Inhibin subunit beta e-related double stranded oligonucleotide compositions and methods relating thereto

Abstract

The present disclosure provides human inhibin subunit beta E (INHBE) gene-targeting double stranded oligonucleotides, compositions, and methods of using the same, either alone or in combination with additional therapeutics, for preventing and / or treating various INHBE-related disorders, including maintenance of weight loss achieved. In some embodiments, the provided double stranded oligonucleotides comprise certain chemistry' modifications, e.g.. stereospecific mtemucleotidic linkage modifications, and have unexpected advantageous properties.

Description

[0001] Attorney Docket No.: 2010581-1677

[0002] INHIBIN SUBUNIT BETA E-RELATED DOUBLE STRANDED OLIGONUCLEOTIDE COMPOSITIO S AND METHODS RELATING THERETO

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] Tills application claims priority to U. S. Provisional Application Nos. 63 / 771.532, filed March 13, 2025, and 63 / 981,820, filed February 12, 2026. the entirety of each of which is incorporated herein by reference.

[0005] This application incorporates herein by reference, in their entireties, U. S. Provisional Application Nos.

[0006] 63 / 691,262, filed September 5, 2024, 63 / 713,955, filed October 30, 2024, 63 / 768,125, filed March 6, 2025, 63 / 645,058, filed May 9, 2024, 63 / 680,974, filed August 8, 2024, and 63 / 713,973, filed October 30, 2024, and 63 / 768,140, filed March 6, 2025, and International Patent Application No. PCT / US2024 / 045826, filed September 9, 2024.

[0007] BACKGROUND

[0008] Gene -targeting oligonucleotides are useful in various applications, e.g., therapeutic, diagnostic, research and nanomatcrials applications The use of naturally-occurring nucleic acids (e.g., unmodified DNA or RNA) in such applications can be limited by. for example, their susceptibility to endo- and exo-nucleases. As such, various synthetic counterparts have been developed to circumvent these shortcomings. These include synthetic oligonucleotides that contain chemical modifications, e g., base modifications, sugar modifications, backbone modifications. There remains, however, a need in the art for double-stranded (ds) oligonucleotides with improved properties for use in connection with the above-described applications.

[0009] SUMMARY

[0010] The present disclosure is directed, in part, to the recognition that controlling structural elements of the oligonucleotides of a double-stranded (ds) oligonucleotide can have a significant impact on the ds oligonucleotide's properties and / or activity. In certain embodiments, such structural elements include one or more of: (1) chemical modifications (e.g,, modifications of a sugar, base and / or internucleotidic linkage) and patterns thereof; and (2) alterations in stereochemistry (e.g., stereochemistry of a backbone chiral internucleotidic linkage) and patterns thereof. One or more of such structural elements can, in certain embodiments, be independently present in one or both oligonucleotides of a ds oligonucleotide. In certain embodiments, the properties and / or activities impacted by such structural elements include, but are not limited to, participation in, direction of a decrease in expression, activity- or level of a gene or a gene product thereof, mediated, for example, by RNA interference (RNAi interference), RNase H-mediated knockdown, steric hindrance of translation, etc. Moreover, in certain embodiments, the properties and / or activities impacted by such structural elements include, but are not limited to, participation in, Ago2 loading, thermal stability, in vivo Page I of 566

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[0012] stability' delivery to tissues and into cells, among others.

[0013] In certain embodiments, the present disclosure demonstrates that compositions comprising ds oligonucleotides (e.g., dsRNAi oligonucleotides, also referred to as dsRNAi agents) with controlled structural elements provide unexpected properties and / or activities.

[0014] In certain embodiments, the present disclosure encompasses the recognition that stereochemistry, e.g., stereochemistry' of backbone chiral centers, can unexpectedly maintain or improve properties of ds oligonucleotides. For example, but not by' way of limitation, the instant disclosure relates, in part, to ds oligonucleotides comprising a guide strand comprising backbone phosphoryl guanidine chiral centers in the Sp configuration.

[0015] In one aspect, the invention relates to a double-stranded RNAi (dsRN Ai) agent capable of directing INHBE (Inhibin pE)-specific RNA interference to, e.g., induce lipolysis while preserving muscle mass, the dsRNAi agent comprising a guide strand and a passenger strand, wherein: the guide strand is complementary or substantially complementary to a target RNA sequence; the guide strand comprises a seed region at its 5’ end region that is capable of mediating the initial recognition of the target RNA sequence, e.g., nucleotides at positions 2-6, at positions 2-7. or at positions 2-8, relative to the 5 ’-end of the guide strand; the guide strand comprises a PN intcmuclcotidic linkage, e.g., a phosphoryl guanidine intcmuclcotidic linkage or a mesyl N o k > N / N-P O I-0 phosphoramidate (MsPA) internucleotidic linkage, composing the structure of. e.g.,

[0016]

[0017] !’ (nOOl), e.g., in the Sp configuration, in the seed region, e g., between the third (+3) nucleotide and the immediately downstream (-1-4) nucleotide, relative to its 5’ terminal nucleotide; and the passenger strand comprises a PN intcmuclcotidic linkage, e.g., a phosphoryl guanidine or MsPA intcmuclcotidic linkage, comprising the

[0018] i A )=N—? P--0

[0019] "

[0020]

[0021] " N 6

[0022] structure of, e.g., 'f(nOOl), between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide in, e.g., the 7?p configuration In an exemplary' embodiment, the passenger strand further comprises a PN internucleotidic linkage, e.g., a phosphoryl guanidine or MsPA internucleotidic linkage,

[0023] ?

[0024] i }-N-P-O

[0025] ''N 6

[0026] comprising the structure of, e.g.. ' ' (nOOl). between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide in, e.g.. the Rp configuration. In an exemplary embodiment, the guide strand comprises a phosphorothioate (PS) internucleotidic linkage, e.g. in the Rp configuration, or a natural phosphate linkage (PO) between the +2 nucleotide and the immediately downstream (+3) nucleotide, relative the 5’ nucleotide of the guide strand. In an exemplary embodiment, the nucleoside 3’ and / or 5’ to the PN internucleotidic linkage, e.g., a phosphoryl guanidine or MsPA internucleotidic linkage, in the seed region Page 2 of 566

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[0028] comprises a 2+F ribose modification. In an exemplary embodiment, the +2 nucleoside, the +3 nucleoside, or the +2 nucleoside and +3 nucleoside, relative to die 5’ terminal nucleoside of the guide strand, comprise a 2’- F modification. In an exemplary embodiment, when the guide strand comprises a PS internucleotidic linkage between the +2 nucleotide and the immediately downstream (+3) nucleotide, relative the 5’ nucleotide of the guide strand, the +3 nucleoside, relative to the 5‘ terminal nucleoside of the guide strand, comprises a 2'-F modification. In an exemplary embodiment, when the guide strand comprises natural phosphate linkage (PO) between the +2 nucleotide and the immediately downstream (+3) nucleotide, relative the 5’ nucleotide of the guide strand, the +2 and the +3 nucleoside, relative to the 5’ terminal nucleoside of the guide strand, comprise a 2’-F modification. In an exemplary embodiment, the guide strand further comprises a PN internucleotidic linkage, e.g., a phosphoryl guanidine or MsPA internucleotidic linkage, comprising the structure of, e.g., A. 9

[0029] | > N — P O

[0030] ~" N' 6

[0031] If(nOOl), between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5’ term inal nucleotide of the guide strand. In an exemplary' embodiment, the nucleoside 3" to the PN internucleotidic linkage, e.g., a phosphoryl guanidine or MsPA internucleotidic linkage, between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5 ’ terminal nucleotide of the guide strand, comprises a 2‘ -F ribose modification. In an exemplary embodiment, the PN internucleotidic linkage, e.g.. a phosphoryl guanidine or MsPA internucleotidic linkage, between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5’ terminal nucleotide of the guide strand, is in, e.g., the Rp configuration. In an exemplary embodiment, the guide strand comprises a 5’ phosphate modification, e.g., a 5’ phosphate mimic modification. In an exemplary’ embodiment, the 5’ phosphate mimic modification O N=N

[0032] -0.-P- Base

[0033] O'

[0034] is, e.g.,

[0035]

[0036] 5, wherein: the base is, e.g., N3U, or is selected from, e.g., A, C, G, T, U, abasic, and modified nucleobases other than N3U; and R1is selected from, e.g., H, OH, O-alkyl, O-methyl (0-Me), F, O-methoxyethyl (MOE), and 2‘-(9,4’C-methylene-bridged or locked nucleic acid (2’,4’-BNA or LNA). In an exemplary- embodiment, R1is, e.g., O-methyl (O-Me). In an exemplary' embodiment, the guide strand:ir-N 9 X=N— P— O further comprises a PN internucleotidic linkage comprising the structure of, e.g.,

[0037]

[0038] ' (nOOl), between, e.g., the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide in, e.g., the Rp configuration,

[0039] p— O

[0040] and / or a PN internucleotidic linkage comprising the structure of, e g..

[0041]

[0042] (nOOl), between, e.g., the Page 3 of 566

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[0044] penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide in, e.g., the / / p configuration. In an exemplary embodiment, the guide strand does not comprise a PN internucleotidic linkage between the 3 ’ terminal (N) nucleotide and the penultimate (N-l) nucleotide. In an exemplary7embodiment, the guide strand does not comprise a PN internucleotidic linkage the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide. In an exemplary embodiment, the guide strand comprises a phosphorothioate (PS) internucleotidic linkage between, e.g.. the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide in. e.g,, the Sp configuration. In an exemplary7embodiment, the guide strand comprises, e.g.. a PN cap, e.g., a phosphoryl guanidine cap at, e g., its 5’-end (5’-end PN cap). In an exemplary7embodiment, the 5 '-end PN cap

[0045] is selected from, e

[0046]

[0047] .g.,, wherein: the base is N3U, or is selected from, e g., A. C, G. T, U. abasic, and modified nucleobases other than N3U; and R1is selected from II, OH, O-alkyl, O-methyl (O-Me), F, O-methoxyethyl (MOE), and 2’ -0,4’ C-methylene -bridged or locked nucleic acid (2’,4’-BNA orLNA). In an exemplary7embodiment, R1is, e.g., O-methyl (O-Me). In an exemplary7embodiment, the guide strand comprises, e.g., a natural phosphate linkage (PO) between, e.g., the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, andor, e.g., a natural phosphate linkage (PO) between, e.g., the +2 nucleotide and the +3 nucleotide, relative to the 5' terminal nucleotide.

[0048] In certain embodiments, the invention relates to an oligonucleotide having the structure of RNAl{p.m(U)[n001R].m(C)p.m(C)p.m(L!)p.m(U)p.m(C)p.[fl2r](C)p.m(A)p.[fl2r|(U)p.[fl2r](U)p.[fl2r](C)p. m(U)p.m(G)p.m(C)p.m(C)p m(G)p.m(D)p.m(C)p.m(U)p.m(U)[Ssp].m(A)}|CHEMl{[nC6o]}^CHEM2[[Gal NAc3 C 12oyl] } SCHEM 1, RNA 1, 1: R 1 - 1: R I |CHEM 1, CHEM2, 1: R2- 1: R 1 $$$V2.0, or RNAl{p.m(U)[n001R].m(C)p.m(C)p.m(U)p.m(U)p.m(C)p.[fl2r](C)p.m(A)p.[fl2r](U)p.[fl2r](U)p.[fl2r](C)p. ni(U)p.m(G)p.m(C)p.m(C)p.m(G)p.m(U)p.m(C)p.m(U)p.in(U)[n001R].m(A)}|CHEMl {[nC6o]}; CHEM2{[

[0049]

[0050] GalNAc3 C 12oyl] } SCHEM 1, RNA 1.1: R 1 - 1: R1 |CHEM 1. CHEM2.1: R2- 1: R1 $$$V2.0 or a pharmaceutically acceptable salt form thereof.

[0051] In certain embodiments, the invention relates to a dsRNAi agent having the structure of RNAl{[d5m](U)[Ssp].[fl2r](A)[Rsp].m(A)[n001S].[fl2r](G)p.m(A)p.[fl2r](C)p.m(G)p.m(G)p.m(C)p.m(A)[ n001R].[f]2r](G)p m(A)p.m(A)p.[fl2r](U)p.m(G)p.[fl2r](G)p.m(A)p.m(A)p.m(G)p.m(G)p.m(A)[Ssp].m(U)[ Ssp].m(U)!|RNA2{p.m(U)[n001R].m(C)p.m(C)p.m(U)p.m(U)p.m(C)p.[fl2r](C)p.m(A)p.[fl2r](U)p.[fl2r](U) p.|fl2r](C)p.m(U)p.m(G)p.m(C)p.m(C)p.m(G)p.m(U)p.m(C)p.m(U)p.m(U)|Ssp].m(z )}iCHEMl{[ptz]}|CH EM2 { | nC6o] } | CHEM3 { [GalN Ac3 C 12oyl] } SCHEM 1, RNA 1, 1: R1 - 1: R11 CHEM2, RNA2, 1: R1 - Page 4 of 566

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[0053] 1: R1 |CHEM2. CHEM3, 1: R2-1: R1$S$V2. O, RNAl{[d5m](U)[SspJ.[f]2r](A)[RspJ.m(A)[n001S] [fl2r](G)p.m(A)p [fl2r](C)p m(G)p.m(G)p.m(C)p.m(A)[ n001R].[fl2r](G)p m(A)p.m(A)p.[fl2r](U)p.m(G)p.[fl2r](G)p.m(A)p.m(A)p.m(G)p.m(G)p.m(A)[Ssp].m(U)[ Ssp].m(U)}|RNA2{p.m(U)[n001R].m(C)p.m(C)p.m(U)p.m(U)p.m(C)p.[fl2r](C)p.m(A)p.[fl2r](U)p.[fl2r](U) p.[fl2r|(C)p.m(U)p.m(G)p.m(C)p.in(C)p.m(G)p.in(U)p.m(C)p.m(U)p.m(U)[n001R].m(A)}|CHEMl{[ptz]}|C HEM2 { [nC6o] } | CHEM3 { [GalNAc3 C 12oyl] } $CHEM 1, RNA 1, 1: R1 - 1: R11 CHEM2. RNA2, 1: R1 - 1: R1 |CHEM2. CHEM3,1: R2-1: R1 $$$V2.0, RNAl{[d5m](U)[Ssp] [fl2r](A)p.[f]2r](A)[n001S].m(G)p.m(A)p.[fl2r](C)p.m(G)p.m(G)p m(C)p.m(A)[n001 R|.|fl2r](G)p.m(A)p.m(A)p.[fl2r](U)p ni(G)p.[fl2r](G)p.m(A)p.m(A)p.m(G)p.m(G)p.m(A)[Ssp].m(U)| Ssp]. m(U)}|RNA2{p.m(U)[n001R].m(C)p.m(C)p.m(U)p m(l;)p.m(C)p.[fl2r](C)p.m(A)p.[fl2r](U)p.[fl2rJ(U)p.[fl 2r](C)p.m(U)p.m(G)p.m(C)p.m(C)p,m(G)p.m(U)p.m(C)p,m(U)p.m(U)[Ssp].m(A)} |CHEM1 { [tz] } |CHEM2 [ [cp] } JCHEM3 { [nC6o] } |CHEM4 { [GalN Ac3C 12oy 1] } $CHEM 1, RNA 1 J: R1 - 1: R11 CHEM3, RNA2, 1: R 1 - 1: R1|CHEM1. CHEM2,1: R2-1: R1 CHEM3. CHEM4.1: R2-1: R1$$$V2. O, or RNAl{|d5m](U)[Ssp] [fl2r|(A)p.[fl2r](A)[n001S|.m(G)p.m(A)p.|fl2r](C)p.m(G)p.m(G)p m(C)p.m(A)[n001 R] [fl2r](G)p.m(A)p m(A)p.[fl2r](U)p m(G)p.[fl2r](G)p m(A)p.m(A)p.m(G)p.m(G)p.m(A)[Ssp].m(U)[Sspj. m(U)}|RNA2{p m(U)[n001R],m(C)p.m(C)p,m(U)p.m(U)p.m(C)p.[fl2r](C)p.m(A)p.[fl2r](U)p.[fl2r](U)p [fl 2r](C)p.m(U)p.m(G)p m(C)p.m(C)p.m(G)p.m(U)p.m(C)p.m(U)p.m(U)[n001R] m(A)}|CHEMl{[tz]}|CHEM 2 { [cp ] } | CHEM3 { [nC6o] } | CHEM4 { [GalNAc3 C 12oy 1 SCHEM 1, RNA 1, 1: R1 - 1: R1! CHEM3. RN A2, 1: R1 -

[0054]

[0055] 1: R1 |CHEM LCHEM2, 1: R2-1: R1 CHEM3, CHEM4, 1: R2-1: R1$S$V2. O or a pharmaceutically acceptable salt form thereof.

[0056] phosphorothioate (PS) internucleotidic linkage

[0057] phosphorothioate (PS) internucleotidic linkage phosphorothioate (PS) internucleotidic linkage

[0058] In particular embodiments of the present disclosure, the dsRNAi agent comprises a 5’ phosphate modification. In certain embodiments, the 5’ phosphate modification is a 5’ phosphate mimic modification. In particular embodiments, the 5’ phosphate mimic modification is selected from

[0059]

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[0080]

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[0083]

[0084]

[0085] wherein the base is A, C, G, T, U, abasic, or a modified nucleobase; R‘ is selected from H, OH, O-alkyl, O-methyl (O-Me), F, O-methoxyethyl (MOE), and 2'-<,4'C-methylene-bridged or locked nucleic acid (2’.4’-BNA or LNA); and R2is selected from alkyl, methyl, ethyl, isopropyl, propyl, cyclohexyl, benzyl, phenyl, tolyl, xylyl, and, and arene group. Tn an exemplary- embodiment, the 5’ terminal modification is triazolyl phosphonate or methyl phosphonate. In an exemplary' embodiment, the 5’ terminal Page 13 of 566

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[0087] modification is triazolyl phosphonate.

[0088] In particular embodiments, the guide strand further comprises a backbone phosphorothioate (PS) internucleotidic linkage in the p configuration between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and a backbone PS internncleotidic linkage in the Sp configuration between the -t-2 nucleotide and immediately downstream (-1-3) nucleotide.

[0089] In particular embodiments, the guide strand further comprises a backbone phosphorothioate (PS) internucleotidic linkage tn the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and wherein the backbone linkage between the +2 nucleotide and the immediately downstream (+3) nucleotide of the guide strand is an unmodified phosphodiester (PO) linkage.

[0090] In accordance with another aspect of the disclosed subject matter, the present disclosure provides a double-stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein the guide strand is complementary or substantially complementary to a target RNA sequence, wherein the guide strand comprises a 5' phosphate modification, and wherein the guide strand further comprises a backbone phosphorothioate (PS) internucleotidic linkage in the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately’ downstream (+2) nucleotide, and a backbone PS internucleotidic linkage in the Sp configuration between the +2 nucleotide and immediately downstream (+3) nucleotide.

[0091] In accordance with another aspect of the disclosed subject mater, the present disclosure provides a double-stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein the guide strand is complementary’ or substantially complementary’ to a target RNA sequence; wherein the guide strand comprises a 5’ phosphate modification; wherein the guide strand further comprises a backbone phosphorothioate (PS) internucleotidic linkage in the Sp configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide; and wherein the backbone linkage between the +2 nucleotide and the immediately downstream (+3) nucleotide of the guide strand is an unmodified phosphodiester (PO) linkage.

[0092] In particular embodiments, the 5’ phosphate modification is a 5’ phosphate mimic modification. In certain embodiments, the 5’ phosphate mimic modification is selected from:

[0093]

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[0096]

[0097] the base is selected from A, C, G, T, U, abasic, and modified nucleobases other than N3U;

[0098] R1is selected from H. OH, O-alkyl, O-methyl (O-Me), F, O-m ethoxy ethyl (MOE), and 2'-(9,4’C- methylene-bridged or locked nucleic acid (2’,4’-BNA or LNA); and

[0099] R2is selected from alkyl, methyl, ethyl, isopropyl, propyl, cyclohexyl, benzyl, phenyl, tolyl, xylyl, aryl, and arene group.

[0100] In certain embodiments, the 5" phosphate mimic modification

[0101]

[0102] is particular embodiments, R1is LNA bridge to the 4’ position. In certain embodiments, R1is MOE. In particular

[0103] embodiments, R1is F. In certain embodiments,

[0104]

[0105] R2in

[0106]

[0107] In particular embodiments, the guide strand further comprises a PN internucleotidic linkage, e.g., a phosphoryl guanidine or MsPA internucleotidic linkage, in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide. In certain embodiments, the guide strand further comprises backbone PS internucleotidic linkages in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and between the penultimate (N-I) nucleotide and the immediately upstream (N-2) nucleotide. In particular embodiments, the guide strand further comprises a PN internucleotidic linkage, e.g,, a phosphoryl guanidine or MsPA internucleotidic linkage, in the Rp configuration between the +10 nucleotide and the +11 nucleotide. In certain embodiments, the guide strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides Page 15 of 566

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[0109] between the second (+2) nucleotide relative to the 5 ’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3' terminal nucleotide. In particular embodiments, the guide strand comprises a 2’ modification, of the 3‘ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage. In particular embodiments, the guide strand comprises an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide. In certain embodiments, the guide strand comprises an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage between the seventh (+7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide. In particular embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between an} two adjacent nucleotides between the penultimate 3 ’ nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide.

[0110] In certain embodiments, the passenger strand comprises one or more Rp, Sp, or stereorandom non- negatively charged internucleotidic linkage occurs upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand In particular embodiments, the passenger strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5" direction, relative to the central nucleotide of the passenger strand. In certain embodiments, the passenger strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3" direction, relative to the central nucleotide of the passenger strand. In particular embodiments, the passenger strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand. In certain embodiments, the passenger strand in combination with one or more of the aforementioned guide strands, comprises one or more modified sugars between the 5'' terminal (+1) nucleotide and the penultimate (N-l) nucleotide.

[0111] In particular embodiments, the passenger strand compnses one or more of 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49, one or more backbone chiral centers in Rp or Sp configuration, one or more backbone phosphoryl guanidine chiral centers in the Rp configuration between the +7 nucleotide and the immediately dowmstream (+8) nucleotide, i.e., in the 3’ direction, one or more backbone phosphoryl guanidine chiral centers in the Rp configuration between the +15 nucleotide and the immediately downstream (+16) nucleotide, i.e., in the 3’ direction, and / or backbone phosphorothioate chiral centers in the Sp configuration between the 5‘ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3" direction, (+2) nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide.

[0112] In particular embodiments, each strand of the dsRNAi agent independently has a length of about 15 to about 49 nucleotides.

[0113] In particular embodiments, the dsRNAi agent of any of the preceding claims, wherein the Rp, Sp, or Page 16 of 566

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[0115] stereorandom non-negatively charged backbone internucleotidic linkages have neutral charge. In particular [CH2]nCH3

[0116] r"'N\ — P

[0117] > 6' -cy

[0118] embodiments, the neutral backbone internucleotidic linkage is

[0119]

[0120] l£H2]mCH3, wherein n is about 0 to 49 and m is about 0 to 49.

[0121] In particular embodiments, the guide strand comprises a linkage having the following structure between the third (+3) and fourth (+4) nucleotides of die guide strand, between the tenth (+10) and eleventh (+11) nucleotides of the guide strand, or both,

[0122] In particular embodiments, guide strand comprises a linkage having the following structure

[0123] -N

[0124] „,0

[0125] N P, C

[0126]

[0127] cC° between the third (+3) and fourth (+4) nucleotides of the guide strand, between the seventh (+7) and eighth (+8) nucleotides of the guide strand, between the tenth (+10) and eleventh (+11) nucleotides of the guide strand, between the eighteenth (+18) and nineteenth (+19) nucleotides of the guide strand, or combinations thereof.

[0128] In particular embodiments, the passenger strand comprises a linkage having the following structure [CH2]nCH3

[0129] V=N—, P<

[0130] o °y.

[0131]

[0132] [CH2]mCH3, where n is about 0 to 49 and m is about 0 to 49, at 5’ to the central nucleotide of the passenger strand, 3' to the central nucleotide of the passenger strand, or both.

[0133] In particular embodiments, the passenger strand comprises a linkage having the following structure,[CH2]nCH3

[0134] L>-N— P;

[0135] — ( N, / o II Q w <

[0136]

[0137] [CH2]mCH3. wherein n is about 11 to 49 and m is 0, or n is 11 or 15.

[0138] In particular embodiments, the passenger strand comprises a non-negatively charged backbone internucleotidic linkage in the Rp configuration between the +1 nucleotide and the +8 nucleotide, a non- negatively charged backbone internucleotidic linkage in the Rp configuration between the +1 nucleotide and the +16 nucleotide and / or a backbone phosphorothioate chiral centers in the Sp configuration between the 5’ terminal (+1 ) nucleotide and the +2 nucleotide and between the 3 ’ terminal nucleotide and the penultimate (N- 1) nucleotide.

[0139] In particular embodiments, the passenger strand comprises:

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[0142] O / p y

[0143] P -0 "

[0144] /

[0145] N II

[0146]

[0147] \ in the Rp configuration between the +7 nucleotide and the +8 nucleotide;.

[0148] P o "

[0149] N

[0150] jl

[0151]

[0152] \ / in the Rp configuration between the +15 nucleotide and the + 16 nucleotide, and a backbone phosphorothioate chiral centers in the Sp configuration betw een the +1 nucleotide and the +2 nucleotide and between the 3" terminal nucleotide and the penultimate (N-l) nucleotide.

[0153] In particular embodiments, the passenger strand comprises:

[0154] 4-o / p \

[0155] p / - o" ’

[0156]

[0157] ' — / in the Rp configuration between the +7 nucleotide and the +8 nucleotide and backbone phosphorothioate chiral centers in the Sp configuration between the +1 nucleotide and the +2 nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide.

[0158] In accordance with another aspect of the disclosed subject matter, the present disclosure provides a method for reducing level and / or activity of atranscript or a protein encoded thereby, comprising administering to a cell expressing the transcript a double-stranded RNAi (dsRNAi) agent of any of the above aspects or embodiments.

[0159] In certain embodiments, the cell is an immune cell, a blood cell, a cardiac cell, a lung cell, an optic cell, a muscle cell, a liver cell, a kidney cell, a cell of the central nervous system, or a cell of the peripheral nervous system. In particular embodiments, the cell of the central nervous system is a brain cell. In certain embodiments, when the double-stranded RNAi (dsRNAi) agent is contacted with a cell comprising transcripts of both the target allele and another allele of the same nucleic acid sequence, it shows suppression of transcripts of the particular allele at a level that is greater than when the double-stranded RNAi (dsRNAi) agent is absent, greater than a level of suppression observed for another allele of the same nucleic acid sequence, both greater than when the double -stranded RNAi (dsRNAi) agent is absent, and greater than a level of suppression observed for another allele of the same nucleic acid sequence.

[0160] It is to be understood that both the foregoing general description and the following detailed description are examples and are provided for purpose of illustration and not intended to limit the scope of the disclosed

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[0163] subject matter in any manner.

[0164] I DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0165] Technologies of the present disclosure may be understood more readily by reference to the following detailed description of certain embodiments.

[0166] Definitions

[0167] As used herein, the following definitions shall apply unless otherwise indicated For purposes of this disclosure, the elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics. 75th Ed. Additionally, general principles of organic chemistry are described in " Organic Chemistry'", Thomas Sorrell, University Science Books, Sausalito: 1999, and " March's Advanced Organic Chemistry", 5th Ed., Ed,: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001.

[0168] As used herein in the present disclosure, unless otherwise clear from context, (i) the term “a” or “an” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising”, “comprise”, “including” (whether used with “not limited to” or not), and “include” (whether used with “not limited to” or not) may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; (iv) the term “another” may be understood to mean at least an additional / second one or more; (v) the terms “about” and “approximately” may be understood to permit standard variation as w ould be understood by those of ordinary skill in the art; and (vi) where ranges are provided, endpoints are included

[0169] Unless otherwise specified, description of oligonucleotides and elements thereof (e.g., base sequence, sugar modifications, internucleotidic linkages, linkage phosphorus stereochemistry, patterns thereof, etc.) is from 5’ to 3’, with the 5’ terminal nucleotide identified as the “+1” position and the 3’ terminal nucleotide identified either by the number of nucleotides of the full sequence or by “N”, with the penultimate nucleotide identified, e.g., as “N-l”, and so on. As those skilled in the art will appreciate, in certain embodiments, oligonucleotides may be provided and / or utilized as salt forms, particularly pharmaceutically acceptable salt forms, e.g., sodium salts. As those skilled in the art will also appreciate, in certain embodiments, individual oligonucleotides within a composition may be considered to be of the same constitution and / or structure even though, within such composition (e.g., a liquid composition), particular such oligonucleotides might be in different salt form(s) (and may be dissolved and the oligonucleotide chain may exist as an anion form when, e.g., m a liquid composition) at a particular moment in time. For example, those skilled in the art wall appreciate that, at a given pH, individual internucleotidic linkages along an oligonucleotide chain may be in an acid (H) form, or in one of a plurality of possible salt forms (e.g., a sodium salt, or a salt of a different cation, depending on which ions might be present in the preparation or composition), and will understand that, so long as their acid forms (e.g., replacing all cations, if any, with H+) are of the same constitution and / or Page 19 of 566

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[0171] structure, such individual oligonucleotides may properly be considered to be of the same constitution and / or structure.

[0172] Aliphatic: As used herein, “aliphatic” means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation (but not aromatic), or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is completely saturated or that contains one or more units of unsaturation (but not aromatic), or combinations thereof. In certain embodiments, aliphatic groups contain 1-50 aliphatic carbon atoms In certain embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms, in other embodiments, aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1 -5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyi groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0173] Alkenyl: As used herein, the term “alkenyl” refers to an aliphatic group, as defined herein, having one or more double bonds.

[0174] Alkyl: As used herein, the term “alkyl” is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkvl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, alkyl has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C1-C20 for straight chain, C2-C2U for branched chain), and alternatively, about 1-10. In certain embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure where such rings are monocyclic, bicyclic, or polycyclic, and alternatively about 5, 6 or 7 carbons in the ring structure. In certain embodiments, an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C1-C4 for straight chain lower alkyls)

[0175] Alkynyi: As used herein, the term “alkynyi” refers to an aliphatic group, as defined herein, having one or more triple bonds.

[0176] Analog: The tenn “analog” includes any chemical moiety which differs structurally from a reference chemical moiety or class of moieties, but which is capable of performing at least one function of such a reference chemical moiety or class of moieties. As non-limiting examples, a nucleotide analog differs structurally from a nucleotide but performs at least one function of a nucleotide; a nucleobase analog differs structurally from a nucleobase but performs at least one function of a nucleobase; etc.

[0177] Animal: As used herein, the tenn “animal” refers to any member of the animal kingdom. In certain embodiments, “animal” refers to humans, at any stage of development. In certain embodiments, “animal”

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[0180] refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate and / or a pig) In certain embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish and / or worms. In certain embodiments, an animal may be a transgenic animal, a genetically-engineered animal and / or a clone.

[0181] Aryl: The term “aryl", as used herein, used alone or as part of a larger moiety as in “aralkyl,"’ “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic, bicyclic or polycyclic ring systems having a total of five to thirty ring members, wherein at least one ring in the system is aromatic. In certain embodiments, an aryl group is a monocyclic, bicyclic or polycyclic ring system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. In certain embodiments, each monocyclic ring unit is aromatic. In certain embodiments, an aryl group is a biaryl group. The term “ary 1” may be used interchangeably with the term 'aryl ring.” In certain embodiments of the present disclosure, “aryl"’ refers to an aromatic ring system which includes, but is not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “and,” as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphth im idyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

[0182] Chiral control: As used herein, “chiral control"’ refers to control of the stereochemical designation of the chiral linkage phosphorus in a chiral internucleotidic linkage within an oligonucleotide. As used herein, a chiral internucleotidic linkage is an internucleotidic linkage whose linkage phosphorus is chiral. In certain embodiments, a control is achieved through a chiral element that is absent from the sugar and base moieties of an oligonucleotide, for example, in certain embodiments, a control is achieved through use of one or more chiral auxiliaries during oligonucleotide preparation, which chiral auxiliaries often are part of chiral phosphoramidit.es used during oligonucleotide preparation. In contrast to chiral control, a person having ordinary' skill in the art will appreciate that conventional oligonucleotide synthesis which does not use chiral auxiliaries cannot control stereochemistry' at a chiral internucleotidic linkage if such conventional oligonucleotide synthesis is used to form the chiral internucleotidic linkage. In certain embodiments, the stereochemical designation of each chiral linkage phosphorus in each chiral internucleotidic linkage within an oligonucleotide is controlled.

[0183] Chirally controlled oligonucleotide composition: The terms “chirally controlled oligonucleotide composition"’, “chirally controlled nucleic acid composition”, and the like, as used herein, refers to a composition that comprises a plurality of oligonucleotides (or nucleic acids) which share a common base sequence, wherein the plurality' of oligonucleotides (or nucleic acids) share the same linkage phosphorus stereochemistry' at one or more chiral internucleotidic linkages (chirally controlled or stereodefined internucleotidic linkages, whose chiral linkage phosphorus is Rp or Sp in the composition (“stereodefined”),

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[0186] not a random Rp and Sp mixture as non-chirally controlled internucleotidic linkages). In certain embodiments, a chirally controlled oligonucleotide composition comprises a plurality of oligonucleotides (or nucleic acids) that share: 1) a common base sequence, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone phosphorus modifications, wherein the plurality of oligonucleotides (or nucleic acids) share the same linkage phosphorus stereochemistry at one or more chiral internucleotidic linkages (chirally controlled or stereodefined internucleotidic linkages, whose chiral linkage phosphorus is Rp or Sp in the composition (“stereodefined”), not a random Rp and Sp mixture as non-chirally controlled internucleotidic linkages). Level of the plurality of oligonucleotides (or nucleic acids) in a chirally controlled oligonucleotide composition is pre-determined / controlled or enriched (e.g., through chirally controlled oligonucleotide preparation to stereoselectively form one or more chiral internucleotidic linkages) compared to a random level in a non- chirally controlled oligonucleotide composition. In certain embodiments, about 1%-100%, (e.g., about 5%- 100%. 10%-l 00%, 20%-l00%, 30%-l 00%. 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%. or about 5%, 10%, 20%, 30%, 40%, 50%. 60%, 70%, 80%, 85%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally’ controlled oligonucleotide composition arc oligonucleotides of the plurality. In certain embodiments, about l%-100%. (e.g., about 5%-100%, 10%-100%, 20%-100%. 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%. 90-100%, 95-100%, 50%-90%, or about 5%, 10%. 20%. 30%. 40%. 50%. 60%. 70%.

[0187] 80%, 85%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, 99%, or 100%. or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 1%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition that share the common base sequence, the common pattern of backbone linkages, and the common pattern of backbone phosphorus modifications are oligonucleotides of the plurality. In certain embodiments, a level is about l%-100%, (e.g.. about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%. 50%-100%, 60%-l00%, 70%-100%, 80-100%, 90-100%.

[0188] 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%. 40%, 50%, 60%. 70%, 80%, 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%. 60%. 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a composition, or of all oligonucleotides in a composition that share a common base sequence (e.g., of a plurality of oligonucleotide or an oligonucleotide type), or of all oligonucleotides in a composition that share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone phosphorus modifications, or of all oligonucleotides in a composition that share a common base sequence, a common patter of base modifications, a common pattern of sugar modifications, a common pattern of internucleotidic linkage types, and / or a common pattern of internucleotidic linkage modifications. In certain embodiments, the plurality- of oligonucleotides share the same stereochemistry at about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30, or about 1, 2, 3, 4, 5. 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17. 18, 19, or 20. or at least 1, 2, 3,

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[0191] 4, 5, 6, 7, 8, 9. 10. 11. 12. 13, 14, 15, 16, 17, 18, 19, or 20) chiral internucleotidic linkages. In certain embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1%- 100% (e.g, about 5%-100?% 10%-100%, 20%-100?% 30%-100?% 40%-100%, 50%-100%, 60%-100?% 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, about 5%, 10%, 15%, 20?% 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60?% 65%, 70%, 75%, 80?% 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%. 65%. 70%, 75%, 80%, 85%, 90%. 95%, or 99%) of chiral internucleotidic linkages. In certain embodiments, oligonucleotides (or nucleic acids) of a plurality share the same pattern of sugar and / or nucleobase modifications, in any. In certain embodiments, oligonucleotides (or nucleic acids) of a plurality are various forms of the same oligonucleotide (e.g., acid and / or various salts of the same oligonucleotide). In certain embodiments, oligonucleotides (or nucleic acids) of a plurality are of the same constitution. In certain embodiments, level of the oligonucleotides (or nucleic acids) of the plurality is about l%-100?% (e.g., about 5%-100?% 10%-100?% 20%-100?% 30%-100?% 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100?% 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50?% 60%, 70?% 80%. 85%, 90%. 91%, 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, or 100%. or at least 5%, 10%, 20?% 30%, 40%, 50%, 60%, 70%, 80%, 85?% 90%, 91%, 92%, 93?% 94%, 95%, 96%, 97%, 98?% or 99%) of all oligonucleotides (or nucleic acids) in a composition that share the same constitution as the oligonucleotides (or nucleic acids) of the plurality. In certain embodiments, each chiral internucleotidic linkage is a chiral controlled internucleotidic linkage, and the composition is a completely chirally controlled oligonucleotide composition. In certain embodiments, oligonucleotides (or nucleic acids) of a plurality are structurally identical. In certain embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of at least 80%, 85%, 90?% 91%, 92%, 93%, 94?% 95?% 96%, 97?% 98%. 99% or 99.5%, typically at least 90%, 91%, 92%, 93%, 94%, 95?% 96%, 97%, 98%. 99% or 99.5?% In certain embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of at least 95%. In certain embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of at least 96%. In certain embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of at least 97%. In certain embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of at least 98%. In certain embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of at least 99%. In certain embodiments, a percentage of a level is or is at least (DS)nc, wherein DS is a diastereopurity as described in the present disclosure (e.g., 90?% 91%, 92%, 93?% 94%, 95?% 96%, 97?% 98?% 99% or 99.5% or more) and nc is the number of chirally controlled internucleotidic linkages as described in the present disclosure (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25. 5-20. i, 2, 3.

[0192] 4, 5. 6, 7, 8, 9. 10, I I, 12, 13, 14. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). In certain embodiments, a percentage of a level is or is at least (DS)nc, wherein DS is 95%-100?% For example, when DS is 99% and nc is 10, the percentage is or is at least 90% ((99%)10~ 090 = 90?o). In certain embodiments, level of a plurality of oligonucleotides in a composition is represented as the product of the diastereopurity of each chirally controlled internucleotidic linkage in the oligonucleotides. In certain embodiments, diastereopurity of Page 23 of 566

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[0194] an internucleotidic linkage connecting two nucleosides m an oligonucleotide (or nucleic acid) is represented by the diastereopurity of an internucleotidic linkage of a dimer connecting the same two nucleosides, wherein the dimer is prepared using comparable conditions, in some instances, identical synthetic cycle conditions (e.g., for the linkage between Nx and Ny in an oligonucleotide.... NxNy, the dimer is NxNy). In certain embodiments, not all chiral internucleotidic linkages are chiral controlled internucleotidic linkages, and the composition is a partially chirally controlled oligonucleotide composition. In certain embodiments, a non¬ chirally controlled internucleotidic linkage has a diastereopurity of less than about 80%, 75%, 70%, 65%, 60%.

[0195] 55%, or of about 50%, as typically observed in stereorandom oligonucleotide compositions (e.g., as appreciated by those skilled in the art, from traditional oligonucleotide synthesis, e.g., the phosphoramidite method). In certain embodiments, oligonucleotides (or nucleic acids) of a plurality are of the same type. In certain embodiments, a chirally controlled oligonucleotide composition comprises non-random or controlled levels of individual oligonucleotide or nucleic acids types. For instance, in certain embodiments a chirally controlled oligonucleotide composition comprises one and no more than one oligonucleotide type. In certain embodiments, a chirally controlled oligonucleotide composition comprises more than one oligonucleotide type. In certain embodiments, a chirally controlled oligonucleotide composition comprises multiple oligonucleotide types. In certain embodiments, a chirally controlled oligonucleotide composition is a composition of oligonucleotides of an oligonucleotide type, which composition comprises a non-random or controlled level of a plurality of oligonucleotides of the oligonucleotide type.

[0196] Comparable: The term “comparable” is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to one another to permit comparison of results obtained or phenomena observed. In certain embodiments, comparable sets of conditions or circumstances are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will appreciate that sets of conditions are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in resul ts ob tained or phenomena observed under the different sets of conditions or circumstances are caused by or indicative of the variation in those features that are varied.

[0197] Cycloaliphatic: The temi “cycloaliphatic,” “carbocycle,” “carbocyclyl,” “carbocyclic radical,” and “carbocyclic ring,” are used interchangeably, and as used herein, refer to saturated or partially unsaturated, but non-aromatic, cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems, as described herein, having, unless otherwise specified, from 3 to 30 ring members. Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cy elooety 1. cyclooctenyl, norbomyl, adamantyl, and cyclooctadienyl. In certain embodiments, a cycloaliphatic group has 3-6 carbons. In certain embodiments, a cycloaliphatic group is saturated and is cycloalkyl. The term “cycloaliphatic” may also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl or tetrahydronaphthyl. In certain embodiments, a cycloaliphatic group is Page 24 of 566

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[0199] bicyclic. In certain embodiments, a cycloaliphatic group is tricyclic. In certain embodiments, a cycloaliphatic group is polycyclic. In certain embodiments, ‘'cycloaliphatic” refers to Cs-Cs monocyclic hydrocarbon, or Cs- Cio bicyclic or polycyclic hydrocarbon, that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule, or a C -Cis polycyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule.

[0200] Heteroaliphatic: The term “heteroaliphatic”, as used herein, is given its ordinary meaning in the art and refers to aliphatic groups as described herein in which one or more carbon atoms are independently- replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like). In certain embodiments, one or more units selected from C, CH, CH2, and CH3 are independently replaced by one or more heteroatoms (including oxidized and / or substituted forms thereof). In certain embodiments, a heteroaliphatic group is heteroalkyl. In certain embodiments, a heteroaliphatic group is heteroalkenyl.

[0201] Heteroalkyl: The term “heteroalkyl”, as used herein, is given its ordinary meaning in the art and refers to alkyl groups as described herein in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like). Examples of heteroalkyl groups include, but arc not limited to, alkoxy-, poly(cthylcnc glycol)-, alkyl-substitutcd amino, tetrahydrofuranyl, piperidmyl, morpholinyl, etc.

[0202] Heteroaryl: The terms “heteroaryl” and “heteroar-”, as used herein, used alone or as part of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer to monocyclic, bicyclic or polycy clic ring systems having a total of five to thirty- ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In certain embodiments, a heteroaryl group is a group having 5 to 10 ring atoms (i.e., monocyclic, bicyclic or polycy clic), in certain embodiments 5, 6, 9, or 10 ring atoms. In certain embodiments, each monocyclic ring unit is aromatic. In certain embodiments, a heteroaryl group has 6, 10, or 14 71 electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl. tetrazolyl, oxazolyl, isoxazolyl. oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl. and pteridiny-1. In certain embodiments, a heteroaryl is a heterobiaryl group, such as bipyridyl and the like. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaroniatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of atachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthal azinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-I,4-oxazin-3(4H)-one. A heteroaryl group may be monocyclic, bicyclic or polycyclic. The term “heteroaryl” may be used interchangeably with the terms '‘heteroaryl ring,”

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[0205] “heteroaryl group / ’ or “heteroaromatic,” any of which terms include rings that are optionally substituted, lire term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl group, wherein the alkyl and heteroaryl portions independently are optionally substituted.

[0206] Heteroatom: The term “heteroatom", as used herein, means an atom that is not carbon or hydrogen. In certain embodiments, a heteroatom is boron, oxygen, sulfur, nitrogen, phosphorus, or silicon (including oxidized fomis of nitrogen, sulfur, phosphorus, or silicon; charged forms of nitrogen (e.g., quatemized forms, forms as in iminium groups, etc.), phosphorus, sulfur, oxygen; etc.). In certain embodiments, a heteroatom is silicon, phosphorus, oxygen, sulfur or nitrogen. In certain embodiments, a heteroatom is silicon, oxygen, sulfur or nitrogen. In certain embodiments, a heteroatom is oxygen, sulfur or nitrogen.

[0207] Heterocycle: As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring", as used herein, are used interchangeably and refer to a monocyclic, bicyclic or polycyclic ring moiety (e.g., 3-30 membered) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In certain embodiments, a heterocyclyl group is a stable 5- to 7-membered monocyclic or 7 - to 10 -membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur and nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl). NH (as in pyrrolidmyl). or+NR (as in N-substituted pyrrohdinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrohdinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle.” “heterocyclyl,” “heterocyclyl ring.” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical,” are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more ary l, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyi, or tetrahydroquinolinyl. A heterocyclyl group may be monocyclic, bicyclic or polycyclic. Tire term “heterocyclyl alkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[0208] Identity: As used herein, the temi “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., oligonucleotides. DNA, RNA, etc.) and / or between polypeptide molecules. In certain embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be perfonned by aligning the two sequences for optimal comparison Page 26 of 566

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[0210] purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%. at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence. Tire nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS. 1989. 4: 11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4 The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna. CMP matrix.

[0211] Intemucleotidic linkage: As used herein, the phrase “internucleotidic linkage" refers generally to a linkage linking nucleoside units of an oligonucleotide or a nucleic acid. In certain embodiments, an internucleotidic linkage is a phosphodiester linkage, as extensively found in naturally occurring DNA and RNA molecules (natural phosphate linkage (-OP(=O)(OH)O-), which as appreciated by those skilled in the art may exist as a salt form). In certain embodiments, an internucleotidic linkage is a modified internucleotidic linkage (not a natural phosphate linkage). In certain embodiments, an internucleotidic linkage is a “'modified internucleotidic linkage" w herein at least one oxygen atom or -OH of a phosphodiester linkage is replaced by a different organic or inorganic moiety. In certain embodiments, such an organic or inorganic moiety is selected from =S, =Se, =NR’, -SR', -SeR’, -N(R B(R')3. -S-, -Se-, and -N(R’)-, wherein each R’ is independently as defined and described in the present disclosure. In certain embodiments, an internucleotidic linkage is a phosphotriester linkage, phosphorothioate linkage (or phosphorothioate diester linkage, OP(=O)(SH)O, which as appreciated by those skilled in the art may exist as a salt form), or phosphorothioate triester linkage. In certain embodiments, a modified internucleotidic linkage is a phosphorothioate linkage. In certain embodiments, a modified internucleotidic linkage is a phosphoryl guanidine linkage. In certain embodiments, an internucleotidic linkage is one of. e.g., PNA (peptide nucleic acid) or PMO (phosphorodiamidate Morpholino oligomer) linkage. In certain embodiments, a modified internucleotidic linkage is a non-negatively charged internucleotidic linkage. In certain embodiments, a modified internucleotidic linkage is a neutral internucleotidic linkage (e.g., nOOl in certain provided oligonucleotides). In certain a embodiments, a phosphoryl guanidine linkage is a non-negatively charged linkage or a neutral Page 27 of 566

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[0213] internucleotidic linkage. In certain embodiments, an internucleotidic linkage is a PN internucleotidic linkage, e.g. a phosphoryl guanidine internucleotidic linkage or a mesyl phosphoramidate (MsPA) internucleotidic linkage. It is understood by a person of ordinary' skill in the art that an internucleotidic linkage may exist as an anion or cation at a given pH due to the existence of acid or base moieties in the linkage. In certain embodiments, a modified internucleotidic linkages is a modified internucleotidic linkages designated as s, si, s2, s3. s4, s5, s6, s7. s8, s9, slO, si 1, s!2, s!3, s!4, s 15, s!6, s!7 and s!8 as described in WO 2017 / 210647.

[0214] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g., animal, plant and / or microbe).

[0215] In vivo: As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant and / or microbe).

[0216] Linkage phosphorus: as defined herein, the phrase “linkage phosphorus” is used to indicate that the particular phosphorus atom being referred to is the phosphorus atom present in the internucleotidic linkage, which phosphorus atom corresponds to the phosphorus atom of a phosphodiester internucleotidic linkage as occurs in naturally occurring DNA and RNA In certain embodiments, a linkage phosphorus atom is in a modified internucleotidic linkage, wherein each oxygen atom of a phosphodiester linkage is optionally’ and independently replaced by an organic or inorganic moiety. In certain embodiments, a linkage phosphorus atom is chiral (e.g., as in phosphorothioate internucleotidic linkages). In certain embodiments, a linkage phosphorus atom is achiral (e.g.. as in natural phosphate linkages).

[0217] Modified nucleobase: The terms "modified nucleobase", "modified base" and the like refer to a chemical moiety which is chemically distinct from a nucleobase, but which is capable of performing at least one function of a nucleobase. In certain embodiments, a modified nucleobase is a nucleobase which comprises a modification. In certain embodiments, a modified nucleobase is capable of at least one function of a nucleobase, e.g.. forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising an at least complemen tary' sequence of bases. In certain embodiments, a modified nucleobase is substituted A, T, C, G, or U, or a substituted tautomer of A, T, C, G. or U. In certain embodiments, a modified nucleobase in the context of oligonucleotides refer to a nucleobase that is not A, T, C, G or U.

[0218] Modified nucleoside: Tire term "modified nucleoside" refers to a moiety derived from or chemically similar to a natural nucleoside, but which comprises a chemical modification which differentiates it from a natural nucleoside. Non -limiting examples of modified nucleosides include those which comprise a modification at the base and / or the sugar. Non-limiting examples of modified nucleosides include those w ith a 2’ modification at a sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (which lack a nucleobase). In certain embodiments, a modified nucleoside is capable of at least one function of a nucleoside, e.g., forming a moiety in a poly-mer capable of base -pairing to a nucleic acid comprising an at least complementary sequence of bases.

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[0221] Modified nucleotide: The term “modified nucleotide” includes any chemical moiety which differs structurally from a natural nucleotide but is capable of performing at least one function of a natural nucleotide. In certain embodiments, a modified nucleotide comprises a modification at a sugar, base and / or internucleotidic linkage. In certain embodiments, a modified nucleotide comprises a modified sugar, modified nucleobase and / or modified internucleotidic linkage. In certain embodiments, a modified nucleotide is capable of at least one function of a nucleotide, e.g., fonning a subunit in a polymer capable of base -pairing to a nucleic acid comprising an at least complementary sequence of bases.

[0222] Modified sugar: Tire term "modified sugar” refers to a moiety that can replace a sugar. A modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical property of a sugar. In certain embodiments, as described in the present disclosure, a modified sugar is substituted ribose or deoxyribose. In certain embodiments, a modified sugar comprises a 2' -modification, Examples of useful 2’-modification are widely utilized in the art and described herein. In certain embodiments, a 2’-modification is 2’-F. In certain embodiments, a 2’ -modification is 2’-OR, wherein R is optionally substituted Cuio aliphatic. In certain embodiments, the 2’ modification is a 2’-O-C16 lipid modification. In certain embodiments, a 2’- modification is 2’-OMe. In certain embodiments, a 2’ -modification is 2 -MOE. In certain embodiments, a modified sugar is a bicyclic sugar (c.g,, a sugar used in LNA, BNA, etc.). In certain embodiments, in the context of oligonucleotides, a modified sugar is a sugar that is not ribose or deoxyribose as typically found in natural RNA or DNA.

[0223] Nucleic acid: The term “nucleic acid”, as used herein, includes any nucleotides and polymers thereof. The term “polynucleotide”, as used herein, refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) or a combination thereof. These terms refer to the primary structure of the molecules and. thus, include double- and single -stranded DNA, and double- and single¬ stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA comprising modified nucleotides and / or modified polynucleotides, such as, though not limited to, methylated, protected and / or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxynbonucleotides (DNA); RNA or DNA derived from N-glycosides or C -glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified internucleotidic linkages. Tire term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified internucleotidic linkages. Examples include, and are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. Unless otherwise specified, the prefix poly- refers to a nucleic acid containing 2 to about 10,000 nucleotide monomer units and wherein the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.

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[0226] Nucleobase: The term "nucleobase" refers to the parts of nucleic acids that are involved in the hydrogen-bonding that binds one nucleic acid strand to another complementary strand m a sequence specific manner. The most common naturally-occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In certain embodiments, a naturally-occurring nucleobases are modified adenine, guanine, uracil, cytosine, or thymine. In certain embodiments, a naturally-occurring nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In certain embodiments, a nucleobase comprises a heteroaryl ring wherein a ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In certain embodiments, a nucleobase comprises a heterocyclic ring wherein a ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In certain embodiments, a nucleobase is a “modified nucleobase,’’ a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T), In certain embodiments, a modified nucleobase is substituted A, T, C, G or U. In certain embodiments, a modified nucleobase is a substituted tautomer of A. T, C, G, or U. In certain embodiments, a modified nucleobase is methylated adenine, guanine, uracil, cytosine, or thymine. In certain embodiments, a modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobase and retains the property of hydrogen -bonding that binds one nucleic acid strand to another in a sequence specific manner In certain embodiments, a modified nucleobase can pair with all of the five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide duplex. As used herein, the term “nucleobase” also encompasses structural analogs used m lieu of natural or naturally-occurring nucleotides, such as modified nucleobases and nucleobase analogs. In certain embodiments, a nucleobase is optionally substituted A. T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U. In certain embodiments, a “nucleobase” refers to a nucleobase unit in an oligonucleotide or a nucleic acid (e.g., A, T, C, G or U as in an oligonucleotide or a nucleic acid).

[0227] Nucleoside: The term “nucleoside” refers to a moiety wherein a nucleobase or a modified nucleobase is covalently bound to a sugar or a modified sugar In certain embodiments, a nucleoside is a natural nucleoside, e.g., adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. In certain embodiments, a nucleoside is a modified nucleoside, e.g., a substituted natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxy cytidine. In certain embodiments, a nucleoside is a modified nucleoside, e.g., a substituted tautomer of a natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxy cytidine. In certain embodiments, a “nucleoside” refers to a nucleoside unit m an oligonucleotide or a nucleic acid.

[0228] Nucleotide: The term “nucleotide” as used herein refers to a monomeric unit of a polynucleotide that consists of a nucleobase, a sugar, and one or more internucleotidic linkages (e.g., phosphate linkages in natural DNA and RNA). Hie naturally occurring bases [guanine. (G), adenine. (A), cytosine, (C), thymine, (T), and Page 30 of 566

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[0230] uracil (LT) ] are derivatives of purine or pyrimidine, though it should be understood that naturally and non-naturally occurring base analogs are also included. The naturally occurring sugar is the pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), though it should be understood that naturally and non-naturally occurring sugar analogs are also included. Nucleotides are linked via internucleotidic linkages to form nucleic acids, or polynucleotides. Many internucleotidic linkages are known in the art (such as, though not limited to, phosphate, phosphorothioates, boranophosphates and the like). Artificial nucleic acids include PNAs (peptide nucleic acids), phosphotriesters, phosphorothionates, H-phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates and other variants of the phosphate backbone of native nucleic acids, such as those described herein. In certain embodiments, a natural nucleotide comprises a naturally occurring base, sugar and internucleotidic linkage. As used herein, the term “nucleotide" also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified nucleotides and nucleotide analogs. In certain embodiments, a “nucleotide’’ refers to a nucleotide unit in an oligonucleotide or a nucleic acid.

[0231] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotides, and may contain any combination of natural and non-natural nucleobases, sugars, and internucleotidic linkages.

[0232] Oligonucleotides can be single-stranded or double-stranded, A single -stranded oligonucleotide can have double -stranded regions (formed by two portions of the single -stranded oligonucleotide) and a double¬ stranded oligonucleotide, which comprises two oligonucleotide chains, can have single-stranded regions for example, at regions where the two oligonucleotide chains are not complementary to each other. Example oligonucleotides include, but are not limited to structural genes, genes including control and termination regions, self-replicating systems such as viral or plasmid DNA, single-stranded and double-stranded RNAi agents and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs. microRNA mimics, supermirs, aptamers, antimirs. antagomirs, U1 adaptors, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

[0233] Oligonucleotides of the present disclosure can be of various lengths. In particular embodiments, oligonucleotides can range from about 2 to about 200 nucleosides in length. In various related embodiments, oligonucleotides, single-stranded, double-stranded, or triple-stranded, can range in length from about 4 to about 10 nucleosides, from about 10 to about 50 nucleosides, from about 20 to about 50 nucleosides, from about 15 to about 30 nucleosides, from about 20 to about 30 nucleosides in length. In certain embodiments, the oligonucleotide is from about 9 to about 39 nucleosides in length. In certain embodiments, the oligonucleotide is at least 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, or 25 nucleosides in length In certain embodiments, the oligonucleotide is at least 4 nucleosides in length. In certain embodiments, the oligonucleotide is at least 5 nucleosides in length. In certain embodiments, the oligonucleotide is at least 6 nucleosides in length. In certain embodiments, the oligonucleotide is at least 7 nucleosides in length. In certain Page 31 of 566

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[0235] embodiments, the oligonucleotide is at least 8 nucleosides m length. In certain embodiments, the oligonucleotide is at least 9 nucleosides in length. In certain embodiments, the oligonucleotide is at least 10 nucleosides in length. In certain embodiments, the oligonucleotide is at least 11 nucleosides in length In certain embodiments, the oligonucleotide is at least 12 nucleosides in length. In certain embodiments, the oligonucleotide is at least 15 nucleosides in length. In certain embodiments, the oligonucleotide is at least 15 nucleosides in length. In certain embodiments, the oligonucleotide is at least 16 nucleosides in length. In certain embodiments, the oligonucleotide is at least 17 nucleosides in length. In certain embodiments, the oligonucleotide is at least 18 nucleosides in length. In certain embodiments, the oligonucleotide is at least 19 nucleosides in length. In certain embodiments, the oligonucleotide is at least 20 nucleosides in length. In certain embodiments, tire oligonucleotide is at least 25 nucleosides in length. In certain embodiments, the oligonucleotide is at least 30 nucleosides in length. In certain embodiments, each nucleoside counted in an oligonucleotide length independently comprises a nucleobase comprising a ring having at least one nitrogen ring atom. In certain embodiments, each nucleoside counted in an oligonucleotide length independently comprises A. T, C. G, or U, or optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G or U.

[0236] Oligonucleotide type: As used herein, the phrase “oligonucleotide type” is used to define an oligonucleotide that has a particular base sequence, pattern of backbone linkages (i.e., pattern of internucleotidic linkage types, for example, phosphate, phosphorothioate, phosphorothioate triester, etc.), pattern of backbone chiral centers (i.e.. pattern of linkage phosphorus stereochemistry (Rp / Sp)), and pattern of backbone phosphorus modifications. In certain embodiments, oligonucleotides of a common designated “type” are structurally identical to one another.

[0237] One of skill in the art will appreciate that synthetic methods of the present disclosure provide for a degree of control during tire synthesis of an oligonucleotide strand such that each nucleotide unit of the oligonucleotide strand can be designed and / or selected in advance to have a particular stereochemistry at the linkage phosphorus and / or a particular modification at the linkage phosphorus, and / or a particular base, and / or a particular sugar. In certain embodiments, an oligonucleotide strand is designed and / or selected in advance to have a particular combination of stereocenters at the linkage phosphorus. In certain embodiments, an oligonucleotide strand is designed and / or determined to have a particular combination of modifications at the linkage phosphorus. In certain embodiments, an oligonucleotide strand is designed and / or selected to have a particular combination of bases. In certain embodiments, an oligonucleotide strand is designed and / or selected to have a particular combination of one or more of the above structural characteristics. In certain embodiments, the present disclosure provides compositions comprising or consisting of a plurality of oligonucleotide molecules (e.g., chirally controlled oligonucleotide compositions). In certain embodiments, all such molecules are of the same type (i.e., are structurally identical to one another). In certain embodiments, however, provided compositions comprise a plurality of oligonucleotides of different types, typically in pre -determined relative Page 32 of 566

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[0239] amounts.

[0240] Optionally Substituted: As described herein, compounds, e g., oligonucleotides, of the disclosure may contain optionally substituted and / or substituted moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at even? position. In certain embodiments, an optionally substituted group is unsubstituted. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Certain substituents are described below.

[0241] Suitable monovalent substituents on a substitutable atom, e.g, a suitable carbon atom, are independently halogen; -(CH2)0-4R°; -(CH2)0-4OR°; -O(CH2)0-4R°, -O(CH2)0-4C(O)OR°: -(CH2)0- CH(OR ) (CH ). Ph which may be substituted with R°: -(CH2)0-4O(CH2)0-1Ph which may be substituted with R: -CH=CHPh, which may be substituted with R°; -(CH2)0-4O(CH2)0-1-pyridyl which may be substituted with R°; ~NO2; -CN; -N3; -(CH2)0-4N(RC)2; -(CH2)0-4N(RC)C(O)R°; -N(R°)C(S)RC: -(CH2)0-4N(RO)C(O)NR°2; ”N(RO)C(S)NR°2; -(CH2)0-4N(R°)C(O)ORC; -N(RC)N(R°)C(O)R°; -N(R°)N(R°)C(O)NRO2; -N(R°)N(R°)C(O)OR°; -(CH2)0-4C(O)RO; -C(S)R°; -(CH2)0-4C(O)ORO; -(CH2)0-4C(O)SR°; -(CH2)0-4C(O)OSiR°3: -(CH2)0-3C(O)R°; -OC(O)(CH2)0-4SR°, -SC(S)SR°; -(CH2)0^SC(O)R°; -(CH2)0-1C(O)NR°2; -C(S)NRO2; -C(S)SR°; -(CH2)0^OC(O)NR°2; -C(O)N(OR°)R°; C(O)C(O)R°; - C(O)CH2C(O)R°; -C(NOR°)R°; -(CH2)0-4SSRO; -(CH2)0-4S(O)2RO; -(CH2)0-4S(O)2OR°; -(CH2)0-4OS(O)2RO; -S(0)2NRO2: -(CH2)0-4S(O)R°; -N(RO)S(O)2NR°2; -N(R°)S(O)2R°; -N(OR°)R°; -C(NH)NRO2; -Si(R°); -OSi(R°)3; -B(RO)2; -0B(RO)2; -OB(OR°)2; -P(RO)2; -P(OR°)2; -P(R°)(OR°): -OP(R°)2; -OP(OR°)2; -OP(R°)(OR°); -P(O)(R°)2; ~P(0)(0RO)2; ~0P(0)(RO)2; ~OP(O)(OR°)2: -OP(O)(OR°)(SR°); -SP(O)(R°)2; -SP(O)(ORO)2; -N(R°)P(O)(RO)2; -N(RO)P(O)(OR°)2; -P(RO)2[B(R°)3]; -P(OR°)2[B(RO)3]: -OP(R°)2[B(RO)3]; -OP(ORO)2[B(R°)3]; -(C1-4straight or branched alkylene)O-N(R°)2; or -(C’1-4 straight or branched alkylene)C(O)O -N(R°)2, wherein each R° may be substituted as defined herein and is independently hydrogen, C1-20aliphatic, C1-20heteroaliphatic having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, -CH2-(C6-14aryl), -O(CH2)0-1(C6-14aryl), -CH2-(5-14 membered heteroaryl ring), a 5-20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening Page 33 of 566

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[0243] atom(s), form a 5-20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below.

[0244] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)0-2R®, -(haloR*), -(CH2)0-2OH, -(CH2)0-2OR®, -(CH2)0-2CH(OR*)2; ~O(haloR*), -CN, -N3, -(CH2)0-2C(O)R*, -(CH2)0-2C(O)OH, -(CH2)0-2C(O)OR*, -(CH2)0-2SR*, -(CH2)0-2SH, -(CH2)0-2NH2, -(CH2)0-2NHR*, -(CH2)O2R’2, -NO2, -SiR*3, -OSiR*3, -C(O)SR* (Ci straight or branched alkylene)C(O)OR®, or SSR® wherein each R* is unsubstituted or where preceded by “halo" is substituted only with one or more halogens, and is independently selected from C1-4aliphatic, -CH2Ph, -O(CH2)0-1Ph, and a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0245] Suitable divalent substituents, e.g., on a suitable carbon atom, are independently the following: O. =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)2R*, =NR*. =NOR*, -O(C(R*2))2-3O-, or -S(C(R*2))2-3S-, wherein each independent occurrence of R* is selected from hydrogen, Ci-g aliphatic which may be substituted as defined below, and an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR*2)2-3O-, wherein each independent occurrence of R* is selected from hydrogen, Ci-e aliphatic which may be substituted as defined below, and an unsubstituted 5-6-membered saturated, partially unsaturated, and aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0246] Suitable substituents on the aliphatic group of R are independently halogen, R®, -(haloR*), OH, -OR®, -O(haloR’), -CN, -C(O)OH, -C(O)OR®, -NH2, -NHR®, -NR®2, or -NO2, wherein each R® is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently CM aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0247] In certain embodiments, suitable substituents on a substitutable nitrogen are independently -R, -NR’2, -C(O)Rt, -C(O)ORt, -C(O)C(O)Rt, -C(O)CH2C(O)Rt, -S(O)2Rt, -S(O)2NRt2, -C(S)NRt2, -C(NH)NRt2, or -N(Rt)S(O)2R1'; wherein each R ' is independently hydrogen, C1-4aliphatic which may be substituted as defined below', unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or ary l ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the definition above, two independent occurrences of Rt, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0248] Suitable substituents on the aliphatic group of R1 are independently halogen, -R®. -(haloR*), -OH, - Page 34 of 566

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[0250] OR*, -O(haloR*), -CN, C(O)OH, C(O)OR”, -NH2, -NHR*. -NR*2. or -NO2, wherein each R* is unsubstituted or where preceded by ‘'halo” is substituted only with one or more halogens, and is independently C1-4aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0251] P -modification: as used herein, the term “P -modification” refers to any modification at the linkage phosphorus other than a stereochemical modification. In certain embodiments, a P -modification comprises addition, substitution, or removal of a pendant moiety covalently attached to a linkage phosphorus.

[0252] Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

[0253] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In certain embodiments, an active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In certain embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g.. those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transderm ally; or nasally, pulmonary, and to other mucosal surfaces.

[0254] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0255] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each earner must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable earners include: sugars, such as Page 35 of 566

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[0257] lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or poly anhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0258] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals ■without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In certain embodiments, pharmaceutically acceptable salt include, but are not limited to. nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. In certain embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate. 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like In certain embodiments, a provided compound comprises one or more acidic groups, e.g., an oligonucleotide, and a pharmaceutically acceptable salt is an alkali, alkaline earth metal, or ammonium (e.g., an ammonium salt of N(R)s, wherein each R is independently defined and described in the present disclosure) salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In certain embodiments, a pharmaceutically acceptable salt is a sodium salt. In certain embodiments, a pharmaceutically acceptable salt is a potassium salt In certain embodiments, a pharmaceutically acceptable salt is a calcium salt. In certain embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate. In certain Page 36 of 566

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[0260] embodiments, a provided compound comprises more than one acid groups, for example, an oligonucleotide may comprise two or more acidic groups (e.g., in natural phosphate linkages and / or modified internucleotidic linkages). In certain embodiments, a pharmaceutically acceptable salt, or generally a salt, of such a compound comprises two or more cations, which can be the same or different. In certain embodiments, in a pharmaceutically acceptable salt (or generally, a salt), all ionizable hydrogen (e.g., in an aqueous solution with a pKa no more than about 11. 10. 9, 8, 7. 6, 5, 4. 3, or 2; in certain embodiments, no more than about 7; in certain embodiments, no more than about 6; in certain embodiments, no more than about 5; in certain embodiments, no more than about 4: in certain embodiments, no more than about 3) in the acidic groups are replaced with cations. In certain embodiments, each phosphorothioate and phosphate group independently exists in its salt form (e.g., if sodium salt, ~O~P(O)(SNa)~O~ and ~O“P(O)(ONa)~O“, respectively). In certain embodiments, each phosphorothioate and phosphate internucleotidic linkage independently exists in its salt form (e.g., if sodium salt, “O”P(O)(SNa)”O~ and “O~P(O)(ONa)~O“. respectively). In certain embodiments, a pharmaceutically acceptable salt is a sodium salt of an oligonucleotide. In certain embodiments, a pharmaceutically acceptable salt is a sodium salt of an oligonucleotide, wherein each acidic phosphate and modified phosphate group (e.g., phosphorothioate, phosphate, etc.), if any, exists as a salt form (all sodium salt).

[0261] Predetermined: By predetermined (or pre -determined) is meant deliberately selected or non-random or controlled, for example as opposed to randomly occurring, random, or achieved without control. Those of ordinary skill in the art. reading the present specification, will appreciate that the present disclosure provides technologies that permit selection of particular chemistry and / or stereochemistry’ features to be incorporated into oligonucleotide compositions, and further permits controlled preparation of oligonucleotide compositions having such chemistry and / or stereochemistry features. Such provided compositions are “predetermined” as described herein. Compositions that may contain certain oligonucleotides because they happen to have been generated through a process that are not controlled to intentionally generate the particular chemistry and / or stereochemistry features are not “predetermined” compositions. In certain embodiments, a predetermined composition is one that can be intentionally reproduced (e.g., through repetition of a controlled process). In certain embodiments, a predetermined level of a plurality of oligonucleotides in a composition means that the absolute amount, and / or the relative amount (ratio, percentage, etc.) of the plurality of oligonucleotides in the composition is controlled. In certain embodiments, a predetermined level of a plurality of oligonucleotides in a composition is achieved through chirally controlled oligonucleotide preparation.

[0262] 5’ phosphate mimic modification: The term “5' phosphate mimic modification” refers to a chemical modification of the 5’-phosphate of, e.g., the 5’ terminus of an oligonucleotide which mimics phosphate properties while maintaining metabolic stability that could maintain or lead to more efficient guide strand incorporation into Ago2 and therefore maintaining or improving RNA silencing (RNAi) activity of tire oligonucleotide.

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[0265] Protecting group: The term '’protecting group,’’ as used herein, is well known in the art and includes those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rdedition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Also included are those protecting groups specially adapted for nucleoside and nucleotide chemistry described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06 / 2012. the entirety of Chapter 2 is incorporated herein by reference. Suitable amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc). 9-(2-sulfo)fluorenylmethyl carbamate. 9-(2,7-dibromo)fluoroenylm ethyl carbamate, 2,7-di-t-butyl-[9-( 10, 10-dioxo- 10, 10,10, 10- tetrahydrothioxanthyl)] methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2- trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1 -di methyl -2-haloethyl carbamate, 1, l-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), l,l-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC). 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), l-(3,5-di-t-butylphenyl)-l-methylethyl carbamate (t-Bumeoc), 2- (2’- and 4’-pyridyl)ethyl carbamate (Pyoc), 2-(N, N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1- isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p- methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p -bromobenzyl carbamate, p -chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(l,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4 -dimethy lthiophenyl carbamate (Bmpc), 2 -phosphonioethyl carbamate (Peoc), 2- triphenylphosphonioisopropyl carbamate (I’poc), l,l-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate. 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate. 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivative, N'-p-toluenesulfonylaminocarbonyl derivative, N’- phenylammothiocarbonyl derivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p- decyloxybenzyl carbamate, 2,2 -dimethoxy carbonyl vinyl carbamate. o-(N, N-dimethylcarboxamido)benzyl carbamate, l,l-dimethyl-3-(N, N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate. di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p’-methoxyphenylazo)benzyl carbamate, 1 -methylcyclobutyl carbamate, 1 -methylcyclohexyl carbamate, 1 -methyl- 1-cyclopropylmethyl carbamate, l-methyl-l-(3,5- dimethoxyphenyl)ethyl carbamate, 1 -methyl- 1 -(p--phenylazophenyl)ethyl carbamate, 1 -methyl- 1- Page 38 of 566

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[0267] phenylethyl carbamate. 1 -methyl-- l--(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(tnmethylammonium)benzyl carbamate. 2,4,6-trim ethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative. benzamide, p -phenylbenzamide, o -nitophenylacetamide, o -nitrophenoxyacetamide, acetoacetamide, (N '-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o- nitrophenyl)propanamide. 2-methyl-2-(o-nitrophenoxy)propanamide. 2-methyl-2-(o-phenylazophenoxyjpropanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N- acetylmethionine derivative, o -nitrobenzamide, o -(benzoyloxymethyl)benzamide, 4,5-diphenyl-3 - oxazolin -2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N- 1,1,4,4-tetramethyldisih lazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl- 1,3,5-triazacyclohexan-2-one, 5-substituted 1.3-dibenzyl-l,3,5-triazacyclohexan-2-one. 1-substituted 3,5- dimtro -4 -pyridone, N-methylamme, --allylamine, N-[2-(trimethylsilyl)ethoxy]methy amine (SEM), N-3- acetox propylamine. N-( l-isopropyl-4-nitro-2-oxo-3-pyroolm-3-yl)amme, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fem), N-2-picolylamino N’-oxide, N- 1, 1 -dimethylthiomethyleneamine. N -benzylideneamine. N-p-methoxybenzylideneamine, N- diphenylmethyieneamme, N-[(2-pyridyl)mesityl]methyleneamine, N-(N’, N'~ dimethylaminomethylene)amine, N, N’-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N- cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N -borane derivative, N- diphenylborimc acid derivative, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp). dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide. o -nitrobenzenesulfenamide (Nps), 2,4- dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6- trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2, 3,5,6- tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzene sulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (Mds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), P-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4’,8’-dimethoxynaphthylmethyl)benzenesulfona ide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

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[0270] Suitably protected carboxylic acids farther include, but are not limited to, silyl-, alkyl-, alkenyl-, ary 1-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, tri ethyl silyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, tetrahydropyran-2- yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include optionally substituted benzyl (e.g., p-methoxybenzyl (MPM). 3,4-dimethoxybenzyl. O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.

[0271] Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl. 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2- trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S. S dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl. 1-ethoxyethyl, 1 -(2-chloroethoxy)ethyl, 1 -methyl- 1 -methoxyethyl, l-methyl-l- benzyloxyethyl. l-methyl-l-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p- cyanobenzyl, p - phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2- picolyl N-oxido, diphenylmethyl, p,p’- dinitrobenzhydryl. 5-dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p- methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4’-bromophenacyloxyphenyl)diphenylmethyl, 4,4’,4”-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4’, 4’"-tris(levulinoyloxyphenyl)methyl, 4,4’,4”-tris(benzoyloxyphenyl)methyl, 3-(imidazol-l-yl)bis(4’,4”-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, l,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p- xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4- methoxycrotonate, benzoate, p-phenylbenzoate. 2,4.6 -trimethylbenzoate (mesitoate), alkyl methyl carbonate,

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[0274] 9 -fluorenylmethyl carbonate (Fmoc). alkyl ethyl carbonate, alkyl 2,2,2 -trichloroethyl carbonate (Troc), 2- (trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyi carbonate, alkyl p -nitrobenzyl carbonate, alkyl S -benzyl thiocarbonate, '¬ ethoxy- 1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate. 4-azidobutyrate, 4-nitro-4- methylpentanoate, o-(dibromomethyl)benzoate. 2-formylbenzenesulfonate, 2-(methylthiomethoxy)etbyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6 -dichloro 4 ( 1.1,3,3 -tetramethylbutyl)phenoxy acetate, 2,4- bis( 1, 1 - dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)- -2- -methyl- -2-butenoate, o-(m ethoxy carbonyl) benzoate, a-naphthoate, nitrate, alkyl N. N, N’, N’-tetramethylphosphorodiamidate. alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4- dimtrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2 - or 1,3- diols, the protecting groups include methylene acetal, ethylidene acetal, 1-t- butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2- trichlorocthylidcnc acetal, acetonide, cyclopcntylidcnc ketal, cyclohcxylidcnc ketal, cyclohcptylidcnc ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethox benzylidene ketal, 3,4- dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho ester, 1 -ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene ortho ester, a-methoxybenzylidene ortho ester, l-(N, N-dimethylamino)etbylidene derivative, a-(N, N’-dimethylarnino)benzylidene derivative, 2-oxacyclopentylidene ortho ester, di— t— butylsilylene group (DTBS), l,3-(l,l,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-t- butoxy disiloxane- 1,3-diylidene derivative (TBDS). cyclic carbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

[0275] In certain embodiments, a hydroxyl protecting group is acetyl, t-butyl, tbutoxymethyl, methoxymethyl, tetrahydropyranyl, 1 -ethoxyethyl, 1 -(2-chloroethoxy)ethyl, 2- trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl. 2,6- dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl), 4,4'-dimetboxytrityl, trimethyl silyl, triethylsilyl, t-butyldimethylsilyl, t- butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifiuoroacetyl, pivaloyl. 9- fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4'-dimethoxytntyl, (DMTr) and 4,4',4"-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl (NPE), 2- (4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4',4"-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9- Page 41 of 566

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[0277] phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthme-9-y 1 (MOX). In certain embodiments, each of the hydroxyl protecting groups is, independently selected from acetyl, benzyl, t- butyldimethylsilyl, t- butyldiphenylsilyl and 4,4’-dimethoxytrityl. In certain embodiments, the hydroxyl protecting group is selected from the group consisting of trityl, monomethoxytrityl and 4,4'-dimethoxytrityl group. In certain embodiments, a phosphorous linkage protecting group is a group attached to the phosphorous linkage (e.g., an internucleotidic linkage) throughout oligonucleotide synthesis. In certain embodiments, a protecting group is atached to a sulfur atom of a phosphorothioate group. In certain embodiments, a protecting group is atached to an oxygen atom of an internucleotide phosphorothioate linkage. In certain embodiments, a protecting group is attached to an oxygen atom of the intemucleotide phosphate linkage. In certain embodiments a protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2- (p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarboxaniido)-l -propyl, 4-oxopentyl, 4-methylthio-l-butyl, 2-cyano-l.1 -dimethylethyl, 4-N -methylaminobutyl, 3 -(2 -pyridyl)-! -propyl, 2-[N-methyl- N-(2-pyridyl)|aminoethyl, 2-(N-formyl, N-methyl)aminoethyl, or 4-[N-methyI-N-(2,2,2- trifluoroacetyl)amino]butyl.

[0278] Subject: As used herein, the term “subject’’ or “test subject” refers to any organism to which a compound (e.g., an oligonucleotide) or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In certain embodiments, a subject is a human. In certain embodiments, a subject may be suffering from and / or susceptible to a disease, disorder and / or condition

[0279] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. A base sequence which is substantially identical or complementary to a second sequence is not folly identical or complementary' to the second sequence, but is mostly or nearly identical or complementary' to the second sequence. In certain embodiments, an oligonucleotide with a substantially complementary sequence to another oligonucleotide or nucleic acid forms duplex with the oligonucleotide or nucleic acid in a similar fashion as an oligonucleotide with a folly complementary' sequence. In addition, one of ordinary skill in the biological and / or chemical arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0280] Sugar: The term “sugar” refers to a monosaccharide or polysaccharide in closed and / or open form, hi certain embodiments, sugars are monosaccharides. In certain embodiments, sugars are polysaccharides. Sugars include, but are not limited to, ribose, deoxyribose, pentoforanose, pentopyranose, and hexopyranose moieties. As used herein, the term “sugar” also encompasses structural analogs used in lieu of conventional sugar molecules, such as glycol, polymer of which forms the backbone of the nucleic acid analog, glycol Page 42 of 566

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[0282] nucleic acid (“GNA”). etc. As used herein, the term 'sugar ’ also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified sugars and nucleotide sugars. In certain embodiments, a sugar is an RNA or DN sugar (ribose or deoxyribose) In certain embodiments, a sugar is a modified ribose or deoxyribose sugar, e.g., 2’-modified, 5’-modified, etc. As described herein, in certain embodiments, when used in oligonucleotides and / or nucleic acids, modified sugars may provide one or more desired properties, activities, etc. In certain embodiments, a sugar is optionally substituted ribose or deoxyribose. In certain embodiments, a “sugar” refers to a sugar unit in an oligonucleotide or a nucleic acid.

[0283] Susceptible to: An individual who is "susceptible to” a disease, disorder and / or condition is one who has a higher risk of developing the disease, disorder and / or condition than does a member of the general public. In certain embodiments, an individual who is susceptible to a disease, disorder and / or condition is predisposed to have that disease, disorder and / or condition. In certain embodiments, an individual who is susceptible to a disease, disorder and / or condition may not have been diagnosed with the disease, disorder and / or condition. In certain embodiments, an individual who is susceptible to a disease, disorder and / or condition may exhibit symptoms of the disease, disorder and / or condition In certain embodiments, an individual who is susceptible to a disease, disorder and / or condition may not exhibit symptoms of the disease, disorder and / or condition. In certain embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In certain embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0284] Therapeutic agent: As used herein, the term “therapeutic agent” in general refers to any agent that elicits a desired effect (e g., a desired biological, clinical, or pharmacological effect) when administered to a subject. In certain embodiments, an agent, e.g., a dsRNAi agent, is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In certain embodiments, an appropriate population is a population of subjects suffering from and / or susceptible to a disease, disorder or condition. In certain embodiments, an appropriate population is a population of model organisms. In certain embodiments, an appropriate population may be defined by one or more criterion such as age group, gender, genetic background, preexisting clinical conditions, prior exposure to therapy. In certain embodiments, a therapeutic agent is a substance that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of, and / or reduces incidence of one or more hepatic symptoms or features of a di sease, disorder, and / or condition in a subject when administered to the subject in an effective amount. In certain embodiments, a “therapeutic agent” is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In certain embodiments, a “therapeutic agent” is an agent for which a medical prescription is required for administration to humans. In certain embodiments, a therapeutic agent is a provided compound, e.g., a provided oligonucleotide.

[0285] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired Page 43 of 566

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[0287] biological response when administered as part of a therapeutic regimen. In certain embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of ordinary’ skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces incidence of one or more symptoms or features of the disease, disorder, and / or condition. In certain embodiments, a therapeutically effective amount is administered in a single dose; in certain embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0288] Treat: As used herein, the term “treat,” ‘“treatment.” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of. reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In certain embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0289] Unsaturated: The term "unsaturated." as used herein, means that a moiety has one or more units of unsaturation

[0290] Wild-type: As used herein, the term “wild-type” has its art-understood meaning that refers to an entity' having a structure and / or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0291] As those skilled in the art will appreciate, methods and compositions described herein relating to provided compounds (e.g., oligonucleotides) generally also apply to pharmaceutically acceptable salts of such compounds.

[0292] 1. Description of Certain Embodiments

[0293] Oligonucleotides are useful tools for a wide variety of applications. For example. RNAi oligonucleotides are useful in therapeutic, diagnostic, and research applications, including the treatment of a variety of conditions, disorders, and diseases. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) is limited, for example, by their susceptibility to endo- and exo-nucleases. As such, various synthetic counterparts have been developed to circumvent these shortcomings and / or to further improve various properties and activities. These include synthetic oligonucleotides that contain chemical modifications, e.g., base modifications, sugar modifications, backbone modifications, etc., which, among other things, render Page 44 of 566

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[0295] these molecules less susceptible to degradation and improve other properties and / or activities. From a structural point of view, modifications to mtemucleotidic linkages can introduce chirality and / or alter charge, and certain properties may be affected by configurations of linkage phosphorus atoms of oligonucleotides. For example, binding affinity, sequence specific binding to complementary RNA, stability against nucleases, cleavage of target nucleic acids, delivery, pharmacokinetics, etc., can be affected by, inter alia, chirality and / or charge of backbone linkage atoms.

[0296] In certain embodiments, the present disclosure demonstrates that compositions comprising ds oligonucleotides (e.g., dsRNAi oligonucleotides, also referred to as dsRNAi agents) with controlled structural elements provide unexpected properties and / or activities.

[0297] In certain embodiments, the present disclosure encompasses the recognition that stereochemistry, e.g., stereochemistry of backbone chiral centers, can unexpectedly maintain or improve properties of ds oligonucleotides. For example, but not by way of limitation, the instant disclosure relates, in part, to ds oligonucleotides comprising a guide strand comprising backbone phosphoryl guanidine chiral centers in Sp configuration.

[0298] In certain embodiments, the guide strand comprises a backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction.

[0299] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center m the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i e., tn the 3’ direction, further comprise a phosphoryl guanidine chiral center in the Sp configuration between the +7 and the immediately downstream (+8) nucleotide, between the +10 nucleotide and the immediately downstream (+11) nucleotide, between the +18 nucleotide and the immediately downstream (+19) nucleotide, or combinations thereof.

[0300] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, further comprise a phosphoryl guanidine chiral center in the Rp configuration between the +7 and the immediately downstream (+8).

[0301] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center Page 45 of 566

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[0303] in the Sp configuration between the +3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fifth (-1-5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (+6) nucleotide In certain embodiments, the modified sugar comprises a 2"-F modification, 2"-H modification, 2 ’-OH modification, 2’-O-alkyl modification, e.g., 2’-O-methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center m the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the t3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3‘

[0304] Page 46 of 566

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[0306] direction, comprise a 2’-F modification of the fourth (-1-4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i e. in the 3’ direction, comprise a 2’-F modification of the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -1-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3‘ direction, comprise a 2’-F modification of the sixth (+6) nucleotide.

[0307] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, further comprises one or more of:

[0308] (1) a guide strand comprising backbone phosphorothioate chiral centers in Sp configuration between the 3Tterminal nucleotide and the penultimate (TN- 1 ) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream, i.e., mthe 5’ direction, (N-2) nucleotide;

[0309] (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

[0310] (3) a guide strand comprising one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5 ’ direction, relative to backbone phosphorothioate chiral centers m Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, where the upstream backbone phosphorothioate chiral centers are in Rp or Sp configuration;

[0311] (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide; and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

[0312] (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any tw7o adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide;

[0313] (6) a guide strand comprising one or more backbone phosphoryl guanidine chiral centers in the Rp configuration between the +7 and the immediately downstream (+8), i.e., in the 3’ direction.

[0314] (7) a guide strand comprising a 5’ terminal modification, e.g., a 5’ phosphate mimic modification: (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more backbone chiral centers in Rp or Sp configuration;

[0315] (9) a passenger strand in combination with one or more of the aforementioned guide strands,

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[0318] comprising one or more backbone phosphoryl guanidine chiral centers in the Rp configuration between the +7 nucleotide and the immediately downstream (+8) nucleotide, • e. in the 3' direction;

[0319] (10) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more backbone phosphoryl guanidine chiral centers in the Rp configuration between the H- 15 nucleotide and the immediately downstream (+16) nucleotide, i.e., m the 3‘ direction: and (11) a passenger strand in combination with one or more of the aforementioned guide strands, composing backbone phosphorothioate chiral centers in the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide;

[0320] wherein the ds oligonucleotide further comprises one or more of:

[0321] (1) a guide strand where one or both of the 5' and 3’ terminal dinucleotides are not linked by a Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, i.e., the guide strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages downstream, i.e.. in the 3’ direction, relative to the linkage between the 5’ temiinal dinucleotide and / or upstream, i.e, in the 5’ direction, relative to the linkage between the 3’ temiinal dinucleotide;

[0322] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5" temiinal nucleotide of the guide strand and the penultimate 3‘ (N-l) nucleotide of the guide strand, w here N is the 3’ terminal nucleotide;

[0323] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide; (4) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the seventh (+7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide; (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide;

[0324] (6) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0325] (7) a passenger strand where one or more Rp. Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0326] (8) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e., in the '+ direction, relative to the central Page 48 of 566

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[0328] nucleotide of the passenger strand; and

[0329] (9) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0330] In certain embodiments, the ds oligonucleotide further comprises a 2' modification, e.g., a 2‘ F modification, of the 3 ' nucleotide of a nucleotide pair linked by a Rp, Sp, or stereorandom non -negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non -negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -3 nucleotide and the immediately downstream (-1-4) nucleotide, i e, in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5‘ temiinal (+ 1) nucleotide, second (+2) nucleotide, third, (t-3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5‘ temiinal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately dowmstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp Page 49 of 566

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[0332] configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (+6) nucleotide In certain embodiments, the modified sugar comprises a 2’-F modification, 2’-H modification, 2’-OH modification, 2’-O-alkyl modification, e.g., 2'-O-methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e,, in the 3’ direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -t-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2 -F modification of the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3" direction, comprise a 2’-F modification of the fifth (+5) nucleotide In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the sixth (+6) nucleotide.

[0333] In certain embodiments, the present disclosure encompasses the recognition that stereochemistry, e.g,, stereochemistry of chiral centers at a 5’ terminal modification, e.g., a 5' phosphate mimic modification of guide strands, can unexpectedly maintain or improve properties of the ds oligonucleotides described herein. For example, but not by w-ay of limitation, the instant disclosure relates, in part, to ds oligonucleotides comprising a guide stranding comprising one or more of: (1) a backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide; (2) a phosphorothioate chiral center in Rp or Sp configuration; (3) an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage where the 3‘ nucleotide of a nucleotide pair linked by an Rp, Sp, or Page 50 of 566

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[0335] stereorandom non-negatively charged internucleotidic linkage comprises a 2‘ modification, e.g., a 2’ F; and (4) a 5’ terminal modification, e.g.. 5’ phosphate mimic modification selected from:

[0336] (a) 5’ PO modifications, such as, but not limited to:

[0337]

[0338] (b) 5’ VP modifications, such as, but not limited to:

[0339]

[0340] (d) 5’ PN and 5’ triazoIe-P modifications, such as, but not limited to:

[0341]

[0342] Wherein Base is selected from A, C, G, T, U, abasic and modified nucleobases;

[0343] R1is selected from an alkyl, methyl, ethyl, isopropyl, propyl, cyclohexyl, benzyl, phenyl, tolyl, xylyl, aryl, or arene group

[0344] R2is selected from H, OH, O-alkyl, O-Me, F. MOE, locked nucleic acid (LN ) bridges and bridged nucleic acid (BNA) bridges to the 4’ C, such as, but not limited to:

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[0347]

[0348] In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non- negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0349] In certain other embodiments, the present disclosure encompasses the recognition that stereochemistry, e.g., stereochemistry of chiral centers at the 5" terminal nucleotide of guide strands, can unexpectedly maintain or improve properties of ds oligonucleotides wherein the guide strand of the ds oligonucleotide also comprises a phosphorothioate chiral center in Rp or Sp configuration. For example, but not by way of limitation, the instant disclosure relates, in part, to ds oligonucleotides comprising a guide stranding comprising: (1) a phosphorothioate chiral center in Rp or Sp configuration; (2) an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage where the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage comprises a 2’ modification, e.g., a 2' F; and (3) a 5’ terminal modification, e.g., a 5’ phosphate mimic modification selected from:

[0350] (a) 5’ PO nucleotides, such as, but not limited to:

[0351] o

[0352]

[0353] (b) 5’ VP nucleotides, such as, but not limited to:

[0354]

[0355] (c) 5’ MeP nucleotides, such as. but not limited to:

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[0358]

[0359] (d) 5" PN and 5’ triazole-P nucleotides, such as, but not limited to:

[0360]

[0361] (e) 5" abasic VP and 5’ abasic MeP nucleotides, such as, but not limited to:

[0362] O" O’

[0363] -O-P-O 'O-P=O

[0364] ° o

[0365]

[0366] and. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0367] In certain other embodiments, the present disclosure encompasses the recognition that stereochemistry, e.g,, stereochemistry of chiral centers at the 5‘ terminal nucleotide of guide strands, can unexpectedly maintain or improve properties of ds oligonucleotides wherein the guide strand of the ds oligonucleotide also comprises a phosphorothioate chiral center in Rp or Sp configuration. For example, but not by way of limitation, the instant disclosure relates, in part, to ds oligonucleotides comprising a guide stranding comprising: (1) a backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide; (2) a backbone phosphoryl guanidine chiral center in the Rp configuration between the +7 nucleotide and the immediately downstream (-1-8) nucleotide; (3) a phosphorothioate chiral center in Rp or Sp configuration; (4) an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage where the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage comprises a 2’ modification, e.g., a T F; and (5) a y phosphate mimic modification selected from:

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[0370] (a) 5‘ PO nucleotides, such as, but not limited to:

[0371] o

[0372]

[0373] (b) 5‘ VP nucleotides, such as. but not limited to:

[0374]

[0375] (b) 5’ abasic VP and 5’ abasic MeP nucleotides, such as, but not limited to:

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[0378] O' O'

[0379] " O-p— o 'O-P=o

[0380] ? £ o £

[0381]

[0382] Jand ’. In certain embodiments, the one or more Rp, Sp, or stcrcorandom non-negatively charged intern uclcotidic linkage incorporated into the guide strand is an Rp non- negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non -negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0383] In certain embodiments, the present disclosure encompasses the recognition that non-naturally occurring internucleotidic linkages, e.g., neutral internucleotidic linkages, can, in certain embodiments, be used to link one or more molecules to the double-stranded oligonucleotides described herein. In certain embodiments, such linked molecules can facilitate targeting and / or delivery of the double -stranded oligonucleotide. For example, but not limitation, such linked molecules an include lipophilic molecules. In certain embodiments, the linked molecule is a molecule comprising one or more GalNAc moieties. In certain embodiments, the linked molecule is a receptor. In certain embodiments, the linked molecule is a receptor ligand

[0384] In certain embodiments, the present disclosure provides technologies for incorporating various additional chemical moieties into ds oligonucleotides. In certain embodiments, the present disclosure provides, for example, reagents and methods for introducing additional chemical moieties through nucleobases (eg. by covalent linkage, optionally via a linker, to a site on a nucleobase).

[0385] In certain embodiments, the present disclosure provides technologies, e.g., ds oligonucleotide compositions and methods thereof, that achieve allele-specific suppression, wherein transcripts from one allele of a particular target gene is selectively knocked down relative to at least one other allele of the same gene.

[0386] Among other things, the present disclosure provides structural elements, technologies and / or features that can be incorporated into ds oligonucleotides and can impart or tune one or more properties thereof (e.g., relative to an otherwise identical ds oligonucleotide lacking the relevant technology or feature). In certain embodiments, the present disclosure documents that one or more provided technologies and / or features can usefully be incorporated into ds oligonucleotides of various sequences.

[0387] In certain embodiments, the present disclosure demonstrates that certain provided structural elements, technologies and / or features are particularly useful for ds oligonucleotides that participate in and / or direct RNAi mechanisms (e.g., RNAi agents). Regardless, however, the teachings of the present disclosure are not limited to ds oligonucleotides that participate in or operate via any particular mechanism. In certain Page 55 of 566

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[0389] embodiments, the present disclosure pertains to any ds oligonucleotide, useful for any purpose, which operates through any mechanism, and which comprises any sequence, structure or format (or portion thereof) described herein In certain embodiments, the present disclosure provides a ds oligonucleotide, useful for any purpose, which operates through any mechanism, and which comprises any sequence, structure or format (or portion thereof) described herein, comprising a guide strand comprising backbone phosphoryl guanidine chiral centers in Sp configuration.

[0390] In certain embodiments, the guide strand comprises a backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction.

[0391] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3" direction, further comprise a phosphoryl guanidine chiral center in the Sp configuration between the +7 and the immediately downstream (+8) nucleotide, between the +10 nucleotide and the immediately downstream (+11) nucleotide, between the +18 nucleotide and the immediately downstream (+19) nucleotide, or combinations thereof.

[0392] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, further comprise a phosphoryl guanidine chiral center in the Sp configuration between the +7 and the immediately downstream (+8) nucleotide, between the +15 nucleotide and the immediately downstream (+16) nucleotide, or combinations thereof.

[0393] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5' terminal (+1) nucleotide, in certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3‘

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[0396] direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -1-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3‘ direction, comprise a modified sugar at the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2’-F modification, 2'-H modification, 2'-OH modification, 2’-O-alkyl modification, e.g., 2'-O-methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5' terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2'-F modification of the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration betyveen the -t-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the sixth (+6) nucleotide.

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[0399] In certain embodiments, the present disclosure provides a ds oligonucleotide, useful for any purpose, which operates through any mechanism, and which comprises comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately’ downstream (- 4) nucleotide, i.e., in the 3’ direction, which further comprises:

[0400] (1) a guide strand comprising backbone phosphorothioate chiral centers in Sp configuration between the 3" terminal nucleotide and the penultimate (N-l) nucleotide and as between tlie penultimate (N-l) nucleotide and the immediately upstream, i.e., in the 5’ direction, (N-2) nucleotide:

[0401] (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3" direction, (+2) nucleotide and between the +2 nucleotide and the immediately’ downstream (+3) nucleotide;

[0402] (3) a guide strand comprising one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5 ’ direction, relative to backbone phosphorothioate chiral centers in Sp configuration betw een the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately7upstream (N-2) nucleotide, where the upstream backbone phosphorothioate chiral centers arc in Rp or Sp configuration;

[0403] (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide; and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

[0404] (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide;

[0405] (6) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more backbone chiral centers in Rp or Sp configuration;

[0406] (7) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more backbone phosphoryl guanidine chiral centers in the Rp configuration betw een the +7 nucleotide and the immediately downstream (+8) nucleotide, i.e,, in the 3’ direction;

[0407] (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more backbone phosphoryl guanidine chiral centers in the Rp configuration between the +15 nucleotide and the immediately downstream (+16) nucleotide, i.e., in the 3’ direction; and (9) a passenger strand in combination witli one or more of the aforementioned guide strands, comprising backbone phosphorothioate chiral centers in the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the 3' terminal nucleotide and the penultimate (N-l) nucleotide:

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[0410] wherein the ds oligonucleotide further comprises one or more of:

[0411] (1) a guide strand where one or both of the 5’ and 3' terminal dinucleotides are not linked by non- negatively charged internucleotidic linkages, i.e., the guide strand comprises one more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between tire 5 ' terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3' terminal dinucleotide;

[0412] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5" terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0413] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide; (4) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the seventh (-1-7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide; (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3' terminal nucleotide, and the upstream N-10 nucleotide;

[0414] (6) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0415] (7) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., m the 3' direction, relative to the central nucleotide of the passenger strand.

[0416] (8) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0417] (9) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0418] In certain embodiments, the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3 ’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non- negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom Page 59 of 566

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[0420] non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ temiinal (+1) nucleotide, second (4-2) nucleotide, third, ( -3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5' temiinal (4-1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 43 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (4-3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (4-6) nucleotide. In certain embodiments, the modified sugar comprises a 2’-F modification, 2'-H modification, 2 '-OH modification, 2’-O-alkyl modification, e.g., 2’-O- methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -i-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5’ temiinal (4-1) nucleotide, second (4-2) nucleotide, third, (4-3) nucleotide, fourth (4-4) nucleotide, fifth (4-5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments,

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[0423] the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2'-F modification of the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2 -F modification of the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e,, in the 3’ direction, comprise a 2"-F modification of the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the sixth ( -1-6) nucleotide.

[0424] In certain embodiments, the provided ds oligonucleotides may participate in (e.g., direct) RNAi mechanisms. In certain embodiments, provided ds oligonucleotides may participate in RNase H (ribonuclease FI) mechanisms. In certain embodiments, provided ds oligonucleotides may act as translational inhibitors (e.g., may provide steric blocks of translation).

[0425] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i e., in the 3’ direction, and the guide strand further comprises backbone phosphorothioate chiral centers in Sp configuration between the 3 ‘ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-I) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of:

[0426] (1) a guide strand comprising backbone phosphorothioate chiral centers in Sp configuration between the 3" terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream, i.e., in the 5’ direction, (N-2) nucleotide;

[0427] (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

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[0430] ( 3) a guide strand comprising one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5 ’ direction, relative to backbone phosphorothioate chiral centers m Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, where the upstream backbone phosphorothioate chiral centers are in Rp or Sp configuration;

[0431] (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide; and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

[0432] (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide;

[0433] (7) a guide strand comprising a 5’ terminal modification, e.g., a 5’ phosphate mimic modification; (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more backbone chiral centers in Rp or Sp configuration;

[0434] (9) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more backbone phosphoryl guanidine chiral centers in the Rp configuration between the +7 nucleotide and the immediately downstream (+8) nucleotide, i.e., in the 3’ direction;

[0435] (10) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more backbone phosphoryl guanidine chiral centers in the Rp configuration between the +15 nucleotide and the immediately downstream (+16) nucleotide, i.e., in the 3’ direction; and (11) a passenger strand in combination with one or more of the aforementioned guide strands, comprising backbone phosphorothioate chiral centers in the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e.. tn the 3' direction, (+2) nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide:

[0436] In certain embodiments, the ds oligonucleotide further comprises a 2' modification, e g., a 2’ F modification, of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non -negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ temiinal (+1) nucleotide, second (+2) nucleotide, third, (+3)

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[0439] nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (-1-6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -1-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3‘ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (-1-4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -1-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2’-F modification, 2’-H modification, 2’-OH modification, 2’-0-alky'l modification, e.g., 2’-O-methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately dowmstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (1-4) nucleotide, fifth (1-5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e,, in the 3’ direction, comprise a 2'-F modification of the 5' tenninal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2"-F modification of the third (+3) nucleotide. In certain embodiments, the ds Page 63 of 566

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[0441] oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -1-3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the fifth (-1-5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the sixth (1-6) nucleotide.

[0442] In certain further embodiments, the passenger strand comprises an Sp backbone phosphorothioate chiral center between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and an Sp backbone phosphorothioate chiral center between the penultimate (N-l) nucleotide and the 3’ terminal (N) nucleotide. In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, and the guide strand further comprises backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (-1-3) nucleotide, and one or more of:

[0443] (1) a guide strand where one or both of the 5’ and 3" terminal dinucleotides are not linked by non- negatively charged internucleotidic linkages, i.e, the guide strand comprises one more non -negative! y charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5' terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the y terminal dinucleotide;

[0444] (2) a guide strand where one or more Rp, Sp, or stereorandom non -negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5" terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0445] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide; (4) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the seventh (+7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide; (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide;

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[0448] (6) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0449] (7) a passenger strand where one or more Rp. Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3‘ direction, relative to the central nucleotide of the passenger strand.

[0450] (8) a passenger strand where one or more backbone phosphorothioate chiral centers m Rp or Sp configuration internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0451] (9) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0452] In certain embodiments, the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non- negatively charged internucleotidic linkages, where n is about 1 to 49.

[0453] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e.. in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+ 1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e,, in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (-1-4) nucleotide. In certain embodiments, the ds Page 65 of 566

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[0455] oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -1-3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2'-F modification, 2’-H modification, 2 ’-OH modification, 2’-O-alkyl modification, e.g.. 2'-O-methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the --3 nucleotide and the immediately downstream (+4) nucleotide, i.e,, in the 3’ direction, comprise a 2’-F modification of the 5' terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (-1-5) nucleotide, and / or sixth (-1-6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center m the Sp configuration between the -t-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2‘-F modification of the 5' terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2 -F modification of the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fifth (+5) nucleotide In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -t-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the sixth (+6) nucleotide.

[0456] In certain further embodiments, the passenger strand comprises an Sp backbone phosphorothioate chiral center between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, an Rp backbone phosphoryl guanidine between the +7 nucleotide and the immediately downstream ( 18) nucleotide, and an Sp backbone phosphorothioate chiral center between the penultimate (N-l) nucleotide Page 66 of 566

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[0458] and tlie 3‘ terminal (N) nucleotide. In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center m the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, a guide strand backbone phosphoryl guanidine chiral center in the Rp configuration between the +7 nucleotide and the immediately downstream (+8) nucleotide, i.e., in the 3" direction and the guide strand further comprises backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and one or more of:

[0459] (1) a guide strand where one or both of the 5’ and 3' terminal dinucleotides are not linked by non- negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3" direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e.. in the + direction, relative to the linkage between the 3’ terminal dinucleotide;

[0460] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0461] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5' terminal nucleotide; (4) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the seventh (+7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide; (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3 ' nucleotide of the guide strand, here N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide;

[0462] (6) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0463] (7) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0464] (8) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0465] (9) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp Page 67 of 566

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[0467] configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0468] In certain embodiments, the ds oligonucleotide further comprises a 2’ modification, e.g., a 2‘ F modification, of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non -negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49.

[0469] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth ( + 6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5' terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2’-F modification, 2’-H modification, 2’-OH modification, 2'-O-alkyl modification, e g., 2'-O-methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp Page 68 of 566

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[0471] configuration between the +3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a 2'-F modification of the 5' terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the second (-1-2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the --3 nucleotide and the immediately downstream (+4) nucleotide, i.e,, in the 3’ direction, comprise a 2’-F modification of the third (+3) nucleotide In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -1-3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the sixth (+6) nucleotide.

[0472] In certain further embodiments, the passenger strand comprises an Sp backbone phosphorothioate chiral center between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, an Rp backbone phosphoryl guanidine between the +7 nucleotide and the immediately downstream (+8) nucleotide, and an Sp backbone phosphorothioate chiral center between the penultimate (N-l ) nucleotide and die 3 / terminal (N) nucleotide. In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center m the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +7 nucleotide and the immediately downstream (+8) nucleotide, i.e., in the 3" direction and the guide strand further comprises backbone phosphorothioate chiral centers in Rp, Sp. or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and betw een the +2 nucleotide and the immediately downstream (+3) nucleotide, and one or more of:

[0473] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by nonnegativ ely charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively Page 69 of 566

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[0475] charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0476] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3' (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0477] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the tenth ( +10) and eleventh (+11) nucleotides, relative to the 5' terminal nucleotide; (4) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the seventh (+7) and eighth (+8) nucleotides, relative to the 5' terminal nucleotide; (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide;

[0478] (6) a passenger strand where one or more Rp, Sp, or stcreorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0479] (7) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3 direction, relative to the central nucleotide of the passenger strand.

[0480] (8) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e.. in the 5' direction, relative to the central nucleotide of the passenger strand; and

[0481] (9) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0482] In certain embodiments, the ds oligonucleotide further comprises a 2" modification, e.g., a 2‘ F modification, of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp. or stereorandom non- negatively charged internucleotidic linkages, where n is about 1 to 49.

[0483] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral Page 70 of 566

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[0485] center m the Sp configuration between the +3 nucleotide and the immediately downstream (44) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a modified sugar at the 5' terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e,, in the 3’ direction, comprise a modified sugar at the third (4-3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3' direction, comprise a modified sugar at the fifth (45) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (4-6) nucleotide. In certain embodiments, the modified sugar comprises a 2’-F modification, 2’-H modification, 2’-OH modification, 2’-O-aIkyl modification, e.g., 2‘-O-methyl (OMe) modification, 2‘- Methoxyethyl (MOE) modification, locked nucleic acid (LN A), unlocked nucleic acid (UNA), glycol nucleic acid (GN ), or Homo-DNA, In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 43 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a 2'-F modification of the 5' terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5’ terminal (4-1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -i-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the second (4-2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp Page 71 of 566

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[0487] configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2'-F modification of the third (+3) nucleotide In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the sixth (+6) nucleotide.

[0488] In certain further embodiments, the passenger strand comprises an Sp backbone phosphorothioate chiral center between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and an Rp backbone phosphoryl guanidine between the +7 nucleotide and the immediately downstream (+8) nucleotide. In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +7 nucleotide and the immediately downstream (+8) nucleotide, i.e., in the 3' direction and the guide strand further comprises backbone phosphorothioate chiral centers in Rp. Sp, or alternating configurations between the 5" terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and one or more of:

[0489] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non- negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3‘ direction, relative to tire linkage between the 5’ terminal dinucleotide and / or upstream, i.e.. in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide,

[0490] (2) a guide strand where one or more Rp. Sp. or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-I) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0491] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5' terminal nucleotide; (4) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the seventh (+7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide; (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3‘

[0492] Page 72 of 566

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[0494] nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide:

[0495] (6) a passenger strand where one or more Rp, Sp. or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0496] (7) a passenger strand where one or more Rp. Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e, in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0497] (8) a passenger strand where one or more backbone phospho rothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0498] (9) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand.

[0499] In certain embodiments, the ds oligonucleotide further comprises a 2’ modification, e g., a 2' F modification, of the 3‘ nucleotide of a nucleotide pair linked by an Rp, Sp, or stcrcorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non- negatively charged internucleotidic linkages, where n is about 1 to 49.

[0500] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream ( 4) nucleotide, i.e., in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., m the 3’ direction, compose a modified sugar at the 5' terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (t-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp Page 73 of 566

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[0502] configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fifth (+5) nucleotide, hr certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2’-F modification, 2'-H modification, 2’-OH modification, 2’-O-alkyl modification, e.g., 2'-O-methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream ( +4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (4-5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the 5" terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3" direction, comprise a 2’-F modification of the third (4-3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center m the Sp configuration between the -3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the sixth (4-6) nucleotide.

[0503] In certain further embodiments, the passenger strand comprises an Sp backbone phosphorothioate chiral center between the 5' terminal (+1) nucleotide and the immediately downstream (+2)

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[0506] nucleotide, an Rp backbone phosphoryl guanidine between the +7 nucleotide and the immediately downstream (+8) nucleotide, and an Rp backbone phosphoryl guanidine between the +15 nucleotide and the immediately downstream (+16) nucleotide. In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +7 nucleotide and the immediately downstream (+8) nucleotide, i.e., in the 3‘ direction and the guide strand further comprises backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and one or more of:

[0507] (1) a guide strand where one or both of the 5’ and 3' terminal dinucleotides are not linked by non- negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3" direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e.. in the '■ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0508] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0509] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5' terminal nucleotide; (4) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the seventh (+7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide; (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3 ' nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide;

[0510] (6) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0511] (7) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0512] (8) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0513] (9) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp Page 75 of 566

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[0515] configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0516] In certain embodiments, the ds oligonucleotide further comprises a 2’ modification, e.g., a 2‘ F modification, of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non -negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49.

[0517] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth ( + 6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5' terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2’-F modification, 2’-H modification, 2’-OH modification, 2'-O-alkyl modification, e g., 2'-O-methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp Page 76 of 566

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[0519] configuration between the +3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a 2'-F modification of the 5' terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the second (-1-2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the --3 nucleotide and the immediately downstream (+4) nucleotide, i.e,, in the 3’ direction, comprise a 2’-F modification of the third (+3) nucleotide In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -1-3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the sixth (+6) nucleotide.

[0520] In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp. Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage. In certain further embodiments, the passenger strand comprises an Sp backbone phosphorothioate chiral center between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and an Sp backbone phosphorothioate chiral center between the penultimate (N-l) nucleotide and the 3’ terminal (N) nucleotide.

[0521] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e, in the 3’ direction, and the guide strand further comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of backbone phosphorothioate chiral centers in Sp configuration between the 3" terminal nucleotide and the penultimate (N-l) nucleotide and as Page 77 of 566

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[0523] between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of:

[0524] (1) a guide strand where one or both of the 5’ and 3' terminal dinucleotides are not linked by non- negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non -negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3' terminal dinucleotide;

[0525] (2) a guide strand where one or more Rp, Sp, or stereorandom non -negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5' terminal nucleotide of the guide strand and the penultimate 3' (N-1) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0526] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide; (4) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the seventh (-1-7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide; (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any tw o adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3' terminal nucleotide, and the upstream N-10 nucleotide;

[0527] (6) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i e.. in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0528] (7) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand.

[0529] (8) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0530] (9) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0531] In certain embodiments, the ds oligonucleotide further comprises a 2’ modification, e.g.. a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp. Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49.

[0532] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl Page 78 of 566

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[0534] guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3" direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e,, in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center m the Sp configuration between the t-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3" direction, comprise a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2’-F modification, 2’-H modification, 2 ’-OH modification, 2’-O-aIkyl modification, e g., 2’-O- methyl (OMe) modification, 2’- Methoxy ethyl (MOE) modification, locked nucleic acid (LN A), unlocked nucleic acid (UNA), glycol nucleic acid (GNA). or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5‘ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2"-F modification of the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp Page 79 of 566

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[0536] configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2'-F modification of the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fifth (4-5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream ( -4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the sixth (4-6) nucleotide.

[0537] In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp. Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage. In certain further embodiments, the passenger strand comprises an Sp backbone phosphorothioate chiral center between the 5’ terminal (4-1) nucleotide and the immediately downstream (4-2) nucleotide and an Sp backbone phosphorothioate chiral center between the penultimate (N-l) nucleotide and the 3’ terminal (N) nucleotide.

[0538] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (44) nucleotide, i.e., in the 3’ direction, and the guide strand further comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the second (4-2) and third (4-3) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and the internucleotidic linkage to the penultimate 3' (N-l) nucleotide, and one or more of:

[0539] (1) a guide strand where one or both of the 5’ and 3‘ terminal dinucleotides are not linked by non- negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e, in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0540] (2) a guide strand where one or more Rp, Sp. or stereorandom non-negatively charged internucleotidic Page 80 of 566

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[0542] linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5" terminal nucleotide of the guide strand and the penultimate 3’ (N-l ) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0543] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5' terminal nucleotide; (4) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the seventh (+7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide; (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide;

[0544] (6) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0545] (7) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand.

[0546] (8) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0547] (9) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand.

[0548] In certain embodiments, the ds oligonucleotide further comprises a 2’ modification, e.g.. a 2’ F modification, of tire 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49.

[0549] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center Page 81 of 566

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[0551] in the Sp configuration between the +3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fifth (-1-5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2"-F modification, 2"-H modification, 2 ’-OH modification, 2’-O-alkyl modification, e.g., 2’-O-methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center m the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the t3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3‘

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[0554] direction, comprise a 2’-F modification of the fourth (-1-4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i e. in the 3’ direction, comprise a 2’-F modification of the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -1-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3‘ direction, comprise a 2’-F modification of the sixth (+6) nucleotide.

[0555] In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage. In certain further embodiments, the passenger strand comprises an Sp backbone phosphorothioate chiral center between the 5’ terminal (-1-1) nucleotide and the immediately downstream (+2) nucleotide and an Sp backbone phosphorothioate chiral center betw een the penultimate (N-l) nucleotide and the 3’ terminal (N) nucleotide

[0556] In certain embodiments, tire ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, and the guide strand further comprises backbone phosphorothioate chiral centers in Sp configuration between the 3' terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of:

[0557] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non- negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3' direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide:

[0558] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3' (N-l) nucleotide of the guide strand, where N is the 3" terminal nucleotide:

[0559] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide; (4) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the seventh (+7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide;

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[0562] (5) a guide strand comprising one or more backbone phosphorotliioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide;

[0563] (6) a passenger strand where one or more Rp. Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e.. in the 5" direction, relative to the central nucleotide of the passenger strand; and

[0564] (7) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3 ' direction, relative to the central nucleotide of the passenger strand.

[0565] (8) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and

[0566] (9) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand.

[0567] In certain embodiments, the ds oligonucleotide further comprises a 2’ modification, e.g., a 2' F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone phosphorotliioate chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non- negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third. (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide. In certain embodiments, the ds Page 84 of 566

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[0569] oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e,, in the 3’ direction, comprise a modified sugar at the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (4-6) nucleotide. In certain embodiments, the modified sugar comprises a 2 -1 modification, 2’-H modification, 2’-OH modification, 2’-O-alkyl modification, c.g,, 2 -0- methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a 2 -1 modification of the 5’ terminal (+1) nucleotide, second (4-2) nucleotide, third, (4-3) nucleotide, fourth (44) nucleotide, fifth (4-5) nucleotide, and / or sixth (4-6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately dowmstream (4-4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the 5’ terminal (4-1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the second (4-2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the third (4-3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -i-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (4-4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp Page 85 of 566

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[0571] configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the sixth (+6) nucleotide.

[0572] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, and the guide strand comprises backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ temiinal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and one or more of:

[0573] (1) a guide strand where one or both of the 5’ and 3' terminal dinucleotides are not linked by non- negatively charged internucleotidic linkages, i.e.. the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e, in the 3 ‘ direction, relative to the linkage between the 5’ temiinal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinuclcotidc;

[0574] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5' temiinal nucleotide of the guide strand and the penultimate 3" (N-l ) nucleotide of the guide strand, here N is the 3’ terminal nucleotide;

[0575] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide; (4) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the seventh (+7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide; (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide;

[0576] (6) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e.. in the 5‘ direction, relative to the central nucleotide of the passenger strand; and

[0577] (7) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e, in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0578] (8) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp Page 86 of 566

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[0580] configuration internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand: and

[0581] (9) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0582] wherein the ds oligonucleotide further comprises a 2' modification, e.g., a 2' F modification, of the 3‘ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non- negatively charged internucleotidic linkage. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e, in the 3' direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fifth (+5) nucleotide. In certain embodiments, the ds Page 87 of 566

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[0584] oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2’-F modification, 2’-H modification, 2 ’-OH modification, 2’-O-alkyl modification, e.g., 2'-O-methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of tire 5" terminal ( 1) nucleotide, second (+2) nucleotide, third, (4-3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5’ terminal ( -1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the second (4-2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (44) nucleotide, i.e., in the 3’ direction, comprise a 2 -F modification of the third (4-3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (4-4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the sixth (4-6) nucleotide.

[0585] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, and the guide strand further comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of backbone chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N- 1 ) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of:

[0586] (1) a guide strand where one or both of the 5’ and 3 / terminal dinucleotides are not linked by non- Page 88 of 566

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[0588] negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0589] (2) a guide strand where one or more Rp, Sp, or stereorandom non -negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l ) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0590] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide; (4) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the seventh (+7) and eighth (+8) nucleotides, relative to the 5' terminal nucleotide; (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3 ' nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide;

[0591] (6) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e.. in the 5' direction, relative to the central nucleotide of the passenger strand; and

[0592] (7) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0593] (8) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0594] (9) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0595] In certain embodiments, the ds oligonucleotide further comprises a 2' modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non- negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain Page 89 of 566

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[0597] embodiments, the one or more Rp, Sp, or stereorandom non -negatively charged internucleotidic linkage is a stereorandom non -negatively charged internucleotidic linkage. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e,, in the 3’ direction, comprise a modified sugar at the 5' terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center m the Sp configuration between the -3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a modified sugar at the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (4-6) nucleotide. In certain embodiments, the modified sugar comprises a 2’-F modification, 2’-H modification, 2’-OH modification, 2’-O-alkyl modification, e.g., 2'-O-methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center m the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3" direction, comprise a 2’-F modification of the 5’ terminal (4-1) nucleotide, second (4-2) nucleotide, third, (4-3) nucleotide, fourth (4-4) nucleotide, fifth (4-5) nucleotide, and / or sixth (4-6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp Page 90 of 566

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[0599] configuration between the +3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5' terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream ( +4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fifth (4-5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the sixth (4-6) nucleotide.

[0600] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center m the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e, in the 3’ direction, and the guide strand further comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the 5’ terminal (4-1) nucleotide and the immediately downstream (42) nucleotide and between the (42) nucleotide and the immediately downstream (4-3) nucleotide, as w'ell as between the (4-5) nucleotide and the (4-6) nucleotide, and one or more of:

[0601] (1) a guide strand where one or both of the 5’ and 3' terminal dinucleotides are not linked by non- negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e, in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0602] (2) a guide strand w here one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3' terminal nucleotide;

[0603] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the tenth (4-10) and eleventh (4-11) nucleotides, relative to the 5’ terminal nucleotide; (4) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage Page 91 of 566

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[0605] occurs between the seventh (+7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide; (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide;

[0606] (6) a passenger strand where one or more Rp. Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0607] (7) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand.

[0608] (8) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e.. in the 5’ direction, relative to the central nucleotide of the passenger strand: and

[0609] (9) a passenger strand where one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0610] In certain embodiments, the ds oligonucleotide further comprises a 2" modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non -negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non- negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5' terminal (+1) nucleotide, second (+2) nucleotide, third. (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the t3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3‘

[0611] Page 92 of 566

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[0613] direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -1-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3‘ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fifth (-1-5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2'-F modification, 2’-H modification, 2'-OII modification, 2’-O-alkyl modification, e.g., 2'-O- methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA). or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (-1-6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the 5' terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration betyveen the -t-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (-1-4) nucleotide. In certain embodiments, the ds Page 93 of 566

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[0615] oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2'-F modification of the fifth (+5) nucleotide In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -1-3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3' direction, comprise a 2'-F modification of the sixth (+6) nucleotide.

[0616] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (-14) nucleotide, i.e., m the 3’ direction, and the guide strand further comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5 ’ terminal nucleotide of the guide strand and the penultimate 3 (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide, a 2' modification, e.g., a 2’ F modification, of the 3 ’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp. Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5‘ terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center m the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the t3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3‘

[0617] Page 94 of 566

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[0619] direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -1-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3‘ direction, comprise a modified sugar at the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2’-F modification, 2'-H modification, 2'-OH modification, 2’-O-alkyl modification, e.g., 2'-O-methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5' terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2'-F modification of the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration betyveen the -t-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the sixth (+6) nucleotide.

[0620] Page 95 of 566

[0621] 13346662vlAttorney Docket No.: 2010581-1677

[0622] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center m the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, and the guide strand farther comprises backbone phosphorothioate chiral centers in Sp configuration between tire 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, a 2' modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration.

[0623] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, further comprise one or a plurality of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the 5’ tenninal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately dowmstream (+4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2’-F modification, 2"-H modification, 2‘-OH modification, 2’-O-alkyl modification, e.g., 2‘-O- Page 96 of 566

[0624] 13346662vlAttorney Docket No.: 2010581-1677

[0625] methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA). or Homo-DNA. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5’ terminal (+1) nucleotide, second (+2) nucleotide, third, (+3) nucleotide, fourth (+4) nucleotide, fifth (+5) nucleotide, and / or sixth (-1-6) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the 5" terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e,, in the 3’ direction, comprise a 2’-F modification of the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the t-3 nucleotide and the immediately downstream (-1-4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, comprise a 2’-F modification of the fifth (+5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -1-3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3" direction, comprise a 2’-F modification of the sixth (+6) nucleotide.

[0626] In certain embodiments, the one or more Rp, Sp, or stereorandom non -negatively charged internucleotidic linkage incorporated into the guide strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non- negatively charged internucleotidic linkage. In certain further embodiments, the passenger strand comprises an Sp backbone phosphorothioate chiral center between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and an Sp backbone phosphorothioate chiral center between the penultimate (N- 1) nucleotide and the 3’ terminal (N) nucleotide.

[0627] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream Page 97 of 566

[0628] 13346662vlAttorney Docket No.: 2010581-1677

[0629] (44) nucleotide, i.e., in the 3 ‘ direction, and the guide strand further comprises backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration.

[0630] In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream ( -4) nucleotide, i.e., in the 3’ direction, further comprise one or a plurality7of modified sugars. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3?direction, comprise a modified sugar at the 5’ terminal (+1 ) nucleotide, second (+2) nucleotide, third, (4-3) nucleotide, fourth (4-4) nucleotide, fifth (45) nucleotide, and / or sixth (+6) nucleotide. In certain embodiments, tire ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately' downstream (4-4) nucleotide, i.e, in the 3’ direction, comprise a modified sugar at the 5' terminal (+1) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the second (+2) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 43 nucleotide and the immediately downstream (44) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the third (+3) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3' direction, comprise a modified sugar at the fourth (+4) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the 4-3 nucleotide and the immediately downstream (4-4) nucleotide, i.e,, in the 3’ direction, comprise a modified sugar at the fifth (4-5) nucleotide. In certain embodiments, the ds oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the -3 nucleotide and the immediately downstream (4-4) nucleotide, i.e., in the 3’ direction, comprise a modified sugar at the sixth (4-6) nucleotide. In certain embodiments, the modified sugar comprises a 2 -1 modification, 2’-H modification, 2’-OH modification, 2’-O-alkyl modification, e.g., 2'-O-methyl (OMe) modification, 2’- Methoxyethyl (MOE) modification, locked nucleic acid (LNA), unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or Homo-DNA. In certain embodiments, the ds Page 98 of 566

[0631] 13346662vlAttorney Docket No.: 2010581-1677

[0632] oligonucleotides comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately do...

Claims

Attorney Docket No.: 2010581-1677CLAIMS1. A double-stranded RNAi (dsRNAi) agent capable of directing INHBE (Inhibin pE)-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein:the guide strand is complementary or substantially complementary to an INHBE target RNA sequence, the guide strand comprises a seed region at its 5 ‘-end region that is capable of mediating the initial recognition of the target RNA sequence: andthe passenger strand comprises a PN internucleotidic linkage between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide.

2. The dsRNAi agent of claim 1, wherein the guide strand comprises a PN internucleotidic linkage in the seed region.

3. Hie dsRNAi agent of claims 1 or 2, wherein the guide strand comprises a phosphorothioate (PS) internucleotidic linkage or a natural phosphate linkage (PO) between the +2 nucleotide and the immediately downstream (+3) nucleotide, relative to the 5’ nucleotide of the guide strand.

4. The dsRNAi agent of any of claims 1-3, wherein the guide strand comprises a phosphorothioate (PS) internucleotidic linkage between the +2 nucleotide and the immediately downstream (+3) nucleotide, relative to the 5’ nucleotide of the guide strand.

5. The dsRNAi agent of claim 4, wherein the PS internucleotidic linkage between the +2 nucleotide and the immediately downstream (+3) nucleotide, relative to the 5’ nucleotide of the guide strand, is in the Rp configuration.

6. Tire dsRNAi agent of claims 1-4, wherein the PN internucleotidic linkage between the 5’ terminal (+1) nucleotide and the immediately downstream (12) nucleotide of the passenger strand is in the Rp configuration.

7. lire dsRNAi agent of any of claims 1-4. wherein the passenger strand further comprises a PN internucleotidic linkage between the 3’ terminal (N) nucleotide and the penultimate (N-l ) nucleotide.

8. The dsRNAi agent of claim 7, wherein the PN internucleotidic linkage between the 3' terminal (N) nucleotide and the penultimate (N-l) nucleotide of the passenger strand is in the Rp configuration.

9. Hie dsRNAi agent of any of the preceding claims, wherein the PN internucleotidic linkage is a phosphoryl guanidine internucleotidic linkage.

10. The dsRNAi agent of claim 9, wherein the PN internucleotidic linkage is a MsPA internucleotidic Page 552 of 56613346662vlAttorney Docket No.: 2010581-1677linkage.

11. Hie dsRNAi agent of any of claims 1-10, wherein the PN internucleotidic linkage comprises the r-N?Iz>=N — P— O'" N 0structure of! f(nOOl).

12. The dsR Ai agent of any of claims 1-11, wherein the PN internucleotidic linkage in the seed region is between the +3 nucleotide and the immediately downstream (+4) nucleotide, relative to the 5' terminal nucleotide of the guide strand.

13. The dsRNAi agent of claim 12, wherein the PN internucleotidic linkage in the seed region of the guide strand is in the Sp configuration.

14. The dsRNAi agent of claims 13, wherein the nucleoside 3’ and / or 5" to the PN internucleotidic linkage in the seed region comprises a 2"-F ribose modification.

15. The dsRNAi agent of claim 14. wherein the +2 nucleoside, the +3 nucleoside, or the +2 nucleoside and +3 nucleoside, relative to the 5’ terminal nucleoside of the guide strand, comprise a 2’-F modification.

16. Hie dsRNAi agent of claims 1-15, wherein when the guide strand comprises a PS internucleotidic linkage between the +2 nucleotide and the immediately downstream (+3) nucleotide, relative the 5' nucleotide of the guide strand, the +3 nucleoside, relative to the 5’ terminal nucleoside of the guide strand, comprises a 2’-F modification.

17. The dsRNAi agent of claims 1-1. wherein when the guide strand a comprises natural phosphate linkage (PO) between the +2 nucleotide and the immediately downstream (+3) nucleotide, relative the 5’ nucleotide of the guide strand, the +2 nucleoside, relative to the 5 ' terminal nucleoside of the guide strand, comprises a 2’-F modification.

18. The dsRNAi agent of any of claims 1-17, wherein the guide strand further comprises a PN internucleotidic linkage between the -t-10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5’ terminal nucleotide of the guide strand.

19. The dsRNAi agent of claim 18, wherein the nucleoside 3' to the PN internucleotidic linkage between the +10 nucleotide and tire immediately downstream (+11) nucleotide, relative to the 5’ terminal nucleotide of the guide strand, comprises a 2’-F ribose modification.Page 553 of 56613346662vlAttorney Docket No.: 2010581-167720. The dsRNAi agent of claim 19, wherein the PN internucleotidic linkage between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5 ’ terminal nucleotide of the guide strand, is in the Rp configuration,21. The dsRNAi agent of any of claims 1-20, wherein the guide strand comprises a 5’ phosphate modification.

22. The dsRNAi agent of claim 21, wherein the 5' phosphate modification is a 5’ phosphate mimic modification.

23. The dsRNAi agent of claim 22, wherein the 5’ phosphate mimic modification isthe base is N3U, or is selected from A, C, G, T, U, abasic, and modified nucleobases other than N3U: andR!is selected from H, OH, O-alkyl, O-methyl (O-Me), F, O-methoxyethyl (MOE), and 2’-f),4’C- methylene-bridged or locked nucleic acid (2’,4’-BNA or LNA).

24. The dsRNAi agent of claim 23, wherein R1is O-methyl (O-Me)25. lire dsRNAi agent of any of claims 1-24, wherein the guide strand further comprises a backbone phosphorothioate (PS) internucleotidic linkage chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l ) nucleotide, and / or a PN internucleotidic linkage between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide26. The dsRNAi agent of claim 25, wherein the PN internucleotidic linkage between the 3' terminal (N) nucleotide and the penultimate (N-l) nucleotide, and / or the PN internucleotidic linkage between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, is / are in the 5p configuration.27 The dsRNAi agent of any of claims 1 -26, wherein the guide strand comprises a phosphoryl guanidine cap at its 5 ’-end ( ’-end PN cap).Page 4 of 56613346662vlAttorney Docket No.: 2010581-1677The dsRNAi agent of claim 27, wherein the 5 ’-end PN cap is selected fromw'herein:the base is N3U, or is selected from A, C, G. T, U, abasic, and modified nucleobases other than N3U; andR1is selected from H, OH, O-alkyl, O-methyl (O-Me), F, O-m ethoxyethyl (MOE), and 2'-O,4'C- methylene-bridged or locked nucleic acid (2’,4’-BNA or LNA).

29. The dsRNAi agent of claim 28, wherein R1is O-methyl (O-Me)30. The dsRNAi agent of any of claims 1-29, wherein the guide strand comprises a natural phosphate linkage (PO) betyveen the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and / or a natural phosphate linkage (PO) between the +2 nucleotide and the +3 nucleotide, relative to the 5’ terminal nucleotide.

31. A double-stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein the guide strand is complementary' or substantially complementary' to a target RNA sequence; wherein the guide strand comprises a 5’ phosphate modification; and wherein the guide strand further comprises a backbone phosphorothioate (PS) internucleotidic linkage in the > S’p configuration between the 5’ terminal (+1) nucleotide and the immediately doyvnstrearo (+2) nucleotide, and a backbone PS internucleotidic linkage in the p configuration betyveen the +2 nucleotide and immediately downstream (+3) nucleotide.

32. A double -stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRN Ai agent comprising a guide strand and a passenger strand, wherein the guide strand is complementary or substantially complementary’ to a target RNA sequence; wherein the guide strand comprises a 5’ phosphate modification; wherein the guide strand further comprises a backbone phosphorothioate (PS) internucleotidic linkage in the Sp configuration betw een the 5’ terminal ( + 1 ) nucleotide and the immediately downstream (+2)Page 555 of 56613346662vlAttorney Docket No.: 2010581-1677nucleotide; and wherein the backbone linkage between the +2 nucleotide and the immediately downstream (+3) nucleotide of the guide strand is an unmodified phosphodiester (PO) linkage33. The dsRNAi agent of claim 1 or 32, wherein the 5’ phosphate modification is a 5’ phosphate mimic modification.

34. Hie dsRNAi agent of claim 33, wherein the 5’ phosphate mimic modification is selected from:wherein:the base is N3U, or is selected from A, C, G, T, U, abasic, and modified nucleobases other than N3U;R1is selected from H, OH, O-alkyl, O-methyl (O-Me), F, O-methoxyethyl (MOE), and 2 ’-(9,4’ C’-m ethylene - bridged or locked nucleic acid (2’,4’-BNA or LNA); andR2is selected from alkyl, methyl, ethyl, isopropyl, propyl, cyclohexyh benzyl, phenyl, tolyl, xylyl, aryl, and arene group.

35. Hie dsRNAi agent of claim 34, wherein the 5’ phosphate mimic modification is36. Hie dsRNAi agent of claim 34 or 35. wherein the R1is LNA bridge to the 4’ position.37 Hie dsR Ai agent of claim 34 or 35, wherein the R1is MOE.

38. Hie dsRNAi agent of claim 34 or 35, wherein the R1is F.O' O P==O39. The dsRNAi agent of claim 34, whereinR2in ’Page 556 of 56613346662vlAttorney Docket No.: 2010581-1677O’40. The dsRNAi agent of any of the preceding claims, wherein the guide strand further comprises a PN internucleotidic linkage in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide.

41. The dsRNAi agent of any of the preceding claims, wherein the guide strand further comprises backbone PS internucleotidic linkages in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide.

42. The dsRNAi agent of any of the preceding claims, wherein the guide strand further comprises a PN internucleotidic linkage in the Rp configuration between the +10 nucleotide and the +11 nucleotide43. The dsRNAi agent of any of the preceding claims, wherein the guide strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5" temiinal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide.

44. The dsRNAi agent of any of the preceding claims, wherein the guide strand comprises a 2' modification, of tire 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage.

45. The dsRNAi agent of any of tire preceding claims, wherein the guide strand comprises an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide.

46. The dsRNAi agent of any of the preceding claims, wherein the guide strand comprises an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage between the seventh (+7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide.

47. The dsRNAi agent of any of the preceding claims, wherein the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3' terminal nucleotide, and the upstream N-10 nucleotide.

48. The dsRNAi agent of any of the preceding claims, wherein the passenger strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand.

49. lire dsRNAi agent of any of the preceding claims, wherein tire passenger strand comprises one or more Page 557 of 56613346662vlAttorney Docket No.: 2010581-1677Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

50. The dsRNAi agent of any of the preceding claims, wherein the passenger strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e., in the 5" direction, relative to the central nucleotide of the passenger strand.

1. The dsRNAi agent of any of the preceding claims, wherein the passenger strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

52. The dsRNAi agent of any of the preceding claims, wherein the passenger strand in combination with one or more of the aforementioned guide strands, comprises one or more modified sugars between the 5 ’ terminal (+1) nucleotide and the penultimate (N-l) nucleotide.

53. The dsRNAi agent of any of the preceding claims, wherein the passenger strand comprises one or more of:a. 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49;b. one or more backbone chiral centers in Rp or Sp configuration;c. one or more backbone phosphoryl guanidine chiral centers in the Rp configuration betw een the +7 nucleotide and the immediately downstream (+8) nucleotide, i.e., in the 3‘ direction;d. one or more backbone phosphoryl guanidine chiral centers in the Rp configuration between the +15 nucleotide and the immediately’ downstream (+16) nucleotide, i.e., in the 3’ direction; and / or e. backbone phosphorothioate chiral centers in the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the 3' terminal nucleotide and the penultimate (N-l) nucleotide.

54. The dsRNAi agent of any of the preceding claims, wherein each strand of the dsRNAi agent independently has a length of about 15 to about 49 nucleotides.55 The dsRNAi agent of any ofthe preceding claims, wherein the Rp, Sp, or stereorandom non-negatively charged backbone internucleotidic linkages have neutral charge.

56. Hie dsRNAi agent of claim 55, wherein the neutral backbone internucleotidic linkage is [CH2]nCH3.0^N ~ P\" N o °><[CHjmCHj wherein n is about 0 to 49 and m is about 0 to 49.

57. Hie dsRNAi agent of claim 56, wherein the guide strand comprises a linkage having the followingPage 558 of 56613346662vlAttorney Docket No.: 2010581-1677structurebetween the third (+3) and fourth (+4) nucleotides of the guide strand, between the tenth ( +10) and eleventh (+11) nucleotides of the guide strand, or both.

58. The dsRNAi agent of claim 56, wherein the guide strand comprises a linkage having the following structurebetween the third (+3) and fourth (+4) nucleotides of the guide strand, between the seventh (+7) and eighth (+8) nucleotides of the guide strand, between the tenth (+10) and eleventh (+11) nucleotides of the guide strand, between the eighteenth (+18) and nineteenth ( + 19) nucleotides of the guide strand, or combinations thereof.

59. The dsRNAi agent of claim 56, wherein the passenger strand comprises a linkage having the following [CH2]nCH3r"\.o 'N o °y.structure[1CHo2lJmCHi3, where n is about Oto 49 and m is about 0 to 49. at 5’ to the central nucleotide of the passenger strand, 3 ’ to the central nucleotide of the passenger strand, or both.

60. The dsRNAi agent of claim 56, wherein the passenger strand comprises a linkage having the following [CH2]nCH3.o^I" N o oy,structure[CH2]mCH3, wherein n is about 11 to 49 and m is 0.

61. The dsRNAi agent of claim 60, wherein n is 11 or 15.

62. Tire dsRNAi agent of any of the preceding claims, wherein the passenger strand comprises:i. a non -negatively charged backbone internucleotidic linkage in the Rp configuration between the +7 nucleotide and the +8 nucleotide;li. a non-negatively charged backbone internucleotidic linkage in the Rp configuration between the +1 nucleotide and the +16 nucleotide; and / oriii. backbone phosphorothioate chiral centers in the Sp configuration between the 5’ terminal (+1) nucleotide and the +2 nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide.

63. The dRNAi agent of any of the preceding claims, wherein the passenger strand comprises:Page 559 of 56613346662vlAttorney Docket No.: 2010581-1677P-O N' N N' — 1 in the Rp configuration between the +7 nucleotide and the +8 nucleotide;1 q p-o' N' " x^- X / Ali. ' — / in the Rp configuration between the + 15 nucleotide and the +16 nucleotide; andiii. backbone phosphorothioate chiral centers in the Sp configuration between the +1 nucleotide and the +2 nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide.

64. The dsRNAi agent of any of the preceding claims, wherein the passenger strand comprises:3 5-o ' / / ,0 \p-o'1N'■ — / in the Rp configuration between the +7 nucleotide and the +8 nucleotide; andbackbone phosphorothioate chiral centers in the Sp configuration between the +1 nucleotide and the +2 nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide.65 An oligonucleotide having the structure ofRNA1 {p.m(U)[n001R].m(C)p.m(C)p.m(U)p.m(U)p.m(C)p.[fl2r](C)p.m(A)p.[fl2r](U)p.[fl2r](U)p.[fl2r](C)p. m(U)p,m(G)p.m(C)p,m(C)p.m(G)p.m(U)p,m(C)p.m(U)p,m(U)[Ssp],m(A)}|CHEMl{[nC6o]}iCHEM2{[Gal NAc3C12oyl] } $CHEM1, RNA1,1: R1-1: R1 |CHEM1, CHEM2:1: R2-1: R1S$$V2. O or a pharmaceutically acceptable salt form thereof.

66. An oligonucleotide having the structure of RNAl{p.m(U)[n001R].m(C)p m(C)p.m(U)p.m(U)p.m(C)p.[fl2r](C)p. (A)p.[fl2r](U)p [fl2r](U)p.[fl2r](C)p. m(U)p.m(G)p.m(C)p.m(C)p.m(G)p.m(U)p.ro(C)p m(U)p.ra(U)[n001R].m(A)}|CHEMl {[nC6o]}: CHEM2{[GalNAc3C12oyl]}$CHEMl, RNAEl: Rl-l: Rl|CHEMl, CHEM2,l: R2-l: Rl$$$V2.0 or a pharmaceutically acceptable salt form thereof.

67. A dsRNAi agent having the structure ofRNA1 {[d5m](U)[Ssp].[fl2r](A)[Rsp].m(A)[n001S].[fl2r](G)p.m(A)p.[fl2r](C)p.m(G)p.m(G)p.m(C)p.m(A)[ n001R].[fl2r](G)p.m(A)p.m(A)p.[fl2r](U)p.m(G)p.[fl2r](G)p.m(A)p.m(A)p.m(G)p.m(G)p.m(A)[Ssp].m(U)[Page 560 of 56613346662vlAttorney Docket No.: 2010581-1677 Ssp].m(U)}|RNA2{p.m(U)|n001R].m(C)p.m(C)p.m(Lr)p.m(U)p.m(C)p.|fl2r](C)p.m(A)p.[tl2r](U)p.[fl2r](U) p.[fl2r](C)p.m(L)p.m(G)p.m(C)p.m(C)p.m(G)p.m(L!)p.m(C)p.m(U)p.m(U)[Ssp].m(A)}iCHEMl{[ptz]}|CEl EM2 { [nC6o] } | CHEM3 { [GalN Ac3 C 12oyl] } $CHEM 1, RN A 1, 1: R 1 - 1: R11 CHEM2, RNA2, 1: R 1 -1: R1|CHEM2, CHEM3,1: R2-1: R1$$$V2. O or a pharmaceutically acceptable salt form thereof.

68. A dsRNAi agent having the structure of RNAl{[d5m](L!)[SspJ.[fl2r](A)[Rsp].m(A)[n00lS].[fl2r](G)p.m(A)p.[fl2r](C)p.m(G)p.m(G)p.m(C)p.m(A)[ n001R].[fl2r](G)p.m(A)p.m(A)p.[fl2r](U)p.m(G)p.[fl2r](G)p.m(A)p.m(A)p m(G)p.m(G)p.m(A)[Ssp].m(LT)[ Ssp].m(LT)}|RNA2{p.m(U)[n001R].m(C)p.m(C)p.m(LT)p.m(U)p.m(C)p.[fl2r](C)p.m(A)p.[fl2r](U)p.[fl2r](Li) p.[fl2r](C)p.m(E)p.m(G)p.m(C)p.m(C)p.m(G)p.m(U)p.m(C)p.m(U)p.m(U)[n001R].m(A)}iCHEMl { [ptz]} |C HEM2{ [nC6o] } |CHEM3 { [GalNAc3C 12oyl] }$CHEM1, RN 1, 1: R1 - 1: R1 |CHEM2, RNA2, 1: R 1 - 1: R1|CHEM2, CHEM3, 1: R2-1: R1$$$V2. Oor a pharmaceutically acceptable salt form thereof.69 A dsRNAi agent having the structure ofRNA1 {[d5m](U)[Ssp].[fl2r](A)p.[fl2r](A)[n001S].m(G)p.m(A)p.[fl2.r](C)p.m(G)p.m(G)p.m(C)p.m(A)[n001 R].[fl2r](G)p.ra(A)p.m(A)p.[fl2r](U)p.m(G)p.[fl2r](G)p.m(A)p.m(A)p.ro(G)p,m(G)p,m(A)[Ssp],m(U)[Ssp]. m(U)}|RNA2{p.m(U)[n001R].m(C)p.m(C)p.m(U)p.m(LT)p.m(C)p.[fl2r](C)p.m(A)p.[fl2r](U)p.[fl2r](U)p.[fl 2r](C)p.in(LT)p.m(G)p.m(C)p.m(C)p.m(G)p.m(LI)p.m(C)p.m(U)p.m(U)[Ssp].m(A)}^CHEMl{[tz][! CElEM2{ [cp]}|CHEM3{[nC6o]}|CHEM4{[GalNAc3C12oyl]}$CHEMl. RNAl,l: Rl-l: Rl|CHEM3, RNA2,l: Rl- 1: R1|CHEM1, CHEM2,1: R2-1: R1|CHEM3, CHEM4,1: R2-1: R1$S$V2. O or a pharmaceutically acceptable salt form thereof.

70. A dsRNAi agent having the structure of RNA 1 { [d5m] (U) [Ssp]. [fl2r](A)p. [f!2r j (A) [nOO 1 S],m(G)p m(A)p. [f!2r] (C)p m(G)p,m(G)p,m(C)p,m(A) [nOO 1 R].[fl2r](G)p.m(A)p.m(A)p.[fl2r](U)p.m(G)p.[fl2r](G)p.m(A)p.m(A)p.m(G)p.m(G)p.m(A)[Ssp],m(U)[Ssp]. m(U)}|RNA2{p.m(U)[n001R].m(C)p.m(C)p.m(U)p.m(LT)p.m(C)p.[fl2r](C)p.m(A)p.[fl2r](U)p.[fl2r](U)p.[fl 2r](C)p.m(U)p.m(G)p.m(C)p.m(C)p.m(G)p.m(U)p m(C)p.m(LT)p.m(U)[n001R] m(A)[|CHEMl {[tz]}|CHEM 2 { [cp] } | CHEM3 { [nC6o] } | CHEM4 { [GalN Ac3 C 12oyl] } $CHEM 1, RNA 1, 1: R1 - 1: R11 CHEM3. RN A2, 1: R1 -1: R1|CHEM1, CHEM2,1: R2-1: R1|CHEM3, CHEM4,1: R2-1: R1$S$V2. O or a pharmaceutically acceptable salt form thereof.

71. An oligonucleotide, wherein the oligonucleotide is selected from Table 1.

72. An oligonucleotide, wherein the oligonucleotide is:5 ’-[cp] [tz] [d5m]B 1 [Ssp] [f!2r] B2 [Rsp]mB3 [nOO 1 S] [fl2r]B4pmB5p[fl2r]B6pmB7pmB8pmB9pmB 10 [n 001 R] [fl2r] B 1 IpmB 12pm B 13p [fl 2r] B 14pmB 15p [fI2r]B 16pmB 17pmB 18pmB 19pmB20pmB21 [Ssp]mB 22 [Ssp]mB23-3' or a salt thereof, wherein:[cp] represents -P(O)(OH)2:Page 561 of 56613346662vlAttorney Docket No.: 2010581-1677. V-A r -N> N>|tz] represents'N, wherein the carbon atom is bonded to [cp] and the nitrogen atom is bonded to the 5 ’-carbon atom of [d5mU];[d5m]Bl represents a 5’-deoxy-2'-O-methylnucleoside (e.g., [d5m]U represents 5’-deoxy-2’-O-methyl uridine);each of Bl, B2, B3. B4, B5, B6, B7, B8, B9. BIO, Bl 1, B12, B13, B14. B15, B16, B17, B18. B19, B20, B21, B22 and B23 is independently a nucleoside;[SspJ represents S'p phosphorothioate linkage;[f!2r] represents a 2’-F modified sugar in a nucleoside (e.g., [f!2r]A represents 2’-fhioro-2’- deoxyadenosine, [fl2r]G represents 2 ’-fluoro-2’ -deoxyguanosine, [f!2r]C represents 2’-fluoro-2’-deoxycytidine, [fl2r]U represents 2’-fluoro-2’-deoxyuridine, etc.);[Rsp] represents p phosphorothioate linkage;m represents a 2’-OMe modified sugar in a nucleoside (e.g., mA represents 2 ’-O-methyl adenosine. mG represents 2’-O-methylguanosine, mC represents 2’-O-methylcytidine, mil represents 2 -0- methyluridine, etc.);[nOOlS] represents > Sp A-(l,3-dimcthylimidazolidin-2-ylidcnc)phosphoramidatc linkage;p represents phosphodiester linkage; and[nOOIR] represents / tp A-(l,3-dimethylimidazolidin-2-ylidene)phosphoramidate linkage.

73. The oligonucleotide of claim 72, wherein B1B2B3B4B5B6B7B8B9B10B11B12B13B14B15B16B17B18B19B20B21B22B23 is or comprises a base sequence selected from Table 174. An oligonucleotide, wherein the oligonucleotide is:5"-[GalNAc3C12oyl]|nC6o]pmDl^Ssp]mD2pmD3pmD4pniD5pmD6p[112rlD7pmD8p 112r]D9p[112 i D 10p[112r]D 11 pm D 12pm D 13 mD 14pmD 15pmD 16pmD 17pm D 18 D 19pmD20[ Ssp]mD21 ~3 ’ or a salt thereof, wherein:[GalNAc3C12oyl] representsO Page 62 of 56613346662vlAttorney Docket No.: 2010581-1677[nC6o] represents -NH(CH2)e-, wherein - H- is bonded to [GalNAc3C12oyl], and the end -CH - is bonded to an oxygen atom that is bonded to the linkage phosphorus of a phosphodiester linkage:p represents phosphodiester linkage;each of DI, D2, D3, D4, D5, D6, D7, D8, D9, DIO, Dll, D12, D13. D14, D15, D16, D17, D18, D19, D20 and D21 is independently a nucleoside:m represents a 2’-0Me modified sugar in a nucleoside (e.g., mA represents 2'-O-methyladenosine. mG represents 2’-O-methylguanosme. mC represents 2’-O-methylcytidine. mU represents 2 -0-methyluridine, etc ):[Ss ] represents 5p phosphorothioate linkage; and[f!2r] represents a 2’-F modified sugar in a nucleoside (e.g., [fl2rjA represents 2’-fluoro-2’- deoxyadenosine, [fl2r]G represents 2'-fluoro-2'-deoxyguanosine, [f!2r]C represents 2’-fhioro-2’- deoxycytidine, [f!2r]U represents 2’-fluoro-2T-deoxyuridine, etc.).

75. An oligonucleotide, wherein the oligonucleotide is:5’-[GalNAc3C12oyl][nC6o]pmDl[n001R]mD2pmD3pmD4pmD5pmD6p fl2r5D7ptnD8p[fl2r|D9p[ fl2r]D10p fl2r]Dl lpmD12pmDl 3pmD14pmD15pi D16pn5D17prftD18pmD19pmD20[Ssp]inD21-3’ or a salt thereof, wherein:NHAc V Y [GalNAc3C12oyl] represents°;[nC6o] represents -NH(CH?)g-, wherein -NH- is bonded to [GalNAc3C12oyl], and the end -CH?-is bonded to an oxygen atom that is bonded to the linkage phosphorus of a phosphodiester linkage:p represents phosphodiester linkage;each of DI, D2, D3, 1)4. D5, D6, D7, D8, D9, DIO, Dll, D12, D13, D14, D15, D16, D17, D18, D19, D20 and D21 is independently a nucleoside;m represents a 2’-0Me modified sugar in a nucleoside (e.g.. mA represents 2’-O-methyladenosine, mG represents 2 -O-methylguanosme, mC represents 2’-O-methylc tidine, mU represents 2 -0-methyluridine, etc );[nOOIR] represents 7?p rV-( 1,3-dimethylimidazolidin-2-ylidene)pbosphoramidate linkage;[Ssp] represents Np phosphorothioate linkage; and[f!2r] represents a 2'4' modified sugar in a nucleoside (e.g., [fl2r]A represents 2’-fluoro-2’- Page 563 of 56613346662vlAttorney Docket No.: 2010581-1677deoxyadenosine, [fl2r]G represents 2’-fluoro-2’-deoxyguanosine. |f!2r]C represents 2’-fluoro-2’-deoxycytidine, [fl2r]U represents 2’-fluoro-2’-deoxyuridine. etc.).

76. An oligonucleotide, wherein the oligonucleotide is:5’-[GalNAc3C12oyl][nC6o]pmDl[n001R]mD2pmD3pmD4pmD5pmD6p[fl2rjD7pmD8p[fl2r]D9p[ fl2r]D10p fl2r]Dl IpmD12prnD13pmD14pmD15pmDl 6prnDr7pmD18pmD19pmD2O nOC- 1 R]mD21“3’ or a salt thereof, wherein:HO / OH| GalNAc3C12oyl] represents0;[nC6o] represents ”NH(CH2)6~. wherein ~NH~ is bonded to [GalNAc3C12oylj, and the end ~CH2“ is bonded to an oxygen atom that is bonded to the linkage phosphorus of a phosphodiester linkage;p represents phosphodiester linkage;each of DI, D2, D3. D4, D5, D6, D7, D8, D9, DIO, Dll, D12, D13, D14, D15, D16, D17, D18, D19, D20 and D21 is independently a nucleoside:m represents a 2’-0Me modified sugar in a nucleoside (e.g., mA represents 2’-O-methyladenosine, raG represents 2 ’-O-m ethylguanosine, mC represents 2’-O-methylcytidine, mU represents 2 -0-methyluridine, etc.);[nOOIR] represents / ip 7V-(l,3-dimethylimidazolidin-2-ylidene)phosphoratnidate linkage; and [fl2r] represents a 2’-F modified sugar in a nucleoside (e.g., [f!2r]A represents 2’-fluoro-2’- deoxyadenosine, [f 2r]G represents 2?-fluoro-2’-deoxyguanosine. [f!2r]C represents 2’-fluoro-2’-deoxycytidine, [fl2r]U represents 2’-fluoro-2’-deoxyuridine, etc ).

77. The oligonucleotide of any one of claims 74-76, whereinD 1D2D3D4D5D6D7D8D9D 10D 1 ID 12D 131) 14D 15D 16D 17D 18D 19D20D21 is or comprises a base sequence selected from Table 1.

78. A dsRNAi agent, wherein the dsRNAi agent comprises the oligonucleotide of claim 72 and / or the oligonucleotide of any one of claims 74-76.79 A dsRNAi agent, wherein the dsRNAi agent is selected from Table 1.

80. A composition comprising the oligonucleotide or dsRNAi agent of any one of the preceding claims.

81. A method for reducing level and / or activity of a transcript or a protein encoded thereby comprising administering to a cell expressing the transcript an oligonucleotide or dsRNAi agent of any of the preceding Page 564 of 56613346662vlAttorney Docket No.: 2010581-1677claims, wherein the guide strand of the dsRNAi agent comprises a targeting -binding sequence that is completely complementary to a target sequence in the transcript.

82. The method of claim 81, wherein the cell is an immune cell, a blood cell, a cardiac cell, a lung cell, an optic cell, a muscle cell, a liver cell, a kidney cell, a cell of the central nervous system, or a cell of the peripheral nervous system, optionally wherein the cell of the central nervous system is a brain cell.

83. lire method of claim 81 or 82. wherein when the oligonucleotide or dsRNAi agent is contacted with a cell comprising transcripts of both the target allele and another allele of the same nucleic acid sequence, it shows suppression of transcripts of the particular allele at a level that is:a. greater than when the oligonucleotide or dsRNAi agent is absent;b. greater than a level of suppression observed for another allele of the same nucleic acid sequence; orc. both greater than when the oligonucleotide or dsRNAi agent is absent, and greater than a level of suppression observed for another allele of the same nucleic acid sequence.84 A method, comprising administering an oligonucleotide. dsRNAi agent, or composition of any one of the preceding claims to a system or subject.

85. A method for preventing or treating a condition, disorder, or disease, comprising administering an effective amount of an oligonucleotide, dsRNAi agent, or composition of any one of the preceding claims to a subject susceptible thereto or suffering therefrom.

86. An oligonucleotide, dsRNAi agent, composition, phosphoramidite, or method described in the specification.Page 565 of 56613346662vl

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