ADVANCED RNA TARGETING (ARNATAR)

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: the text file named “SiRNA_sequence_listing,” which was created on Dec. 18, 2023.

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

FIELD

Certain embodiments are directed to methods and compounds for modulating gene expression by Advanced RNA Targeting (ARNATAR). Such methods and compounds are useful for reducing expression of certain genes, many of which are associated with a variety of diseases and disorders.

BACKGROUND

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

Sequence-specific silencing of gene expression, RNA interference (RNAi), was discovered in 1998 by Fire et al. (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).

RISC comprises a complex of multiple proteins interacting with an oligomeric compound to inhibit gene expression. The oligomeric compound acts as a template for RISC to recognize complementary messenger RNA (mRNA) transcripts to target a specific mRNA transcript 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), certain oligonucleotides, and single strand siRNAs (Lima et al., Single-stranded siRNAs activate RNAi in animals. Cell. 2012, 150(5):883-94), and double stranded oligomeric compounds such as short hairpin RNAs (shRNAs) and small interfering RNAs (siRNAs).

In 2001, Elbashir et al., showed that 21-nucleotide long siRNA duplexes specifically suppress expression of endogenous and heterologous genes in mammalian cell lines and theorized that siRNA may eventually be used as a gene-specific therapeutic (Duplexes of 21-Nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells, Nature, 2001, 411:494-498).

Currently, five siRNA compounds have received U.S. Food and Drug Administration (FDA) marketing approval (patisiran, givosiran, inclisiran, lumasiran and vutrisiran) while several more are in clinical trials (Moumné et al, Oligonucleotide Therapeutics: From Discovery and Development to Patentability, Pharmaceutics, 2022, 14(2):260).

Patisiran (Onpattror™) became the first FDA approved siRNA therapeutic in 2018 (Hoy, Patisiran: First Global Approval, Drugs, 2018, 78:1625-1631). Patisiran is a partially modified siRNA targeting transthyretin (TTR) for the treatment of peripheral nerve disease (polyneuropathy) caused by hereditary transthyretin-mediated amyloidosis (hATTR), where chemically modified nucleosides are scattered along the 21-nucleotide long sense and antisense strands forming the siRNA duplex. Later approved siRNAs such as givosiran introduced additional modified nucleosides, modified inter-nucleoside linkages and a N-Acetylgalactosamine (GalNAc) conjugate to aid cellular delivery. (Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101). The latest FDA approved siRNA, vutrisiran (Amvuttra™), has the same target and indication as patisiran and was developed by the same company (Alnylam Press Release in Businesswire, Alnylam Announces FDA Approval of Amvuttra™ (vitrusiran), an RNAi Therapeutic for the Treatment of the Polyneuropathy of Hereditary Transthyretin-Mediated Amyloidosis in Adults, June 2022). Vutrisiran is a direct competitor to patisiran and was designed with a different sequence, chemical modification pattern and delivery modality to improve its therapeutic efficacy over patisiran.

The field of therapeutic oligomeric compounds has advanced immensely from the discovery of RNAi in 1998, to the first regulatory approval of an siRNA therapeutic in 2018, to the latest improved siRNA therapeutic. However, the field is still constantly seeking improvements to increase therapeutic efficacy even over existing oligomeric compounds. The ideal oligomeric compound should be: 1) specifically deliverable to the cell or organ of interest, 2) stable/durable once administered to a patient e.g., slow to degradation, a long half-life, 3) specific to the target with no off-target effects, 4) safe for the patient with no activation of the immune system and non-toxic, and 5) potent with efficient and specific cleavage of its target.

Improvements in delivery, stability, specificity, safety and potency of oligomeric compounds are still being sought in order to produce a better therapeutic. Disclosed herein are improved oligomeric compounds with Advanced RNA Targeting (ARNATAR) abilities that enhance their gene silencing activity.

SUMMARY

Several embodiments provided herein relate to the discovery of certain modifications to oligomeric compounds that can enhance their effectiveness in modulating gene expression. In several aspects, the oligomeric compound is single-stranded (e.g., a single stranded oligonucleotide, a single stranded RNA (ssRNA)) or double-stranded (e.g., shRNA and siRNA) and is modified. The single-stranded oligomeric compound comprises a sense strand or an antisense strand. The double-stranded oligomeric compound comprises a sense strand and an antisense strand. The antisense strands can be fully or partially complementary to a target nucleic acid.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprises a sense strand having 19 to 23 linked nucleotides, wherein the sense strand sequence is represented by Formula (I): 5′ M-(Y)n-Z-(Y)r-D-D 3′. The 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.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprises an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (IV): 5′ L-M-(D)v-(Y)s-(Z)t-(Y)u-Z-N-(Z)r 3′. The D is a deoxyribonucleoside, N is a modified or unmodified nucleoside, M is a 2′-OMe modified nucleoside, L is a 5′ OH, 5′ vinyl phosphonate or a 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, r is 1-2, and no single modification type modifies more than two consecutive nucleotides.

In one embodiment, an oligomeric compound capable of inhibiting the 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)n-Z-(Y)r-D-D 3′, and (b) an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (IV: 5′ L-M-(D)v-(Y)s-(Z)t-(Y)u-Z-N-(Z)r 3′. A duplex is formed by the sense and antisense strands, where the duplex region is 19 to 23 nucleotide pairs in length. The D is a deoxyribonucleoside, N is a modified or unmodified nucleoside, M is a 2′-OMe modified nucleoside, L is a 5′ OH, 5′ vinyl phosphonate or a 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 6-8, r is 1-2, v is 0-1, s is 2-7, t is 0-2, u is 0-5, and no single modification type modifies more than two consecutive nucleotides.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprises a sense strand having 19 to 23 linked nucleotides, wherein the sense strand sequence is represented by Formula (II): 5′ Y-Z-(Y)q-FFNM-(Y)q-M-(Y)v-D-D 3′. The 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 2-3, v is 0-1, and no single modification type modifies more than two consecutive nucleotides.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprises an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (V): 5′ L-(Y)p-NM-(FMM)r-(Y)p-(Z)r 3′. The M is a 2′-OMe modified nucleoside, F is a 2′-F modified nucleoside, L is a 5′ OH, 5′ vinyl phosphonate or a 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.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprising: (a) a sense strand having 19 to 23 linked nucleotides, wherein the sense strand sequence is represented by Formula (II): 5′ Y-Z-(Y)q-FFNM-(Y)q-M-(Y)v-D-D 3′, and (b) an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (V): 5′ L-(Y)p-NM-(FMM)r-(Y)p-(Z)r 3′. A duplex is formed by the sense and antisense strands, where the duplex region is 19 to 23 nucleotide pairs in length. The 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 a 5′ OH, 5′ vinyl phosphonate or a 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 2-3, p is 3-5, r is 1-2, v is 0-1, and no single modification type modifies more than two consecutive nucleotides. In certain embodiments, the 5′ (Y)p is YYY and the 3′ (Y)p is YYYY.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows a table with LMNA antisense strand sequences with various modifications.

FIG. 2: shows a table with LMNA sense strand sequences with various modifications.

FIG. 3: shows a table with ApoC3 antisense strand sequences with various modifications.

FIG. 4: shows a table with ApoC3 sense strand sequences with various modifications.

FIG. 5: shows tables with NCL antisense strand and sense strand sequences with various modifications.

FIG. 6: shows an assay where siRNAs were incubated with human serum to determine their stability in serum. LMNA-si2. LMNA-si29 and LMNA-si33 were incubated in human serum for various lengths of time then run on a gel to visualize the amount of siRNA duplex present after exposure to serum.

FIG. 7: shows a tritosome stability assay where siRNAs are assessed for their stability. LMNA-si2, LMNA-si31, LMNA-si47, LMNA-si49 and LMNA-si5l were tested for various lengths of time then run on a gel to visualize the amount of siRNA duplex present after exposure to tritosomes.

FIG. 8: shows an assay where siRNAs were incubated with human serum to determine their stability in serum. LMNA-si2, LMNA-si31, LMNA-si47, LMNA-si49 and LMNA-si5l were incubated in human serum for various lengths of time then run on a gel to visualize the amount of siRNA duplex present after exposure to serum.

FIG. 9: shows select ARNATAR design siRNAs assessed for onset of siRNA activity. ARNATAR Design siRNA compounds showed faster onset of siRNA activity compared to benchmark.

FIG. 10: shows the Melting Temperature (Tm) of select ARNATAR designed siRNAs.

FIG. 11: shows select ARNATAR design siRNAs assessed for RISC loading. ARNATAR designed siRNAs showed faster association with Ago2.

FIG. 12: shows RNA inhibition data from an in vivo mouse study with select ARNATAR designed siRNAs.

FIG. 13: shows safety data from an in vivo mouse study with select ARNATAR designed siRNAs. Serum proteins ALB, ALT and BUN were assessed.

FIG. 14: shows safety data from an in vivo mouse study with select ARNATAR designed siRNAs. Liver and spleen weight were measured.

FIG. 15: shows safety data from an in vivo mouse study with select ARNATAR designed siRNAs. Serum analytes were assessed.

FIG. 16: shows data from an in vivo mouse study on the effect of select ARNATAR designed siRNAs on immunogenicity markers NFkB, IL-6 and TNF.

FIG. 17: shows RNA inhibition at Day 7 from an in vivo mouse study with select ARNA TAR designed siRNAs.

FIG. 18: shows data at Day 7 from an in vivo mouse study on the effect of select ARNATAR designed siRNAs on immunogenicity markers NFkB, IL-6 and TNF.

FIG. 19: shows select siRNAs with ARNATAR platform chemistry are stable in serum and in tritosome assays.

FIG. 20: shows in vitro inhibition of LMNA in HeLa cells by select ARNATAR designed siRNAs.

FIG. 21: shows data from an in vitro study in HeLa cells on the effect of select ARNATAR designed siRNAs on immunogenicity marker NFkB.

FIG. 22: shows in vitro inhibition of LMNA in HEK293 cells by select ARNA TAR designed siRNAs.

FIG. 23: shows mRNA inhibition data from an in vivo Balb/c mouse study with select ARNATAR designed siRNAs.

FIG. 24: shows a table with HAO1 antisense and sense strand sequences with various modifications.

FIG. 25: shows a graph of in vitro inhibition of HAO1 RNA in Hep3B cells at 24 hours by modified oligomeric compounds.

FIG. 26: shows graphs of in vitro inhibition of LMNA RNA in HeLa cells at 72 hours and 96 hours by modified oligomeric compounds.

FIG. 27: shows in vivo inhibition data of LMNA RNA in mice liver at 3 days, 1 week, or 2 weeks after treatment by modified oligomeric compounds.

FIG. 28: shows a graph of in vitro inhibition of LMNA RNA in HeLa cells at 20 hours by modified oligomeric compounds.

FIG. 29: shows graphs of in vitro inhibition of LMNA RNA in HeLa cells at 16 hours, 24 hours and 48 hours by modified oligomeric compounds.

FIG. 30: shows graphs of in vitro inhibition of LMNA RNA in Hepa1-6 cells at 16 hours and 48 hours by modified oligomeric compounds.

FIG. 31: shows a graph of in vitro inhibition of LMNA RNA in HeLa cells at 20 hours by modified oligomeric compounds.

FIG. 32: shows in vivo data of LMNA siRNA activity in mice liver at 10 days and 25 days after treatment by modified oligomeric compounds.

FIG. 33: shows graphs of in vitro inhibition by ARNATAR motif siRNAs compared to third party motifs applied to siRNAs targeting LMNA, NCL and ApoC3.

FIG. 34: shows graphs of in vitro inhibition of LMNA RNA in HeLa cells at 16 hours and 36 hours by modified oligomeric compounds.

FIG. 35: shows a graph of in vitro inhibition of NCL RNA in HeLa cells at 36 hours by modified oligomeric compounds.

FIG. 36: shows tables with AGT antisense strand sequences with various modifications.

FIG. 37: shows tables with AGT sense strand sequences with various modifications.

FIG. 38: shows graphs of in vitro inhibition of AGT RNA in Hep3B cells via transfection at 36 hours or in human primary hepatocytes (IHPH) via free uptake at 48 hours by modified oligomeric compounds. The x-axis is siRNA concentration at nM for Hep3B cells and at μM for HPH cells. The y-axis is AGT mRNA percentage after treatment with varying siRNA concentrations.

FIG. 39: shows graphs of in vitro inhibition of AGT RNA in Hep3B cells via transfection at 20 hours. The x-axis is siRNA concentration at nM.

FIG. 40: shows graphs of AGT in vitro inhibition in human primary hepatocytes (HPH) via free uptake at 42 hours by modified oligomeric compounds. The x-axis is siRNA concentration at various μM concentrations. The y-axis is AGT mRNA percentage after treatment with varying siRNA concentrations.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated-by-reference for the portions of the document discussed herein, as well as in their entirety.

Definitions

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 synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated-by-reference for the portions of the document discussed herein, as well as in their entirety.

Unless otherwise indicated, the following terms have the following meanings:

“2% O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to an O-methoxy-ethyl modification at the 2′ position of a furanose ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means a nucleoside comprising a 2′-MOE modified sugar moiety. “2′-MOE nucleotide” (also 2′-O-methoxyethyl nucleotide) means a nucleotide comprising a 2′-MOE modified sugar moiety.

“2′-O-methyl” (also 2′-OCH₃and 2′-OMe) refers to an O-methyl modification at the 2′ position of a furanose ring. A 2′-O-methyl modified sugar is a modified sugar.

“2′-OMe nucleoside” (also 2′-O-methyl nucleoside) means a nucleoside comprising a 2′-OMe modified sugar moiety. “2′-OMe nucleotide” (also 2′-O-methyl nucleotide) means a nucleotide comprising a 2′-OMe modified sugar moiety.

“2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position of the furanosyl ring other than H or OH. In certain embodiments, 2′ substituted nucleosides include nucleosides with a fluoro (2′-F), O-methyl (2′-OMe), 0-methoxyethyl (2′-MOE) or bicyclic sugar modifications.

“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5 position. A 5-methylcytosine is a modified nucleobase.

“About” means within i7% of a value. For example, if it is stated, “the compounds affected at least about 70% inhibition of mRNA”, it is implied that the mRNA levels are inhibited within a range of 63% and 77%.

“Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

“Antibody” refers to a molecule characterized by reacting specifically with an antigen in some way, where the antibody and the antigen are each defined in terms of the other. Antibody may refer to a complete antibody molecule or any fragment or region thereof, such as the heavy chain, the light chain, F_abregion, and F_cregion.

“Antisense oligonucleotide” or “ASO” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid. In certain embodiments, the antisense oligonucleotide comprises one or more ribonucleosides (RNA nucleosides) and/or deoxyribonucleosides (DNA nucleosides).

“Base complementarity” refers to the capacity for the base pairing of nucleobases of an oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases. Base complementarity also refers to canonical (e.g., A:U, A:T, or C:G) or non-canonical base pairings (e.g., A:G, A:U, G:U, I:U, I:A, or I:C).

“Bicyclic sugar” means a furanose ring modified by the bridging of two non-geminal carbon atoms. A bicyclic sugar is a modified sugar.

“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an oligomeric compound.

“Chemical modification” means modification of molecular structure or element from naturally occurred molecules. For example, siRNA compounds are composed of linked ribonucleosides (also sometimes referred to herein as RNA), therefore, substitution of a deoxyribonucleoside (also sometimes referred to herein as DNA nucleoside) for a ribonucleoside is considered a chemical modification of the siRNA compound.

“Chemically distinct region” refers to a region of an oligomeric compound that is in some way chemically different than another region of the same oligomeric compound. For example, a region having 2′-OMe nucleotides is chemically distinct from a region having nucleotides without 2′-OMe modifications.

“Chimeric oligomeric compounds” means oligomeric compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits. For example, as disclosed herein, siRNA can comprise a peripheral region and a central region. The peripheral region comprises motifs with various modified or unmodified nucleobases so as to confer increased stability, specificity, safety and potency, while the central region comprises various modified or unmodified nucleobases to serve as substrate for RISC mediated degradation.

“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

“Comply” means the adherence with a recommended therapy by an individual.

“Comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

“Contiguous nucleobases” means nucleobases immediately adjacent to each other.

“Deoxyribonucleoside” means a nucleoside having a hydrogen at the 2′ position of the sugar portion of the nucleoside. A deoxyribonucleoside is sometimes referred to as DNA nucleoside, “D” or “d” herein. Deoxyribonucleosides may be modified with any of a variety of substituents and may be connected by covalent linkages other than naturally occurring phosphodiester such as phosphorothioate.

“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2′ position of the sugar portion of the nucleotide. A deoxyribonucleotide is sometimes referred to as DNA nucleotide, “D” or “d” herein. Deoxyribonucleotides may be modified with any of a variety of substituents and may be connected by covalent linkages other than naturally occurring phosphodiester such as phosphorothioate.

“Designing” or “Design” refers to the process of designing an oligomeric compound that specifically hybridizes with a target nucleic acid molecule.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.

“Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an oligomeric compound and a target nucleic acid is a second nucleic acid.

“Fully modified motif” refers to an oligomeric compound comprising a contiguous sequence of nucleosides wherein essentially each nucleoside has a chemical modification.

“Hybridization” means 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, a siRNA and a nucleic acid target.

“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment or therapy.

“Induce”, “inhibit”, “potentiate”, “elevate”, “increase”, “decrease” or the like, generally denotes quantitative differences between two states.

“Inhibiting the expression or activity” refers to a reduction, blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.

“Internucleoside linkage” refers to the chemical bond between nucleosides. The 3′ position of a nucleoside is hereby linked to a 5′ position of a subsequent nucleoside via the internucleoside linkage.

“Linked nucleosides” means adjacent nucleosides (e.g., A, G, C, T, or U) linked together by an internucleoside linkage. Examples of linked nucleosides include deoxyribonucleosides (sometimes referred to as DNA nucleosides herein) or ribonucleosides (sometimes referred to as RNA nucleosides herein).

“Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid through Watson-Crick base-pairing (e.g., A:T, A:U, or C:G).

“Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e., a phosphodiester internucleoside bond).

“Modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

“Modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase. As used herein, where the oligomeric compound is RNA based, a substitution of a deoxyribonucleoside (sometimes referred to as DNA nucleoside herein) for a ribonucleoside is considered a modification of the oligomeric compound. Also, where the oligomeric compound is DNA based, a substitution of a ribonucleoside (sometimes referred to as RNA nucleoside herein) for a deoxyribonucleoside is considered a modification of the oligomeric compound.

“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, a deoxyribonucleoside (sometimes referred to as DNA nucleoside herein) for a ribonucleoside (sometimes referred to as RNA nucleoside herein) substitution, and/or a modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, a deoxyribonucleoside (sometimes referred to as DNA nucleoside herein) for a ribonucleoside (sometimes referred to as RNA nucleoside herein) substitution, and/or a modified nucleobase.

“Modified sugar” means substitution and/or any change from a natural sugar moiety.

“Moiety” means one of the portions into which something is divided i.e., a part or component of something. For example, a sugar moiety of a nucleotide is the sugar component of the nucleotide.

“Monomer” refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

“Motif” means a pattern of modification in an oligomeric compound. For example, as disclosed herein, ARNATAR designed oligomeric compounds comprising motifs with various modified nucleobases and internucleoside linkages in order to improve delivery, stability, specificity, safety and potency of the compounds. The motif is independent from the nucleobase contained and identifies only the pattern of modifications.

“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA (2′-OH).

“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.

“Non-complementary nucleobase” refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), messenger RNA (mRNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).

“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

“Nucleobase complementarity” refers to a nucleobase that is capable of base pairing (also known as being complementary) with another nucleobase. If a nucleobase at a certain position of an oligomeric compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligomeric compound and the target nucleic acid is considered to be complementary at that nucleobase pair. For example, in DNA, adenine (A) is complementary to thymine (T); in RNA, adenine (A) is complementary to uracil (U); and, Guanine (G) is complementary to cytosine (C) in both DNA and RNA. Base pairs, or complementary nucleobases, are usually canonical Watson-Crick base pairs (C:G, A:U, A:T), but, non-canonical base pairs such as Hoogsteen base pairs (e.g., A:G, or A:U), Wobble base pairs (e.g., G:U, I:U, I:A, or I:C, wherein I is hypoxanthine) and the like are also included. Nucleobase complementarity facilitates hybridization of the oligomeric compounds described herein to their target nucleic acids.

“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. “Mimetic” refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.

“Nucleotide” means a nucleoside having a linkage group (e.g., a phosphate (p) or phosphorothioate (PS) group) covalently linked to the sugar portion of the nucleoside. Nucleotides include ribonucleotides and deoxyribonucleotides. Ribonucleotides are the linked nucleotide units forming RNA. Deoxyribonucleotides are the linked nucleotide units forming DNA.

“Off-target effect” refers to an unwanted or deleterious biological effect associated with modulation of RNA or protein expression of a gene other than the intended target nucleic acid.

“Oligomeric activity” means any detectable or measurable activity attributable to the hybridization of an oligomeric compound to its target nucleic acid. In certain embodiments, oligomeric activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid. Oligomeric activity can be modulated by an oligomeric compound such as a siRNA.

“Oligomeric compound” means a sequence of linked monomeric subunits that is capable of undergoing hybridization to at least a region of a target nucleic acid through hydrogen bonding. The monomeric subunits can be modified or unmodified nucleotides or nucleosides. The oligomeric compound acts as a template for RISC to recognize complementary messenger RNA (mRNA) transcripts to target a specific mRNA transcript 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, ssRNAs, siRNAs, shRNAs and miRNAs.

“Oligomeric inhibition” means reduction of target nucleic acid levels in the presence of an oligomeric compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the oligomeric compound.

“Oligomeric mechanisms” include RISC or RNase H related mechanisms involving hybridization of an oligomeric compound with target nucleic acid, wherein the outcome or effect of the hybridization is target degradation and inhibition of gene expression.

“Oligonucleotide” as used herein means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another. Oligonucleotides can have a linking group other than a phosphate group (e.g., a phosphorothioate thiophosphate group) used as a linking moiety between nucleosides. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA nucleosides) and/or deoxyribonucleosides (DNA nucleosides).

“Phosphorothioate linkage” or “PS” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate (=thiophosphate or also known as thiophosphate) linkage is a modified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an oligomeric compound “Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

“RNA” or “ribonucleic acid” consists of ribose nucleotides or ribonucleotides (nitrogenous bases attached to a ribose sugar) linked by phosphodiester bonds, forming strands of varying lengths. The nitrogenous bases in RNA are adenine, guanine, cytosine, and uracil. The ribose sugar of RNA is a cyclical structure of five carbons and one oxygen.

“Ribonucleoside” means a nucleoside having a hydroxy at the 2′ position of the sugar portion of the nucleoside. Ribonucleosides can be modified with any of a variety of substituents and may be connected by covalent linkages other than naturally occurring phosphodiester such as phosphorothioate. A ribonucleoside is sometimes referred to as RNA nucleoside, “R” or “r” herein.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ position of the sugar portion of the nucleotide. Ribonucleotides can be modified with any of a variety of substituents and may be connected by covalent linkages other than naturally occurring phosphodiester such as phosphorothioate. A ribonucleotide is sometimes referred to as RNA nucleotide, “R” or “r” herein.

“Segments” are defined as smaller or sub-portions of regions within a target nucleic acid.

“Sites,” as used herein, are defined as unique nucleobase positions within a target nucleic acid.

“Specifically hybridizable” refers to an oligomeric compound having a sufficient degree of complementarity between an oligomeric compound (e.g., siRNA) and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments.

“Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.

“Subject” means a human or non-human animal selected for treatment or therapy.

“Target” refers to a protein or nucleic acid sequence (e.g., mRNA), the modulation of which is desired.

“Target gene” refers to a gene encoding a target.

“Targeting” means the process of design and selection of an oligomeric compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” all mean a nucleic acid capable of being targeted by oligomeric compounds.

“Target region” means a portion of a target nucleic acid to which one or more oligomeric compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleic acid to which an oligomeric compound is targeted. “5′ target site” refers to the 5-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment. In an embodiment, a target segment is at least a 12-nucleobase portion (i.e., at least 12 consecutive nucleobases) of a target region to which an oligomeric compound is targeted.

“Therapeutic efficacy” refers to the effectiveness of a therapeutic compound such as an oligomeric compound. Therapeutic efficacy can be increased by improvements in delivery, stability, specificity, safety and potency of the therapeutic compound.

“Unmodified” RNA nucleobases mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). “Unmodified” DNA nucleobases mean 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 the RNA nucleobase. In certain embodiments, an unmodified DNA nucleobase is considered modified when a RNA nucleobase is substituted for the DNA nucleobase.

“Unmodified nucleoside” means a nucleoside composed of commonly and naturally occurring nucleobases and sugar moieties. For example, an unmodified nucleoside is an RNA nucleoside if used in an RNA sequence, however, in such RNA sequence, any other nucleoside (e.g., DNA nucleoside, 2′-OMe, or 2′-F) is considered a modified nucleoside.

“Unmodified nucleotide” means a nucleotide composed of commonly and naturally occurring nucleobases, sugar moieties, and internucleoside linkages. For example, an unmodified nucleotide is an RNA nucleotide if used in an RNA sequence, however, in such RNA sequence, any other nucleotide (e.g., DNA nucleotide, 2′-OMe, or 2′-F) is considered a modified nucleotide.

Disclosed herein are improved oligomeric compounds with Advanced RNA Targeting (ARNATAR) designs that enhance their gene silencing activity. Several embodiments provided herein relate to the discovery of certain modification motifs to oligomeric compounds that can enhance their effectiveness in modulating gene expression by improving delivery, stability, specificity, safety and potency of the oligomeric compounds.

In several aspects, the oligomeric compound is single-stranded (e.g., a single stranded oligonucleotide, a microRNA (miRNA) or a single stranded RNA (ssRNA)) or double-stranded (e.g., a shRNA or a siRNA) and is modified. The single-stranded oligomeric compound comprises a sense strand or an antisense strand. The double-stranded oligomeric compound comprises a sense strand and an antisense strand. The antisense strands can be fully or partially complementary to a target nucleic acid.

The following embodiments describe oligomeric compounds comprising linked nucleotides that comprise modified and optionally unmodified nucleosides, wherein adjacent nucleosides are either linked by the naturally occurring phosphodiester bond or by a non naturally occurring bond, such as a thiophosphodiester bond (also referred to a “phosphorothioate internucleotide (PS) linkage”). In some embodiments, where the oligomeric compound is RNA based (e.g., miRNA, ssRNA, shRNA and/or siRNA), the sequence is considered an RNA and unmodified nucleosides refer to ribonucleosides. Modified nucleosides may comprise a modified base and/or a modified sugar (e.g., preferably at 2′ position modified sugar). In the RNA based oligomeric compounds, a deoxyribonucleoside (D) is also considered a modified nucleoside.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid comprises a sense strand having 19 to 23 linked nucleotides, wherein the sense strand sequence is represented by Formula (I): 5′ M-(Y)n-Z-(Y)r-D-D 3′. The 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. Preferably, the -D-D is a TT or TA overhang. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkage between 2 nucleosides. In a further embodiment, the sense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkage can be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5′ side, the 3′ side or both sides. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand. In an embodiment, the oligomeric compound comprising Formula (I) is a ssRNA or siRNA. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid comprises an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (IV): 5′ L-M-(D)v-(Y)s-(Z)t-(Y)u-Z-N-(Z)r 3′. The 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′ vinyl phosphonate 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, 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 comprises one or more phosphorothioate internucleotide (PS) linkage between 2 nucleosides. In a further embodiment, the antisense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkage can be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5′ side, the Y side or both sides. The PS linkage modification can be between positions 1-2, positions 2-3, 19-20 and/or positions 20-21 counting from the 5′ end of the antisense strand. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand. In an embodiment, the oligomeric compound comprising Formula (IV) is a ssRNA or siRNA. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the 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)n-Z-(Y)r-D-D 3′, and (b) an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (IV): 5′ L-M-(D)v-(Y)s-(Z)t-(Y)u-Z-N-(Z)r 3′. A duplex is formed by the sense and antisense strands, where the duplex region is 19 to 23 nucleotide pairs in length. The 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′ vinyl phosphonate 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, u is 0-5, and no single modification type modifies more than two consecutive nucleotides. In a further embodiment, the 3′ Z in the antisense strand is a UU or TT overhang and the -D-D in the sense strand is a TT or TA overhang. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkage between 2 nucleosides. In a further embodiment, the strands comprise a phosphorothioate internucleotide (PS) linkage adjacent to deoxyribonucleosides (D) or ribonucleosides (R). The PS linkage can 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 can be between positions 1-2, positions 2-3 and positions 19-20 and/or 20-21 counting from the 5′ end of the antisense strand. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strands and/or 2 nucleosides at the 3′ end of the strands. In an embodiment, the oligomeric compound comprising Formulas (I) and (IV) is a siRNA. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid comprises a sense strand having 19 to 23 linked nucleotides, wherein the sense strand sequence is represented by Formula (II): 5′ Y-Z-(Y)q-FFNM-(Y)q-M-(Y)v-D-D 3′. The 1) 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 2-3, v is 0-1, and no single modification type modifies more than two consecutive nucleotides. Preferably, the -D-D is a TT or TA overhang. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkage between 2 nucleosides. In a further embodiment, the sense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkage can be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5′ side, the 3′ side or both sides. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand. In an embodiment, the oligomeric compound comprising Formula (II) is a ssRNA or siRNA. In certain embodiments, the FFNM is FFMM or FFRM, where R is a ribonucleoside. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid comprises an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (V): 5′ L-(Y)p-NM-(FMM)r-(Y)p-(Z)r 3′. The 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 a 5′ phosphate, 5′ vinyl phosphonate 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 comprises one or more phosphorothioate internucleotide (PS) linkage between 2 nucleosides. In a further embodiment, the antisense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkage can be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5′ side, the 3′ side or both sides. The PS linkage modification can be between positions 1-2, positions 2-3, positions 19-20 and/or positions 20-21 counting from the 5′ end of the antisense strand. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand. In an embodiment, the oligomeric compound comprising Formula (V) is a ssRNA or siRNA. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid comprising: (a) a sense strand having 19 to 23 linked nucleotides, wherein the sense strand sequence is represented by Formula (II): 5′ Y-Z-(Y)q-FFNM-(Y)q-M-(Y)v-D-D 3′, and (b) an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (V): 5′ L-(Y)p-NM-(FMM)r-(Y)p-(Z)r 3′. A duplex is formed by the sense and antisense strands, where the duplex region is 19 to 23 nucleotide pairs in length. The 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 a 5′ phosphate, 5′ vinyl phosphonate 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 2-3, p is 3-5, r is 1-2, v is 0-1, and no single modification type modifies more than two consecutive nucleotides. In a further embodiment, the 3′ Z in the antisense strand is a UU or TT overhang and the -D-D in the sense strand is a T or TA overhang. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkage between 2 nucleosides. In a further embodiment, the strands comprise a phosphorothioate internucleotide (PS) linkage adjacent to deoxyribonucleosides (D) or ribonucleosides (R). The PS linkage can 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 can be between positions 1-2, positions 2-3, positions 19-20 and/or positions 20-21 counting from the 5′ end of the antisense strand. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strands and/or 2 nucleosides at the 3′ end of the strands. In an embodiment, the oligomeric compound comprising Formulas (II) and (V) is a siRNA. In certain embodiments, the FFNM in Formula (II) is FFMM or FFRM, where R is a ribonucleoside. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In certain embodiments, the modifications on the nucleotides comprise modified sugar moieties. In certain embodiments, the modifications on the nucleotides are selected from the group consisting of a deoxyribonucleoside (also referred to herein as a DNA nucleoside) substituted for a ribonucleoside (also referred to herein as a RNA nucleoside), 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, the “N” designation in a formula is a modified or unmodified nucleoside wherein the modifications can be selected from the group consisting of a deoxyribonucleoside (also referred to herein as a DNA nucleoside) substituted for a ribonucleoside (also referred to herein as a RNA nucleoside), 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. The modifications can also be selected from base substitutions described hereinbelow.

In certain embodiments, a FFNM motif occurs at or near a cleavage site of the sense strand and/or a (FMM)r motif occurs at or near a cleavage site of the antisense strand, where N is a modified or unmodified nucleoside (e.g., M, F, R, D, UNA, or LNA), F is a 2′-Fluoro, M is a 2′-OMe and r is 1-2. In certain embodiments, the FFNM is FFMM or FFRM, where R is a ribonucleoside.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid comprises a sense strand having 21 linked nucleotides, wherein the sense strand sequence is represented by Formula (III): 5′ M*F*MMN*MN*MFFNMN*MN*MMFM*D*D 3′. The 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 an embodiment, the oligomeric compound comprising Formula (III) is a ssRNA or siRNA. In certain embodiments, the FFNM is FFMM or FFRM, where R is a ribonucleoside. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid comprises an antisense strand having 21 linked nucleotides, wherein the antisense strand sequence is represented by Formula (VI): 5′ L-M*N*MNMFNMFMMNMFMFMMN*M*M 3′. The 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, 5′ vinyl phosphonate or 5′ OH, and no single modification type modifies more than two consecutive nucleotides. In an embodiment, the oligomeric compound comprising Formula (VI) is a ssRNA or siRNA. In certain embodiments, the FNM is FMM. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid comprises: (a) a sense strand having 21 linked nucleotides, wherein the sense strand sequence is represented by Formula (III): 5′ M*F*MMN*MN*MFFNMN*MN*MMFM*D*D 3′, and (b) antisense strand having 21 linked nucleotides, wherein the antisense strand sequence is represented by Formula (VI): 5′ L-M*N*MNMFNMFMMNMFMFMMN*M*M 3′. A duplex is formed by the sense and antisense strands, where the duplex region is 19 nucleotide pairs in length and each strand has a 2 nucleotide overhang at the 3′ end. The 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, 5′ vinyl phosphonate or 5′ OH, and no single modification type modifies more than two consecutive nucleotides. In an embodiment, the oligomeric compound comprising Formulas (III) and (VI) is a siRNA. In certain embodiments, the FFNM in Formula (III) is FFMM or FFRM, where R is a ribonucleoside. In certain embodiments, the FNM in Formula (VI) is FMM. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid comprises an antisense strand having 21 linked nucleotides, wherein the antisense strand sequence is represented by Formula (VII): 5′ L-M*D*MFMFNMFMMFMFMFMMN*M*M 3′. The 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′ OH, 5′ vinyl phosphonate or a 5′ phosphate (p), and no single modification type modifies more than two consecutive nucleotides. In an embodiment, the oligomeric compound comprising Formula (VII) is a ssRNA or siRNA. In certain embodiments, the FNM is FMM. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid comprises: (a) a sense strand having 21 linked nucleotides, wherein the sense strand sequence is represented by Formula (III): 5′ M*F*MMN*MN*MFFNMN*MN*MMFM*D*D 3′, and (b) antisense strand having 21 linked nucleotides, wherein the antisense strand sequence is represented by Formula (VII): 5′ L-M*D*MFMFNMFMMFMFMFMMN*M*M 3′. A duplex is formed by the sense and antisense strands, where the duplex region is 19 nucleotide pairs in length and each strand has a 2 nucleotide overhang at the 3′ end. The 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, 5′ vinyl phosphonate or 5′ OH, and no single modification type modifies more than two consecutive nucleotides. In an embodiment, the oligomeric compound comprising Formulas (III) and (VII) is a siRNA. In certain embodiments, the FFNM in Formula (III) is FFMM or FFRM, where R is a ribonucleoside. In certain embodiments, the FNM in Formula (VII) is FMM. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid comprises an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (IX): 5′ L-M-(Y)p-Z-(Y)p-(Z)r 3′. The M is a 2′-OMe modified nucleoside, L is a 5′ phosphate, 5′ vinyl phosphonate 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 ITT overhang. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkage between 2 nucleosides. In a further embodiment, the antisense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkage can be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5′ side, the 3′ side or both sides. The PS linkage modification can be between positions 1-2, positions 2-3, 19-20 and/or positions 20-21 counting from the 5′ end of the antisense strand. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand. In an embodiment, the oligomeric compound comprising Formula (IX) is a ssRNA or siRNA. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the 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)n-Z-(Y)r-D-D 3′, and (b) an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (IX): 5′ L-M-(Y)p-Z-(Y)p-(Z)r 3′. A duplex is formed by the sense and antisense strands, where the duplex region is 19 to 23 nucleotide pairs in length. The D is a deoxyribonucleoside, N is a modified or unmodified nucleoside, M is a 2′-OMe modified nucleoside, L is a 5′ phosphate, 5′ vinyl phosphonate 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, p is 3-5, r is 1-2, and no single modification type modifies more than two consecutive nucleotides. In a further embodiment, the 3′ Z in the antisense strand is a UU or TT overhang and the -D-D in the sense strand is a TT or TA overhang. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkage between 2 nucleosides. In a further embodiment, the strands comprise a phosphorothioate internucleotide (PS) linkage adjacent to deoxyribonucleosides (D) or ribonucleosides (R). The PS linkage can 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 can be between positions 1-2, positions 2-3 and positions 19-20 and/or 20-21 counting from the 5′ end of the antisense strand. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strands and/or 2 nucleosides at the 3′ end of the strands. In an embodiment, the oligomeric compound comprising Formulas (I) and (IX) is a siRNA. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprises a sense strand having 21 linked nucleotides, wherein the sense strand sequence is represented by Formula (X): 5′ MFMMNMNMFFNMNNMMNMDD 3′. The 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 no single modification type modifies more than two consecutive nucleotides. In certain embodiments, the sense strand comprises one or more phosphorothioate internucleotide (PS) linkage between 2 nucleosides. In a further embodiment, the sense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkage can be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5′ side, the 3′ side or both sides. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand. In an embodiment, the oligomeric compound comprising Formula (X) is a ssRNA or siRNA. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprises an antisense strand having 21 linked nucleotides, wherein the antisense strand sequence is represented by Formula (VIII): 5′ L-MNMNMFNMFMMNMFMEMMNMM 3′. The 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 a 5′ OH, 5′ vinyl phosphonate or a 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) linkage between 2 nucleosides. In a further embodiment, the antisense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkage can be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5′ side, the 3′ side or both sides. The PS linkage modification can be between positions 1-2, positions 2-3, 19-20 and/or positions 20-21 counting from the 5′ end of the antisense strand. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand. In an embodiment, the oligomeric compound comprising Formula (VIII) is a ssRNA or siRNA. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprises: (a) a sense strand having 21 linked nucleotides, wherein the sense strand sequence is represented by Formula (X): 5′ MFMMNMNMFFNMNMNMMNMDD 3′, and (b) antisense strand having 21 linked nucleotides, wherein the antisense strand sequence is represented by Formula (VIII): 5′ L-MNMNMFNMFMMNMFMFMMNMM 3′. A duplex is formed by the sense and antisense strands, where the duplex region is 19 nucleotide pairs in length and each strand has a 2 nucleotide overhang at the 3′ end. The D is a deoxyribonucleoside, M is a 2′-OMe modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, I), UNA, or LNA), F is a 2′-F modified nucleoside, L is a 5′ OH, 5′ vinyl phosphonate or a 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) linkage between 2 nucleosides. In a further embodiment, the strands comprise a phosphorothioate internucleotide (PS) linkage adjacent to deoxyribonucleosides (D) or ribonucleosides (R). The PS linkage can 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 can be between positions 1-2, positions 2-3 and positions 19-20 and/or 20-21 counting from the 5′ end of the antisense strand. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strands and/or 2 nucleosides at the 3′ end of the strands. In an embodiment, the oligomeric compound comprising Formulas (X) and (VIII) is a siRNA. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprises a sense strand having 21 linked nucleotides, wherein the sense strand sequence is represented by Formula (XI): 5′ MFMMNMNMFFMMNMNMMFMDD 3′. The D is a deoxyribonucleoside, M is a 2′-OMe modified nucleoside, N is a modified or unmodified nucleoside (e.g., M, F, R, I), UNA, or LNA), 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) linkage between 2 nucleosides. In a further embodiment, the sense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkage can be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5′ side, the 3′ side or both sides. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand. In an embodiment, the oligomeric compound comprising Formula (XI) is a ssRNA or siRNA. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprises an antisense strand having 21 linked nucleotides, wherein the antisense strand sequence is represented by Formula (XII): 5′ L-MDMFMFNMFMMFMFMFMMNMM 3′. The 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 a 5′ OH, 5′ vinyl phosphonate or a 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) linkage between 2 nucleosides. In a further embodiment, the antisense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkage can be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5′ side, the 3′ side or both sides. The PS linkage modification can be between positions 1-2, positions 2-3, 19-20 and/or positions 20-21 counting from the 5′ end of the antisense strand. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand. In an embodiment, the oligomeric compound comprising Formula (XII) is a ssRNA or siRNA. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound.

In one embodiment, an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprises: (a) a sense strand having 21 linked nucleotides, wherein the sense strand sequence is represented by Formula (XI): 5′ MFMMNMNMFFMNNMNMMFMDD 3′, and (b) antisense strand having 21 linked nucleotides, wherein the antisense strand sequence is represented by Formula (XII): 5′ L-MDMFMFNMFMMFMFMFMMNMM 3′. A duplex is formed by the sense and antisense strands, where the duplex region is 19 nucleotide pairs in length and each strand has a 2 nucleotide overhang at the 3′ end. The 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 a 5′ OH, 5′ vinyl phosphonate or a 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) linkage between 2 nucleosides. In a further embodiment, the strands comprise a phosphorothioate internucleotide (PS) linkage adjacent to deoxyribonucleosides (D) or ribonucleosides (R). The PS linkage can 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 can be between positions 1-2, positions 2-3 and positions 19-20 and/or 20-21 counting from the 5′ end of the antisense strand. The PS linkage can also be adjacent to 2 nucleosides at the 5′ end of the strands and/or 2 nucleosides at the 3′ end of the strands. In an embodiment, the oligomeric compound comprising Formulas (XI) and (XII) is a siRNA. Modified nucleosides may comprise a modified base and/or a modified sugar (preferably at 2′ position modified sugar). A deoxyribonucleoside (D) is also considered a modified nucleoside in a RNA based oligomeric compound. Oligomeric compounds can be trafficked into target cells by a variety of modalities. In certain embodiments, oligomeric compounds enter cells via viral delivery vectors, lipid-based delivery, polymer-based delivery, and/or conjugate-based delivery.

In certain embodiments, the oligomeric compound described herein further comprises a conjugate. The conjugate can be selected from cholesterols, lipids, carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. In a preferred embodiment, the conjugate is N-Acetylgalactosamine (GalNAc). In an embodiment, the conjugate can be attached to the 3′ end of a sense strand. In a preferred embodiment, the conjugated oligomeric compound is a siRNA-GalNAc conjugate.

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

In certain embodiments, a pharmaceutical composition comprises the oligomeric compound described herein, alone or in combination with a pharmaceutically acceptable carrier or excipient.

Certain embodiments provide a method for inhibiting the expression of a target nucleic acid in a subject comprising the step of administering the oligomeric compound described herein to the subject, in an amount sufficient to inhibit expression of the target nucleic acid. The oligomeric compound can be administered subcutaneously or intravenously to the subject.

Oligomeric Compounds

Oligomeric compounds of the invention include, but are not limited to, single stranded oligomeric compounds such as microRNAs (miRNAs), single stranded RNAs (ssRNA) and antisense oligonucleotides (ASOs); and double stranded oligomeric compounds such as short hairpin RNAs (shRNAs) and small interfering RNAs (siRNAs). An oligomeric compound may be “antisense” or comprise an “antisense strand” to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

In certain embodiments, an oligomeric compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. For example, in certain such embodiments, a siRNA comprises an antisense strand which has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

In certain embodiments, an oligomeric compound is 12-30 subunits in length. In certain embodiments, an oligomeric compound is 18 to 30 subunits in length. In certain embodiments, an oligomeric compound is 12 to 22 subunits in length. In certain embodiments, an oligomeric compound is 14 to 30 subunits in length. In certain embodiments, an oligomeric compound is 14 to 21 subunits in length. In certain embodiments, an oligomeric compound is 15 to 30 subunits in length. In certain embodiments, an oligomeric compound is 15 to 21 subunits in length. In certain embodiments, an oligomeric compound is 16 to 30 subunits in length. In certain embodiments, an oligomeric compound is 16 to 21 subunits in length. In certain embodiments, an oligomeric compound is 17 to 30 subunits in length. In certain embodiments, an oligomeric compound is 17 to 21 subunits in length. In certain embodiments, an oligomeric compound is 18 to 30 subunits in length. In certain embodiments, an oligomeric compound is 18 to 21 subunits in length. In certain embodiments, an oligomeric compound is 20 to 30 subunits in length. In certain embodiments, an oligomeric compound is 15 subunits in length. In certain embodiments, an oligomeric compound is 16 subunits in length. In certain embodiments, an oligomeric compound is 17 subunits in length. In certain embodiments, an oligomeric compound is 18 subunits in length. In certain embodiments, an oligomeric compound is 20 subunits in length. In certain embodiments, an oligomeric compound is 21 subunits in length. In certain embodiments, an oligomeric compound is 22 subunits in length. In certain embodiments, an oligomeric compound is 23 subunits in length. In certain embodiments, an oligomeric compound is 25 subunits in length. In certain embodiments, an oligomeric compound is 25 subunits in length. In other embodiments, the oligomeric compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such 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, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the oligomeric compound is a siRNA.

It is possible to increase or decrease the length of an oligomeric compound, such as a siRNA, and/or introduce base mismatch(s) without eliminating activity (U.S. Pat. No. 7,772,203, incorporated-by-reference herein). For example, it is possible to introduce non-canonical base pairings (e.g., A:G, A:C, G:U, LU, I:A, or I:C) into an oligomeric compound without eliminating activity. In certain embodiments, designing oligomeric compounds with one or more non-canonical base pairings, i.e., mismatch(s), enhances the activity of the oligomeric compound.

The oligomeric compound can comprise a mismatch(es) with the target, between the oligomeric strands within the duplex, or combinations thereof. The mismatch may occur throughout the siRNA such as in the overhang region or the duplex region.

Oligomeric Compound Motifs

A motif refers to a pattern of modification of an oligomeric compound. Various motifs have been described in the art and are incorporated-by-reference herein (e.g., U.S. Pat. Nos. 11,203,755; 10,870,849; EP Patent 1,532,248; U.S. Pat. Nos. 11,406, 716; 10,668,170; 9,796,974; 8,754,201; 10,837,013; 7,732,593; 7,015,315; 7,750,144; 8,420,799; 8,809,516; 8,796,436; 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; USSN 2020/0031862; and USSN 2016/(0272970). However, it would be clear to skilled artisans that new and improved motifs (also referred to herein as chemical modification patterns) are within the scope of the invention.

In certain embodiments, oligomeric compounds disclosed herein have chemically modified subunits arranged into motifs or patterns (i.e. chemical modification motifs/patterns) to confer on to the oligomeric compounds beneficial properties including, but not limited to: enhanced inhibitory activity to increase potency; increased binding affinity to increase specificity for a target nucleic acid, thereby limiting off-target effects and increasing safety; or enhanced resistance to degradation by in vivo nucleases thereby increasing stability and durability. In certain embodiments, the oligomeric compounds are chimeras where the peripheral nucleobases of the oligomeric compounds comprise motifs with various modified or unmodified nucleobases so as to confer increased stability, specificity, safety and potency, while the central region of the compound comprises various modified or unmodified nucleobases to serve as substrates for RISC mediated degradation. Each distinct region can comprise uniform sugar moieties, modified, or alternating sugar moieties. Each region can comprise a varied pattern of phosphate and phosphorothioate linkages.

In certain embodiments, the oligomeric compounds targeted to a nucleic acid comprises a sense strand with motif described by the one of the following formulas:

Formula (I):

5′ M-(Y)n-Z-(Y)r-D-D 3′,

Formula (II):

5′ Y-Z-(Y)q-FFNM-(Y)q-M-(Y)v-D-D 3′,

Formula (III):

5′ M*F*MMN*MN*MFFNMN*MN*MMFM*D*D 3′,

Formula (X):

5′ MFMMNMNMFFNMNMNMMNMDD 3′,

or

Formula (XI):

5′ MFMMNMNMFFMMNMNMMFMDD 3′,

wherein

- each D is a deoxyribonucleoside (D is a modification of R),
- each R is a ribonucleoside,
- each N is a nucleoside, modified or unmodified (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) linkage,
- each Y is two adjacent nucleosides with different modifications (e.g., MD, DM, DF, FD, MF or FM) or a modified nucleoside adjacent to an unmodified nucleoside (e.g., DR, RID, MR or RM),
- each Z is two adjacent unmodified nucleosides or two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides (e.g., MM, DD, RR, or FF),
- each n is 6-8,
- each q is 2-3,
- each r is 1-2,
- each v is 0-1, and
  
  wherein no single modification type modifies more than two consecutive nucleotides. In certain embodiments, the FFNM is FFRM or FFMM.

In certain embodiments, the oligomeric compounds targeted to a nucleic acid comprises an antisense strand with motif described by one of the following formulas:

Formula (IV):

5′-L-M-(D)v-(Y)s-(Z)t-(Y)u-Z-N-(Z)r 3′,

Formula (V):

5′ L-(Y)p-NM-(FMM)r-(Y)p-(Z)r 3′,

Formula (VI):

5′ L-M*N*MNMFNMFMMNMFMFMMN*M*M 3′,

Formula (VII):

5′ L-M*D*MFMFNMFMMFMFMFMMN*M*M 3′,

Formula (VIII):

5′ L-MNMNMFNMFMMNMFMFMMNMM 3′,

Formula (IX):

5′ L-M-(Y)p-Z-(Y)p-(Z)r 3′,

or

Formula (XII):

5′ L-MDMFMFNMFMMFMFMFMMNMM 3′,

wherein

- each D is a deoxyribonucleoside (D is a modification of R),
- each R is a ribonucleoside.
- each N is a nucleoside, modified or unmodified (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, 5′ vinyl phosphonate or 5′ OH
- each * is a phosphorothioate (PS) linkage,
- each Y is two adjacent nucleosides with different modifications (e.g., MD, DM, DF, FD, MF or FM) or a modified nucleoside adjacent to an unmodified nucleoside (e.g., DR, RD, MR or RM),
- each Z is two adjacent unmodified nucleosides or two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides (e.g., MM, DD, RR, or FF), each (5p) is 5′-phosphate,
- each n is 6-8,
- each p is 3-5,
- each r is 1-2,
- each v is 0-1.
- each s is 2-7,
- each t is 0-2,
- and
  
  wherein no single modification type modifies more than two consecutive nucleotides. In certain embodiments, the FNM is FMM.

In certain embodiments, the oligomeric compounds targeted to an mRNA nucleic acid comprises siRNA duplex with any of the following motifs:

- Duplex I with the sense strand of Formula (I) and antisense strand of Formula (IV);
- Duplex II with the sense strand of Formula (II) and antisense strand of Formula (V);
- Duplex III with the sense strand of Formula (III) and antisense strand of Formula (VI);
- Duplex IV with the sense strand of Formula (III) and antisense strand of Formula (VII);
- Duplex V with the sense strand of Formula (I) and antisense strand of Formula (IX);
- Duplex VI with the sense strand of Formula (X) and antisense strand of Formula (VIII);
- Duplex VII with the sense strand of Formula (XI) and antisense strand of Formula (XII);
- Duplex VIII with the sense strand of Formula (X) and antisense strand of Formula (XII); or
- Duplex IX with the sense strand of Formula (XI) and antisense strand of Formula (VIII);
  
  wherein the duplex region is 19 to 23 nucleotide pairs in length, and wherein no single modification type modifies more than two consecutive nucleotides.
  
  Target mRNAs and Associated Gene Expression

Several embodiments are directed to methods of modulating gene expression by oligomeric compound inhibition.

In certain embodiments, a method of inhibiting laminin (LMNA) gene expression in a cell comprises administering to the cell an oligomeric compound targeted to an mRNA transcript of LMNA. In an embodiment, the oligomeric compound is designed to target a 19 nucleotide long sequence of LMNA conserved between human and mouse (SEQ ID NO: 1).

In certain embodiments, a method of inhibiting apolipoprotein C3 (ApoC3) gene expression in a cell comprises administering to the cell an oligomeric compound targeted to an mRNA transcript of ApoC3. In an embodiment, the oligomeric compound is designed to target a 19 nucleotide long sequence of ApoC3 conserved between human and mouse (SEQ ID NO: 177).

In certain embodiments, a method of inhibiting Nucleolin (NCL) gene expression in a cell comprises administering to the cell an oligomeric compound targeted to an mRNA transcript of NCL. In an embodiment, the oligomeric compound is designed to target a 19 nucleotide long sequence of NCL conserved between human and mouse (SEQ ID NO: 98).

Hybridization

In some embodiments, hybridization occurs between an oligomeric compound disclosed herein and an mRNA. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

In Watson-Crick canonical base pairings, 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 usually Watson-Crick base pairs (C:G, A:U, A:T), but, non-canonical base pairs such as Hoogsteen base pairs (e.g., A:G or A:U), Wobble base pairs (e.g., G:U, I:U, I:A, I:C, wherein I is hypoxanthine) and the like are also permitted during hybridization of the oligomeric compound to a target nucleic acid or target region. Wobble base pairs in RNAi agents have previously been described (see e.g., U.S. Pat. Nos. 7,732,593; 7,750,144).

Nucleobase complementarity facilitates hybridization of the oligomeric compounds described herein to their target nucleic acids with the stronger the pairing (e.g., the more base pairs and/or the stronger the hydrogen bond), the stronger the hybridization of the oligomeric compound to the target. Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the oligomeric compound to be hybridized.

Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the oligomeric compounds provided herein are specifically hybridizable with a target mRNA with little to no off-target binding.

Complementarity

An oligomeric compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the oligomeric compound can hybridize with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., inhibition of a target nucleic acid, such as an mRNA nucleic acid).

Non-complementary nucleobases between an oligomeric compound and an mRNA nucleic acid may be tolerated provided that the oligomeric compound remains able to specifically hybridize to a target nucleic acid. Moreover, an oligomeric compound may hybridize over one or more segments of an mRNA nucleic acid such that intervening or adjacent segments is not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

In certain embodiments, the oligomeric compounds provided herein, or a specified portion thereof, are, or 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, a target region, target segment, or specified portion thereof. Percent complementarity of an oligomeric compound with a target nucleic acid can be determined using routine methods.

For example, an oligomeric compound in which 18 of 20 nucleobases of the antisense strand of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. Percent complementarity of an oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., 1990, J. Mol. Biol., 215:403-410; Zhang and Madden, 1997, Genome Res., 7:649-656) and available through the website for the National Center for Biotechnology Information (NCBI, https://blast.ncbi.nlm.nih.gov/Blast.cgi). Percent homology, sequence identity or complementarity, can be determined by, for example, NCBI Blast (Johnson et al., Nucleic Acids Res. 2008, 36 (Web Server issue): W5-W9).

In certain embodiments, the oligomeric compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an oligomeric compound may be fully complementary to an mRNA nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an oligomeric compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase oligomeric compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the oligomeric compound.

Fully complementary can also be used in reference to a specified portion of the oligomeric compound or the nucleic acid target. For example, a 20 nucleobase portion of a 30 nucleobase oligomeric compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligomer is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein 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 the target sequence, depending on whether the remaining 10 nucleobases of the oligomeric compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the oligomeric compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the oligomeric compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous.

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 comprise 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(s) relative to a target nucleic acid, such as an mRNA nucleic acid, or specified portion thereof.

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 comprise 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(s) relative to a target nucleic acid, such as an mRNA nucleic acid, or specified portion thereof.

The oligomeric compounds provided herein also include those which are complementary to a portion of a target nucleic acid. As used herein, “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 of an oligomeric compound. In certain embodiments, the oligomeric compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the oligomeric compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the oligomeric compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the oligomeric compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the oligomeric compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the oligomeric compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the oligomeric compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the oligomeric compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are oligomeric compounds that are 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.

Identity

The oligomeric compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific ARNATAR number, or portion thereof. As used herein, an oligomeric compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the oligomeric compounds described herein as well as compounds having non-identical bases relative to the oligomeric compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the oligomeric compound. Percent identity of an oligomeric compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.

In certain embodiments, a portion of the oligomeric compound is compared to an equal length portion of the target nucleic acid. 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 an equal length portion of the target nucleic acid.

Chemical Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a covalent linkage (e.g., phosphate group or a chemically modified linkage as described infra) to the sugar portion of the nucleoside. Oligonucleotides are formed through the covalent linkage of adjacent nucleotides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the linkage groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide. Oligomeric compounds are made up of one (e.g., ssRNAs, antisense oligonucleotides or miRNAs) or more oligonucleotides (e.g., siRNAs or shRNAs)

Modifications to oligomeric compounds encompass substitutions or changes to nucleobases, internucleoside linkages or sugar moieties. Modified oligomeric compounds are often preferred over native or unmodified forms because of desirable properties such as, for example, enhanced delivery (e.g., increased cellular uptake), enhanced specificity or affinity for a nucleic acid target, increased stability in the presence of nucleases, enhanced safety (e.g., fewer side effects after administration of the compound to a subject) or increased potency (e.g., inhibitory activity).

Nucleobase Modifications

A nucleobase is a heterocyclic moiety capable of base pairing with a nucleobase of another nucleic acid. Modifications to nucleobases can be advantageous to an oligomeric compound for various reasons including, but not limited to, increase stability of the oligomeric compound, increase specificity, decreased immunogenicity of the oligomeric compound, increase affinity of the oligomeric compound, increase potency of the oligomeric compound and other desirable features.

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 comprise substituting nucleobases with nucleobase analogs or modification of a part of the nucleobase. Examples of nucleobase modifications include, but are not limited to, 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 the like.

In certain embodiments, oligomeric compounds targeted to an mRNA nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobases are, for example, deoxyribonucleotides substituted for ribonucleotides. In certain embodiments, a modified nucleobase can be a thymine substitution for an uracil. In certain embodiments, multiple nucleobases of an oligomeric compound are modified. In certain embodiments, each nucleobase of an oligomeric compound is modified.

Internucleoside Linkage Modifications

The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. For nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked 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 selected over oligomeric compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, decrease toxicity, increased stability and durability, decreased degradation and other desirable features for an oligomeric compound. 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).

Oligomeric compounds having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates (e.g., 5′-methylphosphonate (5′-MP)), phosphoramidate, phosphorothioates (e.g., phosphorodithioate Rp isomer (PS,Rp), phosphorodithioate Rp isomer (PS,Sp), or 5′-phosphorothioate (5′-PS)), methoxypropylphosphonate, (S)-5′-C-methyl with Phosphate, peptide nucleic acid (PNA), and 5′-(E)-vinylphosphonate.

In certain embodiments, oligomeric compounds targeted to an mRNA nucleic acid 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 linkage of an oligomeric compound is a phosphorothioate internucleoside linkage. In certain embodiments, the PS linkage is adjacent to a deoxyribonucleoside (sometimes referred to as a DNA nucleoside, “)” or “d” herein) or a ribonucleoside (sometimes referred to as a RNA nucleoside, “R” or “r” herein). In certain embodiments, each internucleoside linkage of an oligomeric compound is a phosphorothioate internucleoside linkage.

Sugar Modifications

Oligomeric compounds provided herein can contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart desirable features such as increased stability, increased durability (e.g., increased half-life), increased binding affinity, decreased off-target effects, decreased immunogenicity, decreased toxicity, increased potency, or some other beneficial biological property to the oligomeric compounds. Sugar modifications and their advantages are known in the art (Friedrich and Aigner, 2022, BioDrugs, 36(5):549-571; Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101; Chiu and Rana, 2003, RNA, 9:1034-1048; Choung et al., Biochem Biophys Res Commun, 2006, 342:919-927; Amarzguioui et al., 2003, Nucleic Acids Res, 31(2):589-595; Braasch et al., 2003, Biochemistry, 42(26):7967-7975; Czauderna et al., 2003, Nucleic Acids Res, 31(11):2705-2716; Allerson et al., 2005, J Med Chem, 48:901-904; Layzer et al., 2004, RNA, 10:766-771; Ui-Tei, et al., 2008, Nucleic Acids Res, 36(7):2136-51; Bramsen and Kjems, 2012, Frontiers in Genetics, 3(154):1-22; Bramsen et al., 2010, Nucleic Acids Res, 38(17):5761-5773; Muhonen et al., 2007, Chem & Biodiversity, 4:858-873; Viel et al., 2008, Oligonucleotides, 18:201-212; which are incorporated-by-reference herein).

In certain embodiments, nucleosides comprise a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings can include, without limitation, addition of substitutent groups (e.g., 5′ sugar modifications, or 2′ sugar modifications); bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA); replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R)2 (R═H, C₁-C₁₂alkyl or a protecting group); nucleoside mimetic; and combinations thereof.

A 2′-modified sugar refers to a furanosyl sugar modified at the 2′ position. A 2′-modified nucleoside refers to a nucleoside comprising a sugar modified at the 2′ position of a furanose ring. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2′ modifications are selected from substituents including, but not limited to: O[(CH₂)_nO]_mCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_nON[(CH₂)_nCH₃]₂, where n and m are from 1 to about 10. Other 2′-substituent groups can also be selected from: C₁-C₁₂alkyl, 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, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, and a group for improving the pharmacodynamic properties of an oligomeric compound, and other substituents having similar properties.

Further examples of nucleosides having modified sugar moieties include, without limitation, nucleosides comprising 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, phosphorodiamidate morpholino (PMO), tricyclo-DNA (tcDNA), 2′-arabino-fluoro, 2′-O-benzyl, glycol nucleic acid (GNA), and unlocked nucleic acid (UNA) substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl. O—C₁-C₁₀alkyl, OCF₃, O(CH₂)2SCH₃, O(CH₂)2-O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C₁-C₁₀alkyl. 2′-OMe or 2′-OCH₃or 2′-O-methyl each refers to a nucleoside comprising a sugar comprising an —OCH₃group at the 2′ position of the sugar ring. 2′-F refers to a sugar comprising a fluoro group at the 2′ position. 2′-O-methoxyethyl or 2′-O-MOE or 2′-MOE each refers to a nucleoside comprising a sugar comprising an —O(CH₂)2OCH₃group at the 2′ position of the sugar ring.

BNAs refer to modified nucleosides comprising a bicyclic sugar moiety wherein a bridge connecting two carbon atoms of the sugar ring connects the 2′ carbon and another carbon of the sugar ring. Examples of bicyclic nucleosides include, without limitation, nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms such as in locked nucleic acid (LNA). In certain embodiments, oligomeric compounds provided herein include one or more bicyclic nucleosides wherein the bridge comprises a 4′ to 2′ bicyclic nucleoside. LNAs and UNAs have been described by Campbell and Wengel (Chem Soc Rev, 2011, 40(12):5680-9) and are incorporated-by-reference herein.

In certain embodiments, oligomeric compounds comprise one or more nucleotides having modified sugar moieties. In certain embodiments, the modified sugar moiety has a 2′-OMe modification. In certain embodiments, the modified sugar moiety has 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)—(VII).

Oligomeric Compound Delivery Systems

Oligomeric compounds require entry into target cells to become active. A variety of modalities have been used to traffic 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).

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

Polymer-based particles are also used in oligomeric compound delivery systems. Such polymers include poly(lactic-co-glycolic acid) (PLGA), polyethylenimine (PEI), poly(l-lysine) (PLL), poly(beta-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 traits desired for delivery of the oligomeric compound.

The oligomeric compounds disclosed herein may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting compound. Conjugate groups can include cholesterols, lipids, carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, dyes, tocopherol (Nishina et al., 2008, Molecular Therapy, 16(4):734-740), etc. Conjugate-based delivery can actively deliver oligomeric compounds to specific cell types.

In an example, N-Acetylgalactosamine (GalNAc) is conjugated to an oligomeric compound and delivers it into hepatocytes. Various GalNAc conjugates can be found in several publications including the following, all of which are incorporated-by-reference herein: 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. Pat. No. 10,087,208; Prakash et al., 2014, Nucleic Acids Res, 42(13):8796-807; Debacker et al., 2020, Molecular Therapy, 28(8):1759-1771; U.S. Pat. Nos. 11,110,174; 9,796,756; 9,181,549; 10,344,275; 10,570,169; 9,506,030; and, U.S. Pat. No. 7,582,744.

In a further example, the following GalNAc is conjugated at the 3′ end of an oligonucleotide, wherein the oligonucleotide comprises the sense strand of an ARNATAR designed siRNA.

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Oligomeric Compound Synthesis

siRNAs were designed, synthesized, and prepared using methods known in the art.

Solid phase syntheses of oligonucleotides were done on a MerMade™ 48× synthesizer (BioAutomation, LGC, Biosearch Technologies. Hoddesdon, UK), which can make up to 48 1 μMole or 5 μMole scale oligonucleotides per run using standard phosphoramidite chemistry. Solid support is controlled pore glass (500-1400 Å) loaded with 3′-GalNAc conjugates (AM Chemicals, Vista, CA, USA; Primetech ALC, Minsk, Belarus; Gene Link. Elmsford, NY, USA; or any GalNAc conjugate disclosed herein) or universal solid support (AM Chemicals, Vista, CA, USA). Ancillary synthesis reagents and standard 2′-cyanoethyl phosphoramidite monomers (2′-fluoro nucleosides, 2′-O-methyl nucleosides, RNA nucleosides, DNA nucleosides) were obtained from various sources (Hongene Biotech, Shanghai, China; Sigma-Aldrich, St. Louis, MO, USA; Glen Research, Sterling, VA, USA; ThermoFisher Scientific, Waltham, MA, USA; LGC Biosearch Technologies, Hoddesdon, UK). Phosphoramidite mixtures were prepared in anhydrous acetonitrile or 30% DMF:acetonitrile and were coupled using 0.25M 4,5-dicyanoimidazole(DCI) (Sigma-Aldrich, St. Louis, MO, USA) with coupling times ranging from 120-360 seconds. Standard phosphodiester linkages were achieved using 0.02M iodine mixture in Tetrahydrofuran (THF), pyridine and water. Phosphorothiate linkages were generated using 0.05M sulfurizing Reagent II (3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT) (40:60, Pyridine/Acetonitrile) (LGC Biosearch Technologies, Hoddesdon, UK) with an oxidation time of 6 minutes. All sequences were synthesized with Dimethoxy Trityl (DMT) protecting group removed.

Upon completion of solid phase synthesis, the oligonucleotides were cleaved from the solid support and deprotection of base labile groups performed by incubation in ammonium hydroxide at 55° C. for 6 hours. Ammonium hydroxide was removed using a centrifugal vacuum concentrator to dryness at room temperature. For sequences containing natural ribonucleotides (2′-OH) protected with tert-butyl dimethyl silyl (TBDMS), a second deprotection was performed using triethylamine; trihydrofluoride (TEA:3HF). To each TBDMS protected oligonucleotide 100 μL DMSO and 125 μL TEA:3HF was added and incubated at 65° C. for 2.5 hours. After incubation 25 μL of 3 M sodium acetate was added to the solution which was subsequently precipitated in butanol at −20′C for 30 minutes. The cloudy solution was centrifuged to a cake at which time the supernatant was carefully decanted with a pipette. The standard precipitation process was then completed with 75% ethanol:water then 100% ethanol as supernatant solutions. The oligonucleotide cake was dried for 30 minutes in a centrifugal vacuum concentrator.

Desalting without HPLC purification was performed after precipitation with 3M sodium acetate with a follow on G25 Sephadex® column (Sigma-Aldrich, St. Louis, MO, USA) elution. Purification of oligonucleotides was afforded by anion exchange chromatography on a Gilson GX271 prep HPLC system (Middleton, WI, USA) using BioWorks Q40 resin (Uppsala, Sweden). Final desalt was performed by Sephadex® G25 column. All oligonucleotides were analyzed by ion pairing reverse phase HPLC for purity an Agilent 1200 analytical HPLC (Santa Clara, CA, USA), negative ion mass spectrometry for intact mass on an Agilent 6130 single quad mass spectrometer (Santa Clara, CA, USA), and A260 quantification by UV/Vis on a Tecan Infinite® M Plex plate reader (Zurich, Switzerland).

Double Stranded Oligomeric Compound Duplex Formation

In general, for a double stranded oligomeric compound such as an siRNA compound, a sense and antisense oligonucleotide is annealed together to form a duplex. Duplex formation of 50-300 μM can be achieved by heating samples at 94° C. for 4 mins in 1× phosphate-buffered saline in a block heater, followed by removal of the heating block containing the samples from the block heater and allowing it to gradually cool down to room temperature over a time course of 1 hr.

Compositions and Methods for Formulating Pharmaceutical Compositions

The oligomeric compounds of the invention, such as siRNA compounds described herein, can be combined with pharmaceutically acceptable active or inert substances, such as a diluent, excipient or carrier, for the preparation of pharmaceutical compositions or formulations.

Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

In certain embodiments, the pharmaceutical carrier or excipient is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acid compounds to an animal. The excipient can be liquid or solid and can be selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone and/or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates and/or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, and/or sodium acetate, etc.); disintegrants (e.g., starch, and/or sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipients, which do not deleteriously react with nucleic acid compounds, suitable for parenteral or non-parenteral administration can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an oligomeric compound and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the oligomeric compound is a siRNA.

Pharmaceutical compositions comprising oligomeric compounds such as siRNAs can encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other dsRNA which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn 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.

In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, and/or intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer (e.g., PBS). In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers.

Dosages

For purposes of the disclosure, the amount or dose of the active agent (oligomeric compound of the invention) administered should be sufficient to e.g., inhibit the expression of a target nucleic acid in an animal. In the animal (e.g., human), dose will be determined by the efficacy of the particular active agent and the condition of the animal, as well as the body weight of the animal to be treated.

Many assays for determining an administered dose are known in the art.

The dose of the active agent of the present disclosure also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular active agent of the present disclosure. Typically, the attending physician will decide the dosage of the active agent of the present disclosure with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, active agent of the present disclosure to be administered, route of administration, and the severity of the condition being treated.

Dosing

In certain embodiments, pharmaceutical compositions are administered according to a dosing regimen (e.g., dose, dose frequency, and duration) wherein the dosing regimen can be selected to achieve a desired effect. The desired effect can be, for example, reduction of a target nucleic acid or the prevention, reduction, amelioration or slowing the progression of a disease, disorder and/or condition, or symptom thereof, associated with the target nucleic acid. In certain embodiments, the variables of the dosing regimen are adjusted to result in a desired concentration of pharmaceutical composition in a subject. “Concentration of pharmaceutical composition” as used with regard to dose regimen can refer to the oligomeric compound or active ingredient of the pharmaceutical composition. For example, in certain embodiments, dose and dose frequency are adjusted to provide a tissue concentration or plasma concentration of a pharmaceutical composition at an amount sufficient to achieve a desired effect.

Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Dosing is also dependent on drug potency and metabolism. In certain embodiments, dosage is from 0.01 μg to 50 mg per kg of body weight, 0.01 μg to 100 mg per kg of body weight, or within a range of 0.001 mg to 1000 mg dosing, and may be given once or more daily, weekly, monthly, quarterly or yearly, or even once every 2 to 20 years. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligomeric compound is administered in maintenance doses, ranging from 0.01 μg to 100 mg per kg of body weight, once or more daily, once or more weekly, once or more monthly, once or more quarterly, once or more yearly, to once every 20 years or ranging from 0.001 mg to 1000 mg dosing. In certain embodiments, it may be desirable to administer the oligomeric compound from at most once daily, once weekly, once monthly, once quarterly, once yearly, once every two years, once every three years, once every four years, once every five years, once every ten years, to once every 20 years.

In certain embodiments, the range of dosing is between any of 1 mg-1500 mg, 100 mg-1400 mg, 100 mg-1300 mg, 100 mg-1200 mg, 100 mg-1100 mg, 100 mg-1000 mg, 100 mg-900 mg, 200 mg-800 mg, 300 mg-700 mg, 400 mg-600 mg, 100 mg-400 mg, 200 mg-500 mg, 300 mg-600 mg, and 400 mg-700 mg. In certain embodiments, a 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.

In certain embodiments, the dsRNA is dosed at any of about 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, or 900 mg twice a year. In certain embodiments, the dsRNA is dosed at about 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, or 900 mg quarterly.

Administration

The oligomeric compounds, such as siRNAs, or pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be oral, inhaled or parenteral.

In certain embodiments, the compounds and compositions as described herein are administered parenterally. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. In certain embodiments, parenteral administration is by infusion. Infusion can be chronic or continuous or short or intermittent. In certain embodiments, infused pharmaceutical agents are delivered with a pump.

In certain embodiments, parenteral administration is by injection. The injection can be delivered with a syringe or a pump. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to a tissue or organ.

In certain embodiments, formulations for parenteral, intrathecal or intraventricular administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

In certain embodiments, formulations for oral administration of the compounds or compositions can include, but is not limited to, pharmaceutical carriers, excipients, powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In certain embodiments, oral formulations are those in which compounds provided herein are administered in conjunction with one or more penetration enhancers, surfactants and chelators.

In Vitro Testing of siRNAs

Described herein are methods for treatment of cells with siRNAs, which can be modified appropriately for treatment with other oligomeric compounds.

Cells may be treated with siRNAs when the cells reach approximately 60-80% confluency in culture.

One reagent commonly used to introduce siRNAs into cultured cells includes the cationic lipid transfection reagent Lipofectamine™ RNAiMAX (Invitrogen, Waltham, MA), siRNAs may be mixed with Lipofectamine™ RNAiMAX in OPTI-MEM 1 (Thermo Fisher Scientific, Waltham, MA) to achieve the desired final concentration of siRNA and a Lipofectamine™ RNAiMAX concentration that may range from 0.001 to 300 nM siRNAs. Transfection procedures are done according to the manufacturer's recommended protocols.

Another technique used to introduce siRNAs into cultured cells includes electroporation.

siRNAs conjugated with GalNAc can be introduced to cells through incubation of the siRNAs with cells without transfection reagents, referenced herein as “free uptake”. The siRNA-GalNAc conjugates are transported into asialoglycoprotein receptor (ASGR) positive cells such as hepatocytes via endocytosis.

Cells are treated with siRNAs by routine methods. Cells may be harvested 4-144 hours after siRNAs treatment, at which time mRNA (harvested at 4-144 hrs) or protein levels (extracted at 24-96 hrs) of target nucleic acids are measured by methods known in the art and described herein. In general, treatments are performed in multiple replicates, and the data are presented as the average of the replicate treatments plus the standard deviation.

The concentration of siRNAs used varies from cell line to cell line and target to target. Methods to determine the optimal siRNAs concentration for a particular target in a particular cell line are well known in the art. In general, cells are treated with siRNAs in a dose dependent manner to allow for the calculation of the half-maximal inhibitory concentration value (IC50). siRNAs are typically used at concentrations ranging from 0.001 nM to 300 nM when transfected with Lipofectamine™ RNAiMAX. siRNAs are used at higher concentrations ranging from 7.5 to 20,000 nM when transfected using electroporation or free uptake.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Thermo Fisher Scientific, Waltham, MA), Qiagen RNeasy kit (Qiagen, Hilden, Germany), or AcroPrep Advance 96-well Filter Plates (Pall Corporation, Port Washington, New York) using Qiagen's RLT, RW1 and RPE buffers. RNA extraction procedures are done according to the manufacturer's recommended protocols.

In Vivo Testing of Oligomeric Compounds

The oligomeric compounds of the invention, for example, siRNAs, are tested in animals to assess their ability to inhibit expression of a target nucleic acid and produce phenotypic changes such as a change in one or more markers affected by the target nucleic acid. Also, the phenotypic change can be a decrease in a disease, disorder, condition, or symptom related to the target nucleic acid. Testing may be performed in normal animals, or in experimental disease models. For administration to animals, oligomeric compounds are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline (PBS). Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Calculation of dosage and dosing frequency depends upon factors such as route of administration and animal body weight. In one embodiment, following a period of treatment with oligomeric compounds of the invention, RNA encoding the target nucleic acid is isolated from liver tissue and changes in the target nucleic acid expression are measured. Changes in protein levels expressed by the target nucleic acid can also be measured.

Kits of the Invention

According to another aspect of the invention, kits are provided. Kits according to the invention include package(s) comprising any of the compositions of the invention or oligomeric compound of the invention. In various aspects, the kit comprises any of the compositions of the invention as a unit dose. For purposes herein “unit dose” refers to a discrete amount dispersed in a suitable carrier.

The phrase “package” means any vessel containing compositions presented herein. In preferred embodiments, the package can be a box or wrapping. Packaging materials for use in packaging pharmaceutical products are well known to those of skill 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 pre-filled syringes), bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

The kit can also contain items that are not contained within the package but are attached to the outside of the package, for example, pipettes.

Kits may optionally contain instructions for administering compositions of the present invention to a subject having a condition in need of treatment. Kits may also comprise instructions for approved uses of components of the composition herein by regulatory agencies, such as the United States Food and Drug Administration. Kits may optionally contain labeling or product inserts for the present compositions. The package(s) and/or any product insert(s) may themselves be approved by regulatory agencies. The kits can include compositions in the solid phase or in a liquid phase (such as buffers provided) in a package. The kits also can include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.

The kit may optionally also contain one or more other compositions for use in combination therapies as described herein. In certain embodiments, the package(s) is a container for any of the means for administration such as intravitreal delivery, intraocular delivery, intratumoral delivery, peritumoral delivery, intraperitoneal delivery, intrathecal delivery, intramuscular injection, subcutaneous injection, intravenous delivery, intra-arterial delivery, intraventricular delivery, intrasternal delivery, intracranial delivery, or intradermal injection.

Methods of Use

The invention provides methods for inhibiting the expression of a target nucleic acid in a subject comprising administering an effective amount of an oligomeric compound of the invention or a pharmaceutical composition of the invention, so as to inhibit the expression of a target nucleic acid in the subject.

In some embodiments of the present disclosure, the subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perissodactyla, including Equines (horses). In some aspects, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In a preferred aspect, the mammal is a human.

Specific Embodiments

Embodiment 1 of the invention comprises an oligomeric compound capable of inhibiting the 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)n-Z-(Y)r-D-D 3′

wherein:

- 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
  
  wherein no single modification type modifies more than two consecutive nucleotides.

The oligomeric compound of embodiment 1, wherein the -D-D is a TT or TA overhang.

The oligomeric compound of embodiment 1, wherein the modifications on the nucleotides are selected from the group consisting of a deoxyribonucleoside (DNA nucleoside) substituted for a ribonucleoside, locked nucleic acid (LNA), 2′-OMe, 2′-F, unlocked nucleic acid (UNA), and combinations thereof.

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

The oligomeric compound of embodiment 1, wherein the sense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand.

Embodiment 2 of the invention comprises an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprising an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (IV):

5′ L-M-(D)v-(Y)s-(Z)t-(Y)u-Z-N-(Z)r 3′

wherein:

- D is a deoxyribonucleoside,
- N is a modified or unmodified nucleoside,
- M is a 2′-OMe modified nucleoside,
- L is a 5′ phosphate, 5′ vinyl phosphonate 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,
- r is 1-2,
- v is 0-1,
- s is 2-7,
- t is 0-2,
- u is 0-5, and
  
  wherein no single modification type modifies more than two consecutive nucleotides.

The oligomeric compound of embodiment 2, wherein the 3′ Z is a UU or TT overhang.

The oligomeric compound of embodiment 2, wherein the modifications on the nucleotides are selected from the group consisting of a deoxyribonucleoside (DNA nucleoside) substituted for a ribonucleoside, locked nucleic acid (LNA), 2′-OMe, 2′-F, unlocked nucleic acid (UNA), and combinations thereof.

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

The oligomeric compound of embodiment 2, wherein the antisense strand comprises a phosphorothioate (PS) internucleotide linkage modification between positions 1-2, positions 2-3, positions 19-20 and positions 20-21 counting from the 5′ end of the antisense strand.

The oligomeric compound of embodiment 2, wherein the antisense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand.

Embodiment 3 of the invention comprises oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprising:

- a) a sense strand having 19 to 23 linked nucleotides, wherein the sense strand sequence is represented by Formula (I):

5′ M-(Y)n-Z-(Y)r-D-D 3′

- b) antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (IV):

5′L-M-(D)v-(Y)s-(Z)t-(Y)u-Z-N-(Z)r3′, and

- c) a duplex formed by the sense and antisense strands,
- wherein
  - D is a deoxyribonucleoside,
  - N is a modified or unmodified nucleoside,
  - M is a 2′-OMe modified nucleoside,
  - L is a 5′ phosphate, 5′ vinyl phosphonate 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,
  - u is 0-5, and
- wherein the duplex region is 19 to 23 nucleotide pairs in length, and
- wherein no single modification type modifies more than two consecutive nucleotides.

The oligomeric compound of embodiment 3, wherein the 3′ Z in the antisense strand is a UU or TT overhang.

The oligomeric compound of embodiment 3, wherein the -D-D in the sense strand is a TT or TA overhang.

The oligomeric compound of embodiment 3, wherein the modifications on the nucleotides are selected from the group consisting of a deoxyribonucleoside (DNA nucleoside) substituted for a ribonucleoside, locked nucleic acid (LNA), 2′-OMe, 2′-F, and unlocked nucleic acid (UNA), or combinations thereof.

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

The oligomeric compound of embodiment 3, wherein the antisense strand comprises a phosphorothioate (PS) internucleotide linkage modification between positions 1-2, positions 2-3, positions 19-20 and positions 20-21 counting from the 5′ end of the antisense strand.

The oligomeric compound of embodiment 3, wherein the strands comprise a phosphorothioate internucleotide (PS) linkage adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand.

Embodiment 4 of the invention comprises an oligomeric compound capable of inhibiting the 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′ Y-Z-(Y)q-FFNM-(Y)q-M-(Y)v-D-D 3′

wherein:

- 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 2-3,
- v is 0-1, and
  
  wherein no single modification type modifies more than two consecutive nucleotides.

The oligomeric compound of embodiment 4, wherein the -D-D is a TT or TA overhang.

The oligomeric compound of embodiment 4, wherein the modifications on the nucleotides are selected from the group consisting of a deoxyribonucleoside (DNA nucleoside) substituted for a ribonucleoside, locked nucleic acid (LNA), 2′-OMe, 2′-F, unlocked nucleic acid (UNA), and combinations thereof.

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

The oligomeric compound of embodiment 4, wherein the sense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand.

Embodiment 5 of the invention comprises an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprising an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (V):

5′ L-(Y)p-NM-(FMM)r-(Y)p-(Z)r 3′

wherein:

- M is a 2′-OMe modified nucleoside,
- F is a 2′-F modified nucleoside,
- L is a 5′ phosphate, 5′ vinyl phosphonate 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
  
  wherein no single modification type modifies more than two consecutive nucleotides.

The oligomeric compound of embodiment 5, wherein the 3′ Z is a UU or TT overhang.

The oligomeric compound of embodiment 5, wherein the modifications on the nucleotides are selected from the group consisting of a deoxyribonucleoside (DNA nucleoside) substituted for a ribonucleoside, locked nucleic acid (LNA), 2′-OMe, 2′-F, unlocked nucleic acid (UNA), and combinations thereof.

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

The oligomeric compound of embodiment 5, wherein the antisense strand comprises a phosphorothioate (PS) internucleotide linkage modification between positions 1-2, positions 2-3, positions 19-20 and positions 20-21 counting from the 5′ end of the antisense strand.

The oligomeric compound of embodiment 5, wherein the antisense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand.

Embodiment 6 of the invention comprises an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprising:

- a) a sense strand having 19 to 23 linked nucleotides, wherein the sense strand sequence is represented by Formula (II):

5′ Y-Z-(Y)q-FFNM-(Y)q-M-(Y)v-D-D 3′

- b) antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (V):

5′L-(Y)p-NM-(FMM)-(Y)p—(Z)r3′, and

- c) a duplex formed by the sense and antisense strands, wherein
  - 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 a 5′ phosphate, 5′ vinyl phosphonate 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 2-3,
  - p is 3-5,
  - r is 1-2,
  - v is 0-1,
- wherein the duplex region is 19 to 23 nucleotide pairs in length, and
- wherein no single modification type modifies more than two consecutive nucleotides.

The oligomeric compound of embodiment 6, wherein the 3′ Z in the antisense strand is a UU or TT overhang.

The oligomeric compound of embodiment 6, wherein the -D-D in the sense strand is a TT or TA overhang.

The oligomeric compound of embodiment 6, wherein the modifications on the nucleotides are selected from the group consisting of a deoxyribonucleoside (DNA nucleoside) substituted for a ribonucleoside, locked nucleic acid (LNA), 2′-OMe, 2′-F, unlocked nucleic acid (UNA), and combinations thereof.

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

The oligomeric compound of embodiment 6, wherein the antisense strand comprises a phosphorothioate (PS) internucleotide linkage modification between positions 1-2, positions 2-3 and positions 20-21 counting from the 5′ end of the antisense strand.

The oligomeric compound of embodiment 6, wherein the strands comprise a phosphorothioate internucleotide (PS) linkage adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand.

Embodiment 7 of the invention comprises an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprising an antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (IX):

5′ L-M-(Y)p-Z-(Y)p-(Z)r 3′

wherein:

- M is a 2′-OMe modified nucleoside,
- L, is a 5′ phosphate, 5′ vinyl phosphonate 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, and
  
  wherein no single modification type modifies more than two consecutive nucleotides.

The oligomeric compound of embodiment 7, wherein the 3′ Z is a UU or TT overhang.

The oligomeric compound of embodiment 7, wherein the modifications on the nucleotides are selected from the group consisting of a deoxyribonucleoside (DNA nucleoside) substituted for a ribonucleoside, locked nucleic acid (LNA), 2′-OMe, 2′-F, unlocked nucleic acid (UNA), and combinations thereof.

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

The oligomeric compound of embodiment 7, wherein the antisense strand comprises a phosphorothioate (PS) internucleotide linkage modification between positions 1-2, positions 2-3, positions 19-20 and positions 20-21 counting from the 5′ end of the antisense strand.

The oligomeric compound of embodiment 7, wherein the antisense strand comprises a phosphorothioate internucleotide (PS) linkage adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand.

Embodiment 8 of the invention comprises an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprising:

- a) a sense strand having 19 to 23 linked nucleotides, wherein the sense strand sequence is represented by Formula (I):

5′ M-(Y)n-Z-(Y)r-D-D 3′

- b) antisense strand having 19 to 23 linked nucleotides, wherein the antisense strand sequence is represented by Formula (IX):

5′L-M-(Y)p-Z-(Y)p—(Z)r3′, and

- c) a duplex formed by the sense and antisense strands,
- wherein
  - D is a deoxyribonucleoside,
  - M is a 2′-OMe modified nucleoside,
  - L is a 5′ phosphate, 5′ vinyl phosphonate 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,
  - p is 3-5,
  - r is 1-2,
- wherein the duplex region is 19 to 23 nucleotide pairs in length, and
- wherein no single modification type modifies more than two consecutive nucleotides,

The oligomeric compound of embodiment 8, wherein the 3′ Z in the antisense strand is a UU or TT overhang.

The oligomeric compound of embodiment 8, wherein the -D-D in the sense strand is a TT or TA overhang.

The oligomeric compound of embodiment 8, wherein the modifications on the nucleotides are selected from the group consisting of a deoxyribonucleoside (DNA nucleoside) substituted for a ribonucleoside, locked nucleic acid (LNA), 2′-OMe, 2′-F, and unlocked nucleic acid (UNA), or combinations thereof.

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

The oligomeric compound of embodiment 8, wherein the antisense strand comprises a phosphorothioate (PS) internucleotide linkage modification between positions 1-2, positions 2-3, positions 19-20 and positions 20-21 counting from the 5′ end of the antisense strand.

The oligomeric compound of embodiment 8, wherein the strands comprise a phosphorothioate internucleotide (PS) linkage adjacent to 2 nucleosides at the 5′ end of the strand and/or 2 nucleosides at the 3′ end of the strand.

Embodiment 9 of the invention comprises an oligomeric compound capable of inhibiting the 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′ MFMMNMNMFFNMNMNMMNMDD 3′,

wherein

- 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, and
  
  wherein no single modification type modifies more than two consecutive nucleotides.

The oligomeric compound of embodiment 9, wherein the N can be a ribonucleoside (R), deoxyribonucleoside (D), 2′-OMe, 2′-MOE, 2′-F, unlocked nucleic acid (UNA) or locked nucleic acid (LNA).

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

Embodiment 10 of the invention comprises an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprising an antisense strand having 21 linked nucleotides, wherein the antisense strand sequence is represented by Formula (VIII):

5′ L-MNMNMFNMFMMNMFMFMMNMM 3′,

wherein

- 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, and
- L is a 5′ phosphate, 5′ vinyl phosphonate or 5′ OH, and
  
  wherein no single modification type modifies more than two consecutive nucleotides.

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

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

Embodiment 11 of the invention comprises an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprising:

- a) a sense strand having 21 linked nucleotides, wherein the sense strand sequence is represented by Formula (X):

5′ MFMMNMNMFFNMNMNMMNMDD 3′

- b) antisense strand having 21 linked nucleotides, wherein the antisense strand sequence is represented by Formula (VIII):

5′L-MNMNMFNMFMMNMFMFMMNMM3′, and

- c) a duplex formed by the sense and antisense strands,
- wherein
  - 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, and
  - L is a 5′ phosphate, 5′ vinyl phosphonate or 5′ OH, and
- wherein the duplex region is 19 nucleotide pairs in length,
- wherein each strand has a 2 nucleotide overhang at the 3′ end, and
- wherein no single modification type modifies more than two consecutive nucleotides,

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

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

Embodiment 12 of the invention comprises an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprising a sense strand having about 21 linked nucleotides, wherein the sense strand sequence is represented by Formula (X):

5′ MFMMNMNMFFMMNMNMMFMDD 3′,

wherein

- 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
  
  wherein no single modification type modifies more than two consecutive nucleotides.

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

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

Embodiment 13 of the invention comprises an oligomeric compound capable of inhibiting the expression of a target nucleic acid, comprising an antisense strand having about 21 linked nucleotides, wherein the antisense strand sequence is represented by Formula (XII):

5′ L-MDMFMFNMFMMFMFMFMMNMM 3′,

wherein

- 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, and
- L is a 5′ phosphate, 5′ vinyl phosphonate or 5′ OH, and
  
  wherein no single modification type modifies more than two consecutive nucleotides.

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

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

Embodiment 14 of the invention comprises an oligomeric compound capable of inhibiting or inhibits the expression of a target nucleic acid, comprising:

- a) a sense strand having 21 linked nucleotides, wherein the sense strand sequence is represented by Formula (XI):

5′ MFMMNMNMFFMMNMNMMFMDD 3′,

- b) antisense strand having 21 linked nucleotides, wherein the antisense strand sequence is represented by Formula (XII):

5′L-MDMFMFNMFMMFMFMFMMNMM3′, and

- c) a duplex formed by the sense and antisense strands,
- wherein
  - 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, and
  - L is a 5′ phosphate, 5′ vinyl phosphonate or 5′ OH, and
- wherein the duplex region is 19 nucleotide pairs in length,
- wherein each strand has a 2 nucleotide overhang at the 3′ end, and
- wherein no single modification type modifies more than two consecutive nucleotides.

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

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

The oligomeric compound of any preceding embodiment, wherein a FFNM motif occurs at or near the cleavage site of the sense strand, optionally wherein the FFNM is FFMM or FFRM, and wherein F is a 2′-Fluoro, N is a modified or unmodified nucleoside, R is a ribonucleoside and M is a 2′-OMe.

The oligomeric compound of any preceding embodiment, wherein a FMM motif occurs at or near the cleavage site of the antisense strand, and wherein F is a 2′-Fluoro and M is a 2′-OMe.

The oligomeric compound of any preceding embodiment, wherein the oligomeric compound is compound is single-stranded or double-stranded.

The oligomeric compound of any preceding embodiment, wherein the single-stranded oligomeric compound is a single stranded oligonucleotide, microRNA (miRNA), or a single stranded RNA (ssRNA).

The oligomeric compound of any preceding embodiment, wherein the double-stranded oligomeric compound is a shRNA or siRNA.

The oligomeric compound of any preceding embodiment, further comprising a conjugate. The conjugate can be selected from cholesterols, lipids, carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, peptides, and dyes. In a preferred embodiment, the conjugate is N-Acetylgalactosamine (GalNAc). The conjugate can be attached to the 3′ end of the sense strand of the oligomeric compound.

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

An embodiment of the invention comprises a pharmaceutical composition comprising the oligomeric compound of any preceding embodiment, alone or in combination with a pharmaceutically acceptable carrier or excipient.

An embodiment of the invention comprises a method for inhibiting the expression of a target nucleic acid in a subject comprising the step of administering the oligomeric compound of any preceding embodiment to the subject, in an amount sufficient to inhibit expression of the target nucleic acid. The oligomeric compound can be administered subcutaneously or intravenously to the subject.

An embodiment of the invention comprises a process for making an oligomeric compound of any of the preceding claims comprising the steps of:

- a. synthesizing a sense strand oligonucleotide on a solid support using phosphoramidite chemistry
- b. synthesizing an antisense oligonucleotide on a solid support using phosphoramidite chemistry; and
- c. annealing the two oligonucleotides so synthesized thereby making the oligomeric compound.

Advantages of the Invention

The field of oligomeric therapeutic compounds is still maturing and improvements in delivery, stability, specificity, safety and potency are still being sought to improve the therapeutic efficacy of the oligomeric compound. Three general aspects of oligomer design subject to improvement are, in no particular order of importance, 1) the sequence of the oligomeric compound, 2) the chemical modifications to the oligomeric compound, and 3) the delivery modality of the oligomeric compound. The importance of the three aspects is exemplified in the progression of oligomeric compounds leading to the development of vutrisiran, the latest FDA approved siRNA therapeutic. The developer of vutrisiran, Alnylam, has taken three iterations of TTR targeting siRNAs to clinical trial: patisiran, revusiran and vutrisiran.

The first siRNA developed to target TTR is patisiran (also known as Onpattror™), where some chemically modified nucleosides are scattered along the 21-nucleotide long sense and antisense strands forming the siRNA duplex in order to stabilize the siRNA and increase inhibition potency over non-modified siRNA with the nucleotide same sequence (Friedrich and Aigner, Therapeutic siRNA: State-of-the-Art and Future Perspectives, 2022, BioDrugs, 36(5):549-571). Patisiran was given FDA approval as the first in class, siRNA, therapeutic.

The second siRNA developed to target TTR is revusiran. Although revusiran also targeted TTR, in this siRNA, a new sequence, additional modified nucleosides, modified inter-nucleoside linkages and a N-Acetylgalactosamine (GalNAc) conjugate was introduced. However, during Phase 3 clinical trial, revusiran treatment was stopped early due to an imbalance in mortality of trial patients and the trial discontinued (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 revusiran program was discontinued.

The third siRNA developed to target TTR is vutrisiran (also known as ALN-TTRSC02 and Amvuttrar™) and combined the sequence of revusiran with additional chemical modifications and 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). The chemical modifications of vitrusiran stabilized it enough to allow a more advantageous dosing schedule and administration method compared to patisiran: 25 mg vitrusiran was administered to a patient once every three months via subcutaneous injection compared to 0.3 mg/kg patisiran administered to patients once every three weeks via intravenous infusion. Vutrisiran received FDA approval after positive data from a Phase 3 clinical trial (HELIOS-A) showing that vitrusiran significantly improved the signs and symptoms of polyneuropathy (Alnylam Press Release in Businesswire, Alnylam Announces FDA Approval of Amvuttrar™ (vitrusiran), an RNAi Therapeutic for the Treatment of the Polyneuropathy of Hereditary Transthyretin-Mediated Amyloidosis in Adults, June 2022).

Accordingly, the development history of TTR siRNAs shows the importance of the oligomeric compound sequence, types of chemical modifications, patterns of chemical modifications and delivery system of oligomeric compounds.

Disclosed herein are thoughtfully designed chemical modification motifs, Advanced RNA Targeting (ARNATAR), for oligomeric compounds. Bound by no particular theory, the chemical modification motifs, irrespective of sequence, increase stability, specificity, safety and potency of oligomeric compounds. The specific chemical modification motifs of the invention, used in conjunction with sequence selection for targets and delivery modality make potent oligmeric therapeutic compounds.

Without being bound by any particular theory, the ARNATAR designed oligomeric compounds disclosed herein optimize chemical modifications motifs for characteristics such as duplex annealing temperature, RISC loading speed, stability, specificity, safety and potency.

In certain embodiments, ARNATAR designed oligomeric compounds with lower annealing temperature and faster RISC loading speed can produce a faster acting therapeutic compound beneficial for acute diseases which require fast relief after administration.

In certain embodiments, ARNATAR designed oligomeric compounds produce more stable and durable therapeutic compounds allowing longer lasting benefits for acute and chronic diseases and/or less frequent dosing of the therapeutic compound.

In certain embodiments, ARNATAR designed oligomeric compounds have enhanced binding irrespective of the oligomeric compound sequence based on the modification motif. This enhanced binding to a target nucleic acid allows the production of a therapeutic compound with enhanced specificity and safety due to fewer off-target effects. Additionally, the ARNATAR motifs have been designed to be less toxic and safer for a therapeutic compound.

In certain embodiments, ARNATAR designed oligomeric compounds are designed to be very potent inhibitors of a target nucleic acid. This potency allows less of the oligomeric compound to be administered to be therapeutically effective, leading to less general toxicity of any oligomeric compound after administration. Also, less compound required for a therapeutic dose, decreases manufacturing costs.

In certain embodiments. ARNATAR designed oligomeric compounds are very potent inhibitors of a target nucleic acid. The high potency allows target nucleic acid reduction in tissues other than liver.

Accordingly, there is a need for improved oligomeric compounds to treat diseases. ARNATAR oligomeric compounds have been designed to improve speed, stability, specificity, safety and potency in order to produce improved therapeutic compounds.

EXAMPLES
Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is herein incorporated-by-reference in its entirety.

When the tables below show both an unmodified sequence (“sequence”) and a modified sequence (“Sequence+Chemistry”) in the same row, the respective SEQ ID NO applies to the modified sequence. For example, SEQ ID NO: 16 in Table 3 stands for the modified sequence

mG*mC*mGmUmCmAfCmCfAfAfAmAmAmGmCmGmCmAmA*T*T.

Example 1: Designing Lmna siRNA with Varying Sense Strand Modifications

Laminin (LMNA) was selected as a target for testing various Advanced RNA Targeting (ARNATAR) designs. A laminin (LMNA) region with a conserved sequence between human and mouse was identified and a 19 nt long sequence (SEQ ID NO: 1) in this homology region was targeted by oligomeric compounds.

TABLE 1

LMNA Target Sequences

Sense or

SEQ ID

Name
Antisense
Sequence (5′ to 3′)
NO:

LMNA DNA Target
Sense
GCGTCACCAAAAAGCGCAA
1

Sequence

LMNA RNA Target
Sense
GCGUCACCAAAAAGCGCAA
2

Sequence

siRNAs were designed to target the LMNA mRNA target sequence (SEQ ID NO: 2) and comprise a 2-nucleotide overhang at the 3′ end of the strand: either TT (SEQ ID NOs: 3-4) or UU (SEQ ID NO: 5). All antisense strands have a 5′-phosphate. A notation is made before each nucleotide indicating the type of chemical modification, if any, made to the nucleotide. If no modification notation is made before a letter designating a nucleotide, the nucleotide is a deoxyribonucleotide. Notations for the chemical modifications to the strands can be found as follows:

- (5p)=5′-phosphate
- r=ribonucleotide (e.g., rA indicates an adenosine)
- d (or no notation made before a nucleotide)=deoxyribonucleotide which has been substituted for a ribonucleotide (e.g., dA or A indicates a 2′-deoxy adenosine)
- f=2′-F (i.e., indicating a 2′-fluoro-modified nucleoside, e.g., fA indicates a 2′-fluoro adenosine)
- m=2′-OMe (i.e., indicating a 2′-O-methyl-modified nucleoside, e.g., mA indicates a 2′-O-methyl adenosine)
- gna=Glycol nucleic acid (e.g., gnaT indicates a 2,3-dihydroxypropyl thymine)
- p=phosphate
- *=phosphorothioate (PS) linkage (i.e., indicates that a 5′-phosphorothioate (=° ° 5′-thiophosphate) is present instead of a 5′-phosphate, e.g., *A indicates a 2′-deoxy adenosine 5′-thiophosphate, *rA indicates an adenosine 5′-thiophosphate and *mA indicates a 2′-O-methyl adenosine 5′-thiophosphate)
- GA=GalNAc (GA1=GalNAc1, GA2=GalNAc2 and GA3=GalNAc3 are specific GalNAc conjugates described herein)

A listing of LMNA siRNAs sequences can be found in the tables, infra, or at FIGS. 1-2.

Cell cultures were grown to approximately 60-80% confluency before varying doses of siRNA were transfected into cells using RNAiMAX (InVitrogen, Waltham, MA) according to the manufacturer's recommended protocol and the cells further cultured for a period of time. siRNA activity was determined by measuring the levels of target mRNA through qRT-PCR using the LMNA primer probe set listed in Table 2. qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents in QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). The target RNA levels detected in qRT-PCR assay were normalized to either total RNA levels measured with Ribogreen™ (ThermoFisher Scientific, Waltham, MA, USA) or GAPDH mRNA levels detected in the aliquotes of RNA samples using qRT-PCR.

TABLE 2

human and mouse LMNA Primer-Probe Sets

Primer

SEQ

Name
Primer Sequence (5′ to 3′)
ID NO:

hsLMNA-S
CGGGTGGATGCTGAGAAC
3

hsLMNA-A
TGCTTCCCATTGTCAATCTCC
4

hsLMNA-P
AGTGAGGAGCTGCGTGAGACCAA
5

msLMNA-S
GGACCAGGTGGAACAGTATAAG
6

msLMNA-A
TCAATGCGGATTCGAGACTG
7

msLMNA-P
CAGCTTGGCGGAGTATGTCTTTTCTAGC
8

A. Minimal Sense Strand Modifications Improves siRNA Activity

As shown in Table 3, LMNA-si1 is a siRNA with TT overhangs attached to an unmodified ribonucleotide sequence targeting LMNA. LMNA-si2 is a modified siRNA where the chemical modification pattern reflects that of lumasiran, a FDA approved siRNA, with the exception that the sense strand of LMNA-si2 has an additional TT overhang connected by PS linkages and does not have a GalNAc conjugate, while the antisense strand is 21 nts with TT overhang rather than 23 nt long. LMNA-si3 has a modified sense strand and an antisense strand with a TT overhand attached to an unmodified ribonucleotide sequence. LMNA-si4 has a sense strand with a TT overhang attached to an unmodified ribonucleotide sequence and a modified antisense strand. In this example, all antisense strands have a 5′-phosphate.

TABLE 3

LMNA Oligomeric Compounds

SEQ

siRNA
Strand
Sense or
Sequence + Chemistry

ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL019
Sense
rGrCrGrUrCrArCrCrArArA
GCGUCACCAAAA
15

si1

rArArGrCrGrCrArATT
AGCGCAATT

ATXL018
Antisense
rUrUrGrCrGrCrUrUrUrUrU
UUGCGCUUUUUG
33

rGrGrUrGrArCrGrCTT
GUGACGCTT

LMNA-
ATXL021
Sense
mG*mC*mGmUmCmAfC
GCGUCACCAAAA
16

si2

mCfAfAfAmAmAmGmCm
AGCGCAATT

GmCmAmA*T*T

ATXL020
Antisense
mU*fU*mGmCmGfCmUfU
UUGCGCUUUUUG
34

fUmUmUmGmGfUmGIAm
GUGACGCTT

CmGmC*T*T

LMNA-
ATXL021
Sense
mG*mC*mGmUmCmAfC
GCGUCACCAAAA
16

si3

mCfAfAfAmAmAmGmCm
AGCGCAATT

GmCmAmA*T*T

ATXL018
Antisense
rUrUrGrCrGrCrUrUrUrUrU
UUGCGCUUUUUG
33

rGrGrUrGrArCrGrCTT
GUGACGCTT

LMNA-
ATXL019
Sense
rGrCrGrUrCrArCrCrArArA
GCGUCACCAAAA
15

314

rArArGrCrGrCrArATT
AGCGCAATT

ATXL020
Antisense
mU*fU*mGmCmGfCmUfU
UUGCGCUUUUUG
34

fUmUmUmGmGfUmGfAm
GUGACGCTT

CmGmC*T*T

siRNA was transfected at 0-10 nM final concentration into HeLa cells (ATCC, Manassas, VA, USA) with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr. siRNA activity was determined by qRT-PCR using the LMNA primer probe set in Table 2 and the half-maximal inhibitory concentration value (IC50) of the siRNAs targeting LMNA was calculated (Table 4).

TABLE 4

LMNA siRNA Inhibition in HeLa Cells after 24 hrs

LMNA-si1
LMNA-si2
LMNA-si3
LMNA-si4

IC50 (nM)
0.094
5.664
12.22
0.095

Fold
60.0
1.00
0.5
59.7

LMNA-si2 was used as the benchmark for activity. The results show that LMNA-si1 and LMNA-si4, with minimal modification on the sense strand, exhibited greater potency than LMNA-si2 and L MNA-si3, suggesting that reducing the modifications to the sense strand improves siRNA activity.

B. Addition of Peripheral Sense Strand Modifications to Protect the siRNA

The previous study, supra, indicated that reducing modifications to the siRNA sense strand improved siRNA activity 24 hr after transfection. However, siRNAs need to be stable metabolically to maintain a more durable activity in vivo. Some chemical modifications have been able to improve such stability without compromising activity (Choung et al., Biochem Biophys Res Commun, 2006, 342:919-927), therefore, strategic modifications 2′-OMe were made to the 3′ and/or 5′ end of the sense strand of LMNA-si4 to determine whether the modifications would affect siRNA activity over time. Also, the effect of mUmU or TT overhangs at the 3′ ends of the sense strands was assessed. In this example, all antisense strands have a 5′-phosphate.

TABLE S

LMNA Oligomeric Compounds with Sense Strand Peripheral Modifications

SEQ

siRNA
Strand
Sense or
Sequence + Chemistry

ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL022
Sense
mGmCrGrUrCrArCrCrArArA
GCGUCACCAAA
35

si5

rArArGrCrGrCrArAmUmU
AAGCGCAAUU

ATXL020
Antisense
mU*fU*mGmCmGfCmUfUf
UUGCGCUUUU
34

UmUmUmGmGfUmGfAmC
UGGUGACGCTT

mGmC*T*T

LMNA-
ATXL023
Sense
mGmCrGrUrCrArCrCrArArA
GCGUCACCAAA
19

si6

rArArGrCrGrCrArATT
AAGCGCAATT

ATXL020
Antisense
mU*fU*mGmCmGfCmUfUf
UUGCGCUUUU
34

UmUmUmGmGfUmGfAmC
UGGUGACGCTT

mGmC*T*T

LMNA-
ATXL024
Sense
rGrCrGrUrCrArCrCrArArAr
GCGUCACCAAA
36

si7

ArArGrCrGrCrArAmUmU
AAGCGCAAUU

ATXL020
Antisense
mU*fU*mGmCmGfCmUfUf
UUGCGCUUUU
34

UmUmUmGmGfUmGfAmC
UGGUGACGCTT

mGmC*T*T

LMNA-
ATXL025
Sense
mGmCmGrUrCrArCrCrArAr
GCGUCACCAAA
37

si8

ArArArGrCrGrCrAmAmUm
AAGCGCAAUU

U

ATXL020
Antisense
mU*fU*mGmCmGfCmUfUf
UUGCGCUUUU
34

UmUmUmGmGfUmGfAmC
UGGUGACGCTT

mGmC*T*T

LMNA-
ATXL026
Sense
mGmCmGrUrCrArCrCrArAr
GCGUCACCAAA
18

si9

ArArArGrCrGrCrArATT
AAGCGCAATT

ATXL020
Antisense
mU*fU*mGmCmGfCmUfUf
UUGCGCUUUU
34

UmUmUmGmGfUmGfAmC
UGGUGACGCTT

mGmC*T*T

LMNA-
ATXL027
Sense
rGrCrGrUrCrArCrCrArArAr
GCGUCACCAAA
38

si10

ArArGrCrGrCrAmAmUmU
AAGCGCAAUU

ATXL020
Antisense
mU*fU*mGmCmGfCmUfUf
UUGCGCUUUU
34

UmUmUmGmGfUmGfAmC
UGGUGACGCTT

mGmC*T*T

LMNA-
ATXL028
Sense
mG*mC*rGrUrCrArCrCrArA
GCGUCACCAAA
39

si11

rArArArGrCrGrCrArAmUm
AAGCGCAAUU

U

ATXL020
Antisense
mU*fU*mGmCmGfCmUfUf
UUGCGCUUUU
34

UmUmUmGmGfUmGfAmC
UGGUGACGCTT

mGmC*T*T

LMNA-
ATXL029
Sense
mG*mC*mG*rUrCrArCrCrA
GCGUCACCAAA
40

si12

rArArArArGrCrGrCrAmAm
AAGCGCAAUU

UmU

ATXL020
Antisense
mU*fU*mGmCmGfCmUfUf
UUGCGCUUUU
34

UmUmUmGmGfUmGfAmC
UGGUGACGCTT

mGmC*T*T

LMNA-
ATXL030
Sense
mG*mC*rGrUrCrArCrCrArA
GCGUCACCAAA
20

si13

rArArArGrCrGrCrArA*T*T
AAGCGCAATT

ATXL020
Antisense
mU*fU*mGmCmGfCmUfUf
UUGCGCUUUU
34

UmUmUmGmGfUmGfAmC
UGGUGACGCTT

mGmC*T*T

LMNA-
ATXL031
Sense
mG*mC*mG*rUrCrArCrCrA
GCGUCACCAAA
21

si14

ArArArArGrCrGrCrA*mA*
AAGCGCAATT

T*T

ATXL020
Antisense
mU*fU*mGmCmGfCmUfUf
UUGCGCUUUU
34

UmUmUmGmGfUmGfAmC
UGGUGACGCTT

mGmC*T*T

LMNA-
ATXL032
Sense
mGmCmGmUmCmArCmCr
GCGUCACCAAA
41

si15

ArArArAmAmGmCmGmCm
AAGCGCAAUU

AmAmUmU

ATXL020
Antisense
mU*fU*mGmCmGfCmUfUf
UUGCGCUUUU
34

UmUmUmGmGfUmGfAmC
UGGUGACGCTT

mGmC*T*T

siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 6).

TABLE 6

LMNA siRNA Inhibition in Hela Cells after 24 hrs

IC50 (nM)
Fold

LMNA-si2
5.781
1.0

LMNA-si5
0.088
65.7

LMNA-si6
0.039
149.1

LMNA-si7
0.084
68.7

LMNA-si8
0.051
113.2

LMNA-si9
0.075
77.4

LMNA-si10
0.236
24.5

LMNA-si11
0.155
37.3

LMNA-si12
0.188
30.8

LMNA-si13
0.123
47

LMNA-si14
0.097
59.8

LMNA-si15
8.336
0.7

All the siRNAs with 3′ and 5′ sense strand modifications were more active than LMNA-si2. However, the addition of more 2′-OMe beyond the 3′ and 5′ periphery of the sense strand did not contribute to siRNA activity as LMNA-si15, which had most of its sense strand modified, was the least active siRNA. 2′-OMe modifications at the 5′ end of the sense strand made the siRNA more active than modification at the 3′ end (compare LMNA-si6 and LMNA-si7, LMNA-si9 and LMNA-si10). No PS linkages at the 5′ end of the sense strand made the siRNA more active than addition of three PS linkages at the 5′ end of the sense strand (compare LMNA-si12 and LMNA-si8). There was no obvious difference between having a mUmU versus a TT overhang to activity.

Selected siRNAs were further tested for a longer time in cell culture and in different cell types: LMNA-si2, LMNA-si8. LMNA-si12 and LMNA-si14.

siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr and 72 hr. siRNA was also transfected into HEK293 cells (ATCC, Manassas, VA, USA) with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr and 120 hr. si RNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 7).

TABLE 7

LMNA siRNA Inhibition in HeLa or HEK293 Cells

HeLa Cells
HeLa Cells
HEK293 Cells
HEK293 Cells

(24 hrs)
(72 hrs)
(24 hrs)
(120 hrs)

siRNA
IC50 (nM)
Fold
IC50 (nM)
Fold
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNA-si2
0.411
1.00
0.182
1
78.93
1.00
0.631
1

LMNA-si8
0.027
15.4
0.029
6.3
0.595
132.7
0.191
3.3

LMNA-si12
0.022
18.8
0.025
7.4
1.132
69.7
0.03
21.1

LMNA-si14
0.026
16
0.029
6.3
1.799
43.9
0.036
17.6

Tables 6-7 show that siRNAs with modifications in the sense strand can increase siRNA activity in different cell types for various time periods.

C. Additional Sense Strand Modifications to Protect the siRNA

The previous study, supra, indicated that peripheral modifications to the siRNA sense strand improved siRNA activity. Additional strategic 2′-OMe modifications were made to the sense strand of LMNA-si4 to determine whether the additional modifications would affect siRNA activity over time (Table 8) with the objective to increase 2′-OMe modifications in the sense strand in order to increase stability, but not compromise on potency. In this example, all antisense strands have a 5′-phosphate.

TABLE 8

LMNA Oligomeric Compounds with Additional 2′-OMe Sense Strand Modifications

SEQ

siRNA
Strand
Sense or
Sequence + Chemistry

ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL048
Sense
mG*mC*mG*mUrCrArCr
GCGUCACCAAA
42

si23

CrArArArArArGrCrGrCm
AAGCGCAAUU

AmAmUmU

ATXL020
Antisense
mU*fU*mGmCmGfCmUf
UUGCGCUUUUU
34

UfUmUmUmGmGfUmGf
GGUGACGCTT

AmCmGmC*T*T

LMNA-
ATXL049
Sense
mG*mC*mG*mUmCrAr
GCGUCACCAAA
43

si24

CrCrArArArArArGrCrOm
AAGCGCAAUU

CmAmAmUmU

ATXL020
Antisense
mU*fU*mGmCmGfCmUf
UUGCGCUUUUU
34

UfUmUmUmGmGfUmGf
GGUGACGCTT

AmCmGmC*T*T

LMNA-
ATXL050
Sense
mG*mC*mG*rUrCrArCr
GCGUCACCAAA
44

si25

CrArArArArArGrCrGrCr
AAGCGCAAUU

AmA*mU*mU

ATXL020
Antisense
mU*fU*mGmCmGfCmUf
UUGCGCUUUUU
34

UfUmUmUmGmGfUmGf
GGUGACGCTT

AmCmGmC*T*T

LMNA-
ATXL051
Sense
mG*mC*mG*mUmCmAr
GCGUCACCAAA
45

si26

CmCrArArAmArAmGrC
AAGCGCAAUU

mOmCmAmA*mU*mU

ATXL020
Antisense
mU*fU*mGmCmGfCmUf
UUGCGCUUUUU
34

UfUmUmUmGmGfUmGf
GGUGACGCTT

AmCmGmC*T*T

LMNA-
ATXL052
Sense
mG*mC*mG*mUrCmAr
GCGUCACCAAA
46

si27

CmCrArArAmArAmGrC
AAGCGCAAUU

mGmCmAmA*mU*mU

ATXL020
Antisense
mU*fU*mGmCmGfCmUf
UUGCGCUUUUU
34

UfUmUmUmGmGfUmGf
GGUGACGCTT

AmCmGmC*T*T

LMNA-
ATXL053
Sense
mG*mC*mG*mUrCmAr
GCGUCACCAAA
48

si28

CmCrArArAmArAmGrC
AAGCGCAAUU

mGrCmAmA*mU*mU

ATXL020
Antisense
mU*fU*mGmCmGfCmUf
UUGCGCUUUUU
34

UfUmUmUmGmGfUmGf
GGUGACGCTT

AmCmGmC*T*T

siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr. siRNA was transfected at 0-10 nM into HEK293 cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 48 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 9).

TABLE 9

LMNA siRNA Inhibition in HeLa or HEK293 Cells

Hela Cells (24 hrs)

HEK293 Cells (48 hrs)

siRNA
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNA-si2
9.287
1
1.088
1

LMNA-si14
0.194
47.8
0.087
12.6

LMNA-si23
0.366
25.4
0.08
13.7

LMNA-si24
0.287
32.4
0.1
10.9

LMNA-si25
0.320
29.0
0.096
11.3

LMNA-si26
1.668
5.6
0.407
2.7

LMNA-si27
0.846
11.0
0.053
20.4

LMNA-si28
0.386
24.1
0.110
9.9

A subset of siRNAs with robust activity and more 2′-OMe modified nucleotides in the sense strand was further tested for activity: LMNA-si27 (sense strand ATXL052), LMNA-si28 (sense strand ATXL053).

siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr or 72 hr. siRNA was transfected at 0-10 nM into HEK293 cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 72 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 10).

TABLE 10

LMNA siRNA Inhibition in HeLa or HEK293 Cells

HeLa Cells
HeLa Cells
HEK293 Cells

(24 hrs)
(72 hrs)
(72 hrs)

siRNA
IC50 (nM)
Fold
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNA-si2
3.228
1.0
0.431
1.0
1.415
1.0

LMNA-si12
0.228
14.1
0.073
5.9
0.364
3.9

LMNA-si27
0.371
8.7
0.075
5.7
0.1
14.2

LMNA-si28
0.42
7.7
0.158
2.7
0.255
5.5

LMNA-si27 is more potent than LMNA-si28 due to the difference in sense strand modifications.

Example 2: Designing LMNA siRNA with Varying Antisense Strand Modifications

In Example 1, supra, siRNA antisense strands were held constant while the sense strands underwent various chemical modifications to find motifs that would convey stability and potency to the siRNAs. In this example, siRNA sense strands are held constant while the antisense strand is variously modified. The modified antisense strand from LMNA-si2 was used as a template from which individual nucleotides were modified. Nucleotide positions are counted starting from the 5′ end of strand; the 5′ most nucleotide is position 1.

Without being bound by any particular theories, it has been previously demonstrated that 2′-F modification increase the melting temperature (Tm) of RNA duplex more than 2′-OMe does (Bramsen and Kjems, 2012, Frontiers in Genetics, 3(154):1-22), and deoxyribonucleotide that further reduce Tm as compared with 2′-OMe at certain positions can be tolerated by RISC (Ui-Tei, et al., Nucl Acids Res, 2008, 36(7): 2136-51). We thus altered the number of 2′-F modifications and incorporated deoxyribonucleotide at certain positions, and evaluated the effect of such modification patterns on siRNA activity.

As shown in Fable 11, in some antisense strands: nucleotide 2 is modified from a ribonucleotide with a 2′-F modification to a deoxyribonucleotide; nucleotide 7 is modified from a ribonucleotide with a 2′-OMe modification to a deoxyribonucleotide; the 2′-F modification at nucleotide 8 is changed to a 2′-OMe modification; nucleotide 16 is modified from a ribonucleotide with a 2′-F modification to a deoxyribonucleotide; PS linkages added to link the deoxyribonucleotides to some adjacent ribonucleotides to determine whether the PS linkage is protective of deoxyribonucleotide; and the TT overhang is replaced with a mUmU overhang at the 3′ end of the antisense strand.

TABLE 11

LMNA Oligomeric Compounds with Antisense Strand Modifications

SEQ

siRNA
Strand
Sense or
Sequence + Chemistry

ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL021
Sense
mG*mC*mGmUmCmAfCmCfAfAf
GCGUCACCAAA
16

si2

AmAmAmGmCmGmCmAmA*T*T
AAGCGCAATT

ATXL020
Antisense
mU*U*mGmCmGfCmUfUfUmU
UUGCGCUUUUU
34

mUmGmGfUmGfAmCmGmC*T*T
GGUGACGCTT

LMNA-
ATXL021
Sense
mG*mC*mGmUmCmAfCmCfAfAf
GCGUCACCAAA
16

si16

AmAmAmGmCmGmCmAmA*T*T
AAGCGCAATT

ATXL041
Antisense
mU*fU*mGmCmGfCmUfUfUmU
UUGCGCUUUUU
57

mUmGmGfUmG*AmCmGmC*mU
GGUGACGCUU

*mU

LMNA-
ATXL021
Sense
mG*mC*mGmUmCmAfCmCfAfAf
GCGUCACCAAA
16

si17

AmAmAmGmCmGmCmAmA*T*T
AAGCGCAATT

ATXL042
Antisense
mU*T*mGmCmGfCmUfUfUmUm
UTGCGCUUUUU
59

UmGmGfUmGfAmCmGmC*mU*
GGUGACGCUU

mU

LMNA-
ATXL021
Sense
mG*mC*mGmUmCmAfCmCfAfAf
GCGUCACCAAA
16

si18

AmAmAmGmCmGmCmAmA*T*T
AAGCGCAATT

ATXL043
Antisense
mU*T*mGmCmGfC*TfUfUmUmU
UTGCGCTUUUU
61

mGmGfUmGfAmCmGmC*mU*m
GGUGACGCUU

U

LMNA-
ATXL021
Sense
mG*mC*mGmUmCmAfCmCfAfAf
GCGUCACCAAA
16

si19

AmAmAmGmCmGmCmAmA*T*T
AAGCGCAATT

ATXL044
Antisense
mU*fU*mGmCmGfC*TfUfUmUm
UUGCGCTUUUU
71

UmGmGfUmGfAmCmGmC*mU*
GGUGACGCUU

mU

LMNA-
ATXI.021
Sense
mG*mC*mGmUmCmAfCmCfAfAf
GCGUCACCAAA
16

si20

AmAmAmGmCmGmCmAmA*T*T
AAGCGCAATT

ATXL045
Antisense
mU*fU*mGmCmGfC*TmUfUmU
UUGCGCTUUUU
72

mUmGmGfUmGfAmCmGmC*mU
GGUGACGCUU

*mU

LMNA-
ATXL021
Sense
mG*mC*mGmUmCmAfCmCfAfAf
GCGUCACCAAA
16

si21

AmAmAmGmCmGmCmAmA*T*T
AAGCGCAATT

ATXL046
Antisense
mU*T*mGmCmGfC*TmUfUmUm
UTGCGCTUUUU
62

UmGmGfUmGfAmCmGmC*mU*
GGUGACGCUU

mU

LMNA-
ATXL021
Sense
mG*mC*mGmUmCmAfCmCfAfAf
GCGUCACCAAA
16

si22

AmAmAmGmCmGmCmAmA*T*T
AAGCGCAATT

ATXL047
Antisense
mU*T*mGmCmGfC*TmUfUmUm
UTGCGCTUUUU
63

UmGmGfUmG*AmCmGmC*mU*
GGUGACGCUU

mU

In two sets of experiments, siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 12).

TABLE 12

LMNA siRNA Inhibition in HeLa Cells

Hela Cells (24 hrs)

siRNA
IC50 (nM)
Fold

LMNA-si2
12.32
1.0

LMNA-si17
186.9
0.07

LMNA-si18
0.529
23.3

LMNA-si20
0.208
59.1

LMNA-si21
0.546
22.5

LMNA-si22
1.129
10.9

LMNA-si2
1.961
1.0

LMNA-si16
1.875
1.0

LMNA-si19
0.086
22.9

The results at 24 hrs show changing the nucleotide in position 7 to a deoxyribonucleotide improved activity of LMNA-si18, LMNA-si19, LMNA-si20, LMNA-si21 and LMNA-si22. The change to a deoxyribonucleotide at position 2 or position 16 was tolerated as shown by the activity of LMNA-si16, LMNA-si17, LMNA-si18, LMNA-si21 and LMNA-si22.

In two sets of experiments, siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen. Waltham, MA) and the cells further cultured for 72 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 13).

TABLE 13

LMNA siRNA Inhibition in Hela Cells

HeLa Cells (72 hrs)

siRNA
IC50 (nM)
Fold

LMNA-si2
0.154
1.0

LMNA-si17
0.59
0.3

LMNA-si18
0.068
2.3

LMNA-si20
0.033
4.7

LMNA-si21
0.059
2.6

LMNA-si22
0.115
1.3

LMNA-si2
0.067
1.0

LMNA-si16
0.296
0.2

LMNA-si19
0.028
2.4

At the 72 hr timepoint, LMNA-si20 and LMNA-si21 showed the best activity.

Example 3: Designing LMNA siRNA with Varying Sense and Antisense Strand Modifications

Modified sense and antisense strands disclosed in the previous examples were mixed and matched to form new siRNA compounds (Table 14) and assessed for stability and activity (Table 15).

TABLE 14

LMNA Oligomeric Compounds with Modifications

to Both Strands

Sequence +

SIRNA
Strand
Sense or
Chemistry

SEQ ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL052
Sense
mG*mC*mG*mUrCmAr
GCGUCACCAAA
46

si29

CmCrArArAmArAmGr
AAGCGCAAUU

CmGmCmAmA*mU*mU

ATXL043
Antisense
mU*T*mGmCmGfC*Tf
UTGCGCTUUUU
61

UfUmUmUmGmGfUmGf
GGUGACGCUU

AmCmGmC*mU*mU

LMNA-
ATXL053
Sense
mG*mC*mG*mUrCmAr
GCGUCACCAAA
48

si30

CmCrArArAmArAmGr
AAGCGCAAUU

CmGrCmAmA*mU*mU

ATXL043
Antisense
mU*T*mGmCmGfC*Tf
UTGCGCTUUUU
61

UfUmUmUmGmGfUmGf
GGUGACGCUU

AmCmGmC*mU*mU

LMNA-
ATXL052
Sense
mG*mC*mG*mUrCmAr
GCGUCACCAAA
46

si31

CmCrArArAmArAmGr
AAGCGCAAUU

CmGmCmAmA*mU*mU

ATXL045
Antisense
mU*fU*mGmCmGfC*T
UUGCGCTUUUU
72

mUfUmUmUmGmGfUmG
GGUGACOCUU

fAmCmGmC*mU*mU

LMNA-
ATXL053
Sense
mG*mC*mG*mUrCmAr
GCGUCACCAAA
48

si32

CmCrArArAmArAmGr
AAGCGCAAUU

CmGrCmAmA*mU*mU

ATXL045
Antisense
mU*fU*mGmCmGfC*T
UUGCGCTUUUU
72

mUfUmUmUmGmGfUmG
GGUGACGCUU

fAmCmGmC*mU*mU

LMNA-
ATXL052
Sense
mG*mC*mG*mUrCmAr
GCGUCACCAAA
46

si33

CmCrArArAmArAmGr
AAGCGCAAUU

CmGmCmAmA*mU*mU

ATXL046
Antisense
mU*T*mGmCmGfC*Tm
UTGCGCTUUUU
62

UfUmUmUmGmGfUmGf
GGUGACGCUU

AmCmGmC*mU*mU

LMNA-
ATXL053
Sense
mG*mC*mG*mUrCmAr
GCGUCACCAAA
48

si34

CmCrArArAmArAmGr
AAGCGCAAUU

CmGrCmAmA*mU*mU

ATXL046
Antisense
mU*T*mGmCmGfC*Tm
UTGCGCTUUUU
62

UfUmUmUmGmGfUmGf
GGUGACGCUU

AmCmGmC*mU*mU

siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr. 96 hr, or 144 hr. siRNA activity was determined by qRT1-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 15).

TABLE 15

LMNA siRNA Inhibition in HeLa Cells

HeLa Cells
HeLa Cells
HeLa Cells

(24 hrs)
(96 hrs)
(144 hrs)

siRNA
IC50 (nM)
Fold
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNA-si2
1.205
1.0
0.39
1.0
0.7
1.0

LMNA-si1
0.131
9.2
0.125
3.1
0.625
1.1

LMNA-si29
0.018
68.1
0.053
7.4
0.135
5.2

LMNA-si30
0.029
41.0
0.098
4.0
0.244
2.9

LMNA-si31
0.021
57.5
0.036
10.8
0.075
9.4

LMNA-si32
0.031
39.1
0.036
11.0
0.081
8.6

LMNA-si33
0.023
51.7
0.042
9.3
0.045
15.5

LMNA-si34
0.043
27.9
0.065
6.0
0.137
5.1

Sense strand ATXL052 was more potent than ATXL053 when used in a si RNA compound. Antisense strands ATXL045 and ATXL046 were more potent than ATXL043. LMNA-si31 and LMNA-si33 had the best activity over time. siRNA was transfected at 0-10 nM into HEK293 cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 96 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 16).

TABLE 16

LMNA siRNA Inhibition in HEK293 Cells

HEK293 Cells (96 hrs)

siRNA
IC50 (nM)
Fold

LMNA-si2
0.528
1

LMNA-si1
0.169
3.1

LMNA-si29
0.077
6.8

LMNA-si30
0.134
3.9

LMNA-si31
0.027
19.2

LMNA-si32
0.08
6.6

LMNA-si33
0.054
9.7

LMNA-si34
0.174
3.0

Similar to the results in HeLa cells, the sense strand ATXL052 is more active than ATXL053. LMNA-si31 and LMNA-si33 are more active than others.

siRNA was transfected at 0-10 nM into murine HePa1-6 cells (ATCC, Manassas, VA, USA) with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr, 72 hr, or 120 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 17).

TABLE 17

LMNA siRNA Inhibition in Mouse HePa1-6 Cells

HePa1-6 Cells
HePa1-6 Cells
HePa1-6 Cells

(24 hrs)
(72 hrs)
(120 hrs)

siRNA
IC50 (nM)
Fold
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNA-si2
8.278
1.0
0.593
1.0
0.562
1.0

LMNA-si29
0.496
16.7
0.219
2.7
0.622
0.9

LMNA-si31
0.103
80.1
0.078
7.6
0.072
7.8

LMNA-si33
0.557
14.9
0.123
4.8
0.756
0.7

LMNA-si31 was found to be more potent than the other compounds in mouse cells. LMNA-si29 and LMNA-si33 were more potent than the benchmark LMNA-si2.

Serum Stability Assay

LMNA-si2, LMNA-si29 and LMNA-si33 were exposed to human serum to test for stability. siRNAs were incubated with human serum (Sigma-Aldrich, St. Louis, MO, USA) at 0.4 uM concentration, at 37° C. for a desired time (0, 8, 16, and 24 h). Then BlueJuice™ gel loading buffer (ThermoFisher Scientific, Waltham, MA, USA) was added and the siRNA mix was loaded onto 4-20% native TBE gel (ThermoFisher Scientific, Waltham, MA, USA). After running the gel, it was stained with 1:10000 SYBR™ Gold (ThermoFisher Scientific, Waltham, MA, USA) in water, at room temperature for 10 min and imaged under UV light.

The siRNA compounds were found to be stable after 24 hr serum treatment (FIG. 6).

Example 4: Designing ApoC3 siRNA with Varying Sense and Antisense Strand Modifications

Based on the previous studies, supra, with LMNA, some of the chemical modification conferring high activity was assessed in a different target to determine whether the chemical modification motifs would also be beneficial. ApoC3 was chosen as the second test target and siRNAs designed, as shown in Table 18 and FIG. 3-4, to target a 19 nt long sequence of human ApoC3: CUCUCGAGUUCUGGGAUUlG (SEQ ID NO: 177). The primer probe sets of human ApoC3 used for RT-PCR is shown in Table 19. ApoC3-si1, is a modified siRNA where the chemical modification pattern reflects that of lumasiran with the exception that the sense strand of ApoC3-si1 has an addition 1 overhang connected by PS linkages and does not have a GalNAc conjugate, and the antisense strand is 21 nt rather than 23 nt. All antisense strands have a 5′-phosphate. The modified siRNAs were tested for activity in a couple of cell lines.

TABLE 18

ApoC3 Oligomeric Compounds with Modifications

to Both Strands

Sequence +

SIRNA
Strand
Sense or
Chemistry

SEQ ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

ApoC3-
ATXL059
Sense
mC*mU*mCmUmGmAfG
CUCUGAGUUCU
74

si1

mUfUfCfUmGmGmGmA
GGGAUUUGTT

mUmUmUmG*T*T

ATXL058
Antisense
mC*fA*mAmAmUfCmC
CAAAUCCCAGA
77

fCfAmGmAmAmCfUmC
ACUCAGAGTT

fAmGmAmG*T*T

ApoC3-
ATXL060
Sense
mC*mU*mC*rUrGrAr
CUCUGAGUUCU
78

si2

GrUrUrCrUrGrGrGr
GGGAUUUG

ArUmUmUmG

ATXL058
Antisense
mC*fA*mAmAmUfCmC
CAAAUCCCAGA
77

fCfAmGmAmAmCfUmC
ACUCAGAGTT

fAmGmAmG*T*T

ApoC3-
ATXL061
Sense
mC*mU*mC*rUrGrAr
CUCUGAGUUCU
79

si3

GrUrUrCrUrGrGrGr
GGGAUUUG

ArUmU*mU*mG

ATXL058
Antisense
mC*fA*mAmAmUfCmC
CAAAUCCCAGA
77

fCfAmGmAmAmCfUmC
ACUCAGAGTT

fAmGmAmG*T*T

ApoC3-
ATXL068
Sense
mC*mU*mC*mUrGmAr
CUCUGAGUUCU
80

si6

GmUrUrCrUmGrGmGr
GGGAUUUGUU

AmUmUmUmG*mU*mU

ATXL067
Antisense
mC*A*mAmAmUfC*Cm
CAAAUCCCAGA
84

CfAmGmAmAmCfUmCf
ACUCAGAGUU

AmGmAmG*mU*mU

ApoC3-
ATXL069
Sense
mC*mU*mC*mUrGmAr
CUCUGAGUUCU
81

si7

GmUrUrCrUmGrGmGr
GGGAUUUGUU

AmUrUmUmG*mU*mU

ATXL067
Antisense
mC*A*mAmAmUfC*Cm
CAAAUCCCAGA
84

CfAmGmAmAmCfUmCf
ACUCAGAGUU

AmGmAmG*mU*mU

ApoC3-
ATXL068
Sense
mC*mU*mC*mUrGmAr
CUCUGAGUUCU
80

si8

GmUrUrCrUmGrGmGr
GGGAUUUGUU

AmUmUmUmG*mU*mU

ATXL072
Antisense
mC*A*mAmAmUfC*Cf
CAAAUCCCAGA
85

CfAmGmAmAmCfUmCf
ACUCAGAGUU

AmGmAmG*mU*mU

ApoC3-
ATXL069
Sense
mC*mU*mC*mUrGmAr
CUCUGAGUUCU
81

si9

GmUrUrCrUmGrGmGr
GGGAUUUGUU

AmUrUmUmG*mU*mU

ATXL072
Antisense
mC*A*mAmAmUfC*Cf
CAAAUCCCAGA
85

CfAmGmAmAmCfUmCf
ACUCAGAGUU

AmGmAmG*mU*mU

TABLE 19

ApoC3 primer probe set sequences

SEQ ID

Name
Sequence
NO:

ApoC3-F
AGGGTTACATGAAGCACGC
9

ApoC3-R
AGAGAACTTGTCCTTAACGGTG
10

ApoC3-P
AGGGAACTGAAGCCATCGGTCAC
11

siRNA was transfected at 0-10 nM into Hep3B3 cells (ATCC, Manassas, VA. USA) with RNAiMAX (invitrogen, Waltham, MA) and the cells further cultured for 20 hr, 60 hr, or 168 hr. siRNA activity was determined by measuring the levels of target mRNA through qRT-PCR using the primer probe set in Table 19 and the IC50 of the siRNAs targeting ApoC3 was calculated (Table 20). qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents in QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). The target RNA levels detected in qRT-PCR assay were normalized to either total RNA levels measured with Ribogreen™ (ThermoFisher Scientific, Waltham, MA, USA) or GAPDH mRNA levels detected in the aliquots of RNA samples using qRT-PCR.

TABLE 20

ApoC3 siRNA Inhibition in Hep3B Cells

Hep3B Cells
Hep3B Cells
Hep3B Cells

(20 hrs)
(60 hrs)
(168 hrs)

siRNA
IC50 (nM)
Fold
IC50 (nM)
Fold
IC50 (nM)
Fold

ApoC3-si1
20.66
1
0.085
1
0.305
1

ApoC3-si3
0.104
199.4
0.013
6.7
0.375
0.8

ApoC3-si6
0.151
137.3
0.014
6
0.132
2.3

ApoC3-si7
0.134
153.9
0.016
5.2
0.054
5.6

ApoC3-si8
0.068
305.4
0.008
10.1
0.072
4.2

ApoC3-si9
0.143
144.5
0.019
4.6
0.133
2.3

siRNA was transfected at 0-10 nM into HepG2 cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr or 72 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting ApoC3 was calculated (Table 21).

TABLE 21

ApoC3 siRNA Inhibition in HepG2 Cells

HepG2 Cells (24 hrs)

HepG2 Cells (72 hrs)

siRNA
IC50 (nM)
Fold
IC50 (nM)
Fold

ApoC3-si1
13.61
1
0.907
1

ApoC3-si3
1.329
10.2
1.074
0.8

ApoC3-si6
0.209
65.2
0.098
9.3

ApoC3-si8
0.413
33
0.736
1.2

The new chemical modification motif initially designed with LMNA targeting siRNAs, when used to modify ApoC3 targeting siRNAs, generally conveyed increased activity irrespective of target or the siRNA sequence.

Example 5: Designing Additional Lmna siRNAs with Varying PS Linkages and 2′-F in Sense and Antisense Strands

Based on the previous studies, supra, with LMNA and ApoC3, showing some patterns of chemical modification confer high activity, additional chemical modification patterns were designed for LMNA and assessed to determine whether the new motifs would also be beneficial. The motif for ATXL046 was the base used to add or change chemical modifications to the antisense strand. Some of the additional modifications that may have been included in the new LMNA siRNAs include substituting more deoxyribonucleotides for ribonucleotides, reducing PS linkages, reducing 2′F, adding more 2′-OMe. The motif for ATXL052 was the base used to add or change chemical modifications to the sense strand. Some of the additional modifications include adding a PS linkage to a non-cleavage site or adding PS linkage to all ribonucleotide. See Table 22, infra. All antisense strands have a 5′-phosphate.

TABLE 22

LMNA Oligomeric Compounds with Modifications

to Both Strands

Sequence +

SIRNA
Strand
Sense or
Chemistry

SEQ ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL052
Sense
mG*mC*mG*mUrCmArCm
GCGUCACCAAA
46

si33

CrArArAmArAmGrCmGm
AAGCGCAAUU

CmAmA*mU*mU

ATXL046
Antisense
mU*T*mGmCmGfC*TmUf
UTGCGCTUUUU
62

UmUmUmGmGfUmGfAmCm
GGUGACGCUU

GmC*mU*mU

LMNA-
ATXL052
Sense
mG*mC*mG*mUrCmArCm
GCGUCACCAAA
46

si35

CrArArAmArAmGrCmGm
AAGCGCAAUU

CmAmA*mU*mU

ATXL074
Antisense
mU*T*mGmCmGfC*TmUf
UTGCGCTUUUU
95

UmUmUmGmG*TmGfAmCm
GGTGACGCUU

GmC*mU*mU

LMNA-
ATXL052
Sense
mG*mC*mG*mUrCmArCm
GCGUCACCAAA
48

si36

CrArArAmArAmGrCmGr
AAGCGCAAUU

CmAmA*mU*mU

ATXL075
Antisense
mU*T*mGmCmGfC*TmUf
UTGCGCTUUUU
96

UmUmUmGmG*TmGfAmCm
GGTGACGCUU

GmCmUmU

LMNA-
ATXL052
Sense
mG*mC*mG*mUrCmArCm
GCGUCACCAAA
46

si37

CrArArAmArAmGrCmGm
AAGCGCAAUU

CmAmA*mU*mU

ATXL076
Antisense
mU*T*mGmCmG*C*TmU*
UTGCGCTUTUU
97

TmUmUmGmG*TmG*AmCm
OGTGACGCUU

GmC*mU*mU

LMNA-
ATXL052
Sense
mG*mC*mG*mUrCmArCm
GCGUCACCAAA
48

si38

CrArArAmArAmGrCmGr
AAGCGCAAUU

CmAmA*mU*mU

ATXL077
Antisense
mU*mU*mGmCmGfC*TmU
UUGCGCTUUUU
73

fUmUmUmGmGfUmGfAmC
GGUGACGCUU

mGmC*mU*mU

LMNA-
ATXL052
Sense
mG*mC*mG*mUrCmArCm
GCGUCACCAAA
46

si39

CrArArAmArAmGrCmGm
AAGCGCAAUU

CmAmA*mU*mU

ATXL078
Antisense
mU*T*mGmCmGfCT*mUf
UTGCGCTUUUU
64

UmUmUmGmOfUmGfAmCm
GGUGACGCUU

GmC*mU*mU

LMNA-
ATXL079
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCAAA
49

si40

mCrArArAmArA*mGrC*
AAGCGCAAUU

mGmCmAmA*mU*mU

ATXL046
Antisense
mU*T*mGmCmGfC*TmUf
UTGCGCTUUUU
62

UmUmUmGmGfUmGfAmCm
GGUGACGCUU

GmC*mU*mU

LMNA-
ATXL080
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCAAA
50

si41

mCrA*rA*rA*mArA*mG
AAGCGCAAUU

rC*mGmCmAmA*mU*mU

ATXL046
Antisense
mU*T*mGmCmGfC*TmUf
UTGCGCTUUUU
62

UmUmUmGmGfUmGfAmCm
GGUGACGCUU

GmC*mU*mU

siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 20 hr. siRNA was transfected at 0-10 nM into HEK293 cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 48 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 23).

TABLE 23

LMNA siRNA Inhibition in HeLa or HEK293 Cells

HeLa Cells (20 hrs)

HEK293 Cells (48 hrs)

siRNA
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNA-si2
4.682
1
3.243
1

LMNA-si31
0.098
47.5
0.181
17.9

LMNA-si33
0.095
49.2
0.185
17.5

LMNAsi35
0.752
6.2
0.48
6.8

LMNAsi36
0.216
21.7
0.316
10.3

LMNAsi37
100.6
0
6.294
0.5

LMNAsi40
0.052
90
0.082
39.6

LMNAsi41
9.32
0.5
9.905
0.3

The sense strand cleavage site is among the central nucleotides of the sense strand. Adding PS linkages to the central nucleotides of the sense strand can inhibit activity (LMNA-si41), while adding PS linkages outside the central cleavage site can increase activity (LMNA-si40). Introducing two PS deoxyribonucleotides at positions 6 and 7 of antisense strand was not beneficial for activity (see LMNA-si37). Selected siRNAs were further tested in another cell line for a longer time period.

siRNA was transfected at 0-10 nM into Hepa1-6 cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr or 72 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 24).

TABLE 24

LMNA siRNA Inhibition in Hepa1-6 Cells

Hepa1-6 Cells (24 hrs)
Hepa1-6 Cells 72 hrs)

SIRNA
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNA-si31
0.862
1
0.148
1

LMNA-si38
96.12
0
6.403
0

LMNA-si39
1.381
0.6
0.224
0.7

LMNA-si40
2.618
0.3
0.740
0.2

In Hepa1-6 cells, LMNA-si39 and LMNA-si31 had similar activity over time, but LMNA-si38 had worse activity and LMNA-si40 had slightly worse activity compared with LMNA-si31.

Example 6: Additional Modifications to Increase Stability of the Sense Strand

Additional chemical modification patterns were designed for LMNA and assessed to determine whether the new motifs would be beneficial. The motif for ATXL046 was the base used to add or change chemical modifications to the antisense strand. The new modifications varied the amount of PS linkages and added 2′-F to the central nucleotides of the sense strand (Table 25). All antisense strands have a 5′-phosphate.

TABLE 25

LMNA Oligomeric Compounds with Modifications

to Both Strands

Sequence +

siRNA
Strand
Sense or
Chemistry

SEQ ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL081
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCA
51

si42

mCfAfAmAmArA*mGrC*
AAAAGCGCA

mOmCmAmA*mU*mU
AUU

ATXL078
Antisense
mU*T*mGmCmGfCT*mUf
UTGCGCTUU
64

UmUmUmGmGfUmGfAmCm
UUGGUGACG

GmC*mU*mU
CUU

LMNA-
ATXL082
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCA
53

si43

mCfAmAfAmArA*mGrC*
AAAAGCGCA

mGmCmAmA*mU*mU
AUU

ATXL078
Antisense
mU*T*mGmCmGfCT*mUf
UTGCGCTUU
64

UmUmUmGmGfUmGfAmCm
UUGGUGACG

GmC*mU*mU
CUU

LMNA-
ATXL083
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCA
54

si44

mCfArA*rA*mArA*mGr
AAAAGCGCA

C*mGmCmAmA*mU*mU
AUU

ATXL078
Antisense
mU*T*mGmCmGfCT*mUf
UTGCGCTUU
64

UmUmUmGmGfUmGfAmCm
UUGGUGACG

GmC*mU*mU
CUU

LMNA-
ATXL084
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCA
55

si45

mCfArA*mAmArA*mGrC
AAAAGCGCA

*mGmCmAmA*mU*mU
AUU

ATXL078
Antisense
mU*T*mGmCmGfCT*mUf
UTGCGCTUU
64

UmUmUmGmGfUmGfAmCm
UUGGUGACG

GmC*mU*mU
CUU

LMNA-
ATXL085
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCA
56

si46

mCfAfArA*mArA*mGrC
AAAAGCGCA

*mGmCmAmA*mU*mU
AUU

ATXL078
Antisense
mU*T*mGmCmGfCT*mUf
UTGCGCTUU
64

UmUmUmGmGfUmGfAmCm
UUGGUGACG

GmC*mU*mU
CUU

LMNA-
ATXL081
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCA
51

si47

mCfAfAmAmArA*mGrC*
AAAAGCGCA

mGmCmAmA*mU*mU
AUU

ATXL045
Antisense
mU*fU*mGmCmGfC*TmU
UUGCGCTUU
72

fUmUmUmGmOfUmGfAmC
UUGGUGACG

mGmC*mU*mU
CUU

LMNA-
ATXL082
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCA
53

si48

mCfAmAfAmArA*mGrC*
AAAAGCGCA

mGmCmAmA*mU*mU
AUU

ATXL045
Antisense
mU*fU*mGmCmGfC*TmU
UUGCGCTUU
72

fUmUmUmGmGfUmGIAmC
UUGGUGACG

mGmC*mU*mU
CUU

LMNA-
ATXL083
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCA
54

si49

mCfArA*rA*mArA*mGr
AAAAGCGCA

C*mGmCmAmA*mU*mU
AUU

ATXL045
Antisense
mU*fU*mGmCmGfC*TmU
UUGCGCTUU
72

fUmUmUmGmGfUmGfAmC
UUGGUGACG

mGmC*mU*mU
CUU

LMNA-
ATXL084
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCA
55

si50

mCfArA*mAmArA*mGrC
AAAAGCOCA

*mGmCmAmA*mU*mU
AUU

ATXL045
Antisense
mU*fU*mGmCmGfC*TmU
UUGCGCTUU
72

fUmUmUmGmGfUmGfAmC
UUGGUGACG

mGmC*mU*mU
CUU

LMNA-
ATXL085
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCA
56

si51

mCfAfArA*mArA*mGrC
AAAAGCGCA

*mGmCmAmA*mU*mU
AUU

ATXL045
Antisense
mU*fU*mGmCmGfC*TmU
UUGCGCTUU
72

fUmUmUmGmGfUmGfAmC
UUGGUGACG

mGmC*mU*mU
CUU

siRNA was transfected at 0-10 nM into HeLa cells with RNAi MAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 26).

TABLE 26

LMNA siRNA Inhibition in HeLa Cells

HeLa Cells (24 hrs)

HeLa Cells (120 hrs)

siRNA
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNA-si2
9.478
1
0.95
1

LMNA-si31
0.176
54
0.148
6.4

LMNA-si42
0.381
25
0.446
2.1

LMNA-si43
0.179
53
0.26
3.7

LMNA-si44
0.44
21.6
0.338
2.8

LMNA-si45
0.267
35.5
0.335
2.8

LMNA-si46
0.235
40.3
0.456
2.1

LMNA-si2
9.478
1
0.95
1

LMNA-si31
0.176
54
0.148
6.4

LMNA-si47
0.104
91.2
0.104
9.2

LMNA-si48
0.44
21.6
0.169
5.6

LMNA-si49
0.337
28.2
0.115
8.3

LMNA-si50
0.391
24.2
0.15
6.4

LMNA-si51
0.256
37
0.079
12.1

siRNA was transfected at 0-10 nM into HEK293 cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 48 hr and 120 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 27).

TABLE 27

LMNA siRNA Inhibition in HEK293 Cells

HEK293 Cells (48 hrs)
HEK293 Cells (120 hrs)

SIRNA
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNA-si2
5.433
1
2.021
1

LMNA-si31
0.055
99.5
0.078
25.9

LMNA-si42
0.031
177
0.578
3.5

LMNA-si43
0.054
100.8
0.087
23.1

LMNA-si44
0.084
64.9
0.193
10.5

LMNA-si45
0.044
123.8
0.129
15.6

LMNA-si46
0.161
33.7
0.108
18.7

LMNA-si2
5.433
1
2.021
1

LMNA-si31
0.055
99.5
0.078
25.9

LMNA-si47
0.031
177
0.077
26.3

LMNA-si48
0.051
105.5
0.118
17.2

LMNA-si49
0.060
89.9
0.087
23.2

LMNA-si50
0.036
148.9
0.135
15

LMNA-si51
0.021
255.8
0.113
17.9

All the newly designed siRNAs in this example showed better activity for a longer duration compared to benchmark siRNA LMNA-si2. LMNA-si39, LMNA-si47, LMNA-si49 and LMNA-si51 have consistently good activity and duration of activity in the different cell types. Selected siRNA designs were re-assessed for activity for over an even longer time period.

siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for different times. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was 1 calculated (Table 28).

TABLE 28

LMNA siRNA Inhibition in HeLa Cells

HeLa Cells
HeLa Cells
HeLa Cells
HeLa Cells

(24 hrs)
(72 hrs)
(120 hrs)
(168 hrs)

siRNA
IC50 (nM)
Fold
IC50 (nM)
Fold
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNA-si31
0.0354
100
0.025
11.1
0.026
11.8
0.144
8.1

LMNA-si42
0.007
472.7
0.021
13.3
0.111
2.7
0.631
1.8

LMNA-si44
0.056
63.6
0.025
10.9
0.095
3.2
0.482
2.4

LMNA-si46
0.068
51.9
0.016
17.2
0.164
1.8
0.581
2

LMNA-si47
0.038
94.1
0.014
19.9
0.055
5.5
0.141
8.2

LMNA-si49
0.188
18.9
0.02
14
0.071
4.3
0.251
4.6

LMNA-si51
0.044
80.1
0.018
15.4
0.095
3.2
0.142
8.2

LMNA-si2
3.538
1
0.277
1
0.303
1
1.164
1

When assessed at the longest time point, 168 hrs, the results show that LMNA-si47 and LMNA-si51 were the most active, while LMNA-si3l was also very active. ARNATAR designed compounds showed faster onset of activity than the benchmark LMNA-si2 and still stayed more active at later time points.

Stability assays were performed on LMNA-si3l, LMNA-si47, LMNA-si49 and LMNA-si51. The first assay exposed the compounds to human serum to test for stability, by mixing siRNA with human serum. The second assay exposed the compounds to rat tritosome to test for stability. The test compounds were found to have comparable stability compared to benchmark LMNA-si2 (FIGS. 7-8).

Serum Stability Assay

siRNAs were incubated with human serum (Sigma-Aldrich, St. Louis, MO, USA) at 0.4 μM concentration, at 37° C. for a desired time (0, 4, 8, and 24 h). Then BlueJuice™ gel loading buffer (ThermoFisher Scientific, Waltham, MA, USA) was added and the siRNA mix was loaded onto 4-20% native TBE gel (ThermoFisher Scientific, Waltham, MA, USA). After running the gel, it was stained with 1:10000 SYBR™ Gold (ThermoFisher Scientific, Waltham, MA, USA) in water, at room temperature for 10 min and imaged under UV light (FIG. 8).

Tritosome Stability Assay

The tritosome stability assay described here is based on an assay by Weingartner et al. (Molecular Therapy, Nucleic Acids, 2020, 21:242-250, which is herein incorporated-by-reference). siRNA was incubated for 0, 4, 24, or 72 h in Rat Liver Tritosomes (R0610.LT; XenoTech, Kansas City, KS, USA). To mimic the acidified environment, tritosome lysates were mixed 10:1 with low pH buffer (1.5M acetic acid, 1.5M sodium acetate, pH 4.75). 4 WL of these acidified tritosomes was mixed with 1 μL siRNA (10 μM) and incubated for the indicated times at 37° C. Then BlueJuice™ gel loading buffer (ThermoFisher Scientific, Waltham, MA, USA) was added and the siRNA mix was loaded onto 4-20% native TBE gel (ThermoFisher Scientific, Waltham, MA, USA). After running the gel, it was stained with 1:10000 SYBR™ Gold (ThermoFisher Scientific, Waltham, MA, USA) in water, at room temperature for 10 min and imaged under UV light (FIG. 7).

Example 7: Designing NCL siRNA with Varying Sense and Antisense Strand Modifications

Based on the previous studies, supra, with LMNA and ApoC3, some of the chemical modification conferring high activity was assessed in a different target to determine whether the chemical modification motifs would also be beneficial. NCL was chosen as the third test target and siRNAs designed, as shown in Table 29 and FIG. 5, to target a 19 nucleotide long sequence of human NCL: GGAUAGUUACUGACCGGGA (SEQ ID NO: 98). The primer probe set sequences are shown in Table 30. NCL-si6, a modified siRNA where the chemical modification pattern reflects that of lumasiran with the exception that the sense strand of NCL-si6 has an addition TT overhang connected by PS linkages and does not have a GalNAc conjugate, and the antisense strand is 21 nt rather than 23 nt. The chemical modification pattern of NCL-si7 is based on that of LMNA-si47 described supra. The chemical modification pattern of NCL-si8 is based on that of LMNA-si51 described supra. The chemical modification pattern of NCL-si9 is based on that of LMNA-si42 described supra. The chemical modification pattern of NCL-si10 is based on that of LMNA-si46 described supra. All antisense strands have a 5′-phosphate. The modified siRNAs were tested for activity in a HeLa cells.

TABLE 29

NCL Oligomeric Compounds with Modifications

to Both Strands

Sequence +

BIRNA
Strand
Sense or
Chemistry

SEQ ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

NCL-si6
ATXL125
Sense
mG*mG*mAmUmAmGfUmU
GGAUAGUUA
89

fAfCfUmGmAmCmCmGmG
CUGACCGGG

mGmA*T*T
ATT

ATXL124
Antisense
(5p)mU*fC*mCmCmGfG
UCCCGGUCA
90

mUfCfAmGmUmAmAfCmU
GUAACUAUC

fAmUmCmC*T*T
CTT

NCL-si7
ATXL128
Sense
mG*mG*mAmUrA*mGrU*
GGAUAGUUA
91

mUfAfCmUmGrA*mCrC*
CUGACCGGG

mGmGmGmA*mU*mU
AUU

ATXL126
Antisense
(5p)mU*fC*mCmCmGfG
UCCCGGTCA
93

T*mCfAmGmUmAmAfCmU
GUAACUAUC

fAmUmCmC*mU*mU
CUU

NCL-si18
ATXL129
Sense
mG*mG*mAmUrA*mGrU*
GGAUAGUUA
92

mUfAfCrU*mGrA*mCrC
CUGACCGGG

*mGmGmGmA*mU*mU
AUU

ATXL126
Antisense
(5p)mU*fC*mCmCmGfG
UCCCGGTCA
93

T*mCfAmGmUmAmAfCmU
QUAACUAUC

fAmUmCmC*mU*mU
CUU

NCL-si9
ATXL128
Sense
mG*mG*mAmUrA*mGrU*
GGAUAGUUA
91

mUfAfCmUmGrA*mCrC*
CUGACCGGG

mGmGmGmA*mU*mU
AUU

ATXL127
Antisense
(5p)mU*C*mCmCmGfGT
UCCCGGTCA
94

*mCfAmGmUmAmAfCmUf
GUAACUAUC

AmUmCmC*mU*mU
CUU

NCL-si10
ATXL129
Sense
mG*mG*mAmUrA*mGrU*
GGAUAGUUA
92

mUfAfCrU*mGrA*mCrC
CUGACCGGG

*mGmGmGmA*mU*mU
AUU

ATXL127
Antisense
(5p)mU*C*mCmCmGIGT
UCCCGGTCA
94

*mCfAmGmUmAmAfCmUf
GUAACUAUC

AmUmCmC*mU*mU
CUU

TABLE 30

The primer probe set sequences

for human NCL mRNA

SEQ ID

Name
Sequence
NO:

hsNCL-F
GCTTGGCTTCTTCTGGACTCA
12

hsNCL-R
TCGCGAGCTTCACCATGA
13

hsNCL-P
CGCCACTTGTCCGCTTCACAC
14

TCC

siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr. siRNA activity was determined by measuring the levels of target mRNA through qRT-PCR using the primer probe set in Table 30 and the IC50 of the siRNAs targeting NCL was calculated (Table 31). qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents in QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). The target RNA levels detected in qRT-PCR assay were normalized to either total RNA levels measured with Ribogreen™ (ThermoFisher Scientific, Waltham, MA, USA) or GAPDH mRNA levels detected in the aliquots of RNA samples using qRT-PCR.

TABLE 31

NCL siRNA Inhibition in HeLa Cells

HeLa Cells (24 hrs)

SIRNA
IC50 (nM)
Fold

NCL-si6
0.528
1

NCL-si7
0.004
136.7

NCL-si8
0.003
207.1

NCL-si9
0.0002
2945.8

NCL-si10
0.002
326.3

The new chemical modification motif initially designed with LMNA targeting siRNAs, when used to modify NCL targeting siRNAs, generally conveyed increased activity irrespective of target or the siRNA sequence.

Example 8: Further Characterization of the ARNATAR Designed Modified Oligomeric Compounds In Vitro

Previous studies, supra, assess the inhibition activity over time of oligomeric compounds after transfection into cell lines. Oligomeric compounds may have different activity over time depending on the mode of transportation into cells e.g., transfection versus free uptake.

When transfected into cells, the oligomeric compound is quickly released into the cytoplasm of the cells and quickly starts actively inhibiting the target. In other words, the oligomeric compounds are released into the cytoplasm in a quick burst, have early onset and robust activity, but the activity period is short.

When oligomeric compounds are transported into cell by free uptake (e.g., endocytosis via a receptor such as asialoglycoprotein receptor (ASGR)), the compounds are initially stored and protected from nucleases in endosomes/lysosomes and slowly released into the cytoplasm. This slow release of the compounds leads to a delayed onset and slower inhibition activity initially, but, allow a prolonged and sustained time period of activity.

When siRNA duplex is released to the cytoplasm, the duplex needs to interact with RISC and undergoes strand separation, in order to allow antisense strand-guided target nucleic acid target degradation. The efficiency of RISC loading and strand separation also affects siRNA activity and kinetics.

Select potent oligomeric compounds from the previous studies were assessed for additional characteristics such as melting temperature (Tm) and loading of the compound into the RISC system. LMNA-si2 was used as a benchmark for comparison.

A. ARNATAR Motifs Show Faster Onset of siRNA Activity after Transfection

The ARNATAR design siRNAs assessed in previous examples showed a faster onset of siRNA activity. Without being bound by any particular theory, this faster onset may be due to faster RISC loading and strand separation than the benchmark compound. Studies were performed with selected oligomeric compounds to assess the time course, melting temperature (Tm) and RISC loading (Eamens et al., 2009, RNA, 15:2219-2235).

Selected siRNAs were transfected at 0-10 nM into HeLa cells for 2 or 4 hrs, and LMNA mRNA levels were detected through qRT-PCR using primer probe sets listed in Table 2. The percentage of mRNA levels relative to the level in the time point 0 of the LMNA-si2 samples is shown in Table 32 and FIG. 9.

TABLE 32

LMNA mRNA Levels in Cells Treated with

Different siRNAs for Different Time

LMNA-
LMNA-
LMNA-
LMNA-
LMNA-

Time
Ctrl
si2
si31
si47
si49
si51

0
106.90
100.00
100.42
100.48
99.25
92.98

2 hr
96.21
97.82
95.43
94.22
97.81
102.12

4 hr
116.49
109.93
35.12
21.43
22.89
27.72

The ARNATAR designed siRNAs showed faster onset of triggering target reduction since substantial reduction of the target LMNA mRNA was observed at 4 hr after transfection ARNATAR designed siRNAs but not the reference siRNA (LMNA-si2).

Melting Temperature (Tm) Determination by Melting Curve

The Tm of a double stranded oligomeric compound is the temperature at which one half of the duplex dissociates and becomes single stranded. Tm is a measure of duplex stability. A lower Tm may facilitate faster dissociation between sense and antisense strands and allow faster availability of the antisense strand to interacting with target RNA.

0.5 μl of 10 μM siRNAs were incubated in 20 μl 1×TE buffer containing 1:1000 Ribogreen (invitrogen, ThermoFisher Scientific, Waltham, MA, USA). Triplicates for each sample were plated in a 96-well qPCR plate and placed in a QuantStudio® 3 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA) and under the following conditions to obtain the melting temperature (Tm): 95° C., 15 see; 40° C., 1 min; melt curve to 95° C., 15 sec/° C.). Tm was analyzed with the QuantStudior™ Design & Analysis Software v1.5.2 and the results shown in Table 33 and FIG. 10.

TABLE 33

Tm value of selected siRNAs

LMNA-
LMNA-
LMNA-
LMNA-
LMNA-
LMNA-
LMNA-
LMNA-
LMNA-
LMNA-
LMNA-
LMNA-

si31
si42
si43
si44
si45
si46
si47
si48
si49
si50
si51
si2

Tm
69.69
69.12
69.09
68.20
68.39
70.36
70.32
70.37
69.28
69.51
70.38
72.71

(° C.)

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

Measurement of siRNA Loaded into RISC

siRNAs must be loaded to RISC and associate with Ago2 to degrade target RNAs. Measurement of RISC-loaded siRNA (antisense strand) in cells was based on the study of Castellanos-Rizaldos et al. (Nucleic Acid Therapeutics, 2020, 30(3): 133-142) using a combination of immunoprecipitation followed by the stem-loop RT-qPCR of the final lysates of the RISC-loaded siRNAs.

i. Sample Preparation

For each siRNA to be tested, 1.5×10⁶HELA cells were transfected with 2 nM siRNA and harvested after 16 hrs. The cells were harvested and the cell pellet washed with 1×PBS then resuspended with 500 μL pre-chilled RIPA Lysis and Extraction Buffer (Thermofisher Scientific, Waltham, MA, USA) supplemented with 5 μl. RNAse Out™ (ThermoFisher Scientific, Waltham, MA, USA) and Protease Inhibitor Cocktail (Roche, Sigma-Aldrich, St. Louis, MO, USA). The cellular mixture was gently mixed by pipetting and incubated on ice for 30 min. The cellular mixture was centrifuged to separate the pellet and 400 μL of the supernatant decanted into a clean tube. 10 μL (2.5%) supernatant was set aside as input and the rest stored at −80° C. until needed.

ii. Ago2 Bead Coating Preparation

40 μL, magnetic beads were washed with 200 μL RIPA Lysis and Extraction Buffer. The washed beads were resuspended in 100 μL RIPA Lysis and Extraction Buffer and 10 μL (5 μg) Ago2 antibody (ab57113, Abcam, Cambridge, UK) and incubated at 4° C. for 2 hrs.

iii. Ago2 Immunoprecipitation

200 μL of the supernatant from the sample preparation was incubated with Ago2 antibody precoated beads for 3 hrs with gentle rotation at 4° C. The mixture was centrifuged, the supernatant removed and the beads washed 7× with 500 μL wash buffer (50 mM Tris-Cl, pH7.5; 150 mM NaCl; 5 mM E DTA; 0.1% NP-40). 250 μL PBS/TritonX100 buffer was added to the washed beads and the mixture incubate at 95° C. for 10 min, vortexed, then iced for 10 min. The mixture was centrifuged at 16,000 g for 10 mins at 4° C., then the supernatant was transferred to a clean tube and set aside for the next step.

iv. Prepare Samples for Reverse Transcription (RTT)

The 10 μL (2.5%) input supernatant was mixed with 250 μL PBS/TritonX100 buffer, heated to 95° C. for 10 min, vortexed, then placed on ice for 10 min before being centrifuged for 10 min at 4° C. The supernatant was transferred to clean tube. 20 μL of the supernatant for each sample was added to a 96-well plate, and heated in a PCR machine at 95° C. for 10 min to allow the duplexes to denature, then held at 4° C. for 5 min. To prepare a control, 250 μL PBST buffer (0.25% TritonX100 in 1×PBS buffer) was added to untreated beads.

v. Reverse Transcription (RT)

For each RT reaction, 5 μL of prepared sample was heated to 90° C. for 3 min to unpair the sense and antisense strands then the samples were iced immediately afterward to keep the strand separated. hsLMNA-A primer was used for antisense strand reaction, hsLMNA-S primer was used for the sense strand reaction. RT was performed using reverse transcription kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's suggested protocol. For a control, in an RT reaction, 5 μL of 0.2 μM siRNA was used as a RNA template to set up a standard curve.

vi. qPCR Quantitation

cDNA from the RT reaction was diluted 1:3 with DEPC water. The control RT reaction was also diluted 1:3 then serially diluted 1:10 an additional 5×. The concentration of cDNA in the serial dilution of the control reaction was: 16.66 nM, 1.666 nM, 0.1666 nM, 0.01666 nM, 0.001666 nM, 0 nM.

4 μL diluted RT cDNA template was mixed with 5 μL of 2× TaqMan™ Universal Master Mix II without UNG (ThermoFisher Scientific, Waltham, MA, USA) and 0.5 μL DDW. Primer hsLMNA-S or hsLMNA-A was used as appropriate. PCR was run under the following conditions: 95° C., 3 min; 40 cycles of 95° C., 15 second; 60° C., 20 second.

In summary, siRNAs associated with Ago2 were isolated using Ago2 antibody through immunoprecipitation from HELA cells transfected with 2 nM of each siRNA at two different time points. The isolated siRNAs and total cellular siRNAs were quantified using a kit from ThermoFisher (Waltham, MA, USA) with their proprietary stem-loop primer probe sets. The recovery rate of antisense siRNA associated with Ago2 relative to the levels of total cellular antisense siRNA was calculated, and normalized to the recovery rate of miRNA-16 as detected through qRT-PCR using stem-loop primer probe sets specific to human miRNA-16 (ThermoFisher Scientific, Waltham, MA, USA). The calculated recovery rates are shown in Table 34.

TABLE 34

The Relative Level of Antisense siRNA Associated with Ago2

in Cells Transfected with the siRNAs for Different Times

LMNA-
LMNA-
LMNA-
LMNA-
LMNA-

si2
si31
si47
si49
si51

48 hr
1.5
2.66
4.12
2.3
3.31

52 hr
1.19
3.60
4.47
2.62
2.64

This study shows more efficient loading of ARNATAR motif siRNAs into RISC than the LMNA-si2 reference siRNA (Table 34 and FIG. 11).

B. ARNATAR Motifs Show Better Activity Upon Free Uptake into Cells In Vitro

LMNA Study

Select LMNA siRNAs were conjugated with various types of N-Acetylgalactosamine (GalNAc) (Table 35) in order to allow free uptake of the compound into cells. GalNAc was attached to the sense strand of the siRNAs.

TABLE 35

LMNA Oligomeric Compounds with Modifications to Both Strands

SIRNA Name
Sequence and Chemistry
GalNAc

LMNA-si52
LMNA-si2
GalNAc 1

LMNA-si55
LMNA-si47
GalNAc 2

LMNA-si64
LMNA-si42
GalNAc 2

LMNA-si52 was designed to be another benchmark to compare ARNATAR design compounds. It has the sequence and chemistry as LMNA-si2 as described in Example 1 and is conjugated to a GalNAc used by Alnylam in their siRNA compounds (Nair et al., J. Am. Chem. Soc. 2014, 136(49):16958-16961, herein incorporated-by-reference): also known as GalNAc 1 or GA1.

LMNA-si55 and LMNA-si64 use the sequence and chemistry of LMNA-si47 and LMNA-si42, respectively, and which were shown in previous examples to have a potent and stable chemical modification motif, conjugated to a GalNAc from AM Chemicals (U.S. Pat. No. 10,087,208, herein incorporated-by-reference): also known as GalNAc 2, GalNAc (AM) or GA2.

GalNAc-conjugated siRNA was directly added to primary hepatocytes in vitro at 6.4, 32, and 160 nM final concentrations and the cells further cultured for 48 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 36).

TABLE 36

LMNA siRNA Inhibition in Primary Hepatocytes

Primary Hepatocytes

(48 hrs)

SIRNA
IC50 (nM)
Fold

LMNA-si52
29.45
1

LMNA-si55
2.343
12.57

LMNA-si64
1.526
19.3

After free uptake into cells, LMNA-si55 and LMNA-si64 showed over 10-fold increased activity over benchmark LMNA-si52.

ApoC3 Study

Select ApoC3 siRNAs were conjugated with N-Acetylgalactosamine (GalNAc) (Table 37) in order to allow free uptake of the compound into cells. GalNAc was attached to the sense strand of the siRNAs.

TABLE 37

ApoC3 Oligomeric Compounds with Modifications to Both Strands

SIRNA Name
Chemical Modifications
GalNAc

ApoC3-si10
LMNA-si2 motif
GalNAc 2

ApoC3-si11
LMNA-si47 motif
GalNAc 2

ApoC3-si12
LMNA-si42 motif
GalNAc 2

ApoC3-si13
LMNA-si52 motif with 2 less
GalNAc 2

PS linkages

ApoC3-si14
LMNA-si46 motif without 3′
GalNAc 2

PS linkages

Chemical Modifications in Table 37 describe the pattern of modifications regardless of the nucleoside sequence. For example, ApoC3-si10—having an LMNA-si2 motif—has the same pattern of chemical modifications as LMNA-si2, while the nucleoside sequence is different from that of LMNA-si2. For the full sequence and chemistry of ApoC3 siRNAs see FIGS. 3-4.

0-4 μM GalNAc-conjugated siRNA was directly added to primary hepatocytes in vitro and the cells further cultured for 48 hr and 72 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 19 and the IC50 of the siRNAs targeting ApoC3 was calculated (Table 38).

TABLE 38

IC50 of Apoe3 siRNA in primary hepatocytes upon free uptake

ApoC3-10

(reference)
ApoC3-11
ApoC3-12
ApoC3-13
ApoC3-14

IC50 (nM)
NA
4.243
2.616
3.007
0.179

Example 9: Characterization of the ARNATAR Designed Modified Oligomeric Compounds 72 Hours In Vivo

LMNA-si55, LMNA-si64 and benchmark LMNA-si52 were previously assessed for their inhibition potency and other characteristics in vitro. In this study, the oligomeric compounds were tested in mice to determine whether in vitro results could be correlated in vivo.

Three animals per group of 7- to 8-week-old Balb/C male mice were injected subcutaneously with 0.6 mg/kg, 3 mg/kg or 15 mg/kg of LMNA-si52, LMNA-si55 or LMNA-si64. For LMNA-si55, the high dose was 12 mg/kg rather than 15 mg/kg due to material limitation. Phosphate buffered saline (PBS) was injected as a control. Post dosing 72 hr, the mice were sacrificed and organs harvested for analysis (Tables 39-40 and FIG. 12).

A. Inhibition Potency

TABLE 39

LMNA siRNA Inhibition in Mouse Liver at 72 hrs

siRNA

LMNA-si52
LMNA-si55
LMNA-si64

Dose

0.6
3
15
0.6
3
12
0.6
3
15

mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg

LMNA
95.74
69.86
48.27
72.27
49.97
24.99
69.77
40.54
26.95

mRNA

TABLE 40

LMNA siRNA Inhibition in Mouse Liver

Mouse Liver at 72 hrs

IC50

SIRNA
(mg/kg)
Fold

LMNA-si52
12.46
1

LMNA-si55
2.664
4.7

LMNA-si64
2.09
6

LMNA-si55 and LMNA-si64 were more potent that benchmark LMNA-si52 and inhibited LMNA mRNA in a dose dependent manner.

B. Safety

As shown in the tables below, stress or damage markers were assessed for various organs. The blood chemistry was measured by ACP Diagnostic Services Laboratory; University of California San Diego. The markers assessed include: serum albumin (ALB), alanine transaminase (also alanine amino transferase or ALT), blood urea nitrogen (BUN), total bilirubin (TBIL), liver weight, spleen weight and other analytes (FIGS. 13-15). ALB, if low, can be a marker for liver disease, kidney disease or other medical issue. ALT is an enzyme mainly found in the liver. High levels of ALT may indicate the presence of liver disease such as acute hepatitis. BUN is a waste product from protein breakdown by the liver and is excreted by the kidneys. A high BUN level is a marker for kidney or liver dysfunction. TBIL measures the amount of bilirubin in the blood. High levels of TBIL may indicate liver disease, blockage of the bile ducts or other medical issue. Body and organ (e.g., liver and spleen) weights were taken to make sure the mice were not feeling a general malaise and off their feed.

Generally, all the assessed markers were found to be within tolerable parameters compared to PBS control after administering the oligomeric compounds (FIGS. 13-15).

C. Immunogenicity

Liver samples from the same animals as used above were analyzed for immunogenetic signals. mRNA levels of some immune response markers were examined using qRT-PCR with primer probe sets specific for mouse NfkB, IL6 and TNF (ThermoFisher Scientific, Waltham, MA, USA) as shown in Table 41 with the results presented in Table 42 and FIG. 16. Primer probe sets were from ThermoFisher Scientific (Waltham, MA, USA).

TABLE 41

Primer-Probe Sets

Primer/

SEQ ID

Probe Name
Sequence
NO

hsNFKB-F
AAACACTGTGAGGATGGGATC
99

hsNFKB-R
TCTGTCATTCGTGCTTCCAG
100

hsNFKB-P
TGTCACATGAAGTATACCCAGGTTTGCG
101

msNfkB-F
GTGTCAGAGCCCTTGTAACTG
102

msNfkB-R
ACATTTGCCCAGTTCCGTAG
103

msNfkB-P
TCGTCTGCCATGGTGAAGATGCG
104

msIL6-F
AAACCGCTATGAAGTTCCTCTC
105

msIL6-R
GTGGTATCCTCTGTGAAGTCTC
106

msIL6-P
TTGTCACCAGCATCAGTCCCAAGAA
107

msTNF-F
AGACCCTCACACTCAGATCA
108

msTNF-R
TGTCTTTGAGATCCATGCCG
109

msTNF-P
CCTGTAGCCCACGTCGTAGCAAA
110

TABLE 42

mRNA Levels in Liver Samples of Animals Treated with siRNAs for 72 Hr

LMNA-
LMNA-
LMNA-
LMNA-
LMNA-
LMNA-
LMNA-
LMNA-
LMNA-

PBS
si52-0.6
si52-3
si52-15
si55-0.6
si55-3
si55-12
si64-0.6
si64-3
si64-15

NFKB
100.0
105.95
109.24
98.69
118.47
147.70
125.26
70.42
81.19
88.11

mRNA %

IL6
100.0
99.75
119.20
107.96
103.17
120.68
122.05
76.71
82.41
89.31

mRNA %

TNF
100.0
85.34
111.30
142.46
137.86
179.44
151.28
89.55
138.90
133.25

mRNA %

In general all the assessed makers were found to be within tolerable levels compared to PBS control 72 hrs after administering the oligomeric compounds (FIG. 16).

Example 10: Characterization of the ARNATAR Designed Modified Oligomeric Compounds 7 Days In Vivo

LMNA-si52, LMNA-si55 or LMNA-si64 were assessed for a longer time in mice.

3 animals per group of 7- to 8-week-old male Balb/C mice were injected subcutaneously with 4 mg/kg or 12 mg/kg of LMNA-si52, LMNA-si55 or LMNA-si64. Phosphate buffered saline (PBS) was injected as a control. Post dosing 7 days, the mice were sacrificed and organs harvested for analysis (Tables 43-44 and FIGS. 17-18).

TABLE 43

LMNA siRNA % Inhibition in Mouse Liver at Day 7

Treatment
Dose
% LMNA mRNA

PBS
—
100.0

LMNA-si52
12 mg/kg
33.5

LMNA-si52
4 mg/kg
60.13

LMNA-si55
12 mg/kg
26.93

LMNA-si55
4 mg/kg
39.1

LMNA-si64
12 mg/kg
20.98

LMNA-si64
4 mg/kg
35.83

LMNA-si55 and LMNA-si64 knocked down LMNA mRNA levels in a dose dependent manner at day 7 after dosing mice. LMNA knockdown by LMNA-si55 and LMNA-si64 was greater than LMNA-si52 at equivalent dose levels Table 43 and FIG. 17).

Immunogenicity was assessed after dosing mice with LMNA-si52, LMNA-si55 and LMNA-si64. In general, the immune markers did not change substantially in the siRNA treated mice at Day 7 (Table 44 and FIG. 18).

TABLE 44

mRNA levels of selected immune response

markers in liver samples

LMNA-
LMNA-
LMNA-
LMNA-
LMNA-
LMNA-

PBS
si52-12
si52-4
si55-12
si55-4
si64-12
si64-4

Il-6
100.0
62.75
76.94
78.46
59.27
44.18
51.6

mRNA

%

NfKB
100.0
80.92
86.44
109.84
108.77
85.20
82.39

mRNA

%

TNF
100.0
106.63
126.69
142.40
133.94
173.45
177.37

Example 11: Additional Modifications to Improve the Chemical Modification Motif

Additional chemical modification patterns were designed for LMNA and assessed to determine whether the new motifs would be beneficial. The motif for LMNA-si47 was the base used to add or change chemical modifications to the antisense strand (Table 45). All antisense strands have a 5′-phosphate.

TABLE 45

LMNA Oligomeric Compounds with Modifications

to Both Strands

Sequence +

SIRNA
Strand
Sense or
Chemistry

SEQ ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL021
Sense
mG*mC*mGmUmCmAfCmC
GCGUCACCA
16

si2

fAfAfAmAmAmGmCmGmC
AAAAGCGCA

mAmA*T*T
ATT

ATXL020
Antisense
mU*[U*mGmCmGfCmUfU
UUGCGCUUU
34

fUmUmUmGmGfUmGfAmC
UUGGUGACG

mGmC*T*T
CTT

LMNA-
ATXL081
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCA
51

si42

mCfAfAmAmArA*mGrC*
AAAAGCGCA

mGmCmAmA*mU*mU
AUU

ATXL078
Antisense
mU*T*mGmCmGfCT*mUf
UTGCGCTUU
64

UmUmUmGmGfUmGfAmCm
UUGGUGACG

GmC*mU*mU
CUU

LMNA-
ATXL081
Sense
mG*mC*mGmUrC*mArC*
GCGUCACCA
51

si47

mCfAfAmAmArA*mGrC*
AAAAGCOCA

mmCmAmA*mU*mU
AUU

ATXL045
Antisense
mU*fU*mGmCmGfC*TmU
UUGCGCTUU
72

fUmUmUmGmGfUmGfAmC
UUGGUGACG

mGmC*mU*mU
CUU

LMNA-
ATXL154
Sense
mG*fC*mGmUrC*mArC*
GCGUCACCA
22

si65

mCfAfAmAmArA*mGrC*
AAAAGCGCA

mGmCfAmA*T*T
ATT

ATXL152
Antisense
mU*T*mGmCmGfCT*mUf
UTGCGCTUU
65

UmUmUfGmGfUmGfAmCm
UUGGUGACG

GC*mU*mU
CUU

LMNA-
ATXLI55
Sense
mG*fC*mGmUrC*mArC*
GCGUCACCA
24

si66

mCfAfAmAmArA*mGrC*
AAAAGCGCA

mGmCrA*mA*T*T
ATT

ATXL152
Antisense
mU*T*mGmCmGfCT*mUf
UTGCGCTUU
65

UmUmUfGmGfUmGfAmCm
UUGGUGACG

GC*mU*mU
CUU

LMNA-
ATXL154
Sense
mG*fC*mGmUrC*mArC*
GCGUCACCA
22

si67

mCfAfAmAmArA*mGrC*
AAAAGCGCA

mGmCfAmA*T*T
ATT

ATXL153
Antisense
mU*T*mGmCmGfCT*mUf
UTGCGCTUU
66

UmUmUG*mGfUmGfAmCm
UUGGUGACG

GC*mU*mU
CUU

LMNA-
ATXL155
Sense
mG*fC*mGmUrC*mArC*
GCGUCACCA
24

si68

mCfAfAmAmArA*mGrC*
AAAAGCGCA

mGmCrA*mA*T*T
ATT

ATXL153
Antisense
mU*T*mGmCmGfCT*mUf
UTGCGCTUU
66

UmUmUG*mGfUmGfAmCm
UUGGUGACG

GC*mU*mU
CUU

siRNA was transfected at 0-10 nM into HeLa or Hepa1-6 cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 46).

TABLE 46

LMNA siRNA Inhibition in HeLa or Hepa1-6 Cells

HeLa Cells (24 hrs)
Hepa 1-6 Cells (24 hrs)

SIRNA
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNAsi2
7.785
1
4.321
1

LMNAsi42
0.126
61.7
0.497
8.7

LMNAsi47
0.025
305.8
0.197
21.9

LMNAsi65
0.086
90.3
0.432
10

LMNAsi66
0.1
78
0.353
12.2

LMNAsi67
0.025
314.5
0.103
41.9

LMNAsi68
0.022
352.4
0.314
13.8

The new design motif for LMNA-si67 demonstrated slightly better activity than LMNA-si47.

Example 12: Additional Modifications to Vary the Chemical Modifications in Oligomeric Compounds

Additional chemical modification patterns were designed for LMNA and assessed to determine whether the new motifs would be beneficial. The motif for LMNA-si68 was the base used to vary chemical modifications in the oligomeric compound to determine if different 2′-F/2′-OMe modification combinations are beneficial for siRNA activity (Table 47). All antisense strands have a 5′-phosphate.

TABLE 47

LMNA Oligomeric Compounds with Modifications

to Both Strands

Sequence +

SIRNA
Strand
Sense or
Chemistry

SEQ ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL171
Sense
mG*fC*mGmUrC*mArC*mC
GCGUCACCAAAAAG
25

si71

fAfArA*mArA*mGrC*mOm
CGCAATT

CrA*mA*T*T

ATXL168
Antisense
mU*T*mGfCmGfCT*mUfUm
UTGCGCTUUUUGGU
67

UmUfGmGfUmGfAmCmGC*m
GACGCUU

U*mU

LMNA-
ATXL171
Sense
mG*fC*mGmUrC*mArC*mC
GCGUCACCAAAAAG
25

si72

fAfArA*mArA*mGrC*mGm
CGCAATT

CrA*mA*T*T

ATXL169
Antisense
mU*T*mGC*mGfCT*mUfUm
UTGCGCTUUUUGGU
68

UmUG*mGfUmGfAmCmGC*m
GACGCUU

U*mU

LMNA-
ATXL171
Sense
mG*fC*mGmUrC*mArC*mC
GCGUCACCAAAAAG
25

si73

fAfArA*mArA*mGrC*mGm
CGCAATT

CrA*mA*T*T

ATXL170
Antisense
mU*T*mGC*mGfCT*mUfUm
UTGCGCTUUUUGOU
69

UmUfGmGfUmGfAmCmGC*m
GACGCUU

U*mU

IMNA-
ATXL154
Sense
mG*fC*mGmUrC*mArC*mC
GCGUCACCAAAAAG
22

si74

fAfAmAmArA*mGrC*mGmC
COCAATT

fAmA*T*T

ATXL168
Antisense
mU*T*mGfCmGfCT*mUfUm
UTGCGCTUUUUGGU
67

UmUfGmGfUmGfAmCmGC*m
GACGCUU

U*mU

LMNA-
ATXL154
Sense
mG*fC*mGmUrC*mArC*mC
GCGUCACCAAAAAG
22

si75

fAfAmAmArA*mGrC*mGmC
CGCAATT

fAmA*T*T

ATXL169
Antisense
mU*T*mGC*mGfCT*mUfUm
UTGCGCTUUUUGGU
68

UmUG*mGfUmGfAmCmGC*m
GACGCUU

U*mU

LMNA-
ATXL154
Sense
mG*fC*mGmUrC*mArC*mC
GCGUCACCAAAAAG
22

si76

fAfAmAmArA*mGrC*mGmC
CGCAATT

fAmA*T*T

ATXL170
Antisense
mU*T*mGC*mGfCT*mUfUm
UTGCGCTUUUUGGU
69

UmUfGmGfUmGfAmCmGC*m
GACGCUU

U*mU

LMNA-
ATXL154
Sense
mG*fC*mGmUrC*mArC*mC
GCGUCACCAAAAAG
22

si77

fAfAmAmArA*mGrC*mGmC
CGCAATT

fAmA*T*T

ATXL186
Antisense
mU*T*mGfCmGfCT*mUfUm
UTGCGCTUUUUGGU
70

UmUfGmGfUmGfAmCmGfCm
GACGCUU

U*mU

LMNA-
ATXL154
Sense
mG*fC*mGmUrC*mArC*mC
GCGUCACCAAAAAG
22

s178

fAfAmAmArA*mGrC*mGmC
CGCAATT

fAmA*T*T

ATXL187
Antisense
mU*T*mGfCmGfCmUmUfUm
UTGCGCUUUUUGGU
60

UmUfGmGfUmGfAmCmGfC*
GACGCUU

mU*mU

LMNA-
ATXL154
Sense
mG*fC*mGmUrC*mArC*mC
GCGUCACCAAAAAG
22

si79

fAfAmAmArA*mGrC*mGmC
CGCAATT

fAmA*T*T

ATXL188
Antisense
mU*fU*mGfCmGfCmUmUfU
UUGCGCUUUUUGGU
58

mUmUfGmGfUmGfAmCmGfC
GACGCUU

mU*mU

siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen. Waltham, MA) and the cells further cultured for 24 hr, 72 and 96 hrs. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 48).

TABLE 48

LMNA siRNA Inhibition in HeLa Cells

HeLa Cells
HeLa Cells
HeLa Cells

(30 hrs)
(72 hrs)
(96 hrs)

IC50

IC50

IC50

siRNA
(nM)
Fold
(nM)
Fold
(nM)
Fold

LMNA-si2
4.112
1
0.322
1
0.519
1

LMNA-si47
0.087
47.4
0.031
10.2
0.115
4.5

LMNA-si71
0.063
65.3
0.032
10.2
0.093
5.6

LMNA-si74
0.062
66.8
0.035
9.1
0.154
3.4

LMNA-si75
0.072
57
0.032
10.1
0.136
3.8

LMNA-si77
0.092
44.7
0.044
7.4
0.117
4.4

LMNA-si78
0.052
79.6
0.039
8.3
0.117
4.4

LMNA-si79
0.064
64.7
0.031
10.4
0.125
4.1

The results show that LMNA-si78 had the highest activity at the earliest time point, 30 hrs, while LMNA-si71, LMNA-si74 and LMNA-si79 also had good activity early on. At the longest time point, 96 hrs, LMNA-si47, LMNA-si7l, LMNA-si77 and LMNA-si78 were the most active. ARNATAR designed compounds showed faster onset of activity and higher activity levels at later time points than the benchmark LMNA-si2 (FIG. 20). The results indicate, altering 2′-OMe/2′-F modification sites as determined here is tolerated for siRNA activity.

Human NFkB mRNA levels were assessed through qRT-PCT using TaqMan primer probe sets (Table 41) in HeLa cells 72 hrs after transfection. The results show that new ARNATAR siRNA designs are at comparable immunogenicity with the benchmark LMNA-si2 when measured by NFkB (FIG. 21).

Stability assays were performed on LMNA-si2, LMNA-si74, LMNA-si75 and LMNA-si78. The first assay exposed the compounds human serum to test for stability. The second assay exposed the compounds tritosome to test for stability. The three compounds have comparable stability in serum and tritosome compared to benchmark LMNA-si2 (FIG. 19).

Example 13: Additional Modifications to Substitute DNA Bases for RNA Bases in the Sense Strand

Additional chemical modification patterns were designed for LMNA siRNA and assessed to determine whether the new motifs would be beneficial. The motif for LMNA-si78 was the base used to modify the sense strand by replacing RNA with DNA nucleotides (Table 49). All antisense strands have a 5′-phosphate.

TABLE 49

LMNA Oligomeric Compounds with Modifications

to Both Strands

Sequence +

SIRNA
Strand
Sense or
Chemistry

SEQ ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL204
sense
mG*(C*mGmUC*mAC*mCfAfAm
GCGUCACCAAA
26

si90

AmAA*mGC*mGmCfAmA*T*T
AAGCGCAATT

ATXL187
antisense
mU*T*mGfCmGfCmUmUfUmUmU
UTGCGCUUUUU
60

fGmGfUmGfAmCmGfC*mU*mU
GGUGACGCUU

LMNA-
ATXL204
sense
mG*[C*mGmUC*mAC*mCfAfAm
GCGUCACCAAA
26

si91

AmAA*mGC*mGmCfAmA*T*T
AAGCGCAATT

ATXL168
antisense
mU*T*mGfCmGfCT*mUfUmUmU
UTGCGCTUUUU
67

fGmGfUmGfAmCmGC*mU*mU
GGUGACGCUU

siRNA was transfected at 0-10 nM into HEK293 cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 30 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 of the siRNAs targeting LMNA was calculated (Table 50).

TABLE 50

LMNA siRNA Inhibition in HEK293 Cells

HEK293 Cells

(30 hrs)

IC50

siRNA
(nM)
Fold

LMNA-si2
4.712
1

LMNA-si74
0.346
13.6

LMNA-si78
0.19
24.9

LMNA-si90
0.439
10.7

LMNA-si91
0.335
14.1

The results show that ARNATAR designed compounds were more potent than benchmark LMNA-si2 (Table 50 and FIG. 22).

NFkB mRNA level was assessed through RT-qPCR in HEK293 cells 48 hrs after transfection. The results show that several new ARNATAR siRNA designs are at least, or less, immunogenic when measured by NFkB than the benchmark LMNA-si2 (FIG. 22).

Example 14: In Vivo Study Characterizing ARNATAR Design Oligomeric Compounds

Select LMNA siRNAs were conjugated with various types of N-acetylgalactosamine (GalNAc) (Table 51) in order to allow free uptake of the compound into cells.

TABLE 51

LMNA Oligomeric Compounds with Modifications to Both Strands

siRNA Name
Sequence and Chemistry
GalNAc

LMNA-si52
LMNA-si2
GalNAc 1

LMNA-si57
LMNA-si2
GalNAc 2

LMNA-si92
LMNA-si74
GalNAc 2

LMNA-si93
LMNA-si75
GalNAc 2

LMNA-si94
LMNA-si78
GalNAc 2

LMNA-si52 was designed to be another benchmark to compare ARNATAR design compounds. It has the sequence and chemistry of LMNA-si2 as described in Example 1 and is conjugated to a GalNAc used by Alnylam in their siRNA compounds such as vutrisiran (Keam, 2022, Drugs, 82:1419-1425; Nair et al., J. Am. Chem. Soc. 2014, 136(49):16958-16961; herein incorporated-by-reference): GalNAc1 (also known as GA1).

LMNA-si57, LMNA-si92, LMNA-si93 and LMNA-si94 use the sequence and chemistry as described in Table 51 and are conjugated to a GalNAc from AM Chemicals (U.S. Pat. No. 10,087,208, herein incorporated-by-reference): GalNAc2 (also known as GalNAc (AM) or GA2).

7- to 8-week old Balb/c mice were injected subcutaneously with 3 mg/kg or 15 mg/kg of GalNAc conjugated siRNA, with 3 mice per group. Phosphate buffered saline (PBS) was injected as a control. Post dosing 3 days, the mice were sacrificed and organs harvested for analysis (Table 52 and FIG. 23).

TABLE 52

Percent LMNA mRNA in Mouse Liver at Day 3

LMNA mRNA %

siRNA
3 mg/kg
15 mg/kg

LMNA-si52
75.96
50.57

LMNA-si92
76.64
30.16

LMNA-si93
83.73
38.86

LMNA-si94
71.98
27.77*

LMNA-si57
87.64
60

*dosed at 13.5 mg/kg due to material limitation

At the 3 mg or 15 mg dose, LMNA-si92, -si93, and -si94 are more potent than the reference siRNA LMNA-si57. These four si RNAs share the same GalNAc2. In addition, LMNA-si52, which is essentially the same as LMNA-si57 but differs only in GalNAc, appears to be more potent than LMNA-si57, suggesting that different GalNAc can affect delivery efficiency. However, this data further suggests that the LMNA-si55 and LMNA-si64 design evaluated in the above described experiment in EXAMPLE 9 (FIG. 12) should have higher increase in potency compared with reference siRNA chemistry, if the same GalNAc is used.

Example 15: Comparison of Benchmark siRNA to ARNATAR siRNAs

Homo Sapiens Hydroxyacid Oxidase (HAO1) was chosen as a fourth test target and siRNAs designed as shown in Table 53 and FIG. 24.

The previous studies used as benchmarks modified siRNAs where the chemical modification pattern reflected that of lumasiran with the exception that the sense strand had an addition TT overhang connected by PS linkages and did not have a GalNAc conjugate, and the antisense strand was 21 nt rather than 23 nt.

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

The HAO-lumasiran benchmark and ARNATAR motif siRNAs were tested head-to-head to compare HAO1 inhibition efficacy. All ARNATAR designed antisense strands have a 5-phosphate.

TABLE 53

HAO1 Oligomeric Compounds with Modifications

to Both Strands

Sequence +

SIRNA
Strand
Sense or
Chemistry

SEQ ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

Hao-
HaoA1-
sense
mG*mA*mCmUmUmUfCmAfUfCf
GACUUUCAUCC
111

Lumasiran
S1

CmUmGmGmAmAmAmUmAmUmA
UGGAAAUAUA

HaoA1-
Antisense
mU*fA*mUmAmUfUmUfCfCmAm
UAUAUUUCCAG
112

A1

GmGmAfUmGfAmAmAmGmUmC*m
GAUGAAAGUCC

C*mA
A

Hao-
Hao-
sense
mG*fA*mCmU*rU*mU*rC*mAf
GACUUUCAUCC
114

ART-1a
ART-

UfCmCmU*rG*mG*rA*mAmAfU
UGGAAAUATA

S97

mA*T*A

Hao-
Antisense
mU*A*mUfAmUfUmUmCfCmAmG
UAUAUUUCCAG
113

ART-

fGmAfUmGfAmAmAfGmUfC*mC
GAUGAAAGUCC

A1

*mA
A

Hao-
Hao-
sense
mG*fA*mCmU*rU*mU*rC*mAf
GACUUUCAUCC
114

ART1b
ART-

UfCmCmU*rG*mG*rA*mAmAfU
UGGAAAUATA

S97

mA*T*A

Hao-
Antisense
mU*A*mUfAmUfUmUmCfCmAmG
UAUAUUUCCAG
115

ART-

fGmAfUmGfAmAmAfG*mU*mC
GAUGAAAGUC

A2

Hao-
Hao-
sense
mG*fA*mCmU*rU*mU*rC*mAf
GACUUUCAUCC
114

ART2a
ART-

UfCmCmU*rG*mG*rA*mAmAfU
UGGAAAUATA

S97

mA*T*A

Hao-
Antisense
mU*A*mUfAmUfUT*mCfCmAmG
UAUAUUTCCAG
116

ART-

fGmAfUmGfAmAmAG*mUfC*mC
GAUGAAAGUCC

A74-1

*mA
A

Hao-
Hao-
sense
mG*fA*mCmU*rU*mU*rC*mAf
GACUUUCAUCC
114

ART2b
ART-

UfCmCmU*rG*mG*A*mAmAfUm
UGGAAAUATA

S97

A*T*A

Hao-
Antisense
mU*A*mUfAmUfUT*mCfCmAmG
UAUAUUTCCAG
117

ART-

fGmAfUmGfAmAmAG*mU*mC
GAUGAAAGUC

A74-2

siRNA was transfected at 0-0.4 nM into Hep3b cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells cultured for 24 hr. siRNA activity was determined by a HAO1 gene expression assay from ThermoFisher Scientific (catalog #Hs00213909_m1; Waltham, MA. USA) and the amount of HAO1 mRNA left after siRNA knockdown was assessed (Table 54 and FIG. 25).

TABLE 54

HAO1 siRNA Inhibition in Hep3b Cells After 24 Hrs

HAO1 mRNA level %

siRNA
0 nM
0.016 nM
0.06 nM
0.4 nM

Hao-Lumasiran
100
73.2
48.9
35

Hao-ART-1a
100
32.3
29.5
29

Hao-ART-1b
100
40.3
23.9
11.3

Hao-ART-2a
100
30.4
27
21.6

Hao-ART-2b
100
29.6
29.6
14.5

The ARNATAR designed siRNAs showed better inhibition rates than the benchmark siRNA.

Example 16: Additional Chemical Modifications to Evaluate the Effects on siRNA Activity by 5′-Phosphate on the Antisense Strand and Phosphorothioate on the Sense Strand

Additional chemical modification patterns were designed for LMNA siRNA (Table 55) and assessed to determine whether the new motifs would be beneficial.

Prior siRNAs evaluated above contain 5′-phosphate on the antisense strand, which is required for Ago2 binding and function. Since siRNAs can be phosphorylated in cells (Weitzer S, Martinez J. Nature, 2007, 447(7141):222-226), the 5′-phosphate may not be necessarily added during siRNA synthesis. Thus, the activity of siRNAs with or without 5′ phosphate was evaluated; LMNA-si94 is previously disclosed LMNA-si78 with a 5′-phosphate on the antisense strand and the addition of a GalNAc conjugate on the sense strand. LMNA-si95 is LMNA-si94 with a 5′-OH instead of a 5′-phosphate on the antisense strand.

Additionally, the effects of the position and number of phosphorothioate (5′ or 3′, or both 5′ and 3′) around DNA or RNA nucleotides in the sense strand was evaluated; LMNA-si96 to LMNA-si100 was designed based on previously disclosed LMNA-si78 or LMNA-si90. LMNA-si96 is LMNA-si90 with a GalNAc conjugate. siRNA compounds LMNA-si97, LMNA-si98, LMNA-si99 and LMNA-si100 comprise the antisense strand of LMNA-si78 and a sense strand with a newly designed motif moving the phosphorothioate to various locations and a GalNAc conjugate.

The GalNAc conjugate used in this study is known herein as GalNAc2 (AM Chemicals, U.S. Pat. No. 10,087,208, herein incorporated-by-reference).

TABLE 55

LMNA Oligomeric Compounds with Modifications

to Both Strands

Sequence +

SIRNA
Strand
Sense or
Chemistry

SEQ ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL154
Sense
mG*fC*mGmUrC*mArC*mCfAf
GCGUCACCAAA
23

si94
GA

AmAmArA*mGrC*mGmCfAmA*T
AAGCGCAATT

*T-GalNAc2

ATXL187
Antisense
mU*T*mGfCmGfCmUmUfUmUmU
UTGCGCUUUUU
60

fGmGfUmGfAmCmGfC*mU*mU
GGUGACGCUU

LMNA-
ATXL154
sense
mG*fC*mGmUrC*mArC*mCfAf
GCGUCACCAAA
23

si95
GA

AmAmArA*mGrC*mGmCfAmA*T
AAGCGCAATT

*T-GalNAc2

ATXL187
antisense
(OH)mU*T*mGfCmGfCmUmUfU
UTGCGCUUUUU
60

OH

mUmUfGmGfUmGfAmCmGfC*mU
GGUGACGCUU

*mU

LMNA-
ATXL206
sense
mG*fC*mGmUC*mAC*mCfAfAm
GCGUCACCAAA
27

si96

AmAA*mGC*mGmCfAmA*T*T-
AAGCGCAATT

GalNAc2

ATXL187
antisense
mU*T*mGfCmGfCmUmUfUmUmU
UTGCGCUUUUU
60

fGmGfUmGfAmCmGfC*mU*mU
GGUGACGCUU

LMNA-
ATXL207
sense
mG*fC*mGmU*rC*mA*rC*mCf
GCGUCACCAAA
28

si97

AfAmAmA*A*mG*C*mGmCfAmA
AAGCGCAATT

*T*T-GalNAc2

ATXL187
antisense
mU*T*mGfCmGfCmUmUfUmUmU
UTGCGCUUUUU
60

fGmOfUmGfAmCmGfC*mU*mU
GOUGACGCUU

LMNA-
ATXL208
sense
mG*fC*mGmUfCmAfCmCfAfAm
GCGUCACCAAA
29

si98

AmAfAmGfCmGmCfAmA*T*T-
AAGCGCAATT

GalNAc2

ATXL187
antisense
mU*T*mGfCmGfCmUmUfUmUmU
UTGCGCUUUUU
60

fGmGfUmGfAmCmGfC*mU*mU
GGUGACGCUU

LMNA-
ATXL209
sense
mG*fC*mGmU*C*mA*C*mCfAf
GCGUCACCAAA
31

si99

AmAmA*A*mG*C*mGmCfAmA*T
AAGCGCAATT

*T-GalNAc2

ATXL187
antisense
mU*T*mGfCmGfCmUmUfUmUmU
UTGCGCUUUUU
60

fGmGfUmGfAmCmGfC*mU*mU
GGUGACGCUU

LMNA-
ATXL210
sense
mG*fC*mGmU*CmA*CmCfAfAm
GCGUCACCAAA
32

si100

AmA*AmG*CmGmCfAmA*T*T-
AAGCGCAATT

GalNAc2

ATXL187
antisense
mU*T*mGfCmGfCmUmUfUmUmU
UTGCOCUUUUU
60

fGmGfUmGfAmCmGfC*mU*mU
GGUGACGCUU

In Vitro Assay

siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 72 hr and 96 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 values of the siRNAs targeting LMNA were calculated (Table 56) and the percent inhibition of LMNA RNA levels graphed (FIG. 26).

TABLE 56

LMNA siRNA Inhibition in HeLa Cells

HeLa Cells

HeLa Cells

(72 hrs)

(96 hrs)

IC50

IC50

siRNA
(nM)
Fold
(nM)
Fold

LMNA-si52
0.3
1
0.18
1

LMNA-si94
0.07
4.31
0.014
12.54

LMNA-si95
0.18
1.63
0.11
1.64

LMNA-si96
0.19
1.56
0.06
2.89

LMNA-si97
0.05
6.33
0.01
13.67

LMNA-si98
0.18
1.64
0.14
1.3

LMNA-si99
0.13
2.27
0.04
4.15

LMNA-si100
0.19
1.6
0.18
1.02

The results indicate that siRNA with an antisense strand with 5′-phosphate (LMNA-si94) showed better activity than siRNA with an antisense strand with 5′-OH(LMNA-si95), and that siRNA can tolerate phosphorothioate in the sense strand at both 5′ and 3′ of DNA or RNA nucleotide. In addition, phosphorothioate in the sense strand at 3′ of a DNA nucleotide (LMNA-si96) appears slightly better than at 5′ of a DNA nucleotide (LMNA-si100). The results also show that ARNATAR designed compounds were more potent than benchmark LMNA-si52 in vitro. (Table 56 and FIG. 26).

In Vivo Assay

Several siRNA compounds found to be potent in the in vitro inhibition assay above was assessed in vivo; 6- to 8-week old male Balb/c mice were injected subcutaneously with 3 mg/kg of siRNA, with 3 mice per group. Groups of mice were assessed at 3-days, 1-week or 2-weeks. Phosphate buffered saline (PBS) was injected as a control. Post dosing 3-days, 1-week or 2-week, the mice were sacrificed and organs harvested for analysis (Table 57 and FIG. 27). No groups of mice treated with LMNA-si94 or LMNA-si95 were taken into the 1-week or 2-week time points. siRNA activity was determined by measuring the levels of target mRNA through qRT-PCR using the LMNA primer probe set listed in Table 2. qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents in QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). The target RNA levels detected in qRT-PCR assay were normalized to GAPDH mRNA levels detected in the aliquots of RNA samples using qRT-PCR.

TABLE 57

Percent LMNA mRNA in Mouse Liver

at Day 3, 1 Week or 2 Week

LMNA Knockdown Levels

(% Inhibition)

siRNA
3-Day
1-Week
2-Week

LMNA-si52
65.5
72.5
71.3

LMNA-si94
59.5
n/a
n/a

LMNA-si95
56.0
n/a
n/a

LMNA-si96
76.5
69.6
73.3

LMNA-si97
52.0
53.0
67.9

LMNA-si98
70.5
59.2
64.3

LMNA-si99
70.5
68.6
72.2

The in vivo results show that, when dosed at 3 mg/kg, the motif designs for LMNA-si94, LMNA-si95 and LMNA-si97 were more potent than the benchmark compound LMNA-si52 at the 3-day period. At the 1- and 2-week time periods, LMNA-si97 and LMNA-si98 were more potent inhibitors than LMNA-si52. No mice treated with LMNA-si94 or LMNA-si95 were carried through more than 3-days post-treatment.

Example 17: Additional Modifications to Increase siRNA Durability

Sense and antisense strands used to design LMNA siRNA in previous examples were mixed and matched in different combinations (Table 58) and assessed to determine whether the new siRNA compounds would be potent. The sense strands from LMNA-si97 (ATXL207) and LMNA-si98 (ATXL208), which showed durable inhibition activity at the 2-week time point in the previous example, was paired with different antisense strands from siRNA previously shown to be potent LMNA inhibitor (LMNA-si74) or stable (LMNA-si79). Each antisense strand has a 5′-phosphate and each sense strand has a GalNAc2 conjugate.

TABLE 58

LMNA Oligomeric Compounds with Modifications to Both Strands

SEQ

SIRNA
Strand
Sense or
Sequence + Chemistry

ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL208
sense
mG*fC*mGmUfCmAfCmCfAfAmA
GCGUCACCAAA
29

si103

mAfAmGfCmGmCfAmA*T*T-
AAGCOCAATT

GalNAc2

ATXL168
antisense
mU*T*mGfCmGfCT*mUfUmUmUf
UTGCOCTUUUU
67

GmGfUmGfAmCmGC*mU*mU
GGUGACGCUU

LMNA-
ATXL208
sense
mG*fC*mGmUfCmAfCmCfAfAmA
GCGUCACCAAA
29

si104

mAfAmGfCmGmCfAmA*T*T-
AAGCGCAATT

GaINAc2

ATXL188
antisense
mU*fU*mGfCmGfCmUmUfUmUm
UUGCGCUUUUU
58

UfGmGfUmGfAmCmGfCmU*mU
GGUGACGCUU

LMNA-
ATXL207
sense
mG*fC*mGmU*rC*mA*rC*mCfAf
GCGUCACCAAA
28

si105

AmAmA*rA*mG*rC*mGmCfAmA
AAGCGCAATT

*T*T-GalNAc2

ATXL168
antisense
mU*T*mGfCmGfCT*mUfUmUmUf
UTGCGCTUUUU
67

GmGfUmGfAmCmGC*mU*mU
GGUGACGCUU

LMNA-
ATXL207
sense
mG*fC*mGmU*rC*mA*rC*mCfAf
GCGUCACCAAA
28

si106

AmAmA*rA*mG*rC*mGmCfAmA
AAGCGCAATT

*T*T-GalNAc2

ATXL188
antisense
mU*fU*mGfCmGfCmUmUfUmUm
UUGCGCUUUUU
58

UfGmGfUmGfAmCmGfCmU*mU
GGUGACGCUU

In Vitro Assay

siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 20 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 values of the siRNAs targeting LMNA were calculated (Table 59) and the percent inhibition of LMNA RNA levels graphed (FIG. 28).

TABLE 59

LMNA siRNA Inhibition in HeLa Cells

HeLa Cell (20 hrs)

siRNA
IC50 (nM)
Fold

LMNA-si52
0.287
1.0

LMNA-si103
0.052
5.5

LMNA-si104
0.194
1.5

LMNA-si105
0.021
14.0

LMNA-si106
0.009
30.9

siRNAs with LMNA-si97 sense strand that contains several RNA nucleotides showed better activity, while the siRNA (LMNA-si103) with a fully 2′-OMe/F modified sense strand and a DNA modified antisense strand also improved activity compared with reference siRNA (LMNA-si52). However, the siRNA (LMNA-si104) that is fully 2′-OMe/F modified in both sense and antisense had slightly better activity compared with the reference siRNA.

Example 18: Additional Modifications to Increase siRNA Durability

Sense and antisense strands used to design LMNA siRNA in previous examples were mixed and matched in different combinations (LMNA-si107 and LMNA-si108) or a non-GalNAc version of LMNA-103 (LMNA-si109) as shown in Table 60, and assessed to determine whether the new siRNA compounds would be potent. Each antisense strand has a 5′-phosphate. The sense strand of LMNA-si107 and LMNA-si108 has a GalNAc2 conjugate.

TABLE 60

LMNA Oligomeric Compounds with Modifications to Both Strands

SEQ

siRNA
Strand
Sense or
Sequence + Chemistry

ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL206
sense
mG*fC*mGmUC*mAC*mCfAfAmA
GCGUCACCAAA
27

si107

mAA*mGC*mGmCfAmA*T*T-
AAGCGCAATT

GalNAc2

ATXL168
antisense
mU*T*mGfCmGfCT*mUfUmUmUf
UTGCGCTUUUU
67

GmGfUmGfAmCmGC*mU*mU
GGUGACGCUU

LMNA-
ATXL154
sense
mG*f C*mGmUrC*mArC*mCfAfAm
GCGUCACCAAA
23

si108
GA

AmArA*mGrC*mGmCfAmA*T*T-
AAGCGCAATT

GalNAc2

ATXL168
antisense
mU*T*mGfCmGfCT*mUfUmUmUf
UTGCGCTUUUU
67

GmGfUmGfAmCmGC*mU*mU
GGUGACGCUU

LMNA-
ATXL232
sense
mG*fC*mGmUfCmAfCmCfAfAmA
GCGUCACCAAA
30

si109

mAfAmGfCmGmCfAmA*T*T
AAGCGCAATT

ATXL168
antisense
mU*T*mGfCmGfCT*mUfUmUmUf
UTGCGCTUUUU
67

GmGfUmGfAmCmGC*mU*mU
GGUGACGCUU

In Vitro Assays

siRNA was transfected at 0-10 nM into HeLa cells or Hepa1-6 cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for various times as indicated in the tables. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 values of the siRNAs targeting LMNA were calculated (Tables 61-63) and the percent inhibition of LMNA RNA levels graphed (FIGS. 29-31).

TABLE 61

LMNA siRNA Inhibition in HeLa Cells

HeLa Cells (16 hrs)

HeLa Cells (48 hrs)

siRNA
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNA-si52
5.76
1
0.15
1

LMNA-si103
0.31
18.4
0.009
15.8

LMNA-si107
0.06
100.3
0.003
44.6

LMNA-si108
0.013
434.1
0.0006
234.8

TABLE 62

LMNA siRNA Inhibition in Hepa1-6 Cells

Hepa1-6 Cells (16 hrs)
Hepa1-6 Cells (48 hrs)

siRNA
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNA-si52
11.83
1
0.343
1

LMNA-si103
1.58
7.5
0.075
4.6

LMNA-si107
0.23
50.6
0.033
10.5

LMNA-si108
0.17
71.2
0.043
8

TABLE 63

LMNA siRNA Inhibition in HeLa Cells

HeLa Cell (20 hrs)

siRNA
IC50 (nM)
Fold

LMNAsi2
15.43
1

LMNAsi109
0.33
47

LMNAsi74
0.04
438

The results indicate that the new siRNA combinations generally showed better activity than the reference siRNAs LMNA-si2 or LMNA-si52, while the two siRNAs containing RNA nucleotides in sense strand (LMNA-si107, LMNA-si108) have even better activity.

Example 19: Characterization of the Duration of siRNA Activity In Vivo

In addition to activity, another important factor for a siRNA therapeutic is the duration of action in vivo. To evaluate the duration of action of si RNAs with newly modified patterns, siRNAs LMNA-si103 and reference siRNA LMNA-si52 were dosed subcutaneously at 5 mg/kg into 7-week BalB/C mice, and sacrificed at 10- or 25-days after siRNA injection. LMNA mRNA levels in total liver were analyzed using qRT-PCR with the primer probe set as described in Table 2. The results are shown in Table 64. Substantially better knockdown was observed for LMNA-103 than for reference siRNA LMNA-si52 in vivo. In addition, LMNA-si103 and reference LMNA-si52 have similar recovery slopes (FIG. 32), suggesting that the newly modified siRNA has a better activity and a comparable duration with the reference siRNA in animals.

TABLE 64

Percent LMNA mRNA in Mouse Liver at Day 10 or 25

LMNA Knockdown Levels

(% Inhibition)

10 days
25 days

PBS
100
100

LMNA-si52
59.42
100.45

LMNA-si103
41.74
74.80

Example 20: Comparison of ARNATAR siRNA Platform Design with Third Party siRNA Platform Design

As disclosed in Example 1, LMNA-si2 benchmark was designed based on the chemical modification design of a commercially available therapeutic, Lumasiran. Benchmark LMNA-si52 is LMNA-si2 with the addition of a GalNAc conjugate (Example 8). However, to minimize variables in the above experiments, LMNA-si2 (and LMNA-si52) differed from Lumasiran in that it was designed to have a LMNA sequence, a structure with overhangs at both ends, and 21 nt length.

In this study, the chemical modification platform ESC Plus (ESC+) (Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101) was used to design benchmark siRNAs for three targets: LMNA, NCL and ApoC3. As described by Hu et al., these new benchmark siRNAs have 21 nt sense strands, 23 nt antisense strands, without 3′ overhangs in the sense strands, and 2 natural nucleotides overhanging at the 3′ end of antisense strands (forming 23 nt base-pairing with mRNA target). Also, the 5′ ends of the antisense strands have no phosphates and position 7 in the antisense strands are “GNA” modified nucleotides when commercially available. To form a better benchmark siRNA, 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, ApoC3-AL as shown in Table 65.

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

The siRNAs were made without GalNAc conjugates in order to only compare the chemical modification motifs.

TABLE 65

Oligomeric Compounds with Different Designs for NCL, LMNA, and

APOC3 mRNAs

SIRNA
Strand
Sense or
Sequence + Chemistry

SEQ ID

Name
Name
Antisense
(5′ to 3′)
Sequence
NO:

LMNA-
ATXL-154
Sense
mG*fC*mGmUrC*mArC*mCfAf
GCGUCACC
22

74

AmAmArA*mGrC*mGmCfAmA*
AAAAAGCG

T*T
CAATT

ATXL-168
Antisense
(Sp)mU*T*mGfCmGfCT*mUfU
UTGCGCTUU
67

mUmUfGmGfUmGfAmCmGC*m
UUGGUGAC

U*mU
GCUU

LMNA-
ATXL-239
Sense
mC*mA*mGmCmGmUfCmAfCf
CAGCGUCA
118

si111

CfAmAmAmAmAmGmCmGmC
CCAAAAAG

mAmA
CGCAA

ATXL238
Antisense
mU*fU*mGmCmGfCgnaTfUfUm
UUGCGCTU
119

UmUmGmGfUmGfAmCmGmCm
UUUGGUGA

UmG*mC*mC
CGCUGCC

NCL-74
NCL-74s
Sense
mG*fG*mAmCrA*mUrU*mCfCf
GGACAUUC
120

AmAmGrA*mCrA*mGmUfAmU*
CAAGACAG

T*T
UAUTT

NCL-74a
Antisense
(5p)mA*T*mAfCmUfGT*mCfU
ATACUGTCU
121

mUmGfGmAfAmUfGmUmCC*m
UGGAAUGU

U*mU
CCUU

NCL-
NCL-Als1
Sense
mG*mA*mGmGmAmCfAmUfUf
GAGGACAU
122

ALN

CfCmAmAmGmAmCmAmGmU
UCCAAGAC

mAmU
AGUAU

NCL-Ala
Antisense
mA*fU*mAmCmUfGgnaTfCfUm
AUACUGTC
123

UmGmGmAfAmUfGmUmCmCm
UUGGAAUG

UmC*mA*mG
UCCUCAG

ApoC3-
Apoc3-74s
Sense
mC*fU*mCmUrG*mArG*mUfUf
CUCUGAGU
76

74

CmUmGrG*mGrA*mUmUfUmG*
UCUGGGAU

T*T
UUGTT

Apoc3-74a
Antisense
(5p)mC*A*mAfAmUICC*mCfA
CAAAUCCC
86

mGmAfAmCfUmCfAmGmAG*m
AGAACUCA

U*mU
GAGUU

ApoC3-
Apoc3-ALs
Sense
mU*mU*mCmUmCmUfGmAfGf
UUCUCUGA
124

AL

UfUmCmUmGmGmGmAmUmU
GUUCUGGG

mUmG
AUUUG

Apoc3-
Antisense
mC*fA*mAmAmUfCgnaCfCfAm
CAAAUCCC
125

ALa

GmAmAmCfUmCfAmGmAmGm
AGAACUCA

AmA*mC*mU
GAGAACU

In Vitro Assays

siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) for LMNA and NCL siRNAs and the cells further cultured for 20 hr. For ApoC3 siRNA, it was transfected into Hep3B cells at 0-10 nM using RNAiMax and cells were incubated for 60 hr. siRNA activity was determined by qRT-PCR using the primer probe sets as listed in Table 2, 30, 19. The IC50 of the siRNAs targeting corresponding mRNAs are shown in Table 66 and the percent inhibition of LMNA RNA levels graphed (FIG. 33).

TABLE 66

The activity of siRNAs targeting different

mRNAs in HeLa or Hep3B cells

Treated

time

Cell type
(hour)
siRNAs
IC50 (nM)
Fold

LMNA
HeLa
20
LMNA-74
~0.0000069
>1000

mRNA

LMNA-si111
10.46
1

NCL
HeLa
20
NCL-74
~0.00000061
>1000

mRNA

NCL-ALN
0.033
1

ApoC3
Hep3B
60
ApoC3-74
0.004
140

mRNA

ApoC3-AL
0.56
1

The results showed that the newly designed siRNAs dramatically improved activity compared with the ESC+ design. Also, the ARNATAR design motif generally conveyed increased activity irrespective of mRNA target or siRNA sequence.

Example 21: Comparison of siRNA with Varying Lengths Targeting LMNA, NCL and AGT mRNAs

Previously tested siRNA compounds were mainly 21 nt long, with two overhangs at both ends. Since the length of siRNAs generated in cells by Dicer cleavage may vary (e.g., from about 21 nt to 23 nt), the effects of varying siRNA length on siRNA activity were evaluated. To this end, siRNAs 21-mer or 23-mer lengths were designed with ARNATAR motifs to target NCL (NCL-74 and NCL-23 nt) and LMNA (LMNA-si110) as shown in Table 67. The ARNATAR design motifs include, among other design elements, 2 nt overhangs at both strands and 5′-phosphate on the antisense strand. 23mer siRNAs from previous Examples with non-ARNATAR chemical modifications were used as benchmarks (LMNA-si111, NCL-ALN). The siRNAs were made without GalNAc conjugates in order to only compare the chemical modification motifs.

TABLE 67

Modified LMNA Oligomeric Compounds of Varying Lengths

SIRNA
Strand
Sense or
Sequence + Chemistry

SEQ ID

Name
Name
Antisense
(S′ to 3′)
Sequence
NO:

LMNA-
ATXL237
sense
mC*fA*mGmCrG*mUrC*mAfCfC
CAGCGUCACC
126

si110

mAmArA*mArA*mGmCfGmCmA
AAAAAGCGCA

fA*T*T
ATT

ATXL236
antisense
(Sp)mU*T*mGfCmGfCT*mUfUm
UTGCGCTUUU
127

UmUfGmGfUmGfAmCmGC*mUm
UGGUGACGCU

G*C*C
GCC

LMNA-
ATXL239
sense
mC*mA*mGmCmGmUfCmAfCfCf
CAGCGUCACC
118

si111

AmAmAmAmAmGmCmGmCmA
AAAAAGCGCA

mA
A

ATXL238
antisense
mU*fU*mGmCmGfCgnaTfUfUmU
UUGCGCTUUU
119

mUmGmGfUmGfAmCmGmCmUm
UGGUGACOCU

G*mC*mC
GCC

NCL-
NCL-Als1
Sense
mG*mA*mGmGmAmCfAmUfUfC
GAGGACAUUC
122

ALN

fCmAmAmGmAmCmAmGmUmA
CAAGACAGUA

mU
U

NCL-ALa
Antisense
mA*fU*mAmCmUfGgnaTfCfUmU
AUACUGTCUU
123

mGmGmAfAmUfGmUmCmCmUm
GGAAUGUCCU

C*mA*mG
CAG

NCL-
NCL-74s
Sense
mG*fG*mAmCrA*mUrU*mCfCfA
GGACAUUCCA
120

74

mAmGrA*mCrA*mGmUfAmU*T*
AGACAGUAUT

T
T

NCL-74a
Antisense
(Sp)mA*T*mAfCmUfGT*mCfUm
ATACUGTCUU
121

UmGfGmAfAmUfGmUmCC*mU*
GGAAUGUCCU

mU
U

NCL-
NCL-
Sense
mG*fA*mGmGrA*mCrA*mUfUfC
GAGGACAUUC
128

23nt
23nt-s

mCmArA*mGrA*mCmAfGmUmA
CAAGACAGUA

fU*T*T
UTT

NCL-
Antisense
(5p)mA*T*mAfCmUfGT*mCfUm
ATACUGTCUU
129

23nt-a

UmGfGmAfAmUfGmUmCfCmUC*
GGAAUGUCCU

mA*mG
CAG

In Vitro Assays—Comparison of LMNA-si110 and LMNA-si74 to Benchmark LMNA-si111

LMNA siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 16 and 36 hr. siRNA activity was determined by qRT-PCR using the primer probe set in Table 2 and the IC50 values of the siRNAs targeting LMNA were calculated (Table 68) and the percent inhibition of LMNA RNA levels graphed (FIG. 34).

TABLE 68

LMNA siRNA Inhibition in HeLa Cells

HeLa Cell (16 hrs)

HeLa Cell (36 hrs)

siRNA
IC50 (nM)
Fold
IC50 (nM)
Fold

LMNA-si110
1.61
22
0.0367
9

LMNA-si111
35.6
1
0.331
1

LMNA-si74
0.0447
796
0.0004
824

In Vitro Assays—Comparison of NCL-74 and NCL-23 nt to NCL-ALN

NCL siRNA was transfected at 0-10 nM into HeLa cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 36 hr. siRNA activity was determined by qRT-PCR using the primer probe sets as listed in Table 30. The results are shown in FIG. 35. Since NCL mRNA reduction was too great to determine the IC50, the percentage mRNA levels at 0.08 nM siRNA concentration are listed in Table 69.

TABLE 69

NCL siRNA Inhibition in HeLa Cells at 36 Hrs

% NCL mRNA at

siRNA
0.08 nM siRNA

NCL-23nt (23 nt)
23%

NCL-ALN (Benchmark)
38%

NCL74 (21 nt)
16%

The results showed that the 23 nt siRNAs with newly designed chemistry also showed better activity compared with benchmark siRNA of the same length, although the 21 nt siRNA appear to have more activity compared with 23mer siRNAs.

In Vitro Assay—Comparison of 21 nt to 23 nt siRNAs Targeting AGT

To further determine if the better activity of 21-mer siRNA over 23-mer siRNA also applies to siRNA sequences targeting other mRNA transcripts, 21-mer and 23-mer siRNAs targeting human angiotensinogen (AGT) mRNA were designed as shown in Table 70 and FIGS. 36-37). A GalNAc (as described in Sharma et al., 2018, Bioconjugate Chem, 29:2478-2488 and known herein as GA3 or GalNAc3) is conjugated to the sense strand. The ARNATAR designed siRNAs have a 5′-phosphate on the antisense strand.

TABLE 70

Oligomeric Compounds with Different Length Targeting AGT mRNA

SEQ

Strand
Sense or
Sequence + Chemistry

ID

siRNA
Name
Antisense
(5′ to 3′)
Sequence
NO:

AT-
ATs484
Sense
mC*fC*mAmAfUmAfGmCfUfUm
CCAAUAGCUU
131

si600

AmAfCmAfAmGmCfCmU*T*T-
AACAAGCCUT

GA3
T

ATa483
Antisense
(Sp)mA*G*mGfCmUfUmGmUfU
AGGCUUGUUA
134

mAmAfGmCfUmAfUmUmGfG*m
AGCUAUUGGG

G*mU
U

AT-
ATs633
Sense
mA*fC*mCmCfAmAfUmAfGfCm
ACCCAAUAGC
136

si633

UmUfAmAfCmAmAfGmCmCfU*
UUAACAAGCC

T*T-GA3
UTT

ATa633
Antisense
(5p)mA*G*mGfCmUfUmGmUfU
AGGCUUGUUA
137

mAmAfGmCfUmAfUmUmGfGm
AGCUAUUGGG

GmU*T*T
UTT

In Vitro Assays

AGT siRNA was transfected at 0-10 nM into Hep3B cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 36 hr. In addition, the siRNAs were incubated with human primary hepatocytes without transfection for 48 hr. siRNA activity was determined by qRT-PCR using the primer probe sets as listed in Table 71. The IC50 of the siRNAs targeting AGT are shown in Table 72 and the percent inhibition of AGT mRNA levels graphed (FIG. 38).

TABLE 71

Sequences of Primer Probe Set for AGT

Name
Sequence
SEQ ID NO:

hsAGT-F
CTGATCCAGCCTCACTATGC
138

hsAGT-R
AGGTCATAAGATCCTTGCAGC
139

hsAGT-P
AGGGTCTCACTTTCCAGCAAAACTCC
140

TABLE 72

siRNA Activity Targeting AGT in Hep3B cells

Human primary

Hep3B Cells (36 hr)
hepatocytes

siRNA
IC50

IC50

siRNA
Length
(nM)
Fold
(μM)
Fold

ATsi600
21mer
0.057
1.62
0.137
11.8

ATsi633
23mer
0.093
1
1.617
1

The results with this target also show that the 21 nt siRNA has better activity than the 23 nt siRNA.

Example 22: New Chemical Modification Motifs Showed Better Activity than Reference Design for Different siRNAs Targeting AGT

The above results suggest that the newly optimized ARNATAR chemistry design increased the activity of siRNAs for different targets or sequences. To further determine if this observation is generalizable, siRNAs were designed to target 5 different regions of human AGT, a therapeutic target for the treatment of resistant hypertension. The siRNA sequences and chemistry are listed in Table 73. For comparison, the siRNAs were designed with ARNATAR motifs, or with a third party's structure and modification design (ESC or ESC+ as described in Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101). The ARNATAR designed siRNAs have a 5′-phosphate on the antisense strand. The siRNAs were made without GalNAc conjugates in order to only compare the chemical modification motifs.

TABLE 73

Oligomeric Compounds Targeting Different Regions of AGT mRNA.

SEQ

Strand
Sense or

ID

SIRNA
Name
Antisense
Sequence + Chemistry
Sequence
NO:

ATsi-
ATs365m
sense
mU*fA*mGmUrC*mGrC*mUfGfC
UAGUCGCUGCAA
141

365m

mAmArA*mArC*mUmUfGmA*T*
AACUUGATT

T

ATa365m
Antisense
(5p)mU*C*mAfAmGfUT*mUfUm
UCAAGUTUUGCA
142

GmCfAmGfCmGfAmCmUA*mG*
GCGACUAGC

mC

643-AN
ATsi-
sense
mU*fA*mGmUfCmGfCmUfGfCm
UAGUCGCUGCGA
143

643s1

GmAfAmAfCmUmUfGmA*T*T
AACUUGATT

ATa643
Antisense
(Sp)m U*C*mAfAmGfUmUmUfCm
UCAAGUUUCGCA
144

GmCfAmGfCmGfAmCmUfA*mG*
GCGACUAGC

mC

643-AL
643-Als
sense
mG*mC*mUmAmGmUfCmGfCfUf
GCUAGUCGCUGC
145

GmCmGmAmAmAmCmUmUmGm
GAAACUUGA

A

643-Ala
Antisense
mU*fC*mAmAmGfUgnaTfUfCmG
UCAAGUUUCGCA
146

mCmAmGfCmGfAmCmUmAmGm
GCGACUAGCAC

C*mA*mC

ATsi565
AT&565
sense
mU*fG*mCmAfAmGfAmCfUfUm
UGCAAGACUUGA
147

GmAfCmAfCmCmGfAmA*T*T
CACCGAATT

ATa565
Antisense
(5p)mU*T*mCfGmGfUG*mUfCm
UTCGOUGUCAAG
149

AmAfGmUfCmUfUmGmCA*mG*
UCUUGCAGC

mC

612-AN
ATs565
sense
mU*fG*mCmAfAmGfAmCfUfUm
UGCAAGACUUGA
147

GmAfCmAfCmCmGfAmA*T*T
CACCGAATT

ATa612
Antisense
(Sp)mU*T*mCfGmGfUmGmUfCm
UTCGGUGUCAAG
151

AmAfGmUfCmUfUmGmCfA*mG*
UCUUGCAGC

mC

612-AL
612-Als
sense
mG*mC*mUmGmCmAfAmGfAfCf
GCUGCAAGACUU
152

UmUmGmAmCmAmCmCmGmAm
GACACCGAA

A

612-Ala⁺⁺
Antisense
mU*fU*mCmGmGgnaTmGfUfCm
UUCGGUGUCAAG
153

AmAmGmUfCmUfUmGmCmAmG
UCUUGCAGCGA

mC*mG*mA

ATsiS79
ATs579
sense
mG*fA*mCmCfCmUfAmCfCfUm
GACCCUACCUUC
154

UmCfAmUfAmCmCfUmG*T*T
AUACCUGTT

ATaS79
Antisense
(Sp)mC*A*mGfGmUfAT*mGfAm
CAGGUATGAAGG
155

AmGfGmUfAmGfGmGmUC*mU*
UAGGGUCUU

mU

579-
579s-ALI
sense
mA*mA*mGmAmCmCfCmUfAfCf
AAGACCCUACCU
156

AL1

CmUmUmCmAmUmAmCmCmUm
UCAUACCUG

G

579a-AL
Antisense
mC*fA*mGmGmUfAgnaTfGfAmA
CAGGUAUGAAGG
157

mGmGmUfAmGfGmGmUmCmUm
UAGGGUCUUUG

U*mU*mG

ATsi597
ATs597
sense
mU*fU*mAmAfCmAfAmGfCfCm
UUAACAAGCCUA
158

UmAfAmGfGmUmCfUmU*T*T
AGGUCUUTT

ATa597
Antisense
(Sp)mA*A*mGfAmCfCT*mUfAm
AAGACCTUAGGC
161

GmGfCm UfUmGfUmUmAA*mG*
UUGUUAAGC

mC

597-AL1
597s-AL1
sense
mG*mC*mUmUmAmAfCmAfAfGf
GCUUAACAAGCC
163

CmCmUmAmAmGmGmUmCmUm
UAAGGUCUU

U

597a-AL
Antisense
mA*fA*mGmAmCfCgnaTfUfAmG
AAGACCUUAGOC
164

mGmCmUfUmGfUmUmAmAmGm
UUGUUAAGCUG

C*mU*mG

ATsi598
ATs598
sense
mC*fC*mAmAfUmAfGmCfUfUm
CCAAUAGCUUAA
130

AmAfCmAfAmGmCfCmU*T*T
CAAGCCUTT

ATa598
Antisense
(Sp)mA*G*mGfCmUfUG*mUfUm
AGGCUUGUUAAG
133

AmAfGmCfUmAfUmUmGG*mG*
CUAUUGGGU

mU

598-AL1
598s-AL1
sense
mA*mC*mCmCmAmAfUmAfGfCf
ACCCAAUAGCUU
165

UmUmAmAmCmAmAmGmCmCm
AACAAGCCU

U

598a-
Antisense
mA*fG*mGmCmUfUmGfUfUmAm
AGGCUUGUUAAG
166

AL⁺⁺⁺

AmGmCfUmAfUmUmGmGmGmU
CUAUUOGGUAG

*mA*mG

⁺⁺Since gnaG was not available, 612-Ala differs from the normal ESC+ chemistry in placement of the gna modification, however, gna at position 6 has also been widely used in drug discovery (see PCT/US2019/032150).

⁺⁺⁺Since gnaG is not available, 598a-AL does not contain gna at position 7, similar to the ESC chemistry.

In Vitro Assays—Comparison of ARNATAR Motifs to Third Party Motifs

AGT siRNAs were transfected at 0-10 nM into Hep3B cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr. siRNA activity was determined by qRT-PCR using the primer probe sets as listed in Table 71. The IC50 of the siRNAs targeting AGT are shown in Table 74 and the percent inhibition of AGT mRNA levels graphed (FIG. 39).

TABLE 74

siRNA Activity Targeting AGT in Hep3B cells

siRNAs
IC50 (nM)

579AL
15.06

ATsi-579
0.0947

597AL
1.105

ATsi-597
0.087

598-AL
0.3776

ATsi-598
0.0604

612-AL
1233

612-AN
0.1821

ATsi-565
0.0284

643-AL
N/A

ATsi-365m
0.1346

643-AN
0.4656

The results indicate that for all these different sequences, the optimized ARNATAR designs are more potent than the third party design.

Example 23: Comparison of siRNA with Different Chemical Motifs Targeting the Same AGT Regions

As shown in prior experiments above, altering chemical modification can affect siRNA activity. We thus evaluated the activities of altering chemical modifications of siRNAs targeting the same sequences of AGT mRNA (see Table 75 and FIGS. 36-37).

TABLE 75

Oligomeric Compounds Targeting Same Region of AGT with

Different Chemical Motif.

SEQ

Strand
Sense or

ID

siRNA
Name
Antisense
Sequence + Chemistry
Sequence
NO:

ATsi603
ATs603
sense
mU*fG*mCmAfAmGfAmCf
UGCAAGACUU
148

UfUmGmAfCmAfCmCmGf
GACACCGAAT

AmA*T*T-GA3
T

ATa603
Antisense
(5p)mU*T*mCfGmGfUG*m
UTCGGUGUCA
150

UfCmAmAfGmUfCmUfUm
AGUCUUGCAG

GmCA*mG*mC
C

ATsi612
ATs565
sense
mU*fG*mCmAfAmGfAmCf
UGCAAGACUU
148

UfUmGmAfCmAfCmCmGf
GACACCGAAT

AmA*T*T-GA3
T

ATa612
Antisense
(Sp)mU*T*mCfGmGfUmGm
UTCGGUGUCA
151

UfCmAmAfGmUfCmUfUm
AGUCUUGCAG

GmCfA*mG*mC
C

ATsi481
ATs481
sense
mU*fU*mAmA*rC*mA*rA*
UUAACAAGCC
160

mGfCfCmUmA*rA*mG*rG*
UAAGGUCUUT

mUmCfUmU*T*T-GA3
T

ATa481
Antisense
(Sp)mA*A*mGfAmCfCmUm
AAGACCUUAG
167

UfAmGmGfCmUfUmGfUm
GCUUGUUAAG

UmAfA*mG*mC
C

ATsi482
Ats482
sense
mU*fU*mAmAfCmAfAmGf
UUAACAAGCC
159

CfCmUmAfAmGfGmUmCf
UAAGGUCUUT

UmU*T*T-GA3
T

ATa482
Antisense
(5p)mA*A*mGfAmCfCmUm
AAGACCUUAG
167

UfAmGmGfCmUfUmGfUm
GCUUGUUAAG

UmAfA*mG*mC
C

ATsi482a
ATs482
sense
mU*fU*mAmAfCmAfAmGf
UUAACAAGCC
159

CfCmUmAfAmGfGmUmCf
UAAGGUCUUT

UmU*T*T-GA3
T

ATa-
Antisense
(5p)mA*A*mGfAmCfCT*m
AAGACCTUAG
162

482a

UfAmGmGfCmUfUmGfUm
GCUUGUUAAG

UmAfA*mG*mC
C

ATsi483
ATs483
sense
mC*fC*mAmA*rU*mA*rG*
CCAAUAGCUU
132

mCfUfUmAmA*rC*mA*rA*
AACAAGCCUT

mGmCfCmU*T*T-GA3
T

ATa483
Antisense
(Sp)mA*G*mGfCmUfUmG
AGGCUUGUUA
134

mUfUmAmAfGmCfUmAfU
AGCUAUUGGG

mUmGfG*mG*mU
U

ATsi484
ATs484
sense
mC*fC*mAmAfUmAfGmCf
CCAAUAGCUU
131

UfUmAmAfCmAfAmGmCf
AACAAGCCUT

CmU*T*T-GA3
T

ATa484
Antisense
(5p)mA*G*mGfCmUfUG*m
AGGCUUGUUA
135

UfUmAmAfGmCfUmAfUm
AGCUAUUGGG

UmGG*mG*mU
U

In Vitro Assays—Comparison of siRNAs with Different Chemical Motifs Targeting the Same AGT Sequences

The siRNAs were incubated at 0-5 μM (ATsi481, ATsi482, ATsi482a, ATsi483, ATsi484) or 0-50 μM (ATsi603, ATsi612) final concentration by free uptake with human primary hepatocytes. Cells were incubated for 42 hr, and total RNA was prepared. siRNA activity was evaluated using qRT-PCR with ACT primer probe sets (Table 71). The IC50 of the siRNAs targeting AGT mRNA are shown in Table 76 and the percent inhibition of AGT mRNA levels graphed (FIG. 40).

TABLE 76

siRNA Activity Targeting AGT in Human Primary Hepatocytes

siRNA
IC50 (μM)

ATsi481
0.0015

ATsi482
0.0353

ATsi482a
0.0085

ATsi603
0.0009

ATsi612
0.0269

ATsi483
0.026

ATsi600
0.096

ATsi484
0.154

TABLE 77

SEQUENCE LISTING

Sequence Listing

SEQ

ID

NO
SEQUENCE
DESCRIPTION

1
GCGTCACCAAAAAGCGCAA
LMNA DNA Target Seq, sense strand

2
GCGUCACCAAAAAGCGCAA
LMNA RNA Target Seq

3
CGGGTGGATGCTGAGAAC
Human LMNA Primer hsLMNA-S

4
TGCTTCCCATTGTCAATCTCC
Human LMNA Primer hsLMNA-A

5
AGTGAGGAGCTGCGTGAGACCAA
Human LMNA Probe hsLMNA-P

6
GGACCAGGTGGAACAGTATAAG
Mouse LMNA Primer msLMNA-S

7
TCAATGCGGATTCGAGACTG
Mouse LMNA Primer msLMNA-A

8
CAGCTTGGCGGAGTATGTCTTTTCTAGC
Mouse LMNA Probe msLMNA-P

9
AGGGTTACATGAAGCACGC
Human ApoC3 Primer ApoC3-F

10
AGAGAACTTGTCCTTAACGOTG
Human ApoC3 Primer ApoC3-R

11
AGGGAACTGAAGCCATCGGTCAC
Human ApoC3 Probe ApoC3-P

12
GCTTGGCTTCTTCTGGACTCA
Human NCL Primer hsNCL-F

13
TCGCGAGCTTCACCATGA
Human NCL Primer hsNCL-R

14
CGCCACTTGTCCGCTTCACACTCC
Human NCL Probe hsNCL-P

168
GCGUCACCAAAAAGCGCAATT
LMNA sense strands ATXL019,

ATXL021, ATXL026, ATXL023,

ATXL030, ATXL031, ATXLIS4,

ATXL155, ATXL171, ATXL204,

ATXL206, ATXL232

169
UUGCGCUUUUUGGUGACGCTT
LMNA antisense strands ATXL018,

ATXL020

170
GCGUCACCAAAAAGCGCAAUU
LMNA sense strand ATXL022,

ATXL024, ATXL025, ATXL027-

ATXL029, ATXL032, ATXL048-

ATXL053, ATXL079-ATXL085

171
UUGCGCUUUUUGGUGACGCUU
LMNA antisense strand ATXL041,

ATXL188

172
UTGCGCUUUUUGGUGACGCUU
LMNA antisense strand ATXL042,

ATXL187

173
UTGCGCTUUUUGGUGACGCUU
LMNA antisense strand ATXL043,

ATXL046, ATXL047, ATXL078,

ATXL081, ATXL152, ATXL152,

ATXL168-ATXL170, ATXL186

174
UUGCGCTUUUUGGUGACGCUU
LMNA antisense strand ATXL044,

ATXL045, ATXL077

175
CUCUGAGUUCUGGGAUUUGTT
ApoC3 sense strand ATXL059.

ATXL116, ApoC3-74s

176
CAAAUCCCAGAACUCAGAGTT
ApoC3 antisense strand ATXL058

177
CUCUGAGUUCUGGGAUUUG
ApoC3 sense strand ATXL060,

ATXL061

178
CUCUGAGUUCUGGGAUUUGUU
ApoC3 sense strand ATXL068,

ATXL069

179
CAAAUCCCAGAACUCAGAGUU
ApoC3 antisense strand ATXL067,

ATXL072, ApoC3-74a

180
GGAUAGUUACUGACCGGGATT
NCL sense strand ATXL125

181
UCCCGGUCAGUAACUAUCCTT
NCL antisense strand ATXL124

182
GGAUAGUUACUGACCGGGAUU
NCL sense strand ATXL128, ATXL129

183
UCCCGGTCAGUAACUAUCCUU
NCL antisense strand ATXL126,

ATXL127

184
CAAAUCCCCAGAACUCAGAGUU
ApoC3 antisense strand ATXL066

185
UTGCGCTUUUUGGTGACGCUU
LMNA antisense strand ATXL074 and

ATXL075

186
UTGCGCTUTUUGGTGACOCUU
LMNA antisense strand ATXL076

98
GGAUAGUUACUGACCGGGA
NCL Target Seq

99
AAACACTGTGAGGATGGGATC
hsNFKB formard primer hsNFKB-F

100
TCTGTCATTCGTGCTTCCAG
hsNFKB reverse primer hsNFKB-R

101
TGTCACATGAAGTATACCCAGGTTTGCG
bsNFkB probe hsNFKB-P

102
GTGTCAGAGCCCTTGTAACTG
msNIkB Forward Primer NfkB-F

103
ACATTTGCCCAGTTCCGTAG
msNfkB Reverse Primer NikB-R

104
TCGTCTGCCATGGTGAAGATGCG
msNfkB Probe NfkB-P

105
AAACCGCTATGAAGTTCCTCTC
msIL-6 Forward Primer IL6-F

106
GTGGTATCCTCTGTGAAGTCTC
msIL-6 Reverse Primer IL6-R

107
TTGTCACCAGCATCAGTCCCAAGAA
msIL-6 Probe IL6-P

108
AGACCCTCACACTCAGATCA
msTNF Forward Primer TNF-F

109
TGTCTTTGAGATCCATGCCG
msTNF Reverse Primer TNF-R

110
CCTGTAGCCCACGTCGTAGCAAA
msTNF Probe TNF-P

187
GACUUUCAUCCUGGAAAUAUA
HaoAl-SI sense strand

188
UAUAUUUCCAGGAUGAAAGUCCA
HaoAl-Al antisense strand

189
GACUUUCAUCCUGGAAAUATA
Hao-ART-S97 sense strand

190
UAUAUUUCCAGGAUGAAAGUC
Hao-ART-A2 antisense strand

191
UAUAUUTCCAGGAUGAAAGUCCA
Hao-ART-A74-1 antisense strand

192
UAUAUUTCCAGGAUGAAAGUC
Hao-ART-A74-2 antisense strand

193
CAGCOUCACCAAAAAGCGCAA
LMNA sense strand ATXL239

194
UUGCGCTUUUUGGUGACGCUGCC
LMNA antisense strand ATXL238

195
GGACAUUCCAAGACAGUAUTT
NCL sense strand NCL-74s

196
ATACUGTCUUGGAAUGUCCUU
NCL antisense strand NCL-74a

197
GAGGACAUUCCAAGACAGUAU
NCL sense strand NCL-Als1

198
AUACUGTCUUGGAAUGUCCUCAG
NCL antisense strand NCL-ALa

199
UUCUCUGAGUUCUGGGAUUUG
ApoC3 sense strand Apoc3-ALs

200
CAAAUCCCAGAACUCAGAGAACU
ApoC3 antisense strand Apoc3-ALa

201
CAGCGUCACCAAAAAGCGCAATT
LMNA sense strand ATXL237

202
UTGCGCTUUUUGGUGACGCUGCC
LMNA antisense strand ATXL236

203
GAGGACAUUCCAAGACAGUAUTT
NCL sense strand NCL-23nt-s

204
ATACUGTCUUGGAAUGUCCUCAG
NCL sense strand NCL-23nt-a

205
CCAAUAGCUUAACAAGCCUTT
AGT sense strand ATs600, ATs598,

ATs483, ATs484

206
AGGCUUGUUAAGCUAUUGGGU
AGT antisense strand ATa600,

ATa598, ATa483, ATa484

207
ACCCAAUAGCUUAACAAGCCUTT
AGT sense strand ATs633

208
AGGCUUGUUAAGCUAUUGGGUTT
AGT antisense strand ATa633

138
CTGATCCAGCCTCACTATGC
hsAGT forward primer hsAGT-F

139
AGGTCATAAGATCCTTGCAGC
hsAGT reverse primer hsAGT-R

140
AGGGTCTCACTTTCCAGCAAAACTCC
hsAGT probe hsAGT-P

209
UAGUCGCUGCAAAACUUGATT
AGT sense strand, ATs365m

210
UCAAGUTUUGCAGCGACUAGC
AGT antisense strand ATa365m

211
UAGUCGCUGCGAAACUUGATT
AGT sense strand ATsi-643sl

212
UCAAGUUUCGCAGCGACUAGC
AGT antisense strand ATa643

213
GCUAGUCGCUGCGAAACUUGA
AGT sense strand 643-Als

214
UCAAGUUUCGCAGCGACUAGCAC
AGT antisense strand 643-ALa

215
UGCAAGACUUGACACCGAATT
AGT sense strand ATs565, ATs612,

ATs603

216
UTCGGUGUCAAGUCUUGCAGC
AGT antisense strands ATa565,

ATa612, ATa603

217
GCUGCAAGACUUGACACCGAA
AGT sense strand 612-Als

218
UUCGGUGUCAAGUCUUGCAGCGA
AGT antisense strand 612-Ala

219
GACCCUACCUUCAUACCUGTT
AGT sense strand, ATs579

220
CAGGUATGAAGGUAGGGUCUU
AGT antisense strand ATa579

221
AAGACCCUACCUUCAUACCUG
AGT sense strand 579s-AL1

222
CAGGUAUGAAGGUAGGGUCUUUG
AGT antisense strand 579a-AL

223
UUAACAAGCCUAAGGUCUUTT
AGT sense strand ATs597

224
AAGACCTUAGGCUUGUUAAGC
AGT antisense strand ATa597,

ATa482a

225
GCUUAACAAGCCUAAGGUCUU
AGT sense strand 597s-AL1

226
AAGACCUUAGGCUUGUUAAGCUG
AGT antisense strand 597a-AL

227
ACCCAAUAGCUUAACAAGCCU
AGT sense strand 598s-ALI

228
AGGCUUGUUAAGCUAUUGGGUAG
AGT antisense strand 598a-AL

229
AAGACCUUAGGCUUGUUAAGC
AGT antisense strand ATa481, ATa482

	Number	Date	Country
	63433706	Dec 2022	US
	63472780	Jun 2023	US

	Number	Date	Country
Parent	PCT/US23/84688	Dec 2023	WO
Child	18791380		US

ADVANCED RNA TARGETING (ARNATAR)

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

Provisional Applications (2)

Continuations (1)