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WO2024196898A1 - Nucléotides non codants chimiquement modifiés - Google Patents

Nucléotides non codants chimiquement modifiés Download PDF

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Publication number
WO2024196898A1
WO2024196898A1 PCT/US2024/020471 US2024020471W WO2024196898A1 WO 2024196898 A1 WO2024196898 A1 WO 2024196898A1 US 2024020471 W US2024020471 W US 2024020471W WO 2024196898 A1 WO2024196898 A1 WO 2024196898A1
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Prior art keywords
nucleic acid
acid molecule
double
linker
target
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Poorya Hosseini
Behfar ARDEHALI
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Camford Labs Inc
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Camford Labs Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/532—Closed or circular
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance

Definitions

  • Double-stranded nucleic acid molecules can be designed to be complementary to target nucleic acid molecules, such as a messenger ribonucleic acid (mRNA) and/or non-coding (e.g., regulatory) region of the RNA.
  • target nucleic acid molecules such as a messenger ribonucleic acid (mRNA) and/or non-coding (e.g., regulatory) region of the RNA.
  • Double-stranded nucleic acid molecules can target an RNA in order to affect expression, stability, translation and/or splicing of the RNA, thereby modulating expression of gene products from the target nucleic acid molecule.
  • mRNA messenger ribonucleic acid
  • non-coding e.g., regulatory region of the RNA.
  • Double-stranded nucleic acid molecules can target an RNA in order to affect expression, stability, translation and/or splicing of the RNA, thereby modulating expression of gene products from the target nucleic acid molecule.
  • mRNA messenger
  • aspects disclosed herein provide synthetic nucleic acid molecules comprising: a double-stranded noncoding ribonucleic acid molecule comprising a sense strand of the double-stranded noncoding ribonucleic acid molecule base paired to an antisense strand of the double-stranded noncoding ribonucleic acid molecule, wherein the antisense strand is complementary to a target nucleic acid sequence, and wherein the sense strand comprises a 5’ end and a 3’ end that are reversibly or irreversibly coupled to generate a structure of the synthetic nucleic acid molecule comprising the sense strand with no free ends base paired to the antisense strand.
  • the target nucleic acid sequence is a ribonucleic acid (RNA) sequence.
  • the target nucleic acid sequence is a gene expression product of a gene from Table 1.
  • the target nucleic acid sequence is a gene expression product from a gene encoding complement C5 (C5).
  • the RNA sequence is a messenger RNA (mRNA) sequence.
  • mRNA messenger RNA
  • a nucleic acid sequence of the antisense strand is complementary to an untranslated or regulatory region of the target nucleic acid sequence.
  • the antisense strand is configured to modulate expression of a gene product expressed from the target nucleic acid sequence.
  • the antisense strand comprises about 19 to about 27 contiguous nucleotides, the sense strand comprises about 19 to about 35 contiguous nucleotides.
  • the doublestranded noncoding nucleic acid molecule comprises a chemically modified nucleotide.
  • the sense strand further comprises a chemical linker, wherein the chemical linker couples the 5’ end of the sense strand to the 3’ end of the sense strand.
  • the chemical linker is substantially cleavable under intracellular conditions.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid- labile linker, a peptide linker, a nucleotide linker, or a glucuronide group, an azide-alkyne linker, an aldehyde-oxamine linker, a phosphorothioate-tosylated linker, a phosphate activation agent mediated phosphate-hydroxyl linkage, or a metal chelation ligation.
  • the chemical linker is substantially cleavable under intracellular conditions by an enzyme. In some embodiments, the chemical linker is independently substantially cleavable under intracellular conditions.
  • the antisense strand comprises a 5’ end and a 3’ end, wherein the 5’ end of the antisense strand is not coupled to the 3’ end of the antisense strand. In some embodiments, the antisense strand comprises a 5’ end and a 3’ end, wherein the 5’ end of the antisense strand is coupled to the 3’ end of the antisense strand. In some embodiments, the antisense strand further comprises a chemical linker, wherein the chemical linker couples the 5’ end of the antisense strand to the 3’ end of the antisense strand. In some embodiments, the chemical linker is substantially non-cleavable under intracellular conditions.
  • the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer. In some embodiments, the chemical linker is substantially cleavable under intracellular conditions by an enzyme. In some embodiments, the chemical linker is independently substantially cleavable under intracellular conditions. In some embodiments, the chemical linker is substantially non-cleavable under intracellular conditions. In some embodiments, the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.
  • the antisense strand comprises a 5’ end, wherein the 5’ end of the antisense strand comprises a modification comprising 5’-(E)-Vinylphosphonate (5 ’-VP), 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), or 6’(phosphonooxy-Butyl-Sulfide)Purine (6-PBuS-purine).
  • the doublestranded noncoding nucleic acid molecule further comprises a targeting moiety, wherein the targeting moiety is specific to a target cell or target tissue. In some embodiments, the targeting moiety is coupled to the sense strand of the double-stranded noncoding nucleic acid molecule.
  • the antisense strand comprises a chemical linker, wherein the chemical linker couples the targeting moiety to the sense strand.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, a glucuronide group, a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, an organic molecule, or a polymer.
  • the target cell is a hepatic cell, a cardiac cell, a neuron, a muscle cell, a blood cell, a photoreceptor cell, a pancreatic cell, or a stem cell.
  • the target tissue is liver tissue, heart tissue, brain tissue, muscle tissue, nervous tissue, epithelial tissue, connective tissue, eye tissue, or pancreatic tissue.
  • the targeting moiety comprises a polypeptide, an RNA molecule, a nucleic acid molecule, a lipophilic moiety, or a small molecule.
  • the polypeptide comprises an antibody, a single-domain antibody, a miniprotein, or an antigenbinding fragment thereof.
  • the polypeptide specifically binds to Glucagon Like Peptide 1 Receptor (GLP1R), Asialoglycoprotein receptor (ASGPR), prostate specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), Epithelial cell adhesion molecule (EpCam), AXL receptor tyrosine kinase (AXL), protein tyrosine kinase 7 (PTK7), Programmed death-ligand 1 (PD-L1), or T-cell immunoglobulin and mucin domain-3 (Tim-3), or any combination thereof.
  • GLP1R Glucagon Like Peptide 1 Receptor
  • ASGPR Asialoglycoprotein receptor
  • PSMA prostate specific membrane antigen
  • hTfR human transferrin receptor
  • EGFR epidermal growth factor receptor
  • Her2 human epidermal growth factor receptor 2
  • EpCam Epithelial cell adhe
  • the RNA molecule comprises an aptamer, a ribozyme, a hairpin RNA, a siRNA, or miRNA.
  • the lipophilic moiety comprises lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, l,3-bis-0(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03- (oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.
  • the small molecule comprises a sugar moiety.
  • the sugar moiety comprises an amino sugar.
  • the amino sugar is N-acetyl Galactosamine (GalNAc).
  • the targeting moiety is specific to an antigen or receptor of the target cell or target tissue.
  • the receptor comprises Asialoglycoprotein receptor (ASGPR).
  • the double stranded noncoding nucleic acid molecule comprises one or more nucleotides comprising a modification.
  • the modification comprises a chemical modification.
  • the chemical modification comprises a modification of the sugar, the phosphate backbone, or the nucleobase.
  • the modification of the nucleobase comprises a 2’-O-Me, a 2’- F, a 2’-M0E, a N(6)-methyladenosine, a 5-methylcytidine, a 5-methyluridine (ribothymidine), a ribose modification with bridged nucleic acids, or a nucleotide with alternative chemistry.
  • the ribose modification with bridged nucleic acids is a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), or a constrained ethyl bridged nucleic acid (cEt) modification.
  • the nucleotide with alternative chemistry is a phosphorodiamidate morpholino oligonucleotide (PMO), a peptide nucleic acid (PNA), a tricyclo DNA (tcDNA), an unlocked nucleic acid (UNA), or a glycol nucleic acid (GNA).
  • PMO phosphorodiamidate morpholino oligonucleotide
  • PNA peptide nucleic acid
  • tcDNA tricyclo DNA
  • UNA unlocked nucleic acid
  • GNA glycol nucleic acid
  • the modification of the phosphate backbone linkage comprises a phosphodiester, phosphorothioate isomers, phosphoryl DMI amidate diester isomers, a phosphorodithioate, a methylphosphonate, a 5'-phosphorothioate, a peptide nucleic acid, a 5'-(E)-vinylphosphonate, or a 5'-methyl phosphonate.
  • the double-stranded noncoding ribonucleic acid molecule comprises a siRNA.
  • the double-stranded noncoding ribonucleic acid molecule comprises a dsRNA that associates with Argonaute-containing protein complexes.
  • the linkage of the 5’ and 3’ ends of the double-stranded nucleic acid allows for the inclusion of fewer chemically-modified nucleotides linked to adverse medical side-effects as compared to a control unlinked double-stranded nucleic acid.
  • the adverse medical side effects are selected from the group consisting of reduced platelets, thrombocytopenia, perturbation of heart rate, increased blood pressure, or increased cardiac output.
  • the synthetic nucleic acid molecule exhibits less immunogenicity as compared with an otherwise identical synthetic nucleic acid molecule that is linear.
  • the immunogenicity is measured by gene expression analysis.
  • the gene expression analysis is RNA-seq gene expression analysis.
  • the synthetic nucleic acid molecule exhibits less off-target effects as compared with an otherwise identical synthetic nucleic acid molecule that is linear. In some embodiments, the off-target effects are measured by gene expression analysis. In some embodiments, the gene expression analysis is RNA-seq gene expression analysis. In some embodiments, the synthetic nucleic acid molecule exhibits less toxicity as compared with an otherwise identical synthetic nucleic acid molecule that is linear. In some embodiments, the toxicity is measured by subchronic or chronic toxicity tests. In some embodiments, the synthetic nucleic acid molecule exhibits more target nucleic acid sequence specificity as compared with an otherwise identical synthetic nucleic acid molecule that is linear.
  • the target nucleic acid sequence specificity is measured by gene expression analysis.
  • the gene expression analysis is RNA-seq gene expression analysis.
  • the synthetic nucleic acid molecule exhibits more stability as compared with an otherwise identical synthetic nucleic acid molecule that is linear. In some embodiments, the stability is measured by the degradation of nucleotides in the presence of a nuclease.
  • the synthetic nucleic acid molecule is isolated. In some embodiments, the synthetic nucleic acid molecule is purified and isolated.
  • a pharmaceutical formulation comprises a synthetic nucleic acid molecule described herein and a pharmaceutically acceptable: excipient, carrier, or diluent.
  • the pharmaceutical formulation is formulated for subcutaneous administration.
  • a cell comprises a synthetic nucleic acid molecule described herein.
  • a method of delivering a synthetic nucleic acid molecule described herein to a cell of a subject comprises: introducing (i) a synthetic nucleic acid molecule described herein, a pharmaceutical formulation described herein or a cell described herein to a cell of a subject in vivo or ex vivo under conditions sufficient to modulate expression of the target nucleic acid molecule in the cell of the subject.
  • nucleic acid molecules comprising: a double-stranded noncoding ribonucleic acid molecule comprising: a) an antisense strand of the double-stranded noncoding ribonucleic acid molecule, wherein the antisense strand comprises a target-binding sequence that is complementary to a target nucleic acid sequence; and b) a sense strand of the double-stranded noncoding ribonucleic acid molecule base paired to the antisense strand of the double-stranded noncoding ribonucleic acid molecule, wherein the sense strand comprises: i) one or more adapters configured to enhance specificity of target binding between the target-binding sequence of the antisense strand and the target nucleic acid sequence, as compared with an otherwise identical double-stranded noncoding ribonucleic acid molecule that does not have a sense strand comprising an adapter of the one or more adapters; and ii) a 5’
  • the target nucleic acid sequence is a ribonucleic acid (RNA) sequence.
  • the target nucleic acid sequence is a gene expression product of a gene from Table 1.
  • the target nucleic acid sequence is a gene expression product from a gene encoding complement C5 (C5).
  • the RNA sequence is a messenger RNA (mRNA) sequence.
  • mRNA messenger RNA
  • a nucleic acid sequence of the antisense strand is complementary to an untranslated or regulatory region of the target nucleic acid sequence.
  • the antisense strand is configured to modulate expression of a gene product expressed from the target nucleic acid sequence.
  • the antisense strand comprises about 19 to about 27 contiguous nucleotides. In some embodiments, the sense strand comprises about 19 to about 35 contiguous nucleotides. In some embodiments, the double-stranded noncoding nucleic acid molecule comprises a chemically modified nucleotide. In some embodiments, the sense strand further comprises a chemical linker, wherein the chemical linker couples the 5’ end of the sense strand to the 3’ end of the sense strand. In some embodiments, the chemical linker is substantially cleavable under intracellular conditions.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, or a glucuronide group, an azide-alkyne linker, an aldehyde-oxamine linker, a phosphorothioate-tosylated linker, a phosphate activation agent mediated phosphate-hydroxyl linkage, or a metal chelation ligation.
  • the chemical linker is substantially cleavable under intracellular conditions by an enzyme. In some embodiments, the chemical linker is independently substantially cleavable under intracellular conditions.
  • the antisense strand comprises a 5’ end and a 3’ end, wherein the 5’ end of the antisense strand is not coupled to the 3’ end of the antisense strand.
  • the antisense strand further comprises a chemical linker, wherein the chemical linker couples the 5’ end of the antisense strand to the 3’ end of the antisense strand.
  • the antisense strand comprises a 5’ end and a 3’ end, wherein the 5’ end of the antisense strand is coupled to the 3’ end of the antisense strand.
  • the chemical linker is substantially non-cleavable under intracellular conditions.
  • the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer. In some embodiments, the chemical linker is substantially cleavable under intracellular conditions by an enzyme. In some embodiments, the chemical linker is independently substantially cleavable under intracellular conditions. In some embodiments, the chemical linker is substantially non-cleavable under intracellular conditions. In some embodiments, the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.
  • the antisense strand comprises a 5’ end, wherein the 5’ end of the antisense strand comprises a modification comprising 5’-(E)-Vinylphosphonate (5 ’-VP), 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), or 6’(phosphonooxy-Butyl-Sulfide)Purine (6-PBuS-purine).
  • the doublestranded noncoding nucleic acid molecule further comprises a targeting moiety, wherein the targeting moiety is specific to a target cell or target tissue. In some embodiments, the targeting moiety is coupled to the sense strand of the double-stranded noncoding nucleic acid molecule.
  • the antisense strand comprises a chemical linker, wherein the chemical linker couples the targeting moiety to the sense strand.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, a glucuronide group, a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, an organic molecule or a polymer.
  • the target cell is a hepatic cell, a cardiac cell, a neuron, a muscle cell, a blood cell, a photoreceptor cell, a pancreatic cell, or a stem cell.
  • the target tissue is liver tissue, heart tissue, brain tissue, muscle tissue, nervous tissue, epithelial tissue, connective tissue, eye tissue or pancreatic tissue.
  • the targeting moiety comprises a polypeptide, an RNA molecule, a nucleic acid molecule, a lipophilic moiety, or a small molecule.
  • the polypeptide comprises an antibody, a single-domain antibody, a miniprotein, or an antigenbinding fragment thereof.
  • the polypeptide specifically binds to Glucagon Like Peptide 1 Receptor (GLP1R), Asialoglycoprotein receptor (ASGPR), prostate specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), Epithelial cell adhesion molecule (EpCam), AXL receptor tyrosine kinase (AXL), protein tyrosine kinase 7 (PTK7), Programmed death-ligand 1 (PD-L1), or T-cell immunoglobulin and mucin domain-3 (Tim-3), or any combination thereof.
  • GLP1R Glucagon Like Peptide 1 Receptor
  • ASGPR Asialoglycoprotein receptor
  • PSMA prostate specific membrane antigen
  • hTfR human transferrin receptor
  • EGFR epidermal growth factor receptor
  • Her2 human epidermal growth factor receptor 2
  • EpCam Epithelial cell adhe
  • the RNA molecule comprises an aptamer, a ribozyme, a hairpin RNA, a siRNA, or miRNA.
  • the lipophilic moiety comprises lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, l,3-bis-0(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.
  • the small molecule comprises a sugar moiety.
  • the sugar moiety comprises an amino sugar.
  • the amino sugar is N-acetyl Galactoseamine (GalNAc).
  • the targeting moiety is specific to an antigen or receptor of the target cell or target tissue.
  • the receptor comprises Asialoglycoprotein receptor (ASGPR).
  • the double stranded noncoding nucleic acid molecule comprises one or more nucleotides comprising a modification.
  • the modification comprises a chemical modification.
  • the chemical modification comprises a modification of the sugar, the phosphate backbone, or the nucleobase.
  • the modification of the nucleobase comprises a 2’-0-Me, a 2’- F, a 2’-M0E, a N(6)-methyladenosine, a 5-methylcytidine, a 5-methyluridine (ribothymidine), a ribose modification with bridged nucleic acids, or a nucleotide with alternative chemistry.
  • the ribose modification with bridged nucleic acids is a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), or a constrained ethyl bridged nucleic acid (cEt) modification.
  • the nucleotide with alternative chemistry is a phosphorodiamidate morpholino oligonucleotide (PMO), a peptide nucleic acid (PNA), a tricyclo DNA (tcDNA), an unlocked nucleic acid (UNA), or a glycol nucleic acid (GNA).
  • PMO phosphorodiamidate morpholino oligonucleotide
  • PNA peptide nucleic acid
  • tcDNA tricyclo DNA
  • UNA unlocked nucleic acid
  • GNA glycol nucleic acid
  • the modification of the phosphate backbone linkage comprises a phosphodiester, phosphorothioate isomers, phosphoryl DMI amidate diester isomers, a phosphorodithioate, a methylphosphonate, a 5'-phosphorothioate, a peptide nucleic acid, a 5'-(E)-vinylphosphonate, or a 5'-methyl phosphonate.
  • the double-stranded noncoding ribonucleic acid molecule comprises a siRNA.
  • the double-stranded noncoding ribonucleic acid molecule comprises a dsRNA that associates with Argonaute-containing protein complexes.
  • the linkage of the 5’ and 3’ ends of the double-stranded nucleic acid allows for the inclusion of fewer chemically-modified nucleotides linked to adverse medical side-effects as compared to a control unlinked double-stranded nucleic acid.
  • the adverse medical side effects are selected from the group consisting of reduced platelets, thrombocytopenia, perturbation of heart rate, increased blood pressure, or increased cardiac output.
  • the adapter is a peptide adapter.
  • the adapter is a nucleotide adapter.
  • the adapter is an antibody.
  • the adapter is a sugar.
  • the adapter is a lipid.
  • the adapter is an aptamer.
  • the synthetic nucleic acid molecule exhibits less immunogenicity as compared with an otherwise identical synthetic nucleic acid molecule that is linear. In some embodiments, the immunogenicity is measured by gene expression analysis. In some embodiments, the gene expression analysis is RNA-seq gene expression analysis. In some embodiments, the synthetic nucleic acid molecule exhibits less off-target effects as compared with an otherwise identical synthetic nucleic acid molecule that is linear. In some embodiments, the off-target effects are measured by gene expression analysis. In some embodiments, the gene expression analysis is RNA-seq gene expression analysis.
  • the synthetic nucleic acid molecule exhibits less toxicity as compared with an otherwise identical synthetic nucleic acid molecule that is linear. In some embodiments, the toxicity is measured by subchronic or chronic toxicity tests. In some embodiments, the synthetic nucleic acid molecule exhibits more target nucleic acid sequence specificity as compared with an otherwise identical synthetic nucleic acid molecule that is linear. In some embodiments, the target nucleic acid sequence specificity is measured by gene expression analysis. In some embodiments, the gene expression analysis is RNA-seq gene expression analysis. In some embodiments, the synthetic nucleic acid molecule exhibits more stability as compared with an otherwise identical synthetic nucleic acid molecule that is linear.
  • the stability is measured by the degradation of nucleotides in the presence of a nuclease.
  • the synthetic nucleic acid molecule is isolated.
  • the synthetic nucleic acid molecule is purified and isolated.
  • a pharmaceutical formulation comprises the synthetic nucleic acid molecule as described herein and a pharmaceutically acceptable: excipient, carrier, or diluent.
  • the pharmaceutical formulation is formulated for subcutaneous administration.
  • a cell comprises the synthetic nucleic acid molecule as described herein.
  • a method of delivering a synthetic nucleic acid molecule to a cell of a subject comprises introducing (i) the synthetic nucleic acid molecule as described herein, (ii) the pharmaceutical formulation as described herein, or (iii) the cell as described herein to a cell of a subject in vivo or ex vivo under conditions sufficient to modulate expression of the target nucleic acid molecule in the cell of the subject.
  • aspects disclosed herein provide methods of delivering nucleic acid molecules to a subject, the method comprising: introducing a double-stranded noncoding ribonucleic acid molecule to the subject under conditions sufficient modulate expression of a target nucleic acid sequence in the subject, wherein the double-stranded noncoding ribonucleic acid molecule comprises a sense strand base paired to an antisense strand, wherein the antisense strand is complementary to a the target nucleic acid sequence, and wherein the sense strand comprises a 5’ end coupled to a 3’ end of the sense strand, wherein the double-stranded noncoding ribonucleic acid molecule has a half-life in vivo that is greater than or equal to about 10 hours when measured using a quantitative nucleic acid detection assay.
  • aspects disclosed herein provide methods of delivering nucleic acid molecules to a subject, the method comprising: introducing a double-stranded noncoding ribonucleic acid molecule to the subject under conditions sufficient modulate expression of a target nucleic acid sequence in the subject, wherein the double-stranded noncoding ribonucleic acid molecule comprises a sense strand base paired to an antisense strand, wherein the antisense strand is complementary to a the target nucleic acid sequence, and wherein the sense strand comprises a 5’ end coupled to a 3’ end of the sense strand, wherein the introducing the double-stranded noncoding ribonucleic acid molecule to the subject induces less immunogenicity in the subject as compared with an otherwise identical double-stranded noncoding ribonucleic acid that does not comprise the sense strand comprising the 5’ end coupled to the 3’ end.
  • aspects disclosed herein provide methods of delivering nucleic acid molecules to a subject, the method comprising: introducing a double-stranded noncoding ribonucleic acid molecule to the subject under conditions sufficient modulate expression of a target nucleic acid sequence in the subject, wherein the double-stranded noncoding ribonucleic acid molecule comprises a sense strand base paired to an antisense strand, wherein the antisense strand is complementary to a the target nucleic acid sequence, and wherein the sense strand comprises a 5’ end coupled to a 3 ’ end of the sense strand, wherein the introducing the double-stranded noncoding ribonucleic acid molecule to the subject causes less toxicity or fewer off-target effects in the subject as compared with an otherwise identical double-stranded noncoding ribonucleic acid that does not comprise the sense strand comprising the 5’ end coupled to the 3’ end.
  • the introducing the double-stranded noncoding ribonucleic acid molecule to the subject causes less toxicity in the subject as compared with an otherwise identical double stranded noncoding ribonucleic acid that does not comprise the sense strand comprising the 5’ end coupled to the 3’ end. In some embodiments, the introducing the double-stranded noncoding ribonucleic acid molecule to the subject causes fewer off-target effects in the subject as compared with an otherwise identical double stranded noncoding ribonucleic acid that does not comprise the sense strand comprising the 5’ end coupled to the 3’ end.
  • the introducing the double-stranded noncoding ribonucleic acid molecule to the subject comprises administering the double-stranded noncoding ribonucleic acid molecule to the subject.
  • the administering comprises subcutaneous administration of the double-stranded noncoding ribonucleic acid molecule to the subject.
  • the administering is performed at a frequency that is less than once per month.
  • modulation of the expression of the target nucleic acid sequence in the subject is therapeutically effective to treat a disease or a condition of the subject.
  • the target nucleic acid sequence is a gene expression product of a gene provided in Table 1. In some embodiments, the gene is C5.
  • the gene expression product is mRNA.
  • disease or the condition is provided in Table 1.
  • the antisense strand comprises about 19 to about 27 contiguous nucleotides.
  • the sense strand comprises about 19 to about 35 contiguous nucleotides.
  • the sense strand further comprises a chemical linker, wherein the chemical linker couples the 5’ end of the sense strand to the 3’ end of the sense strand.
  • the chemical linker is substantially cleavable under intracellular conditions.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, or a glucuronide group, an azide-alkyne linker, an aldehyde-oxamine linker, a phosphorothoate-tosylated linker, a phosphate activation agent mediated phosphate-hydroxyl linkage, or a metal chelation ligation.
  • the chemical linker is substantially cleavable under intracellular conditions by an enzyme. In some embodiments, the chemical linker is independently substantially cleavable under intracellular conditions.
  • the chemical linker is substantially non-cleavable under intracellular conditions.
  • the antisense strand comprises a 5’ end and a 3’ end, wherein the 5’ end of the antisense strand is not coupled to the 3’ end of the antisense strand.
  • the antisense strand comprises a 5’ end and a 3’ end, wherein the 5’ end of the antisense strand is coupled to the 3’ end of the antisense strand.
  • the antisense strand further comprises a chemical linker, wherein the chemical linker couples the 5’ end of the antisense strand to the 3’ end of the antisense strand.
  • the chemical linker is substantially cleavable under intracellular conditions.
  • the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.
  • the chemical linker is substantially cleavable under intracellular conditions by an enzyme.
  • the chemical linker is independently substantially cleavable under intracellular conditions.
  • the chemical linker is substantially non-cleavable under intracellular conditions.
  • the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.
  • the antisense strand comprises a 5’ end, wherein the 5’ end of the antisense strand comprises a modification comprising 5’-(E)-Vinylphosphonate (5’-VP), 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), or
  • the doublestranded noncoding nucleic acid molecule further comprises a targeting moiety, wherein the targeting moiety directs the double-stranded noncoding nucleic acid molecule to a target cell or target tissue in vivo.
  • the targeting moiety is coupled to the sense strand of the double-stranded noncoding nucleic acid molecule.
  • the antisense strand comprises a chemical linker, wherein the chemical linker couples the targeting moiety to the sense strand.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, a glucuronide group, a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.
  • the target cell is a hepatic cell, a cardiac cell, a neuron, a muscle cell, a blood cell, a photoreceptor cell, a pancreatic cell, or a stem cell.
  • the target tissue is liver tissue, heart tissue, brain tissue, muscle tissue, nervous tissue, epithelial tissue, connective tissue, eye tissue, or pancreatic tissue.
  • the targeting moiety comprises a polypeptide, an RNA molecule, a lipophilic moiety, or a small molecule.
  • the polypeptide comprises an antibody, a single-domain antibody, a miniprotein, or an antigen-binding fragment thereof.
  • the polypeptide specifically binds to Glucagon Like Peptide 1 Receptor (GLP1R), Asialoglycoprotein receptor (ASGPR), prostate specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), Epithelial cell adhesion molecule (EpCam), AXL receptor tyrosine kinase (AXL), protein tyrosine kinase 7 (PTK7), Programmed death-ligand 1 (PD-L1), or T-cell immunoglobulin and mucin domain-3 (Tim-3), or any combination thereof.
  • GLP1R Glucagon Like Peptide 1 Receptor
  • ASGPR Asialoglycoprotein receptor
  • PSMA prostate specific membrane antigen
  • hTfR human transferrin receptor
  • EGFR epidermal growth factor receptor
  • Her2 human epidermal growth factor receptor 2
  • EpCam Epithelial cell adhe
  • the RNA molecule comprises an aptamer, a ribozyme, a hairpin RNA, a siRNA, or miRNA.
  • the lipophilic moiety comprises lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, l,3-bis-0(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03- (oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.
  • the small molecule comprises a sugar moiety.
  • the sugar moiety comprises an amino sugar.
  • the amino sugar is N-acetyl Galactosamine (GalNAc).
  • the targeting moiety is specific to an antigen or receptor of the target cell or target tissue.
  • the receptor comprises Asialoglycoprotein receptor (ASGPR).
  • the double stranded noncoding nucleic acid molecule comprises one or more nucleotides comprising a modification.
  • the modification comprises a chemical modification.
  • the chemical modification comprises a modification of the sugar, the phosphate backbone, or the nucleobase.
  • the modification of the nucleobase comprises a 2’-O-Me, or 2’-F, or 2’ -MOE.
  • the modification of the phosphate backbone linkage comprises a phosphodiester, phosphorothioate isomers, phosphoryl DMI amidate diester isomers, a phosphorodithioate, a methylphosphonate, a 5'-phosphorothioate, a peptide nucleic acid, a 5'- (E)-vinylphosphonate, or a 5'-methyl phosphonate.
  • the double-stranded noncoding ribonucleic acid molecule comprises a siRNA.
  • the doublestranded noncoding ribonucleic acid molecule comprises a dsRNA that associates with Argonaute-containing protein complexes.
  • lower immunogenicity is measured by the expression of innate immune response genes.
  • the expression is measured by gene expression analysis.
  • the gene expression analysis is RNA-seq gene expression analysis.
  • aspects disclosed herein provide methods of delivering a nucleic acid molecule to a subject, the methods comprising: introducing a double-stranded noncoding ribonucleic acid molecule to the subject under conditions sufficient modulate expression of a target nucleic acid sequence in the subject, wherein the double-stranded noncoding ribonucleic acid molecule comprises: a) an antisense strand of the double-stranded noncoding ribonucleic acid molecule, wherein the antisense strand comprises a target-binding sequence that is complementary to a target nucleic acid sequence; and b) a sense strand of the double-stranded noncoding ribonucleic acid molecule base paired to the antisense strand of the double-stranded noncoding ribonucleic acid molecule, wherein the sense strand comprises: i) one or more adapters configured to enhance specificity of target binding between the target-binding sequence of the antisense strand and the target nucleic acid sequence
  • aspects disclosed herein provide methods of delivering a nucleic acid molecule to a subject, the methods comprising: introducing a doublestranded noncoding ribonucleic acid molecule to the subject under conditions sufficient modulate expression of a target nucleic acid sequence in the subject, wherein the double-stranded noncoding ribonucleic acid molecule comprises: a) an antisense strand of the double-stranded noncoding ribonucleic acid molecule, wherein the antisense strand comprises a target-binding sequence that is complementary to a target nucleic acid sequence; and b) a sense strand of the double-stranded noncoding ribonucleic acid molecule base paired to the antisense strand of the double-stranded noncoding ribonucleic acid molecule, wherein the sense strand comprises: i) one or more adapters configured to enhance specificity of target binding between the target-binding sequence of the antisense strand and the target nucleic acid sequence, as compared with
  • aspects disclosed herein provide methods of delivering a nucleic acid molecule to a subject, the methods comprising: introducing a double-stranded noncoding ribonucleic acid molecule to the subject under conditions sufficient modulate expression of a target nucleic acid sequence in the subject, wherein the double-stranded noncoding ribonucleic acid molecule comprises: a) an antisense strand of the double-stranded noncoding ribonucleic acid molecule, wherein the antisense strand comprises a target-binding sequence that is complementary to a target nucleic acid sequence; and b) a sense strand of the double-stranded noncoding ribonucleic acid molecule base paired to the antisense strand of the double-stranded noncoding ribonucleic acid molecule, wherein the sense strand comprises: i) one or more adapters configured to enhance specificity of target binding between the target-binding sequence of the antisense strand and the target nucleic acid sequence, as compared
  • the introducing the double-stranded noncoding ribonucleic acid molecule to the subject causes less toxicity in the subject as compared with introducing an otherwise identical double stranded noncoding ribonucleic acid to the subject, wherein the otherwise identical double stranded noncoding ribonucleic acid does not comprise the sense strand comprising the 5’ end coupled to the 3’ end.
  • the introducing the double-stranded noncoding ribonucleic acid molecule to the subject causes fewer off-target effects in the subject as compared with introducing an otherwise identical double stranded noncoding ribonucleic acid to the subject, wherein the otherwise identical double stranded noncoding ribonucleic acid does not comprise the sense strand comprising the 5’ end coupled to the 3’ end.
  • the introducing the double- stranded noncoding ribonucleic acid molecule to the subject comprises administering the doublestranded noncoding ribonucleic acid molecule to the subject.
  • the administering comprises subcutaneous administration of the double-stranded noncoding ribonucleic acid molecule to the subject.
  • the administering is performed at a frequency that is less than once per month.
  • modulation of the expression of the target nucleic acid sequence in the subject is therapeutically effective to treat a disease or a condition of the subject.
  • the target nucleic acid sequence is a gene expression product of a gene provided in Table 1.
  • the gene is C5.
  • the gene expression product is mRNA.
  • the disease or the condition is provided in Table 1.
  • the antisense strand comprises about 19 to about 27 contiguous nucleotides. In some embodiments, the sense strand comprises about 19 to about 35 contiguous nucleotides.
  • the sense strand further comprises a chemical linker, wherein the chemical linker couples the 5’ end of the sense strand to the 3’ end of the sense strand.
  • the chemical linker is substantially cleavable under intracellular conditions.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, or a glucuronide group, an azide-alkyne linker, an aldehyde-oxamine linker, a phosphorothioate-tosylated linker, a phosphate activation agent mediated phosphate-hydroxyl linkage, or a metal chelation ligation.
  • the chemical linker is substantially cleavable under intracellular conditions by an enzyme. In some embodiments, the chemical linker is independently substantially cleavable under intracellular conditions.
  • the chemical linker is substantially non-cleavable under intracellular conditions.
  • the antisense strand comprises a 5’ end and a 3’ end, wherein the 5’ end of the antisense strand is not coupled to the 3’ end of the antisense strand.
  • the antisense strand comprises a 5’ end and a 3’ end, wherein the 5’ end of the antisense strand is coupled to the 3’ end of the antisense strand.
  • the antisense strand further comprises a chemical linker, wherein the chemical linker couples the 5’ end of the antisense strand to the 3’ end of the antisense strand.
  • the chemical linker is substantially cleavable under intracellular conditions.
  • the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.
  • the chemical linker is substantially cleavable under intracellular conditions by an enzyme.
  • the chemical linker is independently substantially cleavable under intracellular conditions.
  • the chemical linker is substantially non-cleavable under intracellular conditions.
  • the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.
  • the antisense strand comprises a 5’ end, wherein the 5’ end of the antisense strand comprises a modification comprising 5’-(E)-Vinylphosphonate (5’-VP), 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), or
  • the doublestranded noncoding nucleic acid molecule further comprises a targeting moiety, wherein the targeting moiety directs the double-stranded noncoding nucleic acid molecule to a target cell or target tissue in vivo.
  • the targeting moiety is coupled to the sense strand of the double-stranded noncoding nucleic acid molecule.
  • the antisense strand comprises a chemical linker, wherein the chemical linker couples the targeting moiety to the sense strand.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, a glucuronide group, a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.
  • the target cell is a hepatic cell, a cardiac cell, a neuron, a muscle cell, a blood cell, a photoreceptor cell, a pancreatic cell, or a stem cell.
  • the target tissue is liver tissue, heart tissue, brain tissue, muscle tissue, nervous tissue, epithelial tissue, connective tissue, eye tissue or pancreatic tissue.
  • the targeting moiety comprises a polypeptide, an RNA molecule, a lipophilic moiety, or a small molecule.
  • the polypeptide comprises an antibody, a single domain antibody, a miniprotein, or an antigen-binding fragment thereof.
  • the polypeptide specifically binds to Glucagon Like Peptide 1 Receptor (GLP1R), Asialoglycoprotein receptor (ASGPR), prostate specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), Epithelial cell adhesion molecule (EpCam), AXL receptor tyrosine kinase (AXL), protein tyrosine kinase 7 (PTK7), Programmed death ligand 1 (PD-L1), or T-cell immunoglobulin and mucin domain-3 (Tim-3), or any combination thereof.
  • GLP1R Glucagon Like Peptide 1 Receptor
  • ASGPR Asialoglycoprotein receptor
  • PSMA prostate specific membrane antigen
  • hTfR human transferrin receptor
  • EGFR epidermal growth factor receptor
  • Her2 human epidermal growth factor receptor 2
  • EpCam Epithelial cell adhe
  • the RNA molecule comprises an aptamer, a ribozyme, a hairpin RNA, a siRNA, or miRNA.
  • the lipophilic moiety comprises lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, l,3-bis-0(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03- (oleoyl)lithocholic acid, 03 (oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.
  • the small molecule comprises a sugar moiety.
  • the amino sugar is N-acetyl Galactosamine (GalNAc).
  • the sugar moiety comprises an amino sugar.
  • the targeting moiety is specific to an antigen or receptor of the target cell or target tissue.
  • the receptor comprises Asialoglycoprotein receptor (ASGPR).
  • the double stranded noncoding nucleic acid molecule comprises one or more nucleotides comprising a modification.
  • the modification comprises a chemical modification.
  • the chemical modification comprises a modification of the sugar, the phosphate backbone, or the nucleobase.
  • the modification of the nucleobase comprises a 2’-0-Me, or 2’-F, or 2’-M0E.
  • the modification of the phosphate backbone linkage comprises a phosphodiester, phosphorothioate isomers, phosphoryl DMI amidate diester isomers, a phosphorodithioate, a methylphosphonate, a 5'-phosphorothioate, a peptide nucleic acid, a 5' (E)-vinylphosphonate, or a 5'-methyl phosphonate.
  • the double-stranded noncoding ribonucleic acid molecule comprises a siRNA.
  • the double-stranded noncoding ribonucleic acid molecule comprises a dsRNA that associate with Argonaute-containing protein complexes.
  • the adapter is a peptide adapter.
  • the adapter is a nucleotide adapter.
  • the adapter is an antibody.
  • the adapter is a sugar.
  • the adapter is a lipid.
  • the adapter is an aptamer.
  • the less immunogenicity is measured by the expression of innate immune response genes.
  • the expression is measured by gene expression analysis.
  • the gene expression analysis is RNA-seq gene expression analysis.
  • aspects disclosed herein provide methods of activating transcription of a gene of interest, the method comprising: introducing a synthetic double-stranded noncoding nucleic acid molecule to cell of a subj ect under conditions sufficient to activate expression of a gene of interest, wherein the synthetic double-stranded noncoding ribonucleic acid molecule comprises a sense strand base paired to an antisense strand, wherein the sense strand comprises a 5’ end of the sense strand that is coupled to a 3’ end of the sense strand, and wherein the antisense strand is a complementary to a target nucleic acid sequence that either encodes the gene of interest or modulates expression of the gene of interest.
  • the antisense strand is complementary to a gene expression product from a gene provided in Table 1. In some embodiments, the gene encodes C5. In some embodiments, the target nucleic acid sequence is an mRNA sequence. In some embodiments, target nucleic acid sequence is an untranslated region or regulatory region that modulates the transcription of the gene of interest. In some embodiments, the antisense strand comprises about 19 to about 27 contiguous nucleotides. In some embodiments, the sense strand comprises about 19 to about 35 contiguous nucleotides. In some embodiments, the method further comprises a chemical linker, wherein the chemical linker couples the 5’ end of the sense strand to the 3’ end of the sense strand.
  • the chemical linker is substantially cleavable under intracellular conditions.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid- labile linker, a peptide linker, a nucleotide linker, or a glucuronide group, an azide-alkyne linker, an aldehyde-oxamine linker, a phosphorothioate-tosylated linker, a phosphate activation agent mediated Phosphate-hydroxyl linkage, or a metal chelation ligation.
  • the chemical linker is substantially cleavable under intracellular conditions by an enzyme.
  • the chemical linker is independently substantially cleavable under intracellular conditions. In some embodiments, the chemical linker is substantially non-cleavable under intracellular conditions. In some embodiments, the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer. In some embodiments, a 5’ end of the antisense strand is not coupled to a 3’ end of the antisense strand. In some embodiments, the antisense strand comprises a 5’ end of the antisense strand coupled to a 3’ end of the antisense strand.
  • the antisense strand comprises a chemical linker, wherein the chemical linker couples the 5’ end of the antisense strand to the 3’ end of the antisense strand.
  • the chemical linker is substantially cleavable under intracellular conditions.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, or a glucuronide group.
  • the chemical linker is substantially cleavable under intracellular conditions by an enzyme.
  • the chemical linker is substantially cleavable under intracellular conditions by an enzyme.
  • the chemical linker is substantially non-cleavable under intracellular conditions.
  • the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.
  • the 5’ end of the antisense strand comprises a modification comprising 5’-(E)-Vinylphosphonate (5’-VP), 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), or 6’(phosphonooxy-Butyl-Sulfide)Purine (6-PBuS-purine).
  • the synthetic double-stranded noncoding nucleic acid molecule comprises a targeting moiety, wherein the targeting moiety guides the synthetic double-stranded noncoding nucleic acid molecule to a target cell or target tissue in vivo.
  • the targeting moiety is coupled to the sense strand of the double-stranded noncoding nucleic acid molecule.
  • the targeting moiety is coupled to the sense strand by a chemical linker.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, a glucuronide group, a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.
  • the target cell is a hepatic cell, a cardiac cell, a neuron, a muscle cell, a blood cell, a photoreceptor cell, a pancreatic cell, or a stem cell.
  • the target tissue is liver tissue, heart tissue, brain tissue, muscle tissue, nervous tissue, epithelial tissue, connective tissue, eye tissue, or pancreatic tissue.
  • the targeting moiety comprises a polypeptide, an RNA molecule, a lipophilic moiety, or a small molecule.
  • the polypeptide comprises an antibody, a single-domain antibody, a miniprotein, or an antigenbinding fragment thereof.
  • the polypeptide comprises an agonist of Glucagon Like Peptide 1 Receptor (GLP1R), Asialoglycoprotein receptor (ASGPR), prostate specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), Epithelial cell adhesion molecule (EpCam), AXL receptor tyrosine kinase (AXL), protein tyrosine kinase 7 (PTK7), Programmed death-ligand 1 (PD-L1), T-cell immunoglobulin and mucin domain-3 (Tim-3).
  • GLP1R Glucagon Like Peptide 1 Receptor
  • ASGPR Asialoglycoprotein receptor
  • PSMA prostate specific membrane antigen
  • hTfR human transferrin receptor
  • EGFR epidermal growth factor receptor
  • Her2 human epidermal growth factor receptor 2
  • EpCam Epithelial cell adhesion molecule
  • the RNA molecule comprises an aptamer, a ribozyme, a hairpin RNA, a siRNA, or miRNA.
  • the lipophilic moiety comprises lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3- bis-0(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, borneol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.
  • the small molecule comprises a sugar moiety.
  • the sugar moiety comprises an amino sugar.
  • the amino sugar is N-acetyl Galactosamine (GalNAc).
  • the targeting moiety is specific to an antigen or receptor of the target cell or target tissue.
  • the receptor comprises Asialoglycoprotein receptor (ASGPR).
  • the synthetic double stranded noncoding nucleic acid molecule comprises one or more nucleotides comprising a modification.
  • the modification comprises a chemical modification.
  • the chemical modification comprises a modification of the sugar, the phosphate backbone, or the nucleobase.
  • the chemical modification of the nucleobase comprises a 2’-0-Me, 2’-F, or a 2’-M0E.
  • the chemical modification of the phosphate backbone linkage comprises a phosphodiester, phosphorothioate isomers, phosphoryl DMI amidate diester isomers, phosphorodithioate, methylphosphonate, 5'- phosphorothioate, peptide nucleic acid, 5'-(E)- vinylphosphonate, 5'-methyl phosphonate.
  • the synthetic double-stranded noncoding nucleic acid molecule exhibits less immunogenicity in vivo as compared with an otherwise identical linear noncoding nucleic acid molecule.
  • immunogenicity in vivo is measured by an immunogenicity assay.
  • the double-stranded noncoding nucleic acid molecule exhibits less toxicity or fewer off-target effects in the subject as compared with an otherwise identical linear noncoding nucleic acid molecule.
  • toxicity is measured by subchronic or chronic toxicity tests.
  • off-target effects are measured by gene expression analysis.
  • the gene expression analysis is RNA-seq gene expression analysis.
  • the double-stranded noncoding nucleic acid molecule exhibits less immunogenicity in the subject as compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the less immunogenicity is measured by the expression of innate immune response genes.
  • the expression is measured by gene expression analysis.
  • the gene expression analysis is RNA-seq gene expression analysis.
  • the double-stranded noncoding nucleic acid molecule exhibits more target nucleic acid sequence specificity in the subject as compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the more target nucleic acid sequence specificity is measured by the expression of the target gene.
  • the expression is measured by gene expression analysis.
  • the gene expression analysis is RNA-seq gene expression analysis.
  • the double-stranded noncoding nucleic acid molecule exhibits more stability in vivo as compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, the synthetic double-stranded noncoding nucleic acid molecule exhibits more durability in vivo as compared with an otherwise identical linear noncoding nucleic acid molecule.
  • aspects disclosed herein provide methods of activating transcription of a gene of interest, the methods comprising: introducing a synthetic double-stranded noncoding nucleic acid molecule to cell of a subject under conditions sufficient to activate expression of a gene of interest, wherein the synthetic double-stranded noncoding ribonucleic acid molecule comprises: a) an antisense strand of the double-stranded noncoding ribonucleic acid molecule, wherein the antisense strand comprises a target-binding sequence that is complementary to a target nucleic acid sequence; and b) a sense strand of the double-stranded noncoding ribonucleic acid molecule base paired to the antisense strand of the double-stranded noncoding ribonucleic acid molecule, wherein the sense strand comprises: i) one or more adapters configured to enhance specificity of target binding between the target-binding sequence of the antisense strand and the target nucleic acid sequence, as compared with an otherwise identical double
  • the antisense strand is complementary to a gene expression product from a gene provided in Table 1. In some embodiments, the gene encodes C5. In some embodiments, the target nucleic acid sequence is an mRNA sequence. In some embodiments, the target nucleic acid sequence is an untranslated region or regulatory region that modulates the transcription of the gene of interest. In some embodiments, the antisense strand comprises about 19 to about 27 contiguous nucleotides. In some embodiments, the sense strand comprises about 19 to about 35 contiguous nucleotides. In some embodiments, the methods further comprise a chemical linker, wherein the chemical linker couples the 5’ end of the sense strand to the 3’ end of the sense strand.
  • the chemical linker is substantially cleavable under intracellular conditions.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid- labile linker, a peptide linker, a nucleotide linker, or a glucuronide group, an azide-alkyne linker, an aldehyde-oxamine linker, a phosphorothioate-tosylated linker, a phosphate activation agent mediated Phosphate-hydroxyl linkage, or a metal chelation ligation.
  • the chemical linker is substantially cleavable under intracellular conditions by an enzyme.
  • the chemical linker is independently substantially cleavable under intracellular conditions. In some embodiments, the chemical linker is substantially non-cleavable under intracellular conditions. In some embodiments, the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer. In some embodiments, a 5’ end of the antisense strand is not coupled to a 3’ end of the antisense strand. In some embodiments, the antisense strand comprises a 5’ end of the antisense strand coupled to a 3’ end of the antisense strand.
  • the antisense strand comprises a chemical linker, wherein the chemical linker couples the 5’ end of the antisense strand to the 3’ end of the antisense strand.
  • the chemical linker is substantially cleavable under intracellular conditions.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, or a glucuronide group.
  • the chemical linker is substantially cleavable under intracellular conditions by an enzyme.
  • the chemical linker is independently substantially cleavable under intracellular conditions.
  • the chemical linker is substantially non-cleavable under intracellular conditions.
  • the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.
  • the 5’ end of the antisense strand comprises a modification comprising 5’-(E)-Vinylphosphonate (5’-VP), 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh- purine), or 6’(phosphonooxy-Butyl-Sulfide)Purine (6-PBuS-purine).
  • the synthetic double-stranded noncoding nucleic acid molecule comprises a targeting moiety, wherein the targeting moiety guides the synthetic double-stranded noncoding nucleic acid molecule to a target cell or target tissue in vivo.
  • the targeting moiety is coupled to the sense strand of the double-stranded noncoding nucleic acid molecule.
  • the targeting moiety is coupled to the sense strand by a chemical linker.
  • the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, a glucuronide group, a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.
  • the target cell is a hepatic cell, a cardiac cell, a neuron, a muscle cell, a blood cell, a photoreceptor cell, a pancreatic cell, or a stem cell.
  • the target tissue is liver tissue, heart tissue, brain tissue, muscle tissue, nervous tissue, epithelial tissue, connective tissue, eye tissue, or pancreatic tissue.
  • the targeting moiety comprises a polypeptide, an RNA molecule, a lipophilic moiety, or a small molecule.
  • the polypeptide comprises an antibody, a single domain antibody, a miniprotein, or an antigen-binding fragment thereof.
  • the polypeptide comprises an agonist of Glucagon Like Peptide 1 Receptor (GLP1R), Asialoglycoprotein receptor (ASGPR), prostate specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), Epithelial cell adhesion molecule (EpCam), AXL receptor tyrosine kinase (AXL), protein tyrosine kinase 7 (PTK7), Programmed death -ligand 1 (PD-L1), T-cell immunoglobulin and mucin domain-3 (Tim-3).
  • GLP1R Glucagon Like Peptide 1 Receptor
  • ASGPR Asialoglycoprotein receptor
  • PSMA prostate specific membrane antigen
  • hTfR human transferrin receptor
  • EGFR epidermal growth factor receptor
  • Her2 human epidermal growth factor receptor 2
  • EpCam Epithelial cell adhesion molecule
  • the RNA molecule comprises an aptamer, a ribozyme, a hairpin RNA, a siRNA, or miRNA.
  • the lipophilic moiety comprises lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, l,3-bis-0(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, borneol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.
  • the small molecule comprises a sugar moiety.
  • the sugar moiety comprises an amino sugar.
  • the amino sugar is N-acetyl Galactosamine (GalNAc).
  • the targeting moiety is specific to an antigen or receptor of the target cell or target tissue.
  • the receptor comprises Asialoglycoprotein receptor (ASGPR).
  • the synthetic double stranded noncoding nucleic acid molecule comprises one or more nucleotides comprising a modification.
  • the modification comprises a chemical modification.
  • the chemical modification comprises a modification of the sugar, the phosphate backbone, or the nucleobase.
  • the chemical modification of the nucleobase comprises a 2’-0-Me, 2’-F, or a 2’ -MOE.
  • the chemical modification of the phosphate backbone linkage comprises a phosphodiester, phosphorothioate isomers, phosphoryl DMI amidate diester isomers, phosphorodithioate, methylphosphonate, 5'- phosphorothioate, peptide nucleic acid, 5'-(E)-vinylphosphonate, 5'-methyl phosphonate.
  • the synthetic double-stranded noncoding nucleic acid molecule exhibits less immunogenicity in vivo as compared with an otherwise identical linear noncoding nucleic acid molecule.
  • immunogenicity in vivo is measured by an immunogenicity assay.
  • the double-stranded noncoding nucleic acid molecule exhibits less toxicity or fewer off-target effects in the subject as compared with an otherwise identical linear noncoding nucleic acid molecule.
  • toxicity is measured by subchronic or chronic toxicity tests.
  • off-target effects are measured by gene expression analysis.
  • the gene expression analysis is RNA-seq gene expression analysis.
  • the double-stranded noncoding nucleic acid molecule exhibits less immunogenicity in the subject as compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the less immunogenicity is measured by the expression of innate immune response genes.
  • the expression is measured by gene expression analysis.
  • the gene expression analysis is RNA-seq gene expression analysis.
  • the double-stranded noncoding nucleic acid molecule exhibits more target nucleic acid sequence specificity in the subject as compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the more target nucleic acid sequence specificity is measured by the expression of the target gene.
  • the expression is measured by gene expression analysis.
  • the gene expression analysis is RNA-seq gene expression analysis.
  • the doublestranded noncoding nucleic acid molecule exhibits more stability in vivo as compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, the synthetic double-stranded noncoding nucleic acid molecule exhibits more durability in vivo as compared with an otherwise identical linear noncoding nucleic acid molecule.
  • aspects disclosed herein provide synthetic nucleic acid molecules comprising: a functional antisense strand complementary to a target mRNA or a noncoding RNA of interest, wherein functionality and stability of the antisense strand is enhanced through hybridization to a partially or fully complementary nucleic acid sequence (sense) with no free ends, wherein the termini of sense have been closed with an adapter element comprising nucleic acid or non-nucleic acid residues that facilitate formation of a looped structure with no free ends, and wherein functionality and stability of the synthetic nucleic acid molecule is enhanced.
  • FIG. 1A shows a non-limiting example of a dsRNA with no free ends on the sense strand termed looped interfering RNA (liRNA), according to some embodiments herein.
  • liRNA looped interfering RNA
  • FIG. IB also shows a non-limiting example of a dsRNA with no free-ends on the sense strand termed looped interfering RNA (liRNA), according to some embodiments herein.
  • liRNA looped interfering RNA
  • FIG. 1C shows examples of common siRNA 2’ sugar rings (top), and backbone modifications (bottom right) incorporated into the sequence of siRNA to improve the nuclease resistance and immunostimulatory profile of the siRNA.
  • FIG. 2A shows non-limiting examples of various monovalent circularized therapeutic double-stranded RNAs (dsRNAs), according to some embodiments herein.
  • dsRNAs monovalent circularized therapeutic double-stranded RNAs
  • FIG. 2B shows non-limiting examples of various multivalent circularized therapeutic double-stranded RNAs (dsRNAs), according to some embodiments herein.
  • dsRNAs multivalent circularized therapeutic double-stranded RNAs
  • FIG. 2C shows a table with examples of reversible (non-covalent) and irreversible bonds (covalent), shown as open and locked conformations, connecting the two ends of the sense strand through adapter/j unction.
  • the antisense strand is also depicted as either an open structure, or a cleavable looped structure capable of forming an open structure once it enters the body or cell.
  • FIG. 3A shows prophetic data of an exemplary exonuclease stability assay comparing linear and circular dsRNAs.
  • FIG. 3B shows prophetic data of an exemplary serum stability assay comparing linear and circular dsRNAs.
  • FIG. 4A depicts a non-limiting example of an experimental method for intravenous or subcutaneous injection of dsRNA in rats, according to some embodiments herein.
  • FIG. 4B shows prophetic data of an exemplary in vivo serum stability assay after an intravenous injection into a mouse.
  • FIG. 5A shows non-limiting example of a locked and open conformations of dsRNAs, according to some embodiments herein.
  • FIG. 5B shows non-limiting example of a modifications such as antibodies, RNA aptamers, small molecules, and lipophilic moieties to improve dsRNA delivery, according to some embodiments herein.
  • FIG. 6 shows an experimental outline for the enzymatic circularization of a sense RNA strand, according to some embodiments herein.
  • FIG. 7A shows a 12% urea PAGE gel displaying a different (faster) migration pattern on the gel for the ligase-mediated looped sense strand of ApoB siRNA (right band).
  • FIG. 7B shows a gel verifying sense strand circularization by RNase R digestion. Ligase reaction products were digested with RNase R, an enzyme that only digests linear RNA. A representative gel displays resistance of faster migrating band to digestion by RNase R (right lane).
  • FIG. 7C shows a substrate concentration optimization reaction for large-scale purification of looped RNA (12% urea PAGE gel, stained with GelRed).
  • FIG. 8A depicts a linear siRNA structure with and without a phosphorothioate nucleotide backbone according to some embodiments herein.
  • FIG. 8B depicts a circular siRNA structure with and without a phosphorothioate nucleotide backbone according to some embodiments herein.
  • FIG. 8C shows the chemical structure of phosphodiester RNA and phosphorothioate (PS) nucleotides according to some embodiments herein.
  • FIG. 9 shows a phosphodiesterase I exonuclease digestion reaction showing improved stability of the sense strand of looped complement C5 siRNA in comparison to the linear siRNA sense strand.
  • Top graph shows ImageJ quantification of intact C5 sense strands of the 12% urea PAGE gel at the bottom panel.
  • FIG. 10 depicts a graph demonstrating efficient depletion of target C5 mRNA in HepG2 cells following lipofectamine RNAiMAX treatment with lOnM of enzymatically looped 21-mer sense siRNA (C521H, also referred to as design 1) and 27-mer sense strand C5 siRNA (C527H, also referred to as design 2).
  • FIG. 11 shows depletion of intercellular adhesion molecule 1 (ICAM1) mRNA in HepG2 cells following treatment with lOnM naked ICAM1 siRNA, containing enzymatically looped ICAM1 sense strand.
  • the level of mRNA depletion by the looped siRNA is comparable to that of linear ICAM1 siRNA.
  • FIG. 12A shows RNA-seq fold-change in gene expression analysis results for linear/open C5 siRNA (-1.5 ⁇ FC>1.5, p-value ⁇ 0.01); siRNA treatment in HepG2 cells, Square (left panel) and cross (right panel) denote downregulated and upregulated genes, respectively.
  • FIG. 12B shows RNA-seq fold-change in gene expression analysis results for looped C5 21-mer sense strand (design 1) siRNA (-1.5 ⁇ FC>1.5, p-value ⁇ 0.01); siRNA treatment in HepG2 cells, Square (left panel) and cross (right panel) denote downregulated and upregulated genes, respectively.
  • C5 displayed with a plus sign.
  • FIG. 12C shows RNA-seq fold-change in gene expression analysis results for looped C5 27-mer sense strand (design 2) siRNA (-1.5 ⁇ FC>1.5, p-value ⁇ 0.01); siRNA treatment in HepG2 cells.
  • Square (left panel) and cross (right panel) denote downregulated and upregulated genes, respectively.
  • FIG. 13A depicts the miRNA-mimicking off-target effect of siRNA strands through partial complementarity at the seed region.
  • FIG. 13B shows seed enrichment scores, and associated p-values, generated from an RNA-seq experiment (FIG. 12A-12C).
  • the seed enrichment scores show that formation of looped sense strand results in a statistically significant decrease in the number of downregulated mRNAs that have 3’-UTR sequence complementarity with sense strand seed region (e.g., lower miRNA- mimicking effect for the sense strand).
  • FIG. 14A shows RNA-seq differential gene expression (DGE) analysis results for downregulated genes in cells treated with linear C5 (C521L) siRNA.
  • DGE differential gene expression
  • FIG. 14B shows RNA-seq DGE analysis results for downregulated genes in cells treated with circular 21-mer sense C5 (C521H, also referred to as design 1) siRNA.
  • Downregulated genes that harbor complementary seed sequences against the antisense strand seed, sense strand seed or both in their 3’-UTR regions are depicted by a cross sign, a square, and a plus sign, respectively.
  • the bottom panel is a zoomed-in display of the upper panel, highlighting the limited number of downregulated genes with sense seed complementarity (light gray solid square) in the looped siRNA.
  • FIG. 14C shows RNA-seq DGE analysis results for downregulated genes in cells treated with looped 27-mer sense C5 (C527H, also referred to as design 2) siRNA.
  • C527H also referred to as design 2
  • Downregulated genes that harbor complementary seed sequences against the antisense strand seed, sense strand seed or both in their 3’-UTR regions are depicted by a cross sign, a square, and a plus sign, respectively.
  • the bottom panel is a zoomed-in display of the upper panel, highlighting the limited number of downregulated genes with sense seed complementarity (solid square, light gray) in the looped siRNA.
  • FIG. 16 shows an illustration of non-limiting examples of adapter (Al) module moieties such as antibodies, nucleic acid aptamers, small molecules, and lipophilic moieties, which enhance siRNA delivery and cellular targeting according to some embodiments herein.
  • FIG. 17 shows non-limiting examples of a monovalent (left) and various multivalent looped therapeutic double- stranded RNAs (dsRNAs) according to some embodiments herein.
  • Two-way or three-way junctions can form bivalent (center) or trivalent (right) siRNA units that target one or multiple mRNA targets.
  • FIG. 18A depicts the miRNA-mimicking off-target effect of siRNA strands through partial complementarity of the siRNA strands at the seed region.
  • FIG. 18B depicts an exemplary miRNA mechanism of action. MicroRNAs could either lead to degradation of target mRNA or inhibition of translation (not shown). Sense strand looping results in steric clash and interferes with loading of the sense strand into the RISC complex and in some cases may not form a functional miRNA-mimicking complex with RISC, therefore reducing the risk of off-target mRNA degradation (detectable in RNA-seq data analysis) and translation inhibition.
  • FIG. 19A shows an outline of in vivo siRNA treatment experiment to measure and compare efficacy and durability of C5 siRNA constructs (linear/open and looped design 1).
  • Five BALB/C mice were injected through the tail vain with vehicle (Invivofectamine), open C5 siRNA/Invivofectamine complex, and C5 looped siRNA (design l)/invivofectamine complex.
  • Plasma samples were collected on day 1, day 5, day 10, day 14 and day 21 post single dose injection on day zero (1.5 mg/kg 200 pL total volume).
  • FIG. 19B shows a mouse complement C5 ELISA test (Abeam ab264609 kit), showing the levels of plasma C5 protein in mice treated with the vehicle (circle), open C5 siRNA (square) and looped C5 siRNA (triangle).
  • compositions, methods, and kits comprising double-stranded noncoding nucleic acid molecules that modulate (e.g., increase or decrease) transcription of a target gene (e.g., a disease-associated gene), that exhibit greater durability (e.g., less instability) in vivo, increased specificity; increased target binding efficiency, less toxicity, less immunogenicity, fewer off-target effects, or any combination thereof.
  • a target gene e.g., a disease-associated gene
  • the double-stranded noncoding nucleic acid molecules disclosed herein comprise a sense strand with 5’ and 3’ ends that are coupled, thereby protecting the antisense (e.g., guide) strand from degradation in vivo.
  • the 5’ and 3’ ends of the sense strand are coupled reversibly, such that the double-stranded noncoding nucleic acid molecule can linearize in vivo at the site of action to induce modulation of transcription of the target gene.
  • the 5’ and 3’ ends of the sense strand are coupled irreversibly.
  • the antisense strand also comprises 3’ and 5’ ends that are reversibly coupled.
  • one or more adapters are attached to the sense strand to improve specificity and increase target binding efficiency.
  • Additional moieties e.g., antibodies, RNA aptamers, small molecules, lipophilic moieties, etc.
  • moieties can be attached to the double-stranded noncoding nucleic acid molecule in order to increase stability, direct targeting of the double-stranded noncoding nucleic acid molecule to a target cell or tissue, lower off-target immunogenic effects, or otherwise improve pharmacological qualities of the double-stranded noncoding nucleic acid molecule.
  • methods of delivering the double-stranded noncoding nucleic acid molecules of the present disclosure to a cell of a subject in vivo or ex vivo comprises administering the double-stranded noncoding nucleic acid molecule to the subject, such as by subcutaneous administration.
  • methods further comprise treating a disease or a condition in the subject by modulating the transcription of the target gene (e.g., disease-associated gene) with the double-stranded noncoding nucleic acid molecule.
  • kits comprising the compositions and systems disclosed herein, and instructions for how to use the double-stranded noncoding nucleic acid molecules disclosed herein to modulate the transcription of a gene of interest.
  • kits may comprise a container to store the system components and instructions.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a sample includes a plurality of samples, including mixtures thereof.
  • determining means determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative, or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.
  • circular as used herein in reference to a strand of a double-stranded noncoding nucleic acid molecule, means that the 5’ and 3’ end of one or more strands of the double-stranded noncoding nucleic acid are coupled to each other directly or indirectly such that the one or more stands has no free ends, irrespective of the shape or conformation of the doublestranded noncoding nucleic acid molecule.
  • zzz vivo is used to describe an event that takes place in a subject’s body.
  • ex vivo is used to describe an event that takes place outside of a subject’s body.
  • An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject.
  • An example of an ex vivo assay performed on a sample is an “zzz vitro" assay.
  • zzz vitro is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained.
  • In vitro assays can encompass cell-based assays in which living or dead cells are employed.
  • In vitro assays can also encompass a cell-free assay in which no intact cells are employed.
  • polynucleotide refers to polymers of nucleotides of any length and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • a polynucleotide may comprise modified nucleotides, such as, but not limited to methylated nucleotides and their analogs or non-nucleotide components. Modifications to the nucleotide structure may be imparted before or after assembly of the polynucleotide.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • nucleic acid encompass double- or triple-stranded nucleic acids, as well as single-stranded molecules.
  • the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).
  • Nucleic acid sequences, when provided, are listed in the 5’ to 3’ direction, unless stated otherwise. Methods described herein provide for the generation of isolated nucleic acids.
  • nucleic acid as referred to herein can comprise at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 375, at least about 400, at least about 425, at least about 450, at least about 475, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000
  • polynucleotides coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • cDNA encoding for a gene or gene fragment referred herein may comprise at least one region encoding for exon sequence
  • nucleic acid molecule refers to production by in vitro chemical and/or enzymatic synthesis.
  • cell generally refers to a biological cell.
  • gene refers to a segment of nucleic acid that encodes an individual protein or RNA (also referred to as a “coding sequence” or “coding region”), optionally together with associated regulatory region such as promoter, operator, terminator, untranslated region (UTR), and the like, which may be located upstream or downstream of the coding sequence.
  • a “genetic locus” referred to herein, is a particular location within a gene.
  • polypeptide may be used interchangeably herein in reference to a polymer of amino acid residues.
  • a protein may refer to a full-length polypeptide as translated from a coding open reading frame, or as processed to its mature form, while a polypeptide or peptide may refer to a degradation fragment or a processing fragment of a protein that nonetheless uniquely or identifiably maps to a particular protein.
  • a polypeptide may be a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Polypeptides may be modified, for example, by the addition of carbohydrate, phosphorylation, etc.
  • homology when used herein to describe to an amino acid sequence or a nucleic acid sequence, relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in roc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J Mol Biol. 1990 Oct 5;215(3):403-10; Nucleic Acids Res. 1997 Sep l;25(17):3389-402).
  • BLAST basic local alignment search tool
  • Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application. Percent identity of sequences can be determined using the most recent version of BLAST, as of the filing date of this application.
  • the term “percent (%) identity”, or “percent sequence identity,” with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • percent (%) identity As used herein, the term “percent (%) identity”, or “percent sequence identity,” with respect to a reference nucleic acid sequence is the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • gene of interest refers to a gene encoding a gene expression product that is detectable directly or indirectly.
  • the double-stranded noncoding nucleic acid molecule is any type of nucleic acid (e.g., ribonucleic acid (RNA), deoxyribonucleic acid (DNA), etc.) that contains two strands.
  • the double-stranded noncoding nucleic acid molecule are double-stranded oligonucleotides, which comprise short pieces of a nucleotide sequence (e.g., DNA, RNA, or DNA and RNA).
  • the double-stranded noncoding nucleic acid molecule comprises an antisense strand configured to silence a target single-stranded nucleic acid sequence, such as a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the double-stranded noncoding nucleic acid molecule comprises an antisense strand configured to enhance expression of a target single-stranded nucleic acid sequence.
  • the double-stranded noncoding nucleic acid molecule is a small interfering RNA (siRNA) e.g., FIG.
  • RNA e.g., mRNA
  • RISC complex an argonaute-containing complex
  • the double-stranded noncoding nucleic acid molecule can be synthetic.
  • the double-stranded noncoding nucleic acid molecule can be a chemically modified nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule can comprise two nucleic acid strands, a sense strand, and an antisense strand.
  • the sense strand of the double-stranded noncoding nucleic acid molecule will have the same or substantially the same sequence as a target nucleic acid sequence.
  • the antisense strand of the double-stranded noncoding nucleic acid molecule can be complementary or substantially complementary to a target nucleic acid sequence.
  • the target nucleic acid sequence is associated with a disease or a condition disclosed herein.
  • modulation of transcription of the target nucleic acid sequence may be therapeutically effective to treat the disease or the condition.
  • modulation of expression of the target nucleic acid sequence e.g., in the case of an RNA target
  • the modulation of the expression of the target nucleic acid sequence comprises post-transcriptional modifications, such as capping, splicing and polyadenylation of the RNA target.
  • Double-stranded nucleic acids can be at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides,
  • Double-stranded nucleic acids can be at most about 40 nucleotides, at most about 39 nucleotides, at most about 38 nucleotides, at most about 37 nucleotides, at most about 36 nucleotides, at most about 35 nucleotides, at most about 34 nucleotides, at most about 33 nucleotides, at most about 32 nucleotides, at most about 31 nucleotides, at most about 30 nucleotides, at most about 29 nucleotides, at most about 28 nucleotides, at most about 27 nucleotides, at most about 26 nucleotides, at most about 25 nucleotides, at most about 24 nucleotides, at most about 23 nucleotides, at most about 22 nucleotides, at most about 21 nucleotides, at most about 20 nucleotides, at most about 19 nucleotides, at most about 18 nucleotides,
  • Double-stranded nucleic acids can be between about 5 to about 40 nucleotides in length. Double-stranded nucleic acids (e.g., siRNAs) can be between about 5 to about 35 nucleotides in length. Double-stranded nucleic acids (e.g., siRNAs) can be between about 5 to about 30 nucleotides in length. Double-stranded nucleic acids (e.g., siRNAs) can be between about 5 to about 25 nucleotides in length. Double-stranded nucleic acids (e.g., siRNAs) can be between about 5 to about 20 nucleotides in length.
  • Double-stranded nucleic acids can be between about 5 to about 15 nucleotides in length. Double-stranded nucleic acids (e.g., siRNAs) can be between about 5 to about 10 nucleotides in length. Double-stranded nucleic acids (e.g., siRNAs) can be between about 10 to about 40 nucleotides in length. Double-stranded nucleic acids (e.g., siRNAs) can be between about 15 to about 40 nucleotides in length. Double-stranded nucleic acids (e.g., siRNAs) can be between about 20 to about 40 nucleotides in length.
  • Doublestranded nucleic acids can be between about 25 to about 40 nucleotides in length. Double-stranded nucleic acids (e.g., siRNAs) can be between about 30 to about 40 nucleotides in length. Double-stranded nucleic acids (e.g., siRNAs) can be between about 35 to about 40 nucleotides in length.
  • the antisense strand of a double-stranded noncoding nucleic acid molecule can form complementary binding with an entire sense strand of the double-stranded noncoding nucleic acid molecule.
  • the antisense strand of a double-stranded noncoding nucleic acid molecule can form complementary binding with a part of a sense strand of a double-stranded noncoding nucleic acid molecule.
  • the antisense strand of a double-stranded noncoding nucleic acid molecule can also form complementary binding with an entire target strand.
  • the antisense strand of a double-stranded noncoding nucleic acid molecule can form complementary binding with a part of a target strand.
  • the antisense strand of a double-stranded noncoding nucleic acid molecule can form complementary binding to one or more portions of a target strand (e.g., an mRNA strand) including a 5’ UTR, a 3’UTR, a regulatory region, and/or a coding sequence.
  • a regulatory region of a target strand e.g., a mRNA
  • the antisense strand of the double-stranded noncoding nucleic acid molecule can form complementary binding to part or all of a portion of an mRNA target strand.
  • the antisense strand of the double-stranded noncoding nucleic acid molecule can be completely complementary (e.g., 100% complementary) to their target strand counterparts.
  • the antisense strand of the double-stranded noncoding nucleic acid molecule and can have imperfect complementarity to their target strand counterparts.
  • An antisense strand of the double-stranded noncoding nucleic acid molecule can have at least about 50% complementarity, at least about 55% complementarity, at least about 60% complementarity, at least about 65% complementarity, at least about 70% complementarity, at least about 75% complementarity, at least about 80% complementarity, at least about 85% complementarity, at least about 90% complementarity, at least about 91% complementarity, at least about 92% complementarity, at least about 93% complementarity, at least about 94% complementarity, at least about 95% complementarity, at least about 96% complementarity, at least about 97% complementarity, at least about 98% complementarity, at least about 99% complementarity, or more to its corresponding target strand.
  • the antisense strand of the double-stranded noncoding nucleic acid molecule can have at most about 99% complementarity, at most about 98% complementarity, at most about 97% complementarity, at most about 96% complementarity, at most about 95% complementarity, at most about 94% complementarity, at most about 93% complementarity, at most about 92% complementarity, at most about 91% complementarity, at most about 90% complementarity, at most about 85% complementarity, at most about 80% complementarity, at most about 75% complementarity, at most about 70% complementarity, at most about 65% complementarity, at most about 60% complementarity, at most about 55% complementarity, at most about 50% complementarity, or less to its corresponding target strand.
  • a double-stranded noncoding nucleic acid molecule can be a DNA oligonucleotide.
  • a double-stranded oligonucleotide can be an RNA oligonucleotide.
  • a double-stranded noncoding nucleic acid molecule can be a chimeric oligonucleotide, which comprises RNA and DNA.
  • a double-stranded noncoding nucleic acid molecule can exist as a single, unfolded double-strand (e.g., linear or open) (e.g., FIG. 8A).
  • doublestranded noncoding nucleic acid molecules can exist in a circularized or looped conformation (e.g., having no free ends) (e.g., FIG. 1A-B, FIG. 2A-B, FIG. 8B, FIG. 17).
  • a 5’ and 3’ end of the sense strand of the double-stranded noncoding nucleic acid can be coupled such that the sense stand has no free ends.
  • a 5’ and 3’ end of the antisense strand of the double-stranded noncoding nucleic acid can be coupled such that the antisense strand has no free ends.
  • Double-stranded noncoding nucleic acid molecules can exist as a single doublestranded noncoding nucleic acid molecule or as a double-stranded noncoding nucleic acid molecule comprised of multiple units or modules (e.g., a double-stranded noncoding nucleic acid molecule comprised of more than one double-stranded noncoding nucleic acid molecule) (FIG. 17).
  • a double-stranded noncoding nucleic acid molecule comprised of more than one doublestranded noncoding nucleic acid molecule can be referred to as a double-stranded noncoding nucleic acid molecule module and/or a multivalent double-stranded noncoding nucleic acid molecule.
  • a multivalent double-stranded noncoding nucleic acid molecule can comprise at least about 2 double-stranded noncoding nucleic acid molecules, at least about 3 double-stranded noncoding nucleic acid molecules, at least about 4 double-stranded noncoding nucleic acid molecules, at least about 5 double-stranded noncoding nucleic acid molecules, at least about 6 double-stranded noncoding nucleic acid molecules, at least about 7 double-stranded noncoding nucleic acid molecules, at least about 8 double-stranded noncoding nucleic acid molecules, at least about 9 double-stranded noncoding nucleic acid molecules, at least about 10 double-stranded noncoding nucleic acid molecules, or more double-stranded noncoding nucleic acid molecules.
  • a multivalent double-stranded noncoding nucleic acid molecule can comprise at most about 10 double-stranded noncoding nucleic acid molecules, at most about 9 double-stranded noncoding nucleic acid molecules, at most about 8 double-stranded noncoding nucleic acid molecules, at most about 7 double-stranded noncoding nucleic acid molecules, at most about 6 double-stranded noncoding nucleic acid molecules, at most about 5 double-stranded noncoding nucleic acid molecules, at most about 4 double-stranded noncoding nucleic acid molecules, at most about 3 double-stranded noncoding nucleic acid molecules, at most about 2 double-stranded noncoding nucleic acid molecules, or fewer double-stranded noncoding nucleic acid molecules.
  • Multivalent double-stranded noncoding nucleic acid molecules can be circularized (FIG. 17).
  • a circularized multivalent double-stranded noncoding nucleic acid molecule comprising two or more double-stranded noncoding nucleic acid molecules can refer to a multivalent looped double-stranded noncoding nucleic acid molecule.
  • a circularized multivalent double-stranded noncoding nucleic acid molecule comprising two double-stranded noncoding nucleic acid molecules can refer to a double-stranded noncoding nucleic acid molecule looped dimer (FIG. 17).
  • a circularized multivalent double-stranded noncoding nucleic acid molecule comprising three ASOs can refer to a double-stranded noncoding nucleic acid molecule looped trimer (FIG. 17).
  • a multivalent double stranded oligonucleotide can be linked by at least about 2 linkers, at least about 3 linkers, at least about 4 linkers, at least about 5 linkers, at least about 6 linkers, at least about 7 linkers, at least about 8 linkers, at least about 9 linkers, at least about 10 linkers, or more linkers.
  • a multivalent double stranded oligonucleotide can be linked by at most about 10 linkers, at most about 9 linkers, at most about 8 linkers, at most about 7 linkers, at most about 6 linkers, at most about 5 linkers, at most about 4 linkers, at most about 3 linkers, or at most about 2 linkers.
  • Circular double-stranded oligonucleotides can be made from their linear counterparts through enzymatic or chemical reactions to link the 5’ and 3’ ends of the linear strand.
  • a circular double-stranded oligonucleotide can be formed through the formation of a phosphodiester bond to link the 5’ and 3’ ends of both the linear antisense strand and the linear sense strand.
  • a circular double-stranded oligonucleotide can be formed through the addition of a linker element to connect the 5’ and 3’ ends of both the linear antisense strand and the linear sense strand.
  • a circular double-stranded oligonucleotide can also be formed through the addition of a linker element to connect the 5’ and 3’ ends of the linear sense strand.
  • a linker can include, but is not limited to a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, a glucuronide group, an azide-alkyne linker, an aldehydeoxamine linker, a phosphorothioate-tosylated linker, a phosphate activation agent mediated phosphate-hydroxyl linkage, or a metal chelation ligation linker.
  • a nucleotide linker can be an oligonucleotide linker, an oligo aptamer linker, an RNA linker, a DNA linker.
  • a peptide linker can be a polypeptide linker (e.g., an antibody linker).
  • a linker can comprise a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer. Additional examples of monovalent or multivalent circularized double-stranded oligonucleotides can be found in FIG.
  • a linker can be permanent (e.g., non-cleavable/covalent) (e.g., FIG. 2C, FIG. 5A).
  • a linker can be reversible (non-covalent).
  • a linker can be cleavable (e.g., FIG. 2C, FIG. 5A).
  • a linker can be cleaved by, for example, an enzyme or a catalyst. Enzymes which have cleaving properties include but are not limited to proteases and endonucleases.
  • a catalyst can be a chemical catalyst (e.g., an acid-based catalyst or a metal catalyst) or a non-chemical catalyst (e.g., light, pH, or heat).
  • a linker can be cleaved through the use of a reversible click chemistry reaction.
  • a linker can be independently cleaved.
  • a click chemistry reaction is a reaction used to join two specific molecular entities in the absence of water.
  • Examples of click-chemistry reactions can include but are not limited to copper(I)-catalyzed azide-alkyne cycloadditions, strain promoted alkyne-nitrone cycloadditions, strain promoted azide-alkyne cycloadditions, alkene and azide cycloadditions, alkene and tetrazine invers-demand Diels-Alder reactions, and alkene and tetrazole photoclick reactions.
  • Amines and thiols can be used to facilitate reversible click chemistry reactions.
  • a linker can be cleavable under certain conditions.
  • a linker can be cleavable or substantially cleavable in intracellular conditions but non-cleavable or substantially non-cleavable in extracellular conditions.
  • a linker can be non-cleavable or substantially non-cleavable in intracellular conditions but cleavable or substantially cleavable in extracellular conditions.
  • Selective cleavability can be related to conditions such as but not limited to pH or ionic concentration.
  • a double-stranded oligonucleotide can comprise an adapter.
  • adapters are peptide adapters, nucleotide adapters, antibody adapters, targeting moiety adapters (e.g., glycans), sugar adapters, lipid adapters, DNA aptamers, or RNA aptamers (FIG. 16).
  • a nucleotide adapter can be an oligonucleotide adapter, an oligo aptamer adapter, an RNA adapter, a DNA adapter.
  • a peptide adapter can be a polypeptide adapter (e.g., a protein adapter).
  • the sense strand comprises an adapter.
  • the antisense strand comprises an adapter.
  • both the sense strand and the antisense strand comprise an adapter.
  • an adapter can be contiguous with a linker.
  • an adapter is not adjacent to a linker.
  • the double stranded oligonucleotide comprises one adapter.
  • the double stranded oligonucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more adapters. Double stranded oligonucleotides can comprise a single adapter or multiple adapters.
  • a double stranded oligonucleotide can comprise at least about 2 adapters, at least about 3 adapters, at least about 4 adapters, at least about 5 adapters, at least about 6 adapters, at least about 7 adapters, at least about 8 adapters, at least about 9 adapters, at least about 10 adapters, or more adapters.
  • an ASO can comprise at most about 10 adapters, at most about 9 adapters, at most about 8 adapters, at most about 7 adapters, at most about 6 adapters, at most about 5 adapters, at most about 4 adapters, at most about 3 adapters, at most about 2 adapters, or fewer adapters.
  • a single double stranded oligonucleotide (e.g., monovalent double stranded oligonucleotide) comprising an adapter can be circularized.
  • Monovalent double stranded oligonucleotides comprising an adapter can be circularized by reversibly or irreversibly joining a free end of the adapter to a free end of the double stranded oligonucleotide through a linker.
  • Multivalent double stranded oligonucleotides comprising two or more adapters can be circularized.
  • Double stranded oligonucleotides can comprise a single adapter and one or more linkers.
  • a first free end of an adapter can be joined to a first free end of a double stranded oligonucleotide through a first linker, and a second free end of the adapter can be joined to a second free end of the double stranded oligonucleotide through a second linker, thus forming a looped double stranded oligonucleotide.
  • Multivalent double stranded oligonucleotides can comprise two or more adapters and two or more linkers.
  • a multivalent double stranded oligonucleotide can be linked by at least about 2 adapters, at least about 3 adapters, at least about 4 adapters, at least about 5 adapters, at least about 6 adapters, at least about 7 adapters at least about 8 adapters, at least about 9 adapters, at least about 10 adapters, or more adapters.
  • a multivalent double stranded oligonucleotide can be linked by at most about 10 adapters, at most about 9 adapters, at most about 8 adapters, at most about 7 adapters, at most about 6 adapters, at most about 5 adapters, at most about 4 adapters, at most about 3 adapters, or at most about 2 adapters.
  • Adapters can comprise sequences that provide additional functionality for the doublestranded noncoding nucleic acid molecule.
  • an adapter can extend the 5’ and 3’ ends of the linear sense strand of a double-stranded noncoding nucleic acid molecule.
  • Adapters can have full or partial complementarity to a flanking region of a gene of interest and/or a target gene (e.g., adapters can comprise hybridizing regions and/or sticky ends).
  • adapters can comprise non-hybridizing regions (e.g., non-flanking regions, blunt ends, etc.).
  • Adapters can add flexibility to a double-stranded noncoding nucleic acid molecule.
  • an adapter connecting the 5’ and 3’ ends of the linear sense strand of a double-stranded noncoding nucleic acid molecule can improve the “steric freedom” of the double-stranded noncoding nucleic acid molecule and allow it to interact with protein(s) involved in downstream events (e.g., degradation by RNase H) in an unrestricted manner.
  • an adapter can change the shape of a double-stranded noncoding nucleic acid molecule.
  • An adapter can vary in length.
  • a glycan adapter can be between about 5 to 10 glycan residues in length to form a glycan chain.
  • an antibody adapter can comprise a single antibody.
  • an adapter can be a nucleotide adapter.
  • a nucleotide adapter can be a nucleotide adapter.
  • nucleotides can be at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleoi ides, at least ab >ut 8 nucleotide ;, at least about 9 nucleotides, a : least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides,
  • DNA, RT [A, etc. can be at most about 4( nucleotides, at most about 39 nucleotides, at most about 38 nucleotides, at most about 37 nucleotides, at most about 36 nucleotides, at most about 35 nucleotides, at most about 34 nucleotides, at most about
  • nucleotides at most about 32 nucleotides, at most about 31 nucleotides, at most about 30 nucleotides, at most about 29 nucleotides, at most about 28 nucleotides, at most about 27 nucleotides, at most about 26 nucleotides, at most about 25 nucleotides, at most about 24 nucleotides, at most about 23 nucleotides, at most about 22 nucleotides, at most about 21 nucleotides, at most about 20 nucleotides, at most about 19 nucleotides, at most about 18 nucleotides, at most about 17 nucleotides, at most about 16 nucleotides, at most about 15 nucleotides, at most about 14 nucleotides, at most about 13 nucleotides, at most about 12 nucleotides, at most about 11 nucleotides, at most about 1( nucleotides, a most about 9 nucleotides, at most about 8 nucle
  • a nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 5 to about 20 nucleotides in length.
  • a nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 5 to about 15 nucleotides in length.
  • a nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 5 to about 10 nucleotides in length.
  • a nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 10 to about 40 nucleotides in length.
  • a nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 15 to about 40 nucleotides in length.
  • a nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 20 to about 40 nucleotides in length.
  • a nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 25 to about 40 nucleotides in length.
  • a nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 30 to about 40 nucleotides in length.
  • a nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 35 to about 40 nucleotides in length.
  • an adapter can be a peptide adapter.
  • a peptide adapter e.g., polypeptide
  • a peptide adapter (e.g., polypeptide) can be between about 2 to about 20 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 2 to about 18 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 2 to about 16 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 2 to about 14 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 2 to about 12 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 2 to about 10 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 2 to about 8 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 2 to about 6 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 2 to about 4 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 4 to about 20 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 6 to about 20 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 8 to about 20 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 10 to about 20 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 12 to about 20 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 14 to about 20 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 16 to about 20 peptides in length.
  • a peptide adapter (e.g., polypeptide) can be between about 18 to about 20 peptides in length.
  • an adapter can enhance the function of a double-stranded noncoding nucleic acid molecule.
  • the enhanced function of a double-stranded noncoding nucleic acid molecule with an adapter can be due to increased steric freedom of the modified ASO, allowing it to interact with proteins involved in downstream events (e.g., degradation by RNAse) in an unrestricted manner.
  • a double-stranded noncoding nucleic acid molecule with an adapter can have increased steric freedom as compared to a double-stranded noncoding nucleic acid molecule without an adapter.
  • a double-stranded noncoding nucleic acid molecule with an adapter can have at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about
  • a doublestranded noncoding nucleic acid molecule with an adapter can have at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less increased steric freedom as compared to a double-stranded noncoding nucleic acid molecule without an adapter.
  • a double-stranded noncoding nucleic acid molecule with an adapter can have increased target specificity as compared to a double-stranded noncoding nucleic acid molecule without an adapter.
  • a double-stranded noncoding nucleic acid molecule with an adapter can have at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, or more increased target specificity as
  • a doublestranded noncoding nucleic acid molecule with an adapter can have at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less increased target specificity as compared to a double-stranded noncoding nucleic acid molecule without an adapter.
  • a double-stranded noncoding nucleic acid molecule with an adapter can have increased target binding as compared to a double-stranded noncoding nucleic acid molecule without an adapter.
  • a double-stranded noncoding nucleic acid molecule with an adapter can have at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about
  • a doublestranded noncoding nucleic acid molecule with an adapter can have at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less increased target binding as compared to a double-stranded noncoding nucleic acid molecule without an adapter.
  • Double-stranded noncoding nucleic acid molecules disclosed herein can comprise a modification of the sugar, the phosphate backbone, or the nucleobase.
  • noncoding nucleic acid molecules can be modified through the addition of moieties onto the molecule.
  • a modification can be a chemical modification, a synthetic modification, or a natural modification.
  • Nucleic acid molecules can be modified at the nucleobase.
  • Nucleobase modifications include but are not limited to 2’-O-methylation (2’-O-Me), conversion of uridine to pseudouridine, N(6)-methyladenosine, 5 -methylcytidine, 5-methyluridine (ribothymidine), 2’-fluoro (2’F), 2’-O- methoxy ethyl (2’ -MOE), ribose modification with bridged nucleic acids (e.g., locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), or constrained ethyl bridged nucleic acid (cEt) modifications), or nucleotides with alternative chemistries (e.g., phosphorodiamidate morpholino oligonucleotides (PMO), peptide nucleic acids (PNA), tricyclo DNA (tcDNA), unlocked nucleic acids (UNA) or glycol nucleic acids (GNA)) .
  • 2’-O-Me conversion of
  • Nucleic acid molecules can be modified at the phosphate backbone (e.g., FIG. 8C).
  • a phosphate backbone can be modified to comprise a phosphodiester, phosphorothioate isomers, phosphoryl DMI amidate diester isomers, a phosphorodithioate, a methylphosphontae, a 5’- phosphorothioate, a peptide nucleic acid, a 5’-(E)-vinylphosphonate, or a 5 ’-methyl phosphonate.
  • Additional moieties can be added or attached onto a double-stranded nucleic acid (e.g., a siRNA).
  • Additional moieties can include, but are not limited to antibodies, lipophilic moieties, small molecules, and RNA aptamers (e.g., ribozymes, docosanoic acid, etc.) (e.g., FIG. 5B, FIG. 16). Additional moieties can be added to double-stranded nucleic acids to alter the pharmacological features (e.g., structural or chemical parameters) of the double-stranded nucleic acid.
  • a double-stranded nucleic acid can be modified with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more moieties.
  • An additional moiety can be added at the 5’ end of a double-stranded nucleic acid.
  • an additional moiety can be added at the 3’ end of a double-stranded nucleic acid.
  • an additional moiety can be added in the middle of a double-stranded nucleic acid (e.g., neither at the 3’ end nor the 5’ end).
  • An additional moiety can be added to the sense strand of an siRNA.
  • an additional moiety can be added to the antisense strand of an siRNA.
  • Modification can be made to double-stranded noncoding nucleic acid molecules (e.g., siRNAs) in order to increase stability.
  • the double-stranded nucleic acids as described herein can have an increased stability of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more as compared to a control double-stranded nucleic acid.
  • Nucleic acid stability can be measured by analyzing the half-life of the double-stranded noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule as described herein can have an increased half-life of at least about 30 minutes, at least about 1 hour, at least about 90 minutes, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, or more
  • Modifications can be made to double-stranded noncoding nucleic acid molecules (e.g., siRNAs) in order to lower off-target effects.
  • the number of off-target effects as measured by sequencing can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more as compared to a control double-stranded nucleic acid.
  • Modifications can be made to double-stranded noncoding nucleic acid molecules (e.g., siRNAs) in order to lower adverse immunogenic effects.
  • the number of adverse immunogenic effects as measured by an immunogenicity assay can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more as compared to a control double-stranded nucleic acid.
  • Modifications can be made to double-stranded noncoding nucleic acid molecules (e.g., siRNAs) in order to lower toxicity.
  • Toxicity as measured by an toxicity assay can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more as compared to a control double-stranded nucleic acid.
  • control double-stranded nucleic acid can be an otherwise identical double-stranded noncoding nucleic acid molecule without the modification.
  • Modifications can be made to double-stranded noncoding nucleic acid molecules (e.g., siRNAs) in order to increase stability and/or durability.
  • Durability and/or stability as measured by nucleic acid detection can be increased by least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more as compared to a control double-stranded nucleic acid.
  • the control double-stranded nucleic acid molecule can be an otherwise identical double-stranded noncoding nucleic acid molecule without the modification.
  • Circularization of the double-stranded noncoding nucleic acid molecules can allow for the inclusion of fewer chemically-modified nucleotides (e.g., nucleotides containing phosphorothioates) which are linked to adverse medical side effects. Circularization can allow for the inclusion of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 fewer chemically-modified nucleotides which are linked to adverse medical side effects.
  • Adverse medical side effects can include but are not limited to reduced platelets, thrombocytopenia, perturbation of heart rate, raised blood pressure, or raised cardiac output through the activation of the complement cascade.
  • Double-stranded noncoding nucleic acid molecules can be used to effect transcriptional regulation.
  • a dsRNA can bind to a target gene or an mRNA of a target gene (e.g., a target RNA) in order to modulate the expression of the target gene.
  • a dsRNA can bind to a specific region of a target gene or a target mRNA such as a regulatory region in order to modulate the target gene.
  • Modulation of a target gene can result in increased expression of the target gene.
  • modulation of a target gene can result in decreased expression of the target gene.
  • modulation of a target gene can maintain the expression of the target gene.
  • a double-stranded noncoding nucleic acid molecule can increase expression of a target gene by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more as compared to a control expression level.
  • a double-stranded noncoding nucleic acid molecule can increase expression of a target gene by at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0.1%, or less as compared to a control expression level.
  • a double-stranded noncoding nucleic acid molecule can increase expression of a target gene by at least or up to about 0.1 -fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3 -fold, at least or up to about 4-fold, at least or up to about 5 -fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9- fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40
  • a double-stranded noncoding nucleic acid molecule can increase expression of a target gene by at most or less than about 10,000-fold, at most or less than about 5,000-fold, at most or less than about 1,000-fold, at most or less than about 500-fold, at most or less than about 100-fold, at most or less than about 90-fold, at most or less than about 80-fold, at most or less than about 70-fold, at most or less than about 60-fold, at most or less than about 50-fold, at most or less than about 40-fold, at most or less than about 30-fold, at most or less than about 20-fold, at most or less than about 10-fold, at most or less than about 9-fold, at most or less than about 8-fold, at most or less than about 7-fold, at most or less than about 6-fold, at most or less than about 5 -fold, at most or less than about 4-fold, at most or less than about 3 -fold, at most or less than about 2-fold, at most or less than
  • a double-stranded noncoding nucleic acid molecule can decrease expression of a target gene by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more as compared to a control expression level.
  • a double-stranded noncoding nucleic acid molecule can decrease expression of a target gene by at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0.1%, or less as compared to a control expression level.
  • a double-stranded noncoding nucleic acid molecule can decrease expression of a target gene by at least or up to about 0.1 -fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3 -fold, at least or up to about 4-fold, at least or up to about 5 -fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9- fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40
  • a double-stranded noncoding nucleic acid molecule can decrease expression of a target gene by at most or less than about 10,000-fold, at most or less than about 5,000-fold, at most or less than about 1,000-fold, at most or less than about 500-fold, at most or less than about 100-fold, at most or less than about 90-fold, at most or less than about 80-fold, at most or less than about 70-fold, at most or less than about 60-fold, at most or less than about 50-fold, at most or less than about 40-fold, at most or less than about 30-fold, at most or less than about 20-fold, at most or less than about 10-fold, at most or less than about 9-fold, at most or less than about 8-fold, at most or less than about 7-fold, at most or less than about 6-fold, at most or less than about 5 -fold, at most or less than about 4-fold, at most or less than about 3 -fold, at most or less than about 2-fold, at most or less than about
  • Modulation of a target mRNA can result in increased expression of the gene product expressed from the target mRNA.
  • modulation of a target mRNA can result in decreased expression of the gene product expressed from the target mRNA.
  • modulation of a target mRNA can maintain expression of the gene product expressed from the target mRNA.
  • a double-stranded noncoding nucleic acid molecule can increase expression of a target mRNA by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more as compared to a control expression level.
  • a double-stranded noncoding nucleic acid molecule can increase expression of a target mRNA by at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0.1%, or less as compared to a control expression level.
  • a double-stranded noncoding nucleic acid molecule can increase expression of a target mRNA by at least or up to about 0.1 -fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3 -fold, at least or up to about 4-fold, at least or up to about 5 -fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9- fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to
  • a double-stranded noncoding nucleic acid molecule can increase expression of a target mRNA by at most or less than about 10,000-fold, at most or less than about 5,000-fold, at most or less than about 1,000-fold, at most or less than about 500-fold, at most or less than about 100-fold, at most or less than about 90-fold, at most or less than about 80-fold, at most or less than about 70-fold, at most or less than about 60-fold, at most or less than about 50-fold, at most or less than about 40-fold, at most or less than about 30-fold, at most or less than about 20-fold, at most or less than about 10-fold, at most or less than about 9-fold, at most or less than about 8-fold, at most or less than about 7-fold, at most or less than about 6-fold, at most or less than about 5 -fold, at most or less than about 4-fold, at most or less than about 3 -fold, at most or less than about 2-fold, at most or
  • a double-stranded noncoding nucleic acid molecule can decrease expression of a target mRNA by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more as compared to a control expression level.
  • a double-stranded noncoding nucleic acid molecule can decrease expression of a target mRNA by at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0.1%, or less as compared to a control expression level.
  • a double-stranded noncoding nucleic acid molecule can decrease expression of a target mRNA by at least or up to about 0.1 -fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3 -fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9- fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40
  • a double-stranded noncoding nucleic acid molecule can decrease expression of a target mRNA by at most or less than about 10,000-fold, at most or less than about 5,000-fold, at most or less than about 1,000-fold, at most or less than about 500-fold, at most or less than about 100-fold, at most or less than about 90-fold, at most or less than about 80-fold, at most or less than about 70-fold, at most or less than about 60-fold, at most or less than about 50-fold, at most or less than about 40-fold, at most or less than about 30-fold, at most or less than about 20-fold, at most or less than about 10-fold, at most or less than about 9-fold, at most or less than about 8-fold, at most or less than about 7-fold, at most or less than about 6-fold, at most or less than about 5 -fold, at most or less than about 4-fold, at most or less than about 3 -fold, at most or less than about 2-fold, at most or
  • a targeting moiety can be used to direct a double-stranded noncoding nucleic acid molecule to a target cell or tissue.
  • a targeting moiety can be but is not limited to a lipophilic moiety, a small molecule, a peptide (e.g., a polypeptide), an RNA molecule, a nanoparticle, an antibody, a single-domain antibody, a miniprotein, or an antigen binding fragment thereof.
  • a targeting moiety can be specific to an antigen or receptor on the target cell or tissue (e.g., Asialoglycoprotein receptor (ASGPR)).
  • a targeting moiety can be specific to an antigen or receptor on the target cell or tissue (e.g., Asialoglycoprotein receptor (ASGPR)).
  • a targeting moiety can be a lipophilic moiety, lipophilic moiety can comprise one or more fatty acid groups or salts thereof.
  • a lipophilic be a lipid. Lipids are fatty acids and their derivatives which are insoluble in water but soluble in organic solvents.
  • a lipophilic moiety can be unsaturated.
  • a lipophilic moiety can be monosaturated.
  • a lipophilic moiety can be polysaturated.
  • a double bond of an unsaturated lipophilic moiety can be in a cis conformation.
  • a double bond of an unsaturated lipophilic moiety can be in a trans conformation.
  • lipophilic moieties can be a triglyceride, a phospholipid, a sterol, an oil, a wax, a hormone, a vitamin, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis- O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, borneol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03- (oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.
  • a targeting moiety can be a small molecule.
  • a small molecule can be a sugar, an amino acid, a phenolic compound, an alkaloid, a sterol, a lipid, a fatty acid, or other small chemical compound.
  • a chemical compound can be a molecule that has a molecular weight of less than 1000 Daltons. Alternatively, or in addition to, a small molecule is a molecule with a size on the order of 1 nm.
  • a targeting moiety can be a sugar or sugar moiety.
  • a sugar can be a monosaccharide.
  • a sugar can be a disaccharide.
  • a sugar can be a polysaccharide.
  • Nonlimiting examples of sugars include glucose, dextrose, fructose, galactose, a sugar alcohol, a pentose, xylose, ribose, sucrose, cellulose, starch, lactose, maltose, trehalose, lactulose, cellobiose, chitobiose, glycogen, or chitin.
  • a small molecule can be an amino sugar such as but not limited to N-acetyl Galactosamine (GalNAc), N-acetylglucosamine or sialic acid.
  • a targeting moiety can be an antibody or an antigen-binding fragment thereof.
  • An antibody also known as an immunoglobulin, is a blood protein produced to counteract a specific antigen.
  • Antibodies can be Y-shaped proteins which comprise variable binding sites that are specific to particular epitopes.
  • An antibody can be a monoclonal antibody.
  • an antibody can be a polyclonal antibody.
  • An antibody can be a single-domain antibody.
  • An antibody can be an antibody fragment.
  • An antibody can be an agonist.
  • an antibody can be an antagonist.
  • an antibody can be an allosteric modulator (e.g., a positive allosteric modulator or a negative allosteric modulator).
  • a targeting moiety can be a polypeptide.
  • polypeptides can be an agonist of Glucagon Like Peptide 1 Receptor (GLP1R), Asialoglycoprotein receptor (ASGPR), prostate specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), Epithelial cell adhesion molecule (EpCam), AXL receptor tyrosine kinase (AXL), protein tyrosine kinase 7 (PTK7), Programmed death-ligand 1 (PD-L1), and T-cell immunoglobulin and mucin domain-3 (Tim-3).
  • GLP1R Glucagon Like Peptide 1 Receptor
  • ASGPR Asialoglycoprotein receptor
  • PSMA prostate specific membrane antigen
  • hTfR human transferrin receptor
  • EGFR epidermal growth factor receptor
  • Her2 human epidermal growth factor receptor 2
  • a targeting moiety can be an RNA molecule.
  • An RNA molecule can comprise an aptamer, a ribozyme, a hairpin RNA, a siRNA, or a miRNA.
  • a double-stranded nucleic acid can be targeted to a target gene.
  • a double-stranded nucleic acid can be targeted to an RNA molecule that encodes a target gene.
  • target genes include AMT, ABCA4, ACADVL, ADA, AGT, ALAS1, ALDH2, ALMS1, ANGPTL3, APOA5, ApoC3, APOL1, APP, AR, ARG1, ASL, ASS1, AT3 (SERPINC1), ATP7B, ATXN2, ATXN3, BCL-xL, MCL-1, Cl-INH (SERPING1), C3, C5, CEBPA, CERS2, CFB, Clcn7, CNGA3, CPS1, CTNS, CYP24A1, DGAT2, DMPK, DNM2, DUX4, Dystrophin, Dystrophin (Exon 44), Dystrophin (Exon 45), Dystrophin (Exon 51), Dystrophin (Exon 53), EPO, FAH
  • C5 also known as complement 5 is a gene which encode a component of the complement system, a part of the innate immune system. C5 can be involved in inflammation, homeostasis, and defense against pathogens.
  • the C5 protein is comprised of the C5 alpha and beta chains, which are linked by a disulfide bridge. Mutations in the C5 gene can cause complement component 5 deficiency, a disease characterized by recurrent bacterial infections.
  • a double-stranded noncoding nucleic acid molecule can be engineered with a targeting moiety to target a target cell.
  • a target cell can be but is not limited to a stem cell (e.g., an induced pluripotent stem cell, an embryonic stem cell), a bone cell, a blood cell, a red blood cell, a white blood cell, a platelet, a dermal cell, a fibroblast, a hepatocyte, a lymphocyte, a bone marrow cell, a glandular cell, a hepatic cell, a cardiac cell, a pancreatic cell, a gall bladder cell, a muscle cell, a sperm cell, an egg cell, a fat cell, a nerve cell, a neuron, a Schwann cell, an interneuron, an immune cell, an osteoblast, a chondrogenic cell, an odontoblast, a cementoblast, a chondrocyte, a mesenchymal cell, an epitheli
  • a double-stranded noncoding nucleic acid molecule can be engineered with a targeting moiety to target a target tissue.
  • a target tissue can be but is not limited to nervous tissue, brain tissue, spinal cord tissue, epithelial tissue, epidermal tissue, intestinal tissue, heart tissue, liver tissue, eye tissue, pancreatic tissue, lung tissue, bladder tissue, muscle tissue, cardiac muscle tissue, smooth muscle tissue, skeletal muscle tissue, connective tissue fat tissue, bone tissue, and tendon tissue.
  • double-stranded noncoding nucleic acid molecules and pharmaceutical formulations thereof that are therapeutically effective to treat a disease or a disorder disclosed herein.
  • the double-stranded nucleic acids disclosed herein can be used to treat a disease or disorder in a subject.
  • diseases or disorders can be cancer, inflammatory diseases or disorder, metabolic diseases or disorders, cardiovascular diseases or disorders, immunodeficiency diseases or disorders, respiratory diseases or disorders, pain, digestive diseases or disorders, reproductive diseases or disorders, endocrine diseases or disorders, immune diseases or disorders, autoimmune diseases or disorders, or neurological diseases or disorders.
  • the double-stranded noncoding nucleic acid molecule comprises an antisense strand configured to modulate expression of a gene expression product expressed from a gene of interest provided in Table 1.
  • the modulation of the expression of the gene of interest is therapeutically effective to treat the indication provided in Table 1 that corresponds with the gene of interest.
  • the modulation can be activation or inhibition.
  • the double-stranded noncoding nucleic acid molecule further comprises a targeting moiety specific to the target tissue provided in Table 1 corresponding with the gene of interest and indication.
  • a subject can be a biological entity containing expressed genetic materials.
  • the biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa.
  • the subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro.
  • the subject can be an animal such as a fish, a bird, a reptile, an insect, an amphibian, or a mammal.
  • the mammal can be a feline, a canine, a primate, an ape, a rodent, a camelid, a pig, a sheep, a cow, a horse, a goat, or a rabbit.
  • the mammal can be a human.
  • the subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.
  • a pharmaceutical formulation can comprise a composition disclosed herein.
  • a pharmaceutical formulation can further comprise an excipient.
  • An excipient can be a buffer, a carrier, a stabilizer, a solubilizer, a filler, a preservative, a dilutant, a vehicle, a detergent, a salt, a peptide, a surfactant, an oligosaccharide, an amino acid, an adjuvant, a carbohydrate, and/or a bulking agent.
  • a pharmaceutical formulation can remain in the subject for at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 28 hours, at least about 32 hours, at least about 36 hours, at least about 40 hours, at least about 44 hours, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least
  • a pharmaceutical formulation can remain in the subject for at most about 4 weeks, at most about 3 weeks, at most about 2 weeks, at most about 13 days, at most about 12 days, at most about 11 days, at most about 10 days, at most about 9 days, at most about 8 days, at most about 7 days, at most about 6 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, at most about 44 hours, at most about 40 hours, at most about 36 hours, at most about 32 hours, at most about 28 hours, at most about 24 hours, at most about 23 hours, at most about 22 hours, at most about 21 hours, at most about 20 hours, at most about 19 hours, at most about 18 hours, at most about 17 hours, at most about 17 hours, at most about 16 hours, at most about 15 hours, at most about 14 hours, at most about 13 hours, at most about 12 hours, at most about 11 hours, at most about 10 hours, at most about 9 hours, at most about 8 hours, at most about 7 hours, at most about 6 hours, at most about 5 days
  • a pharmaceutical formulation can have a half-life as measured by a transcription inhibition assay.
  • a pharmaceutical formulation can have a half-life of at least about 15 minutes, 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 28 hours, at least about 32 hours, at least about 36 hours, at least about 40 hours, at least about 44 hours, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least
  • a pharmaceutical formulation can have a half-life of at most about 4 weeks, at most about 3 weeks, at most about 2 weeks, at most about 13 days, at most about 12 days, at most about 11 days, at most about 10 days, at most about 9 days, at most about 8 days, at most about 7 days, at most about 6 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, at most about 44 hours, at most about 40 hours, at most about 36 hours, at most about 32 hours, at most about 28 hours, at most about 24 hours, at most about 23 hours, at most about 22 hours, at most about 21 hours, at most about 20 hours, at most about 19 hours, at most about 18 hours, at most about 17 hours, at most about 17 hours, at most about 16 hours, at most about 15 hours, at most about 14 hours, at most about 13 hours, at most about 12 hours, at most about 11 hours, at most about 10 hours, at most about 9 hours, at most about 8 hours, at most about 7 hours, at most about 6 hours,
  • the cell comprises the double-stranded nucleic acids as disclosed herein.
  • the cell(s) may be isolated from other sample components, or reaction components.
  • the cell(s) may be purified.
  • a cell sample comprising the cell(s) may have a purity of at least or about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the cell(s) may be formulated in a pharmaceutical composition or formulation, such as for treatment of a disease or a condition provided in Table 1.
  • the cell(s) may be a cell line, or a plurality of cells containing or expressing the one or more systems of the present disclosure.
  • compositions or pharmaceutical formulations disclosed herein are methods of creating, isolating, and/or purifying the double-stranded nucleic acid compositions disclosed herein. Also disclosed herein, in some embodiments, are methods of utilizing the compositions or pharmaceutical formulations disclosed herein to treat diseases and disorders of a subject. The methods disclosed herein may be modified by applying molecular barcodes to the nucleic acid molecules. [0151] In some embodiments, the methods of the present disclosure comprise purifying or isolating the double-stranded noncoding nucleic acid molecules.
  • Double-stranded noncoding nucleic acid molecules can be purified and/or isolated through the use of several processes such as but not limited to phenol -chloroform extraction, a DNA filtration column, salt and proteinase K treatments, and the use of silica-gel membranes.
  • the methods of the present disclosure comprise delivering the double-stranded noncoding nucleic acid molecule to a target cell or tissue.
  • the compositions disclosed herein can be delivered to a target through the use of several means such as viral vector particles (e.g., retrovirus, adenovirus, adeno-associated virus (AAV), or herpes simplex virus), cationic nanoparticles, lipid nanoparticles, cationic polymers, plasmids, cells, or physical methods (e.g., sonication, electroporation, lipofection).
  • viral vector particles e.g., retrovirus, adenovirus, adeno-associated virus (AAV), or herpes simplex virus
  • cationic nanoparticles e.g., lipid nanoparticles, cationic polymers, plasmids, cells, or physical methods (e.g., sonication, electroporation, lipofection).
  • the methods comprise enhancing or silencing expression or translation of a gene of interest by introducing the double-stranded noncoding nucleic acid molecule of the present disclosure to a target cell or tissue.
  • introducing the double-stranded noncoding nucleic acid molecule of the present disclosure to a target cell or tissue enhances or silences expression and/or translation of a gene of interest as compared with introducing an otherwise identical synthetic nucleic acid molecule that is linear to the target cell or tissue (FIG. 12A-12C).
  • introducing the double-stranded noncoding nucleic acid molecule to a target cell or tissue enhances or silences expression and/or translation of a gene of interest by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more as compared with introducing an otherwise identical synthetic nucleic acid molecule that is linear to the target cell or tissue.
  • introducing the double-stranded noncoding nucleic acid molecule to a target cell or tissue enhances or silences expression and/or translation of a gene of interest by at least about 2- fold, at least about 3 -fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20- fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more as compared with introducing an otherwise identical synthetic nucleic acid molecule that is linear to the target cell or tissue.
  • introducing the double-stranded noncoding nucleic acid molecule to a target cell or tissue enhances or silences expression and/or translation of a gene of interest by at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less as compared with introducing an otherwise identical synthetic nucleic acid molecule that is linear to the target cell or tissue.
  • introducing the double-stranded noncoding nucleic acid molecule to a target cell or tissue enhances or silences expression and/or translation of a gene of interest by at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70-fold, at most about 60- fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2- fold, or less as compared with introducing an otherwise identical synthetic nucleic acid molecule that is linear to the target cell or tissue.
  • the methods comprise delivering (e.g., administering) the double-stranded noncoding nucleic acid molecule to a subject, wherein the double-stranded noncoding nucleic acid molecule exhibits less immunogenicity in the target cell or tissue as compared with an otherwise identical linear noncoding nucleic acid molecule (FIG. 15).
  • immunogenicity is reduced in a target cell or tissue with the double-stranded noncoding nucleic acid molecule by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • immunogenicity is reduced in a target cell or tissue with the double-stranded noncoding nucleic acid molecule by at least about 2-fold, at least about 3 -fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • immunogenicity is reduced in a target cell or tissue with the double-stranded noncoding nucleic acid molecule by at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • immunogenicity is reduced in a target cell or tissue with the double-stranded noncoding nucleic acid molecule by at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70-fold, at most about 60-fold, at most about 50-fold, at most about 40- fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, or less when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the methods comprise delivering (administering) the doublestranded noncoding nucleic acid molecule to a subject, wherein the double-stranded noncoding nucleic acid molecules exhibits less off-target effects in the target cell or tissue as compared with an otherwise identical synthetic nucleic acid molecule that is linear (FIG. 13A-14C).
  • off-target effects are reduced in a target cell or tissue with the double-stranded noncoding nucleic acid molecule by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • off-target effects are reduced in a target cell or tissue with the double-stranded noncoding nucleic acid molecule by at least about 2-fold, at least about 3 -fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more.
  • off-target effects are reduced in a target cell or tissue with the double-stranded noncoding nucleic acid molecule by at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70-fold, at most about 60-fold, at most about 50-fold, at most about 40- fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, or less when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule exhibits less toxicity in the target cell or tissue as compared with an otherwise identical synthetic nucleic acid molecule that is linear. In some embodiments, toxicity is reduced in a target cell or tissue with the double-stranded noncoding nucleic acid molecule by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • toxicity is reduced in a target cell or tissue with the double-stranded noncoding nucleic acid molecule by at least about 2-fold, at least about 3 -fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • toxicity is reduced in a target cell or tissue with the double-stranded noncoding nucleic acid molecule by at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • toxicity is reduced in a target cell or tissue with the double-stranded noncoding nucleic acid molecule by at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70-fold, at most about 60-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, or less when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule exhibits more target nucleic acid sequence specificity in the target cell or tissue as compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the doublestranded noncoding nucleic acid molecule has increased target nucleic acid sequence specificity in a target cell or tissue by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule has increased target nucleic acid sequence specificity in a target cell or tissue by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule has increased target nucleic acid sequence specificity in a target cell or tissue by at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule has increased target nucleic acid sequence specificity in a target cell or tissue by at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70-fold, at most about 60-fold, at most about 50- fold, at most about 40-fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3 -fold, at most about 2-fold, or less when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule exhibits more stability in vivo as compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule has increased stability in vivo by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule has increased stability in vivo by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule has increased stability in vivo by at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule has increased stability in vivo by at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70-fold, at most about 60-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90- fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, or less when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule exhibits more stability ex vivo as compared with an otherwise identical linear noncoding nucleic acid molecule (FIG. 9).
  • the double-stranded noncoding nucleic acid molecule has increased stability ex vivo by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule has increased stability ex vivo by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule has increased stability ex vivo by at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the double-stranded noncoding nucleic acid molecule has increased stability ex vivo by at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70-fold, at most about 60-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90- fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, or less when compared with an otherwise identical linear noncoding nucleic acid molecule.
  • the antisense strand of the double-stranded noncoding nucleic acid molecule is complementary to a target nucleic acid sequence that either encodes the gene of interest or modulates expression of the gene of interest.
  • the antisense strand of the double-stranded noncoding nucleic acid molecule is complementary to a transcriptional enhancer, transcriptional silencer, or a promoter of transcription of a gene of interest.
  • the antisense strand of the double-stranded noncoding nucleic acid molecule is complementary to long noncoding RNAs (IncRNAs) or microRNAs (miRNAs) to affect regulation of expression of the gene of interest.
  • the antisense strand of the double-stranded noncoding nucleic acid molecule is complementary to intronic regions of pre- mRNA (prior to splicing) to affect translation of the resulting mRNA isoforms. In some embodiments, the antisense strand of the double-stranded noncoding nucleic acid molecule is complementary to regions of mRNA (after splicing) to affect translation of the mRNA. For example, the antisense strand may silence expression or translation of the gene of interest or enhance expression or translation of the gene of interest.
  • the methods of the present disclosure comprise treating a disease or a condition in a subject by administering the double-stranded noncoding nucleic acid molecule to the subject under conditions sufficient to modulate expression of a gene of interest associated with the disease or condition (e.g., Table 1).
  • the administering can be administered orally, intrathecally, percutaneously, rectally, sublingually, intranasally, intravitreally, subcutaneously, intramuscularly, transdermally, or intravenously.
  • the compositions disclosed herein can be made into a dosage formulation that can vary based on the subject being treated and the mode of administration.
  • the double-stranded nucleic acid active ingredient can comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of a dosage formulation.
  • the double-stranded nucleic acid active ingredient can comprise at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less of a dosage formulation.
  • a dosage formulation for intravenous administration can be at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 110 mg, at least about 120 mg, at least about 130 mg, at least about 140 mg, at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, at least about 200 mg, at least about 210 mg, at least about 220 mg, at least about 230 mg, at least about 240 mg, at least about 250 mg, at least about 260 mg, at least about 270 mg, at least about 280 mg, at least about 290 mg, at least about 300 mg, at least about 310 mg, at least about 320 mg, at least about 330 mg, at least about 340 mg
  • a dosage formulation for intravenous administration can be at most about 350 mg, at most about 340 mg, at most about 330 mg, at most about 320 mg, at most about 310 mg, at most about 300 mg, at most about 290 mg, at most about 280 mg, at most about 270 mg, at most about 260 mg, at most about 250 mg, at most about 240 mg, at most about 230 mg, at most about 220 mg, at most about 210 mg, at most about 200 mg, at most about 190 mg, at most about 180 mg, at most about 170 mg, at most about 160 mg, at most about 150 mg, at most about 140 mg, at most about 130 mg, at most about 120 mg, at most about 110 mg, at most about 100 mg, at most about 90 mg, at most about 80 mg, at most about 70 mg, at most about 60 mg, at most about 50 mg, at most about 45 mg, at most about 40 mg, at most about 35 mg, at most about 30 mg, at most about 25 mg, at most about 20 mg, at most about 15 mg
  • a dosage formulation for intravenous administration can be in a range from 25 to 50 mg, 25 to 100 mg, 25 mg to 150 mg, 25 mg to 200 mg, 25 mg to 250 mg, 25 mg to 300 mg, 25 mg to 300 mg, 30 to 50 mg, 30 to 100 mg, 30 mg to 150 mg, 30 mg to 200 mg, 30 mg to 250 mg, 30 mg to 300 mg, 50 mg to 100 mg, 50 mg to 150 mg, 50 mg to 200 mg, 50 mg to 250 mg, 50 mg to 300 mg, 50 mg to 300 mg, 100 mg to 150 mg, 100 mg to 200 mg, 100 mg to 250 mg, 100 mg to 300 mg, 100 mg to 250 mg, 150 mg to 200 mg, 150 mg to 250 mg, 150 mg to 300 mg, 150 mg to 350 mg, 200 to 250 mg, 200 to 300 mg, 200 to 350 mg, 250 mg to 300 mg, 250 mg to 350 mg, or 300 mg to 350 mg.
  • a dosage formulation for subcutaneous administration can be at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 110 mg, at least about 120 mg, at least about 130 mg, at least about 140 mg, at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, at least about 200 mg, at least about 210 mg, at least about 220 mg, at least about 230 mg, at least about 240 mg, at least about 250 mg, at least about 260 mg, at least about 270 mg, at least about 280 mg, at least about 290 mg, at least about 300 mg, at least about 310 mg, at least about 320 mg, at least about 330 mg, at least about 340 mg,
  • a dosage formulation for subcutaneous administration can be at most about 350 mg, at most about 340 mg, at most about 330 mg, at most about 320 mg, at most about 310 mg, at most about 300 mg, at most about 290 mg, at most about 280 mg, at most about 270 mg, at most about 260 mg, at most about 250 mg, at most about 240 mg, at most about 230 mg, at most about 220 mg, at most about 210 mg, at most about 200 mg, at most about 190 mg, at most about 180 mg, at most about 170 mg, at most about 160 mg, at most about 150 mg, at most about 140 mg, at most about 130 mg, at most about 120 mg, at most about 110 mg, at most about 100 mg, at most about 90 mg, at most about 80 mg, at most about 70 mg, at most about 60 mg, at most about 50 mg, at most about 45 mg, at most about 40 mg, at most about 35 mg, at most about 30 mg, at most about 25 mg, at most about 20 mg, at most about 15 mg,
  • a dosage formulation for subcutaneous administration can be in a range from 25 to 50 mg, 25 to 100 mg, 25 mg to 150 mg, 25 mg to 200 mg, 25 mg to 250 mg, 25 mg to 300 mg, 25 mg to 300 mg, 30 to 50 mg, 30 to 100 mg, 30 mg to 150 mg, 30 mg to 200 mg, 30 mg to 250 mg, 30 mg to 300 mg, 50 mg to 100 mg, 50 mg to 150 mg, 50 mg to 200 mg, 50 mg to 250 mg, 50 mg to 300 mg, 50 mg to 300 mg, 100 mg to 150 mg, 100 mg to 200 mg, 100 mg to 250 mg, 100 mg to 300 mg, 100 mg to 250 mg, 150 mg to 200 mg, 150 mg to 250 mg, 150 mg to 300 mg, 150 mg to 350 mg, 200 to 250 mg, 200 to 300 mg, 200 to 350 mg, 250 mg to 300 mg, 250 mg to 350 mg, or 300 mg to 350 mg.
  • compositions disclosed herein can be administered to a patient at a frequency of at least three times a day, at least twice a day, at least once a day, at least once every other day, at least once every three days, at least once every four days, at least once every five days, at least once every six days, at least once a week, at least once every two weeks, at least once every three weeks, at least once every four weeks, at least once every two months, at least once every three months, at least once every four months, at least once every five months, at least once every six months, at least once every seven months, at least once every eight months, at least once every nine months, at least once every ten months, at least once every eleven months, or at least once a year.
  • compositions disclosed herein can be administered to a patient at a frequency of no more than once a year, no more than once every eleven months, no more than once every ten months, no more than once every nine months, no more than once every eight months, no more than once every seven months, no more than once every six months, no more than once every five months, no more than once every four months, no more than once every three months, no more than once every two months, no more than once every four weeks, no more than once every three weeks, no more than once every two weeks, no more than once every seven days, no more than once every six days, no more than once every five days, no more than once every four days, no more than once every three days, no more than once every two days, no more than once a day, no more than twice a day, or no more than three times a day.
  • kits comprising the one or more compositions or pharmaceutical formulations disclosed herein.
  • the kits comprise one or more double-stranded nucleic acids described herein.
  • the kits further comprise a cell, or a plurality of cells.
  • the kits further comprise cell media, such as growth media.
  • the kits further comprise additional constituents of the cell media, such as mevalonate, antibiotics, and so forth. The exact nature of the components configured in the inventive kit depends on its intended purpose.
  • Instructions for use may be included in the kit.
  • “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to affect a desired outcome, e.g., producing a double-stranded nucleic acid, isolating a doublestranded nucleic acid, or investigating therapeutic potential of the one or more double-stranded nucleic acids.
  • the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, or other useful paraphernalia as will be readily recognized by those of skill in the art.
  • the materials or components assembled in the kit can be provided to the user stored in any convenient and suitable ways that preserve their operability and utility.
  • the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures.
  • the components are typically contained in suitable packaging material(s).
  • packaging material refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like.
  • the packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment.
  • the packaging materials employed in the kit are those customarily utilized in gene expression assays and in the administration of treatments.
  • a package refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components.
  • a package can be a plastic vial or tube used to contain suitable quantities of the genetically-encoded system, and/or cells.
  • the packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.
  • Circular RNAs can exert biological functions by acting as transcriptional regulators.
  • a typical mixture (10 pl) is composed of 1 pM L-RNA, 2 U Rnl2 and 20 U RiboLock RNase Inhibitor in 1 x T4 Rnl2 buffer (50 mM Tris-HCl (pH 7.5), 2 mM MgC12, 1 mM DL- Dithiothreitol (DTT) and 400 pM adenosine triphosphate (ATP)).
  • RNA samples are pre-treated at 80°C for 3 minutes, and cooled to the reaction temperature at a rate of 6°C/min. Then, the reaction is carried out at 25°C for 2 hours and terminated by heating the mixture at 75°C for 10 minutes.
  • the cyclic structures of reaction products are confirmed by treating them with Exonuclease T (5 U) at 25 °C for 6 hours.
  • a normal universal controlled pore glass is used for RNA oligonucleotide synthesis. All the oligonucleotides are synthesized according to the standard RNA synthesis on ABI394 with 2-tert-butyldimethylsilyl (TBDMS) RNA monomers. The concentrations of all oligonucleotides are measured at 260 nm using a Thermo Scientific NanoDrop 2000 Spectrophotometer. High-performance liquid chromatography (HPLC) is performed on a Waters Alliance e2695 system equipped with a Waters Xbridge OST C18 column (2.5 pm, 10.0 x 50 mm). MS data are obtained with a Waters Xevo G2 Q-TOF spectrometer using ESI. Diethyl pyrocarbonate (DEPC)-treated water is used for all solutions and HPLC purification.
  • DEPC Diethyl pyrocarbonate
  • RNA is dissolved in water to make the final 100 pM RNA solution.
  • the final composition of the reaction mixture to circularize the RNA is as follows: 7 pL RNA solution, 1 pL 10 mM ATP, 1 pL 10 * reaction buffer, and 1 pL T4 RNA ligase (10 U/pL).
  • the solution 10 pL/tube is placed in PCR at 4°C for 12 hr. After mixing these liquids together, the crude products are mixed with 6 x RNA loading buffer (0.25% bromophenol blue and 30% glycerol in DEPC-treated water).
  • the solution (18 pL/well) is loaded into 20% native polyacrylamide PAGE (1 mm thick) gels.
  • the gels are then electrophoresed at 220 V for 50 minutes using 1 x Tris-borate-EDTA (TBE) buffer (pH 8.2).
  • TBE Tris-borate-EDTA
  • the two- side sample lanes of the gel are cut and stained with 1 x SYBR Gold (Invitrogen) and then imaged.
  • the images are printed out according to the same size of the gel, which made it possible to mark the location of the gels without SYBR Gold stain.
  • the gel zones at the marked location are cut, crumbled into tiny particles, and immersed into a 1 x TBE buffer at 37°C overnight.
  • RNA oligonucleotides are characterized using ESI-MS, and double-stranded oligonucleotides ( ⁇ 0.2 nmol) are dissolved in water/acetonitrile (50:50, 20 pL) containing 1% tri ethylamine to make a final concentration of 10 pM. The solutions are then analyzed with a Waters Xevo G2 Q-Tof spectrometer with ESI in the negative ion mode. The molecular weight of the circular RNA is 18 less than the linear RNA due to the condensation reaction.
  • the circular RNAs are dissolved in 1 x PBS buffer to make the 6-pmol stock solution.
  • a 10-pL stock solution is mixed with an equal amount of the complementary RNA with 5'- phosphate modification to form the circular RNA.
  • the RNA is annealed by heating to 85°C for 5 min and is subsequently cooled to room temperature for at least 1 hour for further use.
  • Circular RNA or control linear RNA (3 pM, 5 pL) is incubated at 37°C in an enzyme solution to make a final concentration of 1 pmol/L (15 pL).
  • RNase is used for enzymatic stability in this study, respectively.
  • Aliquots of 3 pL (containing 3 pmol siRNA) are aliquoted at different time points (2, 4, 6, and 8 hours) and immediately frozen in liquid nitrogen and then stored at -80°C until assayed.
  • 1 pL 6 x RNA-loading buffer is added to the aliquots.
  • the samples are run on 10% native polyacrylamide gels in TBE buffer according to the procedure mentioned before. It is hypothesized that circular RNA would be more stable when compared with an otherwise identical RNA molecule that is linear (FIG. 3A).
  • Circular RNAs containing a disulfide bond are chemically synthesized as follows. Passenger strands including a disulfide bond with protective groups at both ends are obtained. Thiol -Modifier C6 S-S Amidite and 3 '-Thiol -Modifier C3 S-S CPG are used at the 5' end and at the 3' end, respectively, to synthesize passenger strands containing a disulfide bond at both ends. To deprotect both terminal protecting groups, 50 mM dithiothreitol (DTT) in Tris-buffer is added to the solution of the passenger strand, and the mixture is incubated at room temperature for 18 hours.
  • DTT dithiothreitol
  • the reaction solution is purified using a NAP-10 column (GE Healthcare) to obtain a passenger strand with a thiol group at both terminals in an aqueous solution.
  • HPLC Shimadzu reversed-phase preparative high performance liquid chromatography
  • Liquid chromatography is performed utilizing a XBridge C18 column (Waters; 5 pm, 4.6 x 250 mm) with buffers A (100 mM triethylammonium acetate [TEAA] in distilled water) and B (acetonitrile). Oligonucleotides are separated using a linear gradient of 5%-50% buffer B from 0 to 22.0 min at a flow rate of 1 mL/min at 60°C. Purified oligonucleotides are collected, desalted through an NAP- 10 column (GE Healthcare), and then concentrated by centrifugation.
  • NAP- 10 column GE Healthcare
  • Non- cleavable circular passenger strands are synthesized.
  • 10-20 Cu wires (FUJIFILM Wako Pure Chemical Corporation) are added to the solution of the passenger strand (50 pM) with NaCl (200 mM).
  • the solution is heated to 80°C for 3 minutes and gently cooled to room temperature.
  • CuAAC copper-catalyzed azide-alkyne cycloaddition
  • the solution is purified using an NAP- 10 column (GE Healthcare), and reversed-phase preparative HPLC is carried out as described previously.
  • purified oligonucleotides are collected, desalted through an NAP- 10 column, and concentrated by centrifugation. The annealing is conducted as described previously.
  • LC-MS liquid chromatography-mass spectrometry
  • Agilent 6120 series single quadrupole LC/MS system Agilent Technologies
  • Liquid chromatography is performed using an ACQUITY BEH C18 column (1.7 pm, 2.1 x 50 mm; Waters) with buffers A (8.6 mM trimethylamine and 100 mM hexafluoroisopropanol in water) and B (methanol).
  • the oligonucleotides are separated with a linear gradient of 10%-90% buffer B from 0 to 18.0 min at a flow rate of 0.3 mL/min at 60°C.
  • SEC sizeexclusion chromatography
  • LC-MS/MS analysis is conducted by ACQUITY UPLC H-class for UPLC, Synapt G2 HDMS (Waters) for MS, and MassLynx V4.1 for data processing.
  • Liquid chromatography is performed using an ACQUITY BEH Cl 8 column (1.7 pm, 2.1 x 50 mm) with buffer A (8.6 mM trimethylamine and 100 mM hexafluoroisopropanol in water) and buffer B (methanol).
  • the oligonucleotides are separated with a linear gradient of 10%-90% of buffer B from 0 to 10 minutes at a flow rate of 0.3 mL/min at 50°C.
  • MaxEnt-1 software is used for mass deconvolution of the MS data. Collisional activation of the selected m/z is performed with a collision energy of 25 eV.
  • HeLa cells (ATCC) and RAW264.7 cells (ATCC) are maintained in RPMI 1640 medium (Life Technologies; A10491-01) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37°C.
  • HepG2 cells (ATCC), Huh-7 cells (JCRB), and L929 cells (RCB) cells are maintained in MEM (11095-080; Gibco), Dulbecco's modified Eagle's medium (DMEM) (08458-16; Nacalai Tesque), and Minimum Essential Medium (MEM) (11095-080; Gibco), respectively, in a humidified atmosphere of 5% CO2 at 37°C.
  • Primary mouse hepatocytes (MSCP10; Life Technologies) are cultured in William's E Medium (A1217601; Life Technologies) supplemented with Primary Hepatocyte Thawing and Plating Supplements (CM3000; ThermoFisher Scientific).
  • RNAiMAX RNAiMAX
  • Opti-MEM 31985-070; Life Technologies
  • oligonucleotides/RNAiMAX solutions are prepared according to the manufacturer's protocol.
  • 20 pL solution is treated in a 96-well plate (167008; Nunc), and then 80 pL cell suspension (10,000 cells/well) is added. After gentle shaking, the plate is maintained in a humidified atmosphere of 5% CO2 at 37°C for 24 hours.
  • mouse primary hepatocytes 20 pL diluted oligonucleotide solution is treated in a 96-well Collagen I Multiwell Microplate (#356702; Corning), followed by the addition of 80 pL cell suspension and incubation in a humidified atmosphere of 5% CO2 at 37° C for 24 hours.
  • RNA is extracted and converted to cDNA using SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo) according to the manufacturer's protocol.
  • the reaction conditions are as follows: 37°C for 15 min, 50°C for 5 min, 98°C for 5 min, and 4°C for 5 minutes.
  • mRNA levels are evaluated by quantitative RT-PCR using TaqMan Gene Expression Master Mix (Life Technologies), the TaqMan probes listed above (Table 2), and QuantStudio 12K flex (Thermo Fisher Scientific) with the reaction conditions of 50°C for 2 min, 95°C for 10 min, and (95°C for 15 s and 60°C for 1 min) x 40 cycles.
  • the relative mRNA expression is quantified using the comparative Ct method.
  • Oligonucleotides are synthesized on a MerMade-12 DNA/RNA synthesizer.
  • Sterling solvents/reagents from Glen Research 500-A controlled pore glass (CPG) solid supports from Prime Synthesis, 2'-deoxy 3'-phosphoramidites from Thermo, and 2'-0Me and 2'-F nucleoside 3'- phosphoramidites from Hongene are all used as received.
  • the 2'-OMe-uridine-5'-bis-POM-(E) vinylphosphonate (VP) 3'-phosphoramidite is synthesized, dissolved to 0.15 M in 85% acetonitrile 15% dimethylformamide (DMF), and coupled using standard conditions on the synthesizer.
  • DMF dimethylformamide
  • GalNAc CPG support is prepared.
  • 5-Bromohexyl phosphorami dite (Glen Research, Cat# 10- 1946) is dissolved to 0.15 M in acetonitrile and coupled using standard conditions on the synthesizer.
  • Low-water content acetonitrile is purchased from EMD Chemicals.
  • a solution of 0.6 M 5-(S- ethylthio)-lH-tetrazole in acetonitrile is used as the activator.
  • the phosphoramidite solutions are 0.15 M in anhydrous acetonitrile with 15% DMF as a co-solvent for 2'-0Me uridine and cytidine.
  • the oxidizing reagent is 0.02 M 12 in THF/pyridine/water.
  • N,N-dimethyl-N'-(3-thioxo-3H- 1,2,4- dithiazol-5-yl)methanimidamide (DDTT), 0.09 M in pyridine, is used as the sulfurizing reagent.
  • the detritylation reagent is 3% di chloroacetic acid (DCA) in dichloromethane (DCM).
  • DCA di chloroacetic acid
  • DCM dichloromethane
  • the solution is removed by filtration, and the CPG beads with the resulting 5'-(5-azidohexyl) solid-supported oligonucleotides are washed with DMF (2 x 10 ml) and dried under a stream of argon.
  • the oligonucleotides are released from the solid support and purified and desalted as described previously.
  • the oligonucleotides are then dissolved in water to a concentration of approximately 10 OD260 units/ml.
  • the click cyclization reaction is optimized to proceed at room temperature. It provided almost quantitative (>92.5%) conversion of the starting linear oligonucleotides within 4 hours at room temperature.
  • a representative room-temperature reaction is performed as follows: linear oligonucleotide (9 mg, 0.9 mL, 10 mg/ml) is suspended in 1.8 ml of methanol and 0.4 ml of water. Solvents are degassed with argon. A freshly prepared mixture of 0.25 ml of 0.1 mM sodium L- ascorbate and 0.25 ml of 20 mM copper sulfate in argon-degassed water is added.
  • Cyclic oligonucleotides are HPLC purified quickly in order to minimize complexation of copper to the phosphate backbone. After ion-exchange HPLC purification and size-exclusion HPLC desalting, sterile filtration, and lyophilization, about 3 mg (30% isolated yield, comparable to regular oligonucleotide synthesis and purification) of cyclic oligonucleotide is obtained with the required high purity needed for in vivo experiments. Cyclic oligonucleotides are then annealed with an antisense strand to form the GalNAc-sciRNAs.
  • Modified oligonucleotide is added at 0.1 mg/ml to a solution of 50 mM Tris-HCl (pH 7.2) and 10 mM MgC12. Stability is evaluated in the presence of snake venom phosphodiesterase (SVPD) (Worthington, Cat# LS003926) and of phosphodiesterase II (PDII) from bovine spleen (Worthington, Cat# LS003 602). SVPD is added to the oligonucleotide at 750 mU/ml. Enzyme is prepared as a stock of 1000 mU/mL aliquoted into 1 ml tubes and stored at -20°C. A new aliquot is used each week. PDII is added at 500 mU/ml. Enzyme is prepared as a stock of 2000 mU/ml, aliquoted into 1 ml tubes and stored at -20°C. A new aliquot is used each week.
  • SVPD snake
  • the sample is injected onto a Dionex DNAPac PA200 column (4 mm x 250 mm) at 30°C and run at a flow rate of 1 ml/min with a gradient of 40-55% Buffer B over 7.5 min.
  • Buffer A is 20 mM sodium phosphate, 15% acetonitrile, pH 11;
  • Buffer B is Buffer A containing 1 M sodium bromide (pH 11).
  • Aliquots are analyzed every hour for 24 hours.
  • the area under the peak corresponding to full-length oligonucleotide is normalized to the area from the 0 hour time point (first injection). First order decay kinetics are assumed in calculation of half-lives.
  • control oligonucleotide is dT19»dT, where dT19 is 2'-deoxythymidine and indicates a single 3'-terminal PS linkage.
  • control oligonucleotide is dT»dT19 with a single 5 '-terminal PS linkage.
  • Half-lives are reported relative to the half-life of the control sequence. Experiments are performed in triplicate.
  • Rat plasma (BioIVT, Cat# RAT00PL38NCXNN) and liver homogenate (BioIVT, custom order) are diluted with a 10x cofactor solution to achieve a final concentration of 1 mM MgC12, 1 mM MnC12 and 2 mM CaC12.
  • the sciRNA is added to 50 pl of the plasma or the liver homogenate to achieve the final concertation of 20 pg/ml.
  • the reaction mixture is incubated with gently shaking at 37°C.
  • the reaction is stopped by adding 450 pl of Clarity OTX lysis-loading buffer (Phenomenex, Cat# ALO-8579) containing internal standard (oligonucleotide U21 at 1 pg/ml final concentration) and frozen at -80°C until analysis. Experiments are performed in triplicate.
  • Oligonucleotide enrichment for LC-MS analysis is performed using Clarity OTX 96- well solid-phase extraction plates as described by Liu et al. (49).
  • the SPE columns are conditioned initially with 1 ml of methanol followed by equilibration with 2 ml of 50 mM ammonium acetate with 2 mM sodium azide in HPLC-grade water.
  • the samples are loaded on to SPE column by applying positive pressure.
  • the columns are then washed five times with 1 ml of 50 mM ammonium acetate in 50/50 (v/v) water and acetonitrile (pH 5.5).
  • oligonucleotides are eluted using elution buffer containing 10 mM EDTA, 100 mM ammonium bicarbonate in 40/10/50 (v/v/v) acetonitrile/tetrahydrofuran/water (pH 8.8).
  • the eluant is dried under nitrogen and resuspended in 120 pl of LC-MS grade water for LC-MS analysis.
  • Relative quantitation and metabolite identification of modified oligonucleotides is performed using high-resolution mass spectrometry on a Thermo Scientific Q Exactive coupled to ion-pairing reverse-phase liquid chromatography (Dionex Ultimate 3000) (LC-HRMS).
  • a Waters X-BridgeBEH C8 XP Column (Cat# 176002554,130 A, 2.5 pm, 2.1 mm x 30 mm, 80°C) is used for the chromatographic separation. The injection volume and flow rates are 30 pl and 1 ml/min, respectively.
  • Mobile phase A consisted of 16 mM triethylamine (Sigma, Cat# 471283), 200 mM l,l,l,3,3,3-hexafhioro-2-propanol (Fisher, Cat# 67-56-1) in LC-MS grade water (Fisher, Cat# 7732-18-5); mobile phase B is 100% methanol (Fisher, Cat# 67-56-1).
  • the gradient started with 1% mobile phase B and progressed to 35% B over 4.3 min, then the column is equilibrated with 1% mobile phase B for 1 min.
  • the mass spectrometer data acquisition is performed in full scan mode with a scan range of 500-3000 m/z at a resolution setting of 35 000.
  • Spray voltage is 2.8 kV.
  • the auxiliary gas temperature and the capillary temperature are set to 300°C.
  • RNA is dissolved in a mixture of 10% 2H2O/90% H2O with 20 mMNaCl and 10 mM sodium phosphate buffer (pH 7). Equimolar ratios of sense and antisense strands are mixed to yield linear or circular duplexes. Final concentrations of duplexes in 600 pl are in the range from 20 to 60 pM. All spectra are acquired at 25°C on an Agilent VNMRS 800 MHz NMR spectrometer equipped with a cold probe. [0213] Circular Dichroism Spectroscopy
  • the circular dichroism (CD) spectra are obtained on a Jasco J-815 spectropolarimeter equipped with a Julaba F25 circulating bath. The sample is allowed to equilibrate for 5 minutes at 10 °C in 1 x PBS at a final duplex concentration of 1.57 pM. The spectrum is an average of 5 scans. Spectra are collected at a rate of 50 nm/min, with a bandwidth of 1 nm and sampling wavelength of 0.2 nm using fused quartz cells (Stama 29-Q-10). The CD spectra are recorded from 350 to 200 nm at 10 °C.
  • siRNA is evaluated in primary mouse hepatocytes by transfection and by free uptake of siRNAs.
  • siRNA (5 pl) at the indicated concentration is mixed with 4.9 pl of Opti-MEM and 0.1 pl of Lipofectamine RNAiMAX (Invitrogen, Cat# 13778-150) per well of a 384- well plate and incubated at room temperature. After 15 min, 40 pl of William's E medium or EMEM medium containing approximately 5 * 103 primary mouse hepatocytes are added to the wells. Cells are incubated for 24 h prior to RNA purification.
  • siRNA (5 pl at the indicated concentration) is mixed with 5 pl of Opti-MEM per well of a 384-well plate. After 15 min, 45 pl of William's E medium or EMEM medium containing ⁇ 5 x 103 cells are added to the wells. Cells are incubated for 48 hours prior to RNA purification.
  • RNA is isolated using an automated protocol on a BioTek-EL406 platform using Dynabeads (Invitrogen, Cat# 61012). To each well is added 50 pl of lysis/binding buffer (Tris- HC1 pH 7.5, LiCl, EDTA pH 8.0, DTT) and 25 pl of lysis/binding buffer containing 3 pl of magnetic beads. The plates are incubated on an electromagnetic shaker for 10 minutes at room temperature, and then the magnetic beads are captured, and the supernatant removed.
  • lysis/binding buffer Tris- HC1 pH 7.5, LiCl, EDTA pH 8.0, DTT
  • RNA is then washed twice with 150 pl/well of Buffer A (Tris-HCl pH 7.5, LiCl, EDTA pH 8.0, DTT) and once with 150 pl/well of Buffer B (Tris-HCl pH 7.5, LiCl, EDTA pH 8.0).
  • Buffer A Tris-HCl pH 7.5, LiCl, EDTA pH 8.0
  • Buffer B Tris-HCl pH 7.5, LiCl, EDTA pH 8.0
  • the beads are then washed with 150 pl of Elution Buffer, re-captured, and the supernatant is collected.
  • cDNA synthesis is performed using an ABI kit (Cat# 4368813).
  • RNA isolated using Dynabeads To the wells of a 384- well plate containing the RNA isolated using Dynabeads is added 10 pl of a master mix containing 1 pl 10x Buffer, 0.4 pl 25 x dNTPs, 1 pl 10x random primers, 0.5 pl reverse transcriptase, 0.5 pl RNase inhibitor and 6.4 pl of nuclease free water.
  • the plates are sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 hours at 37°C.
  • RNA quantification is performed using the Life Technologies Taqman gene expression system with dual labeled probes. Target gene expression is normalized to endogenous Gapdh. Ct values are measured using a Light Cycler 480 (Roche). To calculate relative fold change real time data are analyzed using the AACt method and normalized to assays performed with cells treated with a non-targeting siRNA control.
  • mice All procedures involving mice are conducted by certified laboratory personnel using protocols consistent with local, state, and federal regulations. Experimental protocols are approved by the Institutional Animal Care and Use Committee (IACUC), the Association for Assessment and Accreditation of Laboratory Animal Care International (accreditation number: 001345), and the office of Laboratory Animal Welfare (accreditation number: A4517-01). When deciding on sample numbers for animal studies, the final number required to ensure confidence in the resulting data are determined while utilizing the least number of animals, as required by IACUC guidelines. Female C57BL/6 mice approximately 8 weeks of age are obtained and randomly assigned to each group. Mice are acclimated in-house for 48 hours prior to study start.
  • IACUC Institutional Animal Care and Use Committee
  • TTR For analysis of TTR, serum samples are kept at room temperature for 1 hour and then spun in a microcentrifuge at 21 000 x g at room temperature for 10 minutes. Serum is transferred into 1.5 ml microcentrifuge tubes for storage at -80°C until the time of assay. Serum samples are diluted 1 :4000 and assayed using a commercially available kit from ALPCO specific for detection of mouse prealbumin (Cat# 41-PALMS-E01). Protein concentrations (pg/ml) are determined by comparison to a purified TTR standard and the manufacturer's instructions are followed.
  • Serum collected for the analysis of circulating C5 is kept at room temperature for 15 minutes and then immediately transferred to 4°C prior to spinning in a microcentrifuge at 21 000 x g at room temperature for 10 minutes. Serum is transferred into 1.5 ml microcentrifuge tubes for storage at -80°C until the time of assay.
  • the serum samples are diluted 1 : 5000 for analysis by ELISA.
  • the primary antibody is goat-anti-human C5 (Complement Technologies, Cat# A220), and the secondary antibody is bovine anti-goat IgG-HRP (Jackson ImmunoResearch, Cat# 805- 035-180), which had minimal cross-reactivity to other species. Antibodies are used at 0.8 mg/ml.
  • the assay is developed using a TMB substrate kit (R&D Systems, Cat# DY999), and the reaction is stopped using sulfuric acid prior to measurement.
  • Mice are sacrificed on day 7 post-dose, and livers are snap frozen in liquid nitrogen and ground into powder for further analysis. Total siRNA liver levels are measured by reconstituting liver powder at 10 mg/mL in PBS containing 0.25% Triton-X 100. The tissue suspension is further ground with 5-mm steel grinding balls at 50 cycles/s for 5 minutes in a tissue homogenizer (Qiagen TissueLyser LT) at 4°C. Homogenized samples are then heated at 95°C for 5 minutes, briefly vortexed, and allowed to rest on ice for 5 minutes.
  • tissue homogenizer Qiagen TissueLyser LT
  • siRNA-containing supernatants are transferred to new tubes.
  • the siRNA sense and antisense strand levels are quantified by stem loop reverse transcription followed by Taqman PCR (SL-RT QPCR), and adapted to chemically modified siRNAs.
  • Ago2 -bound siRNA from mouse liver is quantified by preparing liver powder lysates at 100 mg/mL in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% Triton- X 100) supplemented with freshly added protease inhibitors (Sigma- Aldrich, Cat# P8340) at 1 : 100 dilution and 1 mM PMSF (Sigma-Aldrich, Cat# P7626). Total liver lysate (10 mg) is used for each Ago2 immunoprecipitation and control immunoprecipitation.
  • the siRNA not bound to Ago2 is purified with pre-saturated QAE Resin (GE Healthcare, Cat# 17-0200-01) in lysis buffer (16 mg/ml) supplemented with protease inhibitors at 1 : 100 dilution and 1 mM PMSF. Samples are filtered through cellulose acetate filter (Fisher, Cat# P169702) to remove the resin before proceeding with the Ago2 immunoprecipitation.
  • Anti-Ago2 antibody is purchased from Wako Chemicals (Clone No. 2D4).
  • Control mouse IgG is from Santa Cruz Biotechnology (Cat# sc- 2025). Protein GDynabeads (Life Technologies, Cat# 10003D) are used to precipitate antibodies.
  • Ago2- associated antisense strands are eluted by heating (50 pl PBS, 0.25% Triton X-100; 95°C, 5 min) and quantified by SL-RT QPCR as described previously, and adapted to chemically modified siRNAs.
  • Oligonucleotides (1 pmol scale) are synthesized on an ABI 381 A or 394 DNA synthesizer using a cycle involving phosphoramidite chemistry. Detritylation is performed with 2.5% DCA in CH2C12 for 60 seconds. Coupling step: BMT (0.3 M in dry acetonitrile) is used as activator; propynyl and bromohexyl phosphoramidites (0.09 M in CH3CN) are introduced with a 45 second coupling time; commercially available phosphoramidites (0.09M in CH3CN) are introduced with a 30 second coupling time.
  • BMT 0.3 M in dry acetonitrile
  • the capping step is performed with acetic anhydride using commercial solution (Cap A: Ac2O, pyridine, THF 10/10/80 and Cap B: 10% N- methylimidazole in THF) for 15 seconds.
  • Oxidation is performed with commercial solution of iodide (0.1 M 12, THF, pyridine/water 90/ 5/5) for 10 seconds.
  • Azidation of 5-hydroxyl oligonucleotides is performed according to known methods. Azidation from bromohexyl oligonucleotides is performed as follows: a solution of NaN3 (13 mg) and Nal (30 mg) in dry DMF (1.5 mL) is applied on the solid-supported bromohexyl oligonucleotide for 1 hour and 15 minutes at 65 °C. Then the CPG beads bearing the oligonucleotide are washed with DMF (2 1 mL) and CH2-C12 (5 mL) and dried in a desiccator under reduced pressure for 30 minutes.
  • Azido-alkyne oligonucleotide (1 pmol) is added to CuSO4 (0.4 equiv, 0.4 pmol, 13.2 pL of a 20 mM solution in H2O), freshly prepared (from degassed water) sodium ascorbate (2 equiv, 2 pmol, 13.2 pL of a 100 mM solution in H2O), methanol (100 pL), and water (23.6pL).
  • the tube containing the resulting preparation is flushed with argon and sealed.
  • the reaction is placed in a microwave synthesizer Initiator from Biotage set at 100 W with a 30 second premixing time for 1 hour to 1 hour and 30 minutes at 60 °C. Temperature is monitored with an internal infrared probe.
  • the solution is then desalted on NAP 10.
  • PTMG passive transfer Myasthenia Gravis
  • siRNA-C5 2.5 mg/kg
  • siRNA-C5 5 mg/kg
  • saline control by subcutaneous injection.
  • Treatment is administered ten, seven, and three days before and on the day of PTMG induction.
  • Blood is obtained prior to the siRNA dose and immediately prior to PTMG induction.
  • PTMG is induced by administration of the rat anti-mouse muscle AChR monoclonal antibody (mAb) McAb3.
  • McAb3 is an IgG2b isotype known to activate complement. Animals underwent euthanasia 48 hours after PTMG induction. Weakness is graded on a standard 1-4 disease severity scale, with a higher score indicating more severe weakness.
  • Blood, liver, diaphragm, and tibialis anterior are harvested for analysis (FIG. 4A).
  • C5 mRNA expression is quantified by qRT-PCR. Flash-frozen livers are pulverized by a 2000 Geno/Grinder (Spex SamplePrep), and RNA is extracted using the RNeasy Mini Kit according to the manufacturer’s protocol (QIAGEN). Reverse transcription is performed to generate cDNA according to the manufacturer’s protocol (Life Technologies). qPCR is performed on cDNA using a Roche LightCycler 480 instrument with a rat C5 Taqman FAM probe (Life Technologies) and a rat glyceraldehyde-3- phosphate dehydrogenase (GAPDH) Taqman VIC probe (Life Technologies) as the control. The C5 level is normalized to GAPDH, and the percentage of C5 mRNA remaining is calculated relative to the average of untreated or saline- treated rats.
  • GAPDH rat glyceraldehyde-3- phosphate dehydrogenase
  • Rat serum samples are analyzed by semiquantitative western blot.
  • Sera are diluted 1 :20 into 50 mM Tris (pH 7) with 1% SDS. Samples are run on 10% Bis-Tris protein gels (Life Technologies) and transferred onto a PVDF membrane (Bio-Rad).
  • a goat anti-human C5 antibody (Complement Technology) which crosses to rat, is used at a 1 : 1,000 dilution with a secondary fluorophore-conjugated donkey anti-goat antibody (LI-COR) followed by imaging with the LI-COR Odyssey imaging system. Analysis and quantification of the western blot is done using LI-COR’ s Image Studio software.
  • the percent of C5 remaining in the PTMG study is calculated by normalizing rat samples from day 8 to their individual pre-bleed sample. It is hypothesized that in serum, circular RNA would be more stable when compared with an otherwise identical RNA molecule that is linear (FIG. 3B).
  • Mouse C5 protein levels are analyzed by ELISA. Serum is diluted 1 :5,000 into 0.05 M carbonate-bicarbonate buffer (pH 9.6). The standard curve consisted of nine 2-fold dilutions beginning at 1 : 1,000 of wild-type C57BL/6 serum. DBA/2 serum (C5-deficient strain) is included at a 1 :5,000 dilution to calculate the background of the assay. Diluted samples (100 pL) and controls are incubated at 4°C overnight in 96-well polystyrene plates (Costar). Plates are washed and blocked for 2 hours at room temperature with l x PBS and 2% BSA.
  • Mouse cross-reactive goat anti-human C5 antibody (Complement Technology) is added at a 1 : 1,000 dilution and incubated for 1 hour at room temperature.
  • HRP-conjugated anti-goat antibody (Jackson ImmunoResearch) is used at a 1 :500 dilution with a TMB substrate.
  • OD is read at 450 nm, and the relative C5 level is calculated. The percentage of C5 remaining is calculated relative to the pre-dose value, per individual mouse. It is hypothesized that in vivo, circular RNA would be more stable when compared with an otherwise identical RNA molecule that is linear (FIG. 4B).
  • siRNA-C5 formulations are administered subcutaneously to human subjects with complement system disorders. After one month, samples are taken from the subjects to measure C5 levels. Additional samples are taken to measure toxicity, siRNA-C5 half-life, and immunogenicity.
  • Example 8 Formation of Circular RNAs
  • Circular RNAs exert biological functions by acting as transcriptional regulators.
  • a typical mixture (10 pl) was composed of 1 pM L-RNA, 2 URnl2 and 20 U RiboLock RNase Inhibitor in 1 * T4 Rnll or Rnl2 buffer (50 mM Tris-HCl (pH 7.5), 2 mM MgC12, 1 mM DL-Dithiothreitol (DTT) and 400 pM adenosine triphosphate (ATP)).
  • RNA samples were pretreated at 80°C for 3 minutes, and cooled to the reaction temperature at a rate of 6°C/min. Then, the reaction was carried out at 25°C for 2 hours and terminated by heating the mixture at 75°C for 10 minutes.
  • the cyclic structures of reaction products were confirmed by treating them with Exonuclease T (5 U) at 25°C for 6 hours.
  • Circular RNA or control linear RNA (3 pM, 5 pL) was incubated at 37°C in an enzyme solution to make a final concentration of 1 pmol/L (15 pL).
  • RNase was used for enzymatic stability in this study, respectively.
  • Aliquots of 3 pL (containing 3 pmol siRNA) were aliquoted at different time points (2, 4, 6, and 8 hours) and immediately frozen in liquid nitrogen and then stored at -80°C until assayed.
  • 1 pL 6 * RNA-loading buffer was added to the aliquots.
  • the samples were run on 10% native polyacrylamide gels in TBE buffer according to the procedure mentioned before (FIG. 7B).
  • Circular RNAs exert biological functions by acting as transcriptional regulators.
  • a typical reaction is composed of 10-50 pM linear sense strand, 0.5-2 U RNA ligase 2, RNA ligase 1, T4 DNA ligase, thermostable phage DNA/RNA ligase, or other ligases optimized for loop formation and 20 U RiboLock RNase Inhibitor in 1 * T4 ligase 1 or 2 buffer (50 mM Tris-HCl (pH 7.5), 2-10 mM MgC12 divalent cation, 1 mM DL-Dithiothreitol (DTT) and 400-1000 pl mM adenosine triphosphate (ATP), 10% PEG 8000 and or, 1-3M betaine).
  • T4 ligase 1 or 2 buffer 50 mM Tris-HCl (pH 7.5), 2-10 mM MgC12 divalent cation, 1 mM DL-Dithiothreitol (DTT) and 400-1000 pl mM adenosine triphosphat
  • RNA sense strands were pre-treated at 65°C for 3 minutes, and gradually cooled to the reaction temperature at a rate of 6°C/minon the thermal cycler machine (0. loC/sec). Ligation reaction was carried out at 25°C for 4 hours. The cyclic structures of reaction products were confirmed by Exonuclease T and or Rnase R treatment (0.2-1 Unit/pL). Reactions were terminated by addition of EDTA pH 8 at 50 mM. Looped sense RNA products were separated on a 12% PAGE denaturing urea gel, visualized by GelRed (Biotium) stain. Bands were excised by UV shadowing. [0248] Synthesis and Purification of Circular RNAs
  • looped RNA was mixed with 2 x RNA loading buffer (0.25% bromophenol blue and 95% formamide in DEPC-treated water for denaturing gels and 10% glycerol, 0.05% bromophenol blue for native gels).
  • 2 x RNA loading buffer 0.25% bromophenol blue and 95% formamide in DEPC-treated water for denaturing gels and 10% glycerol, 0.05% bromophenol blue for native gels.
  • the sample was loaded on a 12% native polyacrylamide PAGE (1 mm thick) gel.
  • the gels were then electrophoresed at 150-200 V for 50 minutes using 1 x Tris-borate-EDTA (TBE) buffer (pH 8.2).
  • TBE Tris-borate-EDTA
  • TBE Tris-borate-EDTA
  • each gel was soaked in 1 x GelRed (Biotium) solution for 30 minutes followed by image acquisition using an iBright imaging system (Thermo Fisher Scientific). (FIG. 7A
  • Looped RNA or control linear RNA (1-3 pL) was incubated at 37°C in a lx reaction buffer and 1U of RNase R (BioVision) for 1 hour. The samples were run on 120% native or urea polyacrylamide gels in TBE buffer according to the procedure mentioned before. (FIG. 7B).
  • HepG2 cells were maintained in EMEM (30-2003, ATCC) supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin in a humidified incubator (5% CO2 at 37°C).
  • Primary mouse hepatocytes (B129-7224F; Cell Biologies) were cultured in Complete hepatocyte medium kit (M1265, Cell Biologies).
  • siRNA and Lipofectamine RNAiMAX (13778-075; Life Technologies) (0.3 pL/well for 96-well plates) were added to 25 pL of Opti-MEM (31985-070; Life Technologies) separately and mixed according to the manufacturer's protocol. 50 pL of this mixture was added to a 96-well plate (167008; Nunc), and 50 pL cell suspension (5-10,000 cells/well) was added to the lipofectamine siRNA mixture. After gentle shaking, the plate was maintained in a humidified atmosphere of 5% CO2 at 37°C for 24 hours.
  • RNA levels were determined by either a single-step or 2-step RT qPCR assay.
  • Reverse transcription with the TOYOBO kit was carried out as follows: 37°C for 15 min, 50°C for 5 min, 98°C for 5 min, and 4°C for 5 minutes, and C5, ICAM1 mRNA levels were measured by quantitative RT-PCR using TaqMan Gene Expression Master Mix (IDT) and or SYBR green dye detection on a QuantStudio 6 or 7 real time PCR machine, for x 40 cycles.
  • the relative mRNA expression was quantified using the comparative Ct method and or serial dilution and standard curve.
  • RPL13A and or RPB1 mRNA served as housekeeping genes for quantification of target mRNAs.
  • Modified oligonucleotide was added at 0.1 mg/ml to a solution of 50 mM Tris-HCl (pH 7.2) and 10 mM MgC12. Stability was evaluated in the presence of snake venom phosphodiesterase I (PDI) (Sigma P4506). PDI was added to the oligonucleotide at 750 mU/ml. Enzyme was prepared as a stock of 1000 mU/mL. PDI was incubated with sense strands at a final concentration of 0.5 mU/mL and incubation was carried out for ’A, 1, 2, 3, 4 and 6 hours.
  • PDI snake venom phosphodiesterase I
  • Rat serum (10-50%) (Sigma R9759) were diluted in PBS in a final concentration of 1 mM MgC12, 1 mM MnC12 and 2 mM CaC12.
  • the siRNA was added to 20 pl of plasma and incubated with gentle shaking at 37°C. At each time point, reactions were stopped by EDTA lOmM and/or incubation at 95°C for 5-10 min followed by treatment with proteinase K at 50°C for 30 min and loading on urea PAGE gel for imaging.
  • mice All procedures involving mice were conducted by certified laboratory personnel using protocols consistent with local, state, and federal regulations. Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC), the Association for Assessment and Accreditation of Laboratory Animal Care International (accreditation number: 001345). Female BALB/c mice approximately 8 weeks of age were obtained from Taconic Biosciences and 5 mice were randomly assigned to each group. Mice were acclimated in-house for 5 days prior to study start. [0263] Animals were dosed intravenously (tail-vein injection) at 10 mL/kg with, siRNA, or with invivofectamine PBS saline control. Doses used in this study were 1.5 mg/kg.
  • test compounds were diluted into phosphate buffered saline (PBS, pH 7.4) and mixed with invivofectamine according to manufacturer's protocol. All solutions were stored at 4°C until time of injection. Animals were sacrificed at either 5- or 7-days post dose. Livers were harvested and snap frozen for analysis. Plasma was collected utilizing the retro-orbital eye bleed procedure 24 hours post the final dose in accordance with the IACUC approved protocol. The sample was collected in Becton Dickinson serum separator tubes (Fisher Scientific, Cat# BD365967).
  • oligonucleotide stability assay was performed to evaluate the stability of circular siRNA against complement C5 (C5) mRNA as compared with a linear siRNA against C5 mRNA.
  • concentration of an exonuclease (Crotalus atrox Phosphodiesterase I; Sigma: P4506-100MG) was adjusted by reaction buffer (Tris-HCl buffer) to 2mU/mL.
  • the diluted exonuclease solution was mixed with eleven pmol of linear, or circular, siRNA C5 sense strand for the indicated time (see FIG. 9) at 37°C.
  • lysis/binding buffer Tris- HC1 pH 7.5, LiCl, EDTA pH 8.0, DTT
  • the beadbound RNA was then washed twice with 150 pl/well of Buffer A (Tris-HCl pH 7.5, LiCl, EDTA pH 8.0, DTT) and once with 150 pl/well of Buffer B (Tris-HCl pH 7.5, LiCl, EDTA pH 8.0). The beads were then washed with 150 pl of Elution Buffer, re-captured, and the supernatant was collected.
  • Buffer A Tris-HCl pH 7.5, LiCl, EDTA pH 8.0, DTT
  • Buffer B Tris-HCl pH 7.5, LiCl, EDTA pH 8.0
  • RNA quantification was performed using probe-based (C5) and SYBR green-based (ICAM1) target gene mRNA levels (FIG. 11) were normalized to endogenous RPL13j A or RPB 1 (housekeeping gene).
  • Ct values were measured using a QuantStudio 7 (Applied Biosystems). To calculate relative fold change real time data were analyzed using the AACt and/or standard curve method normalized to assays performed with cells treated with a non-targeting siRNA control.
  • the C5 and ICAM-targeting siRNAs with linear, and circular, sense strands had similar potency demonstrating that circular siRNAs can have equivalent gene silencing activity to linear siRNAs at 10 nM (high) concentration of siRNA.
  • FIG. 10 demonstrates that at 10 nM (high) concentration of siRNA such potency is independent of the size of the siRNA.
  • Gene expression analysis was performed on a human liver cancer cell line (HepG2). Briefly, lOnM of enzymatically cyclized 21-mer (or 27-mer) sense strands C5 siRNA, or linear 21-mer sense strands C5 siRNA. 1.5 pl siRNA (10 pM stock) was mixed with 4 pl lipofectamine RNAiMAX in 500 pl of Opti-MEM per well of a 6-well plate. After 15 minutes, 1 mL of medium containing ⁇ 2.5 x 10 5 HepG2 cells were added to the wells. Cells were incubated for 48 hours prior to RNA purification.
  • RNA libraries were sequenced and differentially expressed genes from RNA-seq data were identified by pairwise analysis with DESeq2 at an adjusted p value of ⁇ 0.01 and a fold change (FC) of -1.5 ⁇ FC>1.5.
  • FC fold change
  • Example 17 siRNA Administration in vivo and C5 Protein Level Measurement
  • FIG. 19A shows the levels of plasma C5 protein in mice treated with the vehicle (circle), open C5 siRNA (square) and looped C5 siRNA (triangle).

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Abstract

L'invention concerne des molécules d'acides nucléiques non codants bicaténaires synthétiques avec une base de brin sens appariée à un brin antisens qui sont utiles pour des applications thérapeutiques. Le brin antisens est complémentaire d'une séquence d'acide nucléique cible pour moduler l'expression d'un gène d'intérêt qui peut être associé à une maladie particulière ou à une affection particulière. Le brin sens a une extrémité 5' et une extrémité 3' qui peuvent être couplées l'une à l'autre directement ou indirectement. Les molécules d'acides nucléiques non codants bicaténaires synthétiques peuvent également être modifiées pour comprendre une fraction de ciblage qui dirige la molécule d'acide nucléique non codant double brin vers une cellule ou un tissu cible in vivo ou ex vivo. Les molécules d'acides nucléiques non codants bicaténaires peuvent également être chimiquement modifiées.
PCT/US2024/020471 2023-03-17 2024-03-18 Nucléotides non codants chimiquement modifiés Pending WO2024196898A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010084371A1 (fr) * 2009-01-26 2010-07-29 Mitoprod Nouvelles molécules d'arn interférent circulaire
US20150299702A1 (en) * 2012-11-30 2015-10-22 Aarhus Universitet Circular rna for inhibition of microrna
WO2022011214A1 (fr) * 2020-07-10 2022-01-13 Alnylam Pharmaceuticals, Inc. Parni circulaires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010084371A1 (fr) * 2009-01-26 2010-07-29 Mitoprod Nouvelles molécules d'arn interférent circulaire
US20150299702A1 (en) * 2012-11-30 2015-10-22 Aarhus Universitet Circular rna for inhibition of microrna
WO2022011214A1 (fr) * 2020-07-10 2022-01-13 Alnylam Pharmaceuticals, Inc. Parni circulaires

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