Highly RNA-tagged (ARNATAR)

Abstract

Disclosed herein are advanced RNA-targeting (ARNATAR) oligomeric compounds that are carefully designed to inhibit gene expression via the RISC pathway, and are useful in methods for reducing the expression of specific genes, many of which are associated with a variety of diseases and disorders.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This PCT application claims the benefit of priority to U.S. Provisional Application No. 63 / 433,706, filed December 19, 2022, and U.S. Provisional Application No. 63 / 472,780, filed June 13, 2023, the entire contents of each of which are incorporated herein by reference in their entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials Incorporated herein by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing, identified and submitted concurrently with this application as a text file entitled "SiRNA_sequence_listing", created on December 18, 2023.

[0003] Throughout this application, various publications are referenced. All publications, gene transcript identifiers, patents, and patent applications mentioned herein are herein incorporated by reference to the same extent as if each individual publication, gene transcript identifier, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0004] Certain embodiments relate to methods and compounds for modulating gene expression through Advanced RNA Targeting (ARNATAR), which are useful for reducing the expression of specific genes, many of which are associated with various diseases and disorders. [Background technology]

[0005] The use of therapeutic oligomeric compounds was first proposed over 40 years ago by Stephenson and Zamecnik (Inhibition of Rous Sarcoma Viral RNA Translation by a Specific Oligodeoxyribonucleotide, PNAS, 1978, 75:285-288). However, issues regarding delivery, stability, specificity, safety, and efficacy have been barriers to the use of oligomeric compounds as therapeutic agents. Decades have been spent studying the mechanisms underlying the ability of oligomeric compounds to inhibit gene expression, for example, by modulating transcription and translation, in order to enhance the delivery, stability, specificity, safety, and efficacy of oligomeric compounds.

[0006] Sequence-specific silencing of gene expression, or RNA interference (RNAi), was discovered by Fire et al. in 1998 (Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis elegans, Nature, 1998, 391:806-811). RNAi inhibits gene expression via the RNA-induced silencing complex (RISC).

[0007] RISC contains a complex of proteins that interact with oligomeric compounds to inhibit gene expression. These oligomeric compounds serve as templates for RISC to recognize complementary messenger RNA (mRNA) and target specific mRNA transcripts for cleavage. Cleavage of the target mRNA blocks translation of the target mRNA and silences the target gene. Oligomeric compounds utilized by RISC include, but are not limited to, single-stranded oligomeric compounds such as microRNAs (miRNAs), specific oligonucleotides, and single-stranded siRNAs (Lima et al., Single-stranded siRNAs activate RNAi in animals. Cell. 2012, 150(5):883-94), as well as double-stranded oligomeric compounds such as short hairpin RNAs (shRNAs) and small interfering RNAs (siRNAs).

[0008] In 2001, Elbashir et al. showed that 21-nucleotide siRNA double-stranded bodies specifically suppressed endogenous and exogenous gene expression in mammalian cell lines and theorized that siRNAs could ultimately be used as gene-specific therapeutic agents (Duplexes of 21-Nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells, Nature, 2001, 411:494-498). Currently, five siRNA compounds (patisiran, givosiran, inclisiran, lumasiran, and vutrisiran) have received marketing approval from the U.S. Food and Drug Administration (FDA), and several more siRNA compounds are in clinical trials (Moumne et al, Oligonucleotide Therapeutics: From Discovery and Development to Patentability, Pharmaceutics, 2022, 14(2):260).

[0009] Pachisiran (Onpattro TMIn 2018, Patisiran became the first FDA-approved siRNA therapeutic (Hoy, Patisiran: First Global Approval, Drugs, 2018, 78:1625-1631). Patisiran is a partially modified siRNA targeting transthyretin (TTR) for the treatment of peripheral neuropathy (polyneuropathy) caused by hereditary transthyretin-mediated amyloidosis (hATTR). It contains chemically modified nucleosides interspersed along a 21-nucleotide double-stranded siRNA consisting of sense and antisense strands. Later approved siRNAs, such as givosiran, incorporate additional modified nucleosides, modified internucleoside linkages, and N-acetylgalactosamine (GalNAc) conjugates to aid in cellular delivery. (Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101). The most recent FDA-approved siRNA, butrisiran (Amvuttra), TM ) has the same target and indication as patisiran and was developed by the company (Alnylam Press Release in Businesswire, Alnylam Announces FDA Approval of Amvuttra TM Alnylam develops RNAi therapy Amvutrisiran for the treatment of polyneuropathy associated with hereditary transthyretin-mediated amyloidosis in adults TM FDA approval of butrisiran (butrisiran) was announced in June 2022. Butrisiran is a direct competitor to patisiran and is designed with a different sequence, chemical modification pattern, and delivery modality to enhance therapeutic efficacy over patisiran.

[0010] The field of therapeutic oligomeric compounds has progressed significantly, from the discovery of RNAi in 1998 to the first regulatory approval of an siRNA therapeutic in 2018 and the development of more recent improved siRNA therapeutics. However, this field is still continually striving to improve upon existing oligomeric compounds to achieve therapeutic benefits. An ideal oligomeric compound should meet the following requirements: 1) be specifically deliverable to target cells or organs; 2) be stable and long-lasting once administered to a patient, e.g., be resistant to degradation and have a long half-life; 3) be specific to its target and have no off-target effects; 4) be safe for patients, without activating the immune system or becoming toxic; and 5) have potent activity that efficiently and specifically cleaves the target.

[0011] Improvements in the delivery, stability, specificity, safety, and efficacy of oligomeric compounds are still being pursued to create better therapeutics. Disclosed herein are improved oligomeric compounds with advanced RNA targeting (ARNATAR) capabilities that enhance gene silencing activity. Summary of the Invention

[0012] Some embodiments described herein relate to the discovery of specific modifications that can enhance the effectiveness of oligomeric compounds in regulating gene expression. In some embodiments, the oligomeric compounds may be single-stranded (e.g., single-stranded oligonucleotides, single-stranded RNA (ssRNA)) or double-stranded (e.g., shRNA and siRNA), and in either case may be modified. The single-stranded oligomeric compounds include a sense strand or an antisense strand. The double-stranded oligomeric compounds include a sense strand or an antisense strand. The antisense strand may be fully or partially complementary to a target nucleic acid.

[0013] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises a sense strand having 19-23 linked nucleotides, and the sense strand sequence is represented by formula (I): 5'M-(Y)nZ-(Y)rD-D3', where D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, n is 6-8, r is 1-2, and no single modification type modifies more than two consecutive nucleotides.

[0014] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises an antisense strand having 19 to 23 linked nucleotides, and the antisense strand sequence is represented by formula (IV): 5'LM-(D)v-(Y)s-(Z)t-(Y)uZN-(Z)r3'. wherein D is a deoxyribonucleoside, N is a modified or unmodified nucleoside, M is a 2'-OMe-modified nucleoside, L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, v is 0-1, s is 2-7, t is 0-2, u is 0-5, and r is 1-2, and no single modification type modifies more than two consecutive nucleotides.

[0015] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises (a) a sense strand having 19 to 23 linked nucleotides, the sense strand sequence being of the formula (I) 5'M-(Y)nZ-(Y) r -D-D3', and (b) an antisense strand having 19 to 23 linked nucleotides, the antisense strand sequence being represented by formula (IV): 5'LM-(D) v-(Y) s -(Z) t -(Y) u -ZN-(Z) r 3'. The double-stranded region is formed by a sense strand and an antisense strand, and the double-stranded region is 19 to 23 nucleotide pairs in length. Here, D is a deoxyribonucleoside, N is a modified or unmodified nucleoside, M is a 2'-OMe-modified nucleoside, L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, n is 6 to 8, r is 1 to 2, v is 0 to 1, s is 2 to 7, t is 0 to 2, and u is 0 to 5, and no single modification type modifies more than two consecutive nucleotides.

[0016] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises a sense strand having 19 to 23 linked nucleotides, and the sense strand sequence is represented by formula (II): 5'YZ-(Y)q-FFNM-(Y)qM-(Y)vD-D3', where D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside, F is a 2'-F-modified nucleoside, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, q is 2 to 3, v is 0 to 1, and no single modification type modifies more than two consecutive nucleotides.

[0017] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises an antisense strand having 19-23 linked nucleotides, and the antisense strand sequence is represented by formula (V): 5'L-(Y)p-NM-(FMM)r-(Y)p-(Z)r3', where M is a 2'-OMe modified nucleoside, F is a 2'-F modified nucleoside, L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, p is 3-5, r is 1-2, and no single modification type modifies more than two consecutive nucleotides.

[0018] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises: (a) a sense strand having 19 to 23 linked nucleotides, the sense strand sequence being represented by formula (II): 5'Y-Z-(Y)q-FFNM-(Y)qM-(Y)vD-D3'; and (b) an antisense strand having 19 to 23 linked nucleotides, the antisense strand sequence being represented by formula (V): 5'L-(Y)p-NM-(FMM)r-(Y)p-(Z)r3'. A double-stranded entity is formed by the sense strand and the antisense strand, and the double-stranded entity region is 19 to 23 nucleotide pairs in length. D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside, F is a 2'-F-modified nucleoside, L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, q is 2 to 3, p is 3 to 5, r is 1 to 2, and v is 0 to 1, and no single modification type modifies more than two consecutive nucleotides. In certain embodiments, 5'(Y)p is YYY and 3'(Y)p is YYYY.

[0019] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises a sense strand having 21 linked nucleotides, and the sense strand sequence is represented by formula (III): 5'M*F*MMN*MN*MFFNMN*MN*MMFM*D*D3', where D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside, N is a modified or unmodified nucleoside, F is a 2'-F modified nucleoside, * is a phosphorothioate (PS) linkage, and no single modification type modifies more than two consecutive nucleotides.

[0020] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises an antisense strand having 21 linked nucleotides, and the antisense strand sequence is represented by formula (VI): 5'LM*N*MNMFNMFMMNMFMFMMN*M*M3', where M is a 2'-OMe modified nucleoside, N is a modified or unmodified nucleoside, F is a 2'-F modified nucleoside, * is a phosphorothioate (PS) linkage, L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), and no single modification type modifies more than two consecutive nucleotides.

[0021] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises (a) a sense strand consisting of 21 linked nucleotides, having a sense strand sequence represented by formula (III): 5'MFMMNMNMFFNMNMNMMFMDD3', and (b) an antisense strand consisting of 21 linked nucleotides, having an antisense strand sequence represented by formula (VI): 5'LM*N*MNMFNMFMMNMFMFMMN*M*M3'. The double-stranded region is 19 nucleotide pairs in length, with each strand having a 2-nucleotide overhang at its 3' end. where D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside, N is a modified or unmodified nucleoside, F is a 2'-F modified nucleoside, * is a phosphorothioate (PS) linkage, L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), and no single modification type modifies more than two consecutive nucleotides.

[0022] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises an antisense strand having 21 linked nucleotides, and the antisense strand sequence is represented by formula (VII): 5'LM*D*MFMFNMFMMFMFMFMMN*M*M3', where D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside, N is a modified or unmodified nucleoside, F is a 2'-F modified nucleoside, * is a phosphorothioate (PS) linkage, L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), and no single modification type modifies more than two consecutive nucleotides.

[0023] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises a sense strand having 19-23 linked nucleotides, and the sense strand sequence is represented by formula (I): 5'M-(Y)nZ-(Y)rD-D3', where D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, n is 6-8, r is 1-2, and no single modification type modifies more than two consecutive nucleotides.

[0024] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises an antisense strand having 19-23 linked nucleotides, and the antisense strand sequence is represented by formula (IX): 5'LM-(Y)pZ-(Y)p-(Z)r3', where M is a 2'-OMe modified nucleoside, L is 5' phosphate, 5' vinylphosphonate, or 5' OH, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, p is 3-5, r is 1-2, and no single modification type modifies more than two consecutive nucleotides.

[0025] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises (a) a sense strand having 19 to 23 linked nucleotides, the sense strand sequence being of the formula (I): 5'M-(Y)nZ-(Y) r -D-D3', and (b) an antisense strand having 19 to 23 linked nucleotides, the antisense strand sequence being represented by the formula (IX): 5'LM-(Y) p -Z-(Y) p -(Z) r 3'. The double-stranded region is formed by a sense strand and an antisense strand, and the double-stranded region is 19 to 23 nucleotide pairs in length. Here, D is a deoxyribonucleoside, N is a modified or unmodified nucleoside, M is a 2'-OMe-modified nucleoside, L is 5' phosphate, 5' vinylphosphonate, or 5' OH, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, n is 6 to 8, p is 3 to 5, and r is 1 to 2, and no single modification type modifies more than two consecutive nucleotides.

[0026] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises a sense strand having 21 linked nucleotides, and the sense strand sequence is represented by the formula (X): 5'MFMMNMNMFFNMNMNMMNMDD3', where D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside, N is a modified or unmodified nucleoside, and F is a 2'-F modified nucleoside, and no single modification type modifies more than two consecutive nucleotides.

[0027] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises an antisense strand having 21 linked nucleotides, and the antisense strand sequence is represented by formula (VIII): 5'L-MNMNMFNMFMMNMFMFMMNMM3', where M is a 2'-OMe modified nucleoside, N is a modified or unmodified nucleoside, F is a 2'-F modified nucleoside, and L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), and no single modification type modifies more than two consecutive nucleotides.

[0028] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises (a) a sense strand consisting of 21 linked nucleotides, having a sense strand sequence of formula (X): 5'MFMMNMNMFFNMNMNMMMNMDD3', and (b) an antisense strand consisting of 21 linked nucleotides, having an antisense strand sequence of formula (VIII): 5'L-MNMNMFNMFMMNMFMFMMNMM3'. The double-stranded region is 19 nucleotide pairs in length, with each strand having a 2-nucleotide overhang at its 3' end. where D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside, N is a modified or unmodified nucleoside, F is a 2'-F modified nucleoside, L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), and no single modification type modifies more than two consecutive nucleotides.

[0029] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises a sense strand having 21 linked nucleotides, and the sense strand sequence is represented by the formula XI: 5'MFMMNMNMFFMMNMNMMFMDD3', where D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside, N is a modified or unmodified nucleoside, and F is a 2'-F modified nucleoside, and no single modification type modifies more than two consecutive nucleotides.

[0030] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises an antisense strand having 21 linked nucleotides, and the antisense strand sequence is represented by formula (XII): 5'L-MDMFMFNMFMMFMFMFMMNMM3', where M is a 2'-OMe modified nucleoside, N is a modified or unmodified nucleoside, F is a 2'-F modified nucleoside, and L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), and no single modification type modifies more than two consecutive nucleotides.

[0031] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises: (a) a sense strand consisting of 21 linked nucleotides, the sense strand having a sequence of formula (XI): 5'MFMMNMNMFFMMNMNMMFMDD3'; and (b) an antisense strand consisting of 21 linked nucleotides, the antisense strand having a sequence of formula (XII): 5'L-MDMFMFNMFMMFMFMFMMNMM3'. The double-stranded region is 19 nucleotide pairs in length, with each strand having a 2-nucleotide overhang at its 3' end. where D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside, N is a modified or unmodified nucleoside, F is a 2'-F modified nucleoside, L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), and no single modification type modifies more than two consecutive nucleotides. [Brief explanation of the drawings]

[0032] [Figure 1] FIG. 1 is a table showing the LMNA antisense strand sequences with various modifications. [Figure 2] FIG. 2 is a table showing LMNA sense strand sequences with various modifications. [Figure 3]FIG. 3 is a table showing the ApoC3 antisense strand sequences with various modifications. [Figure 4] FIG. 4 is a table showing the ApoC3 sense strand sequences with various modifications. [Figure 5] FIG. 5 is a table showing the NCL antisense and sense strand sequences with various modifications. [Figure 6] Figure 6 shows an assay in which siRNAs were incubated with human serum to measure their stability in serum. LMNA-si2, LMNA-si29, and LMNA-si33 were incubated in human serum for various times and then run on a gel to visualize the amount of siRNA double-stranded forms present after exposure to serum. [Figure 7] Figure 7 shows a tritosomal stability assay in which siRNA stability was assessed. LMNA-si2, LMNA-si31, LMNA-si47, LMNA-si49, and LMNA-si51 were tested for various times and then run on a gel to visualize the amount of siRNA double-stranded bodies present after exposure to tritosomal solutions. [Figure 8] Figure 8 shows an assay in which siRNAs were incubated with human serum to measure their stability in serum. LMNA-si2, LMNA-si31, LMNA-si47, LMNA-si49, and LMNA-si51 were incubated in human serum for various times and then run on a gel to visualize the amount of siRNA double-stranded forms present after exposure to serum. [Figure 9] 9 shows selected ARNATAR-designed siRNAs evaluated for the timing of onset of siRNA activity. ARNATAR-designed siRNA compounds exhibited earlier onset of siRNA activity compared to the benchmark. [Figure 10] FIG. 10 shows the melting temperatures (Tm) of selected ARNATAR-designed siRNAs. [Figure 11]11 shows selected ARNATAR-designed siRNAs that were evaluated for incorporation into RISC. ARNATAR-designed siRNAs bound faster to Ago2. [Figure 12] FIG. 12 shows RNA inhibition data from in vivo studies in mice using selected ARNATAR-designed siRNAs. [Figure 13] 13 shows safety data from an in vivo study in mice with selected ARNATAR-designed siRNAs. Serum proteins ALB, ALT, and BUN were assessed. [Figure 14] 14 shows safety data from an in vivo study in mice with selected ARNATAR-designed siRNAs. Liver and spleen weights were measured. [Figure 15] Figure 15 shows safety data from an in vivo study in mice with selected ARNATAR-designed siRNAs. Serum analytes were assessed. [Figure 16] FIG. 16 shows data from an in vivo study in mice on the effect of selected ARNATAR-designed siRNAs on the immunogenic markers NFkB, IL-6 and TNF. [Figure 17] FIG. 17 shows RNA inhibition at day 7 from an in vivo study in mice with selected ARNATAR designed siRNAs. [Figure 18] FIG. 18 shows day 7 data from an in vivo mouse study of the effect of selected ARNATAR-designed siRNAs on the immunogenic markers NFkB, IL-6 and TNF. [Figure 19] FIG. 19 shows that selected siRNAs with ARNATAR platform chemistry are stable in serum and tritosomal assays. [Figure 20] FIG. 20 shows in vitro inhibition of LMNA in HeLa cells by selected ARNATAR-designed siRNAs. [Figure 21]FIG. 21 shows data from an in vitro study in HeLa cells on the effect of selected ARNATAR-designed siRNAs on the immunogenic marker NFkB. [Figure 22] FIG. 22 shows in vitro inhibition of LMNA in HEK293 cells by selected ARNATAR-designed siRNAs. [Figure 23] FIG. 23 shows mRNA inhibition data from an in vivo study in Balb / c mice using selected ARNATAR designed siRNAs. [Figure 24] FIG. 24 is a table showing the HAO1 antisense and sense strand sequences with various modifications. [Figure 25] FIG. 25 shows a graph of in vitro inhibition of HAO1 RNA in Hep3B cells at 24 hours by modified oligomeric compounds. [Figure 26] FIG. 26 shows a graph of in vitro inhibition of L MNA RNA in HeLa cells at 72 and 96 hours by modified oligomeric compounds. [Figure 27] FIG. 27 shows in vivo inhibition data of LMNA RNA in mouse liver after 3 days, 1 week, or 2 weeks of treatment with modified oligomeric compounds. [Figure 28] FIG. 28 shows a graph of in vitro inhibition of L MNA RNA in HeLa cells at 20 hours by modified oligomeric compounds. [Figure 29] FIG. 29 shows a graph of in vitro inhibition of L MNA RNA in HeLa cells at 16, 24 and 48 hours by modified oligomeric compounds. [Figure 30] FIG. 30 shows a graph of in vitro inhibition of L MNA RNA in Hepa1-6 cells at 16 and 48 hours by modified oligomeric compounds. [Figure 31] FIG. 31 shows a graph of in vitro inhibition of L MNA RNA in HeLa cells at 20 hours by modified oligomeric compounds. [Figure 32]FIG. 32 shows in vivo data of LMNA siRNA activity in mouse liver after 10 and 25 days of treatment with modified oligomeric compounds. [Figure 33] FIG. 33 shows a graph of in vitro inhibition by ARNATAR motif siRNA compared to third party motifs applied to siRNAs targeting LMNA, NCL, and ApoC3. [Figure 34] FIG. 34 shows a graph of in vitro inhibition of L MNA RNA in HeLa cells at 16 and 36 hours by modified oligomeric compounds. [Figure 35] FIG. 35 shows a graph of in vitro inhibition of NCL RNA in HeLa cells at 36 hours by modified oligomeric compounds. [Figure 36] FIG. 36 is a table showing the AGT antisense strand sequences with various modifications. [Figure 37] FIG. 37 is a table showing the AGT sense strand sequences with various modifications. [Figure 38] 38 shows graphs of in vitro inhibition of AGT RNA in Hep3B cells via transfection at 36 hours or in human primary hepatocytes (HPH) via free uptake at 48 hours by modified oligomeric compounds. The x-axis shows siRNA concentration in nM for Hep3B cells and μM for HPH cells, and the y-axis shows the percentage of AGT mRNA after treatment with various siRNA concentrations. [Figure 39] Figure 39 shows a graph of in vitro inhibition of AGT RNA in Hep3B cells via transfection at 20 hours. The x-axis shows siRNA concentration in nM. [Figure 40] Figure 40 shows a graph of in vitro inhibition of AGT in human primary hepatocytes (HPH) via free uptake by modified oligomeric compounds at 42 hours. The x-axis shows siRNA concentrations at various μM concentrations, and the y-axis shows the percentage of AGT mRNA after treatment with various siRNA concentrations. Detailed Description

[0033] It is to be understood that both the foregoing general description and the following detailed description are presented for purposes of illustration and description only and are not intended to be limiting of the invention as claimed. As used herein, the use of the singular includes the plural unless expressly stated otherwise. As used herein, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the use of the term "including" and other forms, such as "includes" and "included," is not limiting. Furthermore, terms such as "element" or "component" encompass both elements and components comprised of a single unit and elements and components comprised of multiple subunits, unless expressly stated otherwise.

[0034] The section headings used herein are provided for convenience of organization and should not be construed as limiting the subject matter described. All documents or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference in their entirety, as well as in the portions referred to herein.

[0035] definition Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses and chemical analyses. Where permitted, all patents, applications, published applications and other publications disclosed herein, GENBANK accession numbers and associated sequence information, and other data available through databases such as NCBI (National Center for Biotechnology Information), are incorporated herein by reference in their entirety as well as in their entirety as referenced.

[0036] Unless otherwise stated, the following terms have the following meanings:

[0037] "2'-O-Methoxyethyl" (also including 2'-MOE and 2'-O(CH2)2-OCH3) refers to an O-methoxyethyl modification at the 2' position of the furanoose ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0038] "2'-MOE nucleoside" (also referred to as 2'-O-methoxyethyl nucleoside) refers to a nucleoside containing a 2'-MOE modified sugar moiety. "2'-MOE nucleotide" (also referred to as 2'-O-methoxyethyl nucleotide) refers to a nucleotide containing a 2'-MOE modified sugar moiety.

[0039] "2'-O-methyl" (including 2'-OCH3 and 2'-OMe) refers to an O-methyl modification at the 2' position of the furanoose ring. A 2'-O-methyl modified sugar is a modified sugar.

[0040] "2'-OMe nucleoside" (also referred to as 2'-O-methyl nucleoside) refers to a nucleoside that includes a 2'-OMe modified sugar moiety. "2'-OMe nucleotide" (also referred to as 2'-O-methyl nucleotide) refers to a nucleotide that includes a 2'-OMe modified sugar moiety.

[0041] "2'-substituted nucleoside" means a nucleoside that includes a substituent at the 2'-position of the furanosyl ring other than H or OH. In certain embodiments, 2'-substituted nucleosides include nucleosides with fluoro (2'-F), O-methyl (2'-OMe), O-methoxyethyl (2'-MOE), or bicyclic sugar modifications.

[0042] "5-methylcytosine" means cytosine with a methyl group added to position 5. 5-Methylcytosine is a modified nucleobase.

[0043] "About" means within ±7% of a value. For example, a statement that "a compound affected at least about 70% inhibition of mRNA" suggests that mRNA levels are inhibited within the range of 63% to 77%.

[0044] "Animal" refers to humans or non-human animals, including mice, rats, rabbits, dogs, cats, pigs, and non-human primates (including, but not limited to, monkeys and chimpanzees).

[0045] "Antibody" refers to a molecule characterized by specifically reacting in some way with an antigen, where antibody and antigen are each defined in relation to the other. An antibody may be a complete antibody molecule or a subunit of heavy chains, light chains, F ab Area, and F c It may refer to any fragment or region thereof, such as a region.

[0046] "Antisense oligonucleotide" or "ASO" refers to a single-stranded oligonucleotide having a nucleobase sequence capable of hybridizing to a corresponding region or segment of a target nucleic acid. In certain embodiments, an antisense oligonucleotide contains one or more ribonucleosides (RNA nucleosides) and / or deoxyribonucleosides (DNA nucleosides).

[0047] "Base complementarity" refers to the ability to base pair (i.e., hybridize) between the nucleobases of an oligonucleotide and the corresponding nucleobases in a target nucleic acid, where this base pairing is mediated by Watson-Crick, Hugstein, or reverse-Hugstein hydrogen bonds between the corresponding nucleobases. Base complementarity also refers to canonical base pairs (e.g., A:U, A:T, or C:G) and non-canonical base pairs (e.g., A:G, A:U, G:U, I:U, I:A, or I:C).

[0048] "Bicyclic sugar" means a furanoose ring modified by bridging two non-geminal carbon atoms. A bicyclic sugar is a modified sugar.

[0049] "Cap structure" or "end-capping moiety" means a chemical modification incorporated at either end of an oligomeric compound.

[0050] " Chemical modification " refers to modifying molecular structure or elements derived from naturally occurring molecules. For example, since siRNA compounds are composed of linked ribonucleosides (sometimes referred to herein as RNA), the substitution of deoxyribonucleosides (sometimes referred to herein as DNA nucleosides) for ribonucleosides is considered a chemical modification of siRNA compounds.

[0051] A "chemically distinct region" refers to a region of an oligomeric compound that is chemically distinct in some way from other regions of the same oligomeric compound, e.g., a region having 2'-OMe nucleotides is chemically distinct from a region having nucleotides without 2'-OMe modifications.

[0052] "Chimeric oligomeric compound" refers to an oligomeric compound having at least two chemically distinct regions, each region being composed of multiple subunits. For example, as disclosed herein, an siRNA may comprise a peripheral region and a central region. The peripheral region comprises motifs with various modified or unmodified nucleobases to enhance stability, specificity, safety, and efficacy, while the central region comprises various modified or unmodified nucleobases to serve as substrates for RISC-mediated degradation.

[0053] "Complementary" means the capacity for pairing between nucleobases of a first nucleic acid and nucleobases of a second nucleic acid.

[0054] "Adherence" means that an individual follows recommended treatment.

[0055] "Comprise", "comprises", and "comprising" are understood to mean the inclusion of a stated step or element or group of steps or elements, but not to the exclusion of any other step or element or group of steps or elements.

[0056] "Contiguous nucleobases" means nucleobases immediately adjacent to each other.

[0057] "Deoxyribonucleoside" refers to a nucleoside having a hydrogen atom at the 2' position of the sugar moiety of the nucleoside. Deoxyribonucleosides are sometimes referred to herein as DNA nucleosides, "D" or "d". Deoxyribonucleosides may be modified with various substituents and may be joined by covalent bonds other than the naturally occurring phosphodiester, such as phosphorothioates.

[0058] "Deoxyribonucleotide" refers to a nucleotide having a hydrogen atom at the 2' position of the sugar moiety of the nucleotide. Deoxyribonucleotides are sometimes referred to herein as DNA nucleotides, "D" or "d." Deoxyribonucleotides may be modified with various substituents and may be joined by covalent bonds other than the naturally occurring phosphodiester, such as phosphorothioates.

[0059] "Design" or "engineering" refers to the process of engineering an oligomeric compound that will hybridize with a particular target nucleic acid molecule.

[0060] "Efficacy" means the ability to produce a desired effect.

[0061] "Expression" includes any process by which a gene's coding information is converted into structures present and functioning in a cell, including, but not limited to, the products of transcription and translation.

[0062] "Fully complementary" or "100% complementary" means that every nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, the first nucleic acid is an oligomeric compound and the target nucleic acid is the second nucleic acid.

[0063] A "fully modified motif" refers to an oligomeric compound that essentially contains a contiguous sequence of nucleosides, where each nucleoside is chemically modified.

[0064] "Hybridization" refers to the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an oligomeric compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an siRNA and a nucleic acid target.

[0065] "Immediately adjacent" means that there are no intervening elements between the immediately adjacent elements.

[0066] "Individual" refers to a human or non-human animal selected for treatment or therapy.

[0067] "Induce," "inhibit," "enhance," "enhance," "increase," "decrease," and the like generally indicate a quantitative difference between two states.

[0068] "Inhibition of expression or activity" refers to a reduction or blocking of expression or activity, and does not necessarily mean that expression or activity is completely eliminated.

[0069] "Internucleoside linkage" refers to the chemical bond between nucleosides. The 3' position of a nucleoside is linked to the 5' position of the next nucleoside via an internucleoside bond.

[0070] "Linked nucleosides" means adjacent nucleosides (e.g., A, G, C, T, or U) linked by an internucleoside bond. Examples of linked nucleosides include deoxyribonucleosides (also referred to herein as DNA nucleosides) and ribonucleosides (also referred to herein as RNA nucleosides).

[0071] A "mismatch" or "non-complementary nucleobase" refers to when a nucleobase of a first nucleic acid cannot pair with the corresponding nucleobase of a second nucleic acid or target nucleic acid through Watson-Crick base pairing (e.g., A:T, A:U, or C:G).

[0072] A "modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside linkage (ie, a phosphodiester internucleoside linkage).

[0073] "Modified nucleobase" means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. "Unmodified nucleobase" means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0074] "Modified nucleoside" refers to a nucleoside having an independently modified sugar moiety and / or a modified nucleobase. As used herein, when an oligomeric compound is RNA-based, the substitution of a deoxyribonucleoside (also referred to herein as a DNA nucleoside) for a ribonucleoside is considered a modification of the oligomeric compound. Also, when an oligomeric compound is DNA-based, the substitution of a ribonucleoside (also referred to herein as an RNA nucleoside) for a deoxyribonucleoside is considered a modification of the oligomeric compound.

[0075] "Modified nucleotide" means a nucleotide having, independently, a modified sugar moiety, a modified internucleoside linkage, a deoxyribonucleoside (also referred to herein as a DNA nucleoside) substitution for a ribonucleoside (also referred to herein as an RNA nucleoside), and / or a modified nucleobase.

[0076] "Modified oligonucleotide" means an oligonucleotide containing at least one modified internucleoside linkage, a modified sugar, deoxyribonucleosides (also referred to herein as DNA nucleosides) for ribonucleosides (also referred to herein as RNA nucleosides), and / or modified nucleobases.

[0077] By "modified sugar" is meant a substitution and / or any change from the natural sugar moiety.

[0078] "Moiety" means a part of something, i.e., one of the components that make up a whole. For example, the sugar portion of a nucleotide is the sugar component of the nucleotide.

[0079] "Monomer" refers to a single building block of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether natural or modified.

[0080] "Motif" refers to the pattern of modification in an oligomeric compound. For example, as disclosed herein, oligomeric compounds designed by ARNATAR have motifs that include variously modified nucleobases and internucleoside linkages to improve compound delivery, stability, specificity, safety, and efficacy. The motif is independent of the nucleobases involved and specifies only the pattern of modification.

[0081] "Natural sugar moiety" refers to a sugar moiety found in DNA (2'-H) or RNA (2'-OH).

[0082] A "naturally occurring internucleoside linkage" refers to a 3' to 5' phosphodiester linkage.

[0083] "Non-complementary nucleobases" refers to a pair of nucleobases that do not form hydrogen bonds or undergo hybridization.

[0084] "Nucleic acid" refers to a molecule composed of monomeric nucleotides. Nucleic acids include, but are not limited to, ribonucleic acid (RNA), messenger RNA (mRNA), deoxyribonucleic acid (DNA), single-stranded nucleic acid, double-stranded nucleic acid, short interfering ribonucleic acid (siRNA), and microRNA (miRNA).

[0085] "Nucleobase" refers to a heterocyclic moiety capable of base pairing with a base of another nucleic acid.

[0086] "Nucleobase complementarity" refers to a nucleobase that can form base pairs (also known as complementarity) with another nucleobase. If a nucleobase at a particular position in an oligomeric compound can hydrogen bond with a nucleobase at a particular position in a target nucleic acid, the hydrogen bonding positions between the oligomeric compound and the target nucleic acid are considered complementary in that nucleobase pair. For example, adenine (A) is complementary to thymine (T) in DNA, and adenine (A) is complementary to uracil (U) in RNA. Also, guanine (G) is complementary to cytosine (C) in both DNA and RNA. Base pairs, or complementary nucleobases, are typically canonical Watson-Crick base pairs (C:G, A:U, A:T), but also include non-canonical base pairs such as Fugsteen base pairs (e.g., A:G or A:U) and wobble base pairs (e.g., G:U, I:U, I:A, or I:C, where I refers to hypoxanthine). Nucleobase complementarity facilitates hybridization of the oligomeric compounds described herein to their target nucleic acids.

[0087] By "nucleobase sequence" is meant the order of consecutive nucleobases, independent of sugar, linkage, and / or nucleobase modifications.

[0088] "Nucleoside" means a nucleobase linked to a sugar.

[0089] The term "nucleoside mimetic" includes structures used to replace sugars or sugar and bases, not necessarily linkages, at one or more positions in an oligomeric compound. Examples include morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic, or tricyclic sugar mimetics, i.e., nucleoside mimetics with non-furanose sugar units. The term "nucleotide mimetic" includes structures used to replace nucleosides and linkages at one or more positions in an oligomeric compound. Examples include peptide nucleic acids and morpholinos (morpholinos linked by an -N(H)-C(=O)-O- or other non-phosphodiester bond). Sugar surrogates overlap with the broader term nucleoside mimetic, but are intended to replace only the sugar unit (furanose ring). The tetrahydropyranyl ring provided herein is an example of a sugar surrogate in which the furanosyl sugar group is replaced with a tetrahydropyranyl ring system. "Mimetic" refers to groups that replace the sugar, nucleobase, and / or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, while the nucleobase is maintained for hybridization with a selected target.

[0090] "Nucleotide" refers to a nucleoside having a linking group (e.g., a phosphate group (p) or a phosphorothioate group (PS)) covalently attached to the sugar portion of the nucleoside. Nucleotides include ribonucleotides and deoxyribonucleotides. Ribonucleotides are the linked nucleotide units that make up RNA. Deoxyribonucleotides are the linked nucleotide units that make up DNA.

[0091] "Off-target effect" refers to an unwanted or adverse biological effect associated with the modulation of RNA or protein expression of a gene other than the intended target nucleic acid.

[0092] "Oligomeric activity" refers to any detectable or measurable activity resulting from hybridization of an oligomeric compound with its target nucleic acid. In certain embodiments, oligomeric activity refers to a decrease in the amount or expression of a target nucleic acid of interest or a protein encoded by the target nucleic acid. Oligomeric activity can be modulated by an oligomeric compound, such as an siRNA.

[0093] "Oligomeric compound" refers to a sequence of linked monomeric subunits that can hybridize with at least one region of a target nucleic acid through hydrogen bonding. The monomeric subunits can be modified or unmodified nucleotides or nucleosides. The oligomeric compound serves as a template for RISC to recognize complementary messenger RNA (mRNA) and target specific mRNA transcripts for cleavage. Cleavage of the target mRNA blocks translation of the target mRNA and silences the target gene. Examples of oligomeric compounds include single-stranded and double-stranded compounds, such as antisense oligonucleotides, ssRNA, siRNA, shRNA, and miRNA.

[0094] "Oligomeric inhibition" means a decrease in the level of a target nucleic acid in the presence of an oligomeric compound complementary to the target nucleic acid compared to the level of the target nucleic acid in the absence of the oligomeric compound.

[0095] "Oligomeric mechanisms" include those involving RISC or RNase H, which involve hybridization of an oligomeric compound with a target nucleic acid, the result or effect of which is degradation of the target and inhibition of gene expression.

[0096] As used herein, "oligonucleotide" refers to a polymer of linked nucleosides, each nucleoside independent of the others, modified or unmodified. Oligonucleotides may have linking groups other than phosphate groups (e.g., phosphorothioate = thiophosphate groups) used as internucleoside linking moieties. In certain embodiments, oligonucleotides contain one or more ribonucleosides (RNA nucleosides) and / or deoxyribonucleosides (DNA nucleosides).

[0097] "Phosphorothioate linkage" or "PS" means an internucleoside linkage modified by replacing one of the non-bridging oxygen atoms of the phosphodiester bond with a sulfur atom. A phosphorothioate (also known as thiophosphate, or thiophosphate) linkage is a modified internucleoside linkage.

[0098] "Portion" refers to a defined number of contiguous (i.e., linked) nucleobases in a nucleic acid. In certain embodiments, a "portion" refers to a defined number of contiguous nucleobases in a target nucleic acid. In certain embodiments, a "portion" refers to a defined number of contiguous nucleobases in an oligomeric compound.

[0099] A "region" is defined as a portion of a target nucleic acid that has at least one distinguishable structure, function, or characteristic.

[0100] "RNA" or "ribonucleic acid" consists of ribose nucleotides or ribonucleotides (nitrogenous bases attached to a ribose sugar) linked by phosphodiester bonds to form strands of various lengths. The nitrogenous bases in RNA are adenine, guanine, cytosine, and uracil. The ribose sugar in RNA is a ring structure consisting of five carbons and one oxygen atom.

[0101] "Ribonucleoside" refers to a nucleoside having a hydroxy group at the 2' position of the sugar moiety of the nucleoside. Ribonucleosides may be modified with various substituents and may be joined by covalent bonds other than the naturally occurring phosphodiester, such as, for example, phosphorothioates. Ribonucleosides may be referred to herein as RNA nucleosides, "R" or "r."

[0102] "Ribonucleotide" refers to a nucleotide having a hydroxy group at the 2' position of the sugar moiety of the nucleotide. Ribonucleotides may be modified with various substituents and may be joined by covalent bonds other than the naturally occurring phosphodiester, such as phosphorothioates. Ribonucleotides may be referred to herein as RNA nucleotides, "R" or "r."

[0103] A "segment" is defined as a small region or subregion of a region within a target nucleic acid.

[0104] As used herein, a "site" is defined as a unique nucleobase position within a target nucleic acid.

[0105] "Specifically hybridizable" refers to an oligomeric compound (e.g., siRNA) that has sufficient complementarity with a target nucleic acid to elicit a desired effect while having little or no effect on non-target nucleic acids under the conditions required for specific binding, i.e., physiological conditions in in vivo assays or treatments.

[0106] "Stringent hybridization conditions" or "stringent conditions" refer to conditions under which an oligomeric compound will hybridize to a target sequence while hybridizing to little or no other sequences.

[0107] "Subject" refers to a human or non-human animal selected for treatment or therapy.

[0108] "Target" refers to a protein or nucleic acid sequence (eg, mRNA) whose modulation is desired.

[0109] "Target gene" refers to a gene that encodes a target.

[0110] "Targeting" refers to the process of designing and selecting oligomeric compounds that will specifically hybridize to a particular target nucleic acid and elicit a desired effect.

[0111] "Target nucleic acid," "target RNA," "target RNA transcript," and "nucleic acid target" all refer to a nucleic acid that can be targeted by an oligomeric compound.

[0112] "Target region" refers to a portion of a target nucleic acid that is targeted by one or more oligomeric compounds.

[0113] "Target segment" refers to the sequence of nucleotides of a target nucleic acid to which an oligomeric compound is targeted. "5' target site" refers to the first nucleotide at the 5' end of a target segment. "3' target site" refers to the first nucleotide at the 3' end of a target segment. In one embodiment, a target segment is at least a 12-nucleobase portion (i.e., at least 12 contiguous nucleobases) of a target region to which an oligomeric compound is targeted.

[0114] "Therapeutic efficacy" refers to the effect of a therapeutic compound, such as an oligomeric compound. Therapeutic efficacy can be enhanced by improving the delivery, stability, specificity, safety, and efficacy of the therapeutic compound.

[0115] "Unmodified" RNA nucleobase refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). "Unmodified" DNA nucleobase refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T) and cytosine (C). In certain embodiments, an unmodified RNA nucleobase is considered modified when a DNA nucleobase is substituted for an RNA nucleobase. In certain embodiments, an unmodified DNA nucleobase is considered modified when an RNA nucleobase is substituted for a DNA nucleobase.

[0116] "Unmodified nucleoside" refers to a nucleoside composed of a common, naturally occurring nucleobase and sugar moiety. For example, an unmodified nucleoside is an RNA nucleoside when used in an RNA sequence, although other nucleosides (e.g., DNA nucleosides, 2'-OMe, or 2'-F) are considered modified nucleosides in such RNA sequences.

[0117] "Unmodified nucleotide" refers to a nucleotide composed of a common and naturally occurring nucleobase, sugar moiety, and internucleoside linkage. For example, an unmodified nucleotide is an RNA nucleotide when used in an RNA sequence, but other nucleotides (e.g., DNA nucleotides, 2'-OMe, or 2'-F) are considered modified nucleotides in such RNA sequences.

[0118] Disclosed herein are improved oligomeric compounds with advanced RNA targeting (ARNATAR) designs that enhance gene silencing activity. Some embodiments described herein relate to the discovery of specific modification motifs for oligomeric compounds that can enhance the effectiveness of oligomeric compounds in modulating gene expression by improving delivery, stability, specificity, safety, and efficacy.

[0119] In some embodiments, the oligomeric compounds may be single-stranded (e.g., single-stranded oligonucleotides, microRNA (miRNA), or single-stranded RNA (ssRNA)) or double-stranded (e.g., shRNA or siRNA), in either case modified. The single-stranded oligomeric compounds comprise a sense strand or an antisense strand. The double-stranded oligomeric compounds comprise a sense strand or an antisense strand. The antisense strand may be fully or partially complementary to the target nucleic acid.

[0120] The following embodiments describe oligomeric compounds comprising linked nucleotides containing modified and, optionally, unmodified nucleosides, where adjacent nucleosides are linked by a naturally occurring phosphodiester bond or a non-natural bond, such as a thiophosphodiester bond (also referred to as a "phosphorothioate internucleotide (PS) linkage"). In some embodiments, when the oligomeric compound is RNA-based (e.g., miRNA, ssRNA, shRNA, and / or siRNA), the sequence is considered RNA, and unmodified nucleosides refer to ribonucleosides. Modified nucleosides may include modified bases and / or modified sugars (e.g., modified sugars, preferably at the 2' position). In RNA-based oligomeric compounds, deoxyribonucleosides (D) are also considered modified nucleosides.

[0121] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises a sense strand having 19 to 23 linked nucleotides, and the sense strand sequence is represented by formula (I): 5'M-(Y)nZ-(Y)rD-D3', where D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, n is 6 to 8, r is 1 to 2, and no single modification type modifies more than two consecutive nucleotides. Preferably, -DD is an overhanging TT or TA. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In a further embodiment, the sense strand contains phosphorothioate internucleotide (PS) linkages adjacent to deoxyribonucleosides (D) or ribonucleosides (R). The PS linkages may be adjacent to the deoxyribonucleosides (D) or ribonucleosides (R) on the 5', 3', or both sides. The PS linkages may be adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of Formula (I) is ssRNA or siRNA. Modified nucleosides may contain modified bases and / or modified sugars (preferably at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0122] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises an antisense strand having 19 to 23 linked nucleotides, and the antisense strand sequence is represented by formula (IV): 5'LM-(D)v-(Y)s-(Z)t-(Y)uZN-(Z)r3'. D is a deoxyribonucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), M is a 2'-OMe-modified nucleoside, L is a 5' phosphate, 5' vinylphosphonate, or 5' OH, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, v is 0-1, s is 2-7, t is 0-2, u is 0-5, and r is 1-2, and no single modification type modifies more than two consecutive nucleotides. 3'Z is preferably a UU or TT overhang. In certain embodiments, the sense strand contains one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In a further embodiment, the antisense strand contains phosphorothioate internucleotide (PS) linkages adjacent to deoxyribonucleosides (D) or ribonucleosides (R). The PS linkages may be adjacent to the deoxyribonucleosides (D) or ribonucleosides (R) on the 5', 3', or both sides. The PS linkage modifications may be located between positions 1-2, 2-3, 19-20, and / or 20-21, counting from the 5' end of the antisense strand. The PS linkages may be adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of formula (IV) is ssRNA or siRNA. The modified nucleosides may contain modified bases and / or modified sugars (preferably at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0123] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises (a) a sense strand having 19-23 linked nucleotides, the sense strand sequence being represented by formula (I): 5'M-(Y)nZ-(Y)rD-D3', and (b) an antisense strand having 19-23 linked nucleotides, the antisense strand sequence being represented by formula (IV): 5'LM-(D)v-(Y)s-(Z)t-(Y)uZNZ)r3'. A double-stranded entity is formed by the sense strand and the antisense strand, and the double-stranded entity region is 19-23 nucleotide pairs in length. D is a deoxyribonucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), M is a 2'-OMe-modified nucleoside, L is 5' phosphate, 5' vinylphosphonate, or 5' OH, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, n is 6-8, r is 1-2, v is 0-1, s is 2-7, t is 0-2, and u is 0-5, and no single modification type modifies more than two consecutive nucleotides. In a further embodiment, 3'Z of the antisense strand is an overhang of UU or TT, and -DD of the sense strand is an overhang of TT or TA. In certain embodiments, the sense strand contains one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In further embodiments, the strand contains phosphorothioate internucleotide (PS) linkages adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkages may be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5' side, the 3' side, or both sides. The PS linkage modifications on the antisense strand may be located between positions 1-2, 2-3, and 19-20 and / or 20-21, counting from the 5' end of the antisense strand.The PS linkages may flank the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of formula (I) and (IV) is an siRNA. The modified nucleosides may contain modified bases and / or modified sugars (preferably modified sugars at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0124] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises a sense strand having 19 to 23 linked nucleotides, and the sense strand sequence is represented by formula (II): 5'YZ-(Y)q-FFNM-(Y)qM-(Y)vD-D3', where D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is a 2'-F-modified nucleoside, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, q is 2 to 3, v is 0 to 1, and no single modification type modifies more than two consecutive nucleotides. Preferably, -DD is an overhanging TT or TA. In certain embodiments, the sense strand contains one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In further embodiments, the sense strand contains phosphorothioate internucleotide (PS) linkages adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R). The PS linkages may be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5', 3', or both sides. The PS linkages may be adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of formula (II) is ssRNA or siRNA. In certain embodiments, FFNM is FFMM or FFRM, where R is a ribonucleoside. The modified nucleoside may contain a modified base and / or a modified sugar (preferably a modified sugar at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0125] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises an antisense strand having 19-23 linked nucleotides, and the antisense strand sequence is represented by formula (V): 5'L-(Y)p-NM-(FMM)r-(Y)p-(Z)r3', where M is a 2'-OMe modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is a 2'-F modified nucleoside, L is 5' phosphate, 5' is vinylphosphonate or 5' OH, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, p is 3-5, r is 1-2, and no single modification type modifies more than two consecutive nucleotides. Preferably, the 3'Z is a UU or TT overhang. In certain embodiments, the sense strand contains one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In further embodiments, the antisense strand contains phosphorothioate internucleotide (PS) linkages adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkages may be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5' side, the 3' side, or both sides. The PS linkage modifications may be located between positions 1-2, 2-3, 19-20, and / or 20-21, counting from the 5' end of the antisense strand. The PS linkages may be adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of formula (V) is ssRNA or siRNA. Modified nucleosides may contain modified bases and / or modified sugars (preferably at the 2'-position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0126] In one embodiment, the oligomeric compound capable of inhibiting expression of a target nucleic acid comprises (a) a sense strand having 19 to 23 linked nucleotides, the sense strand sequence being of the formula (II): 5'YZ-(Y) q -FFNM-(Y) q -M-(Y) v -D-D3', and (b) an antisense strand having 19 to 23 linked nucleotides, the antisense strand sequence being represented by the formula (V): 5'L-(Y) p -NM-(FMM) r -(Y) p -(Z) r3'. The double-stranded region is formed by a sense strand and an antisense strand, and the double-stranded region is 19 to 23 nucleotide pairs in length. Here, D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is a 2'-F-modified nucleoside, L is 5' phosphate, 5' vinylphosphonate, or 5' OH, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, q is 2 to 3, p is 3 to 5, r is 1 to 2, and v is 0 to 1, and no single modification type modifies more than two consecutive nucleotides. In further embodiments, the 3'Z of the antisense strand is an overhanging UU or TT, and the -DD of the sense strand is an overhanging TT or TA. In certain embodiments, the sense strand contains one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In further embodiments, the strand contains a phosphorothioate internucleotide (PS) linkage adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkage may be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5' side, the 3' side, or both sides. The PS linkage modification on the antisense strand may be located between positions 1-2, 2-3, 19-20, and / or 20-21, counting from the 5' end of the antisense strand. The PS bond may be adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of formula (II) and formula (V) is siRNA. In certain embodiments, the FFNM of formula (II) is FFMM or FFRM, where R is a ribonucleoside. The modified nucleoside may include a modified base and / or a modified sugar (preferably a modified sugar at the 2' position).Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0127] In certain embodiments, the modified nucleotide comprises a modified sugar moiety. In certain embodiments, the modified nucleotide is selected from the group consisting of deoxyribonucleosides (also referred to herein as DNA nucleosides) substituted with ribonucleosides (also referred to herein as RNA nucleosides), LNA, 2'-OMe, 2'-F, 2'-MOE, UNA, pseudouridine, 2'-thiouridine, N6'-methyladenosine, and 5'-methylcytidine, 5'-fluoro-2'-deoxyuridine, N-ethylpiperidine 5' triazole-modified adenosine, 5'-nitroindole, 2',4'-difluorotolylribonucleoside, N-ethylpiperidine 7'-EAA triazole-modified adenosine, 6'-phenylpyrrolocytosine, and combinations thereof. In certain embodiments, "N" in the formula is a modified or unmodified nucleoside, the modification being selected from the group consisting of deoxyribonucleosides (also referred to herein as DNA nucleosides) substituted with ribonucleosides (also referred to herein as RNA nucleosides), LNA, 2'-OMe, 2'-F, 2'-MOE, UNA, pseudouridine, 2'-thiouridine, N6'-methyladenosine, and 5'-methylcytidine, 5'-fluoro-2'-deoxyuridine, N-ethylpiperidine 5' triazole-modified adenosine, 5'-nitroindole, 2',4'-difluorotolylribonucleoside, N-ethylpiperidine 7'-EAA triazole-modified adenosine, and 6'-phenylpyrrolocytosine. The modification can also be selected from the base substitutions described below.

[0128] In certain embodiments, an FFNM motif appears at or near the cleavage site on the sense strand, and / or an (FMM)r motif appears at or near the cleavage site on the antisense strand, where N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is 2'-fluoro, M is 2'-OMe, and r is 1 to 2. In certain embodiments, FFNM is FFMM or FFRM, where R is a ribonucleoside.

[0129] In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid comprises a sense strand having 21 linked nucleotides, and the sense strand sequence is represented by formula (III): 5'M*F*MMN*MN*MFFNMN*MN*MMFM*D*D3', where D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is a 2'-F-modified nucleoside, * is a phosphorothioate (PS) linkage, and no single modification type modifies more than two consecutive nucleotides. In one embodiment, the oligomeric compound of formula (III) is ssRNA or siRNA. In certain embodiments, FFNM is FFMM or FFRM, where R is a ribonucleoside. The modified nucleoside may contain a modified base and / or a modified sugar (preferably a modified sugar at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0130] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises an antisense strand having 21 linked nucleotides, and the antisense strand sequence is represented by formula (VI): 5'LM*N*MNMFNMFMMNMFMFMMN*M*M3', where M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is a 2'-F-modified nucleoside, * is a phosphorothioate (PS) linkage, L is a 5' phosphate, and 5' is vinylphosphonate or a 5' OH, and no single modification type modifies more than two consecutive nucleotides. In one embodiment, the oligomeric compound comprising formula (VI) is ssRNA or siRNA. In certain embodiments, FNM is FMM. The modified nucleoside may contain a modified base and / or a modified sugar (preferably a modified sugar at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0131] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises (a) a sense strand consisting of 21 linked nucleotides, having a sense strand sequence represented by formula (III): 5'MFMMNMNMFFNMNMNMMFMDD3', and (b) an antisense strand consisting of 21 linked nucleotides, having an antisense strand sequence represented by formula (VI): 5'L-MNMNMFNMFMMNMFMFMMNMM3'. The double-stranded region is 19 nucleotide pairs in length, with each strand having a 2-nucleotide overhang at its 3' end. wherein D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is a 2'-F-modified nucleoside, * is a phosphorothioate (PS) linkage, L is a 5' phosphate, a 5' vinylphosphonate, or a 5' OH, and no single modification type modifies more than two consecutive nucleotides. In one embodiment, the oligomeric compound consisting of formula (III) and formula (VI) is an siRNA. In certain embodiments, FFNM in formula (III) is FFMM or FFRM, where R is a ribonucleoside. In certain embodiments, FNM in formula (VI) is FMM. The modified nucleoside may contain a modified base and / or a modified sugar (preferably a modified sugar at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0132] In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid comprises an antisense strand having 21 linked nucleotides, and the antisense strand sequence is represented by formula (VII): 5'LM*D*MFMFNMFMMFMFMFMMN*M*M3', where D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is a 2'-F-modified nucleoside, * is a phosphorothioate (PS) linkage, and L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), and no single modification type modifies more than two consecutive nucleotides. In one embodiment, the oligomeric compound represented by formula (VII) is ssRNA or siRNA. In certain embodiments, FNM is FMM. Modified nucleosides may contain modified bases and / or modified sugars (preferably at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0133] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises (a) a sense strand consisting of 21 linked nucleotides, having a sense strand sequence represented by formula (III): 5'MFMMNMNMFFNMNMNMMFMFMDD3', and (b) an antisense strand consisting of 21 linked nucleotides, having an antisense strand sequence represented by formula (VII): 5'L-MDMFMFNMFMMFMFMFMMNMM3'. The double-stranded region is 19 nucleotide pairs in length, with each strand having a 2-nucleotide overhang at its 3' end. wherein D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is a 2'-F-modified nucleoside, * is a phosphorothioate (PS) linkage, L is a 5' phosphate, a 5' vinylphosphonate, or a 5' OH, and no single modification type modifies more than two consecutive nucleotides. In one embodiment, the oligomeric compound consisting of formula (III) and formula (VII) is an siRNA. In certain embodiments, FFNM in formula (III) is FFMM or FFRM, where R is a ribonucleoside. In certain embodiments, FNM in formula (VII) is FMM. The modified nucleoside may contain a modified base and / or a modified sugar (preferably a modified sugar at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0134] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises a sense strand having 19 to 23 linked nucleotides, and the sense strand sequence is represented by formula (I): 5'M-(Y)nZ-(Y)rD-D3', where D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, n is 6 to 8, r is 1 to 2, and no single modification type modifies more than two consecutive nucleotides. Preferably, -DD is an overhanging TT or TA. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In a further embodiment, the sense strand contains phosphorothioate internucleotide (PS) linkages adjacent to deoxyribonucleosides (D) or ribonucleosides (R). The PS linkages may be adjacent to the deoxyribonucleosides (D) or ribonucleosides (R) on the 5', 3', or both sides. The PS linkages may be adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of Formula (I) is ssRNA or siRNA. Modified nucleosides may contain modified bases and / or modified sugars (preferably at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0135] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises an antisense strand having 19 to 23 linked nucleotides, and the antisense strand sequence is represented by formula (IX): 5'LM-(Y)pZ-(Y)p-(Z)r3', where M is a 2'-OMe-modified nucleoside, L is a 5' phosphate, 5' is vinylphosphonate or 5'OH, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, p is 3 to 5, r is 1 to 2, and no single modification type modifies more than two consecutive nucleotides. Preferably, the 3'Z is a UU or TT overhang. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In a further embodiment, the antisense strand contains phosphorothioate internucleotide (PS) linkages adjacent to deoxyribonucleosides (D) or ribonucleosides (R). The PS linkages may be adjacent to the deoxyribonucleosides (D) or ribonucleosides (R) on the 5', 3', or both sides. The PS linkage modifications may be located between positions 1-2, 2-3, 19-20, and / or 20-21, counting from the 5' end of the antisense strand. The PS linkages may be adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of formula (IX) is ssRNA or siRNA. The modified nucleosides may contain modified bases and / or modified sugars (preferably at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0136] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises (a) a sense strand having 19 to 23 linked nucleotides, wherein the sense strand sequence is represented by formula (I): 5'M-(Y)nZ-(Y)rD-D3'; and (b) an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by formula (IX): 5'LM-(Y)pZ-(Y) p -(Z) r3'. The double-stranded region is formed by the sense strand and the antisense strand, and the double-stranded region is 19 to 23 nucleotide pairs in length. Here, D is a deoxyribonucleoside, N is a modified or unmodified nucleoside, M is a 2'-OMe-modified nucleoside, L is a 5' phosphate, 5' is vinylphosphonate or 5' OH, Y is two adjacent nucleosides with different modifications or a modified nucleoside adjacent to an unmodified nucleoside, Z is two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides, n is 6 to 8, p is 3 to 5, and r is 1 to 2, and no single modification type modifies more than two consecutive nucleotides. In a further embodiment, the 3'Z of the antisense strand is an overhang of UU or TT, and the -DD of the sense strand is an overhang of TT or TA. In certain embodiments, the sense strand contains one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In further embodiments, the strand contains phosphorothioate internucleotide (PS) linkages adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkages may be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5', 3', or both sides. The PS linkage modifications on the antisense strand may be located between positions 1-2, 2-3, 19-20, and / or 20-21, counting from the 5' end of the antisense strand. The PS linkages may be adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of Formulae (I) and (IX) is an siRNA. Modified nucleosides may contain modified bases and / or modified sugars (preferably at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0137] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises a sense strand having 21 linked nucleotides, and the sense strand sequence is represented by the formula (X): 5'MFMMNMNMFFNMNMNMMMNMDD3', where D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), and F is a 2'-F-modified nucleoside, and no single modification type modifies more than two consecutive nucleotides. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In further embodiments, the sense strand comprises phosphorothioate internucleotide (PS) linkages adjacent to the deoxyribonucleoside (D) or ribonucleoside (R). The PS linkages may be adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R) on the 5', 3', or both sides. The PS linkages may be adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of formula (X) is ssRNA or siRNA. Modified nucleosides may contain modified bases and / or modified sugars (preferably at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0138] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises an antisense strand having 21 linked nucleotides, and the antisense strand sequence is represented by formula (VIII): 5'L-MNMNMFNMFMMNMFMFMMNMM3', where M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is a 2'-F-modified nucleoside, and L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), and no single modification type modifies more than two consecutive nucleotides. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In further embodiments, the antisense strand comprises phosphorothioate internucleotide (PS) linkages adjacent to deoxyribonucleosides (D) or ribonucleosides (R). The PS linkages may be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5', 3', or both sides. The PS linkage modifications may be located between positions 1-2, 2-3, 19-20, and / or 20-21, counting from the 5' end of the antisense strand. The PS linkages may be adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of formula (VIII) is ssRNA or siRNA. The modified nucleosides may contain modified bases and / or modified sugars (preferably at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0139] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises (a) a sense strand consisting of 21 linked nucleotides, having a sense strand sequence of formula (X): 5'MFMMNMNMFFNMNMNMMMNMDD3', and (b) an antisense strand consisting of 21 linked nucleotides, having an antisense strand sequence of formula (VIII): 5'L-MNMNMFNMFMMNMFMFMMNMM3'. The double-stranded region is 19 nucleotide pairs in length, with each strand having a 2-nucleotide overhang at its 3' end. wherein D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is a 2'-F-modified nucleoside, and L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), and no single modification type modifies more than two consecutive nucleotides. In certain embodiments, the sense strand contains one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In further embodiments, the strand contains phosphorothioate internucleotide (PS) linkages adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R). The PS linkages may be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5' side, the 3' side, or both sides. The PS bond modifications on the antisense strand may be located between positions 1-2, 2-3, 19-20, and / or 20-21, counting from the 5' end of the antisense strand. The PS bond may flank the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of formula (X) and (VIII) is an siRNA. The modified nucleoside may contain a modified base and / or a modified sugar (preferably a modified sugar at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0140] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises a sense strand having 21 linked nucleotides, and the sense strand sequence is represented by the formula XI: 5'MFMMNMNMFFMMNMNMMFMDD3', where D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), and F is a 2'-F-modified nucleoside, and no single modification type modifies more than two consecutive nucleotides. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In further embodiments, the sense strand comprises phosphorothioate internucleotide (PS) linkages adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R). The PS linkages may be adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R) on the 5', 3', or both sides. The PS linkages may be adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of formula (XI) is ssRNA or siRNA. The modified nucleosides may contain modified bases and / or modified sugars (preferably at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0141] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises an antisense strand having 21 linked nucleotides, and the antisense strand sequence is represented by formula (XII): 5'L-MDMFMFNMFMMFMFMFMMNMM3', where M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is a 2'-F-modified nucleoside, and L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), and no single modification type modifies more than two consecutive nucleotides. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In further embodiments, the antisense strand comprises phosphorothioate internucleotide (PS) linkages adjacent to deoxyribonucleosides (D) or ribonucleosides (R). The PS linkages may be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5', 3', or both sides. The PS linkage modifications may be located between positions 1-2, 2-3, 19-20, and / or 20-21, counting from the 5' end of the antisense strand. The PS linkages may be adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound of formula (XII) is ssRNA or siRNA. The modified nucleosides may contain modified bases and / or modified sugars (preferably at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.

[0142] In one embodiment, an oligomeric compound capable of inhibiting expression of a target nucleic acid comprises: (a) a sense strand consisting of 21 linked nucleotides, the sense strand having a sequence of formula (XI): 5'MFMMNMNMFFMMNMNMMFMDD3'; and (b) an antisense strand consisting of 21 linked nucleotides, the antisense strand having a sequence of formula (XII): 5'L-MDMFMFNMFMMFMFMFMMNMM3'. The double-stranded region is 19 nucleotide pairs in length, with each strand having a 2-nucleotide overhang at its 3' end. wherein D is a deoxyribonucleoside, M is a 2'-OMe-modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is a 2'-F-modified nucleoside, and L is 5'OH, 5'vinylphosphonate, or 5'phosphate (p), and no single modification type modifies more than two consecutive nucleotides. In certain embodiments, the sense strand contains one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In further embodiments, the strand contains phosphorothioate internucleotide (PS) linkages adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R). The PS linkages may be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5' side, the 3' side, or both sides. The PS bond modifications on the antisense strand may be located between positions 1-2, 2-3, 19-20, and / or 20-21, counting from the 5' end of the antisense strand. The PS bond may be adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compounds of formula (XI) and formula (XII) are siRNAs. The modified nucleosides may contain modified bases and / or modified sugars (preferably at the 2' position). Deoxyribonucleosides (D) are considered modified nucleosides in RNA-based oligomeric compounds.Oligomeric compounds can be introduced into target cells by a variety of modalities, hi certain embodiments, oligomeric compounds are introduced into cells via viral delivery vectors, lipid-based delivery, polymer-based delivery, and / or conjugate-based delivery.

[0143] In certain embodiments, the oligomeric compounds described herein further comprise a conjugate. The conjugate can be selected from cholesterol, lipids, carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In a preferred embodiment, the conjugate is N-acetylgalactosamine (GalNAc). In one embodiment, the conjugate can be attached to the 3' end of the sense strand. In a preferred embodiment, the conjugated oligomeric compound is an siRNA-GalNAc conjugate.

[0144] In certain embodiments, the oligomeric compounds described herein inhibit expression of a target nucleic acid by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%.

[0145] In certain embodiments, pharmaceutical compositions comprise an oligomeric compound described herein, alone or in combination with a pharmaceutically acceptable carrier or excipient.

[0146] In certain embodiments, there is provided a method of inhibiting expression of a target nucleic acid in a subject, the method comprising administering to the subject an oligomeric compound described herein in an amount sufficient to inhibit expression of the target nucleic acid. The oligomeric compound can be administered to the subject subcutaneously or intravenously.

[0147] Oligomeric Compounds Oligomeric compounds of the present invention include, but are not limited to, single-stranded oligomeric compounds such as microRNA (miRNA), single-stranded RNA (ssRNA), and antisense oligonucleotides (ASO), as well as double-stranded oligomeric compounds such as short hairpin RNA (shRNA) and small interfering RNA (siRNA). Oligomeric compounds may be "antisense" or contain an "antisense strand" to a target nucleic acid, meaning that they can hybridize to a target nucleic acid via hydrogen bonding.

[0148] In certain embodiments, an oligomeric compound has a nucleobase sequence written in a 5' to 3' direction, which nucleobase sequence comprises a reverse complementary sequence to a target segment of a target nucleic acid to which the oligomeric compound is targeted. For example, in certain such embodiments, an siRNA comprises an antisense strand, which has a nucleobase sequence written in a 5' to 3' direction, which nucleobase sequence comprises a reverse complementary sequence to a target segment of a target nucleic acid to which the siRNA is targeted.

[0149] In certain embodiments, the oligomeric compounds have a length of 12 to 30 subunits. In certain embodiments, the oligomeric compounds have a length of 18 to 30 subunits. In certain embodiments, the oligomeric compounds have a length of 12 to 22 subunits. In certain embodiments, the oligomeric compounds have a length of 14 to 30 subunits. In certain embodiments, the oligomeric compounds have a length of 14 to 21 subunits. In certain embodiments, the oligomeric compounds have a length of 15 to 30 subunits. In certain embodiments, the oligomeric compounds have a length of 15 to 21 subunits. In certain embodiments, the oligomeric compounds have a length of 16 to 30 subunits. In certain embodiments, the oligomeric compounds have a length of 16 to 21 subunits. In certain embodiments, the oligomeric compounds have a length of 17 to 30 subunits. In certain embodiments, the oligomeric compounds have a length of 17 to 21 subunits. In certain embodiments, the oligomeric compound has a length of 18 to 30 subunits. In certain embodiments, the oligomeric compound has a length of 18 to 21 subunits. In certain embodiments, the oligomeric compound has a length of 20 to 30 subunits. In certain embodiments, the oligomeric compound has a length of 15 subunits. In certain embodiments, the oligomeric compound has a length of 16 subunits. In certain embodiments, the oligomeric compound has a length of 17 subunits. In certain embodiments, the oligomeric compound has a length of 18 subunits. In certain embodiments, the oligomeric compound has a length of 20 subunits. In certain embodiments, the oligomeric compound has a length of 21 subunits. In certain embodiments, the oligomeric compound has a length of 22 subunits. In certain embodiments, the oligomeric compound has a length of 23 subunits. In certain embodiments, the oligomeric compound has a length of 25 subunits.In certain embodiments, the oligomeric compound has a length of 25 subunits, hi other embodiments, the oligomeric compound has a length of 8-80, 12-50, 13-30, 13-50, 14-30, 14-50, 15-30, 15-50, 16-30, 16-50, 17-30, 17-50, 18-22, 18-24, 18-30, 18-50, 19-22, 19-30, 19-50, or 20-30 linked subunits. In certain embodiments, the oligomeric compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, In some embodiments, the oligomeric compound has a length of 9, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits, or a length range defined by any two of the foregoing values. In some embodiments, the oligomeric compound is an siRNA.

[0150] It is possible to increase or decrease the length of an oligomeric compound, such as an siRNA, and / or introduce base mismatches without losing activity (U.S. Patent No. 7,772,203, incorporated herein by reference).For example, it is possible to introduce non-canonical base pairs (e.g., A:G, A:C, G:U, I:U, I:A, or I:C) into an oligomeric compound without losing activity.In certain embodiments, designing an oligomeric compound to have one or more non-canonical base pairs, i.e., mismatches, improves the activity of the oligomeric compound.

[0151] Oligomeric compounds can contain mismatches between the target, mismatches between oligomer strands within the double-stranded body, or a combination thereof. Mismatches can occur throughout the siRNA, including in the overhanging and double-stranded regions.

[0152] Motifs of oligomeric compounds Motif refers to a modification pattern in an oligomeric compound. Various motifs have been described in the art and are incorporated herein by reference (e.g., U.S. Pat. Nos. 11,203,755; 10,870,849; EP Pat. No. 1,532,248; U.S. Pat. No. 1,140,6716; U.S. Pat. No. 10,668,170; U.S. Pat. No. 9,796,974; U.S. Pat. No. 8,754,201; U.S. Pat. No. 1,083,7013; U.S. Pat. No. 7,732,593; U.S. Pat. No. 7,015,315; U.S. Pat. No. 7,750,144; U.S. Pat. No. 8,420,799; U.S. Pat. No. 8,809,516; U.S. Pat. No. 8,796,436; U.S. Pat. No. 6 ... (US Patent Nos. 8,859,749; 9,708,615; 10,233,448; 10,273,477; 10,612,024; 10,612,027; 10,669,544; 11,401,517; 9,260,471; 9,970,005; 11,193,126; 8,604,183; 9,150,605; 9,708,610; US Publication No. 2020 / 0031862; US Publication No. 2016 / 0272970). However, it will be apparent to one skilled in the art that new and improved motifs (also referred to herein as chemically modified patterns) are within the scope of the present invention.

[0153] In certain embodiments, the oligomeric compounds disclosed herein have chemically modified subunits arranged in a motif or pattern (i.e., chemical modification motif / pattern) that confer beneficial properties to the oligomeric compounds, such as, but not limited to, enhanced inhibitory activity for increased potency, increased binding affinity for the target nucleic acid to reduce off-target effects and improve safety, or improved stability and durability due to enhanced resistance to degradation by in vivo nucleases. In certain embodiments, the oligomeric compounds are chimeric, in which the peripheral nucleobases of the oligomeric compounds comprise motifs with various modified or unmodified nucleobases for increased stability, specificity, safety, and potency, and the central region of the compound comprises various modified or unmodified nucleobases to serve as substrates for RISC-mediated degradation. Each of the distinct regions may contain uniform, modified, or alternating sugar moieties. Each region may contain diverse patterns of phosphate and phosphorothioate linkages.

[0154] In certain embodiments, oligomeric compounds targeted to nucleic acids comprise a sense strand having a motif shown in any of the following formulas: Formula (I): 5'M-(Y)nZ-(Y)rD-D3', Formula (II): 5'YZ-(Y)q-FFNM-(Y)qM-(Y)vD-D3', Formula (III): 5'M*F*MMN*MN*MFFNMN*MN*MMFM*D*D3', Formula (X): 5'MFMMNMNMFFNMNMNMMNMDD3', or Formula (XI): 5'MFMMNMNMFFMMNMNMFMDD3'. where: each D is a deoxyribonucleoside (D is a modification of R); each R is a ribonucleoside; each N is a modified or unmodified nucleoside (e.g., D, R, M, F, UNA-modified, or LNA-modified); each M is a 2'-OMe modified nucleoside; each F is a 2'-F modified nucleoside; Each * is a phosphorothioate (PS) bond, each Y is two adjacent nucleosides with different modifications (e.g., MD, DM, DF, FD, MF, or FM), or an unmodified nucleoside and an adjacent modified nucleoside (e.g., DR, RD, MR, or RM); each Z is two adjacent unmodified nucleosides, two adjacent nucleosides having the same modification, or two adjacent unmodified nucleosides such as (e.g., MM, DD, RR, or FF); Each n is 6 to 8, Each q is 2 to 3, Each r is 1 to 2, Each v is between 0 and 1, No single modification type modifies more than two consecutive nucleotides. In certain embodiments, the FFNM is FFRM or FFMM.

[0155] In certain embodiments, oligomeric compounds targeted to nucleic acids comprise an antisense strand having a motif shown in any of the following formulas: Formula (IV): 5'-LM-(D)v-(Y)s-(Z)t-(Y)uZN-(Z)r3', Formula (V): 5'L-(Y)p-NM-(FMM)r-(Y)p-(Z)r3', Formula (VI): 5'LM*N*MNMFNMMFMMNMFMFMMN*M*M3', Formula (VII): 5'LM*D*MFMFNMFMMFMFMFMMN*M*M3', Formula (VIII): 5'L-MNMNMFNMMFMMNMFMFMMNMM3', Formula (IX): 5'LM-(Y)pZ-(Y)p-(Z)r3', or Formula (XII): 5'L-MDMFMFNMFMMFMFMFMMNMM3', where: each D is a deoxyribonucleoside (D is a modification of R); each R is a ribonucleoside; each N is a modified or unmodified nucleoside (e.g., D, R, M, F, UNA-modified, or LNA-modified); each M is a 2'-OMe modified nucleoside; each F is a 2'-F modified nucleoside; each L is a 5' phosphate, a 5' vinylphosphonate, or a 5' OH; Each * is a phosphorothioate (PS) bond, each Y is two adjacent nucleosides with different modifications (e.g., MD, DM, DF, FD, MF, or FM), or an unmodified nucleoside and an adjacent modified nucleoside (e.g., DR, RD, MR, or RM); each Z is two adjacent unmodified nucleosides, two adjacent nucleosides having the same modification, or two adjacent unmodified nucleosides such as (e.g., MM, DD, RR, or FF); Each (5p) is a 5'-phosphate, Each n is 6 to 8, Each p is 3 to 5, Each r is 1 to 2, Each v is between 0 and 1, Each s is 2 to 7, Each t is between 0 and 2, moreover, No single modification type modifies more than two consecutive nucleotides. In certain embodiments, the FNM is FMM.

[0156] In certain embodiments, the oligomeric compound targeted to an mRNA nucleic acid comprises an siRNA double-stranded entity having any of the following motifs: a double-stranded construct I having a sense strand of formula (I) and an antisense strand of formula (IV); a double-stranded construct II having a sense strand of formula (II) and an antisense strand of formula (V); a double-stranded construct III having a sense strand of formula (III) and an antisense strand of formula (VI); a double-stranded molecule IV having a sense strand of formula (IV) and an antisense strand of formula (VII); a double-stranded molecule V having a sense strand of formula (I) and an antisense strand of formula (IX); a double-stranded molecule VI having a sense strand of formula (X) and an antisense strand of formula (VIII); A double-stranded construct VII having a sense strand of formula (XI) and an antisense strand of formula (XII), a double-stranded molecule VIII having a sense strand of formula (X) and an antisense strand of formula (XII), or a double-stranded construct IX having a sense strand of formula (XI) and an antisense strand of formula (VIII); the double-stranded region has a length of 19 to 23 nucleotide pairs; No single modification type may modify more than two consecutive nucleotides.

[0157] Target mRNA and associated gene expression Some embodiments relate to methods for modulating gene expression through the inhibition of oligomeric compounds.

[0158] In certain embodiments, a method for inhibiting laminin (LMNA) gene expression in a cell comprises administering to the cell an oligomeric compound targeted to the LMNA mRNA transcript. In one embodiment, the oligomeric compound is designed to target a 19-nucleotide sequence of LMNA (SEQ ID NO: 1) that is conserved between humans and mice.

[0159] In certain embodiments, a method for inhibiting apolipoprotein C3 (ApoC3) gene expression in a cell comprises administering to the cell an oligomeric compound targeted to an ApoC3 mRNA transcript. In one embodiment, the oligomeric compound is designed to target a 19-nucleotide sequence of ApoC3 that is conserved between humans and mice (SEQ ID NO: 177).

[0160] In certain embodiments, a method for inhibiting nucleolin (NCL) gene expression in a cell comprises administering to the cell an oligomeric compound targeted to an NCL mRNA transcript. In one embodiment, the oligomeric compound is designed to target a 19-nucleotide sequence of NCL (SEQ ID NO: 98) that is conserved between humans and mice.

[0161] mixed In some embodiments, hybridization occurs between the oligomeric compounds disclosed herein and mRNA. The most common mechanism of hybridization is binding between nucleic acid molecules involving hydrogen bonding (e.g., Watson-Crick, Fugstein, or reversed Fugstein) between complementary nucleobases.

[0162] In canonical Watson-Crick base pairing, adenine (A) is complementary to thymine (T) in DNA, adenine (A) is complementary to uracil (U) in RNA, and guanine (G) is complementary to cytosine (C) in both DNA and RNA. Base pairs, or complementary nucleobases, are typically Watson-Crick base pairs (C:G, A:U, A:T). However, non-canonical base pairs, such as Fugsteen base pairs (e.g., A:G or A:U) and wobble base pairs (e.g., G:U, I:U, I:A, I:C, where I refers to hypoxanthine), are also tolerated when oligomeric compounds hybridize to target nucleic acids or target regions. Wobble base pairs in RNAi agents have been previously described (see, e.g., US Patents 7,732,593 and 7,750,144).

[0163] Nucleobase complementarity promotes hybridization of the oligomeric compounds described herein to their target nucleic acids, and the stronger the pairing (e.g., the greater the number of base pairs and / or the stronger the hydrogen bonds), the stronger the hybridization of the oligomeric compound to the target. Hybridization can occur under a variety of conditions. The stringent conditions are sequence-dependent and determined by the nature and composition of the oligomeric compound to be hybridized.

[0164] Methods for determining whether a sequence can specifically hybridize to a target nucleic acid are known in the art. In certain embodiments, the oligomeric compounds described herein can specifically hybridize to target mRNA, with little or no off-target binding.

[0165] Complementarity An oligomeric compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the oligomeric compound are capable of hybridizing with corresponding nucleobases of the target nucleic acid, such that a desired effect (e.g., inhibition of a target nucleic acid, such as an mRNA nucleic acid) can be achieved.

[0166] The presence of non-complementary nucleobases between an oligomeric compound and an mRNA nucleic acid may be tolerated so long as the oligomeric compound is capable of specifically hybridizing to the target nucleic acid. Additionally, an oligomeric compound may hybridize to one or more segments of an mRNA nucleic acid having intervening or adjacent segments (e.g., loop structures, mismatches, or hairpin structures) that do not participate in hybridization.

[0167] In certain embodiments, the oligomeric compounds described herein, or designated portions thereof, are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to an mRNA nucleic acid, target region, target segment, or designated portion thereof. The percent complementarity between an oligomeric compound and a target nucleic acid can be determined using routine methods.

[0168] For example, if 18 of the 20 nucleobases of the antisense strand of an oligomeric compound are complementary to the target region and specifically hybridize, the oligomeric compound will exhibit 90% complementarity. In this example, the remaining non-complementary nucleobases may be clustered with complementary nucleobases or may be interspersed among complementary nucleobases, and they need not be adjacent to each other or to complementary nucleobases. The percentage of complementarity between an oligomeric compound and a region of a target nucleic acid can be routinely determined using BLAST (Basic Local Alignment Search Tool) and PowerBLAST programs (both known in the art) (Altschul et al., 1990, J. Mol. Biol., 215: 403-410; Zhang and Madden, 1997, Genome Res., 7: 649-656). These programs are available through the website of the National Center for Biotechnology Information (NCBI, https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Percent homology, sequence identity, or complementarity can be determined, for example, by NCBI Blast (Johnson et al., 2008, Nucleic Acids Res., 36(Web Server issue):W5-W9).

[0169] In certain embodiments, the oligomeric compounds described herein, or specific portions thereof, are fully complementary (i.e., 100% complementary) to a target nucleic acid, or specific portions thereof. For example, an oligomeric compound may be fully complementary to an mRNA nucleic acid, or a target region, or a target segment, or a target sequence thereof. As used herein, "fully complementary" means that each nucleobase of an oligomeric compound has the ability to form an exact base pair with the corresponding nucleobase of a target nucleic acid. For example, an oligomeric compound consisting of 20 nucleobases is fully complementary to a target sequence consisting of 400 nucleobases, as long as there is a 20 nucleobase portion in the target nucleic acid that is fully complementary to the oligomeric compound.

[0170] Fully complementary may also be used to refer to a specific portion of an oligomeric compound or a nucleic acid target. For example, a 20 nucleobase portion of a 30 nucleobase oligomeric compound may be "fully complementary" to a 400 nucleobase target sequence. A 20 nucleobase portion of a 30 nucleobase oligomer is fully complementary to a target sequence if the target sequence has a corresponding portion (20 nucleobases) in which each nucleobase is complementary to the 20 nucleobase portion of the oligomeric compound. At the same time, the entire 30 nucleobase oligomeric compound may or may not be fully complementary to a target sequence, depending on whether the remaining 10 nucleobases of the oligomeric compound are also complementary to the target sequence.

[0171] The non-complementary nucleobase position may be at the 5' or 3' end of the oligomeric compound. Alternatively, the non-complementary nucleobase or nucleobases may be at internal positions of the oligomeric compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e., linked) or non-contiguous.

[0172] In certain embodiments, oligomeric compounds that are or are up to 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleobases in length have no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase with respect to a target nucleic acid, such as an mRNA nucleic acid or a specified portion thereof.

[0173] In certain embodiments, oligomeric compounds that are or are up to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length have no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase with a target nucleic acid, such as an mRNA nucleic acid or a specified portion thereof.

[0174] Oligomeric compounds described herein also include those complementary to portions of a target nucleic acid. As used herein, a "portion" refers to a defined number of contiguous (i.e., linked) nucleobases within a region or segment of a target nucleic acid. A "portion" can also refer to a defined number of contiguous nucleobases in an oligomeric compound. In certain embodiments, an oligomeric compound is complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, an oligomeric compound is complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, an oligomeric compound is complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, an oligomeric compound is complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, an oligomeric compound is complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, an oligomeric compound is complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, an oligomeric compound is complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, oligomeric compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are oligomeric compounds complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

[0175] identity The oligomeric compounds described herein may also have a defined percent identity to a compound represented by a specific nucleotide sequence, SEQ ID NO:, or specific ARNATAR number, or a portion thereof. As used herein, an oligomeric compound is identical to a sequence disclosed herein if it has the same nucleic acid base-pairing ability. For example, an RNA containing uracil instead of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence because both uracil and thymidine pair with adenine. Shortened and extended versions of the oligomeric compounds described herein, as well as compounds with non-identical bases relative to the oligomeric compounds described herein, are also contemplated. The non-identical bases may be adjacent or interspersed within the oligomeric compound. The percent identity of an oligomeric compound is calculated based on the number of identical bases in the sequence being compared.

[0176] In certain embodiments, a portion of the oligomeric compound is compared to a portion of the target nucleic acid of the same length. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion of the oligomeric compound is compared to a portion of the target nucleic acid of the same length.

[0177] chemical modification A nucleoside is a combination of a base and a sugar. The nucleobase (also referred to as base) portion of a nucleoside is usually a heterocyclic base portion. A nucleotide is a nucleoside that further comprises a covalent bond (e.g., a phosphate group or a chemically modified bond as described below) to the sugar portion of the nucleoside. An oligonucleotide is formed through the covalent bonding of adjacent nucleotides to each other to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the linking group is usually intended to form the internucleoside linkage of the oligonucleotide. An oligomeric compound may be a single oligonucleotide (e.g., ssRNA, antisense oligonucleotide, or miRNA) or multiple oligonucleotides (e.g., siRNA or shRNA).

[0178] Modifications to oligomeric compounds include substitutions or alterations of nucleobases, internucleoside linkages, or sugar moieties. Modified oligomeric compounds are often preferred over native or unmodified forms because they possess desirable properties, such as improved delivery (e.g., increased cellular uptake), increased specificity or affinity for a nucleic acid target, increased stability in the presence of nucleases, improved safety (e.g., reduced side effects following administration of the compound to a subject), or increased potency (e.g., inhibitory activity).

[0179] Nucleobase Modification Nucleobase refers to a heterocyclic moiety that can form base pairs with the nucleobase of another nucleic acid. Modifications to nucleobases are advantageous for oligomeric compounds for various reasons, including, but not limited to, improving the stability of the oligomeric compounds, improving specificity, reducing the immunogenicity of the oligomeric compounds, improving the affinity of the oligomeric compounds, improving the potency of the oligomeric compounds, and other desirable properties.

[0180] Examples of nucleobase modifications and their advantages are well known in the art (Friedrich and Aigner, Therapeutic siRNA: State-of-the-Art and Future Perspectives, 2022, BioDrugs, 36(5):549-571; Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101). Nucleobase modifications can include replacing the nucleobase with a nucleobase analog or modifying a portion of the nucleobase. Examples of nucleobase modifications include, but are not limited to, pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5'-methylcytidine, 5'-fluoro-2'-deoxyuridine, N-ethylpiperidine 5'-triazole-modified adenosine, 5'-nitroindole, 2',4'-difluorotolylribonucleoside, N-ethylpiperidine 7'-EAA triazole-modified adenosine, 6'-phenylpyrrolocytosine, and the like.

[0181] In certain embodiments, oligomeric compounds targeted to mRNA nucleic acids comprise one or more modified nucleobases. In certain embodiments, the modified nucleobases are, for example, deoxyribonucleotides substituted for ribonucleotides. In certain embodiments, the modified nucleobases may be thymine substitutions for uracil. In certain embodiments, multiple nucleobases of the oligomeric compounds are modified. In certain embodiments, each nucleobase of the oligomeric compounds is modified.

[0182] Internucleoside bond modification The naturally occurring internucleoside linkage in RNA and DNA is a 3' to 5' phosphodiester linkage. In nucleosides containing a pentofuranosyl sugar, the phosphate group can be attached to the 2', 3', or 5' hydroxyl moiety of the sugar. Oligomeric compounds having one or more modified, i.e., non-naturally occurring, internucleoside linkages are often chosen over oligomeric compounds having naturally occurring internucleoside linkages for reasons such as improved cellular uptake, increased affinity for target nucleic acids, reduced toxicity, improved stability and durability, reduced degradation, and other desirable properties of oligomeric compounds. Modified internucleoside linkages and their advantages are well known in the art (Friedrich and Aigner, Therapeutic siRNA: State-of-the-Art and Future Perspectives, 2022, BioDrugs, 36(5):549-571; Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101).

[0183] Oligomeric compounds having modified internucleoside linkages include those that retain a phosphorus atom and those that do not. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates (e.g., 5'-methylphosphonate (5'-MP)), phosphoramidates, phosphorothioates (e.g., phosphorodithioate Rp isomers (PS,Rp), phosphorodithioate Sp isomers (PS,Sp), or 5'-phosphorothioates (5'-PS)), methoxypropylphosphonates, phosphorylated forms with an (S)-5'-C-methyl group, peptide nucleic acids (PNAs), and 5'-(E)-vinylphosphonates.

[0184] In certain embodiments, oligomeric compounds targeted to mRNA nucleic acids comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate (PS) linkages. In certain embodiments, one or more internucleoside linkages of an oligomeric compound are phosphorothioate internucleoside linkages. In certain embodiments, a PS linkage is adjacent to a deoxyribonucleoside (sometimes referred to herein as a DNA nucleoside, "D" or "d") or a ribonucleoside (sometimes referred to herein as an RNA nucleoside, "R" or "r"). In certain embodiments, each internucleoside linkage of an oligomeric compound is a phosphorothioate internucleoside linkage.

[0185] Sugar correction The oligomeric compounds described herein may include one or more nucleosides with modified sugar groups. Such sugar-modified nucleosides may confer on the oligomeric compounds increased stability, increased durability (e.g., increased half-life), increased binding affinity, reduced off-target effects, reduced immunogenicity, reduced toxicity, increased efficacy, or other beneficial biological properties. Sugar modifications and their benefits are known in the art (Friedrich and Aigner, 2022, BioDrugs, 36(5):549-571 (Review of therapeutic applications of sugar-modified nucleic acids); Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101 (Frontiers of therapeutic siRNA); Chiu and Rana, 2003, RNA, 9:1034-1048 (Chemical modification and intracellular dynamics of siRNA); Choung et al., Biochem Biophys Res Commun, 2006, 342:919-927 (Studies on siRNA stability and targeting); Amarzguioui et al., 2003, Nucleic Acids Res, 31(2):589-595 (Structure optimization in RNA interference); Braasch et al. al., 2003, Biochemistry, 42(26):7967-7975 (provisional translation: cellular uptake and gene silencing effect of siRNA); Czauderna et al., 2003, Nucleic Acids Res, 31(11):2705-2716 (provisional translation: chemical modification for improving siRNA stability); Allerson et al., 2005, J Med Chem, 48:901-904 (provisional translation: drug development of chemically modified siRNA); Layzer et al., 2004, RNA, 10:766-771 (provisional translation: functionality of siRNA for in vivo administration); Ui-Tei et al., 2008, Nucleic Acids Res, 36(7):2136-51 (Guidelines for designing highly functional siRNA sequences); Bramsen and Kjems, 2012, Frontiers in Genetics, 3(154):1-22 (Trends in the development of high-performance siRNAs); Bramsen et al., 2010, Nucleic Acids Res, 38(17):5761-5773 (Gene silencing by chemically modified siRNAs); Muhonen et al., 2007, Chem & Biodiversity, 4:858-873 (Chemical diversity of oligonucleotide modifications); Viel et al., 2008, Oligonucleotides, 18:201-212 (Biological activity of sugar-modified oligonucleotides); these are incorporated herein by reference.

[0186] In certain embodiments, a nucleoside comprises a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings can include the addition of substituents (e.g., 5'- or 2'-sugar modifications), bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNAs), replacing oxygen atoms of the ribosyl ring with S, N(R), or C(R)(R) (R = H, C-C alkyl, or protecting groups), nucleoside mimetics, and combinations thereof.

[0187] 2'-modified sugar refers to a furanosyl sugar modified at the 2' position. 2'-modified nucleoside refers to a nucleoside comprising a sugar modified at the 2' position of the furanosyl ring. In certain embodiments, such modifications include substituents selected from halides, including, but not limited to, substituted or unsubstituted alkoxy, substituted or unsubstituted thioalkyl, substituted or unsubstituted aminoalkyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, and substituted or unsubstituted alkynyl. In certain embodiments, the 2' modification is O[(CH2) n O] m CH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) nONH2, OCH2C(=O)N(H)CH3, and O(CH2) n ON[(CH2) n CH3]2, where n and m range from 1 to about 10. Other 2'-substituents include C1-C 12 The substituents may be selected from alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties, groups for improving the pharmacodynamic properties of oligomeric compounds, and other substituents with similar properties.

[0188] Further examples of nucleosides having modified sugar moieties include nucleosides containing 5'-vinyl, 5'-methyl (R or S), 2'-F-5'-methyl, 4'-S, 2'-deoxy-2'-fluoro (2'-F), 2'-OCH3 (2'-O-methyl, 2'-OMe), 2'-O(CH2)2OCH3 (2'-O-methoxyethyl, 2'-O-MOE, 2'-MOE), 2'-O-methyl-4-pyridine, phosphorodiamidic acid morpholino (PMO), tricyclo-DNA (tcDNA), 2'-arabino-fluoro, 2'-O-benzyl, glycol nucleic acid (GNA), and unlocked nucleic acid (UNA) substituents. Also, the 2'-position substituent can be allyl, amino, azido, thio, O-allyl, O-C1-C 10 alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-ON(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H or a substituted or unsubstituted C1-C 10Alkyl. 2'-OMe or 2'-OCH3 or 2'-O-methyl refers to a nucleoside containing a sugar containing an -OCH3 group at the 2' position of the sugar ring. 2'-F refers to a sugar containing a fluoro group at the 2' position. 2'-O-Methoxyethyl, or 2'-O-MOE, or 2'-MOE refers to a nucleoside containing a sugar containing an -O(CH2)2OCH3 group at the 2' position of the sugar ring.

[0189] BNAs refer to modified nucleosides containing a bicyclic sugar moiety with a bridge connecting two carbon atoms, one at the 2' position in the sugar ring and the other at the other position in the sugar ring. Examples of bicyclic nucleosides include nucleosides with a bridge between the 4' and 2' ribosyl ring atoms, such as locked nucleic acids (LNAs). In certain embodiments, the oligomeric compounds described herein contain one or more bicyclic nucleosides, including bicyclic nucleosides where the bridge connects the 4' and 2' positions. LNAs and UNAs are described by Campbell and Wengel (Chem Soc Rev, 2011, 40(12):5680-9), which are incorporated herein by reference.

[0190] In certain embodiments, oligomeric compounds comprise nucleotides having one or more modified sugar moieties. In certain embodiments, the modified sugar moieties have a 2'-OMe modification. In certain embodiments, the modified sugar moieties have a 2'-F modification. In certain embodiments, the 2'-OMe and / or 2'-F modified nucleotides are arranged in a motif. In certain embodiments, the motif is selected from any of Formulas (I) through (VII).

[0191] Oligomeric Compound Delivery Systems Oligomeric compounds require entry into target cells for activation. Various modalities have been used to transport oligomeric compounds into target cells, including viral delivery vectors, lipid-based delivery, polymer-based delivery, and conjugate-based delivery (Paunovska et al., Drug Delivery Systems for RNA Therapeutics, 2022, Nature Reviews Genetics, 23(5):265-280; Chen et al., 2022, Molecular Therapy, Nucleic Acids, 29:150-160).

[0192] Lipid-based particles can form specific structures, such as micelles, liposomes, and lipid nanoparticles (LPNs), to deliver oligomeric compounds into cells. To form these particles, LPNs may contain one or more cationic or ionizable lipids (e.g., DLin-MC3-DMA, SM-102, or ALC-0315), cholesterol, co-lipids, 1,2-distearoyl-sn-glycero-3-phosphatylcholine (DSPC), poly(ethylene glycol) (PEG)-modified lipids (e.g., PEG-2000-C-DMG, PEG-2000-DMG, or ALC-0159), C12-200, cKK-E12, etc. Different combinations of lipids can be formulated to deliver oligomeric compounds to different types of cells. In one example, the therapeutic siRNA patisiran was formulated in the cationic ionizable lipid DLin-MC3-DMA, cholesterol, the polar phospholipid DSPC, and PEG-2000-C-DMG for delivery to hepatocytes.

[0193] Polymer-based particles have also been used as delivery systems for oligomeric compounds. Such polymers include poly(lactic-co-glycolic acid) (PLGA), polyethyleneimine (PEI), poly(l-lysine) (PLL), poly(β-amino ester) (PBAE), dendrimers (e.g., poly(amidoamine) (PAMAM) or PLL), and other polymers or modified polymers thereof. The polymer composition can be varied depending on the properties desired for the delivery of the oligomeric compound.

[0194] The oligomeric compounds disclosed herein may be covalently linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the resulting compound. Conjugates may include cholesterol, lipids, carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, dyes, tocopherol (Nishina et al., 2008, Molecular Therapy, 16(4):734-740), and the like. Conjugate-based delivery can actively deliver oligomeric compounds to specific cell types.

[0195] As an example, N-acetylgalactosamine (GalNAc) is conjugated to an oligomeric compound and delivered to hepatocytes. Various GalNAc conjugates are described in several publications, all of which are incorporated herein by reference, including Sharma et al. 2018 Bioconjugate Chem 29:2478-2488, Nair et al. J. Am. Chem. Soc. 2014 136(49):16958-16961, Keam 2022 Drugs 82:1419-1425, U.S. Patent No. 10,087,208, Prakash et al. 2014 Nucleic Acids Res 42(13):8796-8807, and Debacker et al. 2020 Molecular Therapy. 28(8):1759-1771, U.S. Patent No. 11110174, U.S. Patent No. 9796756, U.S. Patent No. 9181549, U.S. Patent No. 10344275, U.S. Patent No. 10570169, U.S. Patent No. 9506030, and U.S. Patent No. 7582744.

[0196] As yet another example, the following GalNAc is attached to the 3' end of an oligonucleotide, which comprises the sense strand of an ARNATAR-designed siRNA.

[0197] [ka]

[0198] Synthesis of oligomeric compounds The siRNAs were designed, synthesized and prepared using methods known in the art.

[0199] Solid phase synthesis of oligonucleotides is performed by MerMade TMThe synthesis was performed using a 48x synthesizer (BioAutomation, LGC, Biosearch Technologies, Hoddesdon, UK). This synthesizer is capable of synthesizing up to 48 oligonucleotides in a single run at 1 μmole or 5 μmole scales using standard phosphoramidite chemistry. The solid supports were either controlled pore glass (500-1400 Å) or universal solid supports (AM Chemicals, Vista, CA, USA) bearing 3'-GalNAc conjugates (AM Chemicals, Vista, CA, USA; Primetech ALC, Minsk, Belarus; Gene Link, Elmsford, NY, USA; or any of the GalNAc conjugates disclosed herein). Accompanying synthetic reagents and standard 2'-cyanoethyl phosphoramidite monomers (2'-fluoronucleosides, 2'-O-methylnucleosides, RNA nucleosides, and DNA nucleosides) were obtained from multiple sources (Hongene Biotech, Shanghai, China; Sigma-Aldrich, St. Louis, MO, USA; Glen Research, Sterling, VA, USA; ThermoFisher Scientific, Waltham, MA, USA; and LGC Biosearch Technologies, Hoddesdon, UK). Phosphoramidite mixtures were prepared in anhydrous acetonitrile or 30% DMF:acetonitrile and coupled with 0.25 M 4,5-dicyanoimidazole (DCI) (Sigma-Aldrich, St. Louis, MO, USA). Coupling times ranged from 120 to 360 seconds. Standard phosphodiester bonds were formed using a mixture of 0.02 M iodine in tetrahydrofuran (THF), pyridine, and water. Phosphorothioate bonds were generated using 0.05 M sulfurization reagent II (3-((dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT) in 40:60 pyridine / acetonitrile (LGC Biosearch Technologies, Hoddesdon, UK) with an oxidation time of 6 min. All sequences were synthesized with the dimethoxytrityl (DMT) protecting group removed.

[0200] After solid-phase synthesis was completed, the oligonucleotides were cleaved from the solid support, and deprotection of base-labile groups was carried out by incubation in aqueous ammonia at 55°C for 6 hours. The aqueous ammonia solution was removed to dryness using a centrifugal vacuum concentrator at room temperature. For sequences containing tert-butyldimethylsilyl (TBDMS)-protected natural ribonucleotides (2'-OH), secondary deprotection was carried out using triethylamine trihydrofluoride (TEA:3HF). 100 μL of DMSO and 125 μL of TEA:3HF were added to each TBDMS-protected oligonucleotide and incubated at 65°C for 2.5 hours. After incubation, 25 μL of 3 M sodium acetate was added to the solution, followed by precipitation in butanol at -20°C for 30 minutes. The cloudy solution was centrifuged, and when it formed a cake, the supernatant was carefully decanted with a pipette. A standard precipitation process was completed using 75% ethanol:water followed by 100% ethanol as the supernatant solution. The oligonucleotide cake was dried in a centrifugal vacuum concentrator for 30 minutes.

[0201] Desalting without HPLC purification was performed by precipitation with 3 M sodium acetate followed by elution through a G25 Sephadex® column (Sigma-Aldrich, St. Louis, MO, USA). Oligonucleotide purification was performed by anion-exchange chromatography using BioWorks Q40 resin (Uppsala, Sweden) on a Gilson GX271 prep HPLC system (Middleton, WI, USA). Final desalting was performed using a Sephadex® G25 column. All oligonucleotides were analyzed by ion-pair reversed-phase HPLC to confirm purity, which was performed using an Agilent 1200 analytical HPLC (Santa Clara, CA, USA). Complete mass confirmation was performed by negative ion mass spectrometry on an Agilent 6130 Single Quad Mass Spectrometer (Santa Clara, CA, USA), and A260 quantification was performed by UV / Vis measurement on a Tecan Infinite® M Plex Pre-reader (Zurich, Switzerland).

[0202] Double-stranded oligomers - double-stranded formation of compounds Typically, in double-stranded oligomeric compounds, such as siRNA compounds, sense and antisense oligonucleotides are annealed together to form duplexes. Formation of 50-300 μM duplexes is achieved by heating a sample in 1x phosphate-buffered saline in a block heater at 94°C for 4 minutes, then removing the heating block containing the sample from the block heater and allowing it to gradually cool to room temperature over 1 hour.

[0203] Compositions and methods for preparing pharmaceutical compositions The oligomeric compounds of the invention, e.g., the siRNA compounds described herein, can be combined with pharmaceutically acceptable active or inactive substances, such as diluents, excipients, or carriers, to prepare pharmaceutical compositions or formulations.

[0204] The compositions and methods for preparing pharmaceutical compositions depend on several criteria, including, but not limited to, the route of administration, the stage of the disease, or the dose to be administered.

[0205] In certain embodiments, a pharmaceutical carrier or excipient is a pharmaceutically acceptable solvent, suspending agent, or other pharmacologically inert carrier for delivering one or more nucleic acid compounds to an animal. Excipients can be liquid or solid and can be selected to provide the desired bulk, viscosity, etc., when combined with the nucleic acids and other components of a particular pharmaceutical composition, taking into account the intended method of administration. Representative pharmaceutical carriers include, but are not limited to, binders (such as, for example, pregelatinized corn starch, polyvinylpyrrolidone, and / or hydroxypropylmethylcellulose), fillers (such as, for example, lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, and / or calcium hydrogen phosphate), lubricants (such as, for example, magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, and / or sodium acetate), disintegrants (such as, for example, starch and / or sodium starch glycolate), and wetting agents (such as, for example, sodium lauryl sulfate).

[0206] Pharmaceutically acceptable organic or inorganic excipients suitable for injectable or non-injectable administration that do not adversely react with nucleic acid compounds can also be used to prepare the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is a suitable diluent for use in compositions delivered parenterally. Thus, in one embodiment of the methods described herein, a pharmaceutical composition comprising an oligomeric compound and a pharmaceutically acceptable diluent is used. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the oligomeric compound is siRNA.

[0207] Pharmaceutical compositions containing oligomeric compounds such as siRNAs may contain any pharmaceutically acceptable salts, esters, or salts of such esters or other double-stranded RNAs that can provide (directly or indirectly) biologically active metabolites or residues thereof when administered to animals, including humans. Thus, for example, the present disclosure also relates to pharmaceutically acceptable salts of oligomeric compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0208] In certain embodiments, the pharmaceutical composition is prepared for administration by injection (e.g., intravenously, subcutaneously, and / or intramuscularly, etc.). In some such embodiments, the pharmaceutical composition includes a carrier and is formulated in an aqueous solution such as water or Hanks' solution, Ringer's solution, or physiological saline buffer (e.g., PBS). In certain embodiments, other ingredients are included (e.g., ingredients that aid solubility or act as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents, etc. Some injectable pharmaceutical compositions are provided in unit dosage form, for example, in ampoules or multi-dose containers.

[0209] Dosage For purposes of this disclosure, the amount or dose of the active ingredient (oligomeric compound of the invention) administered should be sufficient to inhibit expression of the target nucleic acid in, for example, an animal. In animals (e.g., humans), the dosage will be determined by the efficacy of the particular active ingredient, the condition of the animal, and the body weight of the animal to be treated.

[0210] Many assays for determining dosage are known in the art.

[0211] The dosage of the active ingredients of the present disclosure will also be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular active ingredient of the present disclosure. Typically, the attending physician will determine the dosage of the active ingredients of the present disclosure for each patient, taking into account various factors such as age, weight, general health, diet, sex, the active ingredient of the present disclosure to be administered, the route of administration, and the severity of the disease being treated.

[0212] dosage In certain embodiments, the pharmaceutical composition is administered according to a dosing regimen (e.g., dosage, frequency, and duration) that can be selected to achieve a desired effect. The desired effect can be, for example, reduction of the target nucleic acid, or prevention, alleviation, amelioration, or inhibition of progression of a disease, disorder, and / or condition associated with the target nucleic acid, or a symptom thereof. In certain embodiments, the variables of the dosing regimen are adjusted to achieve a desired concentration of the pharmaceutical composition in the subject. The "concentration of the pharmaceutical composition" as used with respect to a dosing regimen can refer to the oligomeric compound or active ingredient in the pharmaceutical composition. For example, in certain embodiments, the dosage and frequency of administration are adjusted to provide a tissue or plasma concentration of the pharmaceutical composition sufficient to achieve the desired effect.

[0213] Dosing depends on the severity and responsiveness of the condition being treated, with treatment duration ranging from several days to several months, or until a cure is achieved or a reduction in symptoms is achieved. Dosing also depends on the potency and metabolism of the drug. In certain embodiments, dosages range from 0.01 μg to 50 mg per kg of body weight, 0.01 μg to 100 mg per kg of body weight, or 0.001 mg to 1000 mg per kg of body weight, and may be administered daily, weekly, monthly, quarterly, or once or more times per year, or once every two to twenty years. After successful treatment, it may be desirable for patients to undergo maintenance therapy to prevent recurrence of the condition. In this case, the oligomeric compound is administered at a maintenance dose ranging from 0.01 μg to 100 mg per kg of body weight, once or more times per day, once or more times per week, once or more times per month, once or more times per quarter, once or more times per year, up to once every twenty years, or between 0.001 mg and 1000 mg per kg of body weight. In certain embodiments, it may be desirable to administer the oligomeric compound at most once a day, once a week, once a month, once a quarter, once a year, once every two years, once every three years, once every four years, once every five years, once every ten years, or once every twenty years.

[0214] In certain embodiments, the dosage range is any of the following: 1 mg to 1500 mg, 100 mg to 1400 mg, 100 mg to 1300 mg, 100 mg to 1200 mg, 100 mg to 1100 mg, 100 mg to 1000 mg, 100 mg to 900 mg, 200 mg to 800 mg, 300 mg to 700 mg, 400 mg to 600 mg, 100 mg to 400 mg, 200 mg to 500 mg, 300 mg to 600 mg, and 400 mg to 700 mg. In certain embodiments, the dose is about 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, or 1500 mg.

[0215] In certain embodiments, dsRNA is administered twice a year at any dose of about 150mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg or 900mg.In certain embodiments, dsRNA is administered every quarter at any dose of about 150mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg or 900mg.

[0216] Administration The oligomeric compounds (e.g., siRNAs) and pharmaceutical compositions of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be oral, inhaled, or parenteral.

[0217] In certain embodiments, the compounds and compositions described herein are administered parenterally. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion. It also includes intracranial, e.g., intrathecal or intraventricular, administration. In certain embodiments, parenteral administration is performed by infusion. Infusion can be chronic, continuous, short-term, or intermittent. In certain embodiments, pharmaceutical ingredients administered by infusion are delivered by a pump.

[0218] In certain embodiments, parenteral administration is by injection. The injection can be by syringe or pump. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly into a tissue or organ.

[0219] In certain embodiments, formulations for parenteral, intrathecal, or intraventricular administration may comprise sterile aqueous solutions, which may contain suitable additives such as buffers, diluents, and penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0220] In certain embodiments, formulations for oral administration of a compound or composition include, but are not limited to, pharmaceutical carriers, excipients, powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets, etc. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. In certain embodiments, oral formulations are those in which a compound described herein is administered with one or more penetration enhancers, surfactants, and chelating agents.

[0221] In vitro testing of siRNA The methods described herein are for treating cells with siRNA and can be appropriately modified for treatment with other oligomeric compounds.

[0222] When cells reach approximately 60-80% confluency in culture, they can be treated with siRNA.

[0223] One reagent commonly used to introduce siRNA into cultured cells is the cationic lipid transfection reagent Lipofectamine™ RNAiMAX (Invitrogen, Waltham, Massachusetts). siRNA was transfected in OPTI-MEM 1 (Thermo Fisher Scientific, Waltham, Massachusetts) with Lipofectamine. TM Mix with RNAiMAX siRNA and Lipofectamine at the desired final concentration TM The maximum concentration of RNAiMAX (0.001-300 nM siRNA) can be achieved. The transfection procedure is carried out according to the manufacturer's recommended protocol.

[0224] Another technique used to introduce siRNA into cultured cells is electroporation.

[0225] GalNAc-conjugated siRNA can be introduced into cells by incubating the siRNA with cells without using a transfection reagent, a process referred to herein as "free uptake." The siRNA-GalNAc conjugate is transported via endocytosis into asialoglycoprotein receptor (ASGR)-positive cells, such as hepatocytes.

[0226] Cells are treated with siRNA in the usual manner. Cells are harvested 4-144 hours after siRNA treatment, at which time the mRNA (harvested 4-144 hours) or protein levels (extracted 24-96 hours) of the target nucleic acid are measured by methods known in the art and described herein. Treatments are generally performed in multiple replicates, and data are presented as the mean and standard deviation of the replicates.

[0227] The concentration of siRNA used varies depending on the cell line and target. Methods for determining the optimal siRNA concentration for a particular target in a particular cell line are well known in the art. Generally, cells are treated with siRNA in a dose-dependent manner, allowing calculation of the half-maximal inhibitory concentration (IC50). siRNA is usually administered via Lipofectamine. TM For transfection using RNAiMAX, siRNA is used at a concentration ranging from 0.001 nM to 300 nM. For transfection by electroporation or free uptake, siRNA is used at higher concentrations ranging from 7.5 nM to 20,000 nM.

[0228] RNA isolation RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. RNA isolation methods are well known in the art. RNA is prepared using methods well known in the art, such as TRIZOL Reagent (Thermo Fisher Scientific, Waltham, MA), the Qiagen RNeasy kit (Qiagen, Hilden, Germany), or AcroPrep Advance 96-well filter plates (Pall Corporation, Port Washington, NY) using Qiagen's RLT, RW1, and RPE buffers. RNA extraction procedures are performed according to the manufacturer's recommended protocol.

[0229] In vivo testing of oligomeric compounds Oligomeric compounds of the present invention, e.g., siRNAs, are tested in animals to assess their ability to inhibit expression of a target nucleic acid and induce a phenotypic change, such as a change in one or more markers affected by the target nucleic acid. The phenotypic change may also be a reduction in a disease, disorder, condition, or symptom associated with the target nucleic acid. Testing can be performed in normal animals or experimental disease models. For administration to animals, the oligomeric compounds are suspended in a pharmaceutically acceptable diluent, such as phosphate-buffered saline (PBS). Administration can be via parenteral routes, such as intraperitoneal, intravenous, or subcutaneous administration. Calculation of dosage and frequency depends on factors such as the route of administration and the animal's body weight. In one embodiment, after a period of treatment with an oligomeric compound of the present invention, RNA encoding the target nucleic acid is isolated from liver tissue, and changes in expression of the target nucleic acid are measured. Changes in the levels of the protein expressed by the target nucleic acid can also be measured.

[0230] Kits of the Invention According to another aspect of the present invention, a kit is provided. The kit according to the present invention comprises a package containing any of the compositions of the present invention or any of the oligomeric compounds of the present invention. In each aspect of the present invention, the kit contains any of the compositions of the present invention as a single dose. As used herein, a "single dose" refers to a discrete amount dispersed in a suitable carrier.

[0231] The term "package" refers to any container that contains a composition presented herein. In a preferred embodiment, the package may be a box or wrapper. Packaging materials used to package pharmaceutical products are well known to those skilled in the art. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes (including prefilled syringes), bottles, and any packaging material appropriate for the selected formulation and intended mode of administration / treatment.

[0232] The kit may also include items not contained within the package but attached to the outside of the package, such as, for example, a pipette.

[0233] The kit may include instructions for administering the composition of the present invention to a subject having a condition in need of treatment. The kit may include instructions for use of the components of the composition herein approved by a regulatory agency, such as the U.S. Food and Drug Administration. The kit may include a label or product insert for the composition, if desired. The packaging and / or product insert may be approved by a regulatory agency. The kit may include a solid phase or liquid phase composition (e.g., a provided buffer) within the package. The kit may also include buffers for preparing solutions for carrying out the method, as well as pipettes for transferring liquids from one container to another.

[0234] The kit may also include one or more other compositions for use in the combination therapy described herein. In certain embodiments, the package is a container for any of the following administration means: intraocular, intraocular, intratumoral, peritumoral, intraperitoneal, intrathecal, intramuscular, subcutaneous, intravenous, intraarterial, intraventricular, intrasternal, intracranial, or intradermal injection.

[0235] How to use The present invention provides a method for inhibiting expression of a target nucleic acid in a subject, the method comprising inhibiting expression of a target nucleic acid in the subject by administering an effective amount of an oligomeric compound of the invention or a pharmaceutical composition of the invention.

[0236] In some embodiments of the present disclosure, the subject is a mammal, including, but not limited to, rodents such as mice and hamsters, lagomorphs such as rabbits, carnivora including cats (Felidae) and dogs (Canidae), artiodactyla including cats (Bovidae) and pigs (Porcineae), or perissodactyla including horses (Equidae). In some aspects, the mammal is from the order Primates, Ceboids, or Simoids (monkeys), or the order Anthropoids (humans and apes). In preferred aspects, the mammal is a human.

[0237] Specific Embodiments Embodiment 1 of the present invention includes an oligomeric compound capable of inhibiting expression of a target nucleic acid, comprising a sense strand having 19 to 23 linked nucleotides, wherein the sense strand sequence is represented by formula (I). 5' M-(Y)nZ-(Y)rDD 3' where: D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside; Y is two adjacent nucleosides having different modifications or a modified nucleoside adjacent to an unmodified nucleoside; Z is two adjacent nucleosides having the same modification or two adjacent unmodified nucleosides; n is 6 to 8, r is 1 to 2, No single modification type may modify more than two consecutive nucleotides.

[0238] The oligomeric compound of embodiment 1, wherein -DD is an overhang of TT or TA.

[0239] The oligomeric compound of embodiment 1, wherein the nucleotide modifications are selected from the group consisting of deoxyribonucleosides substituted with ribonucleosides (DNA nucleosides), locked nucleic acids (LNA), 2'-OMe, 2'-F, unlocked nucleic acids (UNA), and combinations thereof.

[0240] 2. The oligomeric compound of embodiment 1, wherein the sense strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0241] The oligomeric compound of embodiment 1 is one in which the sense strand contains phosphorothioate internucleotide (PS) linkages adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand.

[0242] Embodiment 2 of the present invention includes an oligomeric compound capable of inhibiting expression of a target nucleic acid, comprising an antisense strand having 19 to 23 linked nucleotides, the sequence of the antisense strand being represented by formula (IV). 5' LM-(D)v-(Y)s-(Z)t-(Y)uZN-(Z)r 3' where: D is a deoxyribonucleoside, N is a modified or unmodified nucleoside; M is a 2'-OMe modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; Y is two adjacent nucleosides having different modifications or a modified nucleoside adjacent to an unmodified nucleoside; Z is two adjacent nucleosides having the same modification or two adjacent unmodified nucleosides; r is 1 to 2, v is between 0 and 1, s is 2 to 7, t is between 0 and 2, u is 0 to 5, No single modification type may modify more than two consecutive nucleotides.

[0243] The oligomeric compound of embodiment 2, wherein 3'Z is an overhang of UU or TT.

[0244] The oligomeric compound of embodiment 2, wherein the nucleotide modifications are selected from the group consisting of deoxyribonucleosides substituted with ribonucleosides (DNA nucleosides), locked nucleic acids (LNA), 2'-OMe, 2'-F, unlocked nucleic acids (UNA), and combinations thereof.

[0245] The oligomeric compound of embodiment 2, wherein the antisense strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0246] The oligomeric compound of embodiment 2, wherein the antisense strand contains phosphorothioate (PS) type internucleotide bond modifications between positions 1-2, 2-3, 19-20, and 20-21, counting from the 5' end of the antisense strand.

[0247] The oligomeric compound of embodiment 2 is one in which the antisense strand contains phosphorothioate internucleotide (PS) linkages adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand.

[0248] Embodiment 3 of the present invention provides an oligomeric compound capable of inhibiting expression of a target nucleic acid, comprising: a) a sense strand having 19 to 23 linked nucleotides and having a sense strand sequence represented by formula (I); 5' M-(Y)nZ-(Y)rDD 3', b) an antisense strand having 19 to 23 linked nucleotides and having an antisense strand sequence represented by formula (IV); 5' LM-(D)v-(Y)s-(Z)t-(Y)uZN-(Z)r 3', c) comprising a double-stranded entity formed by the sense strand and the antisense strand; where: D is a deoxyribonucleoside, N is a modified or unmodified nucleoside; M is a 2'-OMe modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; Y is two adjacent nucleosides having different modifications or a modified nucleoside adjacent to an unmodified nucleoside; Z is two adjacent nucleosides having the same modification or two adjacent unmodified nucleosides; n is 6 to 8, r is 1 to 2, v is between 0 and 1, s is 2 to 7, t is between 0 and 2, u is 0 to 5, the double-stranded region has a length of 19 to 23 nucleotide pairs; No single modification type may modify more than two consecutive nucleotides.

[0249] The oligomeric compound of embodiment 3, wherein 3'Z of the antisense strand is an overhang of UU or TT.

[0250] The oligomeric compound of embodiment 3, wherein -DD of the sense strand is an overhang of TT or TA.

[0251] The oligomeric compound of embodiment 3, wherein the nucleotide modifications are selected from the group consisting of deoxyribonucleosides substituted with ribonucleosides (DNA nucleosides), locked nucleic acids (LNA), 2'-OMe, 2'-F, and unlocked nucleic acids (UNA), or combinations thereof.

[0252] The oligomeric compound of embodiment 3, wherein each strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0253] The oligomeric compound of embodiment 3, wherein the antisense strand contains phosphorothioate (PS) type internucleotide bond modifications between positions 1-2, 2-3, 19-20, and 20-21, counting from the 5' end of the antisense strand.

[0254] The oligomeric compound of embodiment 3 is one in which the strands contain phosphorothioate internucleotide (PS) linkages adjacent to the two nucleosides at the 5' end of the strand and / or adjacent to the two nucleosides at the 3' end of the strand.

[0255] Embodiment 4 of the present invention includes an oligomeric compound capable of inhibiting expression of a target nucleic acid, comprising a sense strand having 19 to 23 linked nucleotides, wherein the sense strand sequence is represented by formula (II): 5' YZ-(Y)q-FFNM-(Y)qM-(Y)vDD 3' where: D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; Y is two adjacent nucleosides having different modifications or a modified nucleoside adjacent to an unmodified nucleoside; Z is two adjacent nucleosides having the same modification or two adjacent unmodified nucleosides; q is 2 to 3, v is between 0 and 1, No single modification type may modify more than two consecutive nucleotides.

[0256] The oligomeric compound of embodiment 4, wherein -DD is an overhang of TT or TA.

[0257] The oligomeric compound of embodiment 4, wherein the nucleotide modifications are selected from the group consisting of deoxyribonucleosides substituted with ribonucleosides (DNA nucleosides), locked nucleic acids (LNA), 2'-OMe, 2'-F, unlocked nucleic acids (UNA), and combinations thereof.

[0258] The oligomeric compound of embodiment 4, wherein the sense strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0259] The oligomeric compound of embodiment 4 is one in which the sense strand contains phosphorothioate internucleotide (PS) linkages adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand.

[0260] A fifth embodiment of the present invention comprises an oligomeric compound capable of inhibiting expression of a target nucleic acid, comprising an antisense strand having 19 to 23 linked nucleotides, the sequence of which is represented by formula (V). 5' L-(Y)p-NM-(FMM)r-(Y)p-(Z)r 3' where: M is a 2'-OMe modified nucleoside; F is a 2'-F modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; Y is two adjacent nucleosides having different modifications or a modified nucleoside adjacent to an unmodified nucleoside; Z is two adjacent nucleosides having the same modification or two adjacent unmodified nucleosides; p is 3 to 5; r is 1 to 2, No single modification type may modify more than two consecutive nucleotides.

[0261] The oligomeric compound of embodiment 5, wherein 3'Z is an overhang of UU or TT.

[0262] The oligomeric compound of embodiment 5, wherein the nucleotide modifications are selected from the group consisting of deoxyribonucleosides substituted with ribonucleosides (DNA nucleosides), locked nucleic acids (LNA), 2'-OMe, 2'-F, unlocked nucleic acids (UNA), and combinations thereof.

[0263] 6. The oligomeric compound of embodiment 5, wherein the antisense strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0264] The oligomeric compound of embodiment 5, wherein the antisense strand comprises internucleotide bond modifications of phosphorothioate (PS) type between positions 1-2, 2-3, 19-20 and 20-21 counting from the 5' end of the antisense strand.

[0265] The oligomeric compound of embodiment 5 is one in which the antisense strand contains phosphorothioate internucleotide (PS) linkages adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand.

[0266] Embodiment 6 of the present invention provides an oligomeric compound capable of inhibiting expression of a target nucleic acid, comprising: a) a sense strand having 19 to 23 linked nucleotides and having a sense strand sequence represented by formula (II), 5' YZ-(Y)q-FFNM-(Y)qM-(Y)vDD 3', b) an antisense strand having 19 to 23 linked nucleotides and having an antisense strand sequence represented by formula (V); 5' L-(Y)p-NM-(FMM)-(Y)p-(Z)r 3', c) comprising a double-stranded entity formed by the sense strand and the antisense strand; where: D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; Y is two adjacent nucleosides having different modifications or a modified nucleoside adjacent to an unmodified nucleoside; Z is two adjacent nucleosides having the same modification or two adjacent unmodified nucleosides; q is 2 to 3, p is 3 to 5; r is 1 to 2, v is between 0 and 1, the double-stranded region has a length of 19 to 23 nucleotide pairs; No single modification type may modify more than two consecutive nucleotides.

[0267] The oligomeric compound of embodiment 6, wherein 3'Z of the antisense strand is an overhang of UU or TT.

[0268] The oligomeric compound of embodiment 6, wherein -DD of the sense strand is an overhang of TT or TA.

[0269] The oligomeric compound of embodiment 6, wherein the modification of the nucleotide is selected from the group consisting of deoxyribonucleosides substituted with ribonucleosides (DNA nucleosides), locked nucleic acids (LNA), 2'-OMe, 2'-F, unlocked nucleic acids (UNA), and combinations thereof.

[0270] The oligomeric compound of embodiment 6, wherein each strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0271] The oligomeric compound of embodiment 6, wherein the antisense strand comprises internucleotide bond modifications of the phosphorothioate (PS) type between positions 1-2, 2-3, and 20-21, counting from the 5' end of the antisense strand.

[0272] The oligomeric compound of embodiment 6 is one in which the strands contain phosphorothioate internucleotide (PS) linkages adjacent to the two nucleosides at the 5' end of the strand and / or adjacent to the two nucleosides at the 3' end of the strand.

[0273] Embodiment 7 of the present invention comprises an oligomeric compound capable of inhibiting expression of a target nucleic acid, comprising an antisense strand having 19 to 23 linked nucleotides, the sequence of the antisense strand being represented by formula (IX). 5' LM-(Y)pZ-(Y)p-(Z)r 3', where: M is a 2'-OMe modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; Y is two adjacent nucleosides having different modifications or a modified nucleoside adjacent to an unmodified nucleoside; Z is two adjacent nucleosides having the same modification or two adjacent unmodified nucleosides; n is 6 to 8, r is 1 to 2, No single modification type may modify more than two consecutive nucleotides.

[0274] The oligomeric compound of embodiment 7, wherein 3'Z is an overhang of UU or TT.

[0275] The oligomeric compound of embodiment 7, wherein the modification of the nucleotide is selected from the group consisting of deoxyribonucleosides substituted with ribonucleosides (DNA nucleosides), locked nucleic acids (LNA), 2'-OMe, 2'-F, unlocked nucleic acids (UNA), and combinations thereof.

[0276] 8. The oligomeric compound of embodiment 7, wherein the antisense strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0277] The oligomeric compound of embodiment 7, wherein the antisense strand comprises internucleotide bond modifications of phosphorothioate (PS) type between positions 1-2, 2-3, 19-20 and 20-21 counting from the 5' end of the antisense strand.

[0278] The oligomeric compound of embodiment 7 is one in which the antisense strand contains phosphorothioate internucleotide (PS) linkages adjacent to the two nucleosides at the 5' end of the strand and / or the two nucleosides at the 3' end of the strand.

[0279] Embodiment 8 of the present invention provides an oligomeric compound capable of inhibiting expression of a target nucleic acid, comprising: a) a sense strand having 19 to 23 linked nucleotides and having a sense strand sequence represented by formula (I); 5' M-(Y)nZ-(Y)rDD 3', b) an antisense strand having 19 to 23 linked nucleotides and having an antisense strand sequence represented by formula (IX); 5' LM-(Y)pZ-(Y)p-(Z)r 3', c) comprising a double-stranded entity formed by the sense strand and the antisense strand; where: D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; Y is two adjacent nucleosides having different modifications or a modified nucleoside adjacent to an unmodified nucleoside; Z is two adjacent nucleosides having the same modification or two adjacent unmodified nucleosides; n is 6 to 8, p is 3 to 5; r is 1 to 2, the double-stranded region has a length of 19 to 23 nucleotide pairs; No single modification type may modify more than two consecutive nucleotides.

[0280] The oligomeric compound of embodiment 8, wherein 3'Z of the antisense strand is an overhang of UU or TT.

[0281] The oligomeric compound of embodiment 8, wherein -DD of the sense strand is an overhang of TT or TA.

[0282] The oligomeric compound of embodiment 8, wherein the nucleotide modifications are selected from the group consisting of deoxyribonucleosides substituted with ribonucleosides (DNA nucleosides), locked nucleic acids (LNA), 2'-OMe, 2'-F, and unlocked nucleic acids (UNA), or combinations thereof.

[0283] The oligomeric compound of embodiment 8, wherein each strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0284] The oligomeric compound of embodiment 8, wherein the antisense strand comprises internucleotide bond modifications of phosphorothioate (PS) type between positions 1-2, 2-3, 19-20 and 20-21 counting from the 5' end of the antisense strand.

[0285] The oligomeric compound of embodiment 8 is one in which the strands contain phosphorothioate internucleotide (PS) linkages adjacent to the two nucleosides at the 5' end of the strand and / or adjacent to the two nucleosides at the 3' end of the strand.

[0286] Embodiment 9 of the present invention comprises an oligomeric compound capable of inhibiting expression of a target nucleic acid, comprising a sense strand having 21 linked nucleotides, wherein the sense strand sequence is represented by formula (X). 5' MFMMNMNMFFNMNMNMNMNMDD 3', where: D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; No single modification type may modify more than two consecutive nucleotides. The oligomeric compound of embodiment 9, wherein N can be a ribonucleoside (R), a deoxyribonucleoside (D), 2'-OMe, 2'-MOE, 2'-F, an unlocked nucleic acid (UNA) or a locked nucleic acid (LNA).

[0287] The oligomeric compound of embodiment 9, wherein each strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0288] Embodiment 10 of the present invention comprises an oligomeric compound capable of inhibiting expression of a target nucleic acid, comprising an antisense strand having 21 linked nucleotides, the sequence of the antisense strand being represented by formula (VIII). 5' L-MNMNMFNMMFMMNMFMFMMNMM 3', where: D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; No single modification type may modify more than two consecutive nucleotides.

[0289] The oligomeric compound of embodiment 10, wherein N can be a ribonucleoside (R), a deoxyribonucleoside (D), 2'-OMe, 2'-MOE, 2'-F, an unlocked nucleic acid (UNA) or a locked nucleic acid (LNA).

[0290] 11. The oligomeric compound of embodiment 10, wherein each strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0291] Embodiment 11 of the present invention relates to an oligomeric compound capable of inhibiting expression of a target nucleic acid, comprising: a) a sense strand having 21 linked nucleotides and having a sense strand sequence represented by formula (X), 5' MFMMNMNMFFNMNMNMNMNMDD 3', b) an antisense strand having 21 linked nucleotides and having an antisense strand sequence represented by formula (VIII); 5' L-MNMNMFNMMFMMNMFMFMMNMM 3', c) comprising a double-stranded entity formed by the sense strand and the antisense strand; where: D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; the double-stranded region is 19 nucleotide pairs in length; Each strand has a two-nucleotide overhang at the 3' end, No single modification type may modify more than two consecutive nucleotides.

[0292] The oligomeric compound of embodiment 11, wherein N can be a ribonucleoside (R), a deoxyribonucleoside (D), 2'-OMe, 2'-MOE, 2'-F, an unlocked nucleic acid (UNA) or a locked nucleic acid (LNA).

[0293] 12. The oligomeric compound of embodiment 11, wherein each strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0294] Embodiment 12 of the present invention comprises an oligomeric compound capable of inhibiting expression of a target nucleic acid, comprising a sense strand having about 21 linked nucleotides, wherein the sense strand sequence is represented by formula (XI). 5' MFMMNMNMFFMMNMNMMFMDD 3', where: D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; No single modification type may modify more than two consecutive nucleotides.

[0295] The oligomeric compound of embodiment 12, wherein N can be a ribonucleoside (R), a deoxyribonucleoside (D), 2'-OMe, 2'-MOE, 2'-F, an unlocked nucleic acid (UNA) or a locked nucleic acid (LNA).

[0296] 13. The oligomeric compound of embodiment 12, wherein each strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0297] Embodiment 13 of the present invention comprises an oligomeric compound capable of inhibiting expression of a target nucleic acid, comprising an antisense strand having about 21 linked nucleotides, the sequence of the antisense strand being represented by formula (XII). 5' L-MDMFMFNMFMMFMFMFMMNMM 3', where: D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; No single modification type may modify more than two consecutive nucleotides.

[0298] The oligomeric compound of embodiment 13, wherein N can be a ribonucleoside (R), a deoxyribonucleoside (D), 2'-OMe, 2'-MOE, 2'-F, an unlocked nucleic acid (UNA) or a locked nucleic acid (LNA).

[0299] 14. The oligomeric compound of embodiment 13, wherein each strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0300] Embodiment 14 of the present invention relates to an oligomeric compound capable of inhibiting or inhibiting expression of a target nucleic acid, comprising: a) a sense strand having 21 linked nucleotides and having a sense strand sequence represented by formula (XI), 5' MFMMNMNMFFMMNMNMMFMDD 3', b) an antisense strand having 21 linked nucleotides and having an antisense strand sequence represented by formula (XII); 5' L-MDMFMFNMFMMFMFMFMMNMM 3', c) comprising a double-stranded entity formed by the sense strand and the antisense strand; where: D is a deoxyribonucleoside, M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; the double-stranded region is 19 nucleotide pairs in length; Each strand has a two-nucleotide overhang at the 3' end, No single modification type may modify more than two consecutive nucleotides.

[0301] The oligomeric compound of embodiment 14, wherein N can be a ribonucleoside (R), a deoxyribonucleoside (D), 2'-OMe, 2'-MOE, 2'-F, an unlocked nucleic acid (UNA) or a locked nucleic acid (LNA).

[0302] 15. The oligomeric compound of embodiment 14, wherein each strand comprises at least one phosphorothioate internucleotide (PS) linkage, and the phosphorothioate internucleotide (PS) linkage is adjacent to a deoxyribonucleoside (D) or a ribonucleoside (R).

[0303] The oligomeric compound of any of the preceding embodiments, wherein the FFNM motif appears at or near the cleavage site of the sense strand, and optionally, the FFNM is FFMM or FFRM, F is 2'-fluoro, N is a modified or unmodified nucleoside, R is a ribonucleoside, and M is 2'-OMe.

[0304] The oligomeric compound of any of the previous embodiments, wherein the FMM motif appears at or near the cleavage site in the antisense strand, where F is 2'-fluoro and M is 2'-OMe.

[0305] The oligomeric compound of any of the previous embodiments, wherein the oligomeric compound is single-stranded or double-stranded.

[0306] The oligomeric compound of any of the previous embodiments, wherein the single-stranded oligomeric compound is a single-stranded oligonucleotide, a microRNA (miRNA), or a single-stranded RNA (ssRNA).

[0307] The oligomeric compound of any of the previous embodiments, wherein the double-stranded oligomeric compound is an shRNA or an siRNA.

[0308] The oligomeric compound of any of the previous embodiments further comprises a conjugate. The conjugate may be selected from cholesterol, lipids, carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, peptides, and dyes. In a preferred embodiment, the conjugate is N-acetylgalactosamine (GalNAc). The conjugate may be attached to the 3' end of the sense strand of the oligomeric compound.

[0309] The oligomeric compound of any of the preceding embodiments, wherein the compound inhibits expression of the target nucleic acid by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%.

[0310] One embodiment of the present invention includes a pharmaceutical composition comprising an oligomeric compound of any of the preceding embodiments, alone or in combination with a pharmaceutically acceptable carrier or excipient.

[0311] One embodiment of the present invention includes a method of inhibiting expression of a target nucleic acid in a subject, the method comprising administering to the subject an oligomeric compound of any of the preceding embodiments in an amount sufficient to inhibit expression of the target nucleic acid. The oligomeric compound can be administered to the subject subcutaneously or intravenously.

[0312] An embodiment of the present invention is a process for preparing an oligomeric compound according to any of the preceding claims, comprising: a. synthesizing a sense strand oligonucleotide on a solid support using the phosphoramidite method; b. synthesizing antisense oligonucleotides on a solid support using the phosphoramidite method; c. Annealing the two synthesized oligonucleotides, Create oligomeric compounds.

[0313] Advantages of the Invention The field of oligomeric therapeutic compounds is still immature, and improvements in delivery, stability, specificity, safety, and potency are still being pursued to improve the therapeutic effectiveness of oligomeric compounds. Three general aspects targeted for improvement in oligomer design, in order of importance, are: 1) the sequence of the oligomeric compound, 2) chemical modifications to the oligomeric compound, and 3) the mode of delivery of the oligomeric compound. The importance of these three aspects is illustrated in the evolution of oligomeric compounds leading to the development of butrisiran, the most recent FDA-approved siRNA therapeutic. Alnylam, the developer of butrisiran, has advanced three versions of siRNA targeting TTR into clinical trials: patisiran, revusiran, and butrisiran.

[0314] The first siRNA targeting TTR was patisiran (Onpattro TM Patisiran is a 21-nucleotide siRNA consisting of two 21-nucleotide sense and antisense strands interspersed with chemically modified nucleosides, which stabilizes the siRNA and enhances its inhibitory effect compared to unmodified siRNA of the same sequence (Friedrich and Aigner, Therapeutic siRNA: State-of-the-Art and Future Perspectives, 2022, BioDrugs, 36(5):549-571). Patisiran is the first siRNA therapeutic to be approved by the FDA.

[0315] The second siRNA developed to target TTR is Revusiran. Revusiran also targets TTR, but this siRNA incorporates a novel sequence, additional modified nucleosides, modified internucleoside linkages, and an N-acetylgalactosamine (GalNAc) conjugate. Revusiran treatment was discontinued early during a Phase 3 clinical trial due to disparity in mortality among study patients (Judge et al., Phase 3 Multicenter Study of Revusiran in Patients with Hereditary Transthyretin-Mediated (hATTR) Amyloidosis with Cardiomyopathy (ENDEAVOUR)), Cardiovascular Drugs and Therapy, 2020, 34:357-370). Ultimately, the Revsiran program was discontinued.

[0316] A third siRNA developed to target TTR, butrisiran (also known as ALN-TTRSC02 and Amvuttra™), combines the revusiran sequence with additional chemical modifications and a GalNAc conjugate delivery modality (Janas et al., Safety evaluation of 2'-deoxy-2'-fluoro nucleotides in GalNAc-siRNA conjugates, Nucleic Acids Research, 2019, 47(7):3306-3320). Butrisiran's chemical modifications stabilized it, allowing for a more favorable dosing schedule and administration method compared with patisiran. Patients received 25 mg of butrisiran via subcutaneous injection every three months, whereas patients received 0.3 mg / kg of patisiran via intravenous infusion every three weeks. Butrisiran received FDA approval following positive data from a Phase 3 clinical trial (HELIOS-A) demonstrating significant improvement in the signs and symptoms of polyneuropathy (Alnylam Press Release in Businesswire, Alnylam Announces FDA Approval of Amvuttra TM Alnylam Announces RNAi Therapeutic Amvutrisiran for the Treatment of the Polyneuropathy of Hereditary Transthyretin-Mediated Amyloidosis in Adults TM (butrisiran) FDA approval announced, June 2022).

[0317] Therefore, the history of TTR siRNA development demonstrates the importance of the sequence of the oligomeric compound, the type of chemical modification, the pattern of chemical modification, and the delivery system of the oligomeric compound.

[0318] Disclosed herein are carefully designed chemical modification motifs for advanced RNA targeting (ARNATAR) for oligomeric compounds. Without being bound by any particular theory, the chemical modification motifs improve the stability, specificity, safety, and efficacy of oligomeric compounds, regardless of sequence. The specific chemical modification motifs of the present invention are used in combination with target sequence selection and delivery modalities to form potent oligomeric therapeutic compounds.

[0319] Without being bound by any particular theory, the oligomeric compounds designed by ARNATAR disclosed herein optimize chemical modification motifs for properties such as double-stranded annealing temperature, RISC loading rate, stability, specificity, safety, and efficacy.

[0320] In certain embodiments, oligomeric compounds designed by ARNATAR with low annealing temperatures and fast RISC loading rates may generate faster acting therapeutic compounds beneficial for acute diseases requiring rapid relief after administration.

[0321] In certain embodiments, the oligomeric compounds designed by ARNATAR produce more stable and durable therapeutic compounds that provide a more sustained effect against acute and chronic diseases and / or allow for less frequent administration of the therapeutic compounds.

[0322] In certain embodiments, oligomeric compounds designed by ARNATAR are based on modified motifs that enhance binding regardless of the sequence of the oligomeric compound. This enhanced binding to target nucleic acids allows for the generation of therapeutic compounds with reduced off-target effects, improved specificity, and safety. Furthermore, the ARNATAR motif is designed to result in less toxicity and safer therapeutic compounds.

[0323] In certain embodiments, the oligomeric compounds designed by ARNATAR are designed to be highly potent inhibitors of target nucleic acids. This potency allows for the administration of small amounts of oligomeric compounds to achieve therapeutic effects, reducing the general toxicity of oligomeric compounds after administration. Furthermore, a smaller amount of compound is required for a therapeutic dose, reducing manufacturing costs.

[0324] In certain embodiments, the oligomeric compounds designed by ARNATAR are highly potent inhibitors of target nucleic acids, with high potency allowing for reduction of target nucleic acids in tissues other than the liver.

[0325] Therefore, improved oligomeric compounds are needed to treat diseases. ARNATAR oligomeric compounds are designed to improve speed, stability, specificity, safety, and efficacy to produce improved therapeutic compounds.

[0326] Example Non-Limiting Disclosure and Incorporation by Reference While certain compounds, compositions, and methods are specifically described in accordance with certain embodiments described herein, the following examples are intended to illustrate, but not limit, the compounds described herein. Each reference cited in this application is incorporated herein by reference in its entirety.

[0327] In the tables below, when an unmodified sequence ("Seq") and a modified sequence ("Seq+Chem") are presented on the same line, the corresponding SEQ ID NO: applies to the modified sequence. For example, SEQ ID NO: 16 in Table 3 represents the modified sequence mG*mC*mGmUmCmAfCmCfAfAfAmAmAmGmCmGmCmAmA*T*T.

[0328] Example 1: Design of LMNA siRNAs with different sense strand modifications Laminin (LMNA) was chosen as a target to test various advanced RNA targeting (ARNATAR) designs. A region of laminin (LMNA) with a conserved sequence between humans and mice was identified, and a 19-nt long sequence (SEQ ID NO: 1) in this homologous region was targeted with oligomeric compounds.

[0329] Table 1: LMNA target sequences JPEG2026500220000003.jpg206159

[0330] The siRNA is designed to target the target sequence of LMNA mRNA (SEQ ID NO: 2) and contains a two-nucleotide overhang at the 3' end of the strand. TT (SEQ ID NO: 3-4) or UU (SEQ ID NO: 5) is used. All antisense strands have a 5'-phosphate. Each nucleotide is preceded by a notation indicating the type of chemical modification of that nucleotide, if any. If no modification notation is preceded by the letter indicating the nucleotide, the nucleotide is a deoxyribonucleotide. The notation of chemical modifications for the strand is as follows: (5p) = 5'-phosphate r = ribonucleotide (e.g., rA indicates adenosine) d (or no notation before the nucleotide) = a deoxyribonucleotide substituted for a ribonucleotide (e.g., dA or A indicates 2'-deoxyadenosine) f=2′-F (i.e., 2′-fluoro-modified nucleoside, e.g., fA represents 2′-fluoroadenosine) m=2'-OMe (i.e., 2'-O-methyl modified nucleoside, e.g., mA denotes 2'-O-methyl adenosine) gna = glycolic acid (e.g., gnaT for 2,3-dihydroxypropylthymine) p=phosphate * = phosphorothioate (PS) bond (i.e., a 5'-phosphorothioate (= 5'-thiophosphate) is present in place of the 5'-phosphate. For example, *A indicates 2'-deoxyadenosine 5'-thiophosphate, *rA indicates adenosine 5'-thiophosphate, *mA indicates 2'-O-methyladenosine 5'-thiophosphate). GA=GalNAc (GA1=GalNAc1, GA2=GalNAc2 and GA3=GalNAc3 are specific GalNAc conjugates described herein)

[0331] The sequences of the LMNA siRNAs are shown in the table below or in Figures 1-2.

[0332] Cell cultures were grown to approximately 60-80% confluency before transfection with various doses of siRNA using RNAiMAX (InVitrogen, Waltham, MA) according to the manufacturer's recommended protocol, and the cells were further cultured for a certain period. The activity of the siRNAs was assessed by measuring the amount of target mRNA by qRT-PCR using the LMNA primer-probe set shown in Table 2. qRT-PCR was performed on a QS3 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA) using AgPath-ID. TM The target RNA levels detected by the qRT-PCR assay were measured using One-Step RT-PCR Reagents. TM (ThermoFisher Scientific, Waltham, MA) or GAPDH mRNA levels detected in an aliquot of the RNA sample using qRT-PCR.

[0333] Table 2: Human and mouse LMNA primer-probe sets JPEG2026500220000004.jpg206159

[0334] A. Minimal sense strand modifications improve siRNA activity As shown in Table 3, LMNA-si1 is an LMNA-targeting siRNA with an unmodified ribonucleotide sequence plus a TT overhang. LMNA-si2 is a modified siRNA with a chemical modification pattern that mirrors that of the FDA-approved siRNA, Lumasiran. The sense strand of LMNA-si2 has an additional TT overhang connected by a PS bond and does not contain a GalNAc conjugate. Meanwhile, the antisense strand is 21 nucleotides long and has a TT overhang, not 23 nucleotides. LMNA-si3 has a modified sense strand and an antisense strand with an unmodified ribonucleotide sequence plus a TT overhang. LMNA-si4 has a sense strand with an unmodified ribonucleotide sequence plus a TT overhang and a modified antisense strand. In this example, all antisense strands have a 5'-phosphate.

[0335] Table 3: LMNA oligomer compounds JPEG2026500220000005.jpg206159

[0336] siRNAs were transfected into HeLa cells (ATCC, Manassas, VA, USA) at final concentrations of 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA, USA) and then cultured for 24 hours. siRNA activity was measured by qRT-PCR using the LMNA primer-probe set shown in Table 2, and the half-maximal inhibitory concentrations (IC50) of LMNA-targeting siRNAs were calculated (Table 4).

[0337] Table 4: LMNA siRNA inhibition in HeLa cells after 24 hours JPEG2026500220000006.jpg206159

[0338] LMNA-si2 was used as a benchmark for activity. The results show that LMNA-si1 and LMNA-si4, which have minimal modifications to the sense strand, exhibit higher potency than LMNA-si2 and LMNA-si3, suggesting that reducing modifications to the sense strand can improve siRNA activity.

[0339] B. Addition of peripheral sense-strand modifications to protect siRNA Previous studies have shown that reducing modifications to the siRNA sense strand improves siRNA activity 24 hours after transfection. However, siRNAs need to be metabolically stable to maintain longer-lasting activity in vivo. Some chemical modifications can improve stability without impairing activity (Choung et al., Biochem Biophys Res Commun, 2006, 342:919-927). Therefore, 2'-OMe modifications were strategically made to the 3' and / or 5' ends of the sense strand of LMNA-si4 to assess whether these modifications affect siRNA activity over time. Furthermore, the effects of mUmU or TT overhangs at the 3' end of the sense strand were evaluated. In this example, all antisense strands have a 5'-phosphate.

[0340] Table 5: LMNA oligomer compounds with sense strand peripheral modifications JPEG2026500220000007.jpg206159

[0341] siRNAs were transfected into HeLa cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 24 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 6).

[0342] Table 6: LMNA siRNA inhibition in HeLa cells after 24 hours JPEG2026500220000008.jpg206159

[0343] siRNAs with all 3' and 5' sense strand modifications were more active than LMNA-si2. However, adding 2'-OMe residues beyond the 3' and 5' ends of the sense strand did not contribute to siRNA activity. LMNA-si15, in which the majority of the sense strand was modified, was the least active siRNA. 2'-OMe modifications at the 5' end of the sense strand enhanced siRNA activity compared with modifications at the 3' end (compare LMNA-si6 and LMNA-si7, and LMNA-si9 and LMNA-si10). The absence of a PS bond at the 5' end of the sense strand enhanced siRNA activity compared with the addition of three PS bonds at the 5' end of the sense strand (compare LMNA-si12 and LMNA-si8). There was no clear difference in activity between the presence of mUmU and the presence of a TT overhang.

[0344] Selected siRNAs (LMNA-si2, LMNA-si8, LMNA-si12, LMNA-si14) were further tested long-term in cell culture and different cell types.

[0345] siRNAs were transfected into HeLa cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA), and the cells were cultured for an additional 24 and 72 hours. siRNAs were also transfected into HEK293 cells (ATCC, Manassas, VA) using RNAiMAX (Invitrogen, Waltham, MA), and the cells were cultured for an additional 24 and 120 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 7).

[0346] Table 7: LMNA siRNA inhibition in HeLa or HEK293 cells JPEG2026500220000009.jpg206159

[0347] Tables 6 and 7 show that siRNAs with modifications to the sense strand can enhance siRNA activity in different cell types and for various time periods.

[0348] C. Additional sense-strand modifications to protect siRNA Previous studies have shown that peripheral modifications to the siRNA sense strand improve siRNA activity. The sense strand of LMNA-si4 was strategically modified to include additional 2'-OMe modifications to examine whether these modifications affect siRNA activity over time (Table 8). This was done to increase the 2'-OMe modifications on the sense strand to enhance stability without compromising potency. In this example, all antisense strands have a 5'-phosphate.

[0349] Table 8: LMNA oligomer compounds with further 2'-OMe sense strand modifications JPEG2026500220000010.jpg206159

[0350] siRNA was transfected into HeLa cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA), and the cells were cultured for an additional 24 h. siRNA was also transfected into HEK293 cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA), and the cells were cultured for an additional 48 h. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 of LMNA-targeting siRNA was calculated (Table 9).

[0351] Table 9: LMNA siRNA inhibition in HeLa or HEK293 cells JPEG2026500220000011.jpg206159

[0352] A subset of siRNAs with strong activity and containing more 2'-OMe modified nucleotides on the sense strand were further targeted for activity validation: LMNA-si27 (sense strand ATXL052) and LMNA-si28 (sense strand ATXL053).

[0353] siRNAs were transfected into HeLa cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA), and the cells were cultured for an additional 24 or 72 hours. siRNAs were also transfected into HEK293 cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA), and the cells were cultured for an additional 72 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 10).

[0354] Table 10: LMNA siRNA inhibition in HeLa or HEK293 cells JPEG2026500220000012.jpg206159

[0355] Due to differences in sense strand modifications, LMNA-si27 has higher activity than LMNA-si28.

[0356] Example 2: Design of LMNA siRNAs with different modifications in the antisense strand In Example 1, the antisense strand of the siRNA was kept constant, and the sense strand was subjected to various chemical modifications to find motifs that confer stability and efficacy to the siRNA. In this example, the sense strand of the siRNA was kept constant, and the antisense strand was modified in various ways. The modified antisense strand derived from LMNA-si2 was used as a template for modifying individual nucleotides. The nucleotide positions were counted starting from the 5' end of the strand, with the outermost nucleotide at the 5' end being position 1.

[0357] Without being bound by any particular theory, it has been shown that 2'-F modifications increase the melting temperature (Tm) of RNA duplexes compared to 2'-OMe (Bramsen and Kjems, 2012, Frontiers in Genetics, 3(154):1-22). Furthermore, deoxyribonucleotides that further reduce Tm compared to 2'-OMe at specific positions can be tolerated by RISC (Ui-Tei, et al., Nucl Acids Res, 2008, 36(7):2136-51). Therefore, we varied the number of 2'-F modifications and incorporated deoxyribonucleotides at specific positions to evaluate the effect of these modification patterns on siRNA activity.

[0358] As shown in Table 11, in some antisense strands, nucleotide 2 was modified from a ribonucleotide with a 2'-F modification to a deoxyribonucleotide, nucleotide 7 was modified from a ribonucleotide with a 2'-OMe modification to a deoxyribonucleotide, the 2'-F modification of nucleotide 8 was changed to a 2'-OMe modification, and nucleotide 16 was modified from a ribonucleotide with a 2'-F modification to a deoxyribonucleotide; PS bonds were added to link the deoxyribonucleotides to some adjacent ribonucleotides, and whether the PS bonds protect the deoxyribonucleotides was examined; and the TT overhang at the 3' end of the antisense strand was replaced with an mUmU overhang.

[0359] Table 11: LMNA oligomer compounds with antisense strand modifications JPEG2026500220000013.jpg206159

[0360] In two sets of experiments, siRNAs were transfected into HeLa cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 24 h. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 12).

[0361] Table 12: LMNA siRNA inhibition in HeLa cells JPEG2026500220000014.jpg206159

[0362] Results after 24 hours show that changing the nucleotide at position 7 to a deoxyribonucleotide improved the activity of LMNA-si18, LMNA-si19, LMNA-si20, LMNA-si21, and LMNA-si22. Changes to deoxyribonucleotides at positions 2 or 16 were tolerated, as shown by the activity of LMNA-si16, LMNA-si17, LMNA-si18, LMNA-si21, and LMNA-si22.

[0363] In two sets of experiments, siRNAs were transfected into HeLa cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 72 h. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 13).

[0364] Table 13: LMNA siRNA inhibition in HeLa cells JPEG2026500220000015.jpg206159

[0365] At 72 hours, LMNA-si20 and LMNA-si21 showed the highest activity.

[0366] Example 3: Design of LMNA siRNAs with different modifications in the sense and antisense strands The modified sense and antisense strands disclosed in the previous examples were combined to form new siRNA compounds (Table 14), and their stability and activity were evaluated (Table 15).

[0367] Table 14: LMNA oligomers with modifications on both strands - Compounds JPEG2026500220000016.jpg206159

[0368] siRNAs were transfected into HeLa cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 24, 96, or 144 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 15).

[0369] Table 15: LMNA siRNA inhibition in HeLa cells JPEG2026500220000017.jpg206159

[0370] The sense strand ATXL052 had higher activity than ATXL053 when used as an siRNA compound. The antisense strands ATXL045 and ATXL046 had higher activity than ATXL043. LMNA-si31 and LMNA-si33 showed the highest activity over time.

[0371] siRNAs were transfected into HEK293 cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 96 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 16).

[0372] Table 16: LMNA siRNA inhibition in HEK293 cells JPEG2026500220000018.jpg206159

[0373] Similar to the results in HeLa cells, the sense strand ATXL052 showed higher activity than ATXL053. LMNA-si31 and LMNA-si33 showed higher activity than the others.

[0374] siRNAs were transfected into mouse HePa1-6 cells (ATCC, Manassas, VA, USA) at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 24, 72, or 120 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 17).

[0375] Table 17: LMNA siRNA inhibition in mouse HePa1-6 cells JPEG2026500220000019.jpg206159

[0376] LMNA-si31 was found to have higher activity in mouse cells than the other compounds, while LMNA-si29 and LMNA-si33 had higher activity than the benchmark LMNA-si2.

[0377] Serum stability test LMNA-si2, LMNA-si29, and LMNA-si33 were exposed to human serum to assess their stability. The siRNAs were incubated with human serum (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 0.4 μM for the indicated times (0, 8, 16, and 24 hours) at 37°C. Then, BlueJuice TMGel loading buffer (ThermoFisher Scientific, Waltham, MA, USA) was added, and the siRNA mixture was loaded onto a 4-20% native TBE gel (ThermoFisher Scientific, Waltham, MA, USA). After electrophoresis, the gel was stained with SYBR™ Gold (ThermoFisher Scientific, Waltham, MA, USA) diluted 1:10,000 in water for 10 minutes at room temperature and photographed under UV light.

[0378] The siRNA compounds were confirmed to be stable after 24 hours of serum treatment (FIG. 6).

[0379] Example 4: Design of ApoC3 siRNA with different modifications in the sense and antisense strands Based on the aforementioned studies on LMNA, some of the chemical modifications that confer high activity were evaluated in different targets to determine whether these chemical modification motifs would be similarly beneficial. ApoC3 was selected as the second test target, and siRNAs targeting the 19-nt sequence CUCUGAGUUCUGGGAUUUG (SEQ ID NO: 177) of human ApoC3 were designed as shown in Table 18 and Figures 3-4. The human ApoC3 primer probe set used for RT-PCR is shown in Table 19. ApoC3-sil is a modified siRNA whose chemical modification pattern mirrors that of lumasiran. The sense strand of ApoC3-sil has an additional TT overhang connected by a PS bond and does not contain a GalNAc conjugate. Meanwhile, the antisense strand is 21 nucleotides long, not 23 nucleotides long. All antisense strands have a 5'-phosphate. The modified siRNAs were tested for activity in two cell lines.

[0380] Table 18: ApoC3 oligomer - modification of both strands of the compound JPEG2026500220000020.jpg206159

[0381] Table 19: Sequences of ApoC3 primer-probe sets JPEG2026500220000021.jpg206159

[0382] siRNAs were transfected into Hep3B cells (ATCC, Manassas, VA, USA) at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA, USA) and then cultured for 20, 60, or 168 hours. siRNA activity was determined by measuring target mRNA levels by qRT-PCR using the primer-probe sets shown in Table 19, and the IC50 of siRNAs targeting ApoC3 was calculated (Table 20). qRT-PCR was performed using a QS3 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA) with AgPath-ID. TM The target RNA levels detected by the qRT-PCR assay were determined using the One-Step RT-PCR reagents. TM (ThermoFisher Scientific, Waltham, MA) or GAPDH mRNA levels detected in an aliquot of the RNA sample using qRT-PCR.

[0383] Table 20: Inhibition of ApoC3 siRNA in Hep3B cells JPEG2026500220000022.jpg206159

[0384] siRNAs were transfected into HepG2 cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 24 or 72 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of siRNAs targeting ApoC3 were calculated (Table 21).

[0385] Table 21: Inhibition of ApoC3 siRNA in HepG2 cells JPEG2026500220000023.jpg206159

[0386] New chemical modification motifs initially designed for LMNA-targeting siRNAs generally enhanced activity when used to modify ApoC3-targeting siRNAs, regardless of target or siRNA sequence.

[0387] Example 5: Design of additional LMNA siRNAs with various PS linkages and 2'-F on the sense and antisense strands Based on the aforementioned studies on LMNA and ApoC3, several chemical modification patterns were shown to confer high activity. Therefore, additional chemical modification patterns were designed for LMNA and new motifs were evaluated to determine whether they would be similarly beneficial. The ATXL046 motif was used as the basis for adding or modifying chemical modifications to the antisense strand. Additional modifications that could be included in the new LMNA siRNAs include substituting more deoxyribonucleotides for ribonucleotides, reducing PS linkages, reducing 2'F, and adding more 2'-OMe. The ATXL052 motif was used as the basis for adding or modifying chemical modifications to the sense strand. Some of the additional modifications include adding PS linkages to non-cleavable sites or adding PS linkages to all ribonucleotides. See Table 22 below. All antisense strands have a 5'-phosphate.

[0388] Table 22: LMNA oligomer compounds with modifications on both strands JPEG2026500220000024.jpg206159

[0389] siRNAs were transfected into HeLa cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA), and the cells were cultured for an additional 20 hours. siRNAs were also transfected into HEK293 cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA), and the cells were cultured for an additional 48 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 23).

[0390] Table 23: LMNA siRNA inhibition in HeLa or HEK293 cells JPEG2026500220000025.jpg206159

[0391] The sense strand cleavage site is within the central nucleotide of the sense strand. Adding a PS bond to the central nucleotide of the sense strand can inhibit activity (LMNA-si41), whereas adding a PS bond outside the central cleavage site can enhance activity (LMNA-si40). Introducing two PS deoxyribonucleotides at positions 6 and 7 of the antisense strand was not beneficial to activity (see LMNA-si37). Selected siRNAs were tested for longer periods in different cell lines.

[0392] siRNAs were transfected into Hepa1-6 cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 24 or 72 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 24).

[0393] Table 24: LMNA siRNA inhibition in Hepa1-6 cells JPEG2026500220000026.jpg206159

[0394] In Hepa1-6 cells, LMNA-si39 and LMNA-si31 showed similar activity over time, whereas LMNA-si38 was less active than LMNA-si31, and LMNA-si40 was slightly less active than LMNA-si31.

[0395] Example 6: Additional modifications that increase sense strand stability Additional chemical modification patterns were designed for LMNA and new motifs were evaluated to see if they were beneficial. The ATXL046 motif was used as the basis for adding or altering chemical modifications to the antisense strand. The new modifications changed the amount of PS linkage and added a 2'-F to the central nucleotide of the sense strand (Table 25). All antisense strands have a 5'-phosphate.

[0396] Table 25: LMNA oligomer compounds with modifications on both strands JPEG2026500220000027.jpg206159

[0397] siRNAs were transfected into HeLa cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 24 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 26).

[0398] Table 26: LMNA siRNA inhibition in HeLa cells JPEG2026500220000028.jpg206159

[0399] siRNAs were transfected into HEK293 cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 48 and 120 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 27).

[0400] Table 27: LMNA siRNA inhibition in HEK293 cells JPEG2026500220000029.jpg206159

[0401] All newly designed siRNAs in this example demonstrated superior activity over a longer period compared to the benchmark siRNA LMNA-si2. LMNA-si39, LMNA-si47, LMNA-si49, and LMNA-si51 consistently demonstrated superior activity and duration of activity in different cell types. Selected siRNA designs were reevaluated for activity over a longer period.

[0402] siRNAs were transfected into HeLa cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for different periods of time. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 28).

[0403] Table 28: LMNA siRNA inhibition in HeLa cells JPEG2026500220000030.jpg206159

[0404] When evaluated for up to 168 hours, LMNA-si47 and LMNA-si51 showed the highest activity, with LMNA-si31 also showing very high activity. The ARNATAR-designed compounds showed a faster onset of activity than the benchmark LMNA-si2 and maintained higher activity at later time points.

[0405] Stability assays were performed on LMNA-si31, LMNA-si47, LMNA-si49, and LMNA-si51. In the first assay, stability was tested by exposing the compounds to human serum and mixing the siRNA with human serum. In the second assay, stability was tested by exposing the compounds to rat tritosomes. The test compounds were found to have comparable stability to the benchmark LMNA-si2 (Figures 7-8).

[0406] Serum stability test The siRNA was incubated with human serum (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 0.4 μM for the indicated times (0, 4, 8, and 24 hours) at 37°C. TM Gel loading buffer (ThermoFisher Scientific, Waltham, MA, USA) was added, and the siRNA mixture was loaded onto a 4-20% native TBE gel (ThermoFisher Scientific, Waltham, MA, USA). After electrophoresis, the gel was stained with SYBR™ Gold (ThermoFisher Scientific, Waltham, MA, USA) diluted 1:10,000 in water for 10 minutes at room temperature and photographed under UV light (Figure 8).

[0407] Tritosome stability assay The tritosome stability assay described here is based on the assay by Weingartner et al. (Molecular Therapy, Nucleic Acids, 2020, 21:242-250), which is incorporated herein by reference. siRNA was incubated in rat liver tritosomes (R0610.LT; XenoTech, Kansas City, KS, USA) for 0, 4, 24, or 72 hours. To mimic an acidified environment, tritosome lysates were mixed with low pH buffer (1.5 M acetic acid, 1.5 M sodium acetate, pH 4.75) at a 10:1 ratio. Four microliters of these acidified tritosomes were mixed with 1 microliter of 10 μM siRNA and incubated at 37°C for the indicated time. Next, BlueJuice TM Gel loading buffer (ThermoFisher Scientific, Waltham, MA, USA) was added, and the siRNA mixture was loaded onto a 4-20% native TBE gel (ThermoFisher Scientific, Waltham, MA, USA). After electrophoresis, the gel was stained with SYBR™ Gold (ThermoFisher Scientific, Waltham, MA, USA) diluted 1:10,000 in water for 10 minutes at room temperature and photographed under UV light (Figure 7).

[0408] Example 7: Design of NCL siRNA with different modifications in the sense and antisense strands Based on the aforementioned studies on LMNA and ApoC3, some of the chemical modifications that confer high activity were evaluated in different targets to determine whether these chemical modification motifs would be similarly beneficial. NCL was selected as the third test target, and siRNAs targeting the 19-nucleotide sequence GGAUAGUUACUGACCGGGA (SEQ ID NO: 98) of human NCL were designed as shown in Table 29 and Figure 5. The sequences of the primer-probe set are shown in Table 30. NCL-si6 is a modified siRNA whose chemical modification pattern mirrors that of lumasiran. The sense strand of NCL-si6 has an additional TT overhang connected by a PS bond and does not contain a GalNAc conjugate. Meanwhile, the antisense strand is 21 nucleotides long, rather than 23 nucleotides long. The chemical modification pattern of NCL-si7 is based on that of the previously described LMNA-si47. The chemical modification pattern of NCL-si8 is based on that of the previously described LMNA-si51. The chemical modification pattern of NCL-si9 was based on that of LMNA-si42 described above. The chemical modification pattern of NCL-si10 was based on that of LMNA-si46 described above. All antisense strands contained a 5'-phosphate. The modified siRNAs were tested for activity in HeLa cells.

[0409] Table 29: NCL oligomer compounds with modifications on both strands JPEG2026500220000031.jpg206159

[0410] Table 30: Primer probe set sequences for human NCL mRNA JPEG2026500220000032.jpg206159

[0411] siRNAs were transfected into HeLa cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA, USA) and then cultured for 24 hours. siRNA activity was determined by measuring target mRNA levels by qRT-PCR using the primer-probe sets shown in Table 30, and the IC50 of siRNAs targeting NCL was calculated (Table 31). qRT-PCR was performed using a QS3 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA) with AgPath-ID. TM The target RNA levels detected by the qRT-PCR assay were determined using the One-Step RT-PCR reagents. TM (ThermoFisher Scientific, Waltham, MA) or GAPDH mRNA levels detected in an aliquot of the RNA sample using qRT-PCR.

[0412] Table 31: NCL siRNA inhibition in HeLa cells JPEG2026500220000033.jpg206159

[0413] New chemical modification motifs initially designed for LMNA-targeting siRNAs generally enhanced activity when used to modify NCL-targeting siRNAs, regardless of target or siRNA sequence.

[0414] Example 8: Further characterization of ARNATAR-designed modified oligomeric compounds in vitro The aforementioned studies evaluated the inhibitory activity of oligomeric compounds over time after transfection into cell lines. Oligomeric compounds may have different activities over time depending on the mode of delivery into the cells, e.g., transfection versus free uptake.

[0415] Upon transfection into cells, the oligomeric compounds are rapidly released into the cytoplasm of the cells and begin to actively inhibit the target. In other words, the oligomeric compounds are rapidly released into the cytoplasm, have a rapid onset of activity, are potent, but have a short duration of activity.

[0416] When oligomeric compounds are transported into cells by free uptake (e.g., endocytosis mediated by receptors such as the asialoglycoprotein receptor (ASGR)), these compounds initially accumulate in endosomes / lysosomes, where they are protected from nucleases and slowly released into the cytoplasm. This slow release of compounds initially results in delayed onset and slow onset of inhibitory activity, but allows for long-term and sustained activity.

[0417] Once released into the cytoplasm, the siRNA duplex must interact with RISC and undergo strand separation to allow degradation of the target nucleic acid by the antisense strand. The efficiency of RISC loading and strand separation also affects siRNA activity and kinetics.

[0418] Potential oligomeric compounds selected from previous studies were evaluated for additional properties, such as melting temperature (Tm) and incorporation of the compounds into the RISC system. LMNA-si2 was used as a benchmark for comparison.

[0419] A. The ARNATAR motif demonstrates earlier onset of siRNA activity after transfection. The ARNATAR-designed siRNAs evaluated in the previous example exhibited a faster onset of siRNA activity. Without being bound by theory, this faster onset may be due to faster incorporation into RISC and strand separation than benchmark compounds. Studies were performed with selected oligomeric compounds to assess time course, melting temperature (Tm), and incorporation into RISC (Eamens et al., 2009, RNA, 15:2219-2235).

[0420] HeLa cells were transfected with the selected siRNAs at 0-10 nM for 2 or 4 hours, and LMNA mRNA levels were detected by qRT-PCR using the primer-probe sets listed in Table 2. The ratio of mRNA levels to the levels at time 0 for the LMNA-si2 sample is shown in Table 32 and Figure 9.

[0421] Table 32: LMNA mRNA levels in cells treated with different siRNAs for different times JPEG2026500220000034.jpg206159

[0422] ARNATAR-designed siRNAs demonstrated an earlier onset of expression inducing target reduction, as substantial reduction of target LMNA mRNA was observed 4 h after transfection with ARNATAR-designed siRNA but not with reference siRNA (LMNA-si2).

[0423] Measurement of melting temperature (Tm) by melting curve

[0424] The Tm of a double-stranded oligomeric compound is the temperature at which half of the double-stranded oligomer dissociates into single strands. Tm is a measure of the stability of the double-stranded oligomer. A lower Tm may promote faster dissociation of the sense and antisense strands, allowing the antisense strand to become more readily available to interact with the target RNA.

[0425] 0.5 μl of 10 μM siRNA was incubated in 20 μl of 1X TE buffer containing 1:1000 Ribogreen (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). Triplicate samples were dispensed into a 96-well qPCR plate and placed in a QuantStudio® 3 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA). Melting temperatures (Tm) were acquired using the following conditions: 95°C for 15 seconds, 40°C for 1 minute, and a melting curve was acquired at 15 seconds per 1°C up to 95°C. Tm was measured using the QuantStudio® 3 Real-Time PCR System. TM The analysis was performed using Design & Analysis Software v1.5.2, and the results are shown in Table 33 and Figure 10.

[0426] Table 33: Tm values ​​of selected siRNAs JPEG2026500220000035.jpg206159

[0427] Most of the siRNAs that show increased activity have a lower Tm than the reference siRNA si2.

[0428] Measurement of RISC-loaded siRNA siRNA must be incorporated into RISC and associated with Ago2 to degrade the target RNA. Measurement of RISC-incorporated siRNA (antisense strand) in cells was based on the study by Castellanos-Rizaldos et al. (Nucleic Acid Therapeutics, 2020, 30(3):133-142) and used a combination of immunoprecipitation and subsequent stem-loop RT-qPCR on the final lysate of RISC-incorporated siRNA.

[0429] i. Sample preparation 1.5 × 10 for each siRNA tested 6HELA cells were transfected with 2 nM siRNA and harvested 16 hours later. The cells were harvested, the cell pellet was washed with 1X PBS, and then RNAse Out was added. TM The cells were resuspended in pre-chilled RIPA Lysis and Extraction Buffer (500 μL, ThermoFisher Scientific, Waltham, MA, USA) supplemented with RIPA (5 μL, ThermoFisher Scientific, Waltham, MA, USA) and protease inhibitor cocktail (Roche, Sigma-Aldrich, St. Louis, MO, USA). The cell mixture was gently mixed by pipetting and incubated on ice for 30 minutes. The cell mixture was centrifuged to separate the pellet, and 400 μL of the supernatant was decanted into a clean tube. 10 μL (2.5%) of the supernatant was set aside as input, and the remainder was stored at -80°C until needed.

[0430] ii. Preparation of Ago2 bead coatings 40 μL of magnetic beads were washed with 200 μL of RIPA Lysis and Extraction Buffer, resuspended in 100 μL of RIPA Lysis and Extraction Buffer and 10 μL (5 μg) of Ago2 antibody (ab57113, Abcam, Cambridge, UK), and incubated at 4°C for 2 hours.

[0431] iii.Ago2 immunoprecipitation Two hundred microliters of supernatant from the sample preparation was incubated with Ago2 antibody-precoated beads for 3 hours at 4°C with gentle rotation. The mixture was centrifuged to remove the supernatant, and the beads were washed seven times with 500 μL of wash buffer (50 mM Tris Cl, pH 7.5; 150 mM NaCl; 5 mM EDTA; 0.1% NP-40). 250 μL of PBS / TritonX100 buffer was added to the washed beads, and the mixture was incubated at 95°C for 10 minutes, vortexed, and then placed on ice for 10 minutes. The mixture was centrifuged at 16,000 g for 10 minutes at 4°C, and the supernatant was transferred to a clean tube and saved for the next step.

[0432] iv. Preparation of samples for reverse transcription (RT) Ten μL (2.5%) of the input supernatant was mixed with 250 μL of PBS / TritonX100 buffer and heated at 95°C for 10 minutes, vortexed, placed on ice for 10 minutes, and centrifuged at 4°C for 10 minutes. The supernatant was transferred to a clean tube. ChatGPT: 20 μL of the supernatant from each sample was added to a 96-well plate and heated in a PCR machine at 95°C for 10 minutes to denature the double-stranded fragments, followed by a 5-minute incubation at 4°C. To prepare a control, 250 μL of PBST buffer (0.25% TritonX100 in 1X PBS buffer) was added to untreated beads.

[0433] v. Reverse transcription (RT) For each RT reaction, 5 μL of the prepared sample was heated at 90°C for 3 minutes to unpair the sense and antisense strands and then immediately placed on ice to maintain strand separation. The hsLMNA-A primer was used for the antisense strand reaction, and the hsLMNA-S primer was used for the sense strand reaction. RT was performed using a reverse transcription kit from ThermoFisher Scientific (Waltham, MA, USA) according to the manufacturer's recommended protocol. For control purposes, a standard curve was generated using 5 μL of 0.2 μM siRNA as the RNA template in the RT reaction.

[0434] vi.qPCR quantification The cDNA from the RT reaction was diluted 1:3 with DEPC water. The control RT reaction was also diluted 1:3 and then serially diluted 5-fold to 1:10. The concentrations of cDNA in the serial dilutions of the control reaction were 16.66 nM, 1.666 nM, 0.1666 nM, 0.01666 nM, 0.001666 nM, and 0 nM.

[0435] Add 4 μL of diluted RT cDNA template to 2X TaqMan PCR without UNG. TM The PCR product was mixed with Universal Master Mix II (5 μL, ThermoFisher Scientific, Waltham, MA, USA) and 0.5 μL of DDW. Primer hsLMNA-S or hsLMNA-A was used as appropriate. PCR was performed at 95°C for 3 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 20 seconds.

[0436] Briefly, Ago2-bound siRNA was isolated by immunoprecipitation using an Ago2 antibody from HELA cells transfected with 2 nM of each siRNA at two different time points. Isolated siRNA and total cellular siRNA were quantified using a kit from ThermoFisher (Waltham, MA, USA) and its proprietary stem-loop primer probe set. The recovery rate of Ago2-bound antisense siRNA relative to the level of total cellular antisense siRNA was calculated and normalized to the recovery rate of miRNA-16 detected by qRT-PCR using a stem-loop primer probe set specific for human miRNA-16 (ThermoFisher Scientific, Waltham, MA, USA). The calculated recovery rates are shown in Table 34.

[0437] Table 34: Relative levels of antisense siRNA bound to Ago2 in cells transfected with multiple siRNAs for different times. JPEG2026500220000036.jpg206159

[0438] This study demonstrates that ARNATAR motif siRNAs are more efficiently incorporated into RISC than the LMNA-si2 reference siRNA (Table 34 and Figure 11).

[0439] B. The ARNATAR motif exhibits higher activity in spontaneous cell uptake in vitro. LMNA research Selected LMNA siRNAs were conjugated with various types of N-acetylgalactosamine (GalNAc) (Table 35) to allow free uptake of the compounds into cells. GalNAc was attached to the sense strand of the siRNA.

[0440] Table 35: LMNA oligomers with modifications on both strands - Compounds JPEG2026500220000037.jpg206159

[0441] LMNA-si52 was designed as another benchmark for comparison with ARNATAR-designed compounds. LMNA-si52 has the same sequence and chemical properties as LMNA-si2, as described in Example 1, and is conjugated to GalNAc, which Alnylam uses in its siRNA compounds (Nair et al., J. Am. Chem. Soc. 2014, 136(49):16958-16961, incorporated herein by reference). This GalNAc is also known as GalNAc 1 or GA1.

[0442] LMNA-si55 and LMNA-si64 use the sequences and chemistries of LMNA-si47 and LMNA-si42, respectively, which have been shown in previous examples to have strong and stable chemical modification motifs, and have been conjugated to GalNAc from AM Chemicals (U.S. Pat. No. 10,087,208, incorporated herein by reference), also known as GalNAc 2, GalNAc(AM), or GA2.

[0443] GalNAc-conjugated siRNAs were added directly to primary hepatocytes in vitro at final concentrations of 6.4, 32, or 160 nM and then cultured for 48 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 36).

[0444] Table 36: Inhibition of LMNA siRNA in primary hepatocytes JPEG2026500220000038.jpg206159

[0445] LMNA-si55 and LMNA-si64 showed over 10-fold higher activity than the benchmark LMNA-si52 after free uptake into cells.

[0446] APOC3 research Selected ApoC3 siRNAs were conjugated with N-acetylgalactosamine (GalNAc) (Table 37) to allow free uptake of the compounds into cells. GalNAc was attached to the sense strand of the siRNA.

[0447] Table 37: ApoC3 oligomer - modification of both strands of the compound JPEG2026500220000039.jpg206159

[0448] The chemical modifications in Table 37 represent the modification pattern regardless of the nucleoside sequence. For example, ApoC3-si10, which has the LMNA-si2 motif, has the same chemical modification pattern as LMNA-si2, but the nucleoside sequence is different from LMNA-si2. See Figures 3 and 4 for the complete sequence and chemical properties of ApoC3 siRNA.

[0449] GalNAc-conjugated siRNAs at 0-4 μM were directly added to primary hepatocytes in vitro and then cultured for 48 and 72 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 19, and the IC50 values ​​of siRNAs targeting ApoC3 were calculated (Table 38).

[0450] Table 38: IC50 of ApoC3 siRNA in primary hepatocytes upon free uptake JPEG2026500220000040.jpg206159

[0451] Example 9: In vivo 72 hour characterization of ARNATAR-designed modified oligomeric compounds LMNA-si55, LMNA-si64, and the benchmark LMNA-si52 were previously evaluated in vitro for their inhibitory potency and other properties. In this study, the oligomeric compounds were tested in mice to determine whether the in vitro results correlated in vivo.

[0452] Groups of 7-8 week-old Balb / C male mice (3 mice per group) were subcutaneously injected with LMNA-si52, LMNA-si55, or LMNA-si64 at 0.6 mg / kg, 3 mg / kg, or 15 mg / kg. For LMNA-si55, the high dose was 12 mg / kg instead of 15 mg / kg due to material limitations. Phosphate-buffered saline (PBS) was injected as a control. Mice were sacrificed 72 hours after administration, and organs were harvested for analysis (Tables 39-40 and Figure 12).

[0453] A. Inhibitory Efficacy Table 39: LMNA siRNA inhibition in mouse liver after 72 hours JPEG2026500220000041.jpg206159

[0454] Table 40: LMNA siRNA inhibition in mouse liver JPEG2026500220000042.jpg206159

[0455] LMNA-si55 and LMNA-si64 were more potent than the benchmark LMNA-si52, inhibiting LMNA mRNA in a dose-dependent manner.

[0456] B. Safety Stress or injury markers were assessed for various organs, as shown in the table below. Blood chemistry was measured by the ACP Diagnostic Services Laboratory at the University of California, San Diego. Markers assessed included serum albumin (ALB), alanine aminotransferase (ALT), blood urea nitrogen (BUN), total bilirubin (TBIL), liver weight, spleen weight, and other analytes (Figures 13-15). Low ALB can be a marker for liver disease, kidney disease, or other medical problems. ALT is an enzyme found primarily in the liver. High levels of ALT may indicate the presence of liver disease, such as acute hepatitis. BUN is a waste product of protein breakdown in the liver and is excreted by the kidneys. High BUN levels are a marker for kidney or liver dysfunction. TBIL measures the amount of bilirubin in the blood. High levels of TBIL may indicate liver disease, bile duct blockage, or other medical problems. Body weights and organ weights (eg, liver and spleen) were measured to ensure that the mice were not experiencing general lethargy or failing to eat.

[0457] In general, after administration of the oligomeric compounds, all markers evaluated were found to be within acceptable parameters compared to the PBS control (Figures 13-15).

[0458] C. Immunogenicity Liver samples from the same animals were used to analyze immunogenetic signals. The mRNA levels of several immune response markers were examined by qRT-PCR using primer probe sets specific for mouse NfkB, IL6, and TNF (ThermoFisher Scientific, Waltham, MA, USA). The results are shown in Table 41, Table 42, and Figure 16. The primer probe sets were from ThermoFisher Scientific (Waltham, MA, USA).

[0459] Table 41: Primer-probe set JPEG2026500220000043.jpg206159

[0460] Table 42: mRNA levels in liver samples from animals treated with siRNA for 72 hours JPEG2026500220000044.jpg206159

[0461] In general, 72 hours after administration of the oligomeric compounds, all markers evaluated were found to be within acceptable levels compared to the PBS control (Figure 16).

[0462] Example 10: In vivo 7-day characterization of ARNATAR-designed modified oligomeric compounds LMNA-si52, LMNA-si55, or LMNA-si64 were evaluated for longer periods in mice.

[0463] Groups of 3 7- to 8-week-old male Balb / C mice were subcutaneously injected with LMNA-si52, LMNA-si55, or LMNA-si64 at 4 mg / kg or 12 mg / kg. Phosphate-buffered saline (PBS) was injected as a control. Seven days after administration, mice were sacrificed, and organs were harvested for analysis (Tables 43 and 44 and Figures 17 and 18).

[0464] Table 43: LMNA siRNA inhibition rate in mouse liver after 7 days JPEG2026500220000045.jpg206159

[0465] LMNA-si55 and LMNA-si64 dose-dependently reduced LMNA mRNA levels on day 7 after administration to mice. LMNA knockdown by LMNA-si55 and LMNA-si64 was greater than that by LMNA-si52 at equivalent dose levels (Table 43 and Figure 17).

[0466] Mice were administered LMNA-si52, LMNA-si55, and LMNA-si64, and then immunogenicity was assessed. In general, no significant changes in immune markers were observed in siRNA-treated mice at day 7 (Table 4 and Figure 18).

[0467] Table 44: mRNA levels of selected immune response markers in liver samples JPEG2026500220000046.jpg206159

[0468] Example 11: Additional modifications to improve chemically modified motifs Additional chemical modification patterns were designed for LMNA to evaluate whether the new motifs were beneficial. The LMNA-si47 motif was used as the basis for adding or altering chemical modifications to the antisense strand (Table 45). All antisense strands have a 5'-phosphate.

[0469] Table 45: LMNA oligomer compounds with modifications on both strands JPEG2026500220000047.jpg206159

[0470] siRNAs were transfected into HeLa or Hepa1-6 cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 24 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 4).

[0471] Table 46: LMNA siRNA inhibition in HeLa or Hepa1-6 cells JPEG2026500220000048.jpg206159

[0472] The new design motif of LMNA-si67 showed slightly higher activity than LMNA-si47.

[0473] Example 12: Additional Modifications to Vary Chemical Modifications in Oligomeric Compounds Additional chemical modification patterns were designed for LMNA to evaluate whether the new motifs were beneficial. The LMNA-si68 motif was used as the basis for varying the chemical modifications of oligomeric compounds to examine whether different 2'-F / 2'-OMe modification combinations would be beneficial to siRNA activity (Table 47). All antisense strands have a 5'-phosphate.

[0474] Table 47: LMNA oligomers with modifications on both strands - Compounds JPEG2026500220000049.jpg206159

[0475] siRNAs were transfected into HeLa cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 24, 72, and 96 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 48).

[0476] Table 48: LMNA siRNA inhibition in HeLa cells JPEG2026500220000050.jpg206159

[0477] The results show that LMNA-si78 showed the highest activity at the earliest time point, 30 hours, with LMNA-si71, LMNA-si74, and LMNA-si79 also demonstrating good activity at early stages. At the longest time point, 96 hours, LMNA-si47, LMNA-si71, LMNA-si77, and LMNA-si78 showed the highest activity. The ARNATAR-designed compounds showed a faster onset of activity than the benchmark LMNA-si2 and demonstrated higher activity levels at later time points (Figure 20). The results, as shown herein, demonstrate that altering the 2'-OMe / 2'-F modification site is tolerant to siRNA activity.

[0478] Human NFkB mRNA levels were assessed by qRT-PCR using TaqMan primer probe sets (Table 41) in HeLa cells 72 hours after transfection. The results show that the new ARNATAR siRNA designs have comparable immunogenicity, as measured by NFkB, to the benchmark LMNA-si2 (Figure 21).

[0479] Stability assays were performed on LMNA-si2, LMNA-si74, LMNA-si75, and LMNA-si78. In the first assay, the compounds were exposed to human serum and tested for stability. In the second assay, the compounds were exposed to tritosomes and tested for stability. The three compounds have comparable stability in serum and tritosomes compared to the benchmark LMNA-si2 (Figure 19).

[0480] Example 13: Additional modifications to replace RNA bases with DNA bases in the sense strand Additional chemical modification patterns were designed for LMNA siRNA and evaluated to determine whether the new motifs were beneficial. The motif for LMNA-si78 was the base used to modify the sense strand, replacing RNA with DNA nucleotides (Table 49). All antisense strands have a 5'-phosphate.

[0481] Table 49: LMNA oligomer compounds with modifications on both strands JPEG2026500220000051.jpg206159

[0482] siRNAs were transfected into HEK293 cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 30 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2, and the IC50 values ​​of LMNA-targeting siRNAs were calculated (Table 5).

[0483] Table 50: LMNA siRNA inhibition in HEK293 cells JPEG2026500220000052.jpg206159

[0484] The results show that the ARNATAR-designed compounds exhibited higher activity than the benchmark LMNA-si2 (Table 50 and Figure 22).

[0485] NFkB mRNA levels were assessed by RT-qPCR in HEK293 cells 48 hours after transfection. Results indicate that several new ARNATAR siRNA designs are at least as immunogenic as, or less immunogenic than, the benchmark LMNA-si2, as measured by NFkB (Figure 22).

[0486] Example 14: In vivo characterization of ARNATAR-designed oligomeric compounds Selected LMNA siRNAs were conjugated with various types of N-acetylgalactosamine (GalNAc) (Table 51) to allow free uptake of the compounds into cells.

[0487] Table 51: LMNA oligomer compounds with modifications on both strands JPEG2026500220000053.jpg206159

[0488] LMNA-si52 was designed as another benchmark for comparison with ARNATAR-designed compounds. LMNA-si2 has the same sequence and chemical properties as LMNA-si2, as described in Example 1, and is conjugated to GalNAc, which Alnylam uses in its siRNA compounds, such as butrisilane (Keam, 2022, Drugs, 82:1419-1425; Nair et al., J. Am. Chem. Soc. 2014, 136(49):16958-16961; incorporated herein by reference). GalNAc1 (also known as GA1).

[0489] LMNA-si57, LMNA-si92, LMNA-si93, and LMNA-si94 were conjugated to GalNAc from AM Chemicals (U.S. Pat. No. 10,087,208, incorporated herein by reference) using the sequences and chemistries set forth in Table 51. This GalNAc2 is also known as GalNAc(AM) or GA2.

[0490] Seven- to eight-week-old Balb / c mice (three mice per group) were subcutaneously injected with 3 mg / kg or 15 mg / kg GalNAc-conjugated siRNA. Phosphate-buffered saline (PBS) was injected as a control. Three days after administration, the mice were sacrificed, and organs were harvested for analysis (Table 52 and Figure 23).

[0491] Table 52: Percentage of LMNA mRNA in mouse liver after 3 days *Due to material restrictions, administered at 13.5mg / kg JPEG2026500220000054.jpg206159

[0492] At doses of 3 mg or 15 mg, LMNA-si92, -si93, and -si94 exhibit greater activity than the reference siRNA, LMNA-si57. These four siRNAs share the same GalNAc2. In addition, LMNA-si52, which is essentially the same as LMNA-si57 but differs only in GalNAc, exhibits greater activity than LMNA-si57, suggesting that the different GalNAc may affect delivery efficiency. However, this data further suggests that when the same GalNAc is used, the LMNA-si55 and LMNA-si64 designs evaluated in Example 9 (Figure 12) should exhibit greater potency gains compared to the reference siRNA chemistry.

[0493] Example 15: Comparison of benchmark siRNA and ARNATAR siRNA Homo Sapiens hydroxyacid oxidase (HAO1) ​​was selected as the fourth test target, and siRNAs were designed as shown in Table 53 and FIG.

[0494] Previous studies used modified siRNA as a reference, whose chemical modification pattern was similar to that of lumasiran, but the sense strand had an additional TT overhang connected by a PS bond, did not contain a GalNAc conjugate, and the antisense strand was 21 nucleotides instead of 23 nucleotides.

[0495] In this example, the exact chemical modification motif of lumasiran without the GalNAc conjugate was used to modify siRNA targeting HAO1 (see Friedrich and Aigner (BioDrugs, 2022, 36(5):549-571) for a description of the chemical modification of lumasiran). Additional siRNAs targeting the same HAO1 target sequence were designed based on the ARNATAR motif.

[0496] We conducted a direct comparison test of the HAO-lumasiran benchmark and ARNATAR motif siRNAs to compare their HAO1 inhibitory effects. All ARNATAR-design antisense strands have a 5'-phosphate.

[0497] Table 53: HAO1 oligomers with modifications on both strands - Compounds JPEG2026500220000055.jpg206159

[0498] siRNAs were transfected into Hep3b cells at 0–0.4 nM using RNAiMAX (Invitrogen, Waltham, MA, USA) and then cultured for 24 hours. siRNA activity was measured using the ThermoFisher Scientific HAO1 Gene Expression Assay (Cat. No. Hs00213909_m1; Waltham, MA, USA) to assess the amount of HAO1 mRNA remaining after siRNA knockdown (Table 54 and Figure 25).

[0499] Table 54: Inhibition of HAO1 siRNA in Hep3B cells (after 24 hours) JPEG2026500220000056.jpg206159

[0500] ARNATAR-designed siRNAs showed superior inhibition rates to benchmark siRNAs.

[0501] Example 16: Additional chemical modifications to assess the effect of antisense strand 5'-phosphate and sense strand phosphorothioate on siRNA activity Additional chemical modification patterns for LMNA siRNA (Table 55) were designed and evaluated to determine whether the new motifs were beneficial.

[0502] The previous siRNAs evaluated above contain a 5'-phosphate on the antisense strand, which is required for Ago2 binding and function. Because siRNAs can be phosphorylated in cells (Weitzer S, Martinez J. Nature, 2007, 447 (7141):222-226), the 5'-phosphate may not necessarily be added during siRNA synthesis. Therefore, the activity of siRNAs was evaluated both with and without the 5'-phosphate. LMNA-si94 is the previously described LMNA-si78, which has a 5'-phosphate on the antisense strand and a GalNAc conjugate on the sense strand. LMNA-si95 is LMNA-si94 with a 5'-OH instead of the 5'-phosphate on the antisense strand.

[0503] Furthermore, the effects of the position and number of phosphorothioates (5', 3', or both 5' and 3') around the DNA or RNA nucleotides on the sense strand were evaluated. LMNA-si96 through LMNA-si100 were designed based on the previously described LMNA-si78 or LMNA-si90. LMNA-si96 is LMNA-si90 with a GalNAc conjugate. The siRNA compounds LMNA-si97, LMNA-si98, LMNA-si99, and LMNA-si100 contain the antisense strand of LMNA-si78 and the sense strand with newly designed motifs in which the phosphorothioates were shifted to various positions and GalNAc conjugates.

[0504] The GalNAc conjugate used in this study is known herein as GalNAc2 (AM Chemicals, US Pat. No. 10,087,208, incorporated herein by reference) and was used.

[0505] Table 55: LMNA oligomer compounds with modifications on both strands JPEG2026500220000057.jpg206159

[0506] In vitro assay siRNAs were transfected into HeLa cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 72 and 96 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2. IC50 values ​​for LMNA-targeting siRNAs were calculated (Table 56), and the percent inhibition of LMNA RNA levels was plotted (Figure 26).

[0507] Table 56: LMNA siRNA inhibition in HeLa cells JPEG2026500220000058.jpg206159

[0508] The results show that siRNA with a 5'-phosphate on the antisense strand (LMNA-si94) exhibits superior activity to siRNA with a 5'-OH on the antisense strand (LMNA-si95), and that siRNAs can tolerate phosphorothioates at both the 5' and 3' ends of DNA or RNA nucleotides on the sense strand. Furthermore, phosphorothioate modifications at the 3' position of the sense strand of DNA nucleotides (LMNA-si96) appear to be slightly better than modifications at the 5' position of DNA nucleotides (LMNA-si100). The results also show that the ARNATAR-designed compounds exhibited higher in vitro activity than the benchmark LMNA-si52 (Table 56 and Figure 26).

[0509] In vivo assay Several siRNA compounds demonstrated high activity in the in vitro inhibition assay described above and were subsequently evaluated in vivo. Male Balb / c mice aged 6–8 weeks were subcutaneously injected with 3 mg / kg siRNA, with three mice per group. Mice were evaluated at 3 days, 1 week, or 2 weeks. Phosphate-buffered saline (PBS) was injected as a control. Mice were sacrificed 3 days, 1 week, or 2 weeks after administration, and organs were harvested for analysis (Table 57 and Figure 27). Mice treated with LMNA-si94 or LMNA-si95 were not evaluated at 1 and 2 weeks.

[0510] The activity of the siRNA was evaluated by measuring the amount of target mRNA by qRT-PCR using the LMNA primer-probe set shown in Table 2. qRT-PCR was performed on a QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA) using AgPath-ID TM One-Step RT-PCR reagents were used. Target RNA levels detected in the qRT-PCR assay were normalized to GAPDH mRNA levels detected in an aliquot of the RNA sample using qRT-PCR.

[0511] Table 57: Percentage of LMNA mRNA in mouse liver after 3 days, 1 week, or 2 weeks JPEG2026500220000059.jpg206159

[0512] In vivo results showed that at a dose of 3 mg / kg, the LMNA-si94, LMNA-si95, and LMNA-si97 motif designs exhibited greater activity than the benchmark compound, LMNA-si52, over a 3-day period. At 1- and 2-week periods, LMNA-si97 and LMNA-si98 exhibited more potent inhibitory activity than LMNA-si52. Mice treated with LMNA-si94 or LMNA-si95 were not evaluated beyond 3 days after treatment.

[0513] Example 17: Additional modifications to enhance siRNA durability The sense and antisense strands used to design the LMNA siRNAs in the previous examples were combined in different combinations (Table 58) to evaluate the potency of the new siRNA compounds. The sense strands of LMNA-si97 (ATXL207) and LMNA-si98 (ATXL208), which showed sustained inhibitory activity at 2 weeks in the previous examples, were paired with different antisense strands from LMNA inhibitors (LMNA-si74) that previously showed high inhibitory activity or LMNA-si79 siRNAs that showed stability. Each antisense strand has a 5'-phosphate, and each sense strand has a GalNAc2 conjugate.

[0514] Table 58: LMNA oligomer compounds with modifications on both strands JPEG2026500220000060.jpg206159

[0515] In vitro assay siRNAs were transfected into HeLa cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 20 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2. IC50 values ​​for LMNA-targeting siRNAs were calculated (Table 59), and the percent inhibition of LMNA RNA levels was plotted (Figure 28).

[0516] Table 59: LMNA siRNA inhibition in HeLa cells JPEG2026500220000061.jpg206159

[0517] siRNA with a sense strand of LMNA-si97 containing several RNA nucleotides showed superior activity. Meanwhile, siRNA with a fully 2'-OMe / F-modified sense strand and a DNA-modified antisense strand (LMNA-si103) also showed improved activity compared to the reference siRNA (LMNA-si52). However, siRNA with fully 2'-OMe / F-modified sense and antisense strands (LMNA-si104) showed slightly superior activity compared to the reference siRNA.

[0518] Example 18: Additional modifications to enhance siRNA durability The sense and antisense strands used to design the LMNA siRNAs in the previous examples were combined in different combinations (LMNA-silO7 and LMNA-silO8) or the non-GalNAc version of LMNA-103 (LMNA-silO9) shown in Table 60 and examined to assess the potency of the new siRNA compounds. Each antisense strand carries a 5'-phosphate. The sense strands of LMNA-silO7 and LMNA-silO8 carry a GalNAc2 conjugate.

[0519] Table 60: LMNA oligomer compounds with modifications on both strands JPEG2026500220000062.jpg206159

[0520] In vitro assay siRNAs were transfected into HeLa or Hepa1-6 cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for various times as indicated in the table. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2. IC50 values ​​for LMNA-targeting siRNAs were calculated (Tables 61–63), and the percent inhibition of LMNA RNA levels was plotted (Figures 29–31).

[0521] Table 61: LMNA siRNA inhibition in HeLa cells JPEG2026500220000063.jpg206159

[0522] Table 62: LMNA siRNA inhibition in Hepa1-6 cells JPEG2026500220000064.jpg206159

[0523] Table 63: LMNA siRNA inhibition in HeLa cells JPEG2026500220000065.jpg206159

[0524] The results show that the new siRNA combinations generally exhibited better activity than the reference siRNAs LMNA-si2 or LMNA-si52, and two siRNAs containing RNA nucleotides in the sense strand (LMNA-si107, LMNA-si108) exhibited even higher activity.

[0525] Example 19: Characterization of in vivo duration of siRNA activity In addition to activity, another important factor in siRNA therapy is the in vivo duration of action. To evaluate the duration of action of the newly modified siRNAs, 7-week-old Balb / c mice were subcutaneously administered 5 mg / kg of siRNA LMNA-si103 and reference siRNA LMNA-si52, and sacrificed 10 or 25 days after administration. LMNA mRNA levels in whole liver were analyzed by qRT-PCR using the primer-probe set listed in Table 2. The results are shown in Table 64. LMNA-si103 demonstrated substantially superior knockdown efficacy in vivo compared with the reference siRNA LMNA-si52. Furthermore, LMNA-si103 and reference LMNA-si52 showed similar recovery trends (Figure 32), suggesting that the newly modified siRNAs have higher activity and comparable duration of action in animals compared with the reference siRNA.

[0526] Table 64: Percentage of LMNA mRNA in mouse liver after 10 or 25 days JPEG2026500220000066.jpg206159

[0527] Example 20: Comparison of ARNATAR siRNA Platform Design with Third-Party siRNA Platform Designs As described in Example 1, the LMNA-si2 benchmark was designed based on the chemical modification design of the commercially available therapeutic drug lumasiran. The benchmark LMNA-si52 is LMNA-si2 with an additional GalNAc conjugate (Example 8). However, to minimize the variables in the above experiments, LMNA-si2 (and LMNA-si52) differ from lumasiran in that they have the LMNA sequence, a structure with overhangs at both ends, and a length of 21 nt.

[0528] In this study, we used the chemical modification platform ESC Plus (ESC+) (Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101) to design benchmark siRNAs for three targets: LMNA, NCL, and ApoC3. As described by Hu et al., these new benchmark siRNAs have a 21-nt sense strand and a 23-nt antisense strand. The sense strand has no 3' overhang, and the antisense strand has two natural overhanging nucleotides at its 3' end, forming 23 base pairs with the mRNA target. The antisense strand also lacks a phosphate group at its 5' end, and the seventh position of the antisense strand is a "GNA" modified nucleotide, if commercially available. To create better benchmark siRNAs, the antisense siRNAs share the same 21-nt sequence from the 5' end to ensure similar seed sequences. The benchmark siRNAs are LMNA-si111, NCL-ALN, and ApoC3-AL, as shown in Table 65.

[0529] ARNATAR-designed 21 nt siRNAs targeting NCL and ApoC3 were designed using the LMNA-74 chemical motif, as shown in Table 65. ARNATAR-designed siRNAs have a 5'-phosphate on the antisense strand.

[0530] To compare only the chemical modification motif, siRNA was generated without GalNAc conjugates.

[0531] Table 65: Differently designed oligomeric compounds against NCL, LMNA, and APOC3 mRNA JPEG2026500220000067.jpg206159

[0532] In vitro assay siRNAs were transfected into HeLa cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA) for LMNA and NCL siRNAs and then cultured for 20 hours. For ApoC3 siRNA, RNAiMax was used to transfect Hep3B cells at 0-10 nM, and the cells were incubated for 60 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets listed in Tables 2, 30, and 19. The IC50s of siRNAs targeting the corresponding mRNAs are shown in Table 66, and the percent inhibition of LMNA RNA levels is graphed in Figure 33.

[0533] Table 66: Activity of siRNAs targeting different mRNAs in HeLa or Hep3B cells JPEG2026500220000068.jpg206159

[0534] Results showed that the newly designed siRNAs had dramatically improved activity compared to the ESC+ designs, and the ARNATAR design motif generally showed increased activity regardless of the mRNA target or siRNA sequence.

[0535] Example 21: Comparison of siRNAs of different lengths targeting LMNA, NCL, and AGT mRNA Previously tested siRNA compounds were primarily 21 nt in length with two overhangs at both ends. Because the length of siRNAs generated intracellularly by Dicer cleavage can vary (e.g., approximately 21 nt to 23 nt), we evaluated the effect of varying siRNA length on siRNA activity. To this end, we designed 21-mer or 23-mer siRNAs using the ARNATAR motif targeting NCL (NCL-74 and NCL-23nt) and LMNA (LMNA-sil10), as shown in Table 67. The ARNATAR design motif contains, in addition to other design elements, 2-nt overhangs on both strands and a 5'-phosphate on the antisense strand. 23-mer siRNAs from previous examples with non-ARNATAR chemical modifications were used as benchmarks (LMNA-sil11, NCL-ALN). To compare only the chemical modification motifs, siRNAs were also generated without GalNAc conjugates.

[0536] Table 67: Modified LMNA oligomers of different lengths - Compounds JPEG2026500220000069.jpg206159

[0537] In vitro assay - Comparison of LMNA-si110 and LMNA-si74 with benchmark LMNA-si111

[0538] LMNA siRNA was transfected into HeLa cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 16 and 36 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 2. IC50 values ​​for LMNA-targeting siRNAs were calculated (Table 68), and the percent inhibition of LMNA RNA levels was plotted (Figure 34).

[0539] Table 68: LMNA siRNA inhibition in HeLa cells JPEG2026500220000070.jpg206159

[0540] In vitro assay - Comparison of NCL-ALN with NCL-74 and NCL-23nt

[0541] NCL siRNA was transfected into HeLa cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 36 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets shown in Table 30. The results are shown in Figure 35. Because the reduction in NCL mRNA was too great to determine an IC50, the percentage of mRNA levels at an siRNA concentration of 0.08 nM is shown in Table 69.

[0542] Table 69: Inhibition by NCL siRNA in HeLa cells after 36 hours JPEG2026500220000071.jpg206159

[0543] The results showed that the newly designed 23-nt siRNAs with chemical modifications also showed superior activity compared to benchmark siRNAs of the same length, but the 21-nt siRNAs appeared to have higher activity compared to the 23-mer siRNAs.

[0544] In vitro assay - Comparison of 21nt siRNA and 23nt siRNA using AGT as standard

[0545] To further explore whether the higher activity of 21mer siRNAs over 23mer siRNAs also applies to siRNA sequences targeting other mRNA transcripts, we designed 21mer and 23mer siRNAs targeting human angiotensinogen (AGT) mRNA, as shown in Table 70 and Figures 36-37. GalNAc (described in Sharma et al., 2018, Bioconjugate Chem, 29:2478-2488, and known herein as GA3 or GalNAc3) is conjugated to the sense strand. ARNATAR-designed siRNAs have a 5'-phosphate on the antisense strand.

[0546] Table 70: Oligomeric compounds of different lengths targeting AGT mRNA JPEG2026500220000072.jpg206159

[0547] In vitro assay AGT siRNA was transfected into Hep3B cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 36 hours. Additionally, siRNA was incubated with human primary hepatocytes for 48 hours without transfection. siRNA activity was measured by qRT-PCR using the primer-probe set shown in Table 71. The IC50 values ​​of siRNA targeting AGT are shown in Table 72, and the percent inhibition of AGT mRNA levels is graphed in Figure 38.

[0548] Table 71: Sequence of primer-probe set for AGT JPEG2026500220000073.jpg206159

[0549] Table 72: Activity of siRNA targeting AGT in Hep3B cells JPEG2026500220000074.jpg206159

[0550] The results for this target also show that the 21 nt siRNA has superior activity to the 23 nt siRNA.

[0551] Example 22: New chemical modification motifs that showed superior activity to reference designs against different siRNAs targeting AGT The above results suggest that the newly optimized ARNATAR chemical design increased the activity of siRNAs against different targets or sequences. To further explore whether this observation is generalizable, siRNAs were designed to target five different regions of human AGT, a therapeutic target for the treatment of resistant hypertension. The siRNA sequences and chemical modifications are listed in Table 73. For comparison, siRNAs were designed using the ARNATAR motif or third-party structure and modification designs (ESC or ESC+, as described in Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101). ARNATAR-designed siRNAs have a 5'-phosphate on the antisense strand. To compare only the chemical modification motif, siRNAs were generated without GalNAc conjugates.

[0552] Table 73: Oligomeric compounds targeted to different regions of AGT mRNA JPEG2026500220000075.jpg206159

[0553] + + Because gnaG was unavailable, 612-Ala differs from typical ESC+ chemistry in the placement of the gna modification, but gna at position 6 is also widely used in drug discovery (see PCT / US2019 / 032150).

[0554] +++ Because gnaG is unavailable, 598a-AL does not contain gna at position 7, as does ESC chemistry.

[0555] In vitro assay - Comparison of ARNATAR and third-party motifs

[0556] AGT siRNA was transfected into Hep3B cells at 0-10 nM using RNAiMAX (Invitrogen, Waltham, MA) and then cultured for 24 hours. siRNA activity was measured by qRT-PCR using the primer-probe set shown in Table 71. The IC50 of siRNA targeting AGT is shown in Table 74, and the percent inhibition of AGT mRNA levels is graphed in Figure 39.

[0557] Table 74: Activity of siRNA targeting AGT in Hep3B cells JPEG2026500220000076.jpg206159

[0558] The results show that the optimized ARNATAR design has higher activity than the third-party design across all these different sequences.

[0559] Example 23: Comparison of siRNAs with different chemical motifs targeting the same AGT region As shown in the previous experiments above, changes in chemical modification can affect the activity of siRNA. Therefore, we evaluated the activity of siRNAs targeting the same AGT mRNA sequence when the chemical modification was changed (see Table 75 and Figures 36-37).

[0560] Table 75: Oligomeric compounds with different chemical motifs targeting the same region of AGT JPEG2026500220000077.jpg206159

[0561] In vitro assay: Comparison of siRNAs with different chemical motifs targeting the same AGT sequence

[0562] siRNAs were incubated with human primary hepatocytes at final concentrations of 0-5 μM (ATsi481, ATsi482, ATsi482a, ATsi483, ATsi484) or 0-50 μM (ATsi603, ATsi612) by free uptake. Cells were incubated for 42 hours, and total RNA was prepared. siRNA activity was assessed by qRT-PCR using an AGT primer probe set (Table 71). The IC50 values ​​of siRNAs targeting AGT mRNA are shown in Table 76, and the percent inhibition of AGT mRNA levels is graphed in Figure 40.

[0563] Table 76: Activity of siRNAs targeting AGT in human primary hepatocytes JPEG2026500220000078.jpg206159

Claims

1. An oligomeric compound that inhibits expression of a target nucleic acid, comprising a sense strand having 21 linked nucleotides, the sequence of said sense strand being represented by formula (X). 5' MFMMNMNMMMFFNMNMNMMMNMDD 3', where: D is a deoxyribonucleoside; M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; No single modification type may modify more than two consecutive nucleotides.

2. An oligomeric compound that inhibits expression of a target nucleic acid, comprising an antisense strand having 21 linked nucleotides, the sequence of said antisense strand being represented by formula (VIII). 5' L-MNMNMFNMFMMMNMMM 3', where: D is a deoxyribonucleoside; M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; No single modification type may modify more than two consecutive nucleotides.

3. 1. An oligomeric compound that inhibits expression of a target nucleic acid, comprising: a) a sense strand having 21 linked nucleotides and having a sense strand sequence represented by formula (X), 5' MFMMNMNMMMFFNMNMNMMMNMDD 3', b) an antisense strand having 21 linked nucleotides and having an antisense strand sequence represented by formula (VIII); 5' L-MNMNMFNMFMMMNMMM 3', c) a double-stranded entity formed by the sense strand and the antisense strand; where: D is a deoxyribonucleoside; M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; the double-stranded region is 19 nucleotide pairs in length; Each strand has a two nucleotide overhang at the 3' end, No single modification type may modify more than two consecutive nucleotides.

4. An oligomeric compound that inhibits expression of a target nucleic acid, comprising a sense strand having 21 linked nucleotides, the sequence of said sense strand being represented by formula (XI). 5' MFMMNMNMFFMMMMNMNMMFMDD 3', where: D is a deoxyribonucleoside; M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; No single modification type may modify more than two consecutive nucleotides.

5. An oligomeric compound that inhibits expression of a target nucleic acid, comprising an antisense strand having 21 linked nucleotides, the sequence of said antisense strand being represented by formula (XII). 5' L-MDMFMFNMFMMMFMFMFMMNMM 3', where: D is a deoxyribonucleoside; M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; No single modification type may modify more than two consecutive nucleotides.

6. 1. An oligomeric compound that inhibits expression of a target nucleic acid, comprising: a) a sense strand having 21 linked nucleotides and having a sense strand sequence represented by formula (XI), 5' MFMMNMNMFFMMMMNMNMMFMDD 3', b) an antisense strand having 21 linked nucleotides and having an antisense strand sequence represented by formula (XII); 5' L-MDMFMFNMFMMMFMFMFMMNMM 3', c) a double-stranded entity formed by the sense strand and the antisense strand; where: D is a deoxyribonucleoside; M is a 2'-OMe modified nucleoside; N is a modified or unmodified nucleoside; F is a 2'-F modified nucleoside; L is 5' phosphate, 5' vinylphosphonate, or 5' OH; the double-stranded region is 19 nucleotide pairs in length; Each strand has a two nucleotide overhang at the 3' end, No single modification type may modify more than two consecutive nucleotides.

7. An oligomeric compound according to any of the preceding claims, wherein N is a ribonucleoside (R), a deoxyribonucleoside (D), 2'-OMe, 2'-MOE, 2'-F, an unlocked nucleic acid (UNA) or a locked nucleic acid (LNA).

8. An oligomeric compound according to any of the preceding claims, wherein said oligomeric compound comprises at least one phosphorothioate internucleotide (PS) linkage.

9. An oligomeric compound according to any of the preceding claims, wherein said phosphorothioate internucleotide (PS) linkages are adjacent to deoxyribonucleosides (D) or ribonucleosides (R).

10. An oligomeric compound according to any of the preceding claims, wherein said oligomeric compound is an siRNA.

11. An oligomeric compound according to any of the preceding claims, further comprising a conjugate.

12. 10. The oligomeric compound of claim 9, wherein said conjugate is N-acetylgalactosamine (GalNAc).

13. 10. The oligomeric compound of any of the preceding claims, wherein said compound inhibits expression of a target nucleic acid by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%.

14. A pharmaceutical composition comprising an oligomeric compound according to any of the preceding claims, alone or in combination with a pharmaceutically acceptable carrier or excipient.

15. A method of inhibiting expression of a target nucleic acid in a subject, comprising administering to the subject an oligomeric compound according to any of the preceding claims in an amount sufficient to inhibit expression of the target nucleic acid in the subject.

16. A process for producing an oligomeric compound according to any of the preceding claims, comprising the steps of: a. synthesizing a sense strand oligonucleotide on a solid support using the phosphoramidite method; b. synthesizing antisense oligonucleotides on a solid support using the phosphoramidite method; c. Annealing the two synthesized oligonucleotides, An oligomeric compound is prepared.

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