as an acetyl group, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or tert-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. Suitable protecting groups for hydroxyl groups or alkylhydroxyl groups can be, 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 ether (e.g. trimethylsilyl (TMS) ether, tert-butyldimethylsilyl (TBDMS) ether, triisopropylsilyloxymethyl (TOM) ether and triisopropylsilyl (TIPS) ether), methyl ether or ethoxyethyl ether (EE). Suitable protecting groups for 1,2-diol can be, for example, acetal. A suitable protecting group for 1,3-diol may be, for example, tetraisopropyldisiloxanylidene (TIPDS).

[0374] The deprotection conditions for the blocking group above necessarily change with the selection of blocking group.Therefore, for example, acyl groups such as alkanoyl groups or alkoxycarbonyl groups or aroyl groups can be removed by, for example, hydrolysis with suitable alkali such as alkali metal hydroxides such as lithium hydroxide or sodium hydroxide.Selectively, acyl groups such as tert-butoxycarbonyl groups can be removed by, for example, processing with suitable acid such as hydrochloric acid, sulfuric acid or phosphoric acid or trifluoroacetic acid, and arylmethoxycarbonyl groups such as benzyloxycarbonyl groups can be removed by, for example, hydrogenation of palladium on carbon or by processing with, for example, the Lewis acid of tri(trifluoroacetic acid) borane through catalyst.The suitable selectable blocking group for primary amino groups is, for example, the phthaloyl group that can be removed by, for example, processing with alkylamines such as dimethylaminopropylamine or processing with hydrazine.

[0375] Suitable protecting groups for hydroxyl groups are, for example, acyl groups, for example alkanoyl groups such as acetyl, aroyl groups such as benzoyl; or arylmethyl groups, for example benzyl. The deprotection conditions for the protecting groups above will necessarily vary with the selection of the protecting groups. Thus, for example, acyl groups such as alkanoyl groups or aroyl groups can be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide (e.g., lithium hydroxide, sodium hydroxide) or ammonia. Alternatively, arylmethyl groups such as benzyl groups can be removed, for example, by hydrogenation with a catalyst such as palladium on carbon.

[0376] Suitable protecting groups for carboxyl groups are, for example, esterifying groups, for example a methyl group or an ethyl group which can be removed, for example, by hydrolysis with a base such as sodium hydroxide, or for example a tert-butyl group which can be removed, for example, by treatment with an acid such as an organic acid such as trifluoroacetic acid, or for example a benzyl group which can be removed, for example, by hydrogenation over a catalyst such as palladium on carbon.

[0377] Conveniently, the reaction of the compounds is carried out in the presence of a suitable solvent, which is preferably inert under the corresponding reaction conditions. Examples of suitable solvents include, but are not limited to, hydrocarbons such as hexane, petroleum ether, benzene, toluene or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, tetrachloromethane, 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 mono Methyl or 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) or N-methylpyrrolidone (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 the solvents or mixtures with water.

[0378] The reaction temperature is suitably between about -100°C and 300°C, depending on the reaction steps and conditions used.

[0379] The reaction time of the present invention is usually in the range of a fraction of a minute and a few days. ...

[0380] In addition, by utilizing the procedures described herein, in combination with ordinary skills in the art, additional compounds of the present disclosure can be readily prepared. Those skilled in the art will readily appreciate that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds.

[0381] As will be appreciated by those skilled in the art of organic synthesis, the isolated oligonucleotides of the present disclosure or compounds, conjugates or scaffolds comprising the isolated oligonucleotides of the present disclosure are easily obtained by various synthetic routes, some of which are illustrated in the accompanying examples. The technician will readily recognize which reagents and reaction conditions to use, and how to apply and adjust them in any particular case (when necessary or useful) to obtain the compounds of the present disclosure. In addition, some of the isolated oligonucleotides of the present disclosure or compounds, conjugates or scaffolds comprising the isolated oligonucleotides of the present disclosure can be easily 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, a specific functional group present in the compounds of the present disclosure or its suitable precursor molecules is converted into another functional group; those methods are well known to the technician. Likewise, the skilled artisan will employ synthetic protecting (or protective) groups where necessary or useful; suitable protecting groups and methods for introducing and removing them are well known to those skilled in the art of chemical synthesis and are described in more detail in, e.g., PGM Wuts, TW Greene, "Greene's Protective Groups in Organic Synthesis", 4th Edition (2006) (John Wiley & Sons).

[0382] Biological assays

[0383] The isolated oligonucleotides or compounds, conjugates or scaffolds comprising the isolated oligonucleotides of the present disclosure designed, selected, prepared and / or optimized by the methods described above can be characterized after production using a variety of assays known to those skilled in the art to determine whether the compounds, scaffolds or conjugates have biological activity. For example, isolated oligonucleotides or compounds, conjugates or scaffolds comprising the isolated oligonucleotides of the present disclosure can be characterized by conventional assays (including but not limited to those described below) to determine whether they have the desired activity, such as target binding activity and / or specificity and / or stability.

[0384] In addition, high throughput screening can be used to speed up the analysis using such assays. Therefore, it is possible to rapidly screen the activity of molecules described herein using techniques known in the art. The general method for performing high throughput screening is described in, for example, 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.

[0385] 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 may include, but are not limited to, enzymatic activity assays, electrophoretic mobility shift assays, reporter gene assays, in vitro cell viability assays, and assays described herein.

[0386] In some embodiments, biological assays are described in the Examples herein.

[0387] Pharmaceutical composition

[0388] In some aspects, the present disclosure provides a pharmaceutical composition comprising the isolated oligonucleotide of the present disclosure or a compound, conjugate or scaffold comprising the isolated oligonucleotide of the present disclosure as an active ingredient.

[0389] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0390] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid that reaches the degree of easy injection. It must be stable under manufacturing and storage conditions, and must be preserved against the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol and the like) and a suitable mixture thereof. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion and by using a surfactant, suitable fluidity can be maintained. The effect of preventing microorganisms can be achieved by a variety of antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, isotonic agents, such as sugars, polyols such as mannitol and sorbitol and sodium chloride, will preferably be included in the composition. By including an agent that delays absorption in the composition, such as aluminum monostearate and gelatin, the extended absorption of injectable compositions can be achieved.

[0391] Sterile injectable solutions can be prepared by incorporating the active compound in the desired amount into a suitable solvent (having a combination of one or more components listed above as required), followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle comprising an alkaline dispersion medium and other components required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0392] The formulations of the present disclosure may be in the form of an aqueous solution comprising an aqueous vehicle. The aqueous vehicle component may comprise water and at least one pharmaceutically acceptable excipient. Suitable acceptable excipients include excipients selected from the group consisting of solubilizers, chelating agents, preservatives, tonicity agents, viscosity / suspending agents, buffers, and pH adjusters and mixtures thereof.

[0393] Any suitable solubilizing agent can be used. The example of solubilizing agent includes cyclodextrin, such as cyclodextrin selected from the group consisting of hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, random methylation-β-cyclodextrin, ethylation-β-cyclodextrin, triacetyl-β-cyclodextrin, peracetylation-β-cyclodextrin, carboxymethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 2-hydroxy-3-(trimethylammonium)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, random methylation-γ-cyclodextrin and trimethyl-γ-cyclodextrin and mixtures thereof.

[0394] Any suitable chelating agent may be used. Examples of suitable chelating agents include chelating agents selected from the group consisting of ethylenediaminetetraacetic acid and its metal salts, disodium edetate, trisodium edetate and tetrasodium edetate and mixtures thereof.

[0395] Any suitable preservative may be used. Examples of preservatives include preservatives selected from the group consisting of quaternary ammonium salts such as benzalkonium halide (preferably benzalkonium chloride), chlorhexidine gluconate, benzethonium chloride, cetylpyridinium chloride, benzyl bromide, phenylmercuric nitrate, phenylmercuric acetate, phenylmercuric neodecanoate, thimerosal, methylparaben, propylparaben, sorbic acid, potassium sorbate, sodium benzoate, sodium propionate, ethylparaben, propylaminopropyl biguanide and butylparaben and sorbic acid and mixtures thereof.

[0396] The aqueous vehicle may also include a tonicity agent to adjust tonicity (osmotic pressure). The tonicity agent may be selected from the group consisting of glycols (e.g., propylene glycol, diethylene glycol, triethylene glycol), glycerol, dextrose, glycerin, mannitol, potassium chloride and sodium chloride and mixtures thereof.

[0397] In order to adjust the formulation to an acceptable pH (typically 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 pH range), the formulation may contain a pH adjusting agent. The pH adjusting agent is typically an inorganic acid or metal hydroxide base selected from the group consisting of potassium hydroxide, sodium hydroxide and hydrochloric acid and mixtures thereof, and preferably sodium hydroxide and / or hydrochloric acid. These acidic and / or alkaline pH adjusting agents are added to adjust the formulation to a target acceptable pH range. Therefore, it may not be necessary to use both an acid and a base—depending on the formulation, adding one of the acid or base may be sufficient to bring the mixture to a desired pH range.

[0398] 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 a salt thereof, including disodium tetraborate), citrate buffers (such as citric acid or a salt thereof, including sodium citrate) and ε-aminocaproic acid and mixtures thereof.

[0399] According to a further aspect of the present disclosure, there is provided a pharmaceutical composition comprising a compound of the present disclosure as defined above, or a pharmaceutically acceptable salt, hydrate or solvate thereof, in combination with a pharmaceutically acceptable diluent or carrier.

[0400] The compositions of the present disclosure may be in a form suitable for oral use (e.g., as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), topical use (e.g., as creams, ointments, gels, or aqueous or oily solutions or suspensions), administration by inhalation (e.g., as finely divided powders or liquid aerosols), administration by insufflation (e.g., as finely divided powders), or parenteral administration (e.g., as sterile aqueous or oily solutions for intravenous, subcutaneous, intramuscular or intraperitoneal administration or as suppositories for rectal administration).

[0401] 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 contain, for example, one or more coloring agents, sweeteners, flavoring agents and / or preservatives.

[0402] An effective amount of a compound of the disclosure for use in therapy is an amount sufficient to treat or prevent, slow the progression of, and / or reduce the symptoms associated with an inflammasome-associated condition mentioned herein.

[0403] An effective amount of a compound of the disclosure for use in therapy is an amount sufficient to treat, slow the progression of, and / or reduce the symptoms associated with an inflammasome-associated condition mentioned herein.

[0404] According to well-known medical principles, the size of the dosage of the compound of formula (I) or formula (II) for therapeutic or preventive purposes will vary naturally according to the nature and severity of the condition, the age and sex of the animal or patient, and the route of administration. It should be understood that the present disclosure also provides a pharmaceutical composition comprising any compound, scaffold or conjugate described herein in combination with at least one pharmaceutically acceptable excipient or carrier.

[0405] As used herein, the term "pharmaceutical composition" is a preparation in a form suitable for administration to a subject comprising a compound, a stent or a conjugate of the present disclosure. In some embodiments, the pharmaceutical composition is in bulk dosage form or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The amount of the active ingredient (e.g., a preparation of a disclosed compound or its salt, hydrate, solvate or isomer) in the composition of a unit dose is an effective amount and changes according to the specific treatment involved. It will be appreciated by those skilled in the art that it is sometimes necessary to make conventional changes to the dosage according to the age and condition of the patient. The dosage will also depend on the route of administration. It is envisioned that a variety of routes, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intrathecal, intranasal and similar routes. 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 with any preservatives, buffers or propellants required.

[0406] As used herein, the term "pharmaceutically acceptable" refers to those compounds, scaffolds, conjugates, anions, cations, materials, compositions, carriers and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0407] As used herein, the term "pharmaceutically acceptable excipient" means an excipient useful in preparing a pharmaceutical composition that is generally safe, non-toxic and not biologically or otherwise undesirable, and includes excipients acceptable for veterinary use and human pharmaceutical use. "Pharmaceutically acceptable excipient" as used in this specification and claims includes one and more than one such excipient.

[0408] It should be understood that the pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, for example, intravenous, intradermal, subcutaneous, oral (e.g., ingestion), inhalation, transdermal (surface), and transmucosal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous applications may include the following components: sterile diluents, such as water for injection, saline solutions, non-volatile oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for adjusting tension, such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral products may be encapsulated in ampoules, disposable syringes, or multidose vials made of glass or plastic.

[0409] It should be understood that the compounds or pharmaceutical compositions of the present disclosure can be applied to the subject with many well-known methods currently used for chemotherapy. For example, the compounds of the present disclosure can be injected into the bloodstream or body cavity, or orally or applied through the skin with a patch. The selected dosage should be sufficient to constitute an effective treatment, but not so high as to cause unacceptable side effects. The patient's disease condition (e.g., disease or disorder disclosed herein) and healthy state should preferably be closely monitored during treatment and within a reasonable time period after treatment.

[0410] It will be appreciated that the compounds, scaffolds and conjugates of the present disclosure can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature or from readily prepared intermediates by utilizing standard synthetic methods and procedures known to those skilled in the art or which will be apparent to the skilled artisan in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or standard textbooks in the field. Although not limited to any one or a few sources, classic textbooks such as Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition, John Wiley & Sons: New York, 2001; Greene, TW, Wuts, PGM, 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 for organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for organic Synthesis, John Wiley and Sons (1995), which are incorporated herein by reference, are useful and recognized reference textbooks on organic synthesis known to those skilled in the art.

[0411] Those of ordinary skill in the art will note that during the reaction sequence and synthesis scheme described herein, the order of some steps can be changed, such as introducing and removing blocking groups. Those of ordinary skill in the art will recognize that some groups may need to be protected from the impact of reaction conditions via the use of blocking groups. Blocking groups can also be used to distinguish similar functional groups in molecules. The list of blocking groups and how to introduce and remove these groups can be found in Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, the 3rd edition, John Wiley & Sons: New York, 1999.

[0412] Techniques for preparing and administering 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 an embodiment, the compounds described herein and pharmaceutically acceptable salts thereof are used in combination with a pharmaceutically acceptable carrier or diluent in a pharmaceutical preparation. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous organic solutions. The compound will be present in such a pharmaceutical composition in an amount sufficient to provide the desired dose within the range described herein.

[0413] The pharmaceutical composition comprising the active compound of the present disclosure can be manufactured in a generally known manner, for example by means of conventional mixing processes, dissolution processes, granulation processes, dragee manufacturing (dragee-making) processes, levigating (levigating) processes, emulsification processes, encapsulation processes, embedding processes or freeze-drying processes. The pharmaceutical composition can be prepared in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and / or adjuvants, and the pharmaceutically acceptable carriers help to process the active compound into a product that can be used pharmaceutically. Of course, the appropriate formulation depends on the selected route of administration.

[0414] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid that reaches the degree of easy injection. It must be stable under manufacturing and storage conditions, and must be preserved against the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol and the like) and a suitable mixture thereof. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion and by using a surfactant, suitable fluidity can be maintained. The effect of preventing microorganisms can be achieved by a variety of antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, isotonic agents, such as sugars, polyols such as mannitol and sorbitol and sodium chloride, will preferably be included in the composition. By including an agent that delays absorption in the composition, such as aluminum monostearate and gelatin, the extended absorption of injectable compositions can be achieved.

[0415] Sterile injectable solutions can be prepared by incorporating the active compound in the desired amount into a suitable solvent (having a combination of one or more components listed above as required), followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle comprising an alkaline dispersion medium and other components required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0416] Oral compositions generally include an inert diluent or an edible pharmaceutically acceptable carrier. They can be encapsulated in a gelatin capsule or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of a tablet, lozenge or capsule. Oral compositions can also be prepared using a fluid carrier to be used as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and spit out or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as a part of the composition. Tablets, pills, capsules, lozenges and the like may contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrant such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate, orange flavoring.

[0417] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0418] For intranasal administration, the compound is delivered as a solution or solid formulation. In some embodiments, the compound is delivered as a solution as a mist, drops, or swab. In some embodiments, the compound is delivered as a powder. In some embodiments, the compound is included in a kit that also includes an intranasal applicator.

[0419] Systemic administration can also be by transmucosal means or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, as generally known in the art, the active compound is formulated into an ointment, salves, gels, or creams.

[0420] The active compound can be prepared with a pharmaceutically acceptable carrier that will protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microcapsule 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 preparations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0421] It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved.

[0422] In therapeutic applications, the dosage of the pharmaceutical composition used according to the present disclosure depends on the dosage, the age, weight and clinical condition of the patient receiving, and the experience and judgment of the clinician or practitioner administering the therapy, as well as other factors affecting the selected dosage. Generally, the dosage should be sufficient to cause the symptoms of the disease or disorder disclosed herein to slow down and preferably eliminate the symptoms of the disease or disorder disclosed herein, and also preferably cause the complete disappearance of the disease or disorder. The dosage can be in the range of from about 0.01 mg / kg per day to about 5000 mg / kg per day. The effective amount of a medicament is the amount of an objectively identifiable improvement as indicated by a clinician or other qualified observer. Improved indications of survival and growth disappear. As used herein, the term "dosage effective manner" refers to the amount of an active compound that produces a desired biological effect in a subject or cell.

[0423] It will be appreciated that the pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration.

[0424] It should be understood that for compounds, scaffolds or conjugates of the present disclosure that are capable of further forming salts, all such forms are also contemplated to be within the scope of the claimed disclosure.

[0425] 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 preparing an acid or base salt thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali metal or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, salts derived from inorganic and organic acids selected from 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, hyd rabamic), hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, naphthalenesulfonic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicylic, stearic, basic acetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluenesulfonic, and common amino acids such as glycine, alanine, phenylalanine, arginine, etc.

[0426] In some embodiments, the pharmaceutically acceptable salt is a sodium salt, potassium salt, calcium salt, magnesium salt, diethylamine salt, choline salt, meglumine salt, benzathine penicillin salt, tromethamine salt, ammonium salt, arginine salt, or lysine salt.

[0427] Other examples of pharmaceutically acceptable salts include salts of 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 similar acids. The present disclosure also encompasses salts formed when acidic protons present in the parent compound are replaced by metal ions, such as alkali metal ions, alkaline earth metal ions, or aluminum ions; or coordinated with organic bases such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. In salt form, it should be understood that the ratio of compound to cation or anion in the salt can be 1:1, or any ratio other than 1:1, such as 3:1, 2:1, 1:2, or 1:3.

[0428] It should be understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystal forms (polymorphs) as defined herein, of the same salt.

[0429] The compound or its pharmaceutically acceptable salt is administered orally, nasally, transdermally, pulmonary, inhaled, buccal, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, intrarectally, intrapleurally, intrathecally, and parenterally. In some embodiments, the compound is administered orally. Those skilled in the art will recognize the advantages of certain routes of administration.

[0430] The dosage regimen utilizing the compound is selected based on a variety of 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 patient's renal and liver function; and the specific compound or salt thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counteract, or arrest the progression of the condition. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to counteract or arrest the progression of the condition.

[0431] How to use

[0432] In some aspects, the 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 or a compound, conjugate or scaffold comprising the isolated oligonucleotide of the disclosure.

[0433] 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 the subject an isolated oligonucleotide or a compound, conjugate or scaffold comprising the isolated oligonucleotide of the disclosure.

[0434] In some aspects, the present disclosure provides a method of delivering an isolated oligonucleotide or a compound, conjugate or scaffold comprising the isolated oligonucleotide or an isolated oligonucleotide compound, conjugate or scaffold comprising the present disclosure to a subject, the method comprising administering the isolated oligonucleotide or a compound, conjugate or scaffold comprising the isolated oligonucleotide of the present disclosure to the subject.

[0435] In some aspects, the disclosure provides a method of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated oligonucleotide or a compound, conjugate or scaffold comprising the isolated oligonucleotide of the disclosure.

[0436] In some aspects, the disclosure provides an isolated oligonucleotide or a compound, conjugate, or scaffold comprising the isolated oligonucleotide of the disclosure for modulating (eg, reducing or eliminating) expression of a target gene in a subject.

[0437] In some aspects, the disclosure provides an isolated oligonucleotide or a compound, conjugate or scaffold comprising an isolated oligonucleotide of the disclosure for modulating (e.g., reducing or eliminating) expression of a target gene in a cell or tissue of a subject.

[0438] In some aspects, the disclosure provides compounds, conjugates or scaffolds comprising the isolated oligonucleotides of the disclosure for use in delivering the isolated oligonucleotides of the disclosure.

[0439] In some aspects, the disclosure provides an isolated oligonucleotide or a compound, conjugate or scaffold comprising an isolated oligonucleotide of the disclosure for use in treating or preventing a disease in a subject in need thereof.

[0440] In some aspects, the disclosure provides use of an isolated oligonucleotide or a compound, conjugate or scaffold comprising an isolated oligonucleotide of the disclosure in the preparation of a medicament for modulating (eg, reducing or eliminating) expression of a target gene in a subject.

[0441] In some aspects, the disclosure provides the use of an isolated oligonucleotide or a compound, conjugate or scaffold comprising the isolated oligonucleotide of the disclosure in the preparation of a medicament for modulating (e.g., reducing or eliminating) target gene expression in a cell or tissue of a subject.

[0442] In some aspects, the disclosure provides use of a compound, conjugate or scaffold comprising an isolated oligonucleotide of the disclosure in the preparation of a medicament for delivering an isolated oligonucleotide of the disclosure to a subject.

[0443] In some aspects, the present disclosure provides use of an isolated oligonucleotide of the present disclosure or a compound, conjugate or scaffold comprising the isolated oligonucleotide of the present disclosure in the preparation of a medicament for treating or preventing a disease in a subject in need thereof.

[0444] In some embodiments, the subject is a cell. In some embodiments, the subject is a tissue. In some embodiments, the subject is a human.

[0445] In some embodiments, the modified siRNA molecules having the chemically modified sense and antisense strands of the present disclosure can be directed to 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, PDGFβ gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, P CNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, cyclin D gene, VEGF gene, EGFR gene, cyclin A gene, cyclin E gene, WNT-1 gene, β-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase II gene, p73 gene, p21 (WAF1 / CIP1) gene, p27 (KIP1) gene, PPM1D gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68 gene, tumor suppressor gene mutation, p53 tumor suppressor gene, LDHA or any combination thereof.

[0446] 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 expression of a target gene in a subject.

[0447] In some embodiments, the disease is a viral infection, such as an HCV, HBV, HPV, HSV, or HIV infection.

[0448] 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, ocular melanoma, uveal melanoma, gallbladder cancer, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinoma, Wilms tumor, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), liver cancer, hepatoepithelial carcinoma (liver carcinoma), hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B-cell lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, T-cell lymphoma, non-Hodgkin's 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 tumor, acinar cell carcinoma, prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, stomach cancer, gastric carcinoma, gastrointestinal stromal tumor (GIST), uterine cancer or uterine sarcoma. Prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, skin melanoma, small intestine cancer, stomach cancer, gastric carcinoma, gastrointestinal stromal tumor (GIST), uterine cancer or uterine sarcoma.

[0449] In some embodiments, the cancer is liver cancer, hepatoepithelial cancer, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, or hepatoblastoma.

[0450] In some embodiments, the disease is a proliferative disease, an inflammatory disease, an autoimmune disease, a neurological disease, an eye disease, a respiratory disease, a metabolic disease, a skin disease, an auditory disease, a liver disease, a kidney disease, or an infectious disease. In some embodiments, the disease is a liver disease. In some embodiments, the liver disease is cirrhosis, steatosis, or a combination thereof.

[0451] In some embodiments, the disease is a metabolic disorder. In some embodiments, the metabolic disorder is hypercholesterolemia, hypobetalipoproteinemia, coronary heart disease, peripheral arterial disease, stroke, type 2 diabetes, obesity, or hypertension, or a combination thereof.

[0452] It should be understood that, unless otherwise stated, any description of a method of treatment or prevention includes the use of compounds, scaffolds and conjugates to provide such treatment or prevention as described herein. It should also be understood that, unless otherwise stated, any description of a method of treatment or prevention includes the use of compounds, scaffolds and conjugates to prepare a medicament for treating or preventing such a condition. Treatment or prevention includes treatment or prevention in humans or non-human animals, including rodents and other disease models.

[0453] It should be understood that, unless otherwise stated, any description of a method of treatment includes the use of compounds, scaffolds and conjugates to provide such treatment as described herein. It should also be understood that, unless otherwise stated, any description of a method of treatment includes the use of compounds, scaffolds and conjugates to prepare a medicament for treating such a condition. Treatment includes treatment of humans or non-human animals, including rodents and other disease models.

[0454] As used herein, the term "subject" is interchangeable with the term "subject in need", both of which refer to subjects with a disease or with an increased risk of developing a disease. "Subjects" include mammals. The mammal can be, for example, a human or an appropriate non-human mammal, such as a primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or pig. The subject can also be a bird or poultry. In some embodiments, the mammal is a human. A subject in need can be a subject that has previously been diagnosed or identified as having a disease or disorder disclosed herein. A subject in need can also be a subject suffering from a disease or disorder disclosed herein. Alternatively, a subject in need can be a subject with an increased risk of developing such a disease or disorder relative to the general population (i.e., a subject that is prone to developing such a disorder relative to the general population). A subject in need can suffer from a refractory or resistant 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 beginning of treatment, or may become resistant during treatment. In some embodiments, the subject in need thereof has received all known effective therapies for the diseases or disorders disclosed herein, but all have failed. In some embodiments, the subject in need thereof has received at least one prior therapy.

[0455] 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 include the administration of a compound of the disclosure, or a pharmaceutically acceptable salt, polymorph, or solvate thereof, to alleviate the symptoms or complications of, or to eliminate, a disease, condition, or disorder. The term "treatment" may also include treatment of cells in vitro or in animal models. It should be understood that references to "treating" or "treatment" include the alleviation of established symptoms of a condition. Thus, "treating" or "treatment" of a state, disorder or condition includes: (1) preventing or delaying the development of clinical symptoms of a state, disorder or condition in a human who may be suffering from or susceptible to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the disease or its recurrence (in the case of maintenance treatment) or the development of at least one clinical or subclinical symptom thereof; or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.

[0456] It will be appreciated that the compounds, scaffolds and conjugates of the present disclosure, or pharmaceutically acceptable salts, polymorphs or solvates thereof, can also or may also be used to prevent related diseases, conditions or disorders, or to identify suitable candidates for such purposes.

[0457] As used herein, the terms "preventing," "prevent," or "protecting against" describe the reduction or elimination of the onset of symptoms or complications of such a disease, condition, or disorder.

[0458] As used herein, the term "therapeutically effective amount" refers to an amount of a pharmaceutical agent that is used to treat, alleviate or prevent an established disease or condition, or that exhibits a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend on the subject's weight, size, and health; the nature and extent of the condition; and the 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.

[0459] As used herein, the term "therapeutically effective amount" refers to an amount of a pharmaceutical agent that is used to treat or alleviate an established disease or condition, or that exhibits a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend on the subject's weight, size, and health; the nature and extent of the condition; and the 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.

[0460] It will be appreciated that for any compound, the therapeutically effective amount can be estimated initially in a cell culture assay, for example, of neoplastic cells, or in an animal model (typically 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 doses and routes useful for human administration. Therapeutic / prophylactic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal to 50% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD 50 / ED 50 Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending on the dosage form employed, sensitivity of the patient, and the route of administration.

[0461] Dosage and administration are adjusted to provide sufficient levels of 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, one or more drug combinations, reaction sensitivity and tolerance / response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, weekly or once every two weeks, depending on the half-life and clearance rate of the particular formulation.

[0462] All publications and patent documents cited herein are incorporated herein by reference, as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. The reference to publications and patent documents is not intended as an admission that any one is relevant prior art, nor does it constitute any admission of its content or date. The present invention has now been described by way of written description, and those skilled in the art will recognize that the present invention can be practiced in a variety of embodiments, and the above description and the following examples are for illustrative purposes and are not intended to limit the appended claims. Example

[0463] Example 1: Optimization of chemical modification patterns to improve stability and efficacy of siRNA targeting gene 1.

[0464] This article describes a study showing that the stability and tolerance of siRNA are further improved by reducing the 2'-fluoro nucleotide content and increasing the 2'-O-methyl content. The 2'-fluoro and 2'-O-methyl content of the sense and antisense strands of the siRNA are systematically examined, and an advanced modification design with improved in vitro activity and metabolic stability is identified, which can be converted into enhanced in vivo efficacy, duration and tolerance. In the study described herein, the positional effects of 2'-O-methyl and 2'-fluoro modifications on the sense and antisense strands are systematically examined, and several advanced designs with significantly improved in vitro activity are identified for the 21 / 23mer and 20 / 22mer platforms.

[0465] The studies described herein used a publicly reported prototype compound (21 / 23mer) that is a clinically approved drug. Based on chemical modifications of the prototype compound, the positional effects of 2'-O-methyl and 2'-fluoro modifications on both the sense and antisense strands were systematically examined for the 21 / 23mer and 20 / 22mer platforms. Several advanced designs are described herein that have up to 60-fold improvements in in vitro activity (IC) compared to designs of the prototype compound. 50 4pm compared to 245pm).

[0466] Materials and methods

[0467] Compounds (eg, nucleic acid agents, nucleic acid agents containing nucleotide-based enhancers, and conjugates) are prepared by solid phase synthesis according to standard synthetic protocols.

[0468] In brief, oligonucleotide synthesis is carried out on a solid support to incorporate each nucleoside phosphoramidite from the 3'-end to the 5'-end to prepare an oligonucleotide (oligo) single strand. ETT or BTT is used as an activator for the coupling reaction. Iodine oxidation of phosphite triester (P(III)) in water / pyridine / THF is used to provide a phosphate backbone, and DDTT is used to prepare a thiophosphate bond. Ammonium aqueous solution is used to crack the oligonucleotide from the solid support and to remove the protecting group as a whole. The crude oligonucleotide is then concentrated and purified by strong anion exchange or reversed phase HPLC. The purified fractions are merged and concentrated.

[0469] In some cases, the oligonucleotide single strand is then conjugated to a targeting ligand (e.g., sugar, peptide, antibody) by post-synthetic conjugation to provide a conjugate. The conjugation reaction is carried out using a standard conjugation method. The conjugate crude product is further purified by strong anion exchange or reverse phase HPLC. The purified fractions are combined and concentrated. The synthesized single strand is then dialyzed against water, concentrated, and their OD amounts are measured. The sense strand and antisense strand are annealed based on a 1:1 molar ratio. The annealed duplex solution is lyophilized to provide the desired siRNA molecule.

[0470] To measure in vitro activity, all test articles in the optimization series were transfected into Huh7 cells using Lipofectamine RNAiMax. In a 96-well plate, 20,000 cells / well were transfected with each compound at a concentration range of 5000pM to 2pM according to the manufacturer's protocol (Invitrogen-13778-150). All transfections were performed in triplicate. Before RNA isolation, the cells were incubated at 37°C for 24 hours. The intracellular RNA was then isolated using the RNeasy96 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 draw graphs and calculate IC 50 .

[0471] In the studies described herein, the positional effects of 2'-F and 2'-OMe modifications of the sense and antisense strands of siRNA molecules against target gene 1 were examined on their stability and tolerance.

[0472] Testing of chemical modifications of the 21 / 23mer prototype siRNA molecules targeting target gene 1

[0473] In the first set of optimizations described in this paper ( Figure 1 ), the modification pattern of the exemplary siRNA prototype compound against target gene 1 was used as the parent design [SEQ ID NO: 1: SEQ ID NO: 2], which had an IC of 245 pm 50 Inhibits target gene 1. Another modified siRNA molecule [SEQ ID NO:3:SEQ ID NO:4] showed a comparable IC of 243 pm 50 , and [SEQ ID NO:5:SEQ ID NO:6] showed a ~10-fold increase in RNAi activity compared to the parental design (IC 50 25pm vs. 245pm)( Figure 2A-2C ).

[0474] Subsequent optimization of prototype siRNA molecules: positional effects of 2′-OMe and 2′-F modifications of the sense strand

[0475] In a subsequent set of optimizations described in this paper ( Figure 3 and Figure 5 ), determining the improved potency (IC) via refinement of the modification content (2'-OMe and 2'-F) on the sense strand 50 ), with the focus on the range of 10 to 15. Figure 4A-4E As shown in Figure 2, the in vitro activities of 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, and IC 50 33 pm, 30 pm and 31 pm respectively. However, [SEQ ID NO:10:SEQ ID NO:6] showed an IC of 14 pm 50 , which is a ~2-fold improvement in potency compared to the potency of siRNA molecules [SEQ ID NO:7 and SEQ ID NO:6], and also a ~2-fold improvement compared to siRNA molecules [SEQ ID NO:5:SEQ ID NO:6]. The results described herein indicate that 2'-F modifications at positions 10 and 14 can additively improve in vitro activity in combination with adjacent modifications.

[0476] In addition, when the 2'-OMe modification was moved to position 11 of the siRNA molecule [SEQ ID NO: 11: SEQ ID NO: 6] ( Figure 5 ), the in vitro potency was substantially impaired, providing an IC of 110 pm 50 ,like Figure 6A-6C However, additional modification of the siRNA molecule [SEQ ID NO: 12: SEQ ID NO: 6] showed an IC of 67 pm. 50 Partial rescue efficacy. In addition, the siRNA molecule [SEQ ID NO: 13: SEQ ID NO: 6] showed an excellent IC of 14 pm 50 , and a ~2-fold improvement in in vitro activity compared to the in vitro activity of [SEQ ID NO: 5 and SEQ ID NO: 6].

[0477] Further optimization of the 2'-F content of the antisense strand of siRNA molecules

[0478] The results described here describe the optimization of the 2'-F content on the antisense strand to generate a third set of siRNAs ( Figure 7 and Fig. 9). The design of siRNA containing sense strand and antisense strand according to siRNA molecule [SEQ ID NO:5:SEQ ID NO:6] was used as the parent design, and the effect of 2'-F modification mainly in the seed region (positions 2-8) was examined. 2'-F modification was added at position 3, position 5, position 7, position 8 and position 9, respectively. Among these individual modifications, siRNA molecules [SEQ ID NO:5:SEQID NO:15] and [SEQ ID NO:5:SEQ ID NO:14] showed IC values ​​of 7 pm and 9 pm. 50 , which is a 2-3 fold improvement in activity compared to the activity of the parental design, as Figure 8A-8F 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 marginal effects on in vitro activity.

[0479] like Fig. 9 As shown in , the combined effects of 2'-F modifications at some of these key positions were further examined. [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 designed siRNA molecule [SEQ ID NO:5:SEQ ID NO:6]. See Figure 10A-10C .

[0480] Fourth Optimization of Combinatorial Modifications on Sense and Antisense Strands A subsequent set of optimizations is described here, namely antisense strands with further optimized 2'-F content to provide antisense strands according to SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25. The antisense strand was then paired with the identified best sense strand according to SEQ ID NO: 13 or the suboptimal sense strand according to SEQ ID NO: 9 ( Figure 7 and Figure 8A-8F ) to examine their overall impact on efficacy ( Fig.11 ).like Figures 12A-12F As shown in Figure 2, the siRNA molecules [SEQ ID NO: 9 and SEQ ID NO: 22] provided an IC of 9 pm. 50 , which is comparable to the in vitro activity of siRNA molecules [SEQ ID NO:9 and SEQ ID NO:6] (IC 50) showed a ~3-fold increase in in vitro activity compared to the parental design [SEQ ID NO:5:SEQ ID NO:6] (IC of 25 pm 50 ) in vitro activity compared to 3-fold improvement. Pairing the best sense strand according to SEQ ID NO: 13 with 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, IC 50 12-14pm.

[0481] Optimizing chemical modifications on 20 / 22mers to improve stability and potency of siRNA targeting gene 1

[0482] Guided by the results of 2'-F and 2'-OMe modifications in the 21 / 23mer platform described above, position effects in the 20 / 22mer platform following the same siRNA modification design were examined. To generate a 20 / 22mer duplex based on a 21 / 23mer, two nucleosides at position 21 of the sense strand according to SEQ ID NO: 1 and at position 23 of the antisense strand according to SEQ ID NO: 2 were cleaved to provide a design comprising the sense strand and antisense strand according to the siRNA molecule [SEQ ID NO: 26: SEQ ID NO: 27]. Therefore, [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 used as the parent design in the following 20 / 22mer platform optimization. Although the number of nucleoside positions on the 20mer sense strand is one less than the corresponding position on the 21mer sense strand, the basic siRNA modification trends and position effects observed in the 21 / 23mer platform can be translated to the 20 / 22mer platform.

[0483] In the study described here, the first siRNA modification set of the 20 / 22mer platform [SEQ ID NO:26:SEQ ID NO:27] was used as the parental design, which showed an IC of 58 pm. 50 ( Fig.13 ). The modified siRNA molecule [SEQ ID NO: 28: SEQ ID NO: 27] showed comparable efficacy, such as Figure 14A-Figure 14C The further chemically modified siRNA molecule [SEQ ID NO: 29: SEQ ID NO: 30] showed an IC of 15 pm 50, which is a ~4-fold improvement in RNAi activity compared to that of [SEQ ID NO:26:SEQ ID NO:27]. The significant improvement in in vitro activity is consistent with the trend observed for [SEQ ID NO:5:SEQ ID NO:6] when the same chemical modification was applied to the 21 / 23mer sequence.

[0484] Second modification of 20 / 22mer: position effect of 2'-OMe and 2'-F modifications on the sense strand

[0485] In the study described here, the 2'-OMe content on the second SAR group of the sense strand was refined ( Fig.15 and Fig.17 ). The studies described here identified that the 2'-OMe modification at position 15 can maintain good in vitro potency regardless of the modification at position 14 [SEQ ID NO:31:SEQ ID NO:30] or [SEQ ID NO:32:SEQ ID NO:30]. siRNA molecules [SEQ ID NO:33:SEQ ID NO:30] and [SEQ ID NO:36:SEQ ID NO:30] showed excellent IC values ​​of 4-5 pm 50 ( Figure 16A-16E ). These combined effects of modifications at positions 10, 14, and 15 on the 20mer sense strand are consistent with those on the 21mer sense strand.

[0486] In addition, 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 values ​​of 5-10 pm. 50 ,like Figures 18A-18C The third modification of the 20 / 22mer: Positional effect of 2'-OMe and 2'-F modifications on the antisense strand

[0487] Here we describe the generation of a third set of siRNA modifications to refine the 2'-F content on the antisense strand for improved potency ( Fig.19 In the results described here, it was shown that position 3 of the siRNA molecule [SEQ ID NO:29:SEQ ID NO:45] exhibited an IC of 5 pm 502'-F modifications at other positions in the seed region, including position 3 of the siRNA molecule [SEQ ID NO:29:SEQ ID NO:50], position 5 of the siRNA molecule [SEQ ID NO:29:SEQ ID NO:46], position 7 of the siRNA molecule [SEQ ID NO:29:SEQ ID NO:47], position 8 of the siRNA molecule [SEQ ID NO:29:SEQ ID NO:47], and position 9 of the siRNA molecule [SEQ ID NO:29:SEQ ID NO:48], also showed a positive effect on in vitro activity, IC 50 The range is from 7pm to 11pm ( Fig. 20B and Figure 20D-Figure 20F ).

[0488] In addition, the combinatorial effects of 2'-F modifications at some of these key positions in the seed region were examined ( Fig.21 ).like Figure 22A-22C As shown in , the siRNA molecule [SEQ ID NO:29:SEQ ID NO:37] showed a 2-fold potency improvement compared to the potency of the parental design [SEQ ID NO:29:SEQ ID NO:30]. In addition, the intrinsic potency of the siRNA molecule [SEQ ID NO:29:SEQ ID NO:38] was well maintained.

[0489] The fourth modification of 20 / 22mer: the combined effect of the optimized sense strand and the optimized (or further optimized) antisense strand

[0490] Here we describe the design of a fourth set of siRNA modifications to examine the combined effects of an optimized sense strand with an optimized (or further optimized) antisense strand ( Fig.23 ). In the results described here, all doubly optimized duplexes showed IC 50 ≤7 pm, which shows a 2-fold improvement in potency compared to the parental design ( Figure 24A-24F Among these molecules, the design comprising the sense and antisense strands of the siRNA molecule [SEQ ID NO:44:SEQ ID NO:42] showed the lowest IC of 4 pm. 50 , which is the in vitro activity (IC 50 245 pm) compared to ~60-fold improvement in in vitro activity.

[0491] Summarize

[0492] In the results described herein, the 2'-O-methyl and 2'-fluorine content of the sense and antisense strands across the siRNA molecules were systematically refined in both the 21 / 23mer and 20 / 22mer platforms, and advanced modification designs for improved in vitro activity and stability were identified. The improved in vitro activity and stability disclosed herein can be translated into enhanced in vivo efficacy, duration, and tolerability.

[0493] Studies described herein also show that optimized pairings of sense and antisense strands confer combinatorial improvements in activity, leading to the identification of several advanced designs, such as [SEQ ID NO:44:SEQ ID NO:42], which have up to 60-fold improvement in in vitro activity compared to the prototype benchmark.

[0494] Additional embodiments

[0495] Additional embodiments of the present disclosure include the following:

[0496] Embodiment 1. An isolated oligonucleotide comprising:

[0497] (a) a sense strand comprising X1 nucleotides, wherein at least one nucleotide is modified with a first modification,

[0498] Each of the remaining nucleotides is independently modified with a second modification, and

[0499] X1 is an integer selected from 13-36,

[0500] wherein the first modification and the second modification are different; and

[0501] (b) an antisense strand comprising X2 nucleotides, wherein at least one nucleotide is modified with a third modification,

[0502] Each of the remaining nucleotides is independently modified with a fourth modification, and

[0503] X2 is an integer selected from 18-31,

[0504] Wherein, the third modification and the fourth modification are different.

[0505] Embodiment 2. An isolated oligonucleotide according to embodiment 1, wherein the first modification is a modification at the 2'-position of the sugar portion of at least one nucleotide selected from a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, a 2'-deoxy modification and its equivalents, and combinations thereof.

[0506] Embodiment 3. An 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 a stereoisomer thereof.

[0507] Embodiment 4. An isolated oligonucleotide according to any one of embodiments 1-3, wherein the first modification is a 2'-F modification, a 2'-CN modification or a 2'-N3 modification, or a stereoisomer thereof.

[0508] Embodiment 5. The isolated oligonucleotide according to any one of embodiments 1-4, wherein the first modification is a 2'-F modification or a stereoisomer thereof.

[0509] Embodiment 6. An isolated oligonucleotide according to any one of embodiments 1-5, wherein the second modification is a modification at the 2'-position of the sugar portion of one or more of the remaining nucleotides selected from 2'-C1-C6 alkyl, 2'-OR modification, 2'-amino and morpholino substitution, and equivalents and combinations thereof, in which R is a C1-C6 alkyl optionally substituted with: C1-C6 alkoxy, acetamide, phenyl or a heteroaryl comprising a 5-membered ring or a 6-membered ring and 1 or 2 heteroatoms selected from N, O and S.

[0510] Embodiment 7. The isolated oligonucleotide according to any one of embodiments 1-6, wherein the second modification is a 2'-OR modification.

[0511] Embodiment 8. The isolated oligonucleotide according to any one of embodiments 1-7, wherein the second modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification.

[0512] Embodiment 9. The isolated oligonucleotide according to any one of embodiments 1-8, wherein the second modification is a 2'-O-methyl modification.

[0513] Embodiment 10. The isolated oligonucleotide according to any one of embodiments 1-8, 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.

[0514] Embodiment 11. The isolated oligonucleotide according to any one of embodiments 1-10, wherein the first modification is a 2'-F modification or a stereoisomer thereof, and the second modification is a 2'-O-methyl modification.

[0515] Embodiment 12. An isolated oligonucleotide according to any one of embodiments 1-11, wherein the third modification is a modification at the 2'-position of the sugar portion of at least one nucleotide selected from a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, a 2'-deoxy modification and its equivalents, and combinations thereof.

[0516] Embodiment 13. An isolated oligonucleotide according to any one of embodiments 1-12, 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.

[0517] Embodiment 14. The isolated oligonucleotide of any one of Embodiments 1-13, wherein the third modification is a 2'-F modification, a 2'-CN modification, or a 2'-N3 modification, or a stereoisomer thereof.

[0518] Embodiment 15. The isolated oligonucleotide of any one of Embodiments 1-14, wherein the third modification is a 2'-F modification or a stereoisomer thereof.

[0519] Embodiment 16. An isolated oligonucleotide according to any one of embodiments 1-15, wherein the fourth modification is a modification at the 2'-position of the sugar portion of one or more of the remaining nucleotides selected from 2'-C1-C6 alkyl, 2'-OR modification, 2'-amino and morpholino substitution, and equivalents and combinations thereof, in which R is a C1-C6 alkyl optionally substituted with: C1-C6 alkoxy, acetamide, phenyl or a heteroaryl comprising a 5-membered ring or a 6-membered ring and 1 or 2 heteroatoms selected from N, O and S.

[0520] Embodiment 17. The isolated oligonucleotide of any one of Embodiments 1-16, wherein the fourth modification is a 2'-OR modification.

[0521] Embodiment 18. The isolated oligonucleotide of any one of Embodiments 1-17, wherein the fourth modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification.

[0522] Embodiment 19. The isolated oligonucleotide of any one of Embodiments 1-18, wherein the fourth modification is a 2'-O-methyl modification.

[0523] Embodiment 20. The isolated oligonucleotide of any one of Embodiments 1-18, 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.

[0524] Embodiment 21. The isolated oligonucleotide of any one of Embodiments 1-20, wherein the third modification is a 2'-F modification or a stereoisomer thereof, and the fourth modification is a 2'-O-methyl modification.

[0525] Embodiment 22. The isolated oligonucleotide of any one of embodiments 1-21, wherein in the sense strand at least three nucleotides are modified with the first modification.

[0526] Embodiment 23. An isolated oligonucleotide according to embodiment 22, wherein at least two of the at least three nucleotides modified with the first modification are positioned consecutively.

[0527] Embodiment 24. The isolated oligonucleotide according to embodiment 22 or 23, wherein at least three of the at least three nucleotides modified with the first modification are positioned consecutively.

[0528] Embodiment 25. The isolated oligonucleotide of any one of embodiments 1-24, wherein in the sense strand at least four nucleotides are modified with the first modification.

[0529] Embodiment 26. The isolated oligonucleotide according to embodiment 25, wherein at least three of the at least four nucleotides modified with the first modification are positioned consecutively.

[0530] Embodiment 27. The isolated oligonucleotide according to embodiment 25 or 26, wherein at least four of the at least four nucleotides modified with the first modification are positioned consecutively.

[0531] Embodiment 28. The isolated oligonucleotide of any one of embodiments 1-27, wherein in the sense strand at least five nucleotides are modified with the first modification.

[0532] Embodiment 29. The isolated oligonucleotide according to embodiment 28, wherein at least three of the at least five nucleotides modified with the first modification are positioned consecutively.

[0533] Embodiment 30. The isolated oligonucleotide according to embodiment 28 or 29, wherein at least four of the at least five nucleotides modified with the first modification are positioned consecutively.

[0534] Embodiment 31. An isolated oligonucleotide according to any one of embodiments 22-30, wherein the at least three nucleotides, the at least four nucleotides or the at least five nucleotides modified with the first modification are positioned from position 10 to position 15 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0535] Embodiment 32. An isolated oligonucleotide according to any one of embodiments 22-31, wherein two of the at least three nucleotides modified with the first modification are positioned at a position selected from position 10, position 11, position 12 and position 13 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0536] Embodiment 33. An isolated oligonucleotide according to any one of embodiments 22-32, wherein three of the at least three nucleotides modified with the first modification are positioned at positions selected from position 10, position 11, position 12 and position 13 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0537] Embodiment 34. An isolated oligonucleotide according to any one of embodiments 22-33, wherein one of the at least three nucleotides modified with the first modification is positioned at position 11 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0538] Embodiment 35. The isolated oligonucleotide according to any one of embodiments 22-34, wherein one of the at least four nucleotides modified with the first modification is positioned at position 10 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand.

[0539] Embodiment 36. An isolated oligonucleotide according to any one of embodiments 22-34, wherein one of the at least four nucleotides modified with the first modification is positioned at position 14 from the nucleotide that is complementary to the first nucleotide at the 5' end of the antisense strand.

[0540] Embodiment 37. An isolated oligonucleotide according to any one of embodiments 22-34, wherein two of the at least four nucleotides modified with the first modification are positioned at position 10 to position 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0541] Embodiment 38. The isolated oligonucleotide according to any one of embodiments 22-37, wherein the at least three nucleotides modified with the first modification are not all positioned consecutively.

[0542] Embodiment 39. The isolated oligonucleotide of any one of Embodiments 22-38, wherein the at least three nucleotides, the at least four nucleotides, or the at least five nucleotides are modified with a 2'-F modification.

[0543] Embodiment 40. An isolated oligonucleotide according to embodiment 31, wherein in the sense strand, at most three nucleotides at positions 12, 13 and 15 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand are modified with the first modification.

[0544] Embodiment 41. An isolated oligonucleotide according to embodiment 31, wherein in the sense strand, at position 11, position 12, position 13 and position 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to four nucleotides are modified with the first modification.

[0545] Embodiment 42. An isolated oligonucleotide according to embodiment 31, wherein in the sense strand, at most four nucleotides at positions 10, 11, 12 and 13 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand are modified with the first modification.

[0546] Embodiment 43. An isolated oligonucleotide according to embodiment 31, wherein in the sense strand, at position 10, position 12, position 13 and position 15 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to four nucleotides are modified with the first modification.

[0547] Embodiment 44. An isolated oligonucleotide according to embodiment 31, wherein in the sense strand, at position 10, position 11, position 12, position 13 and position 15 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to five nucleotides are modified with the first modification.

[0548] Embodiment 45. An isolated oligonucleotide according to embodiment 31, wherein in the sense strand, at position 10, position 11, position 12, position 13 and position 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to five nucleotides are modified with the first modification.

[0549] Embodiment 46. The isolated oligonucleotide of any one of Embodiments 1-45, wherein the sense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following formula:

[0550] 5'(M) g (F) f (M) e (F) d (M) c (F) b (M) a 3',

[0551] 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 0-16 and indicates the number of consecutive nucleotides modified with the modification, and wherein the sense strand is any one of:

[0552] a)5'(M)0(F)0(M)0(F)0(M)8(F)3(M) 10 3' (SEQ ID NO:52);

[0553] b)5'(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3' (SEQ ID NO:53);

[0554] c)5'(M)0(F)0(M)0(F)0(M)7(F)3(M) 10 3' (SEQ ID NO:54);

[0555] d)5'(M)0(F)0(M)5(F)1(M)1(F)2(M) 11 3' (SEQ ID NO:55);

[0556] e)5'(M)0(F)0(M)6(F)1(M)1(F)2(M) 11 3' (SEQ ID NO:56);

[0557] f)5'(M)6(F)1(M)1(F)2(M)1(F)1(M)9 3' (SEQ ID NO:57);

[0558] g)5'(M)5(F)1(M)1(F)2(M)1(F)1(M)9 3' (SEQ ID NO:58);

[0559] h)5'(M)0(F)0(M)0(F)0(M)7(F)4(M) 10 3' (SEQ ID NO:59);

[0560] i)5'(M)0(F)0(M)6(F)1(M)1(F)4(M)9 3'(SEQ ID NO:60);

[0561] j)5'(M)0(F)0(M)0(F)0(M)8(F)4(M)9 3'(SEQ ID NO:61);

[0562] k)5'(M)0(F)0(M)0(F)0(M)6(F)4(M) 10 3' (SEQ ID NO: 62);

[0563] l)5'(M)0(F)0(M)5(F)1(M)1(F)4(M)9 3'(SEQ ID NO:63);

[0564] m)5'(M)0(F)0(M)0(F)0(M)7(F)4(M)9 3'(SEQ ID NO:64);

[0565] n)5'(M)0(F)0(M)0(F)0(M)7(F)5(M)9 3' (SEQ ID NO:65); and

[0566] o)5'(M)0(F)0(M)0(F)0(M)6(F)5(M)9 3' (SEQ ID NO: 66).

[0567] Embodiment 47. The isolated oligonucleotide of embodiments 1-46, wherein in the antisense strand, up to seven nucleotides are modified with the third modification.

[0568] Embodiment 48. An isolated oligonucleotide according to embodiment 47, wherein at most four of the at most seven nucleotides modified with the third modification are positioned at position 2 to position 8 from the first nucleotide at the 5' end of the antisense strand.

[0569] Embodiment 49. The isolated oligonucleotide according to embodiment 47 or 48, wherein at most two of the at most seven nucleotides modified with the third modification are positioned consecutively.

[0570] Embodiment 50. An isolated oligonucleotide according to any one of embodiments 47-49, wherein three or four of the up to seven nucleotides modified with the third modification are positioned at a position selected from position 2, position 3, position 5, position 6, position 7 and position 8 from the first nucleotide at the 5' end of the antisense strand.

[0571] Embodiment 51. An isolated oligonucleotide according to any one of embodiments 47-50, wherein at least one of the up to seven nucleotides modified with the third modification is positioned at position 14 from the first nucleotide at the 5' end of the antisense strand.

[0572] Embodiment 52. An isolated oligonucleotide according to any one of embodiments 47-51, wherein two or three of the up to seven nucleotides modified with the third modification are positioned at a position selected from position 2, position 3, position 5 and position 6 from the first nucleotide at the 5' end of the antisense strand.

[0573] Embodiment 53. An isolated oligonucleotide according to any one of embodiments 47-52, wherein three of the up to seven nucleotides modified with the third modification are positioned at a position selected from position 2, position 3, position 5, and position 6 from the first nucleotide at the 5' end of the antisense strand.

[0574] Embodiment 54. An isolated oligonucleotide according to any one of embodiments 47-53, wherein two of the up to seven nucleotides modified with the third modification are positioned at position 8 and position 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

[0575] Embodiment 55. An isolated oligonucleotide according to any one of embodiments 47-54, wherein one or two of the up to seven nucleotides modified with the third modification are positioned at a position selected from position 14 and position 16 from the first nucleotide at the 5' end of the antisense strand.

[0576] Embodiment 56. An isolated oligonucleotide according to any one of embodiments 47-55, wherein two of the up to seven nucleotides modified with the third modification are positioned at position 14 and position 16 from the first nucleotide at the 5' end of the antisense strand.

[0577] Embodiment 57. The isolated oligonucleotide of any one of Embodiments 47-56, wherein the up to seven nucleotides are modified with a 2'-F modification.

[0578] Embodiment 58. The isolated oligonucleotide according to any one of embodiments 47-57, wherein up to six nucleotides are modified with the third modification.

[0579] Embodiment 59. The isolated oligonucleotide of any one of Embodiments 47-58, wherein the up to six nucleotides are modified with a 2'-F modification.

[0580] Embodiment 60. The isolated oligonucleotide according to any one of embodiments 47-59, wherein at most five nucleotides are modified with the third modification.

[0581] Embodiment 61. The isolated oligonucleotide of any one of Embodiments 47-60, wherein the up to five nucleotides are modified with a 2'-F modification.

[0582] Embodiment 62. The isolated oligonucleotide according to any one of embodiments 47-61, wherein at most four nucleotides are modified with the third modification.

[0583] Embodiment 63. The isolated oligonucleotide of any one of embodiments 47-62, wherein up to four nucleotides are modified with a 2'-F modification.

[0584] Embodiment 64. The isolated oligonucleotide according to any one of embodiments 47-63, wherein at most three nucleotides are modified with the third modification.

[0585] Embodiment 65. The isolated oligonucleotide of any one of Embodiments 47-64, wherein the at least three nucleotides are modified with a 2'-F modification.

[0586] Embodiment 66. An isolated oligonucleotide according to any one of embodiments 60-61, wherein in the antisense strand, at most five nucleotides at position 2, position 3, position 6, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand are modified by the third modification.

[0587] Embodiment 67. An isolated oligonucleotide according to embodiments 60-61, wherein in the antisense strand, at position 2, position 6, position 7, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to five nucleotides are modified with the third modification.

[0588] Embodiment 68. An isolated oligonucleotide according to any one of embodiments 58-59, wherein in the antisense strand, at position 2, position 3, position 6, position 8, position 9 and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to six nucleotides are modified by the third modification.

[0589] Embodiment 69. An isolated oligonucleotide according to any one of embodiments 58-59, wherein in the antisense strand, at most six nucleotides at position 2, position 3, position 5, position 8, position 9 and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand are modified by the third modification.

[0590] Embodiment 70. An isolated oligonucleotide according to any one of embodiments 47-57, wherein in the antisense strand, at position 2, position 3, position 5, position 7, position 9, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to seven nucleotides are modified by the third modification.

[0591] Embodiment 71. An isolated oligonucleotide according to embodiments 47-57, wherein in the antisense strand, at position 2, position 3, position 5, position 7, position 10, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to seven nucleotides are modified with the third modification.

[0592] Embodiment 72. The isolated oligonucleotide of any one of Embodiments 1-71, wherein the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following formula:

[0593] 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',

[0594] 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 0-16 and indicates the number of consecutive nucleotides modified with the modification, and wherein the antisense strand is any one of:

[0595] 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)1 5'(SEQ IDNO:14);

[0596] 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)1 5'(SEQ IDNO:15);

[0597] 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)1 5’(SEQ IDNO:16);

[0598] 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)1 5’(SEQ IDNO:17);

[0599] 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)1 5’(SEQ IDNO:18);

[0600] 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)1 5’(SEQ IDNO:20);

[0601] 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)1 5’(SEQ IDNO:21);

[0602] 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)1 5’(SEQ IDNO:22);

[0603] 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)1 5’(SEQ IDNO:23);

[0604] 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)1 5’(SEQID NO:24);

[0605] 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);

[0606] 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);

[0607] 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)1 5’(SEQID NO:30);

[0608] 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)1 5’(SEQID NO:50);

[0609] 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)1 5’(SEQID NO:45);

[0610] 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)1 5’(SEQID NO:46);

[0611] 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)1 5’(SEQID NO:47);

[0612] 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)1 5’(SEQID NO:48);

[0613] 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)1 5’(SEQID NO:49);

[0614] 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)1 5’(SEQID NO:37);

[0615] 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)1 5'(SEQID NO:38);

[0616] 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)1 5'(SEQID NO:39);

[0617] 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)1 5'(SEQID NO:40);

[0618] and

[0619] 25) 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)1 5' (SEQ ID NO: 42).

[0620] Embodiment 73. The isolated oligonucleotide of Embodiment 72, wherein the sense strand comprises

[0621] Nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following formula:

[0622] 5'(M) g (F) f (M) e (F) d (M) c (F) b (M) a 3',

[0623] 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 0-16 and indicates the number of consecutive nucleotides modified with the modification, and wherein the sense strand is any one of:

[0624] a)5'(M)0(F)0(M)0(F)0(M)8(F)3(M) 10 3' (SEQ ID NO:52);

[0625] b)5’(M)0(F)0(M)5(F)1(M)1(F)3(M) 10 3’(SEQ ID NO:53);

[0626] c)5’(M)0(F)0(M)0(F)0(M)7(F)3(M) 10 3’(SEQ ID NO:54);

[0627] d)5’(M)0(F)0(M)5(F)1(M)1(F)2(M) 11 3’(SEQ ID NO:55);

[0628] e)5’(M)0(F)0(M)6(F)1(M)1(F)2(M) 11 3’(SEQ ID NO:56);

[0629] f)5’(M)6(F)1(M)1(F)2(M)1(F)1(M)9 3’(SEQ ID NO:57);

[0630] g)5’(M)5(F)1(M)1(F)2(M)1(F)1(M)9 3’(SEQ ID NO:58);

[0631] h)5’(M)0(F)0(M)0(F)0(M)7(F)4(M) 10 3’(SEQ ID NO:59);

[0632] i)5’(M)0(F)0(M)6(F)1(M)1(F)4(M)9 3’(SEQ ID NO:60);

[0633] j)5’(M)0(F)0(M)0(F)0(M)8(F)4(M)9 3’(SEQ ID NO:61);

[0634] k)5’(M)0(F)0(M)0(F)0(M)6(F)4(M) 10 3’(SEQ ID NO:62);

[0635] l)5’(M)0(F)0(M)5(F)1(M)1(F)4(M)9 3’(SEQ ID NO:63);

[0636] m)5’(M)0(F)0(M)0(F)0(M)7(F)4(M)9 3’(SEQ ID NO:64);

[0637] n)5'(M)0(F)0(M)0(F)0(M)7(F)5(M)9 3' (SEQ ID NO:65); and

[0638] o)5'(M)0(F)0(M)0(F)0(M)6(F)5(M)9 3' (SEQ ID NO: 66).

[0639] Embodiment 74. An isolated oligonucleotide according to any one of embodiments 40-45, wherein in the antisense strand, at position 2, position 3, position 6, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to five nucleotides are modified by the third modification.

[0640] Embodiment 75. An isolated oligonucleotide according to embodiments 40-45, wherein in the antisense strand, at position 2, position 6, position 7, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to five nucleotides are modified with the third modification.

[0641] Embodiment 76. An isolated oligonucleotide according to any one of embodiments 40-45, wherein in the antisense strand, at most six nucleotides at position 2, position 3, position 6, position 8, position 9 and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand are modified by the third modification.

[0642] Embodiment 77. An isolated oligonucleotide according to any one of embodiments 40-45, wherein in the antisense strand, at position 2, position 3, position 5, position 8, position 9 and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to six nucleotides are modified by the third modification.

[0643] Embodiment 78. An isolated oligonucleotide according to any one of embodiments 40-45, wherein in the antisense strand, at position 2, position 3, position 5, position 7, position 9, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to seven nucleotides are modified by the third modification.

[0644] Embodiment 79. An isolated oligonucleotide according to embodiments 40-45, wherein in the antisense strand, at position 2, position 3, position 5, position 7, position 10, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, up to seven nucleotides are modified with the third modification.

[0645] Embodiment 80. The isolated oligonucleotide of any one of embodiments 1-79, wherein X1 is 18-21, and X2 is 20-23.

[0646] Embodiment 81. The isolated oligonucleotide of embodiment 80, wherein X1 is 20 or 21, and X2 is 22 or 23.

[0647] Embodiment 82. An isolated oligonucleotide according to any one of embodiments 1-80, wherein X2 is equal to X1 plus 2.

[0648] Embodiment 83. An isolated oligonucleotide according to any one of Embodiments 1-82, wherein the sense strand comprises at least one nucleotide having a modified phosphate backbone.

[0649] Embodiment 84. An isolated oligonucleotide according to any one of Embodiments 1-83, wherein the antisense strand comprises at least one nucleotide having a modified phosphate backbone.

[0650] Embodiment 85. An isolated oligonucleotide according to embodiment 83 or 84, wherein the modified phosphate backbone comprises a modified phosphodiester bond.

[0651] Embodiment 86. An isolated oligonucleotide according to embodiment 85, wherein the modified phosphodiester bond is modified by replacing one or more oxygen atoms with a moiety or by forming a 2'-5' bond, wherein the moiety is bonded to the phosphorus atom in the phosphodiester bond through a carbon, nitrogen or sulfur atom in the moiety.

[0652] Embodiment 87. An isolated oligonucleotide according to embodiment 85, wherein the modified phosphodiester bond comprises phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate diester, methylsulfonylphosphoramidate or phosphonoacetate.

[0653] Embodiment 88. The isolated oligonucleotide of any one of Embodiments 1-84, comprising one or more non-natural base-containing nucleotides, locked nucleotides, or abasic nucleotides.

[0654] Embodiment 89. An isolated oligonucleotide according to any one of Embodiments 1-88, wherein the terminal nucleotide at the 5' end comprises a phosphate mimetic.

[0655] Embodiment 90. The isolated oligonucleotide of embodiment 89, wherein the 5'-phosphate mimetic is ethylphosphonate, vinylphosphonate, or an analog thereof.

[0656] Embodiment 91. The isolated oligonucleotide of any one of Embodiments 1-90, wherein the antisense strand comprises at least two single-stranded nucleotides at the 3' end.

[0657] Embodiment 92. The isolated oligonucleotide of any one of embodiments 1-91, wherein in the sense strand or the antisense strand or both, a terminal or internal nucleotide is linked to a targeting ligand.

[0658] Embodiment 93. An isolated oligonucleotide according to embodiment 92, wherein 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.

[0659] Embodiment 94. An isolated oligonucleotide according to Embodiment 93, wherein the targeting ligand is GalNAc.

[0660] Embodiment 95. An isolated oligonucleotide according to any one of Embodiments 1-94, wherein the antisense strand is complementary to an mRNA, wherein sequence-specific hybridization of the antisense strand to the mRNA results in degradation of the mRNA.

[0661] Embodiment 96. The isolated oligonucleotide of any one of embodiments 1-95, wherein the isolated oligonucleotide is capable of targeting any specific gene target, including but not limited to PCSK9, ANGPTL-3, AGT, HSD17b3, C3, CFB, and HBV.

[0662] Equivalent

[0663] The details of one or more embodiments of the present disclosure are set forth in the above attached description. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are now described. Other features, objects and advantages of the present disclosure will be apparent from the description, as well as from the claims. In this description and the appended claims, unless otherwise clearly specified in the context, singular forms include plural indicators. Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the field to which the present disclosure belongs. All patents and publications cited in this description are incorporated by reference.

[0664] The foregoing description is presented for purposes of illustration only and is not intended to limit the present disclosure to the precise form disclosed, except as defined in the appended claims.

Claims

1. An isolated oligonucleotide comprising: (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, and X1 is an integer selected from 13-36, wherein 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, Each of the remaining nucleotides is independently modified with a fourth modification, and X2 is an integer selected from 18-31, in, The third modification and the fourth modification are different.

2. The isolated oligonucleotide according to claim 1, wherein the first modification is a modification at the 2'-position of the sugar portion of the at least one nucleotide selected from the group consisting of a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, a 2'-deoxy modification and equivalents thereof, and combinations thereof, Preferably, wherein the first modification is a 2'-F modification, a 2'-CN modification, a 2'-N3 modification or a 2'-deoxy modification, or a stereoisomer thereof, More preferably, wherein the first modification is a 2'-F modification, a 2'-CN modification or a 2'-N3 modification, or a stereoisomer thereof, even more preferably, wherein the first modification is a 2'-F modification or a stereoisomer thereof.

3. The isolated oligonucleotide according to any one of claims 1-2, wherein the second modification is one or more modifications at the 2'-position of the sugar portion of the remaining nucleotides selected from 2'-C1-C6 alkyl, 2'-OR modification, 2'-amino and morpholino substitution, and equivalents thereof, and combinations thereof, in which 2'-OR modification, R is a C1-C6 alkyl optionally substituted with a C1-C6 alkoxy, acetamide, phenyl, or a heteroaryl comprising a 5-membered ring or a 6-membered ring and 1 or 2 heteroatoms selected from N, O and S, Preferably, wherein the second modification is a 2'-OR modification, more preferably wherein the second modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification, Even more preferably, wherein said second modification is a 2'-O-methyl modification.

4. The isolated oligonucleotide according to any one of claims 1 to 3, 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, Preferably, wherein the first modification is a 2'-F modification or a stereoisomer thereof, and the second modification is a 2'-O-methyl modification.

5. The isolated oligonucleotide according to any one of claims 1 to 4, wherein the third modification is a modification at the 2'-position of the sugar portion of at least one nucleotide selected from a 2'-F modification, a 2'-CN modification, a 2'-N3 modification, a 2'-deoxy modification and equivalents thereof, and combinations thereof, Preferably, 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, More preferably, wherein the third modification is a 2'-F modification, a 2'-CN modification or a 2'-N3 modification, or a stereoisomer thereof, Even more preferably, wherein the third modification is a 2'-F modification or a stereoisomer thereof.

6. The isolated oligonucleotide according to any one of claims 1 to 5, wherein the fourth modification is a modification at the 2'-position of the sugar portion of one or more of the remaining nucleotides selected from 2'-C1-C6 alkyl, 2'-OR modification, 2'-amino and morpholino substitution, and equivalents thereof, and combinations thereof, in which 2'-OR modification, R is a C1-C6 alkyl optionally substituted with a C1-C6 alkoxy, acetamide, phenyl, or a heteroaryl comprising a 5-membered ring or a 6-membered ring and 1 or 2 heteroatoms selected from N, O, and S, Preferably, wherein the fourth modification is a 2'-OR modification, More preferably, wherein the fourth modification is a 2'-O-methyl modification or a 2'-methoxyethoxy modification, Even more preferably, wherein said fourth modification is a 2'-O-methyl modification.

7. The isolated oligonucleotide according to any one of claims 1 to 6, 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, Preferably, wherein the third modification is a 2'-F modification or a stereoisomer thereof, and the fourth modification is a 2'-O-methyl modification.

8. The isolated oligonucleotide according to any one of claims 1 to 7, wherein in the sense strand, at least three nucleotides are modified with the first modification, optionally wherein at least two of the at least three nucleotides modified with the first modification are positioned consecutively, or optionally wherein at least three of the at least three nucleotides modified with the first modification are positioned consecutively.

9. The isolated oligonucleotide according to any one of claims 1 to 8, wherein in the sense strand, at least four nucleotides are modified with the first modification, optionally wherein at least three of the at least four nucleotides modified with the first modification are positioned consecutively, or optionally wherein at least four of the at least four nucleotides modified with the first modification are positioned consecutively.

10. The isolated oligonucleotide according to any one of claims 1 to 9, wherein in the sense strand, at least five nucleotides are modified with the first modification, optionally wherein at least three of the at least five nucleotides modified with the first modification are positioned consecutively, or optionally wherein at least four of the at least five nucleotides modified with the first modification are positioned consecutively.

11. The isolated oligonucleotide according to any one of claims 8 to 10, wherein the at least three nucleotides, the at least four nucleotides or the at least five nucleotides modified with the first modification are positioned at position 10 to position 15 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, Optionally, wherein two of the at least three nucleotides modified with the first modification are positioned at a position selected from position 10, position 11, position 12 and position 13 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, or Optionally, wherein three of the at least three nucleotides modified with the first modification are positioned at positions selected from position 10, position 11, position 12 and position 13 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, or Optionally, wherein one of the at least three nucleotides modified with the first modification is positioned at position 11 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

12. The isolated oligonucleotide according to any one of claims 8 to 11, wherein one of the at least four nucleotides modified with the first modification is positioned at position 10 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, or Optionally, wherein one of the at least four nucleotides modified with the first modification is located at position 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, or Optionally, wherein two of the at least four nucleotides modified with the first modification are positioned at position 10 to position 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand.

13. The isolated oligonucleotide according to any one of claims 8 to 12, wherein the at least three nucleotides modified with the first modification are not all positioned consecutively.

14. The isolated oligonucleotide according to any one of claims 8-13, wherein the at least three nucleotides, the at least four nucleotides or the at least five nucleotides are modified with a 2'-F modification.

15. The isolated oligonucleotide according to any one of claims 8 to 14, wherein in the sense strand at position 12, position 13 and position 15 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to three nucleotides are modified with the first modification, At position 11, position 12, position 13 and position 14, starting from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to four nucleotides are modified with the first modification, at position 10, position 11, position 12 and position 13 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to four nucleotides are modified with the first modification, at position 10, position 12, position 13 and position 15 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to four nucleotides are modified with the first modification, At position 10, position 11, position 12, position 13 and position 15 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to five nucleotides are modified with the first modification, or At position 10, position 11, position 12, position 13 and position 14 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to five nucleotides are modified with the first modification.

16. The isolated oligonucleotide of any one of claims 1-15, wherein the sense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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 0-16 and indicates the number of consecutive nucleotides modified with the modification, and wherein the sense strand is any one of: 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)5'(M)0(F)0(M)0(F)0(M)6(F)5(M)9 3' (SEQ ID NO: 66).

17. The isolated oligonucleotide according to any one of claims 1 to 16, wherein in the antisense strand up to seven nucleotides are modified with said third modification, Optionally, wherein at most four of the at most seven nucleotides modified with the third modification are positioned at position 2 to position 8 from the first nucleotide at the 5' end of the antisense strand, or Optionally, wherein at most two of said at most seven nucleotides modified with said third modification are positioned contiguously, or Optionally, wherein three or four of the up to seven nucleotides modified with the third modification are positioned at a position selected from position 2, position 3, position 5, position 6, position 7 and position 8 from the first nucleotide at the 5' end of the antisense strand, or Optionally, wherein at least one of the up to seven nucleotides modified with the third modification is located at position 14 from the first nucleotide at the 5' end of the antisense strand, or Optionally, wherein two or three of the up to seven nucleotides modified with the third modification are located at a position selected from position 2, position 3, position 5 and position 6 from the first nucleotide at the 5' end of the antisense strand, or Optionally, wherein three of the up to seven nucleotides modified with the third modification are positioned at a position selected from position 2, position 3, position 5 and position 6 from the first nucleotide at the 5' end of the antisense strand, or Optionally, wherein two of the up to seven nucleotides modified with the third modification are located at position 8 and position 14 from the nucleotide complementary to the first nucleotide at the 5' end of the antisense strand, or Optionally, wherein one or two of the up to seven nucleotides modified with the third modification are located at a position selected from position 14 and position 16 from the first nucleotide at the 5' end of the antisense strand, or Optionally, wherein two of the up to seven nucleotides modified with the third modification are positioned at position 14 and position 16 from the first nucleotide at the 5' end of the antisense strand.

18. The isolated oligonucleotide according to claim 17, wherein at most six nucleotides are modified with the third modification.

19. The isolated oligonucleotide according to any one of claims 17-18, wherein at most five nucleotides are modified with the third modification.

20. The isolated oligonucleotide according to any one of claims 17-19, wherein at most four nucleotides are modified with the third modification.

21. The isolated oligonucleotide according to any one of claims 17 to 20, wherein at most three nucleotides are modified with the third modification.

22. The isolated oligonucleotide of any one of claims 17-21, wherein the at least seven nucleotides, at least six nucleotides, at least five nucleotides, at least four nucleotides, or at least three nucleotides are modified with a 2'-F modification.

23. The isolated oligonucleotide according to any one of claims 17 to 22, wherein in the antisense strand: At position 2, position 3, position 6, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to five nucleotides are modified with the third modification, or At position 2, position 6, position 7, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to five nucleotides are modified with the third modification.

24. The isolated oligonucleotide according to any one of claims 17-23, wherein in the antisense strand: At position 2, position 3, position 6, position 8, position 9 and position 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to six nucleotides are modified with the third modification, or At position 2, position 3, position 5, position 8, position 9 and position 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to six nucleotides are modified with the third modification.

25. The isolated oligonucleotide according to any one of claims 17 to 24, wherein in the antisense strand At position 2, position 3, position 5, position 7, position 9, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to seven nucleotides are modified with the third modification, or At position 2, position 3, position 5, position 7, position 10, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to seven nucleotides are modified with the third modification.

26. The isolated oligonucleotide of any one of claims 1-25, wherein the antisense strand comprises nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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 0-16 and indicates the number of consecutive nucleotides modified with the modification, wherein the antisense strand is any one of the following: 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)1 5'(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)1 5'(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)1 5'(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)1 5' (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)1 5'(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)1 5'(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)1 5' (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)1 5’(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)1 5’(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)1 5’(SEQ IDNO: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)1 5’(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)1 5’(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)1 5’(SEQ IDNO: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)1 5’(SEQ IDNO: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)1 5’(SEQ IDNO: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)1 5’(SEQ IDNO: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)1 5’(SEQ IDNO: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)1 5’(SEQ IDNO: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)1 5'(SEQ IDNO: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)1 5'(SEQ IDNO: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)1 5'(SEQ IDNO: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)1 5'(SEQ IDNO: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)1 5'(SEQ IDNO:40); and 25)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)1 5' (SEQ ID NO: 42).

27. The isolated oligonucleotide of claim 26, wherein the sense strand comprises Nucleotides modified with a 2'-F modification and nucleotides modified with a 2'-O-methyl modification according to the following 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 0-16 and indicates the number of consecutive nucleotides modified with the modification, and wherein the sense strand is any one of: 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)5'(M)0(F)0(M)0(F)0(M)6(F)5(M)9 3' (SEQ ID NO: 66).

28. The isolated oligonucleotide of claim 15, wherein in the antisense strand: at position 2, position 3, position 6, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to five nucleotides are modified with the third modification, at position 2, position 6, position 7, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to five nucleotides are modified with the third modification, at position 2, position 3, position 6, position 8, position 9 and position 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to six nucleotides are modified with the third modification, at position 2, position 3, position 5, position 8, position 9 and position 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to six nucleotides are modified with the third modification, At position 2, position 3, position 5, position 7, position 9, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to seven nucleotides are modified with the third modification, or At position 2, position 3, position 5, position 7, position 10, position 14 and position 16 from the nucleotide complementary to the first nucleotide at the 5'-end of the antisense strand, up to seven nucleotides are modified with the third modification.

29. The isolated oligonucleotide of any one of claims 1-28, wherein the sense strand comprises at least one nucleotide having a modified phosphate backbone, and / or the antisense strand comprises at least one nucleotide having a modified phosphate backbone.

30. The isolated oligonucleotide of any one of claims 1-29, comprising one or more non-natural base-containing nucleotides, locked nucleotides, or abasic nucleotides.

31. The isolated oligonucleotide of any one of claims 1-30, wherein the terminal nucleotide at the 5' end comprises a phosphate mimetic.

32. The isolated oligonucleotide of any one of claims 1-31, wherein in the sense strand or the antisense strand or both, a terminal or internal nucleotide is linked to a targeting ligand, Optionally, the targeting ligand is selected from one or more of carbohydrates, peptides, lipids, antibodies or fragments thereof, aptamers, albumin, fibrinogen and folic acid, or Optionally, wherein the targeting ligand is GalNAc.

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