Modified polynucleic acid molecules targeting AGT and uses thereof

Abstract

Disclosed herein are polynucleic acid molecules that can be utilized for suppressing the expression of angiotensinogen (AGT). Also, described herein are pharmaceutical compositions comprising polynucleic acid molecules targeting angiotensinogen (AGT) mRNA. Further, provided herein are methods for suppressing the expression of angiotensinogen (AGT) by utilizing the polynucleic acid molecules described herein.

Description

WSGR Docket No.61382-734.601 MODIFIED POLYNUCLEIC ACID MOLECULES TARGETING AGT AND USES THEREOF CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 661,488, filed on June 18, 2024, and U.S. Provisional Application No.63 / 767,372, filed on March 5, 2025, each of which is incorporated herein by reference in its entirety. BACKGROUND OF THE DISCLOSURE

[0002] Angiotensinogen (AGT) plays an important role in regulating blood pressure and other metabolic processes. Accordingly, there is a need for developing an effective AGT inhibitor without cytotoxicity. The polynucleic acid molecules, conjugates thereof, and methods described herein satisfy this need and provide related advantages. INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. SUMMARY OF THE DISCLOSURE

[0004] To meet the need for a more effective AGT inhibitor, disclosed herein, in certain aspects, are polynucleic acid molecules for modulating expression of angiotensinogen (AGT) gene, wherein the polynucleic acid molecule comprising a passenger strand and a guide strand, and the polynucleic acid molecule comprises a nucleic acid sequence that is at least 85%, at least 90%, or at least 95% identical to a nucleic acid sequence in Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33.

[0005] In some instances, the guide strand comprises a nucleic acid sequence that is at least 85%, at least 90%, or at least 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 1-18 and 160. In some instances, the passenger strand comprises a nucleic acid sequence that is at least 85%, at least 90%, or at least 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 21-33. 1WSGR Docket No.61382-734.601

[0006] In some instances, the guide strand comprises a nucleic acid sequence comprising at least 16, 17, 18, 19, or 20 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 1-18 and 160, with no more than 1, 2, 3, or 4 mismatches. In some instances, the passenger strand comprises a nucleic acid sequence comprising at least 16, 17, 18, 19, or 20 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 21-33, with no more than 1, 2, 3, or 4 mismatches.

[0007] In some instances, the passenger strand comprises a nucleic acid sequence selected from SEQ ID NOs: 21-33, and the guide strand comprises a nucleic acid sequence selected from SEQ ID NOs: 1-18 and 160. In some instances, the passenger strand comprises a nucleic acid sequence selected from SEQ ID NOs: 21-22, and 25-27 and the guide strand comprises a nucleic acid sequence selected from SEQ ID NOs: 1-2, 7, and 10-11.

[0008] In some instances, the polynucleic acid molecule comprises (1) a 2’-fluoro modified nucleotides; (2) a 2’-O-methyl modified nucleotides; or (3) a modified internucleotide linkage.

[0009] In some instances, the polynucleic acid molecule comprises at least two consecutive modified internucleotide linkages at the 5’ end. In some instances, the polynucleic acid molecule comprises at least two internucleotide linkages among three internucleotide linkages at the 3’end substituted with modified internucleotide linkages. In some instances, the modified internucleotide linkage is a phosphorothioate internucleotide linkage or a phosphorodithioate internucleotide linkage. In some instances, the phosphorothioate internucleotide linkage is a stereochemically enriched phosphorothioate internucleotide linkage. In some instances, the stereochemically enriched phosphorothioate internucleotide linkage is an SP chiral internucleotide phosphorothioate linkage.

[0010] In some instances, the polynucleic acid comprises a plurality of modified internucleotide linkages, and at least 1, 2, 3, or 4 of the plurality of modified internucleotide linkages are stereochemically enriched phosphorothioate internucleotide linkages. In some instances, the stereochemically enriched phosphorothioate internucleotide linkages comprise an RPchiral internucleotide phosphorothioate linkage or an SPchiral internucleotide phosphorothioate linkage. In some instances, the stereochemically enriched phosphorothioate internucleotide linkage(s) is disposed between two consecutive nucleosides that are two of six 5’ or 3’ end nucleosides of the passenger strand or the guide strand.

[0011] In some instances, the polynucleic acid molecule comprises the phosphorothioate internucleotide linkage located between the two nucleotides at position 11 and 12 from the 5’ end of the guide strand. In some instances, the polynucleic acid molecule comprises the phosphorodithioate internucleotide linkage located between the two nucleotides at position 11 and 12 from the 5’ end of the guide strand. 2WSGR Docket No.61382-734.601

[0012] In some instances, the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnnnnnnnn - 3’, wherein the guide strand comprises ‘5- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, “n” stands for a 2’-O-methyl modified nucleotide, “s” stands for a 3'-phosphorothioate internucleotide linkage.

[0013] In some instances, the guide strand comprises a vinyl phosphonate modified nucleotide at the 5’ end. In some instances, the vinyl phosphonate modified nucleotide is 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4) or 5'(E)vinylphosphonate-2-O-methyluridine-3'phosphate (vpu). In some instances, the vinyl phosphonate modified nucleotide substitutes a 5’ end nucleotide of SEQ ID NOs: 1-18 and 160.

[0014] In some instances, the polynucleic acid molecule comprises a nucleotide analogue substituting any one of modified nucleotides in the guide strand or the passenger strand, wherein the nucleotide analogue comprises a hypoxanthine nucleobase-containing nucleoside substitution, abasic substitution, acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2- thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’-phosphate (U3f), 2-amino-2’-O- methyladenosine-3’-phosphate (a1), 2'-methoxyethyladenosine-3'phosphate (A6), 2'- methoxyethyluridine-3'-phosphate (U6), or 2’-O-methylinosine-3’-phosphate (i).

[0015] In some instances, the hypoxanthine nucleobase-containing nucleoside substitution is an inosine substitution. In some instances, the nucleotide analogue is located within a seed region of the guide strand. In some instances, the nucleotide analogue is located within 7 nucleotides from the 5’ end of the guide strand. In some instances, the passenger strand comprises an inverted abasic moiety at the 5’ end or 3’ end.

[0016] In some instances, the guide strand comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 36-80, 110-142, 148-152, and 161-164.

[0017] In some instances, the guide strand comprises a nucleic acid sequence comprising at least 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 36-80, 110-142, 148-152, and 161-164 with no more than 1, 2, 3, or 4 mismatches.

[0018] In some instances, the passenger strand comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 81-107, 143-147, 153-157, and 165-166. In some instances, the passenger strand comprises a nucleic acid sequence comprising at least 15, 16, 17, 18, 19, or 20 consecutive 3WSGR Docket No.61382-734.601 nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 81-107, 143-147, 153-157, and 165-166, with no more than 1, 2, 3, or 4 mismatches. In some instances, the passenger strand comprises a nucleic acid sequence selected from SEQ ID NOs: 81-107, 143-147, 153- 157, and 165-166, and the guide strand comprises a nucleic acid sequence selected from SEQ ID NOs: 36-80, 110-142, 148-152, and 161-164.

[0019] In some instances, the cytotoxicity of the polynucleic acid molecule is decreased compared to an unmodified polynucleic acid molecule.

[0020] In some instances, provided herein is a polynucleic acid molecule for modulating expression of angiotensinogen (AGT) gene, comprising: a guide strand comprising a nucleic acid sequence of UAGACCAAGGAGAAACGGCUCCU (SEQ ID NO: 7) and a passenger strand comprising a nucleic acid sequence of GAGCCGUUUCUCCUUGGUCUA (SEQ ID NO: 25).

[0021] In some instances, provided herein is a polynucleic acid molecule for modulating expression of angiotensinogen (AGT) gene, comprising: a) a guide strand comprising a nucleic acid sequence of u(Rps)Af(Rps)uugcUfcaauUfuUfuGfcaggu(Sps)u(Sps)c (SEQ ID NO: 49) and a passenger strand comprising a nucleic acid sequence of ascscugcAfaAfaAfuugagcaaua (SEQ ID NO: 81); b) a guide strand comprising a nucleic acid sequence of u(Rps)Af(Rps)uugcUfcaauUfuUfuGfcaggu(Sps)u(Rps)c (SEQ ID NO: 53) and a passenger strand comprising a nucleic acid sequence of ascscugcAfaAfaAfuugagcaaua (SEQ ID NO: 81); c) a guide strand comprising a nucleic acid sequence of usAfsuugcUfcaaussUfuUfuGfcaggususc (SEQ ID NO: 77) and a passenger strand comprising a nucleic acid sequence of ascscugcAfaAfaAfuugagcaaua (SEQ ID NO: 81); d) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggaGfaAfaCfggcucscsu (SEQ ID NO: 57) and a passenger strand comprising a nucleic acid sequence of gsasgccgUfuUfcUfccuuggucua (SEQ ID NO: 85; SEQ ID NO: 154; SEQ ID NO: 156); 4WSGR Docket No.61382-734.601 e) a guide strand comprising a nucleic acid sequence of u(Rps)Af(Rps)gaccAfaggaGfaAfaCfggcuc(Sps)c(Sps)u (SEQ ID NO: 148; SEQ ID NO: 151) and a passenger strand comprising a nucleic acid sequence of gsasgccgUfuUfcUfccuuggucua (SEQ ID NO: 85; SEQ ID NO: 156); f) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggasGfaAfaCfggcucscsu (SEQ ID NO: 164) and a passenger strand comprising a nucleic acid sequence of gsasgccgUfuUfcUfccuuggucua (SEQ ID NO: 85; SEQ ID NO: 156); g) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggaGfaAfaCfggcucscsu (SEQ ID NO: 57) and a passenger strand comprising a nucleic acid sequence of sgsagccgUfuUfcUfccuuggucuas(invAb) (SEQ ID NO: 155); h) a guide strand comprising a nucleic acid sequence of u(Rps)Af(Rps)gaccAfaggassGfaAfaCfggcuc(Sps)c(Sps)u (SEQ ID NO: 152) and a passenger strand comprising a nucleic acid sequence of gsasgccgUfuUfcUfccuuggucua (SEQ ID NO: 156); i) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggassGfaAfaCfggcucscsu (SEQ ID NO: 149; SEQ ID NO: 150) and a passenger strand comprising a nucleic acid sequence of gsasgccgUfuUfcUfccuuggucua (SEQ ID NO: 85; SEQ ID NO: 156); j) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggaGfaAfaCfggcucscsu (SEQ ID NO: 57) and a passenger strand comprising a nucleic acid sequence of sgsagccgUfuUfcUfccuuggucua (SEQ ID NO: 165); k) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggassGfaAfaCfggcucscsu (SEQ ID NO: 150) and a passenger strand comprising a nucleic acid sequence of sgsagccgUfuUfcUfccuuggucua (SEQ ID NO: 165); l) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggasGfaAfaCfggcucscsu (SEQ ID NO: 164) and a passenger strand 5WSGR Docket No.61382-734.601 comprising a nucleic acid sequence of sgsagccgUfuUfcUfccuuggucua (SEQ ID NO: 165); m) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggassGfaAfaCfggcucscsu (SEQ ID NO: 150) and a passenger strand comprising a nucleic acid sequence of sgsagccgUfuUfcUfccuuggucuas(invAb) (SEQ ID NO: 155); n) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggaGfaAfaCfggcucscsu (SEQ ID NO: 57) and a passenger strand comprising a nucleic acid sequence of gssasgccgUfuUfcUfccuuggucua (SEQ ID NO: 166); or o) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggassGfaAfaCfggcucscsu (SEQ ID NO: 150) and a passenger strand comprising a nucleic acid sequence of gssasgccgUfuUfcUfccuuggucua (SEQ ID NO: 166); wherein smaller case “n” stands for 2’-O-methyl modified nucleotide, upper case followed with an “f” (i.e., “Nf”) stands for 2’-fluoro modified nucleotide, “s” stands for 3'-phosphorothioate, “ss” stands for 3'-phosphorodithioate, “(Rps)” stands for 3'- phosphorothioate, Rp diastereomer, “(Sps)” stands for 3' phosphorothioate, Sp diastereomer, and "(invAb)" stands for inverted abasic deoxyribonucleotide.

[0022] In another aspects, the present disclosure provides a polynucleic acid molecule conjugate for modulating expression of angiotensinogen (AGT) gene, wherein the polynucleic acid molecule conjugate comprises a polynucleic acid molecule described herein and an asialoglycoprotein receptor targeting moiety. In some instances, the polynucleic acid molecule and the asialoglycoprotein receptor targeting moiety is coupled via a linker. In some instances, the linker comprises formula (IV) below, 6WSGR Docket No.61382-734.601wherein at least one of Y1 and Y2 is a nucleotide in the polynucleic acid molecule, and wherein theis connected to the asialoglycoprotein receptor targeting moiety.

[0023] In some instances, the Y1 is the last nucleotide on the 3’ end of the passenger strand of the polynucleic acid molecule. In some instances, the Y1 and Y2 are two consecutive nucleotides in the polynucleic acid molecule.

[0024] In some instances, the asialoglycoprotein receptor targeting moiety comprises N- Acetylgalactosamine (GalNAc) or galactose.

[0025] In some instances, the linker and the asialoglycoprotein receptor targeting moiety with the last nucleotide on the 3’ end of the passenger strand of the polynucleic acid molecule are shown in:7WSGR Docket No.61382-734.6018WSGR Docket No.61382-734.6019WSGR Docket No.61382-734.601wherein Z in formula (V’), (V’’’’’’’), (VI), (VI’), (VI’’), or (VI’’’) is -H, -OH, -O-Methyl, -F, or -O-methoxyethyl, R in formula (V’), (V’’’’’’’), (VI), (VI’), (VI’’), or (VI’’’) is adenine, uracil, guanine, cytosine, thymine, abasic, or others, and theis to connect to the next nucleotide of the polynucleic acid molecule.

[0026] In some instances, the linker and the asialoglycoprotein receptor targeting moiety with the first nucleotide on the 5’ end of the passenger strand of the polynucleic acid molecule are shown in:10WSGR Docket No.61382-734.601wherein Z in formula (V’’’) or (X’) is -H, -OH, -O-Methyl, -F, or -O-methoxyethyl, R in formula (V’’’) or (X’) is adenine, uracil, guanine, cytosine, thymine, abasic, or others, and the is to connect to the next nucleotide of the polynucleic acid molecule.

[0027] In some instances, the polynucleic acid molecule conjugate described herein further comprises a lipid, wherein the lipid is coupled with the first nucleotide on the 5’ end of the passenger strand of the polynucleic acid molecule. In some instances, the lipid is shown in:

[0028] In one aspect, the present disclosure provides a pharmaceutical composition comprising a polynucleic acid molecule described herein or a polynucleic acid molecule conjugate described herein, and a pharmaceutically acceptable excipient. In some instances, the pharmaceutical composition is formulated as a nanoparticle formulation. In some instances, the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, transdermal, intravenous, subcutaneous, or intrathecal administration.

[0029] In another aspect, the present disclosure provides a method of modulating expression of angiotensinogen (AGT) gene in a subject, comprising: administering to the subject a polynucleic acid molecule described herein, a polynucleic acid molecule conjugate described herein, or a pharmaceutical composition described herein, thereby modulating the expression of AGT gene in the subject. 11WSGR Docket No.61382-734.601

[0030] In one aspect, the present disclosure provides a method of preventing, alleviating, or treating hypertension in a subject in need thereof, comprising: administering to the subject a polynucleic acid molecule described herein, a polynucleic acid molecule conjugate described herein, or a pharmaceutical composition described herein, wherein the polynucleic acid molecule described herein, the polynucleic acid molecule conjugate described herein, or the pharmaceutical composition described herein reduces the expression of AGT gene in the subject.

[0031] In another aspect, the present disclosure provides a method of preventing, alleviating, or treating atherosclerosis in a subject in need thereof, comprising: administering to the subject a polynucleic acid molecule described herein, a polynucleic acid molecule conjugate described herein, or a pharmaceutical composition described herein, wherein the polynucleic acid molecule described herein, the polynucleic acid molecule conjugate described herein, or the pharmaceutical composition described herein reduces the expression of AGT gene in the subject. In some instances, the subject suffers from a coronary artery disease or a symptom thereof.

[0032] In one aspect, the present disclosure provides a method of preventing, alleviating, or treating obesity in a subject in need thereof, comprising: administering to the subject a polynucleic acid molecule described herein, a polynucleic acid molecule conjugate described herein, or a pharmaceutical composition described herein, wherein the polynucleic acid molecule described herein, the polynucleic acid molecule conjugate described herein, or the pharmaceutical composition described herein reduces the expression of AGT gene in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Various aspects of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative aspects, in which the principles of the disclosure are utilized, and the accompanying drawings below.

[0034] FIG.1 depicts % change in serum AGT following single 2 mg / kg dose in cynomolgus monkeys as described in Example 3.

[0035] FIG.2 depicts the % change in serum AGT following a single 2 mg / kg subcutaneous dose in cynomolgus monkeys as described in Example 4.

[0036] FIG.3 depicts the % change in serum AGT following single 2 mg / kg dose in cynomolgus monkeys as described in Example 13. DETAILED DESCRIPTION OF THE DISCLOSURE

[0037] Angiotensinogen (AGT) a glycoprotein in the rennin-angiotensin system. After various cleavages by different enzymes, AGT is converted to a varieties of angiotensin peptides, among 12WSGR Docket No.61382-734.601 which angiotensin (Ang) II regulates blood pressure and sodium / water homeostasis. Human AGT is mainly synthesized in hepatocytes. After cleavage and processing, AGT can be converted to AngI which is secreted into plasma or extracellular compartments.

[0038] AGT mRNA is mainly detected in many organs or tissues such as liver, adipose, brain, heart, kidneys, and vessels, and is most abundant in livers. At cellular level, AGT is synthesized in hepatocytes, adipocytes, proximal tubule epithelial cells, and astrocytes.

[0039] M235T was first identified from a screening of AGT’s single nucleotide polymorphisms (SNPs) to implicate a causal relationship between the AGT gene and hypertension in humans. See Jeunemaitre X et al. Molecular basis of human hypertension: role of angiotensinogen. Cell. 1992;71:169–180. With more investigation, M235T of AGT is shown to be associated with atherosclerosis in different populations. See, e.g., Katsuya T et al. Association of angiotensinogen gene T235 variant with increased risk of coronary heart disease. Lancet. 1995;345:1600–1603. In addition, T174M may also be related to risk factors or prevalence of coronary artery disease. See, e.g., Gardemann A et al. Angiotensinogen T174M and M235T gene polymorphisms are associated with the extent of coronary atherosclerosis. Atherosclerosis. 1999;145:309–314. AGT may also play an important role in obesity in humans.

[0040] Described herein is a polynucleic acid molecule for modulating expression of AGT gene, wherein the polynucleic acid molecule comprises a passenger strand and a guide strand, and wherein the polynucleic acid molecule comprises a nucleic acid sequence in Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33. Accordingly, provided herein are various target regions of human AGT mRNA the polynucleic acid molecule described herein hybridizes to. In some embodiments, provided herein is the sequences of the polynucleic acid molecule described herein. In some embodiments, provided herein is the possible modifications of the polynucleic acid molecule described herein. In some embodiments, provided herein is the possible conjugates of the polynucleic acid molecule described herein.

[0041] Also described herein is a method of modulating expression of angiotensinogen (AGT) gene in a subject. Described further herein is a method of modulating LDL and / or cholesterol in a subject in need thereof. Definitions

[0042] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B.” 13WSGR Docket No.61382-734.601

[0043] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0044] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0045] “Percent (%) sequence identity” or “Percent (%) identity” with respect to the nucleic acid sequences identified herein is defined as the percentage of nucleic acid in a candidate sequence that are identical with the nucleic acid sequence being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity.

[0046] All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so forth. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and the like. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the polynucleic acid molecules, the polynucleic acid molecule conjugates, the pharmaceutical compositions, the methods and other aspects belong. 14WSGR Docket No.61382-734.601

[0048] As used herein, the term “complementary” indicates a sufficient degree of complementarity between two nucleic acid molecules that bind stably and specifically to avoid nonspecific binding.

[0049] As used herein, the term “polynucleic acid” and the term “polynucleotide” are interchangeably used to refer a chain of nucleotides. The term “nucleotide” includes a sequence “G”, “C”, “A”, “T”, and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base. In some instances, the “nucleotide” can refer to a modified nucleotide (e.g., with modified sugar moiety, modified base, modified internucleotide linkage, or combination thereof, including, but not limited to 2’-modified nucleotide, LNA, ENA, BNA, UNA, GNA etc.) In some instances, the “nucleotide” can refer to a modified nucleotide with a non-canonical base (e.g. including, but not limited to, 2- thiouridine, 2-thiothymidine, inosine, 2-aminopurine, 2,6-diaminopurine, dihydrouridine, 4- thiouridine, 4-thiothymidine, 2-thiocytidine).

[0050] As used herein, a “subject” can be any mammal, including a human and a non-human primate.

[0051] The term “condition,” as used herein, includes diseases, disorders, and susceptibilities. In some cases, the condition is an AGT related disorder or symptoms thereof.

[0052] As used herein, the term “treat”, “treating” or “treatment” of any disease or disorder refers, in one instance, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another instance, “treat”, “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another instance, “treat”, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.

[0053] The terms “prevent,” “preventing,” and “prevention,” as used herein, refer to a decrease in the occurrence of pathology of a condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition. The prevention may be complete, e.g., the total absence of pathology of a condition in a subject. The prevention may also be partial, such that the occurrence of pathology of a condition in a subject is less than that which would have occurred without the present disclosure.

[0054] “Administering” and its grammatical equivalents as used herein can refer to providing pharmaceutical compositions described herein to a subject or a patient. Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the composition to the subject, depending upon the type of disease to be treated or the site of the 15WSGR Docket No.61382-734.601 disease. For example, the composition can be administered, e.g., orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, or via infusion. One or more such routes can be employed.

[0055] The terms “pharmaceutical composition” and its grammatical equivalents as used herein can refer to a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients, carriers, and / or a therapeutic agent to be administered to a subject, e.g., a human in need thereof.

[0056] The term “pharmaceutically acceptable” and its grammatical equivalents as used herein can refer to an attribute of a material which is useful in preparing a pharmaceutical composition that is generally safe, non–toxic, and neither biologically nor otherwise undesirable and is acceptable for veterinary as well as human pharmaceutical use. “Pharmaceutically acceptable” can refer a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the pharmaceutical composition in which it is contained.

[0057] A “pharmaceutically acceptable excipient” refers to an excipient that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

[0058] The term “therapeutic agent” can refer to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. Therapeutic agents can also be referred to as “actives” or “active agents.” Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0059] As used herein, the term “sense strand” can be interchangeably used with the term “passenger strand”, and the tern “antisense strand” can be interchangeably used with the term “guide strand”. In some instances, a nucleic acid sequence described herein for a sense strand and a passenger strand can be interchangeably used. Also, in some instances, a nucleic acid sequence described herein for an antisense strand and a guide strand can be interchangeably used.

[0060] As used herein, the term “consecutive nucleotides” refers to a sequence contains a number of consecutive nucleotides from a reference sequence. For example, if a reference sequence is N1N2N3N4N5N6N7, a consecutive nucleotides can be N1N2N3N4or N3N4N5N6, but a sequence of N1N3N4N5 or N3N4N7 cannot be a consecutive nucleotides. 16WSGR Docket No.61382-734.601

[0061] As used herein, the term “negative control” refers to a subject or a cell receiving no treatment or placebo.

[0062] It is appreciated that certain features of the polynucleic acid molecules, and / or polynucleic acid molecule conjugates, pharmaceutical composition comprising the polynucleic acid molecules or the polynucleic acid molecule conjugates, methods and other aspects, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the polynucleic acid molecules, and / or polynucleic acid molecule conjugates, pharmaceutical composition comprising the polynucleic acid molecules or the polynucleic acid molecule conjugates, methods and other aspects, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present polynucleic acid molecules, and / or polynucleic acid molecule conjugates, pharmaceutical composition comprising the polynucleic acid molecules or the polynucleic acid molecule conjugates, methods and other aspects and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. Polynucleic Acid Molecules Target Regions of Polynucleic Acid Molecules

[0063] Described herein is a polynucleic acid molecule for modulating expression of AGT gene. In some aspects, the polynucleic acid molecule is a single-stranded nucleic acid molecule that hybridizes to certain regions of mRNA. In some aspects, the polynucleic acid molecule is a double-stranded nucleic acid molecule. In some instances, the polynucleic acid molecule comprises a passenger strand (a sense strand) and a guide strand (an antisense strand), and wherein the guide strand hybridizes to certain regions of AGT mRNA.

[0064] In some aspects, the polynucleic acid molecule described herein hybridizes to certain regions of human AGT mRNA. In some aspects, the polynucleic acid molecule described herein hybridizes to certain regions of non-human AGT mRNA.

[0065] In some aspects, the polynucleic acid molecule described herein hybridizes to the 5’ UTR region of human AGT mRNA. In some aspects, the polynucleic acid molecule described herein hybridizes to the coding region of human AGT mRNA. Human AGT gene contains 5 exons and 4 introns. Accordingly, in some aspects, the polynucleic acid molecule described 17WSGR Docket No.61382-734.601 herein hybridizes to the coding region in exon 1 of human AGT mRNA. In some aspects, the polynucleic acid molecule described herein hybridizes to exon 2 of human AGT mRNA. In some aspects, the polynucleic acid molecule described herein hybridizes to exon 3 of human AGT mRNA. In some aspects, the polynucleic acid molecule described herein hybridizes to exon 4 of human AGT mRNA. In some aspects, the polynucleic acid molecule described herein hybridizes to exon 5 of human AGT mRNA. In some aspects, the polynucleic acid molecule described herein hybridizes to the 3’ UTR region of human AGT mRNA. In some aspects, the polynucleic acid molecule described herein hybridizes to human AGT mRNA (NCBI Reference Sequence: NM_ 001384479.1) with a range of transcription starting sites specified in Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33. In some aspects, the polynucleic acid molecule described herein hybridizes to human AGT mRNA (NCBI Reference Sequence: NM_ 001384479.1) with a range of transcription starting sites ranging from at least 1, at least 36, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 2089, or at least 2100.

[0066] In some aspects, the target region that the polynucleic acid molecule described herein hybridizes to is determined by an algorithm that predicts the maximal AGT silencing effectiveness and lowest possible off-target effects. In some specific embodiment, the algorithm is disclosed in He et al., 2017, Scientific Reports, 7, 44836. In some specific embodiment, the algorithm is disclosed in Han et al., 2018, BMC Genomics 19, 669. In some specific embodiment, the algorithm is siRNArules, siRNA-Finder, siRNA Wizard™, siDirect, sirna wizard, Dharmacon siRNA designing tool, White head siRNA designing tool, or Genscript siRNA software.

[0067] In some aspects, the target region is from positions 1269-1292 of AGT mRNA (NM_ 001384479.1) counting from the 5’ end. In some aspects, the target region is from positions 1652-1675 of AGT mRNA (NM_ 001384479.1) counting from the 5’ end. In some aspects, the target region is from positions 1817-1840 of AGT mRNA (NM_ 001384479.1) counting from the 5’ end.

[0068] In some instances, the polynucleic acid molecule hybridizes to nucleotides at positions 1269-1292 of AGT mRNA (NM_ 001384479.1) counting from the 5’ end. In some instances, the polynucleic acid molecule hybridizes to nucleotides at positions 1652-1675 of AGT mRNA (NM_ 001384479.1) counting from the 5’ end. In some instances, the polynucleic acid molecule hybridizes to nucleotides at positions 1817-1840 of AGT mRNA (NM_ 001384479.1) counting from the 5’ end. In some instances, the polynucleic acid molecule hybridizes to a subregion of 18WSGR Docket No.61382-734.601 the target gene (e.g., AGT mRNA) and comprises a nucleic acid sequence that are 80%, 85%, 90%, 95%, 98%, or 100% complementary to the subregion in its at least 80%, 85%, 90%, 95%, or 100% of its entire length. In some instances, the polynucleic acid molecule hybridizes to a subregion of the target gene (e.g., AGT mRNA) and comprises a nucleic acid sequence having at least 16, 17, 18, 19, or 20 consecutive nucleotide sequences that are fully complementary to the subregion. Structure of Polynucleic Acid Molecules Single-stranded nucleic acid molecule

[0069] Described herein is a polynucleic acid molecule for modulating expression of AGT gene, wherein the polynucleic acid molecule single-stranded nucleic acid molecule that is reverse complementary to the target region of AGT mRNA as described above.

[0070] In some aspects, the polynucleic acid molecule described herein is not 100% complementary to the target region of AGT mRNA. Accordingly, in some instances, the polynucleic acid molecule described herein is about 95% complementary to the target region of AGT mRNA. In some aspects, the polynucleic acid molecule described herein is about 90% complementary to the target region of AGT mRNA. In some aspects, the polynucleic acid molecule described herein is about 85% complementary to the target region of AGT mRNA. In some aspects, the polynucleic acid molecule described herein is about 80% complementary to the target region of AGT mRNA. In some aspects, the polynucleic acid molecule described herein is about 75% complementary to the target region of AGT mRNA. In some aspects, the polynucleic acid molecule described herein is about 70% complementary to the target region of AGT mRNA.

[0071] In some aspects, the polynucleic acid molecule described herein comprises a nucleic acid sequence in Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33. In other aspects, the polynucleic acid molecule described herein comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to a sequence in Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33. In some instances, the polynucleic acid molecule described herein comprises at least 80%, at least 85%, at least 90%, at least 95% complementary to a nucleic acid sequence selected from SEQ ID NOs:21-33. In some instances, the polynucleic acid molecule described herein comprises at least 80%, at least 85%, at least 90%, at least 95% complementary to a nucleic acid sequence selected from SEQ ID NOs: 21-22, and 25-27.

[0072] In yet other aspects, the polynucleic acid molecule described herein comprises a nucleic acid sequence that is 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides of the sequences in 19WSGR Docket No.61382-734.601 Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33 with no more than 2, 3, or 4 mismatches. In some aspects, the polynucleic acid molecule described herein comprises a nucleic acid sequence that is 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides that are complementary to a nucleic acid sequence of SEQ ID NOs: 21-33 with no more than 2, 3, or 4 mismatches.

[0073] In some aspects, the polynucleic acid molecule described herein comprises a nucleic acid sequence that is 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides that are complementary to a nucleic acid sequence of SEQ ID NOs: 21-22, and 25-27 with no more than 1, 2, or 3 mismatches.

[0074] In some aspects, the polynucleic acid molecule described herein comprises about 15-30, 16-30, 17-30, 18-30, 18-27, 18-25, 18-23, 19-23, 20-23, or 21-23 nucleotides in length. In some aspects, the polynucleic acid molecule described herein comprises about 15, 16, 17, 18, 19, 20 nucleotides in length. In some aspects, the polynucleic acid molecule described herein comprises about 21, 22, 23, 24, 25 nucleotides in length. In some aspects, the polynucleic acid molecule described herein comprises about 26, 27, 28, 29, 30 nucleotides in length. In some aspects, the polynucleic acid molecule described herein comprises 19 nucleotides in length. In some aspects, the polynucleic acid molecule described herein comprises 21 nucleotides in length. In some aspects, the polynucleic acid molecule described herein comprises 23 nucleotides in length. Double-stranded nucleic acid molecule

[0075] Described herein is a polynucleic acid molecule for modulating expression of AGT gene, wherein the polynucleic acid molecule is a double-stranded molecule that comprises a passenger strand and a guide strand, and wherein the guide strand is reverse complementary to the target region of AGT mRNA as described above.

[0076] In some aspects, the guide strand described herein is 100% complementary to the target region of AGT mRNA. In some aspects, the guide strand described herein is not 100% complementary to the target region of AGT mRNA. Accordingly, in some instances, the guide strand described herein is about 95% complementary to the target region of AGT mRNA. In some aspects, the guide strand described herein is about 90% complementary to the target region of AGT mRNA. In some aspects, the guide strand described herein is about 85% complementary to the target region of AGT mRNA. In some aspects, the guide strand described herein is about 80% complementary to the target region of AGT mRNA. In some aspects, the guide strand described herein is about 75% complementary to the target region of AGT mRNA. 20WSGR Docket No.61382-734.601 In some aspects, the guide strand described herein is about 70% complementary to the target region of AGT mRNA.

[0077] In some aspects, the polynucleic acid molecule described herein comprises a nucleic acid sequence in Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33. In other aspects, the polynucleic acid molecule described herein comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to a sequence in Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33. In some instances, the passenger strand described herein comprises at least 80%, at least 85%, at least 90%, at least 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 21-33. In some instances, the guide strand described herein comprises at least 80%, at least 85%, at least 90%, at least 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 1-18 and 160. In some instances, the passenger strand described herein comprises at least 80%, at least 85%, at least 90%, at least 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 21-22, and 25-27. In some instances, the guide strand described herein comprises at least 80%, at least 85%, at least 90%, at least 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 1-2, 7, and 10-11.

[0078] In yet other aspects, the polynucleic acid molecule described herein comprises a nucleic acid sequence that is 14, 15, 16, or 17 consecutive nucleotides out of the sequences in Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33 with no more than 1, 2, 3, or 4 mismatches. In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 14, 15, 16, or 17 consecutive nucleotides of SEQ ID NOs: 21-33 with no more than 1, 2, 3, or 4 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 14, 15, 16, or 17 consecutive nucleotides of SEQ ID NOs: 1-18 and 160 with no more than 1, 2, 3, or 4 mismatches. In yet other aspects, the polynucleic acid molecule described herein comprises a nucleic acid sequence that is 18 consecutive nucleotides out of the sequences in Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33 with no more than 1, 2, 3, or 4 mismatches. In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 18 consecutive nucleotides of SEQ ID NOs: 21-33 with no more than 1, 2, 3, or 4 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 18 consecutive nucleotides of SEQ ID NOs: 1-18 and 160 with no more than 1, 2, 3, or 4 mismatches. In yet other aspects, the polynucleic acid molecule described herein comprises a nucleic acid sequence that is 19 consecutive nucleotides out of the sequences in 21WSGR Docket No.61382-734.601 Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33 with no more than 1, 2, 3, or 4 mismatches. In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 19 consecutive nucleotides of SEQ ID NOs: 21-33 with no more than 1, 2, 3, or 4 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 19 consecutive nucleotides of SEQ ID NOs: 1-18 and 160 with no more than 1, 2, 3, or 4 mismatches. In yet other aspects, the polynucleic acid molecule described herein comprises a nucleic acid sequence that is 20 consecutive nucleotides out of the sequences in Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33 with no more than 1, 2, 3, or 4 mismatches. In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 20 consecutive nucleotides of SEQ ID NOs: 21-33 with no more than 1, 2, 3, or 4 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 20 consecutive nucleotides of SEQ ID NOs: 1-18 and 160 with no more than 1, 2, 3, or 4 mismatches. In yet other aspects, the polynucleic acid molecule described herein comprises a nucleic acid sequence that is 21 consecutive nucleotides out of the sequences in Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33 with no more than 1, 2, 3, or 4 mismatches. In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 21 consecutive nucleotides of SEQ ID NOs: 21-33 with no more than 1, 2, 3, or 4 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 21 consecutive nucleotides of SEQ ID NOs: 1-18 and 160 with no more than 1, 2, 3, or 4 mismatches. In yet other aspects, the polynucleic acid molecule described herein comprises a nucleic acid sequence that is 22 consecutive nucleotides out of the sequences in Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33 with no more than 1, 2, 3, or 4 mismatches. In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 22 consecutive nucleotides of SEQ ID NOs: 21- 33 with no more than 1, 2, 3, or 4 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 22 consecutive nucleotides of SEQ ID NOs: 1- 18 and 160 with no more than 1, 2, 3, or 4 mismatches.

[0079] In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 14 consecutive nucleotides of SEQ ID NOs: 21-22, and 25-27 with no more than 1, 2, or 3 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 14 consecutive nucleotides of SEQ ID NOs: 1-2, 7, and 10-11 with no more than 1, 2, or 3 mismatches. In some aspects, the passenger strand described herein comprises a 22WSGR Docket No.61382-734.601 nucleic acid sequence that is 15 consecutive nucleotides of SEQ ID NOs: 21-22, and 25-27 with no more than 1, 2, or 3 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 15 consecutive nucleotides of SEQ ID NOs: 1-2, 7, and 10-11 with no more than 1, 2, or 3 mismatches. In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 16 consecutive nucleotides of SEQ ID NOs: 21-22, and 25-27 with no more than 1, 2, or 3 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 16 consecutive nucleotides of SEQ ID NOs: 1-2, 7, and 10-11 with no more than 1, 2, or 3 mismatches. In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 17 consecutive nucleotides of SEQ ID NOs: 21-22, and 25-27 with no more than 1, 2, or 3 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 17 consecutive nucleotides of SEQ ID NOs: 1-2, 7, and 10-11 with no more than 1, 2, or 3 mismatches. In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 18 consecutive nucleotides of SEQ ID NOs: 21-22, and 25-27 with no more than 1, 2, or 3 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 18 consecutive nucleotides of SEQ ID NOs: 1-2, 7, and 10-11 with no more than 1, 2, or 3 mismatches. In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 19 consecutive nucleotides of SEQ ID NOs: 21-22, and 25-27 with no more than 1, 2, or 3 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 19 consecutive nucleotides of SEQ ID NOs: 1- 2, 7, and 10-11 with no more than 1, 2, or 3 mismatches. In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 20 consecutive nucleotides of SEQ ID NOs: 21-22, and 25-27 with no more than 1, 2, or 3 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 20 consecutive nucleotides of SEQ ID NOs: 1-2, 7, and 10-11 with no more than 1, 2, or 3 mismatches. In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 21 consecutive nucleotides of SEQ ID NOs: 21-22, and 25-27 with no more than 1, 2, or 3 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 21 consecutive nucleotides of SEQ ID NOs: 1-2, 7, and 10-11 with no more than 1, 2, or 3 mismatches. In some aspects, the passenger strand described herein comprises a nucleic acid sequence that is 22 consecutive nucleotides of SEQ ID NOs: 21-22, and 25-27 with no more than 1, 2, or 3 mismatches. In some aspects, the guide strand described herein comprises a nucleic acid sequence that is 22 consecutive nucleotides of SEQ ID NOs: 1-2, 7, and 10-11 with no more than 1, 2, or 3 mismatches. 23WSGR Docket No.61382-734.601

[0080] In some aspects, the polynucleic acid molecule described herein comprises a passenger strand and a guide strand of about 15-30, 16-30, 17-30, 18-30, 18-27, 18-25, 18-23, 19-23, 20- 23, or 21-23 nucleotides in length. In some aspects, the polynucleic acid molecule described herein comprises a passenger strand and a guide strand of about 15, 16, 17, 18, 19, 20 nucleotides in length. In some aspects, the polynucleic acid molecule described herein comprises a passenger strand and a guide strand of about 21, 22, 23, 24, 25 nucleotides in length. In some aspects, the polynucleic acid molecule described herein comprises a passenger strand and a guide strand of about 26, 27, 28, 29, 30 nucleotides in length. In some aspects, the polynucleic acid molecule described herein comprises a passenger strand of 19 nucleotides in length, and a guide strand of about 21 nucleotides long. In some aspects, the polynucleic acid molecule described herein comprises a passenger strand of 21 nucleotides long, and a guide strand of about 23 nucleotides in length.

[0081] In some aspects, the passenger strand and the guide strand described herein are reverse complementary to each other and form a duplex with a 3’ overhang on the guide strand. In some aspects, the passenger strand and the guide strand described herein are reverse complementary to each other and form a duplex with a 5’ overhang on the guide strand. In some aspects, the passenger strand and the guide strand described herein are reverse complementary to each other and form a duplex with a 3’ overhang on the passenger strand. In some aspects, the passenger strand and the guide strand described herein are reverse complementary to each other and form a duplex with a 5’ overhang on the passenger strand. Modifications of Polynucleic Acid Molecules

[0082] In some aspects, described herein is the polynucleic acid molecule described herein with modifications. In some aspects, the modifications described herein occurs one or more different structures of the polynucleotide acid molecule described herein (e.g., modifications on sugar ring(s), backbone(s), base(s)). In some aspects, the modifications described herein comprise substitutions of one or more nucleotide in the polynucleic acid molecule described herein. In some aspects, different percentages of the polynucleic acid molecule described herein comprise the modifications described herein. In some aspects, different positions of the polynucleic acid molecule described herein comprise the modifications described herein. For example, WO / 2018 / 035380 is herein incorporated by reference in its entirety. Types of modifications

[0083] In some aspects, the polynucleotide acid molecule described herein comprises one or more sugar-modified nucleotide. In some aspects, the sugar-modified nucleotide is a 2’-fluoro modified nucleotide. In some instances, the 2’-fluoro modified nucleotide comprises thio- 24WSGR Docket No.61382-734.601 modified base containing nucleotide, e.g., 2'-fluoro-2-thiouridine-3'-phosphate (U3f). In some instances, the sugar-modified nucleotide includes a modification at a 2’ hydroxyl group of the ribose moiety. In some instances, the sugar-modified nucleotide includes modification with an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. In some aspects, the sugar-modified nucleotide is a 2’-O-methyl modified nucleotide or 2’-alkoxy modified nucleotide (e.g., 2’-methoxy modified nucleotide). In some instances, 2' hydroxyl group modification includes 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O- dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA). In some instances, the alkyl moiety comprises a hetero substitution. In some instances, the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur. In some aspects, the sugar-modified nucleotide is a 2’- amino modified nucleotide. In some aspects, the sugar- modified nucleotide is a 2’- azido modified nucleotide. In some aspects, the sugar-modified nucleotide is a 2’- deoxy modified nucleotide. In some aspects, the sugar-modified nucleotide is a 2’-O-methoxythyl (2’-MOE). In some aspects, the sugar-modified nucleotide is a locked nucleic acid (LNA). In some aspects, the sugar-modified nucleotide is an ethylene-bridged nucleic acid (ENA). In some aspects, the sugar-modified nucleotide is a (S)-constrained ethyl (cEt). In some aspects, the sugar-modified nucleotide is a tricyclo-DNA (tcDNA). In some aspects, the sugar-modified nucleotide is a 2’-NH2 nucleic acid.

[0084] In some aspects, the polynucleotide acid molecule described herein comprises one or more sugarphosphate-modified nucleotide. In some aspects, the modified sugarphosphate is phosphorodiamidate morpholino (PMO). In some aspects, the modified sugarphosphate is phosphoramidate. In some instances, the heterocyclic substitution includes imidazole, and pyrrolidino. In some aspects, the modified sugarphosphate is thiophosphoramidate. In some aspects, the modified sugarphosphate is peptide nucleic acid (PNA).

[0085] In some aspects, the polynucleotide acid molecule described herein comprises one or more backbone-modified nucleotide. In some aspects, the modified backbone is a methylphosphonate. In some aspects, the modified backbone is phosphorothioate. In some aspects, the modified backbone is phosphorodithioate, e.g., 3’phosphorodithioate (ss). In some aspects, the modified backbone is a guanidinopropyl phosphoramidate. In some aspects, the modified backbone is a mesyl-phosphoramidate (MsPA) linkages. In some instances, the modified backbone comprises one or more of phosphorodithioates, methylphosphonates, 5'- alkylenephosphonates, 5'-methylphosphonate, 3'-alkylene phosphonates, borontrifluoridates, borano phosphate esters and selenophosphates of 3'-5' linkage or 2'-5' linkage, phosphotriesters, 25WSGR Docket No.61382-734.601 thionoalkylphosphotriesters, hydrogen phosphonate linkages, alkyl phosphonates, alkylphosphonothioates, arylphosphonothioates, phosphoroselenoates, phosphoramidates.

[0086] In some instances, the guide strand comprises one or more phosphorothioate internucleotide linkage. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 1st nucleoside (e.g., the 3'-carbon atom of the 1st nucleoside) and the 2nd nucleoside (e.g., the 5'-carbon atom of the 2nd nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 2nd nucleoside (e.g., the 3'-carbon atom of the 2nd nucleoside) and the 3rd nucleoside (e.g., the 5'-carbon atom of the 3rd nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 3rd nucleoside (e.g., the 3'-carbon atom of the 3rd nucleoside) and the 4th nucleoside (e.g., the 5'-carbon atom of the 4th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 4th nucleoside (e.g., the 3'-carbon atom of the 4th nucleoside) and the 5th nucleoside (e.g., the 5'-carbon atom of the 5th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 5th nucleoside (e.g., the 3'-carbon atom of the 5th nucleoside) and the 6th nucleoside (e.g., the 5'-carbon atom of the 6th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 6th nucleoside (e.g., the 3'-carbon atom of the 6th nucleoside) and the 7th nucleoside (e.g., the 5'-carbon atom of the 7th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 7th nucleoside (e.g., the 3'-carbon atom of the 7th nucleoside) and the 8th nucleoside (e.g., the 5'-carbon atom of the 8th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 8th nucleoside (e.g., the 3'-carbon atom of the 8th nucleoside) and the 9th nucleoside (e.g., the 5'-carbon atom of the 9th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 9th nucleoside (e.g., the 3'-carbon atom of the 9th nucleoside) and the 10th nucleoside (e.g., the 5'-carbon atom of the 10th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 10th nucleoside (e.g., the 3'-carbon atom of the 10th nucleoside) and the 11th nucleoside (e.g., the 5'-carbon atom of the 11th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 11th nucleoside (e.g., the 3'-carbon atom of the 11th nucleoside) and the 12th nucleoside (e.g., the 5'- carbon atom of the 12th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 12th nucleoside (e.g., the 26WSGR Docket No.61382-734.601 3'-carbon atom of the 12th nucleoside) and the 13th nucleoside (e.g., the 5'-carbon atom of the 13th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 13th nucleoside (e.g., the 3'-carbon atom of the 13th nucleoside) and the 14th nucleoside (e.g., the 5'-carbon atom of the 14th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 14th nucleoside (e.g., the 3'-carbon atom of the 14th nucleoside) and the 15th nucleoside (e.g., the 5'-carbon atom of the 15th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 15th nucleoside (e.g., the 3'-carbon atom of the 15th nucleoside) and the 16th nucleoside (e.g., the 5'-carbon atom of the 16th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 16th nucleoside (e.g., the 3'-carbon atom of the 16th nucleoside) and the 17th nucleoside (e.g., the 5'-carbon atom of the 17th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 17th nucleoside (e.g., the 3'-carbon atom of the 17th nucleoside) and the 18th nucleoside (e.g., the 5'- carbon atom of the 18th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 18th nucleoside (e.g., the 3'-carbon atom of the 18th nucleoside) and the 19th nucleoside (e.g., the 5'-carbon atom of the 19th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 19th nucleoside (e.g., the 3'-carbon atom of the 19th nucleoside) and the 20th nucleoside (e.g., the 5'-carbon atom of the 20th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 20th nucleoside (e.g., the 3'-carbon atom of the 20th nucleoside) and the 21st nucleoside (e.g., the 5'-carbon atom of the 21st nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 21st nucleoside (e.g., the 3'-carbon atom of the 21st nucleoside) and the 22nd nucleoside (e.g., the 5'-carbon atom of the 22nd nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 22nd nucleoside (e.g., the 3'-carbon atom of the 22nd nucleoside) and the 23rd nucleoside (e.g., the 5'-carbon atom of the 23rd nucleoside) from the 5’ end of the guide strand.

[0087] In some instances, the passenger strand comprises one or more phosphorothioate internucleotide linkage. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 1st nucleoside (e.g., the 3'-carbon atom of the 1st nucleoside) and the 2nd nucleoside (e.g., the 5'-carbon atom of the 2nd nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to 27WSGR Docket No.61382-734.601 the 2nd nucleoside (e.g., the 3'-carbon atom of the 2nd nucleoside) and the 3rd nucleoside (e.g., the 5'-carbon atom of the 3rd nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 3rd nucleoside (e.g., the 3'-carbon atom of the 3rd nucleoside) and the 4th nucleoside (e.g., the 5'- carbon atom of the 4th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 4th nucleoside (e.g., the 3'-carbon atom of the 4th nucleoside) and the 5th nucleoside (e.g., the 5'-carbon atom of the 5th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 5th nucleoside (e.g., the 3'-carbon atom of the 5th nucleoside) and the 6th nucleoside (e.g., the 5'-carbon atom of the 6th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 6th nucleoside (e.g., the 3'-carbon atom of the 6th nucleoside) and the 7th nucleoside (e.g., the 5'-carbon atom of the 7th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 7th nucleoside (e.g., the 3'-carbon atom of the 7th nucleoside) and the 8th nucleoside (e.g., the 5'-carbon atom of the 8th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 8th nucleoside (e.g., the 3'-carbon atom of the 8th nucleoside) and the 9th nucleoside (e.g., the 5'-carbon atom of the 9th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 9th nucleoside (e.g., the 3'-carbon atom of the 9th nucleoside) and the 10th nucleoside (e.g., the 5'- carbon atom of the 10th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 10th nucleoside (e.g., the 3'-carbon atom of the 10th nucleoside) and the 11th nucleoside (e.g., the 5'-carbon atom of the 11th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 11th nucleoside (e.g., the 3'-carbon atom of the 11th nucleoside) and the 12th nucleoside (e.g., the 5'-carbon atom of the 12th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 12th nucleoside (e.g., the 3'-carbon atom of the 12th nucleoside) and the 13th nucleoside (e.g., the 5'-carbon atom of the 13th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 13th nucleoside (e.g., the 3'-carbon atom of the 13th nucleoside) and the 14th nucleoside (e.g., the 5'-carbon atom of the 14th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 14th nucleoside (e.g., the 28WSGR Docket No.61382-734.601 3'-carbon atom of the 14th nucleoside) and the 15th nucleoside (e.g., the 5'-carbon atom of the 15th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 15th nucleoside (e.g., the 3'-carbon atom of the 15th nucleoside) and the 16th nucleoside (e.g., the 5'-carbon atom of the 16th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 16th nucleoside (e.g., the 3'-carbon atom of the 16th nucleoside) and the 17th nucleoside (e.g., the 5'-carbon atom of the 17th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 17th nucleoside (e.g., the 3'-carbon atom of the 17th nucleoside) and the 18th nucleoside (e.g., the 5'-carbon atom of the 18th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 18th nucleoside (e.g., the 3'-carbon atom of the 18th nucleoside) and the 19th nucleoside (e.g., the 5'-carbon atom of the 19th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 19th nucleoside (e.g., the 3'-carbon atom of the 19th nucleoside) and the 20th nucleoside (e.g., the 5'-carbon atom of the 20th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorothioate internucleotide linkage, is covalently bonded to the 20th nucleoside (e.g., the 3'-carbon atom of the 20th nucleoside) and the 21st nucleoside (e.g., the 5'-carbon atom of the 21st nucleoside) from the 5’ end of the passenger strand.

[0088] In some instances, the amidite structure of 3’phosphorodithioate (ss) is shown as below:.

[0089] In some instances, the 3’phosphorodithioate (ss) is incorporated into the polynucleic acid molecule. In some instances, the 3’phosphorodithioate (ss) is incorporated into the siRNA. In some instances, the 3’phosphorodithioate (ss) is incorporated into a guide strand. In some instances, the 3’phosphorodithioate (ss) is incorporated into a passenger strand. In some instances, an incorporated 3’phosphorodithioate (ss) has a structure shown as below, where the base can be any suitable base or modified base that can make Watson-Crick binding with the base on the opposite strand: 29WSGR Docket No.61382-734.601.

[0090] In some instances, the guide strand comprises one or more phosphorothioate internucleotide linkage. In some instances, the guide strand comprises one or more phosphorothioate internucleotide linkage and one or more phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss). In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 1st nucleoside (e.g., the 3'-carbon atom of the 1st nucleoside) and the 2nd nucleoside (e.g., the 5'- carbon atom of the 2ndnucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 2nd nucleoside (e.g., the 3'-carbon atom of the 2nd nucleoside) and the 3rd nucleoside (e.g., the 5'-carbon atom of the 3rd nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 3rd nucleoside (e.g., the 3'-carbon atom of the 3rd nucleoside) and the 4th nucleoside (e.g., the 5'-carbon atom of the 4th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 4th nucleoside (e.g., the 3'-carbon atom of the 4th nucleoside) and the 5th nucleoside (e.g., the 5'-carbon atom of the 5th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 5th nucleoside (e.g., the 3'-carbon atom of the 5th nucleoside) and the 6th nucleoside (e.g., the 5'-carbon atom of the 6th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 6th nucleoside (e.g., the 3'-carbon atom of the 6th nucleoside) and the 7th nucleoside (e.g., the 5'- carbon atom of the 7th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 7th nucleoside (e.g., the 3'-carbon atom of the 7th nucleoside) and the 8th nucleoside (e.g., the 5'-carbon atom of the 8th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 8th nucleoside (e.g., the 3'-carbon atom of the 8th nucleoside) and the 9th 30WSGR Docket No.61382-734.601 nucleoside (e.g., the 5'-carbon atom of the 9th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 9th nucleoside (e.g., the 3'-carbon atom of the 9th nucleoside) and the 10th nucleoside (e.g., the 5'-carbon atom of the 10th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 10th nucleoside (e.g., the 3'-carbon atom of the 10th nucleoside) and the 11th nucleoside (e.g., the 5'-carbon atom of the 11th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 11th nucleoside (e.g., the 3'- carbon atom of the 11th nucleoside) and the 12th nucleoside (e.g., the 5'-carbon atom of the 12th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 12th nucleoside (e.g., the 3'-carbon atom of the 12th nucleoside) and the 13th nucleoside (e.g., the 5'- carbon atom of the 13th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 13th nucleoside (e.g., the 3'-carbon atom of the 13th nucleoside) and the 14th nucleoside (e.g., the 5'-carbon atom of the 14th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 14th nucleoside (e.g., the 3'-carbon atom of the 14th nucleoside) and the 15th nucleoside (e.g., the 5'-carbon atom of the 15th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 15th nucleoside (e.g., the 3'-carbon atom of the 15th nucleoside) and the 16th nucleoside (e.g., the 5'-carbon atom of the 16th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 16th nucleoside (e.g., the 3'- carbon atom of the 16th nucleoside) and the 17th nucleoside (e.g., the 5'-carbon atom of the 17th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 17th nucleoside (e.g., the 3'-carbon atom of the 17th nucleoside) and the 18th nucleoside (e.g., the 5'- carbon atom of the 18th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 18th nucleoside (e.g., the 3'-carbon atom of the 18th nucleoside) and the 19th nucleoside (e.g., the 5'-carbon atom of the 19th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 19th nucleoside (e.g., the 3'-carbon atom of the 19th nucleoside) and 31WSGR Docket No.61382-734.601 the 20th nucleoside (e.g., the 5'-carbon atom of the 20th nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 20th nucleoside (e.g., the 3'-carbon atom of the 20th nucleoside) and the 21st nucleoside (e.g., the 5'-carbon atom of the 21st nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 21st nucleoside (e.g., the 3'- carbon atom of the 21st nucleoside) and the 22nd nucleoside (e.g., the 5'-carbon atom of the 22nd nucleoside) from the 5’ end of the guide strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 22nd nucleoside (e.g., the 3'-carbon atom of the 22nd nucleoside) and the 23rd nucleoside (e.g., the 5'- carbon atom of the 23rd nucleoside) from the 5’ end of the guide strand.

[0091] In some instances, the guide strand comprises one or more phosphorothioate internucleotide linkage and one phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss). In some instances, the guide strand comprises one or more phosphorothioate internucleotide linkages and two phosphorodithioate internucleotide linkages, e.g., 3’phosphorodithioate (ss). In some instances, the guide strand comprises one or more phosphorothioate internucleotide linkages and three phosphorodithioate internucleotide linkages, e.g., 3’phosphorodithioate (ss). In some instances, the guide strand comprises one or more phosphorothioate internucleotide linkages and four phosphorodithioate internucleotide linkages, e.g., 3’phosphorodithioate (ss).

[0092] In some instances, the guide strand comprises one or more phosphorothioate internucleotide linkages and one phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), wherein the one phosphorodithioate internucleotide linkage is covalently bonded to two consecutive nucleosides between positions 9-12 of the guide strand from the 5’ end.

[0093] In some instances, the passenger strand comprises one or more phosphorothioate internucleotide linkage. In some instances, the passenger strand comprises one or more phosphorothioate internucleotide linkage and one or more phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss). In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 1st nucleoside (e.g., the 3'-carbon atom of the 1st nucleoside) and the 2nd nucleoside (e.g., the 5'- carbon atom of the 2ndnucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 2nd nucleoside (e.g., the 3'-carbon atom of the 2nd nucleoside) and the 3rd nucleoside (e.g., the 5'-carbon atom of the 3rd nucleoside) from the 5’ end of the passenger 32WSGR Docket No.61382-734.601 strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 3rd nucleoside (e.g., the 3'-carbon atom of the 3rd nucleoside) and the 4th nucleoside (e.g., the 5'-carbon atom of the 4th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 4th nucleoside (e.g., the 3'- carbon atom of the 4th nucleoside) and the 5th nucleoside (e.g., the 5'-carbon atom of the 5th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 5th nucleoside (e.g., the 3'-carbon atom of the 5th nucleoside) and the 6th nucleoside (e.g., the 5'- carbon atom of the 6th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 6th nucleoside (e.g., the 3'-carbon atom of the 6th nucleoside) and the 7th nucleoside (e.g., the 5'-carbon atom of the 7th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 7th nucleoside (e.g., the 3'-carbon atom of the 7th nucleoside) and the 8th nucleoside (e.g., the 5'-carbon atom of the 8th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 8th nucleoside (e.g., the 3'- carbon atom of the 8th nucleoside) and the 9th nucleoside (e.g., the 5'-carbon atom of the 9th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 9th nucleoside (e.g., the 3'-carbon atom of the 9th nucleoside) and the 10th nucleoside (e.g., the 5'- carbon atom of the 10th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 10th nucleoside (e.g., the 3'-carbon atom of the 10th nucleoside) and the 11th nucleoside (e.g., the 5'-carbon atom of the 11th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 11th nucleoside (e.g., the 3'-carbon atom of the 11th nucleoside) and the 12th nucleoside (e.g., the 5'-carbon atom of the 12th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 12th nucleoside (e.g., the 3'-carbon atom of the 12th nucleoside) and the 13th nucleoside (e.g., the 5'- carbon atom of the 13th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 13th nucleoside (e.g., the 3'-carbon atom of the 13th nucleoside) and the 14th 33WSGR Docket No.61382-734.601 nucleoside (e.g., the 5'-carbon atom of the 14th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 14th nucleoside (e.g., the 3'-carbon atom of the 14th nucleoside) and the 15th nucleoside (e.g., the 5'-carbon atom of the 15th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 15th nucleoside (e.g., the 3'-carbon atom of the 15th nucleoside) and the 16th nucleoside (e.g., the 5'- carbon atom of the 16th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 16th nucleoside (e.g., the 3'-carbon atom of the 16th nucleoside) and the 17th nucleoside (e.g., the 5'-carbon atom of the 17th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 17th nucleoside (e.g., the 3'-carbon atom of the 17th nucleoside) and the 18th nucleoside (e.g., the 5'-carbon atom of the 18th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 18th nucleoside (e.g., the 3'-carbon atom of the 18th nucleoside) and the 19th nucleoside (e.g., the 5'- carbon atom of the 19th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 19th nucleoside (e.g., the 3'-carbon atom of the 19th nucleoside) and the 20th nucleoside (e.g., the 5'-carbon atom of the 20th nucleoside) from the 5’ end of the passenger strand. In some instances, the phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), is covalently bonded to the 20th nucleoside (e.g., the 3'-carbon atom of the 20th nucleoside) and the 21st nucleoside (e.g., the 5'-carbon atom of the 21st nucleoside) from the 5’ end of the passenger strand.

[0094] In some instances, the passenger strand comprises one or more phosphorothioate internucleotide linkage and one phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss). In some instances, the passenger strand comprises one or more phosphorothioate internucleotide linkage and two phosphorodithioate internucleotide linkages, e.g., 3’phosphorodithioate (ss). In some instances, the passenger strand comprises one or more phosphorothioate internucleotide linkage and three phosphorodithioate internucleotide linkages, e.g., 3’phosphorodithioate (ss). In some instances, the passenger strand comprises one or more phosphorothioate internucleotide linkage and four phosphorodithioate internucleotide linkages, e.g., 3’phosphorodithioate (ss). 34WSGR Docket No.61382-734.601

[0095] In some aspects, the modified nucleotide comprises a modified guanine (e.g., inosine) or one or more of any types of unnatural nucleic acids.

[0096] In some aspects, the modified backbone is phosphorothioate, and the phosphorothioate is a stereochemically enriched phosphorothioate. In certain aspects, the strand contains at least one stereochemically enriched phosphorothioate. In some aspects, the strand comprises at least 1, 2, 3 stereochemically enriched phosphorothioates. In some aspects, the strand comprises only 1, 2, 3, or 4 stereochemically enriched phosphorothioates. In further aspects, at least one (e.g., one or two) stereochemically enriched phosphorothioate is disposed between two consecutive nucleosides that are two of six 5’ end nucleosides of the strand. In yet further aspects, at least one (e.g., one or two) stereochemically enriched phosphorothioate is disposed between two consecutive nucleosides that are two of six 3’ end nucleosides of the strand. In still further aspects, one stereochemically enriched phosphorothioate is covalently bonded to the first nucleoside and the second nucleoside from the 5’ end within the strand. In some aspects, one stereochemically enriched phosphorothioate is covalently bonded to the twenty first nucleoside and the twenty second nucleoside from the 5’ end within the strand. In certain aspects, one stereochemically enriched phosphorothioate is covalently bonded to the twenty second nucleoside and the twenty third nucleoside from the 5’ end within the strand. In particular aspects, the stereochemically enriched phosphorothioate has RP stereochemical identity. In certain aspects, the stereochemically enriched phosphorothioate has SPstereochemical identity. In some instances, the modified backbone connects the linker and the targeting moieties to the polynucleic acid molecule.

[0097] In some aspects, the modified backbone comprises 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps), 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps), or combinations thereof. In some instances, an incorporated 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps) has a structure shown as below, where the base can be any suitable base or modified base that can make Watson-Crick binding with the base on the opposite strand:. 35WSGR Docket No.61382-734.601

[0098] In some instances, an incorporated Sp diastereomer (SP-enriched phosphorothioate or Sps) has a structure shown as below, where the base can be any suitable base or modified base that can make Watson-Crick binding with the base on the opposite strand:.

[0099] In some aspects, the polynucleic acid molecules described herein comprises one or more (e.g., from 1 to 20, from 1 to 10, or from 1 to 5) stereochemically enriched (e.g., internucleoside) phosphorothioates (e.g., having diastereomeric excess of at least 10%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, e.g., up to about 99%, for the P-stereogenic center). The polynucleic acid molecules described herein comprises one or more (e.g., from 1 to 20, from 1 to 10, or from 1 to 5; e.g., internucleoside) phosphorodithioates. The phosphorodithioates may be non-P-stereogenic in the polynucleic acid molecules described herein. Phosphorothioates and phosphorodithioates may enhance the stability of the polynucleic acid molecules described herein to exonuclease activity of serum. Non-P-stereogenic phosphorodithioates may simplify the synthesis of the polynucleic acid molecule described herein by reducing the number of possible diastereomers. Typically, the phosphorothioate or phosphorodithioate may connect two contiguous nucleosides within the six 3’ end nucleosides and the six 5’ end nucleosides of the polynucleic acid molecules described herein.

[0100] In some aspects, the stereochemically enriched phosphorothioate internucleotide linkages (e.g., RP-enriched phosphorothioate or SP-enriched phosphorothioate) connect the three nucleosides at the 5’ end of the nucleic acid (positions 1, 2, 3 from the 5’ end) and three nucleosides at the 3’ end (positions 1, 2, 3 from the 3’ end) of the nucleic acid. In some aspects, the stereochemically enriched phosphorothioate internucleotide linkages (e.g., RP-enriched phosphorothioate or SP-enriched phosphorothioate) connect the three nucleosides at the 5’ end of the guide strand and three nucleosides at the 3’ end of the guide strand. In certain aspect, the stereochemically enriched phosphorothioate internucleotide linkage (e.g., RP-enriched phosphorothioate or SP-enriched phosphorothioate) may be covalently bonded to the first nucleoside (e.g., the 3’-carbon atom of the first nucleoside) and the second nucleoside (e.g., the 36WSGR Docket No.61382-734.601 5’-carbon atom of the second nucleoside) from the 5’ end of the guide strand. In some instances, the stereochemically enriched phosphorothioate (e.g., RP-enriched phosphorothioate or SP-enriched phosphorothioate) may be covalently bonded to the second nucleoside (e.g., the 3’-carbon atom of the second nucleoside) and the third nucleoside (e.g., the 5’-carbon atom of the third nucleoside) from the 5’ end of the guide strand. Additionally or alternatively, the stereochemically enriched phosphorothioate (e.g., RP-enriched phosphorothioate or SP-enriched phosphorothioate) may be covalently bonded to the 21stnucleoside from the 5’ end or the 3rdnucleoside from the 3’ end (e.g., the 3’-carbon atom of the 21stnucleoside from the 5’ end, or the 3’-carbon atom of the 3rdnucleoside from the 3’ end) and the 22ndnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5’-carbon atom of the 22ndnucleoside from the 5’ end, or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the guide strand. Further, additionally or alternatively, the stereochemically enriched phosphorothioate (e.g., SP- enriched phosphorothioate or RP-enriched phosphorothioate) may be covalently bonded to the 22ndnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3’-carbon atom of the 22ndnucleoside from the 5’ end, or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5’-carbon atom of the 23rdnucleoside from the 5’ end, or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the guide strand.

[0101] Combinations of a specific 5’ stereochemically enriched phosphorothioate internucleotide linkage and a specific 3’ stereochemically enriched phosphorothioate internucleotide linkage in specific locations within the double stranded siRNA can provide beneficial effect to the function of the siRNA. For example, specific 5’ stereochemically enriched phosphorothioate internucleotide linkages and specific 3’ stereochemically enriched phosphorothioate internucleotide linkages in specific locations within the double stranded siRNA can produce superior efficacy and / or duration of action, e.g., as measured by the reduction in the activity of the target relative to a reference guide strand that lacks the combination of a 5’ stereochemically enriched phosphorothioate and a 3’ stereochemically enriched phosphorothioate internucleotide linkages. In some instances, the double stranded siRNA comprises: (i) RP-enriched phosphorothioate or SP-enriched phosphorothioate covalently bonded to the first nucleoside from the 5’ end (e.g., the 3’-carbon atom of the first nucleoside from the 5’ end) and the second nucleoside from the 5’ end (e.g., the 5’-carbon atom of the second nucleoside from the 5’ end); (ii) RP-enriched phosphorothioate or SP-enriched phosphorothioate covalently bonded to the 2ndnucleoside from the 5’ end (e.g., the 3’ carbon atom of the second nucleoside from the 5’ end) and the third nucleoside from the 5’ end (e.g., the 5’-carbon atom of the 3rdnucleoside from the 5’-end); (iii) RP-enriched phosphorothioate or 37WSGR Docket No.61382-734.601 SP-enriched phosphorothioate covalently bonded to the 21stnucleoside from the 5’ end or the 3rdnucleoside from the 3’ end (e.g., the 3’-carbon atom of the 21stnucleoside from the 5’ end, or the 3’-carbon atom of the 3rdnucleoside from the 3’ end) and the 22ndnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5’-carbon atom of the 22ndnucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end); and (iv) RP-enriched phosphorothioate or SP-enriched phosphorothioate covalently bonded to the 22ndnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3’-carbon atom of the 22ndnucleoside from the 5’ end, or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’-end or the 1stnucleoside from the 3’ end (e.g., the 5’-carbon atom of the 23rd nucleoside from the 5’ end, or the 5’-carbon atom of the 1stnucleoside from the 3’ end) in the guide strand.

[0102] In some embodiments, the configuration of stereochemically enriched phosphorothioates in the guide strand comprises RpsRpsSpsSps (RpsRps at the 5’ end and SpsSps at the 3’ end of the guide strand, for example, RpsRps at the positions 1 and 2 of the guide strand from the 5’ end and SpsSps at the positions 21 and 22 of the guide strand from the 5’ end, or RpsRps at the positions 1 and 2 of the guide strand from the 5’ end and SpsSps at the positions 2 and 1 of the guide strand from the 3’ end) or RpsRpsSpsRps (RpsRps at the 5’ end and SpsRps at the 3’ end, for example, RpsRps at the positions 1 and 2 of the guide strand from the 5’ end and SpsRps at the positions 21 and 22 of the guide strand from the 5’ end, or, RpsRps at the positions 1 and 2 of the guide strand from the 5’ end and SpsRps at the positions 2 and 1 of the guide strand from the 3’ end). In some aspects, the polynucleic acid molecules described herein comprises four stereochemically enriched phosphorothioates: (1) a Rp-enriched phosphorothioate covalently bonded to the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1stnucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 2ndnucleoside from the 5’ end) of the antisense strand; (2) a Rp-enriched phosphorothioate covalently bonded to the 2ndnucleoside from the 5’ end (e.g., the 3’-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 3rdnucleoside from the 5’ end) of the antisense strand; (3) a Sp-enriched phosphorothioate covalently bonded to the 21stnucleoside from the 5’ end or the 3rd nucleoside from the 3’ end (e.g., the 3'-carbon atom of the 21stnucleoside from the 5’ end or the 3’-carbon atom of the 3rdnucleoside from the 3’end) and the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22thnucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’end) of the antisense strand; and (4) a Sp-enriched phosphorothioate covalently bonded to the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3’-carbon atom of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’end) 38WSGR Docket No.61382-734.601 and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1st nucleoside from the 3’end) of the antisense strand. In some aspects, the polynucleic acid molecules described herein comprises four stereochemically enriched phosphorothioates: (1) a Rp-enriched phosphorothioate covalently bonded to the 1stnucleoside from the 5’ end (e.g., the 3’-carbon atom of the 1stnucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'- carbon atom of the 2ndnucleoside from the 5’ end) of the antisense strand; (2) a Rp-enriched phosphorothioate covalently bonded to the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'- carbon atom of the 3rdnucleoside from the 5’ end) of the antisense strand; (3) a Sp-enriched phosphorothioate covalently bonded to the 21stnucleoside from the 5’ end or the 3rdnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 21stnucleoside from the 5’ end or the 3’-carbon atom of the 3rdnucleoside from the 3’ end) and the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22thnucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the antisense strand; and (4) a Rp- enriched phosphorothioate covalently bonded to the 22thnucleoside from the 5’ end, or the 2ndnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleotide from the 3’ end) and the 23rdnucleoside from the 5’ end, or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the antisense strand.

[0103] In some embodiments, the configuration of stereochemically enriched phosphorothioates in the guide strand comprises SpsSpsSpsSps (SpsSps at the 5’ end and SpsSps at the 3’ end of the guide strand, for example, SpsSps at the positions 1 and 2 of the guide strand from the 5’ end and SpsSps at the positions 21 and 22 of the guide strand from the 5’ end, or, SpsSps at the positions 1 and 2 of the guide strand from the 5’ end and SpsSps at the positions 2 and 1 of the guide strand from the 3’ end) or SpsSpsSpsRps (SpsSps at the 5’ end and SpsRps at the 3’ end, for example, SpsSps at the positions 1 and 2 of the guide strand from the 5’ end and SpsRps at the positions 21 and 22 of the guide strand from the 5’ end, or, SpsSps at positions 1 and 2 of the guide strand from the 5’ end and SpsRps at positions 2 and 1 of the guide strand from the 3’ end). In some aspects, the polynucleic acid molecules described herein comprises four stereochemically enriched phosphorothioates: (1) a Sp-enriched phosphorothioate covalently bonded to the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1stnucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 2ndnucleoside from the 5’ end) of the antisense strand; (2) a Sp-enriched phosphorothioate covalently bonded to the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ 39WSGR Docket No.61382-734.601 end) and the 3rdnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 3rdnucleoside from the 5’ end) of the antisense strand; (3) a Sp-enriched phosphorothioate covalently bonded to the 21stnucleoside from the 5’ end or the 3rd nucleoside from the 3’ end(e.g., the 3'-carbon atom of the 21stnucleoside from the 5’ end or the 3’-carbon atom of the 3rdnucleoside from the 3’ end) and the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22thnucleoside from the 5’ end or the 5’-carbon of the 2ndnucleoside from the 3’ end) of the antisense strand; and (4) a Sp-enriched phosphorothioate covalently bonded to the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the antisense strand. In some aspects, the polynucleic acid molecules described herein comprises four stereochemically enriched phosphorothioates: (1) a Sp-enriched phosphorothioate covalently bonded to the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1stnucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'- carbon atom of the 2ndnucleoside from the 5’ end) of the antisense strand; (2) a Sp-enriched phosphorothioate covalently bonded to the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'- carbon atom of the 3rdnucleoside from the 5’ end) of the antisense strand; (3) a Sp-enriched phosphorothioate covalently bonded to the 21stnucleoside from the 5’ end or the 3rd nucleoside from the 3’ end (e.g., the 3'-carbon atom of the 21stnucleoside from the 5’ end or the 3’-carbon atom of the 3rdnucleoside from the 3’ end) and the 22thnucleoside from the 5’ end or the 2nd nucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22thnucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the antisense strand; and (4) a Rp- enriched phosphorothioate covalently bonded to the 22thnucleoside from the 5’ end or the 2nd nucleoside from the 3’ end (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the antisense strand.

[0104] In some embodiments, the configuration of stereochemically enriched phosphorothioates in the guide strand comprises RpsSpsSpsSps (RpsSps at the 5’ end and SpsSps at the 3’ end of the guide strand, for example, RpsSps at the positions 1 and 2 of the guide strand from the 5’ end and SpsSps at the positions 21 and 22 of the guide strand from the 5’ end, or RpsSps at the positions 1 and 2 of the guide strand from the 5’ end and SpsSps at the positions 2 and 1 of the guide strand from the 3’ end) or RpsSpsSpsRps (RpsSps at the 5’ end and SpsRps at the 3’ end 40WSGR Docket No.61382-734.601 of the guide strand, for example, RpsSps at the positions 1 and 2 of the guide strand from the 5’ end and SpsRps at the positions 21 and 22 of the guide strand from the 5’ end, or, RpsSps at the positions 1 and 2 of the guide strand from the 5’ end and SpsRps at positions 2 and 1 of the guide strand from the 3’ end). In some aspects, the polynucleic acid molecules described herein comprises four stereochemically enriched phosphorothioates: (1) a Rp-enriched phosphorothioate covalently bonded to the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1stnucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'- carbon atom of the 2ndnucleoside from the 5’ end) of the antisense strand; (2) a Sp-enriched phosphorothioate covalently bonded to the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'- carbon atom of the 3rdnucleoside from the 5’ end) of the antisense strand; (3) a Sp-enriched phosphorothioate covalently bonded to the 21stnucleoside from the 5’ end or the 3rd nucleoside from the 3’ end (e.g., the 3'-carbon atom of the 21stnucleoside from the 5’ end or the 3'-carbon atom of the 3rdnucleoside from the 3’ end) and the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22thnucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the antisense strand; and (4) a Sp- enriched phosphorothioate covalently bonded to the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the antisense strand. In some aspects, the polynucleic acid molecules described herein comprises four stereochemically enriched phosphorothioates: (1) a Rp-enriched phosphorothioate covalently bonded to the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1stnucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 2ndnucleoside from the 5’ end) of the antisense strand; (2) a Sp-enriched phosphorothioate covalently bonded to the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 3rdnucleoside from the 5’ end) of the antisense strand; (3) a Sp-enriched phosphorothioate covalently bonded to the 21stnucleoside from the 5’ end or the 3rd nucleoside from the 3’ end (e.g., the 3’-carbon atom of the 21stnucleoside from the 5’ end or the 3’-carbon atom of the 3rdnucleoside from the 3’ end) and the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22thnucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the antisense strand; and (4) a Rp-enriched phosphorothioate covalently bonded to the 22thnucleoside from the 5’ end or the 2nd nucleoside from the 3’ end (e.g., the 3'-carbon atom 41WSGR Docket No.61382-734.601 of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5’- carbon atom of the 23rdnucleoside from the 5’ end or the 5'-carbon atom of the 1stnucleoside from the 3’ end) of the antisense strand.

[0105] In some embodiments, the configuration of stereochemically enriched phosphorothioates in the guide strand comprises SpsRpsSpsSps (SpsRps at the 5’ end and SpsSps at the 3’ end of the guide strand, for example, SpsRps at the positions 1 and 2 of the guide strand from the 5’ end and SpsSps at the positions 21 and 22 of the guide strand from the 5’ end, or SpsRps at positions 1 and 2 of the guide strand from the 5’ end and SpsSps at positions 2 and 1 of the guide strand from the 3’ end) or SpsRpsSpsRps (SpsRps at the 5’ end and SpsRps at the 3’ end of the guide strand, for example, SpsRps at the positions 1 and 2 of the guide strand from the 5’ end and SpsRps at the positions 21 and 22 of the guide strand from the 5’ end, or SpsRps at positions 1 and 2 of the guide strand from the 5’ end and SpsRps at positions 2 and 1 of the guide strand from the 3’ end). In some aspects, the polynucleic acid molecules described herein comprises four stereochemically enriched phosphorothioates: (1) a Sp-enriched phosphorothioate covalently bonded to the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1stnucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'- carbon atom of the 2ndnucleoside from the 5’ end) of the antisense strand; (2) a Rp-enriched phosphorothioate covalently bonded to the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'- carbon atom of the 3rdnucleoside from the 5’ end) of the antisense strand; (3) a Sp-enriched phosphorothioate covalently bonded to the 21stnucleoside from the 5’ end or the 3rd nucleoside from the 3’ end (e.g., the 3'-carbon atom of the 21stnucleoside from the 5’ end or the 3’-carbon atom of the 3rdnucleoside from the 3’ end) and the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22thnucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the antisense strand; and (4) a Sp- enriched phosphorothioate covalently bonded to the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the antisense strand. In some aspects, the polynucleic acid molecules described herein comprises four stereochemically enriched phosphorothioates: (1) a Sp-enriched phosphorothioate covalently bonded to the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1stnucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 2ndnucleoside from the 5’ 42WSGR Docket No.61382-734.601 end) of the antisense strand; (2) a Rp-enriched phosphorothioate covalently bonded to the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 3rdnucleoside from the 5’ end) of the antisense strand; (3) a Sp-enriched phosphorothioate covalently bonded to the 21stnucleoside from the 5’ end or the 3rd nucleoside from the 3’ end (e.g., the 3'-carbon atom of the 21stnucleoside from the 5’ end or the 3’-carbon atom of the 3rdnucleoside from the 3’ end) and the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22thnucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the antisense strand; and (4) a Rp-enriched phosphorothioate covalently bonded to the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the antisense strand.

[0106] In some instances, the polynucleic acid molecule described herein comprises one or more phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), and one or more stereochemically enriched phosphorothioate, e.g., 3’phosphorothioate, Rp diastereomer (RP- enriched phosphorothioate or Rps), 3’phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps), or combinations thereof. In some instances, the guide strand comprises one or more phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), and one or more stereochemically enriched phosphorothioate, e.g., 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps), 3’phosphorothioate, Sp diastereomer (SP- enriched phosphorothioate or Sps), or combinations thereof. In some instances, the passenger strand comprises one or more phosphorodithioate internucleotide linkage, e.g., 3’phosphorodithioate (ss), and one or more stereochemically enriched phosphorothioate, e.g., 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps), 3’phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps), or combinations thereof.

[0107] In some aspects, the polynucleic acid molecule described herein comprises one or more purine modification. In some aspects, the purine modification described herein is 2,6- diaminopurine. In some aspects, the purine modification described herein is 3-deaza-adenine. In some aspects, the purine modification described herein is 7-deaza-guanine. In some aspects, the purine modification described herein is 8-azido-adenine.

[0108] In some aspects, the polynucleic acid molecule described herein comprises one or more pyrimidine modification. In some aspects, the pyrimidine modification described herein is 2- 43WSGR Docket No.61382-734.601 thio-thymidine. In some aspects, the pyrimidine modification described herein is 5- carboxamide-uracil. In some aspects, the pyrimidine modification described herein is 5-methyl- cytosine. In some aspects, the pyrimidine modification described herein is 5-ethynyl uracil.

[0109] In some embodiment, the polynucleic acid molecule described herein comprises an abasic substitution. In those cases where a hybridized polynucleotide construct is contemplated for use as siRNA, a reduction of miRNA-like off-target effects is desirable. The inclusion of one or more (e.g., one or two) abasic substitutions in the hybridized polynucleotide constructs may reduce or even eliminate miRNA-like off-target effects, as the abasic substitutions lack nucleobases that are capable of engaging in base-pairing interactions and alleviate steric hindrance. Thus, the polynucleic acid molecule disclosed herein may include one or more (e.g., one or two) abasic substitutions. In some aspects, abasic substitution is at the 5thnucleotide from the 5’ end of the guide strand described herein. In some aspects, abasic substitution is at the 7thnucleotide from the 5’ end of the guide strand described herein.

[0110] When the polynucleic acid molecule disclosed herein includes two or more of the abasic substitutions, their structures may be same or different. In certain aspects, a passenger strand contains one abasic substitution (e.g., a guide strand may be free of abasic substitutions). In other aspects, a guide strand contains one abasic substitution (e.g., a passenger strand may be free of abasic substitutions). In yet other aspects, a guide strand contains one abasic substitution, and a passenger strand contains one abasic substitution. In further aspects, a passenger strand includes an abasic substitution between a nucleoside number (x) and a nucleoside number (x+1), where x is an integer from 2 to 7. In yet further aspects, a guide strand includes an abasic substitution between a nucleoside number (x) and a nucleoside number (x+1), where x is an integer from 2 to 7.

[0111] The abasic substitution may be of formula (III): ,where L is a sugar analogue, or is substituted with a heteroacyl from A, U ,C, G, or is any other substituted nucleic acid (e.g., locked or unlocked nucleic acid, glycol nucleic acid, etc.; each X4is independently O or S; each X5is independently O, S, NH, or a bond; each R9is independently H, optionally substituted C1-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted (C1-9 heterocyclyl)-C1-6-alkyl, 44WSGR Docket No.61382-734.601 optionally substituted (C6-10 aryl)-C1-6-alkyl, optionally substituted (C3-8 cycloalkyl)-C1-6-alkyl, – LinkA(–T)p, or a conjugation moiety; each LinkA is independently a multivalent linker (e.g., including –C(O)–N(H)–); each T is independently an auxiliary moiety; R10is a bond to a 3’-carbon atom of a nucleoside (x) in the strand; R11is a bond to a 5’-oxygen atom of a nucleoside (x+1) in the strand; p is an integer from 1 to 6; and t is an integer from 1 to 6.

[0112] In some aspects, the abasic substitution described herein is attached to the guide strand of the polynucleic acid molecule described herein. In particular aspects, an abasic substitution (e.g., an internucleotide, abasic spacer of formula (III) in which t is 1) may be included in the guide strand described herein (e.g., within the seed region of the guide strand). In some aspects, an abasic substitution (e.g., an internucleotide, abasic spacer of formula (III) in which t is 1) may be bonded to the 3’ carbon atom of the second, third, fourth, or fifth nucleoside from the 5’ end of the guide strand described herein. In certain aspects, an abasic substitution (e.g., an internucleotide, abasic spacer of formula (III) in which t is 1) may be bonded to the 3’ carbon atom of the thirteenth, fourteenth, fifteenth, or sixteenth nucleoside from the 5’ end of the guide strand described herein. In some aspects, an abasic substitution fourth, fifth, sixth, seventh, eighth, and / or ninth nucleoside from the 5’ end of the guide strand described herein.

[0113] The polynucleic acid molecule described herein may contain a strand including a seed region including a hypoxanthine nucleobase-containing nucleoside (e.g., inosine).

[0114] In certain aspects, the hypoxanthine nucleobase-containing nucleoside is a second nucleoside from the 5’ end in the strand. In further aspects, the hypoxanthine nucleobase- containing nucleoside is a third nucleoside from the 5’ end in the strand. In yet further aspects, the hypoxanthine nucleobase-containing nucleoside is a fourth nucleoside from the 5’ end in the strand. In still further aspects, the hypoxanthine nucleobase-containing nucleoside is a fifth nucleoside from the 5’ end in the strand. In particular aspects, the hypoxanthine nucleobase- containing nucleoside is a sixth nucleoside in the strand. In particular aspects, the hypoxanthine nucleobase-containing nucleoside is a seventh nucleoside in the strand. Nucleotide Analogue

[0115] In some aspects, the present disclosure provides a polynucleic acid molecule incorporating a nucleotide analogue.

[0116] In some instances, modifications of the nucleotide with the nucleotide analogue described herein can alter base pairings and structural changes of the inhibitory polynucleic acid molecule. In some instances, the nucleotide analogue can be incorporated into the polynucleic 45WSGR Docket No.61382-734.601 acid molecule, thereby suppressing off-target effects. In some instances, the nucleotide analogue can be incorporated into the polynucleic acid molecule, thereby improving stability and efficacy of the polynucleic acid molecule. In some instances, the nucleotide analogue described herein can be incorporated into a guide strand, a passenger strand, or a combination thereof.

[0117] In some instances, the nucleotide analogue can be placed in the polynucleic acid molecule or be a substitute for a nucleotide in the polynucleic acid molecule, thereby suppressing the off-target effects. In some instances, the nucleotide analogue can be substituted for a nucleotide in the polynucleic acid molecule, thereby improving stability and / or efficacy of the polynucleic acid molecule. In some instances, the nucleotide analogue described herein can be placed in a guide strand, a passenger strand, or both. In some instances, the nucleotide analogue is located within a seed region of the guide strand. In some instances, the nucleotide analogue is located within 7 nucleotides from the 5’ end of the guide strand.

[0118] In some aspects, the present disclosure provides a polynucleic acid molecule comprising a passenger strand (sense strand) and a guide strand (antisense strand), wherein the guide strand comprises a nucleotide analogue as described herein. In some instances, the nucleotide analogue comprises acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid- 3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D- threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4), 5'(E) vinylphosphonate-2-O-methyluridine-3'phosphate (vpu), 2'-methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), 2’-deoxyadenosine-3-phosphate (dA), 2’-deoxycytidine-3’-phosphate (dC), 2’-deoxyguanosine- 3’-phosphate (dG), 2’-deoxythymidine-3’-phosphate (dT), or 2’-O-methylinosine-3’-phosphate (i). In some instance, the nucleotide analogue is selected from a group consisting of the nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T- T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2- thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’-phosphate (U3f), 2-amino-2’-O- methyladenosine-3’-phosphate (a1), 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5- methyluridine-3’-phosphate (vpt4), 5'(E) vinylphosphonate-2-O-methyluridine-3'phosphate 46WSGR Docket No.61382-734.601 (vpu), 2'-methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), 2’-deoxyadenosine-3-phosphate (dA), 2’- deoxycytidine-3’-phosphate (dC), 2’-deoxyguanosine-3’-phosphate (dG), 2’-deoxythymidine- 3’-phosphate (dT), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the nucleotide analogue is selected from a group consisting of the nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D- threoninol nucleic acid-thymine-3’-phosphate (DT-T), 5’(E) vinylphosphonate-2’-O-methoxy- ethyl-5-methyluridine-3’-phosphate (vpt4), 5'(E) vinylphosphonate-2-O-methyluridine- 3'phosphate (vpu), 2'-methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'- phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), 2’-deoxyadenosine-3-phosphate (dA), 2’-deoxycytidine-3’-phosphate (dC), 2’-deoxyguanosine-3’-phosphate (dG), 2’- deoxythymidine-3’-phosphate (dT), and 2’-O-methylinosine-3’-phosphate (i).

[0119] In some instances, the acyclic L-threoninol nucleic acid- 3'-phosphate (T-T) has a generic representation shown as below, where the base can be any suitable base or modified base that can make Watson-Crick binding with the base on the opposite strand:.

[0120] In some instances, the amidite structure of acyclic L-threoninol nucleic acid-thymine-3'- phosphate (T-T) is shown as below:. 47WSGR Docket No.61382-734.601

[0121] In some instances, the acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T) is incorporated into the polynucleic acid molecule. In some instances, the acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T) is incorporated into the siRNA. In some instances, the acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T) is incorporated into a guide strand. In some instances, the acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T) is incorporated into a passenger strand. In some instances, an incorporated acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T) has a structure shown as below:.

[0122] In some instances, the acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T) is placed in the polynucleic acid molecule. In some instances, the acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T) substitutes one or more nucleotide in siRNA. In some instances, the acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T) substitutes one or more nucleotide in a passenger strand and / or a guide strand. In some instances, the acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T) substitutes one or more nucleotide in a passenger strand. In some instances, the acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T) substitutes one or more nucleotide in a guide strand.

[0123] In some instances, the acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A) has a generic representation shown as below, where the base can be any suitable base or modified base that can make Watson-Crick binding with the base on the opposite strand:. 48WSGR Docket No.61382-734.601

[0124] In some instances, the amidite structure of acyclic L-threoninol nucleic acid-adenine-3’- phosphate (T-A) is shown as below:.

[0125] In some instances, the acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A) is incorporated into the polynucleic acid molecule. In some instances, the acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A) is incorporated into the siRNA. In some instances, the acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A)is incorporated into a guide strand. In some instances, the acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A) is incorporated into a passenger strand. In some instances, an incorporated acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A) has a structure shown as below:.

[0126] In some instances, the acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A) is placed in the polynucleic acid molecule. In some instances, the acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A) substitutes one or more nucleotide in siRNA. In some instances, the acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A) substitutes one or more nucleotide in a passenger strand and / or a guide strand. In some instances, the acyclic L- threoninol nucleic acid-adenine-3’-phosphate (T-A) substitutes one or more nucleotide in a passenger strand. In some instances, the acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A) substitutes one or more the nucleotide in a guide strand. 49WSGR Docket No.61382-734.601

[0127] In some instances, the amidite structure of acyclic N-Acetyl L-threoninol abasic nucleic acid-3’-phosphate (T-NAc) is shown as below:.

[0128] In some instances, the acyclic N-Acetyl L-threoninol abasic nucleic acid-3’-phosphate (T-NAc) is incorporated into the polynucleic acid molecule. In some instances, the acyclic N- Acetyl L-threoninol abasic nucleic acid-3’-phosphate (T-NAc) is incorporated into the siRNA. In some instances, the acyclic N-Acetyl L-threoninol abasic nucleic acid-3’-phosphate (T-NAc) is incorporated into a guide strand. In some instances, the acyclic N-Acetyl L-threoninol abasic nucleic acid-3’-phosphate (T-NAc) is incorporated into a passenger strand. In some instances, an incorporated acyclic N-Acetyl L-threoninol abasic nucleic acid-3’-phosphate (T-NAc) has a structure shown as below:.

[0129] In some instances, the acyclic N-Acetyl L-threoninol abasic nucleic acid-3’-phosphate (T-NAc) is placed in the polynucleic acid molecule. In some instances, the acyclic N-Acetyl L- threoninol abasic nucleic acid-3’-phosphate (T-NAc) substitutes one or more nucleotide in siRNA. In some instances, the acyclic N-Acetyl L-threoninol abasic nucleic acid-3’-phosphate (T-NAc) substitutes one or more nucleotide in a passenger strand and / or a guide strand. In some instances, acyclic N-Acetyl L-threoninol abasic nucleic acid-3’-phosphate (T-NAc) substitutes one or more nucleotide in a passenger strand. In some instances, the acyclic N-Acetyl L- threoninol abasic nucleic acid-3’-phosphate (T-NAc) substitutes one or more nucleotide in a guide strand.

[0130] In some instances, the acyclic L-threoninol nucleic acid-uracil-3'-phosphate (T-U) has a generic representation shown as below, where the base can be any suitable base or modified base that can make Watson-Crick binding with the base on the opposite strand: 50WSGR Docket No.61382-734.601.

[0131] In some instances, the amidite structure of acyclic L-threoninol nucleic acid-uracil-3'- phosphate (T-U) is shown as below:.

[0132] In some instances, the acyclic L-threoninol nucleic acid-uracil-3'-phosphate (T-U) is incorporated into the polynucleic acid molecule. In some instances, the acyclic L-threoninol nucleic acid-uracil-3'-phosphate (T-U) is incorporated into the siRNA. In some instances, the acyclic L-threoninol nucleic acid-uracil-3'-phosphate (T-U) is incorporated into a guide strand. In some instances, the acyclic L-threoninol nucleic acid-uracil-3'-phosphate (T-U) is incorporated into a passenger strand. In some instances, an incorporated acyclic L-threoninol nucleic acid-uracil-3'-phosphate (T-U) has a structure shown as below:. 51WSGR Docket No.61382-734.601

[0133] In some instances, the acyclic acyclic L-threoninol nucleic acid-uracil-3'-phosphate (T- U) is placed in the polynucleic acid molecule. In some instances, the acyclic L-threoninol nucleic acid-uracil-3'-phosphate (T-U) substitutes one or more nucleotide in siRNA. In some instances, the acyclic L-threoninol nucleic acid-uracil-3'-phosphate (T-U) substitutes one or more nucleotide in a passenger strand and / or a guide strand. In some instances, the acyclic L- threoninol nucleic acid-uracil-3'-phosphate (T-U) substitutes one or more nucleotide in a passenger strand. In some instances, the acyclic L-threoninol nucleic acid-uracil-3'-phosphate (T-U) substitutes one or more nucleotide in a guide strand.

[0134] In some instances, the acyclic D-threoninol nucleic acid- 3'-phosphate (DT-T) has a generic representation shown as below, where the base can be any suitable base or modified base that can make Watson-Crick binding with the base on the opposite strand:.

[0135] In some instances, the amidite structure of acyclic D-threoninol nucleic acid-thymine-3'- phosphate (DT-T) is shown as below:.

[0136] In some instances, the acyclic D-threoninol nucleic acid-thymine-3'-phosphate (DT-T) is incorporated into the polynucleic acid molecule. In some instances, the acyclic D-threoninol nucleic acid-thymine-3'-phosphate (DT-T) is incorporated into the siRNA. In some instances, the acyclic D-threoninol nucleic acid-thymine-3'-phosphate (DT-T) is incorporated into a guide strand. In some instances, the acyclic D-threoninol nucleic acid-thymine-3'-phosphate (DT-T) is 52WSGR Docket No.61382-734.601 incorporated into a passenger strand. In some instances, an incorporated acyclic D-threoninol nucleic acid-thymine-3'-phosphate (DT-T) has a structure shown as below:.

[0137] In some instances, the acyclic D-threoninol nucleic acid-thymine-3'-phosphate (DT-T) is placed in the polynucleic acid molecule. In some instances, the acyclic D-threoninol nucleic acid-thymine-3'-phosphate (DT-T) substitutes one or more nucleotide in siRNA. In some instances, the acyclic D-threoninol nucleic acid-thymine-3'-phosphate (DT-T) substitutes one or more nucleotide in a passenger strand and / or a guide strand. In some instances, the acyclic D- threoninol nucleic acid-thymine-3'-phosphate (DT-T) substitutes one or more nucleotide in a passenger strand. In some instances, the acyclic D-threoninol nucleic acid-thymine-3'-phosphate (DT-T) substitutes one or more nucleotide in a guide strand.

[0138] In some instances, the amidite structure of 1’,2’-Dideoxyribose-3’-phosphate (dAB) is shown as below:.

[0139] In some instances, the 1’,2’-Dideoxyribose-3’-phosphate (dAB) is incorporated into the polynucleic acid molecule. In some instances, the 1’,2’-Dideoxyribose-3’-phosphate (dAB) is incorporated into the siRNA. In some instances, the 1’,2’-Dideoxyribose-3’-phosphate (dAB) is incorporated into a guide strand. In some instances, the 1’,2’-Dideoxyribose-3’-phosphate (dAB) is incorporated into a passenger strand. In some instances, an incorporated 1’,2’-Dideoxyribose- 3’-phosphate (dAB) has a structure shown as below: 53WSGR Docket No.61382-734.601.

[0140] In some instances, the 1’,2’-Dideoxyribose-3’-phosphate (dAB) is placed in the polynucleic acid molecule. In some instances, the 1’,2’-Dideoxyribose-3’-phosphate (dAB) substitutes one or more nucleotide in siRNA. In some instances, the 1’,2’-Dideoxyribose-3’- phosphate (dAB) substitutes one or more nucleotide in a passenger strand and / or a guide strand. In some instances, the 1’,2’-Dideoxyribose-3’-phosphate (dAB) substitutes one or more nucleotide in a passenger strand. In some instances, the 1’,2’-Dideoxyribose-3’-phosphate (dAB) substitutes one or more nucleotide in a guide strand.

[0141] In some instances, the amidite structure of thymidine-glycol nucleic acid (GNA) S- isomer (Tgn) is shown as below:.

[0142] In some instances, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) is incorporated into the polynucleic acid molecule. In some instances, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) is incorporated into the siRNA. In some instances, the thymidine- glycol nucleic acid (GNA) S-isomer (Tgn) is incorporated into a guide strand. In some instances, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) is incorporated into a passenger strand. In some instances, an incorporated thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) has a structure shown as below: 54WSGR Docket No.61382-734.601.

[0143] In some instances, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) is placed in the polynucleic acid molecule. In some instances, the thymidine-glycol nucleic acid (GNA) S- isomer (Tgn) substitutes one or more nucleotide in siRNA. In some instances, the thymidine- glycol nucleic acid (GNA) S-isomer (Tgn) substitutes one or more nucleotide in a passenger strand and / or a guide strand. In some instances, the thymidine-glycol nucleic acid (GNA) S- isomer (Tgn) substitutes one or more nucleotide in a passenger strand. In some instances, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) substitutes one or more nucleotide in a guide strand.

[0144] In some instances, the amidite structure of 2’-O-methyl-2-thiouridine-3’-phosphate (u3) is shown as below:.

[0145] In some instances, the 2’-O-methyl-2-thiouridine-3’-phosphate (u3) is incorporated into the polynucleic acid molecule. In some instances, the 2’-O-methyl-2-thiouridine-3’-phosphate (u3) is incorporated into the siRNA. In some instances, the 2’-O-methyl-2-thiouridine-3’- phosphate (u3) is incorporated into a guide strand. In some instances, the 2’-O-methyl-2- thiouridine-3’-phosphate (u3) is incorporated into a passenger strand. In some instances, an incorporated 2’-O-methyl-2-thiouridine-3’-phosphate (u3) has a structure shown as below: 55WSGR Docket No.61382-734.601.

[0146] In some instances, the 2’-O-methyl-2-thiouridine-3’-phosphate (u3) is placed in the polynucleic acid molecule. In some instances, the 2’-O-methyl-2-thiouridine-3’-phosphate (u3) substitutes one or more nucleotide in siRNA. In some instances, the 2’-O-methyl-2-thiouridine- 3’-phosphate (u3) substitutes one or more nucleotide in a passenger strand and / or a guide strand. In some instances, the 2’-O-methyl-2-thiouridine-3’-phosphate (u3) substitutes one or more nucleotide in a passenger strand. In some instances, the 2’-O-methyl-2-thiouridine-3’-phosphate (u3) substitutes one or more nucleotide in a guide strand.

[0147] In some instances, the amidite structure of 2’-fluoro-2-thiouridine-3’-phosphate (U3f) is shown as below:.

[0148] In some instances, the 2’-fluoro-2-thiouridine-3’-phosphate (U3f) is incorporated into the polynucleic acid molecule. In some instances, the 2’-fluoro-2-thiouridine-3’-phosphate (U3f) is incorporated into the siRNA. In some instances, the 2’-fluoro-2-thiouridine-3’-phosphate (U3f) is incorporated into a guide strand. In some instances, the 2’-fluoro-2-thiouridine-3’-phosphate (U3f) is incorporated into a passenger strand. In some instances, an incorporated 2’-fluoro-2- thiouridine-3’-phosphate (U3f) has a structure shown as below: 56WSGR Docket No.61382-734.601.

[0149] In some instances, the 2’-fluoro-2-thiouridine-3’-phosphate (U3f) is placed in the polynucleic acid molecule. In some instances, the 2’-fluoro-2-thiouridine-3’-phosphate (U3f) substitutes one or more nucleotide in siRNA. In some instances, the 2’-fluoro-2-thiouridine-3’- phosphate (U3f) substitutes one or more nucleotide in a passenger strand and / or a guide strand. In some instances, the 2’-fluoro-2-thiouridine-3’-phosphate (U3f) substitutes one or more the nucleotide in a passenger strand. In some instances, the 2’-fluoro-2-thiouridine-3’-phosphate (U3f) substitutes one or more the nucleotide in a guide strand.

[0150] In some instances, the amidite structure of 2-amino-2’-O-methyladenosine-3’-phosphate (a1) is shown as below:.

[0151] In some instances, the 2-amino-2’-O-methyladenosine-3’-phosphate (a1) is incorporated into the polynucleic acid molecule. In some instances, the 2-amino-2’-O-methyladenosine-3’- phosphate (a1) is incorporated into the siRNA. In some instances, the 2-amino-2’-O- methyladenosine-3’-phosphate (a1) is incorporated into a guide strand. In some instances, the 2- amino-2’-O-methyladenosine-3’-phosphate (a1) is incorporated into a passenger strand. In some instances, an incorporated 2-amino-2’-O-methyladenosine-3’-phosphate (a1) has a structure shown as below: 57WSGR Docket No.61382-734.601.

[0152] In some instances, the 2-amino-2’-O-methyladenosine-3’-phosphate (a1) is placed in the polynucleic acid molecule. In some instances, the 2-amino-2’-O-methyladenosine-3’-phosphate (a1) substitutes one or more nucleotide in siRNA. In some instances, the 2-amino-2’-O- methyladenosine-3’-phosphate (a1) substitutes one or more nucleotide in a passenger strand and / or a guide strand. In some instances, the 2-amino-2’-O-methyladenosine-3’-phosphate (a1) substitutes one or more nucleotide in a passenger strand. In some instances, the 2-amino-2’-O- methyladenosine-3’-phosphate (a1) substitutes one or more nucleotide in a guide strand.

[0153] In some instances, the nucleotide analogue comprises hypoxanthine nucleobase- containing nucleoside (e.g., inosine).

[0154] In some instances, the amidite structure of 5’(E) vinylphosphonate-2’-O-methoxy-ethyl- 5-methyluridine-3’-phosphate (vpt4) is shown as below:.

[0155] In some instances, the 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’- phosphate (vpt4) is incorporated into the polynucleic acid molecules. In some instances, the 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4) is incorporated into the siRNA. In some instances, the 5’(E) vinylphosphonate-2’-O-methoxy- ethyl-5-methyluridine-3’-phosphate (vpt4) is incorporated into a guide strand. In some instances, the 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4) is incorporated into a passenger strand. In some instances, the 5’(E) vinylphosphonate-2’-O- methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4) is located at the 5’ end of the guide strand. 58WSGR Docket No.61382-734.601 In some instances, the 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’- phosphate (vpt4) is located at the 5’ end of the passenger strand. In some instances, an incorporated 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4) has a structure shown as below:.

[0156] In some instances, the 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’- phosphate (vpt4) substitutes one or more nucleotide in siRNA. In some instances, the 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4) substitutes one or more nucleotide in a passenger strand and / or a guide strand. In some instances, the 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4) substitutes one or more nucleotide in a passenger strand. In some instances, the 5’(E) vinylphosphonate-2’-O- methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4) substitutes one or more nucleotide in a guide strand.

[0157] In some instances, the amidite structure of 5’(E) vinylphosphonate-2-O-methyluridine- 3’phosphate (vpu) is shown as below:.

[0158] In some instances, the 5’(E) vinylphosphonate-2-O-methyluridine-3’phosphate (vpu) is incorporated into the polynucleic acid molecule. In some instances, the 5’(E) vinylphosphonate- 2-O-methyluridine-3’phosphate (vpu) is incorporated into the siRNA. In some instances, the 5’(E) vinylphosphonate-2-O-methyluridine-3’phosphate (vpu) is incorporated into a guide 59WSGR Docket No.61382-734.601 strand. In some instances, the 5’(E) vinylphosphonate-2-O-methyluridine-3’phosphate (vpu) is incorporated into a passenger strand. In some instances, the 5’(E) vinylphosphonate-2-O- methyluridine-3’phosphate (vpu) is located at the 5’ end of a guide strand. In some instances, the 5’(E) vinylphosphonate-2-O-methyluridine-3’phosphate (vpu) is located at the 5’ end of a passenger strand. In some instances, an incorporated 5’(E) vinylphosphonate-2-O- methyluridine-3’phosphate (vpu) has a structure shown as below:.

[0159] In some instances, the 5’(E) vinylphosphonate-2-O-methyluridine-3’phosphate (vpu) substitutes one or more nucleotide in siRNA. In some instances, the 5’(E) vinylphosphonate-2- O-methyluridine-3’phosphate (vpu) substitutes one or more nucleotide in a passenger strand and / or a guide strand. In some instances, the 5’(E) vinylphosphonate-2-O-methyluridine- 3’phosphate (vpu) substitutes one or more nucleotide in a passenger strand. In some instances, the 5’(E) vinylphosphonate-2-O-methyluridine-3’phosphate (vpu) substitutes one or more the nucleotide in a guide strand. The Amount and Location of Modifications

[0160] In some aspects, the polynucleic acid molecule described herein comprises one or more type of modifications as described above. Accordingly, in some aspects, about 10% of the nucleotides from the polynucleic acid molecule described herein are modified with one or more type of modifications as described above. In other aspects, about 20% of the nucleotides from the polynucleic acid molecule described herein are modified with one or more type of modifications as described above. In other aspects, about 30% of the nucleotides from the polynucleic acid molecule described herein are modified with one or more type of modifications as described above. In other aspects, about 40% of the nucleotides from the polynucleic acid molecule described herein are modified with one or more type of modifications as described above. In other aspects, about 50% of the nucleotides from the polynucleic acid molecule described herein are modified with one or more type of modifications as described above. In 60WSGR Docket No.61382-734.601 other aspects, about 60% of the nucleotides from the polynucleic acid molecule described herein are modified with one or more type of modifications as described above. In other aspects, about 70% of the nucleotides from the polynucleic acid molecule described herein are modified with one or more type of modifications as described above. In other aspects, about 80% of the nucleotides from the polynucleic acid molecule described herein are modified with one or more type of modifications as described above. In other aspects, about 90% of the nucleotides from the polynucleic acid molecule described herein are modified with one or more type of modifications as described above. In other aspects, 100% of the nucleotides from the polynucleic acid molecule described herein are modified with one or more type of modifications as described above.

[0161] In some aspects, the one or more types of modifications described herein occurs at different positions within the polynucleic acid molecule described herein. In some aspects, the one or more types of modifications described herein occurs in the seed region within the polynucleic acid molecule described herein. In some aspects, the one or more types of modifications described herein occurs at 3’ end of the polynucleic acid molecule described herein. In some aspects, the one or more types of modifications described herein occurs at 5’ end of the polynucleic acid molecule described herein. In some aspects, the one or more types of modifications described herein occurs dispersedly within the polynucleic acid molecule described herein. In some aspects, the one or more types of modifications described herein occurs in clusters within the polynucleic acid molecule described herein.

[0162] In some aspects, the polynucleic acid molecule is modified with a nucleotide analogue described herein by incorporating the nucleotide analogue in a seed region of the guide strand (positions 2-8 from 5’ end of the guide strand). As described herein, “seed region” refers to a region on the polynucleic acid molecule that comprises sequence that is essential for the binding of the polynucleic acid molecule described herein to the target RNA.

[0163] In some instances, the polynucleic acid molecule is modified with a nucleotide analogue described herein at positions 3-8, positions 4-8, positions 5-8, positions 6-8, or positions 7-8 from the 5’ end of the guide strand. In some instances, the polynucleic acid molecule is modified with a nucleotide analogue described herein at position 2, position 3, position 4, position 5, position 6, position 7, position 8, from the 5’ end of the guide strand or combination thereof. In some instances, the polynucleic acid molecule is modified with one, two, three, four, five, six, or seven nucleotide analogues. In some instances, two or more consecutive positions in the guide strand of the polynucleic acid molecule are modified with nucleotide analogues. In some instances, two or more nucleotide analogue modifications can be placed in the polynucleic acid 61WSGR Docket No.61382-734.601 molecule at alternative positions (e.g., positions 3 and 5, positions 4 and 6, positions 5 and 7, etc.).

[0164] In some instances, the polynucleic acid molecule comprises a polynucleic acid molecule. In some instances, the polynucleic acid molecule is an siRNA comprising a guide strand and a passenger strand. In some instances, the nucleotide analogue is located at the seed region of the guide strand at positions 2-8 from the 5’ end. In some instances, the nucleotide analogue is located at positions 3-8 from the 5’ end. In some instances, the nucleotide analogue is located at positions 4-8 from the 5’ end. In some instances, the nucleotide analogue is located at positions 5-8 from the 5’ end. In some instances, the nucleotide analogue is located at positions 6-8 from the 5’ end. the nucleotide analogue is located at positions 6-7 from the 5’ end. In some instances, the nucleotide analogue is located at positions 7-8 from the 5’ end.

[0165] In some instances, the nucleotide analogue is located at any one of positions 2-8 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at any one of positions 3-8 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at any one of positions 4-8 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at any one of positions 5-8 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at any one of positions 6-8 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at any one of positions 6- 7 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at any one of positions 7-8 from the 5’ end of the guide strand.

[0166] In some instances, the nucleotide analogue is located at position 1 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 2 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 3 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 4 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 5 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 6 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 7 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 8 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 9 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 10 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 11 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 12 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 13 from the 62WSGR Docket No.61382-734.601 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 14 from the 5’ end of the guide strand.

[0167] In some instances, the nucleotide analogue is located at position 3 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 3 from the 5’ end of the guide strand, and the guide strand further comprises 2-thiouridine-3’-phosphate nucleotide. In some instances, the nucleotide analogue located at position 3 from the 5’ end of the guide strand, and the guide strand further comprises 2’-O-methyl-2-thiouridine-3’-phosphate (u3). In some instances, the nucleotide analogue is located at position 3 from the 5’ end of the guide strand, and the guide strand further comprises at least one, at least two, at least three, or at least four 2’-fluoro modified nucleotides. In some instances, the nucleotide analogue is located at position 3 from the 5’ end of the guide strand, and the guide strand further comprises 2’- fluoro modified nucleotides at at least one of positions 2, 7, 12, 14, and 16 from the 5’ end. In some instances, the nucleotide analogue is located at position 3 from the 5’ end of the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at positions 2, 7, 12, 14, and 16 from the 5’ end. Specific Modification Patterns

[0168] In some aspects, described herein is a specific modification pattern for the polynucleic acid molecule which is a double-stranded nucleic acid molecule comprising a passenger strand and a guide strand. In some aspects, the guide strand comprises a 2’-fluoro modified nucleotide in position 2 from the 5’ end. In some aspects, the guide strand comprises a 2’-fluoro modified nucleotide in position 14 from the 5’ end. In some aspects, the guide strand comprises 2’-fluoro modified nucleotides in positions 2 and 14 from the 5’ end. In some aspects, the guide strand comprises a 2’-fluoro modified nucleotide in position 12 from the 5’ end. In some aspects, the guide strand comprises a 2’-fluoro modified nucleotide in position 16 from the 5’ end. In other aspects, the guide strand comprises a 2’-fluoro modified nucleotide in position 6 from the 5’ end. In other aspects, the guide strand comprises a 2’-fluoro modified nucleotide in position 7 from the 5’ end. In other aspects, the guide strand comprises a 2’-fluoro modified nucleotide in position 8 from the 5’ end. In other aspects, the guide strand comprises a 2’-fluoro modified nucleotide in position 9 from the 5’ end. In other aspects, the guide strand comprises a 2’-fluoro modified nucleotide in position 4 from the 5’ end. In some aspects, the guide strand comprises a 2’-fluoro modified nucleotide in position 5 from the 5’ end.

[0169] In some aspects, described herein is a specific modification pattern for the polynucleic acid molecule which is a double-stranded nucleic acid molecule comprising a passenger strand and a guide strand. In some aspects, the passenger strand comprises a 2’-fluoro modified 63WSGR Docket No.61382-734.601 nucleotide in position 9 from the 5’ end. In some aspects, the passenger strand comprises a 2’- fluoro modified nucleotide in position 11 from the 5’ end. In some aspects, the passenger strand comprises 2’-fluoro modified nucleotides in positions 9 and 11 from the 5’ end. In some aspects, the passenger strand comprises a 2’-fluoro modified nucleotide in position 7 from the 5’ end. In some aspects, the passenger strand comprises a 2’-fluoro modified nucleotide in position 10 from the 5’ end. In some aspects, the passenger strand comprises 2’-fluoro modified nucleotides in positions 9, 11, and 7 from the 5’ end. In some aspects, the passenger strand comprises 2’-fluoro modified nucleotides in positions 9 and 11, and 10 from the 5’ end. In some aspects, the passenger strand comprises 2’-fluoro modified nucleotides in positions 9 and 7 from the 5’ end. In some aspects, the passenger strand comprises 2’-fluoro modified nucleotides in positions 9 and 10 from the 5’ end. In some aspects, the passenger strand comprises 2’-fluoro modified nucleotides in positions 9, 11, 7, and 10 from the 5’ end. In other aspects, the passenger strand comprises a 2’-fluoro modified nucleotide in position 8 from the 5’ end. In other aspects, the passenger strand comprises a 2’-fluoro modified nucleotide in position 12 from the 5’ end. In some aspects, the passenger strand comprises 2’-fluoro modified nucleotides in positions 9, 11, and 12 from the 5’ end. In other aspects, the passenger strand comprises a 2’- fluoro modified nucleotide in position 13 from the 5’ end. In some aspects, the passenger strand comprises 2’-fluoro modified nucleotides in positions 9, 11, and 13 from the 5’ end. In other aspects, the passenger strand comprises a 2’-fluoro modified nucleotide in position 16 from the 5’ end.

[0170] In some aspects, the passenger strand and the guide strand of the polynucleic acid molecule comprises any combination of two or more 2’-fluoro modified nucleotides at the positions described in the above two paragraphs.

[0171] In some aspects, the guide strand comprises 5’ - nNfnnnNfnNfNfnnnnNfnNfnnnnnnn-3’. In some aspects, the guide strand comprises 5’ - nNfnnnNfnnnnnnnNfnNfnnnnnnn-3’. In some aspects, the guide strand comprises 5’ - nNfnnnnNfnnnnNfnNfnnnnnnnnn-3’. In some aspects, the guide strand comprises 5’- nNfnnnnNfnnnnNfnNfnNfnnnnnnn-3’. In some aspects, the guide strand comprises 5’- nNfnNfnnnnnnnNfnNfnNfnnnnnnn -3’. In some aspects, the guide strand comprises 5’- nNfnnNfnnnnnnNfnNfnNfnnnnnnn -3’. In some aspects, the guide strand comprises 5’- nNfnnnnnnNfnnNfnNfnNfnnnnnnn -3’. In some aspects, the guide strand comprises 5’-nNfnnnnNfnnnnnnNfnNfnnnnnnn -3’. In some aspects, the guide strand comprises 5’- nNfnnnnnnnnnNfnNfnNfnnnnnnn -3’. In the modification patterns described above, “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide. 64WSGR Docket No.61382-734.601

[0172] In some aspects, the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnnnnnnnn-3’. In some aspects, the passenger strand comprises 5’- nnnnnnNfnNfNfNfnnnnnnnnnn-3’. In some aspects, the passenger strand comprises 5’- nnnnnnnnNfNfNfnnnnnnnnnn-3’. In some aspects, the passenger strand comprises 5’- nnnnnnnnNfnNfNfnnnnnnnnn-3’. In some aspects, the passenger strand comprises 5’- nnnnnnnnNfnNfnNfnnnnnnnn -3’. In the modification patterns described above, “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0173] In some aspects, described herein is a specific modification pattern for the polynucleic acid molecule which is a double-stranded nucleic acid molecule comprising a passenger strand and a guide strand, wherein the passenger strand comprises about twelve 2’-fluoro modified nucleotides and about nine 2’-O-methyl modified nucleotides, and wherein the guide strand comprises about nine 2’-fluoro modified nucleotides and about fourteen 2’-O-methyl modified nucleotides.

[0174] In some aspects, described herein is a specific modification pattern, wherein the passenger strand is fully modified and comprises twelve 2’-fluoro modified nucleotides, nine 2’- O-methyl modified nucleotides, and wherein the guide strand is fully modified and comprises nine 2’-fluoro modified nucleotides and fourteen 2’-O-methyl modified nucleotides.

[0175] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- NfnNfnNfnNfnNfNfNfnNfnNfnNfnNfnNf-3’, wherein the guide strand comprises 5’-nNfnNfnNfnNfnNfnnnNfnNfnNfnNfnnn-3’, wherein “Nf” stands for a 2’- fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0176] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- NfnNfnNfnNfnNfNfNfnNfnNfnNfnNfnNf -3’, wherein the guide strand comprises 5’- nNfnNfnNfnNfnNfnnnNfnNfnNfnNfnnn -3’, wherein the passenger and / or guide strand comprises one or more phosphorothioate internucleotide linkage, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide. In other aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- NfnNfnNfnNfnNfNfNfnNfnNfnNfnNfnNf -3’, wherein the guide strand comprises 5’- nNfnNfnNfnNfnNfnnnNfnNfnNfnNfnnn -3’, wherein the passenger comprises two phosphorothioate internucleotide linkages, wherein the guide comprises four phosphorothioate internucleotide linkages, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0177] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, wherein the guide strand comprises 5’-nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, wherein the passenger comprises two 65WSGR Docket No.61382-734.601 phosphorothioate internucleotide linkages, wherein “s” stands for phosphorothioate internucleotide linkages, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0178] In some aspects, described herein is a specific modification pattern, wherein the passenger strand and / or guide strand is modified as Type I in Table 17. Table 17. Nucleotide Modification PatternsNote: “Nf” stands for a 2’-fluoro modified nucleotide, “n” stands for a 2’-O-methyl modified nucleotide, “s” stands for a 3'-phosphorothioate, “invdN” stands for an inverted deoxy- nucleotide.

[0179] In some aspects, the polynucleic acid molecule provided herein comprises a passenger strand from a nucleic acid sequence of SEQ ID NOs: 21-33 and a guide strand comprises a nucleic acid sequence of SEQ ID NOs: 1-18 and 160. In other aspects, the polynucleic acid molecule provided herein comprises a passenger strand from a nucleic acid sequence of SEQ ID NOs: 21-33, a guide strand comprises a nucleic acid sequence of SEQ ID NOs: 1-18 and 160, and wherein the passenger strand and / or guide strand is modified in Type I, II, III, IV, V, or VI modification pattern specified in Table 17. In some aspects, the polynucleic acid molecule provided herein comprises a passenger strand from a nucleic acid sequence of SEQ ID NOs: 21- 22, and 25-27 and a guide strand comprises a nucleic acid sequence of SEQ ID NOs: 1-2, 7, and 10-11. In other aspects, the polynucleic acid molecule provided herein comprises a passenger strand from a nucleic acid sequence of SEQ ID NOs: 21-22, and 25-27, a guide strand comprises 66WSGR Docket No.61382-734.601 a nucleic acid sequence of SEQ ID NOs: 1-2, 7, and 10-11, and wherein the passenger strand and / or guide strand is modified in Type I modification pattern specified in Table 17.

[0180] In some aspects, described herein is a specific modification pattern, wherein the passenger strand is fully modified and comprises three 2’-fluoro modified nucleotides, eighteen 2’-O-methyl modified nucleotides, and wherein the guide strand is fully modified and comprises four 2’-fluoro modified nucleotides, nineteen 2’-O-methyl modified nucleotides.

[0181] In some aspects, described herein is a specific modification pattern, wherein the passenger strand is fully modified and comprises three 2’-fluoro modified nucleotides and eighteen 2’-O-methyl modified nucleotides, and wherein the guide strand is fully modified and comprises five 2’-fluoro modified nucleotides, eighteen 2’-O-methyl modified nucleotides.

[0182] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnnnnnnnn -3’, wherein the guide strand comprises 5’- nNfnnnnNfnnnnNfnNfnNfnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0183] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnnnnnnnn -3’, wherein the guide strand comprises 5’- nNfnnnnNfnnnnNfnNfnNfnnnnnnn -3’, wherein the passenger strand and / or guide strand comprises one or more phosphorothioate internucleotide linkage, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide. In other aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnnnnnnnn -3’, wherein the guide strand comprises 5’- nNfnnnnNfnnnnNfnNfnNfnnnnnnn -3’, wherein the passenger strand comprises two phosphorothioate internucleotide linkages, wherein the guide comprises four phosphorothioate internucleotide linkages, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0184] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnnnnnnnn -3’, wherein the guide strand comprises 5’- nNfnNfnnnnnnnNfnNfnNfnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0185] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnnnnnnnn -3’, wherein the guide strand comprises 5’- nNfnnNfnnnnnnNfnNfnNfnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0186] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnnnnnnnn -3’, wherein the guide strand 67WSGR Docket No.61382-734.601 comprises 5’- nNfnnnnnnNfnnNfnNfnNfnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0187] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnnnNfnNfNfnnnnnnnnn -3’, wherein the guide strand comprises 5’- nNfnnnnNfnnnnNfnNfnNfnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0188] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnnnNfnNfNfnnnnnnnnn -3’, wherein the guide strand comprises 5’- nNfnNfnnnnnnnNfnNfnNfnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0189] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnnnNfnNfNfnnnnnnnnn -3’, wherein the guide strand comprises 5’- nNfnnNfnnnnnnNfnNfnNfnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0190] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnnnNfnNfNfnnnnnnnnn -3’, wherein the guide strand comprises 5’- nNfnnnnnnNfnnNfnNfnNfnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0191] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnnnNfnNfnNfnnnnnnnn -3’, wherein the guide strand comprises 5’- nNfnnnnNfnnnnNfnNfnNfnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0192] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnnnNfnNfnNfnnnnnnnn -3’, wherein the guide strand comprises 5’- nNfnNfnnnnnnnNfnNfnNfnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0193] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnnnNfnNfnNfnnnnnnnn -3’, wherein the guide strand comprises 5’- nNfnnNfnnnnnnNfnNfnNfnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide.

[0194] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnnnNfnNfnNfnnnnnnnn -3’, wherein the guide strand comprises 5’- nNfnnnnnnNfnnNfnNfnNfnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, and wherein “n” stands for a 2’-O-methyl modified nucleotide. 68WSGR Docket No.61382-734.601

[0195] In some aspects, described herein is a specific modification pattern, wherein the passenger strand and / or guide strand is modified as Type V in Table 17.

[0196] In some aspects, the polynucleic acid molecule provided herein comprises a passenger strand comprising a nucleic acid sequence of SEQ ID NOs: 21-33, and / or a guide strand comprising a nucleic acid sequence of SEQ ID NOs: 1-18 and 160, and wherein the passenger strand and / or guide strand is modified in Type V modification pattern specified in Table 17. In other aspects, the polynucleic acid molecule provided herein comprises a passenger strand comprising a nucleic acid sequence of SEQ ID NOs: 21-22, and 25-27, and / or a guide strand comprising a nucleic acid sequence of SEQ ID NOs: 1-2, 7, and 10-11, and wherein the passenger strand and / or guide strand is modified in Type V modification pattern specified in Table 17.

[0197] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises about three 2’-fluoro modified nucleotides and about eighteen 2’-O- methyl modified nucleotides, with one or more inverted deoxy-nucleotides on the 3’ end as an overhang.

[0198] In some aspects, described herein is a specific modification pattern, wherein the passenger strand is fully modified and comprises three 2’-fluoro modified nucleotides and eighteen 2’-O-methyl modified nucleotides, with two inverted deoxy-nucleotides on the 3’ end as an overhang.

[0199] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’-nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, wherein the passenger strand comprises 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for phosphorothioate internucleotide linkage, e.g., 3’phosphorothioate. In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, wherein the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for modified phosphorothioate internucleotide linkage. In some instances, the modified phosphorothioate internucleotide linkage comprises stereochemically enriched phosphorothioates comprising 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps), 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps), or combinations thereof.

[0200] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’-nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, wherein the passenger strand 69WSGR Docket No.61382-734.601 comprises 5’-nsnsnnnnnnNfnNfNfnnnnnnnnn-3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for phosphorothioate internucleotide linkage, e.g., 3’phosphorothioate.

[0201] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’-nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, wherein the passenger strand comprises 5’- nsnsnnnnnnNfnNfnNfnnnnnnnn-3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for phosphorothioate internucleotide linkage, e.g., 3’phosphorothioate.

[0202] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’- nsNfsnNfnnnnnnnNfnNfnNfnnnnnsnsn -3’, wherein the passenger strand comprises 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for phosphorothioate internucleotide linkage, e.g., 3’phosphorothioate.

[0203] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’- nsNfsnnNfnnnnnnNfnNfnNfnnnnnsnsn-3’, wherein the passenger strand comprises 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for phosphorothioate internucleotide linkage, e.g., 3’phosphorothioate.

[0204] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’- nsNfsnnnnnnNfnnNfnNfnNfnnnnnsnsn -3’, wherein the passenger strand comprises 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for phosphorothioate internucleotide linkage, e.g., 3’phosphorothioate.

[0205] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’- nsNfsnNfnnnnnnnNfnNfnNfnnnnnsnsn -3’, wherein the passenger strand comprises 5’- nsnsnnnnnnNfnNfNfnnnnnnnnn-3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for phosphorothioate internucleotide linkage, e.g., 3’phosphorothioate.

[0206] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’- nsNfsnnNfnnnnnnNfnNfnNfnnnnnsnsn-3’, wherein the passenger strand comprises 5’-nsnsnnnnnnNfnNfNfnnnnnnnnn-3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for phosphorothioate internucleotide linkage, e.g., 3’phosphorothioate.

[0207] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’- nsNfsnnnnnnNfnnNfnNfnNfnnnnnsnsn -3’, wherein the passenger strand 70WSGR Docket No.61382-734.601 comprises 5’-nsnsnnnnnnNfnNfNfnnnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for phosphorothioate internucleotide linkage, e.g., 3’phosphorothioate.

[0208] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’- nsNfsnNfnnnnnnnNfnNfnNfnnnnnsnsn -3’, wherein the passenger strand comprises 5’- nsnsnnnnnnNfnNfnNfnnnnnnnn-3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for phosphorothioate internucleotide linkage, e.g., 3’phosphorothioate.

[0209] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’- nsNfsnnNfnnnnnnNfnNfnNfnnnnnsnsn-3’, wherein the passenger strand comprises 5’- nsnsnnnnnnNfnNfnNfnnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for phosphorothioate internucleotide linkage, e.g., 3’phosphorothioate.

[0210] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’- nsNfsnnnnnnNfnnNfnNfnNfnnnnnsnsn -3’, wherein the passenger strand comprises 5’- nsnsnnnnnnNfnNfnNfnnnnnnnn -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for phosphorothioate internucleotide linkage, e.g., 3’phosphorothioate.

[0211] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’-nsNfsnnnnNfnnnnsNfnNfnNfnnnnnsnsn-3’, wherein the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and wherein “s” stands for phosphorothioate internucleotide linkage, e.g., 3’phosphorothioate

[0212] In some instances, the guide strand comprises a vinyl phosphonate modified nucleotide at the 5’ end. In some instances, the vinyl phosphonate modified nucleotide comprises 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4) or 5'(E) vinylphosphonate-2-O-methyluridine-3'phosphate (vpu). In some instances, the vinyl phosphonate modified nucleotide is 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5- methyluridine-3’-phosphate (vpt4) or 5'(E) vinylphosphonate-2-O-methyluridine-3'phosphate (vpu). In some instances, the first nucleotide on the 5’ end of the guide strand is substituted with the vinyl phosphonate modified nucleotide. In some instances, the vinyl phosphonate modified nucleotide substitutes a 5’ end nucleotide of SEQ ID NOs: 1-18 and 160.

[0213] In some instances, the stereochemically enriched phosphorothioate covalently bonded to the 1stnucleoside (e.g., the 3'-carbon atom of the 1st nucleoside) and the 2ndnucleoside (e.g., the 5'-carbon atom of the 2nd nucleoside) from the 5’ end of the guide strand is 3’phosphorothioate, 71WSGR Docket No.61382-734.601 Rp diastereomer (RP-enriched phosphorothioate or Rps). In some instances, the stereochemically enriched phosphorothioate covalently bonded to the 1stnucleoside (e.g., the 3'-carbon atom of the 1st nucleoside) and the 2ndnucleoside (e.g., the 5'-carbon atom of the 2nd nucleoside) from the 5’ end of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps).

[0214] In some instances, the stereochemically enriched phosphorothioate covalently bonded to the 2ndnucleoside (e.g., the 3'-carbon atom of the 2ndnucleoside) and the 3rdnucleoside (e.g., the 5'-carbon atom of the 3rdnucleoside) from the 5’ end of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps). In some instances, stereochemically enriched phosphorothioate covalently bonded to the 2ndnucleoside (e.g., the 3'-carbon atom of the 2ndnucleoside) and the 3rdnucleoside (e.g., the 5'-carbon atom of the 3rdnucleoside) from the 5’ end of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps).

[0215] In some instances, the stereochemically enriched phosphorothioate covalently bonded to the 21st nucleoside (e.g., the 3'-carbon atom of the 21st nucleoside from the 5’ end or the 3rd nucleotide from the 3’end) and the 22th nucleoside (e.g., the 5'-carbon atom of the 22th nucleoside from the 5’ end or the 2ndnucleotide from the 3’end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps). In some instances, stereochemically enriched phosphorothioate covalently bonded to the 21st nucleoside (e.g., the 3'-carbon atom of the 21st nucleoside from the 5’ end or the 3rd nucleotide from the 3’end) and the 22th nucleoside (e.g., the 5'-carbon atom of the 22th nucleoside from the 5’ end or the 2ndnucleotide from the 3’end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP- enriched phosphorothioate or Sps).

[0216] In some instances, the stereochemically enriched phosphorothioate covalently bonded to the 22thnucleoside (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 2ndnucleotide from the 3’end) and the 23rdnucleoside (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 1stnucleotide from the 3’end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps). In some instances, the stereochemically enriched phosphorothioate covalently bonded to the 22thnucleoside (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 2ndnucleotide from the 3’end) and the 23rdnucleoside (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 1stnucleotide from the 3’end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP- enriched phosphorothioate or Sps).

[0217] In some instances, the stereochemically enriched phosphorothioate covalently bonded to (1) the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1st nucleoside from the 5’ 72WSGR Docket No.61382-734.601 end) and the 2ndnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 2nd nucleoside from the 5’ end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps); (2) the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 3rdnucleoside from the 5’ end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps); (3) the 21st nucleoside from the 5’ end or the 3rd nucleoside from the 3’ end (e.g., the 3’-carbon atom of the 21st nucleoside from the 5’ end or the 3’-carbon atom of the 3rdnucleoside from the 3’end) and the 22th nucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22th nucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); and (4) the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2n nucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps).

[0218] In some instances, the stereochemically enriched phosphorothioate covalently bonded to (1) the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1st nucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 2nd nucleoside from the 5’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); (2) the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 3rdnucleoside from the 5’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); (3) the 21st nucleoside from the 5’ end or the 3rd nucleoside from the 3’ end (e.g., the 3'-carbon atom of the 21st nucleoside from the 5’ end or the 3’-carbon atom of the 3rdnucleoside from the 3’end) and the 22th nucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22th nucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); and (4) the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ 73WSGR Docket No.61382-734.601 end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps).

[0219] In some instances, the stereochemically enriched phosphorothioate covalently bonded to (1) the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1st nucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 2nd nucleoside from the 5’ end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps); (2) the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 3rdnucleoside from the 5’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); (3) the 21st nucleoside from the 5’ end or the 3rd nucleoside from the 3’ end (e.g., the 3'-carbon atom of the 21st nucleoside from the 5’ end or the 3’-carbon atom of the 3rd nucleoside from the 3’ end) and the 22th nucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22th nucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); and (4) the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps).

[0220] In some instances, the stereochemically enriched phosphorothioate covalently bonded to (1) the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1st nucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 2nd nucleoside from the 5’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); (2) the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 3rdnucleoside from the 5’ end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps); (3) the 21st nucleoside from the 5’ end or the 3rd nucleoside from the 3’ end (e.g., the 3'-carbon atom of the 21st nucleoside from the 5’ end or the 3’-carbon atom of the 3rd nucleoside from the 3’ end) and the 22th nucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22th nucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); and (4) the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 74WSGR Docket No.61382-734.601 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps).

[0221] In some instances, the stereochemically enriched phosphorothioate covalently bonded to (1) the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1st nucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 2nd nucleoside from the 5’ end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps); (2) the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 3rdnucleoside from the 5’ end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps); (3) the 21st nucleoside from the 5’ end or the 3rd nucleoside from the 3’ end (e.g., the 3'-carbon atom of the 21st nucleoside from the 5’ end or the 3’-carbon atom of the 3rd nucleoside from the 3’ end) and the 22th nucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22th nucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); and (4) the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps).

[0222] In some instances, the stereochemically enriched phosphorothioate covalently bonded to (1) the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1st nucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 2nd nucleoside from the 5’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); (2) the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 3rdnucleoside from the 5’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); (3) the 21st nucleoside from the 5’ end or the 3rd nucleoside from the 3’ end (e.g., the 3'-carbon atom of the 21st nucleoside from the 5’ end or the 3’-carbon atom of the 3rd nucleoside from the 3’ end) and the 22th nucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22th nucleoside from the 5’ 75WSGR Docket No.61382-734.601 end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); and (4) the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps).

[0223] In some instances, the stereochemically enriched phosphorothioate covalently bonded to (1) the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1st nucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 2nd nucleoside from the 5’ end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps); (2) the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 3rdnucleoside from the 5’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); (3) the 21st nucleoside from the 5’ end or the 3rd nucleoside from the 3’ end (e.g., the 3'-carbon atom of the 21st nucleoside from the 5’ end or the 3’-carbon atom of the 3rd nucleoside from the 3’ end) and the 22th nucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22th nucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); and (4) the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps).

[0224] In some instances, the stereochemically enriched phosphorothioate covalently bonded to (1) the 1stnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 1st nucleoside from the 5’ end) and the 2ndnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 2nd nucleoside from the 5’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); (2) the 2ndnucleoside from the 5’ end (e.g., the 3'-carbon atom of the 2ndnucleoside from the 5’ end) and the 3rdnucleoside from the 5’ end (e.g., the 5'-carbon atom of the 3rdnucleoside from the 5’ end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps); (3) the 21st nucleoside from the 5’ end or the 3rd 76WSGR Docket No.61382-734.601 nucleoside from the 3’ end (e.g., the 3'-carbon atom of the 21st nucleoside from the 5’ end or the 3’-carbon atom of the 3rd nucleoside from the 3’ end) and the 22th nucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 22th nucleoside from the 5’ end or the 5’-carbon atom of the 2ndnucleoside from the 3’ end) of the guide strand is 3'phosphorothioate, Sp diastereomer (SP-enriched phosphorothioate or Sps); and (4) the 22thnucleoside from the 5’ end or the 2ndnucleoside from the 3’ end (e.g., the 3'-carbon atom of the 22thnucleoside from the 5’ end or the 3’-carbon atom of the 2ndnucleoside from the 3’ end) and the 23rdnucleoside from the 5’ end or the 1stnucleoside from the 3’ end (e.g., the 5'-carbon atom of the 23rdnucleoside from the 5’ end or the 5’-carbon atom of the 1stnucleoside from the 3’ end) of the guide strand is 3’phosphorothioate, Rp diastereomer (RP-enriched phosphorothioate or Rps).

[0225] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and where the last two “n” on the 3’ end comprises 2’-deoxythymidine- 3’-phosphate (dT).

[0226] In some instances, the guide strand comprises a nucleic acid sequence comprising 21 nucleotides. In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage.

[0227] In some aspects, described herein is a specific modification pattern, wherein the guide strand comprises 5’- nssNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, where “Nf” stands for a 2’- fluoro modified nucleotide, “n” stands for a 2’-O-methyl modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate. In some instances, the guide strand comprises 5’- nssNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, where “Nf” stands for a 2’-fluoro modified nucleotide, “n” stands for a 2’-O-methyl modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate, and the first “n” from the 5’ end of the guide strand comprises 2’- deoxythymidine-3’-phosphate (dT).

[0228] In some instances, the guide strand comprises 5’- nsNfsnnnnNfnnssnnNfnNfnNfnnnnnsnsn-3’, where “Nf” stands for a 2’-fluoro modified nucleotide, “n” stands for a 2’-O-methyl modified nucleotide, “s” stands for phosphorothioate 77WSGR Docket No.61382-734.601 internucleotide linkage, and “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate.

[0229] In some instances, the guide strand comprises 5’- nsNfsnnnnNfnnnnssNfnNfnNfnnnnnsnsn-3’, where “Nf” stands for a 2’-fluoro modified nucleotide, “n” stands for a 2’-O-methyl modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate.

[0230] In some instances, the guide strand comprises 5’- nsNfsnnnnNfnnnnsNfnNfnNfnnnnnsnsn-3’, where “Nf” stands for a 2’-fluoro modified nucleotide, “n” stands for a 2’-O-methyl modified nucleotide, and “s” stands for phosphorothioate internucleotide linkage.

[0231] In some instances, the guide strand comprises 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn -3’. In some instances, the guide strand comprises 5’- nsNfsnnnnNfnnnnssNfnNfnNfnnnnnsnsn -3’. In some instances, the guide strand comprises 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn- 3’. In some instances, the guide strand comprises 5’- n(Rps)Nf(Rps)nnnnNfnnnnNfnNfnNfnnnnn(Sps)n(Sps)n-3’. In some instances, the guide strand comprises 5’- n(Rps)Nf(Rps)nnnnNfnnnnssNfnNfnNfnnnnn(Sps)n(Sps)n-3’. In some instances, the guide strand comprises 5’- n(Rps)Nf(Rps)nnnnNfnnnnNfnNfnNfnnnnn(Sps)n(Rps)n-3’. In some instances, the guide strand comprises 5’- nsNfsnnnnNfnnnnssNfnNfnNfnnnnnsnsn-3’. In some instances, the guide strand comprises 5’- nsNfsnnnnNfnnnnsNfnNfnNfnnnnnsnsn-3’. In the modification patterns described above, “Nf” stands for a 2’-fluoro modified nucleotide, “n” stands for a 2’-O-methyl modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate, "(Rps)" stands for 3'-phosphorothioate, Rp diastereomer, and "(Sps)" stands for 3' phosphorothioate, Sp diastereomer.

[0232] In some instances, the passenger strand comprises 5’- nsnnnnnNfnNfnNfnnnnnnnnnns(invAb)-3’. In some instances, the passenger strand comprises 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’. In some instances, the passenger strand comprises 5’- nsnnnnnNfnNfnNfnnnnnnnnnn-3’. In some instances, the passenger strand comprises 5’- nssnsnnnnNfnNfnNfnnnnnnnnnn-3’. In the modification patterns described above, “Nf” stands for a 2’-fluoro modified nucleotide, “n” stands for a 2’-O-methyl modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate, and “(invAb)” stands for an inverted abasic deoxyribonucleotide. 78WSGR Docket No.61382-734.601

[0233] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnnnnnnnn-invdN-invdN -3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, wherein “n” stands for a 2’-O-methyl modified nucleotide, and “invdN” stands for an inverted deoxy-nucleotide. In some instances, the invdN is an inverted deoxyl-thymine. In some aspects, the linker conjugated with one or more targeting moieties as shown in Formula (IV’’) or (IV’’’) is added to the first nucleic acid on the 5’ end. In some aspects, the linker conjugated with one or more GalNAc as shown in Formula (V’’) or (V’’’) is added to the first nucleic acid on the 5’ end. In some aspects, the modification pattern comprises one or more phosphorothioate internucleotide linkages. In some aspects, the modification pattern comprises an inverted deoxy-nucleotide, e.g., Formula (VII). In some aspects, the 5’ end modification or the 3’ end modification known in the art is applied to the one or more inverted nucleotides, as shown below:wherein R is a moiety that corresponds to the sugar modification described herein, in some instances, R is -O-methyl; wherein R’ is thymine, abasic, or others; wherein A is -O or -S; and wherein A’ is -O or -S.

[0234] In some aspects, described herein is a specific modification pattern, wherein the passenger strand comprises 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for a 2’-O- methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “s” stands for phosphorothioate internucleotide linkage.

[0235] In some instances, the first nucleotide on the 5’ end of the passenger strand comprises an inverted abasic deoxyribonucleotide (invAb). In some instances, the passenger strand comprises a nucleic acid sequence of 5’-(invAb)snsnnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “(invAb)” stands for an inverted abasic deoxyribonucleotide. 79WSGR Docket No.61382-734.601

[0236] In some instances, the last nucleotide on the 3’ end of the passenger strand comprises an inverted abasic deoxyribonucleotide (invAb). In some instances, the passenger strand comprises a nucleic acid sequence of 5’-nsnnnnnNfnNfnNfnnnnnnnnnns(invAb)-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “(invAb)” stands for an inverted abasic deoxyribonucleotide.

[0237] In some instances, the first nucleotide on the 5’ end of the passenger strand and the last nucleotide on the 3’ end of the passenger strand comprise an inverted abasic deoxyribonucleotide (invAb). In some instances, the passenger strand comprises a nucleic acid sequence of 5’-(invAb)snnnnnnNfnNfnNfnnnnnnnnnns(invAb)-3’, where “n” stands for a 2’-O- methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “(invAb)” stands for an inverted abasic deoxyribonucleotide. In some instances, the passenger strand comprises a nucleic acid sequence of 5’-(invAb)snnnnnnnnNfnNfnNfnnnnnnnns(invAb)-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “(invAb)” stands for an inverted abasic deoxyribonucleotide.

[0238] In some aspects, the polynucleic acid molecule provided herein comprises a passenger strand comprising a nucleic acid sequence of SEQ ID NOs: 21-33, and / or a guide strand comprising a nucleic acid sequence of SEQ ID NOs: 1-18 and 160, and wherein the passenger strand is modified in Type VI modification pattern specified in Table 17 or as described in the preceding paragraph. In other aspects, the polynucleic acid molecule provided herein comprises a passenger strand comprising a nucleic acid sequence of SEQ ID NOs: 21-22, and 25-27, and / or a guide strand comprising a nucleic acid sequence of SEQ ID NOs: 1-2, 7, and 10-11, and wherein the passenger strand is modified in Type VI modification pattern specified in Table 17 or as described in the preceding paragraph.

[0239] Described herein is a polynucleic acid molecule, whose passenger strand comprises a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from SEQ ID NOs: 81-107, 143-147, 153-157, and 165-166. Described herein is a polynucleic acid molecule, whose passenger strand comprises a nucleic acid sequence that is at least 85% identical to a nucleic acid sequence selected from SEQ ID NOs: 81-107, 143-147, 153-157, and 165-166. Described herein is a polynucleic acid molecule, whose passenger strand comprises a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 81-107, 143-147, 153-157, and 165-166. Described herein is a polynucleic acid molecule, whose passenger strand comprises a nucleic acid sequence that is at least 95% 80WSGR Docket No.61382-734.601 identical to a nucleic acid sequence selected from SEQ ID NOs: 81-107, 143-147, 153-157, and 165-166.

[0240] Described herein is a polynucleic acid molecule, which guide strand comprises a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from SEQ ID NOs: 34-80, 110-142, 148-152, and 161-164. Described herein is a polynucleic acid molecule, which guide strand comprises a nucleic acid sequence that is at least 85% identical to a nucleic acid sequence selected from SEQ ID NOs: 34-80, 110-142, 148-152, and 161-164. Described herein is a polynucleic acid molecule, which guide strand comprises a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 34-80, 110-142, 148-152, and 161-164. Described herein is a polynucleic acid molecule, which guide strand comprises a nucleic acid sequence that is at least 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 34-80, 110-142, 148-152, and 161-164.

[0241] In one aspect, described herein is a specific modification motif or pattern for the double- stranded inhibitory polynucleic acid molecule comprising a passenger strand and a guide strand.

[0242] In some aspects, the guide strand comprises a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid- adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T- NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4), 5'(E) vinylphosphonate-2-O-methyluridine-3'phosphate (vpu), 2'-methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), or 2'-O-methylinosine-3'-phosphate (i). In some instance, the nucleotide analogue is selected from a group consisting of the nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S- isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’- phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 5’(E) vinylphosphonate- 2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4), 5'(E) vinylphosphonate-2-O- methyluridine-3'phosphate (vpu), 2'-methoxyethyladenosine-3'phosphate (A6), 2'- methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), and 2'-O- 81WSGR Docket No.61382-734.601 methylinosine-3'-phosphate (i). In some instances, the nucleotide analogue is selected from a group consisting of the nucleotide analogue selected from acyclic L-threoninol nucleic acid- thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’- phosphate (vpt4), 5'(E) vinylphosphonate-2-O-methyluridine-3'phosphate (vpu), 2'- methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'- methoxyethylguanosine-3'-phosphate (G6), and 2'-O-methylinosine-3'-phosphate (i).

[0243] In some aspects, the guide strand comprises a nucleotide analogue at one of positions 2-8 from the 5’ end, and further comprises a 2’-fluoro modified nucleotide at position 2 from the 5’ end. In some aspects, the guide strand comprises a nucleotide analogue at one of positions 2-8 from the 5’ end, and further comprises a 2’-fluoro modified nucleotide at position 7 from the 5’ end. In some aspects, the guide strand comprises a nucleotide analogue at one of positions 2-8 from the 5’ end, and further comprises 2’-fluoro modified nucleotides at position 12 from the 5’ end. In some aspects, the guide strand comprises a nucleotide analogue at one of positions 2-8 from the 5’ end, and further comprises a 2’-fluoro modified nucleotide at position 14 from the 5’ end. In some aspects, the guide strand comprises a nucleotide analogue at one of positions 2-8 from the 5’ end, and further comprises a 2’-fluoro modified nucleotide at position 16 from the 5’ end. In other aspects, the guide strand comprises a nucleotide analogue at one of positions 2-8 from the 5’ end, and further comprises a 2’-fluoro modified nucleotide at positions 2, 12, 14, 16, or combination thereof from the 5’ end. In other aspects, the guide strand comprises a nucleotide analogue at one of positions 2-8 from the 5’ end, and further comprises a 2’-fluoro modified nucleotide at positions 2, 7, 12, 14, 16, or combination thereof from the 5’ end. In other aspects, the guide strand comprises a nucleotide analogue at one of positions 2-8 from the 5’ end, and further comprises a 2’-fluoro modified nucleotide at at least three of positions 2, 12, 14, and 16 from the 5’ end. In other aspects, the guide strand comprises a nucleotide analogue at one of positions 2-8 from the 5’ end, and further comprises a 2’-fluoro modified nucleotide at at least three of positions 2, 7, 12, 14, and 16 from the 5’ end.

[0244] In some instances, the nucleotide analogue is located at position 6 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 6 from the 5’ end of the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at at least one of positions 2, 7, 12, 14, and 16 from the 5’ end. In some instances, the nucleotide analogue is located at position 6 from the 5’ end of the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at at least two of positions 2, 7, 12, 14, and 16 from the 5’ end. In some instances, the nucleotide analogue is located at position 6 from the 5’ end of 82WSGR Docket No.61382-734.601 the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at at least three of positions 2, 7, 12, 14, and 16 from the 5’ end. In some instances, the nucleotide analogue is located at position 6 from the 5’ end of the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at positions 2, 7, 12, 14, and 16 from the 5’ end.

[0245] In some instances, the nucleotide analogue is located at position 7 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 7 from the 5’ end of the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at at least one of positions 2, 6, 8, 12, 14, and 16 from the 5’ end. In some instances, the nucleotide analogue is located at position 7 from the 5’ end of the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at at least one of positions 2, 12, 14, and 16 from the 5’ end. In some instances, the nucleotide analogue is located at position 7 from the 5’ end of the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at at least two of positions 2, 12, 14, and 16 from the 5’ end. In some instances, the nucleotide analogue is located at position 7 from the 5’ end of the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at at least three of positions 2, 12, 14, and 16 from the 5’ end. In some instances, the nucleotide analogue is located at position 7 from the 5’ end of the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at positions 2, 12, 14, and 16 from the 5’ end. In some instances, the nucleotide analogue is located at position 7 from the 5’ end of the guide strand, and the guide strand comprises 2’-fluoro modified nucleotides at positions 2, 6, 12, 14, and 16 from the 5’ end. In some instances, the nucleotide analogue is located at position 7 from the 5’ end of the guide strand, and the guide strand comprises 2’-fluoro modified nucleotides at positions 2, 8, 12, 14, and 16 from the 5’ end.

[0246] In some instances, the nucleotide analogue is located at position 8 from the 5’ end of the guide strand. In some instances, the nucleotide analogue is located at position 8 from the 5’ end of the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at at least one of positions 2, 7, 12, 14, and 16 from the 5’ end. In some instances, the nucleotide analogue is located at position 8 from the 5’ end of the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at at least two of positions 2, 7, 12, 14, and 16 from the 5’ end. In some instances, the nucleotide analogue is located at position 8 from the 5’ end of the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at at least three of positions 2, 7, 12, 14, and 16 from the 5’ end. In some instances, the nucleotide analogue is located at position 8 from the 5’ end of the guide strand, and the guide strand further comprises 2’-fluoro modified nucleotides at positions 2, 7, 12, 14, and 16 from the 5’ end.

[0247] In some instances, the nucleotides of the guide strand comprises DNA or RNA. As described herein, in some instances, the DNA nucleotide comprises an unmodified DNA 83WSGR Docket No.61382-734.601 comprising: an unmodified adenine nucleotide (A), an unmodified guanine nucleotide (G), an unmodified thymine nucleotide (T), or an unmodified cytosine nucleotide (C). As described herein, in some instances, the RNA comprises an unmodified RNA comprising: an unmodified adenine nucleotide (A), an unmodified guanine nucleotide (G), an unmodified uracil nucleotide (U), or an unmodified cytosine nucleotide (C).

[0248] In some instances, the nucleotides of the guide strand comprises the DNA, RNA, nucleotide analogue, 2’-fluoro modified nucleotide, or 2’-O-alkyl modified nucleotide. In some instances, the 2’-O-alkly modified nucleotide comprises 2’-O-methyl modified nucleotides. In some instances, the nucleotides of the guide strand that are not the nucleotide analogue or 2’- fluoro modified nucleotide are selected from DNA nucleotide, RNA nucleotide, and 2’-O-alkly modified nucleotide. In some instances, the nucleotides of the guide strand that are not the nucleotide analogue or 2’-fluoro modified nucleotide are 2’-O-methyl modified nucleotides.

[0249] In some instances, the guide strand comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight phosphorothioate modified internucleotide linkages. In some instances, the guide strand comprises at least two, at least three, or at least four phosphorothioate modified internucleotide linkages. In some instances, the guide strand comprises at most one, at most two, at most three, at most four, at most five, at most six, at most seven, or at most eight phosphorothioate modified internucleotide linkages. In some instances, the guide strand comprises one, two, three, four, five, six, seven, or eight phosphorothioate modified internucleotide linkages. In some instances, the guide strand comprises from 1 to 8, from 2 to 8, from 3 to 8, from 4 to 8, from 5 to 8, or from 6 to 8 phosphorothioate modified internucleotide linkages. In some instances, the guide strand comprises from 1 to 4, from 2 to 4, or from 3 to 4 phosphorothioate modified internucleotide linkages.

[0250] In some instances, the guide strand comprises one phosphorothioate modified internucleotide linkages at the 5’ end and one phosphorothioate modified internucleotide linkages at the 3’ end. In some instances, the guide strand comprises two phosphorothioate modified internucleotide linkages at the 5’ end and two phosphorothioate modified internucleotide linkages at the 3’ end. In some instances, the guide strand comprises three phosphorothioate modified internucleotide linkages at the 5’ end and three phosphorothioate modified internucleotide linkages at the 3’ end. In some instances, the guide strand comprises four phosphorothioate modified internucleotide linkages at the 5’ end and four phosphorothioate modified internucleotide linkages at the 3’ end.

[0251] In some instances, the guide strand comprises 2’-O-methyl modified nucleotide, 2’- fluoro modified nucleotide, and / or nucleotide analogue described herein. In some instances, the 84WSGR Docket No.61382-734.601 guide strand comprises a nucleic acid sequence of 5’- nNfnnnXNfnnnnNfnNfnNfnnnnnnn -3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S- isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’- phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 2'-methoxyethyladenosine- 3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'- phosphate (G6), and 2'-O-methylinosine-3'-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’-nNfnnnXNfnnnnNfnNfnNfnnnnnnn -3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid- thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S- isomer (Tgn).

[0252] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnnXnnnnNfnNfnNfnnnnnnn-3, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3’-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3’phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1’,2’-Dideoxyribose-3’- phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2- thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’-phosphate (U3f), 2-amino-2’-O- methyladenosine-3’-phosphate (a1), 2'-methoxyethyladenosine-3'phosphate (A6), 2'- methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), and 2'-O- methylinosine-3'-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’-nNfnnnnXnnnnNfnNfnNfnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T- T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol 85WSGR Docket No.61382-734.601 abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn).

[0253] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnNfXnnnnNfnNfnNfnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2- thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’-phosphate (U3f), 2-amino-2’-O- methyladenosine-3’-phosphate (a1), 2'-methoxyethyladenosine-3'phosphate (A6), 2'- methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), and 2'-O- methylinosine-3'-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’-nNfnnnNfXnnnnNfnNfnNfnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T- T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn).

[0254] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnnXNfnnnNfnNfnNfnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L- threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2- thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’-phosphate (U3f), 2-amino-2’-O- methyladenosine-3’-phosphate (a1), 2'-methoxyethyladenosine-3'phosphate (A6), 2'- methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), and 2'-O- methylinosine-3'-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’-nNfnnnnXNfnnnNfnNfnNfnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl 86WSGR Docket No.61382-734.601 modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T- T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn).

[0255] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnnNfXnnnNfnNfnNfnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2- thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’-phosphate (U3f), 2-amino-2’-O- methyladenosine-3’-phosphate (a1), 2'-methoxyethyladenosine-3'phosphate (A6), 2'- methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), and 2'-O- methylinosine-3'-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’-nNfnnnnNfXnnnNfnNfnNfnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T- T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn).

[0256] In some instances, the guide strand comprises 2’-O-methyl modified nucleotide, 2’- fluoro modified nucleotide, nucleotide analogue described herein, or phosphorothioate internucleotide linkage. In some instances, the guide strand comprises one or more phosphorothioate modified internucleotide linkages at the 5’ end and one or more phosphorothioate modified internucleotide linkages at the 3’ end. In some instances, the guide strand comprises two phosphorothioate modified internucleotide linkages at the 5’ end and two phosphorothioate modified internucleotide linkages at the 3’ end.

[0257] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnXNfnnnnNfnNfnNfnnnnnsnsn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate 87WSGR Docket No.61382-734.601 internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 2'- methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'- methoxyethylguanosine-3'-phosphate (G6), and 2'-O-methylinosine-3'-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnXNfnnnnNfnNfnNfnnnnnsnsn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn).

[0258] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnXnnnnNfnNfnNfnnnnnsnsn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 2'- methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'- methoxyethylguanosine-3'-phosphate (G6), and 2'-O-methylinosine-3'-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnXnnnnNfnNfnNfnnnnnsnsn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 88WSGR Docket No.61382-734.601 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn).

[0259] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfXnnnNfnNfnNfnnnnnsnsn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 2'- methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'- methoxyethylguanosine-3'-phosphate (G6), and 2'-O-methylinosine-3'-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfXnnnNfnNfnNfnnnnnsnsn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn).

[0260] In some instances, the nucleotide analogue is located at at least one of positions 2-12 from the 5’ end of the guide strand, and the nucleotide analogue comprises 2’-O-methyl-2- thiouridine-3’-phosphate (u3) or 2’-fluoro-2-thiouridine-3’-phosphate (U3f). In some instances, the nucleotide analogue that is located at position 12 of the 5’ end of the guide strand, and the nucleotide analogue consists of 2’-fluoro-2-thiouridine-3’-phosphate (U3f). In some instances, the nucleotide analogue is located at position 3 of the 5’ end of the guide strand, and the nucleotide analogue comprises 2’-O-methyl-2-thiouridine-3’-phosphate (u3). In some instances, the nucleotide analogue is located at position 3 of the 5’ end of the guide strand, and the nucleotide analogue consists of 2’-O-methyl-2-thiouridine-3’-phosphate (u3). 89WSGR Docket No.61382-734.601

[0261] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnnNfXnnnF’nNfnNfnnnnnnn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT- T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), and “F’ ” stands for 2’-fluoro-2-thiouridine-3’-phosphate (U3f). In some instances, the guide strand comprises a nucleic acid sequence of 5’-nsNfsnnnnNfXnnnF’nNfnNfnnnnnsnsn-3’ , where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT- T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), and “F’ ” stands for 2’-fluoro-2-thiouridine-3’-phosphate (U3f).

[0262] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfXnnnNfnnnnNfnNfnNfnnnnnnn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue, e.g., 2’-O-methyl-2-thiouridine-3’-phosphate (u3). In some instances, the guide strand comprises a nucleic acid sequence of 5’-nsNfsXnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., 2’-O-methyl-2-thiouridine-3’-phosphate (u3).

[0263] In some instances, the guide strand comprises 2’-O-methyl modified nucleotide, 2’- fluoro modified nucleotide, and / or nucleotide analogue described herein. In some instances, the guide strand comprises the nucleotide analogue at location 1 from the 5’ end. In some instances, the guide strand comprises 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’- phosphate (vpt4) at location 1 from the 5’ end. In some instances, the guide strand comprises 5'(E) vinylphosphonate-2-O-methyluridine-3'phosphate (vpu) at location 1 from the 5’ end.

[0264] In some instances, the guide strand comprises a nucleic acid sequence of 5’- XsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 90WSGR Docket No.61382-734.601 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4), 5'(E) vinylphosphonate-2-O-methyluridine-3'phosphate (vpu), 2'-methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’-XsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4) and 5’(E) vinylphosphonate-2-O-methyluridine-3’phosphate (vpu).

[0265] In some instances, the guide strand comprises a nucleic acid sequence of 5’- XsNfsnnnnNfnnnnNfnNfnNfnsnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4), 5'(E) vinylphosphonate-2-O-methyluridine-3'phosphate (vpu), 2'-methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’-XsNfsnnnnNfnnnnNfnNfnNfnsnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., 5’(E) vinylphosphonate-2-O-methyluridine-3’phosphate (vpu).

[0266] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnXnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate 91WSGR Docket No.61382-734.601 internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 2'- methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'- methoxyethylguanosine-3'-phosphate (G6), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnXnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A). In some instances, the guide strand comprises acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A) at location 14 from the 5’ end.

[0267] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnXnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 2'- methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'- methoxyethylguanosine-3'-phosphate (G6), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnXnnnsnsn -3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., 2’-O-methylinosine-3’- phosphate (i).

[0268] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnXnnNfnNfnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate 92WSGR Docket No.61382-734.601 internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 2'- methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'- methoxyethylguanosine-3'-phosphate (G6), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnXnnNfnNfnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., 2'- methoxyethyladenosine-3'phosphate (A6) or 2'-methoxyethylguanosine-3'-phosphate (G6).

[0269] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnXnNfnNfnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 2'- methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'- methoxyethylguanosine-3'-phosphate (G6), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnXnNfnNfnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., 2'- methoxyethyladenosine-3'phosphate (A6).

[0270] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnXXnNfnNfnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- 93WSGR Docket No.61382-734.601 threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 2'- methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'- methoxyethylguanosine-3'-phosphate (G6), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnXXnNfnNfnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., 2'- methoxyethyladenosine-3'phosphate (A6).

[0271] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnXNfnNfnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 2'- methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'- methoxyethylguanosine-3'-phosphate (G6), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnXNfnNfnNfnnnnnsnsn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., 2'-methoxyethyluridine- 3'-phosphate (U6).

[0272] In some instances, the passenger strand comprises acyclic L-threoninol nucleic acid- thymine-3'-phosphate (T-T) at location 8 from the 5’ end. In some instances, the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfXNfnNfnnnnnnnnnn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide 94WSGR Docket No.61382-734.601 analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L- threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2- thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’-phosphate (U3f), 2-amino-2’-O- methyladenosine-3’-phosphate (a1), 2'-methoxyethyladenosine-3'phosphate (A6), 2'- methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), and 2’- O-methylinosine-3’-phosphate (i). In some instances, the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfXNfnNfnnnnnnnnnn-3’, where “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T).

[0273] In some aspects, described herein is a specific modification motif of pattern for the double-stranded polynucleic acid molecule comprising a passenger strand and a guide strand. In some aspects, described herein is a specific modification pattern of the passenger strand.

[0274] In some instances, the passenger strand comprises one or more nucleotide analogue. In some instances, the passenger strand comprises one or more nucleotide analogue at locations opposite to the seed region of the guide strand. In some instances, the passenger strand comprises one or more nucleotide analogue at locations 12-22 from the 5’ end. In some instances, the passenger strand comprises one or more nucleotide analogue at locations 2-10 from the 3’ end. In some instances, the passenger strand comprises one or more 2-amino-2’-O- methyladenosine-3’-phosphate (a1). In some instances, the passenger strand comprises 2-amino- 2’-O-methyladenosine-3’-phosphate (a1) at location 11, 12, 13, 14, or 15 from the 5’ end. In some instances, the passenger strand comprises 2-amino-2’-O-methyladenosine-3’-phosphate (a1) at location 13 from the 5’ end. In some instances, the passenger strand comprises 2-amino- 2’-O-methyladenosine-3’-phosphate (a1) at location 14 from the 5’ end. In some instances, the passenger strand comprises 2-amino-2’-O-methyladenosine-3’-phosphate (a1) at location 15 from the 5’ end.

[0275] In some instances, the passenger strand comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight 2’-fluoro modified nucleotides. In some instances, the passenger strand comprises at least two, at least three, or at least four 2’-fluoro modified nucleotides. In some instances, the passenger strand comprises at most one, at most two, at most three, at most four, at most five, at most six, at most seven, or at most eight 2’-fluoro modified nucleotides. In some instances, the passenger strand comprises 95WSGR Docket No.61382-734.601 from one to eight, from two to eight, from three to eight, from four to eight, from five to eight, from six to eight, or from seven to eight 2’-fluoro modified nucleotides. In some instances, the passenger strand comprises one, two, three, four, five, six, seven, or eight 2’-fluoro modified nucleotides. In some instances, the passenger strand comprises one 2’-fluoro modified nucleotides. In some instances, the passenger strand comprises two 2’-fluoro modified nucleotides. In some instances, the passenger strand comprises three 2’-fluoro modified nucleotides. In some instances, the passenger strand comprises four 2’-fluoro modified nucleotides. In some instances, the passenger strand comprises five 2’-fluoro modified nucleotides.

[0276] In some aspects, the passenger strand comprises a 2’-fluoro modified nucleotide at position 7 from the 5’ end. In some aspects, the passenger strand comprises a 2’-fluoro modified nucleotide at position 9 from the 5’ end. In some aspects, the passenger strand comprises 2’- fluoro modified nucleotides at position 11 from the 5’ end. In some aspects, the passenger strand comprises a 2’-fluoro modified nucleotide at at least one of positions 7, 9, and 11 from the 5’ end. In some aspects, the passenger strand comprises a 2’-fluoro modified nucleotide at positions 7, 9, 11, or combination thereof from the 5’ end. In some aspects, the passenger strand comprises a 2’-fluoro modified nucleotide at positions 7, 9, and 11 from the 5’ end.

[0277] In some instances, the nucleotides of the passenger strand comprises DNA nucleotide or RNA nucleotide. As described herein, in some instances, the DNA nucleotide comprises an unmodified DNA nucleotide comprising: an unmodified adenine nucleotide (A), an unmodified guanine nucleotide (G), an unmodified thymine nucleotide (T), or an unmodified cytosine nucleotide (C). As described herein, in some instances, the RNA nucleotide comprises an unmodified RNA nucleotide comprising: an unmodified adenine nucleotide (A), an unmodified guanine nucleotide (G), an unmodified uracil nucleotide (U), or an unmodified cytosine nucleotide (C).

[0278] In some instances, the nucleotides of the passenger strand comprises the DNA nucleotide, RNA nucleotide, nucleotide analogue, 2’-fluoro modified nucleotide, or 2’-O-alkyl modified nucleotide. In some instances, the 2’-O-alkly modified nucleotide comprises 2’-O- methyl modified nucleotides. In some instances, the nucleotides of the passenger strand that are not the nucleotide analogue or 2’-fluoro modified nucleotide are selected from 2′-O-alkyl modified nucleotide, 2′-alkoxy modified nucleotide, 2′-alkyl modified nucleotide, 2′-halo modified nucleotide, DNA nucleotide, RNA nucleotide, ENA, BNA, LNA, and UNA. In some instances, the nucleotides in the passenger strand that are not 2’-fluoro modified nucleotide are 2’-O-methyl modified nucleotides. 96WSGR Docket No.61382-734.601

[0279] In some instances, the passenger strand comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight phosphorothioate modified internucleotide linkages. In some instances, the passenger strand comprises at least two, at least three, or at least four phosphorothioate modified internucleotide linkages. In some instances, the passenger strand comprises at least one phosphorothioate modified internucleotide linkage. In some instances, the passenger strand comprises at most one, at most two, at most three, at most four, at most five, at most six, at most seven, or at most eight phosphorothioate modified internucleotide linkages. In some instances, the passenger strand comprises one, two, three, four, five, six, seven, or eight phosphorothioate modified internucleotide linkages. In some instances, the passenger strand comprises from 1 to 8, from 2 to 8, from 3 to 8, from 4 to 8, from 5 to 8, or from 6 to 8 phosphorothioate modified internucleotide linkages. In some instances, the passenger strand comprises from 1 to 4, from 2 to 4, or from 3 to 4 phosphorothioate modified internucleotide linkages.

[0280] In some instances, the passenger strand comprises at least one, at least two, at least three, or at least four phosphorothioate modified internucleotide linkage at the 5’ end. In some instances, the passenger strand comprises at least one phosphorothioate modified internucleotide linkage at the 5’ end. In some instances, the passenger strand comprises at least one, at least two, at least three, or at least four phosphorothioate modified internucleotide linkage at the 3’ end. In some instances, the passenger strand comprises at least one phosphorothioate modified internucleotide linkage at the 3’ end.

[0281] In some instances, the passenger strand comprises one phosphorothioate modified internucleotide linkage at the 5’ end. In some instances, the passenger strand comprises two phosphorothioate modified internucleotide linkage at the 5’ end. In some instances, the passenger strand comprises three phosphorothioate modified internucleotide linkage at the 5’ end. In some instances, the passenger strand comprises four phosphorothioate modified internucleotide linkage at the 5’ end.

[0282] In some instances, the passenger strand comprises one phosphorothioate modified internucleotide linkage at the 3’ end. In some instances, the passenger strand comprises two phosphorothioate modified internucleotide linkage at the 3’ end. In some instances, the passenger strand comprises three phosphorothioate modified internucleotide linkage at the 3’ end. In some instances, the passenger strand comprises four phosphorothioate modified internucleotide linkage at the 3’ end.

[0283] In some instances, the passenger strand comprises one phosphorothioate modified internucleotide linkages at the 5 end and one phosphorothioate modified internucleotide linkages at the 3’ end. In some instances, the passenger strand comprises two phosphorothioate modified 97WSGR Docket No.61382-734.601 internucleotide linkages at the 5’ end and two phosphorothioate modified internucleotide linkages at the 3’ end. In some instances, the passenger strand comprises three phosphorothioate modified internucleotide linkages at the 5’ end and three phosphorothioate modified internucleotide linkages at the 3’ end. In some instances, the passenger strand comprises four phosphorothioate modified internucleotide linkages at the 5’ end and four phosphorothioate modified internucleotide linkages at the 3’ end.

[0284] In some aspects, described herein is a specific modification pattern for a double-stranded polynucleic nucleic acid molecule comprising a passenger strand and a guide strand. In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnXNfnnnnNfnNfnNfnnnnnnn-3’, and the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnnnnnnnn-3’ , wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L- threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2- thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’-phosphate (U3f), or 2-amino-2’-O- methyladenosine-3’-phosphate (a1). In some instances, the guide strand comprises a nucleic acid sequence of 5’-nNfnnnXNfnnnnNfnNfnNfnnnnnnn-3’, and the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L- threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn).

[0285] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnXNfnnnnNfnNfnNfnnnnnnn-3’, and the passenger strand comprises 5’- nnnnnnNfnNfnNfnnX’nnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and 98WSGR Docket No.61382-734.601 thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), and “X’ ” stands for 2-amino-2’-O- methyladenosine-3’-phosphate (a1).

[0286] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnXNfnnnnNfnNfnNfnnnnnnn-3’ 5’-, and the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnX’nnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), and X’ is 2-amino-2’-O- methyladenosine-3’-phosphate (a1).

[0287] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnXNfnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), or 2-amino-2’-O-methyladenosine-3’-phosphate (a1). In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnXNfnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S- isomer (Tgn).

[0288] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnXNfnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises 5’- 99WSGR Docket No.61382-734.601 nsnsnnnnNfnNfnNfnnX’nnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3’-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’- phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3’phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1’,2’-Dideoxyribose-3’-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), and “X’ ” stands for 2-amino-2’-O-methyladenosine-3’- phosphate (a1).

[0289] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnXNfnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnX’nnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’- phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), and “X’ ” stands for 2-amino-2’-O-methyladenosine-3’- phosphate (a1).

[0290] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnnXnnnnNfnNfnNfnnnnnnn-3’, and the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L- threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2- thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’-phosphate (U3f), or 2-amino-2’-O- methyladenosine-3’-phosphate (a1). In some instances, the guide strand comprises a nucleic acid sequence of 5’-nNfnnnnXnnnnNfnNfnNfnnnnnnn-3’, and the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L- 100WSGR Docket No.61382-734.601 threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn).

[0291] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnnXnnnnNfnNfnNfnnnnnnn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnX’nnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’- phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), and “X’ ” stands for 2-amino-2’-O-methyladenosine-3’- phosphate (a1).

[0292] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnnXnnnnNfnNfnNfnnnnnnn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnX’nnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid- thymine-3’-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3’phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1’,2’-Dideoxyribose-3’-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S- isomer (Tgn), and “X’ ” stands for 2-amino-2’-O-methyladenosine-3’-phosphate (a1).

[0293] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnXnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L- threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2- 101WSGR Docket No.61382-734.601 thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’-phosphate (U3f), and 2-amino-2’-O- methyladenosine-3’-phosphate (a1). In some instances, the guide strand comprises a nucleic acid sequence of 5’-nsNfsnnnnXnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn).

[0294] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnXnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnX’nnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’- phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), and X’ is 2-amino-2’-O-methyladenosine-3’-phosphate (a1).

[0295] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnXnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnX’nnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’- phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), and X’ is 2-amino-2’-O-methyladenosine-3’-phosphate (a1).

[0296] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnnNfXnnnNfnNfnNfnnnnnnn-3’, and the passenger strand comprises 5’- 102WSGR Docket No.61382-734.601 nnnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L- threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2- thiouridine-3’-phosphate (u3), 2’-fluoro-2-thiouridine-3’-phosphate (U3f), or 2-amino-2’-O- methyladenosine-3’-phosphate (a1). In some instances, the guide strand comprises a nucleic acid sequence of 5’-nNfnnnnNfXnnnNfnNfnNfnnnnnnn-3’, and the passenger strand comprises 5’- nnnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L- threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'- phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn).

[0297] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnnNfXnnnNfnNfnNfnnnnnnn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnX’nnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’- phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), and “X’ ” stands for 2-amino-2’-O-methyladenosine-3’- phosphate (a1).

[0298] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nNfnnnnNfXnnnNfnNfnNfnnnnnnn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnX’nnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’- phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), 103WSGR Docket No.61382-734.601 acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), and “X’ ” stands for 2-amino-2’-O-methyladenosine-3’- phosphate (a1).

[0299] In some instances, wherein the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfXnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a is 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), and 2-amino-2’-O-methyladenosine-3’-phosphate (a1). In some instances, wherein the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfXnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a is 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn).

[0300] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfXnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnX’nnnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’- phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol 104WSGR Docket No.61382-734.601 nucleic acid (GNA) S-isomer (Tgn), and “X’ ” stands for 2-amino-2’-O-methyladenosine-3’- phosphate (a1).

[0301] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfXnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnX’nnnnnn-3’, wherein “n” stands for a 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “X” stands for a nucleotide analogue selected from acyclic L-threoninol nucleic acid- thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), and thymidine-glycol nucleic acid (GNA) S- isomer (Tgn), and “X’ ” stands for 2-amino-2’-O-methyladenosine-3’-phosphate (a1).

[0302] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “s” stands for phosphorothioate internucleotide linkage.

[0303] In some instances, the guide strand comprises a nucleic acid sequence of 5’- XsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4), 5'(E) vinylphosphonate-2-O-methyluridine-3'phosphate (vpu), 2'-methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- XsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” 105WSGR Docket No.61382-734.601 stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4) and 5’(E) vinylphosphonate-2-O-methyluridine-3’phosphate (vpu).

[0304] In some instances, the guide strand comprises a nucleic acid sequence of 5’- XsNfsnnnnNfnnnnNfnNfnNfnsnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4), 5'(E) vinylphosphonate-2-O-methyluridine-3'phosphate (vpu), 2'-methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'-methoxyethylguanosine-3'-phosphate (G6), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- XsNfsnnnnNfnnnnNfnNfnNfnsnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4) and 5’(E) vinylphosphonate-2-O-methyluridine-3’phosphate (vpu).

[0305] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnXnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 2'- 106WSGR Docket No.61382-734.601 methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'- methoxyethylguanosine-3'-phosphate (G6), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnXnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., acyclic L-threoninol nucleic acid-adenine-3’-phosphate (T-A).

[0306] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn -3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfXNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 2'- methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'- methoxyethylguanosine-3'-phosphate (G6), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn -3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfXNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., acyclic L-threoninol nucleic acid-thymine-3’-phosphate (T-T).

[0307] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnXnnnsnsn -3’, and the passenger strand comprises a nucleic acid sequence of 5’- nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue selected from acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid-adenine- 3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid- 107WSGR Docket No.61382-734.601 thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’-phosphate (u3), 2’-fluoro-2- thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine-3’-phosphate (a1), 2'- methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'-phosphate (U6), 2'- methoxyethylguanosine-3'-phosphate (G6), and 2’-O-methylinosine-3’-phosphate (i). In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnXnnnsnsn -3’, and the passenger strand comprises a nucleic acid sequence of 5’- nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., 2’-O-methylinosine-3’- phosphate (i).

[0308] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and the last two “n” on the 3’ end comprises 2’-deoxythymidine-3’- phosphate (dT).

[0309] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’- (invAb)snsnnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “(invAb)” stands for an inverted abasic deoxyribonucleotide.

[0310] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn -3’, and the passenger strand comprises a nucleic acid sequence of 5’- nsnnnnnNfnNfnNfnnnnnnnnnns(invAb)-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “(invAb)” stands for an inverted abasic deoxyribonucleotide.

[0311] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnssNfnNfnNfnnnnnsnsn -3’, and the passenger strand comprises a nucleic acid sequence of 5’- nsnnnnnNfnNfnNfnnnnnnnnnns(invAb)-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “(invAb)” stands for an inverted abasic 108WSGR Docket No.61382-734.601 deoxyribonucleotide, and “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate.

[0312] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn -3’, and the passenger strand comprises a nucleic acid sequence of 5’- (invAb)snnnnnnNfnNfnNfnnnnnnnnnns(invAb)-3’, where “n” stands for 2’-O- methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “(invAb)” stands for an inverted abasic deoxyribonucleotide.

[0313] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnsnsn -3’, and the passenger strand comprises a nucleic acid sequence of 5’- (invAb)snnnnnnNfnNfnNfnnnnnnnnnns(invAb)-3’, where “n” stands for 2’-O- methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “(invAb)” stands for an inverted abasic deoxyribonucleotide.

[0314] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nssNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate. In some instances, the guide strand comprises a nucleic acid sequence of 5’- nssNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate, and the first “n” from the 5’ end of the guide strand comprises 2’- deoxythymidine-3’-phosphate (dT).

[0315] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnXnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., 2'- methoxyethyladenosine-3'phosphate (A6) or 2'-methoxyethylguanosine-3'-phosphate (G6).

[0316] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnXnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified 109WSGR Docket No.61382-734.601 nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., 2'- methoxyethyladenosine-3'phosphate (A6).

[0317] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnXXnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., 2'- methoxyethyladenosine-3'phosphate (A6).

[0318] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnXNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “X” stands for a nucleotide analogue, e.g., 2'-methoxyethyluridine- 3'-phosphate (U6).

[0319] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnssnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate.

[0320] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “s” stands for phosphorothioate internucleotide linkage.

[0321] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “s” stands for phosphorothioate internucleotide linkage.

[0322] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nssnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate 110WSGR Docket No.61382-734.601 internucleotide linkage, and “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate.

[0323] In some instances, the guide strand comprises a nucleic acid sequence of 5’- n(Rps)Nf(Rps)nnnnNfnnnnNfnNfnNfnnnnn(Sps)n(Sps)n-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O- methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, "(Rps)" stands for 3'-phosphorothioate, Rp diastereomer, and "(Sps)" stands for 3' phosphorothioate, Sp diastereomer.

[0324] In some instances, the guide strand comprises a nucleic acid sequence of 5’- n(Rps)Nf(Rps)nnnnNfnnnnssNfnNfnNfnnnnn(Sps)n(Sps)n-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate, "(Rps)" stands for 3'-phosphorothioate, Rp diastereomer, and "(Sps)" stands for 3' phosphorothioate, Sp diastereomer.

[0325] In some instances, the guide strand comprises a nucleic acid sequence of 5’- n(Rps)Nf(Rps)nnnnNfnnnnNfnNfnNfnnnnn(Sps)n(Rps)n-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, "(Rps)" stands for 3'-phosphorothioate, Rp diastereomer, and "(Sps)" stands for 3' phosphorothioate, Sp diastereomer.

[0326] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnssNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate.

[0327] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnssNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate.

[0328] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnssNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid 111WSGR Docket No.61382-734.601 sequence of 5’-nssnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “ss” stands for phosphorodithioate internucleotide linkage, e.g., 3’ phosphorodithioate.

[0329] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnsNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnsnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “s” stands for phosphorothioate internucleotide linkage.

[0330] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnsNfnNfnNfnnnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-nsnnnnnNfnNfnNfnnnnnnnnnn-3’, where “n” stands for 2’-O-methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, and “s” stands for phosphorothioate internucleotide linkage.

[0331] In some instances, the guide strand comprises a nucleic acid sequence of 5’- nsNfsnnnnNfnnnnNfnNfnNfnnnsnsn-3’, and the passenger strand comprises a nucleic acid sequence of 5’-(invAb)snnnnnnnnNfnNfnNfnnnnnnnns(invAb)-3’, where “n” stands for 2’-O- methyl modified nucleotide, “Nf” stands for a 2’-fluoro modified nucleotide, “s” stands for phosphorothioate internucleotide linkage, and “(invAb)” stands for an inverted abasic deoxyribonucleotide.

[0332] A polynucleic acid molecule for modulating expression of AGT gene, wherein polynucleic acid molecule comprises a guide strand comprising the nucleotide sequence of usAfsuugcUfcaauUfuUfuGfcaggususc (SEQ ID NO: 34) and a passenger strand comprising the nucleotide sequence of ascscugcAfaAfaAfuugagcaaua (SEQ ID NO: 81), where smaller case “n” stands for 2’-O-methyl modified nucleotide, upper case followed with an “f” (i.e., “Nf”) stands for 2’-fluoro modified nucleotide, and “s” stands for 3'-phosphorothioate.

[0333] A polynucleic acid molecule for modulating expression of AGT gene, wherein polynucleic acid molecule comprises a guide strand comprising the nucleotide sequence of u(Rps)Af(Rps)uugcUfcaauUfuUfuGfcaggu(Sps)u(Sps)c (SEQ ID NO: 49) and a passenger strand comprising the nucleotide sequence of ascscugcAfaAfaAfuugagcaaua (SEQ ID NO: 81), where smaller case “n” stands for 2’-O-methyl modified nucleotide, upper case followed with an “f” (i.e., “Nf”) stands for 2’-fluoro modified nucleotide, “s” stands for 3'-phosphorothioate, "(Rps)" stands for 3'-phosphorothioate, Rp diastereomer, and "(Sps)" stands for 3' phosphorothioate, Sp diastereomer. 112WSGR Docket No.61382-734.601

[0334] A polynucleic acid molecule for modulating expression of AGT gene, wherein polynucleic acid molecule comprises a guide strand comprising the nucleotide sequence of u(Sps)Af(Sps)uugcUfcaauUfuUfuGfcaggu(Sps)u(Sps)c (SEQ ID NO: 50) and a passenger strand comprising the nucleotide sequence of ascscugcAfaAfaAfuugagcaaua (SEQ ID NO: 81), where smaller case “n” stands for 2’-O-methyl modified nucleotide, upper case followed with an “f” (i.e., “Nf”) stands for 2’-fluoro modified nucleotide, “s” stands for 3'-phosphorothioate, "(Rps)" stands for 3'-phosphorothioate, Rp diastereomer, and "(Sps)" stands for 3' phosphorothioate, Sp diastereomer.

[0335] A polynucleic acid molecule for modulating expression of AGT gene, wherein polynucleic acid molecule comprises a guide strand comprising the nucleotide sequence of u(Rps)Af(Sps)uugcUfcaauUfuUfuGfcaggu(Sps)u(Sps)c (SEQ ID NO: 51) and a passenger strand com...

Claims

WSGR Docket No.61382-734.601 CLAIMS WHAT IS CLAIMED IS:

1. A polynucleic acid molecule for modulating expression of angiotensinogen (AGT) gene, wherein the polynucleic acid molecule comprising a passenger strand and a guide strand, and the polynucleic acid molecule comprises a nucleic acid sequence that is at least 85%, at least 90%, or at least 95% identical to a nucleic acid sequence in Table 1, Table 3, Table 6, Table 8, Table 10, Table 12, Table 18, Table 20, Table 22, Table 24, Table 26, Table 28, Table 30, or Table 33.

2. The polynucleic acid molecule of claim 1, wherein the guide strand comprises a nucleic acid sequence that is at least 85%, at least 90%, or at least 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 1-18 and 160.

3. The polynucleic acid molecule of claim 1 or 2, wherein the passenger strand comprises a nucleic acid sequence that is at least 85%, at least 90%, or at least 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 21-33.

4. The polynucleic acid molecule of any one of claims 1-3, wherein the guide strand comprises a nucleic acid sequence comprising at least 16, 17, 18, 19, or 20 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 1-18 and 160, with no more than 1, 2, 3, or 4 mismatches.

5. The polynucleic acid molecule of any one of claims 1-4, wherein the passenger strand comprises a nucleic acid sequence comprising at least 16, 17, 18, 19, or 20 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 21-33, with no more than 1, 2, 3, or 4 mismatches.

6. The polynucleic acid molecule of any one of claims 1-5, wherein the passenger strand comprises a nucleic acid sequence selected from SEQ ID NOs: 21-33, and the guide strand comprises a nucleic acid sequence selected from SEQ ID NOs: 1-18 and 160.

7. The polynucleic acid molecule of any one of claims 1-6, wherein the passenger strand comprises a nucleic acid sequence selected from SEQ ID NOs: 21-22, and 25-27 and the guide strand comprises a nucleic acid sequence selected from SEQ ID NOs: 1-2, 7, and 10- 11. 197WSGR Docket No.61382-734.601 8. The polynucleic acid molecule of any one of claims 1-7, wherein the polynucleic acid molecule comprises (1) a 2’-fluoro modified nucleotides; (2) a 2’-O-methyl modified nucleotides; or (3) a modified internucleotide linkage.

9. The polynucleic acid molecule of any one of claims 1-8, wherein the polynucleic acid molecule comprises at least two consecutive modified internucleotide linkages at the 5’ end.

10. The polynucleic acid molecule of any one of claims 1-9, wherein the polynucleic acid molecule comprises at least two internucleotide linkages among three internucleotide linkages at the 3’end substituted with modified internucleotide linkages.

11. The polynucleic acid molecule of any one of claims 8-10, wherein the modified internucleotide linkage is a phosphorothioate internucleotide linkage or a phosphorodithioate internucleotide linkage.

12. The polynucleic acid molecule of claim 11, wherein the phosphorothioate internucleotide linkage is a stereochemically enriched phosphorothioate internucleotide linkage.

13. The polynucleic acid molecule of claim 12, wherein the stereochemically enriched phosphorothioate internucleotide linkage is an SP chiral internucleotide phosphorothioate linkage.

14. The polynucleic acid molecule of any one of claims 8-12, wherein the polynucleic acid comprises a plurality of modified internucleotide linkages, and at least 1, 2, 3, or 4 of the plurality of modified internucleotide linkages are stereochemically enriched phosphorothioate internucleotide linkages.

15. The polynucleic acid molecule of claim 14, wherein the stereochemically enriched phosphorothioate internucleotide linkages comprise an Rp chiral internucleotide phosphorothioate linkage or an SPchiral internucleotide phosphorothioate linkage.

16. The polynucleic acid molecule of any one of claims 14-15, wherein the stereochemically enriched phosphorothioate internucleotide linkage(s) is disposed between two consecutive nucleosides that are two of six 5’ or 3’ end nucleosides of the passenger strand or the guide strand.

17. The polynucleic acid molecule of any one of claims 11-16, wherein the polynucleic acid molecule comprises the phosphorothioate internucleotide linkage located between the two nucleotides at position 11 and 12 from the 5’ end of the guide strand. 198WSGR Docket No.61382-734.601 18. The polynucleic acid molecule of any one of claims 11-16, wherein the polynucleic acid molecule comprises the phosphorodithioate internucleotide linkage located between the two nucleotides at position 11 and 12 from the 5’ end of the guide strand.

19. The polynucleic acid molecule of any one of claims 1-18, wherein the passenger strand comprises 5’- nsnsnnnnNfnNfnNfnnnnnnnnnn -3’, wherein the guide strand comprises ‘5- nsNfsnnnnNfnnnnNfnNfnNfnnnnnsnsn-3’, wherein “Nf” stands for a 2’-fluoro modified nucleotide, “n” stands for a 2’-O-methyl modified nucleotide, “s” stands for a 3'- phosphorothioate.

20. The polynucleic acid molecule of claim 19, wherein the guide strand comprises a vinyl phosphonate modified nucleotide at the 5’ end.

21. The polynucleic acid molecule of claim 20, wherein the vinyl phosphonate modified nucleotide is 5’(E) vinylphosphonate-2’-O-methoxy-ethyl-5-methyluridine-3’-phosphate (vpt4) or 5'(E) vinylphosphonate-2-O-methyluridine-3'phosphate (vpu).

22. The polynucleic acid molecule of claim 20 or claim 21, wherein the vinyl phosphonate modified nucleotide substitutes a 5’ end nucleotide of SEQ ID NOs: 1-18 and 160.

23. The polynucleic acid molecule of any one of claims 1-22, wherein the polynucleic acid molecule comprises a nucleotide analogue substituting any one of modified nucleotides in the guide strand or the passenger strand, wherein the nucleotide analogue comprises a hypoxanthine nucleobase-containing nucleoside substitution, abasic substitution, acyclic L- threoninol nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threoninol nucleic acid- adenine-3’-phosphate (T-A), acyclic N-acetyl L-threoninol abasic nucleic acid-3'phosphate (T-NAc), acyclic L-threoninol nucleic acid-uracil-3’-phosphate (T-U), acyclic D-threoninol nucleic acid-thymine-3’-phosphate (DT-T), 1',2'-Dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2’-O-methyl-2-thiouridine-3’- phosphate (u3), 2’-fluoro-2-thiouridine-3’-phosphate (U3f), 2-amino-2’-O-methyladenosine- 3’-phosphate (a1), 2'-methoxyethyladenosine-3'phosphate (A6), 2'-methoxyethyluridine-3'- phosphate (U6), or 2’-O-methylinosine-3’-phosphate (i).

24. The polynucleic acid molecule of claim 23, wherein the hypoxanthine nucleobase-containing nucleoside substitution is an inosine substitution.

25. The polynucleic acid molecule of claim 23, wherein the nucleotide analogue is located within a seed region of the guide strand. 199WSGR Docket No.61382-734.601 26. The polynucleic acid molecule of claim 23, wherein the nucleotide analogue is located within 7 nucleotides from the 5’ end of the guide strand.

27. The polynucleic acid molecule of any one of claims 1-26, wherein the passenger strand comprises an inverted abasic moiety at the 5’ end or 3’ end.

28. The polynucleic acid molecule of any one of claims 1-27, wherein the guide strand comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 36-80, 110-142, 148- 152, and 161-164.

29. The polynucleic acid molecule of any one of claims 1-28, wherein the guide strand comprises a nucleic acid sequence comprising at least 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 36-80, 110-142, 148-152, and 161-164 with no more than 1, 2, 3, or 4 mismatches.

30. The polynucleic acid molecule of any one of claims 1-29, wherein the passenger strand comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 81-107, 143-147, 153- 157, and 165-166.

31. The polynucleic acid molecule of any one of claims 1-30, wherein the passenger strand comprises a nucleic acid sequence comprising at least 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 81-107, 143-147, 153- 157, and 165-166, with no more than 1, 2, 3, or 4 mismatches.

32. The polynucleic acid molecule of one of claims 1-31, wherein the passenger strand comprises a nucleic acid sequence selected from SEQ ID NOs: 81-107, 143-147, 153-157, and 165-166, and the guide strand comprises a nucleic acid sequence selected from SEQ ID NOs: 36-80, 110-142, 148-152, and 161-164.

33. The polynucleic acid molecule of any one of claims 1-32, wherein the cytotoxicity of the polynucleic acid molecule is decreased compared to an unmodified polynucleic acid molecule.

34. A polynucleic acid molecule for modulating expression of angiotensinogen (AGT) gene, comprising: 200WSGR Docket No.61382-734.601 a guide strand comprising a nucleic acid sequence of UAGACCAAGGAGAAACGGCUCCU (SEQ ID NO: 7) and a passenger strand comprising a nucleic acid sequence of GAGCCGUUUCUCCUUGGUCUA (SEQ ID NO: 25).

35. A polynucleic acid molecule for modulating expression of angiotensinogen (AGT) gene, comprising: a) a guide strand comprising a nucleic acid sequence of u(Rps)Af(Rps)uugcUfcaauUfuUfuGfcaggu(Sps)u(Sps)c (SEQ ID NO: 49) and a passenger strand comprising a nucleic acid sequence of ascscugcAfaAfaAfuugagcaaua (SEQ ID NO: 81); b) a guide strand comprising a nucleic acid sequence of u(Rps)Af(Rps)uugcUfcaauUfuUfuGfcaggu(Sps)u(Rps)c (SEQ ID NO: 53) and a passenger strand comprising a nucleic acid sequence of ascscugcAfaAfaAfuugagcaaua (SEQ ID NO: 81); c) a guide strand comprising a nucleic acid sequence of usAfsuugcUfcaaussUfuUfuGfcaggususc (SEQ ID NO: 77) and a passenger strand comprising a nucleic acid sequence of ascscugcAfaAfaAfuugagcaaua (SEQ ID NO: 81); d) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggaGfaAfaCfggcucscsu (SEQ ID NO: 57) and a passenger strand comprising a nucleic acid sequence of gsasgccgUfuUfcUfccuuggucua (SEQ ID NO: 85; SEQ ID NO: 154; SEQ ID NO: 156); e) a guide strand comprising a nucleic acid sequence of u(Rps)Af(Rps)gaccAfaggaGfaAfaCfggcuc(Sps)c(Sps)u (SEQ ID NO: 148; SEQ ID NO: 151) and a passenger strand comprising a nucleic acid sequence of gsasgccgUfuUfcUfccuuggucua (SEQ ID NO: 85; SEQ ID NO: 156); f) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggasGfaAfaCfggcucscsu (SEQ ID NO: 164) and a passenger strand comprising a nucleic acid sequence of gsasgccgUfuUfcUfccuuggucua (SEQ ID NO: 85; SEQ ID NO: 156); 201WSGR Docket No.61382-734.601 g) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggaGfaAfaCfggcucscsu (SEQ ID NO: 57) and a passenger strand comprising a nucleic acid sequence of sgsagccgUfuUfcUfccuuggucuas(invAb) (SEQ ID NO: 155); h) a guide strand comprising a nucleic acid sequence of u(Rps)Af(Rps)gaccAfaggassGfaAfaCfggcuc(Sps)c(Sps)u (SEQ ID NO: 152) and a passenger strand comprising a nucleic acid sequence of gsasgccgUfuUfcUfccuuggucua (SEQ ID NO: 156); i) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggassGfaAfaCfggcucscsu (SEQ ID NO: 149; SEQ ID NO: 150) and a passenger strand comprising a nucleic acid sequence of gsasgccgUfuUfcUfccuuggucua (SEQ ID NO: 85; SEQ ID NO: 156); j) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggaGfaAfaCfggcucscsu (SEQ ID NO: 57) and a passenger strand comprising a nucleic acid sequence of sgsagccgUfuUfcUfccuuggucua (SEQ ID NO: 165); k) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggassGfaAfaCfggcucscsu (SEQ ID NO: 150) and a passenger strand comprising a nucleic acid sequence of sgsagccgUfuUfcUfccuuggucua (SEQ ID NO: 165); l) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggasGfaAfaCfggcucscsu (SEQ ID NO: 164) and a passenger strand comprising a nucleic acid sequence of sgsagccgUfuUfcUfccuuggucua (SEQ ID NO: 165); m) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggassGfaAfaCfggcucscsu (SEQ ID NO: 150) and a passenger strand comprising a nucleic acid sequence of sgsagccgUfuUfcUfccuuggucuas(invAb) (SEQ ID NO: 155); n) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggaGfaAfaCfggcucscsu (SEQ ID NO: 57) and a passenger strand 202WSGR Docket No.61382-734.601 comprising a nucleic acid sequence of gssasgccgUfuUfcUfccuuggucua (SEQ ID NO: 166); or o) a guide strand comprising a nucleic acid sequence of usAfsgaccAfaggassGfaAfaCfggcucscsu (SEQ ID NO: 150) and a passenger strand comprising a nucleic acid sequence of gssasgccgUfuUfcUfccuuggucua (SEQ ID NO: 166); wherein smaller case “n” stands for 2’-O-methyl modified nucleotide, upper case followed with an “f” (i.e., “Nf”) stands for 2’-fluoro modified nucleotide, “s” stands for 3'-phosphorothioate, “ss” stands for 3'-phosphorodithioate, “(Rps)” stands for 3'- phosphorothioate, Rp diastereomer, “(Sps)” stands for 3' phosphorothioate, Sp diastereomer, and "(invAb)" stands for inverted abasic deoxyribonucleotide.

36. A polynucleic acid molecule conjugate for modulating expression of angiotensinogen (AGT) gene, wherein the polynucleic acid molecule conjugate comprises a polynucleic acid molecule of any one of claims 1-35 and an asialoglycoprotein receptor targeting moiety.

37. The polynucleic acid molecule conjugate of claim 36, wherein the polynucleic acid molecule and the asialoglycoprotein receptor targeting moiety is coupled via a linker.

38. The polynucleic acid molecule conjugate of claim 37, wherein the linker comprises formula (IV) below,wherein at least one of Y1 and Y2 is a nucleotide in the polynucleic acid molecule, and wherein theis connected to the asialoglycoprotein receptor targeting moiety.

39. The polynucleic acid molecule conjugate of claim 38, wherein the Y1 is the last nucleotide on the 3’ end of the passenger strand of the polynucleic acid molecule. 203WSGR Docket No.61382-734.601 40. The polynucleic acid molecule conjugate of claim 38, wherein the Y1 and Y2 are two consecutive nucleotides in the polynucleic acid molecule.

41. The polynucleic acid molecule conjugate of any one of claims 36-40, wherein the asialoglycoprotein receptor targeting moiety comprises N-Acetylgalactosamine (GalNAc) or galactose.

42. The polynucleic acid molecule conjugate of any one of claims 37-41, wherein the linker and the asialoglycoprotein receptor targeting moiety with the last nucleotide on the 3’ end of the passenger strand of the polynucleic acid molecule are shown in:204WSGR Docket No.61382-734.601205WSGR Docket No.61382-734.601wherein Z in formula (V’), (V’’’’’’’), (VI), (VI’), (VI’’), or (VI’’’) is -H, -OH, -O-Methyl, - F, or -O-methoxyethyl, R in formula (V’), (V’’’’’’’), (VI), (VI’), (VI’’), or (VI’’’) is adenine, uracil, guanine, cytosine, thymine, abasic, or others, and theis to connect to the next nucleotide of the polynucleic acid molecule.

43. The polynucleic acid molecule conjugate of any one of claims 37-42, wherein the linker and the asialoglycoprotein receptor targeting moiety with the first nucleotide on the 5’ end of the passenger strand of the polynucleic acid molecule are shown in: 206WSGR Docket No.61382-734.601wherein Z in formula (V’’’) or (X’) is -H, -OH, -O-Methyl, -F, or -O-methoxyethyl, R in formula (V’’’) or (X’) is adenine, uracil, guanine, cytosine, thymine, abasic, or others, and the is to connect to the next nucleotide of the polynucleic acid molecule.

44. The polynucleic acid molecule conjugate of claim 42, further comprises a lipid, wherein the lipid is coupled with the first nucleotide on the 5’ end of the passenger strand of the polynucleic acid molecule.

45. The polynucleic acid molecule conjugate of claim 44, wherein the lipid is shown in:207WSGR Docket No.61382-734.60146. A pharmaceutical composition comprising a polynucleic acid molecule of any one of claims 1-35 or a polynucleic acid molecule conjugate of any one of claims 36-45, and a pharmaceutically acceptable excipient.

47. The pharmaceutical composition of claim 46, wherein the pharmaceutical composition is formulated as a nanoparticle formulation.

48. The pharmaceutical composition of claim 46 or claim 47, wherein the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, transdermal, intravenous, subcutaneous, or intrathecal administration.

49. A method of modulating expression of angiotensinogen (AGT) gene in a subject, comprising: administering to the subject a polynucleic acid molecule of any one of claims 1- 35, a polynucleic acid molecule conjugate of any one of claims 36-45, or a pharmaceutical composition of claims 46-48, thereby modulating the expression of AGT gene in the subject.

50. A method of preventing, alleviating, or treating hypertension in a subject in need thereof, comprising: administering to the subject a polynucleic acid molecule of any one of claims 1- 35, a polynucleic acid molecule conjugate of any one of claims 36-45, or a pharmaceutical composition of claims 46-48, wherein the polynucleic acid molecule of any one of claims 1- 35, the polynucleic acid molecule conjugate of any one of claims 36-45, or the pharmaceutical composition of claims 46-48 reduces the expression of AGT gene in the subject.

51. A method of preventing, alleviating, or treating atherosclerosis in a subject in need thereof, comprising: administering to the subject a polynucleic acid molecule of any one of claims 1- 35, a polynucleic acid molecule conjugate of any one of claims 36-45, or a pharmaceutical composition of claims 46-48, wherein the polynucleic acid molecule of any one of claims 1- 35, the polynucleic acid molecule conjugate of any one of claims 36-45, or the pharmaceutical composition of claims 46-48 reduces the expression of AGT gene in the subject.

52. The method of claim 51, wherein the subject suffers from a coronary artery disease or a symptom thereof. 208WSGR Docket No.61382-734.60153. A method of preventing, alleviating, or treating obesity in a subject in need thereof,comprising: administering to the subject a polynucleic acid molecule of any one of claims 1- 35, a polynucleic acid molecule conjugate of any one of claims 36-45, or a pharmaceutical composition of claims 46-48, wherein the polynucleic acid molecule of any one of claims 1- 35, the polynucleic acid molecule conjugate of any one of claims 36-45, or the pharmaceutical composition of claims 46-48 reduces the expression of AGT gene in the subject. 209

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