Chemically modified nucleic acids

Abstract

Provided herein are synthetic monovalent or polyvalent single-stranded non-coding nucleic acid molecules comprising a non-coding molecule complementary to the coding or regulatory region of a target gene for regulating gene expression or splicing, wherein the 5' and 3' ends of the single-stranded nucleic acid are joined to create a structure of a single-stranded non-coding nucleic acid molecule without free ends.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Application No. 63 / 490,978, filed Mar. 17, 2023, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Single-stranded nucleic acid molecules (e.g., antisense oligonucleotides (ASOs)) are versatile oligonucleotide therapeutics designed to target nucleic acid molecules such as messenger ribonucleic acid (mRNA), mRNA precursors, and / or non-coding regions of RNA (e.g., intron regions and regulatory regions) via Watson-Crick base pairing. Broadly speaking, the mechanisms of action of these therapeutics fall into two distinct categories: steric blocking ASOs are single-stranded oligonucleotides complementary to the target sequence, mediated solely by occupation, and can modulate splicing (exon inclusion or skipping), RNA processing, and mRNA translation. A second class of ASOs act as guides for enzymes that can degrade mRNA (e.g., RNA-degrading enzymes H or Ago2), edit target mRNA (e.g., ADAR), or edit DNA sequences (e.g., Cas9). The latter two are often classified as guide RNAs rather than ASOs due to their longer size. Occupied ASOs can upregulate or downregulate the expression of target mRNA through diverse mechanisms such as splicing regulation, alteration of nonsense-mediated degradation (NMD), inhibition of miRNA function, and regulation of upstream ORF (uORF) and translation inhibitory element (TIE) utilization (all activated), or they can lead to downregulation of targets by inhibiting the translation mechanism or altering polyadenylation. Splicing oligonucleotides (SSOs) can also regulate the splicing (exon inclusion or exclusion / skipping) of mRNA precursors by binding to exons, introns, or exon / intron junctions. Steric blockade by ASOs can also regulate gene expression by binding to non-protein-coding regions or gene regulatory regions (e.g., UTR regions), thereby regulating the expression of gene expression products from target nucleic acid molecules. There are numerous applications of single-stranded nucleotides, including, but not limited to, their use as therapeutic agents to treat various diseases or disorders. These single-stranded nucleic acid ASOs can be DNA, RNA, chimeric gapmer (RNA:DNA hybrid) based molecules and / or chemically modified variants thereof. [Overview of the project]

[0003] Embodiments disclosed herein provide a synthetic single-stranded nucleic acid molecule comprising a single-stranded non-coding nucleic acid molecule complementary to the coding or regulatory region of a target gene (RNA or DNA) for regulating the expression from the gene, wherein the 5' and 3' ends of the single-stranded nucleic acid are reversibly or irreversibly ligated to generate a single-stranded nucleic acid molecule structure without free ends. In some embodiments, the single-stranded non-coding nucleic acid molecule comprises a nucleic acid sequence complementary to the target nucleic acid sequence.

[0004] Embodiments disclosed herein provide synthetic nucleic acid molecules comprising a single-stranded non-coding ribonucleic acid molecule, the single-stranded non-coding ribonucleic acid molecule comprising: (a) a target-binding sequence complementary to the coding or regulatory region of a target nucleic acid sequence; (b) 5' and 3' ends of a single-stranded nucleic acid molecule reversibly or irreversibly ligated to form a single-stranded nucleic acid molecule structure without free ends; and (c) one or more adapters configured to enhance the specificity of target binding between the target-binding sequence of the single-stranded non-coding ribonucleic acid molecule and the target nucleic acid sequence compared to an otherwise identical single-stranded non-coding ribonucleic acid molecule without one of the one or more adapters. In some embodiments, the target nucleic acid sequence is a ribonucleic acid (RNA) sequence, a deoxyribonucleic acid (DNA) sequence, or a DNA / RNA hybrid sequence. In some embodiments, the target nucleic acid sequence encodes one of the targets in Table 1. In some embodiments, the target nucleic acid sequence is a gene expression product from a gene encoding complement factor B (CFB), complement factor C5 (C5), or ApoB. In some embodiments, the target RNA sequence includes a messenger RNA (mRNA) sequence. In some embodiments, the target binding sequence of the single-stranded non-coding nucleic acid molecule is complementary to an exon, intron, exon / intron junction, intron / exon junction, untranslated region, or regulatory region of the target nucleic acid sequence. In some embodiments, the single-stranded non-coding nucleic acid molecule is configured to regulate the expression level of a gene product expressed from the target nucleic acid sequence. In some embodiments, the single-stranded non-coding nucleic acid molecule is configured to regulate the outcome of a splicing event from the target nucleic acid sequence. In some embodiments, the regulation is an increase in expression level. In some embodiments, the regulation is a decrease in expression level. In some embodiments, the single-stranded non-coding nucleic acid molecule is a chemically modified nucleic acid molecule. In some embodiments, the single-stranded non-coding nucleic acid molecule contains about 16 to about 100 consecutive nucleotides. In some embodiments, the 5' end of the single-stranded non-coding nucleic acid molecule is ligated to the 3' end of the single-stranded non-coding nucleic acid molecule by a chemical linker. In some embodiments, the chemical linker is substantially cleavable under intracellular conditions.In some embodiments, the chemical linker includes a disulfide bond, a photocleavable linker, a diazo linker, an acid-unstable linker, a peptide linker, a nucleotide linker, a glucuronide group, an azide-alkyne linker, an aldehyde-oxamine linker, a phosphorothioate-tosylated linker, a phosphate-activator-mediated phosphate-hydroxyl bond, or a metal chelate ligation linker. In some embodiments, the chemical linker is substantially cleavable by enzymes under intracellular conditions. In some embodiments, the chemical linker is substantially cleavable independently under intracellular conditions. In some embodiments, the chemical linker is not substantially cleavable under intracellular conditions. In some embodiments, the chemical linker includes a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer. In some embodiments, the single-stranded non-coding nucleic acid molecule includes a targeting moiety, where the targeting moiety is specific to a target cell or target tissue. In some embodiments, the target cells are hepatocytes, cardiac cells, neurons, muscle cells, blood cells, photoreceptor cells, pancreatic cells, or stem cells. In some embodiments, the target tissue is liver tissue, cardiac tissue, brain tissue, muscle tissue, nerve tissue, epithelial tissue, connective tissue, eye, or pancreatic tissue. In some embodiments, the targeting moiety comprises polypeptides, macrocyclic peptides, RNA molecules, lipophilic moieties, nanoparticles, or small molecules. In some embodiments, the polypeptide comprises antibodies, single-domain antibodies, miniproteins, or antigen-binding fragments thereof. In some embodiments, the polypeptide comprises glucagon-like peptide 1 receptor (GLP1R) agonists, 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). In some embodiments, the RNA molecule includes an aptamer, ribozyme, hairpin RNA, siRNA, or miRNA.In some embodiments, the lipophilic moiety includes lipids, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, vomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In some embodiments, the low molecular weight includes a sugar moiety. In some embodiments, the low molecular weight includes an amino sugar. In some embodiments, the amino sugar is N-acetylgalactosamine (GalNAc). In some embodiments, the targeting moiety is specific to an antigen or receptor in a target cell or target tissue. In some embodiments, the receptor includes an asialoglycoprotein receptor (ASGPR). In some embodiments, the single-stranded non-coding nucleic acid molecule includes one or more nucleotides with modifications. In some embodiments, the modifications include chemical modifications. In some embodiments, the chemical modifications include modifications of sugars, phosphate backbones, or nucleic acid bases. In some embodiments, nucleotide modifications include 2'-O-Me, 2'-F, 2'-MOE, N(6)-methyladenosine, 5-methylcytidine, 5-methyluridine (ribothymidine), ribose modifications by cross-linked nucleic acids, or nucleotides with alternative chemical structures. In some embodiments, ribose modifications by cross-linked nucleic acids are locked nucleic acids (LNA), ethylene-cross-linked nucleic acids (ENA), or restricted ethyl-cross-linked nucleic acids (cEt). In some embodiments, nucleotides with alternative chemical structures are phosphorodiamidate morpholino oligonucleotides (PMO), thiophosphoamides, peptide nucleic acids (PNA), tricycloDNA (tcDNA), unlocked nucleic acids (UNA), or glycol nucleic acids (GNA). In some embodiments, modifications to the phosphate backbone include phosphodiesters, phosphorothioate isomers (Sp or Rp), phosphoryl DMI amide diester isomers, phosphorodithioates, methylphosphonates, 5'-phosphorothioates, peptide nucleic acids, 5'-(E)-vinylphosphonates, or 5'-methylphosphonates.In some embodiments, ligation of the 5' and 3' ends of a single-stranded nucleic acid allows for a further reduction in the amount of chemically modified nucleotides associated with adverse medical side effects compared to an unligated control single-stranded nucleic acid. In some embodiments, adverse medical side effects are selected from the group consisting of thrombocytopenia, thrombocytopenia, heart rate disturbance, increased blood pressure, or increased cardiac output. In some embodiments, one or more adapters include a peptide adapter or a polypeptide adapter, or a nucleotide adapter or an oligonucleotide adapter. In some embodiments, the peptide adapter or polypeptide adapter is an antibody adapter. In some embodiments, the oligonucleotide adapter has a length containing about 10 to about 25 consecutive nucleotides. In some embodiments, the length contains about 15 to about 20 consecutive nucleotides. In some embodiments, the synthetic nucleic acid molecule is isolated. In some embodiments, the synthetic nucleic acid molecule is purified and isolated. In some embodiments, one or more adapters include sugars, lipids, peptides, antibodies, nucleotide sequences, aptamers, or combinations thereof. In some embodiments, the pharmaceutical formulation consists of a synthetic nucleic acid molecule and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the cells contain synthetic nucleic acid molecules.

[0005] The embodiments disclosed herein provide synthetic nucleic acid molecules comprising a single-stranded non-coding ribonucleic acid molecule complementary to the coding or regulatory region of a target nucleic acid sequence, wherein the 5' and 3' ends of the single-stranded nucleic acid are reversibly or irreversibly ligated to produce a single-stranded nucleic acid molecule structure without free ends. In some embodiments, the target nucleic acid sequence is a ribonucleic acid (RNA) sequence, a deoxyribonucleic acid (DNA) sequence, or a DNA / RNA hybrid sequence or chemically modified variant thereof. In some embodiments, the target nucleic acid sequence is a gene expression product of a gene listed in Table 1. In some embodiments, the target nucleic acid sequence is a gene expression product from a gene encoding complement factor B (CFB), complement factor C5 (C5), or ApoB. In some embodiments, the RNA sequence is a messenger RNA (mRNA) sequence. In some embodiments, the nucleic acid sequence of the single-stranded non-coding nucleic acid molecule is complementary to the exons, introns, exon / intron junctions, intron / exon junctions, untranslated regions, or regulatory regions of the target nucleic acid sequence. In some embodiments, single-stranded non-coding nucleic acid molecules are configured to regulate the outcome of a gene product expressed from a target nucleic acid sequence. In some embodiments, single-stranded non-coding nucleic acid molecules are configured to regulate the expression level of a splicing event from a target nucleic acid sequence. In some embodiments, the regulation is an increase in the expression level. In some embodiments, the regulation is a decrease in the expression level. In some embodiments, the single-stranded non-coding nucleic acid molecule is a chemically modified nucleic acid molecule. In some embodiments, the single-stranded non-coding nucleic acid molecule contains about 16 to about 100 consecutive nucleotides. In some embodiments, the 5' end of the single-stranded non-coding nucleic acid molecule is linked to the 3' end of the single-stranded non-coding nucleic acid molecule by a linker. In some embodiments, the linker is a nucleotide linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a chemical linker. In some embodiments, the chemical linker is substantially cleavable under intracellular conditions.In some embodiments, the chemical linker includes a disulfide bond, a photocleavable linker, a diazo linker, an acid-unstable linker, a peptide linker, a nucleotide linker, a glucuronide group, an azide-alkyne linker, an aldehyde-oxamine linker, a phosphorothioate-tosylated linker, a phosphate-activator-mediated phosphate-hydroxyl bond, or a metal chelate ligation linker. In some embodiments, the chemical linker is substantially cleavable by enzymes under intracellular conditions. In some embodiments, the chemical linker is substantially cleavable independently under intracellular conditions. In some embodiments, the chemical linker is not substantially cleavable under intracellular conditions. In some embodiments, the chemical linker includes a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer. In some embodiments, the single-stranded non-coding nucleic acid molecule includes a targeting moiety, where the targeting moiety is specific to a target cell or target tissue. In some embodiments, the target cells are hepatocytes, cardiac cells, neurons, muscle cells, blood cells, photoreceptor cells, pancreatic cells, or stem cells. In some embodiments, the target tissue is liver tissue, cardiac tissue, brain tissue, muscle tissue, nerve tissue, epithelial tissue, connective tissue, eye, or pancreatic tissue. In some embodiments, the targeting moiety comprises polypeptides, macrocyclic peptides, RNA molecules, lipophilic moieties, nanoparticles, or small molecules. In some embodiments, the polypeptide comprises antibodies, single-domain antibodies, miniproteins, or antigen-binding fragments thereof. In some embodiments, the polypeptide comprises glucagon-like peptide 1 receptor (GLP1R) agonists, 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). In some embodiments, the RNA molecule includes an aptamer, ribozyme, hairpin RNA, siRNA, or miRNA.In some embodiments, the lipophilic moiety includes lipids, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, vomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In some embodiments, the low molecular weight includes a sugar moiety. In some embodiments, the low molecular weight includes an amino sugar. In some embodiments, the amino sugar is N-acetylgalactosamine (GalNAc). In some embodiments, the targeting moiety is specific to an antigen or receptor in a target cell or target tissue. In some embodiments, the receptor includes an asialoglycoprotein receptor (ASGPR). In some embodiments, the single-stranded non-coding nucleic acid molecule includes one or more nucleotides with modifications. In some embodiments, the modifications include chemical modifications. In some embodiments, the chemical modifications include modifications of sugars, phosphate backbones, or nucleic acid bases. In some embodiments, nucleotide modifications include 2'-O-Me, 2'-F, 2'-MOE, N(6)-methyladenosine, 5-methylcytidine, 5-methyluridine (ribothymidine), ribose modifications by cross-linked nucleic acids, or nucleotides with alternative chemical structures. In some embodiments, ribose modifications by cross-linked nucleic acids are locked nucleic acids (LNA), ethylene-cross-linked nucleic acids (ENA), or restricted ethyl-cross-linked nucleic acids (cEt). In some embodiments, nucleotides with alternative chemical structures are phosphorodiamidate morpholino oligonucleotides (PMO), thiophosphoamides, peptide nucleic acids (PNA), tricycloDNA (tcDNA), unlocked nucleic acids (UNA), or glycol nucleic acids (GNA). In some embodiments, modifications to the phosphate backbone include phosphodiesters, phosphorothioate isomers (Sp or Rp), phosphoryl DMI amide diester isomers, phosphorodithioates, methylphosphonates, 5'-phosphorothioates, peptide nucleic acids, 5'-(E)-vinylphosphonates, or 5'-methylphosphonates.In some embodiments, ligation of the 5' and 3' ends of single-stranded nucleic acids allows for a further reduction in the amount of chemically modified nucleotides associated with adverse medical side effects compared to an unligated control single-stranded nucleic acid. In some embodiments, adverse medical side effects are selected from the group consisting of thrombocytopenia, thrombocytopenia, heart rate disturbance, increased blood pressure, or increased cardiac output. In some embodiments, the synthetic nucleic acid molecule is isolated. In some embodiments, the synthetic nucleic acid molecule is purified and isolated. In some embodiments, the pharmaceutical formulation comprises a synthetic nucleic acid molecule, such as those disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the cell comprises a synthetic nucleic acid molecule, such as those disclosed herein.

[0006] Embodiments disclosed herein provide a method for delivering a synthetic nucleic acid molecule to a subject, the method comprising administering a single-stranded non-coding nucleic acid molecule complementary to the coding and / or regulatory regions of a target gene to the subject for the purpose of regulating the expression from the target gene, wherein the 5' and 3' ends of the single-stranded non-coding nucleic acid molecule are ligated to create a structure without free ends, and the single-stranded non-coding nucleic acid molecule has an in vivo half-life of about 10 hours or more, as measured using a quantitative nucleic acid detection assay. Embodiments disclosed herein provide a method for delivering a synthetic nucleic acid molecule to a subject; the method comprises administering a single-stranded non-coding ribonucleic acid molecule to a subject, the single-stranded non-coding ribonucleic acid molecule comprising (a) a target-binding sequence complementary to the coding or regulatory region of a target nucleic acid sequence; (b) 5' and 3' ends of a single-stranded nucleic acid that are reversibly or irreversibly ligated to form a single-stranded nucleic acid molecule structure without free ends; and (c) one or more adapters configured to enhance the specificity of target binding between the target-binding sequence of the single-stranded non-coding ribonucleic acid molecule and the target nucleic acid sequence compared to an otherwise identical single-stranded non-coding ribonucleic acid molecule without one of the one or more adapters, wherein the single-stranded non-coding ribonucleic acid molecule has an in vivo half-life of about 10 hours or more, as measured using a quantitative nucleic acid detection assay. In some embodiments, the administration includes subcutaneous, intravenous, intravitreal, or intrathecal administration of the single-stranded non-coding ribonucleic acid molecule to the subject. In some embodiments, the administration is performed at a frequency of less than once a month.

[0007] Embodiments disclosed herein provide a method for activating the transcription of a gene of interest; the method comprises (a) providing a single-stranded non-coding nucleic acid molecule complementary to a coding region, intron region, or regulatory region of a target gene, wherein the 5' and 3' ends of the single-stranded non-coding nucleic acid molecule are ligated to produce a single-stranded nucleic acid molecule structure without free ends; and (b) introducing the single-stranded non-coding nucleic acid molecule into a sample containing the gene of interest under conditions sufficient to activate the transcription of the gene of interest. Embodiments disclosed herein provide a method for activating the transcription of a gene of interest; the method comprises (a) a target binding sequence complementary to the coding or regulatory region of a target nucleic acid sequence; (b) 5' and 3' ends of a single-stranded nucleic acid reversibly or irreversibly ligated to form a single-stranded nucleic acid molecule structure without free ends; (c) one or more adapters configured to enhance the specificity of target binding between the target binding sequence of the single-stranded non-coding ribonucleic acid molecule and the target nucleic acid sequence compared to an otherwise identical single-stranded non-coding ribonucleic acid molecule without one of the one or more adapters; and introducing the single-stranded non-coding nucleic acid molecule into a sample containing the gene of interest under conditions sufficient to activate the transcription of the gene of interest. In some embodiments, the single-stranded non-coding nucleic acid molecule comprises an antisense strand including the 5' end of an antisense strand ligated to the 3' end of the antisense strand. In some embodiments, the antisense strand comprises a nucleic acid sequence complementary to the nucleic acid sequence of the target RNA, wherein the target RNA encodes one of the targets in Table 1. In some embodiments, the target RNA comprises mRNA. In some embodiments, the nucleic acid sequence of the antisense strand is complementary to the untranslated region, intron, exon, intron / exon junction, exon / intron junction, promoter, enhancer, or regulatory element of the target RNA. In some embodiments, the single-stranded non-coding nucleic acid molecule contains about 19 to about 27 consecutive nucleotides. In some embodiments, the single-stranded non-coding nucleic acid molecule is an RNA molecule or a chemically modified nucleic acid. In some embodiments, the single-stranded non-coding nucleic acid molecule contains about 16 to about 100 consecutive nucleotides.In some embodiments, the 5' end of a single-stranded non-coding nucleic acid molecule is ligated to the 3' end of the single-stranded non-coding nucleic acid molecule by a linker. In some embodiments, the linker is a nucleotide adapter. In some embodiments, the linker is a peptide adapter. In some embodiments, the linker is a chemical linker. In some embodiments, the chemical linker is substantially cleavable under intracellular conditions. In some embodiments, the chemical linker includes a disulfide bond, a photocleavable linker, a diazo linker, an acid-unstable linker, a peptide linker, a nucleotide linker, a glucuronide group, an azide-alkyne linker, an aldehyde-oxamine linker, a phosphorothioate-tosylated linker, a phosphate-activator-mediated phosphate-hydroxyl bond, or a metal chelate ligation linker. In some embodiments, the chemical linker is substantially cleavable enzymatically under intracellular conditions. In some embodiments, the chemical linker is substantially cleavable independently under intracellular conditions. In some embodiments, the chemical linker is not substantially 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, the 5' end of the antisense chain comprises modifications including 5'-(E)-vinylphosphonate (5'-VP), 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), or 6'(phosphonoxy-butyl-sulfide)purine (6-PBuS-purine). In some embodiments, the single-stranded non-coding nucleic acid molecule comprises a targeting moiety, where the targeting moiety is specific to target cells or target tissues. In some embodiments, the target cells are hepatocytes, cardiac cells, neurons, muscle cells, blood cells, photoreceptor cells, pancreatic cells, or stem cells. In some embodiments, the target tissue is liver tissue, cardiac tissue, brain tissue, muscle tissue, nerve tissue, epithelial tissue, connective tissue, eye, or pancreatic tissue. In some embodiments, the targeting moiety comprises a polypeptide, a macrocyclic peptide, an RNA molecule, a lipophilic moiety, or a small molecule.In some embodiments, the polypeptide comprises an antibody, a single-domain antibody, a miniprotein, or an antigen-binding fragment thereof. In some embodiments, the polypeptide comprises a glucagon-like peptide 1 receptor (GLP1R) agonist, 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). In some embodiments, the RNA molecule comprises an aptamer, ribozyme, hairpin RNA, siRNA, or miRNA. In some embodiments, the lipophilic moiety includes lipids, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, vomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In some embodiments, the low molecular weight includes a sugar moiety. In some embodiments, the low molecular weight includes an amino sugar. In some embodiments, the amino sugar is N-acetylgalactosamine (GalNAc). In some embodiments, the targeting moiety is specific to an antigen or receptor in a target cell or target tissue. In some embodiments, the receptor includes an asialoglycoprotein receptor (ASGPR). In some embodiments, the single-stranded non-coding nucleic acid molecule includes one or more nucleotides with modifications. In some embodiments, the modification includes chemical modification. In some embodiments, the chemical modification includes modification of sugars, phosphate backbones, or nucleic acid bases. In some embodiments, nucleotide modification includes 2'-O-Me, 2'-F, 2'-MOE, N(6)-methyladenosine, 5-methylcytidine, 5-methyluridine (ribothymidine), ribose modification by cross-linked nucleic acids, or nucleotides with alternative chemical structures.In some embodiments, the ribose modification by cross-linked nucleic acids is locked nucleic acid (LNA), ethylene-cross-linked nucleic acid (ENA), or restricted ethyl-cross-linked nucleic acid (cEt). In some embodiments, nucleotides with alternative chemical structures are phosphorodiamidate morpholinonucleotides (PMO), thiophosphoamide, peptide nucleic acid (PNA), tricycloDNA (tcDNA), unlocked nucleic acid (UNA), or glycol nucleic acid (GNA). In some embodiments, single-stranded non-coding nucleic acid molecules exhibit lower immunogenicity in vivo compared to otherwise identical linear non-coding nucleic acid molecules. In some embodiments, immunogenicity in vivo is measured by immunogenicity assays. In some embodiments, single-stranded non-coding nucleic acid molecules exhibit lower toxicity to the target compared to otherwise identical linear non-coding nucleic acid molecules. In some embodiments, toxicity is measured by subchronic or chronic toxicity tests. In some embodiments, single-stranded non-coding nucleic acid molecules exhibit less miss-target effect to the target compared to otherwise identical linear non-coding nucleic acid molecules. In some embodiments, the miss-target effect is measured by gene expression analysis. In some embodiments, single-stranded non-coding nucleic acid molecules exhibit higher in vivo durability compared to otherwise identical linear non-coding nucleic acid molecules. In some embodiments, in vivo durability is measured using nucleic acid detection techniques. In some embodiments, single-stranded non-coding nucleic acid molecules without free ends include cyclized oligonucleotides. In some embodiments, the cyclized oligonucleotides constitute a single or polyvalent ASO, which may or may not be separated by linker sequences of different lengths. In some embodiments, a polyvalent ASO includes two or more ASOs. In some embodiments, the two or more ASOs of a polyvalent ASO target the same sequence and / or gene. In some embodiments, the two or more ASOs of a polyvalent ASO target different sequences and / or genes. In some embodiments, the two or more ASOs of a polyvalent ASO have the same mechanism of action. In some embodiments, the two or more ASOs of a polyvalent ASO have different mechanisms of action. In some embodiments, one or more adapters include sugars, lipids, peptides, antibodies, nucleotide sequences, aptamers, or combinations thereof.

[0008] Embodiments disclosed herein provide synthetic nucleic acid molecules comprising a single-stranded nucleic acid molecule without free ends, comprising (a) a functionally active oligonucleotide that targets a congeneral mRNA, (b) an adapter element that enhances the function, and (c) a moiety that reversibly or irreversibly binds to the ends of (a) and (b). In some embodiments, the synthetic nucleic acid molecule further comprises (d) a second moiety that reversibly or irreversibly binds to the ends of (a) and (b).

[0009] Built-in by reference All publications, patents, and patent applications described herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually directed to be incorporated herein by reference. To the extent that any publications and patents or patent applications incorporated herein by reference conflict with any disclosures contained herein, this Specification is intended to supersede and / or take precedence over any such conflicting material.

[0010] Novel features of the concept of the present invention are described in particular in the appended claims. A deeper understanding of the features and advantages of the concept of the present invention can be obtained by referring to the following detailed description, which describes embodiments illustrating specific examples in which the principles of the concept of the present invention are utilized, and the appended drawings include the following: [Brief explanation of the drawing]

[0011] [Figure 1A] Some non-limiting examples of loop-type antisense oligonucleotides (ASOs) according to several embodiments of this specification are shown below. [Figure 1B] An exemplary ASO is shown, which forms a loop structure operated by an adapter element (A1) that connects to the end of the loop via junctions J1 and J2 that facilitate irreversible and / or reversible bonding between A1 and L1. [Figure 1C]This paper presents non-limiting examples of nucleotide backbone, ribose ring, and base modifications that can improve the stability, immunogenicity, and potency of loop-type ASO constructs. [Figure 2A] Some non-limiting examples of various monovalent or polyvalent cyclized therapeutic ASOs according to several embodiments herein are shown. [Figure 2B] The versatility and modularity of the manipulated loop-type ASO construct are shown (top row). Multiple L modules (here, L1-L3) can be linked via adapters (A1-A3) to form a multivalent functional loop-type unit that can act on one or more target genes. [Figure 2C] A non-limiting illustrative explanation of divalent (left) and trivalent (right) ASOs is shown. Each ASO unit (L) of a polyvalent ASO can target an independent gene or RNA, thereby enhancing the valence of the loop construct. [Figure 2D] A non-limiting example of a portion embedded in the adapter section of a loop-type construct is shown. This can act as either a linker or a cell-specific targeting portion (e.g., an antibody, aptamer, small molecule, or lipophilic portion for improving loop-type ASO delivery) according to some embodiments herein. [Figure 3] Non-limiting examples of the structure of the locked and unlocked states of the ASO according to some embodiments herein are shown. [Figure 4A] A linear ASO structure with a phosphorothioate nucleotide backbone is illustrated. [Figure 4B] A cyclic ASO structure with a phosphodiester skeleton is shown in the diagram. [Figure 4C] The chemical structures of phosphodiester bonds and phosphorothioate bonds are shown. [Figure 5A] The diagram shows the enzymatic cyclization of ASO, and the separation and purification of ASO on a denatured urea PAGE gel. Note the slow migration pattern of cyclized ASO on the PAGE (right lane). Open and loop-type ASO were visualized using GelRED. [Figure 5B]A flowchart illustrating a non-exclusive example of the processes involved in the formation of cyclized ASO is shown. [Figure 6A] The accessible open end of ASO is susceptible to degradation by exonucleases (Pac-Man figure). [Figure 6B] This diagram illustrates the resistance of loop-forming / cyclized ASOs to degradation by exonucleases, which is due to the absence of an open end. [Figure 7A] The results of experiments using linear and cyclized ASOs targeting CFB and ApoB are presented. mRNA was treated with exonuclease T, a 3'-to-5' nuclease, for the indicated period. Cyclized oligonucleotides remained largely intact up to 2 hours after digestion with 0.16 U / μL exonuclease T, confirming the formation of loop-type ASOs and the resistance of loop-type ASO constructs to digestion by exonuclease T. ASOs were separated on a 12% urea PAGE gel and subsequently stained with GelRED. [Figure 7B] The graph shows the resistance of loop-type CFB ASO after treatment with exonuclease T at the indicated time. The intensity of the intact ASO band was quantified using ImageJ and expressed as the percentage at 0 minutes. Error bars indicate the standard deviation of three independent experiments. [Figure 8A] This shows that open-type and loop-type chemically modified single-stranded oligonucleotides were digested with rattlesnake phosphodiesterase I (PDI), a nuclease primarily exhibiting exonuclease activity towards nucleic acids. The loop-type construct showed even greater resistance to digestion by PDI. [Figure 8B] The images show gel images of intact open (left) or loop-type (right) structures after digestion with PDI at the indicated time. [Figure 9A]Absence of RNase H-mediated CFB degradation by the standard loop structure after treatment of human hepatoblastoma HepG2 cells with 50 nM of standard CFB ASO or standard loop-type ASO for 24 h. Isolated RNA was reverse transcribed and the cellular levels of CFB mRNA were quantified by probe-based RT qPCR. Rpb1 was used as a housekeeping gene for normalization of CFB transcripts. [Figure 9B] Shows open standard ASO (top) and loop standard (adapter-free) ASO (bottom). [Figure 10] Demonstrates that the engineered loop-type ASO (L1-A1 loop construct) targeting CFB is functional, targets cognate CFB mRNA, and can degrade it. Human hepatoblastoma HepG2 cells were transfected with 50 nM of open or loop engineered ASO (L1-A1 containing the ASO) for 24 h using Lipofectamine, and the effect on mRNA knockdown was measured by probe-based RT-qPCR. [Figure 11A] Demonstrates that loop formation by the standard ApoB ASO (adapter-free, L1 only) abolishes the activity of the standard ApoB ASO. The panel shows the loss of RNase H-mediated ApoB degradation activity by the standard loop structure (adapter-free). [Figure 11B] Addition of A1 to the standard structure of L1 ApoB (engineered loop-type ASO) restores the function of the ASO and results in degradation of target ApoB mRNA in HepG2 cells after 24 h of treatment with 50 nM of Lipofectamine reagent (error bars indicate standard deviation). [Figure 11C]Summarize the activity (functionality) of an open standard ASO (top), a loop standard (adapter-free) ASO (center), and an engineered loop ASO (L1-A1) (bottom). Experiments were conducted in human hepatoblastoma HepG2 cells, demonstrating that removal of the A1 adapter from the looped L1 ASO structure abolishes depletion of target ApoB mRNA degradation. Cells were treated with 50 nM of each ApoB ASO for 24 hours. Isolated RNA was reverse transcribed, and the cellular levels of ApoB mRNA were quantified by SYBR-based RT qPCR. RPL13A was used as a housekeeping gene for normalization of ApoB transcripts. [Figure 12] Addition of adapter A1 has been shown to restore the function of the engineered loop ASO targeting ApoB (L1-A1 loop construct), demonstrating the importance of adapter inclusion and optimization for the function of loop ASOs. The figure also demonstrates the generalizability of the RNase H-mediated KD effect by another loop ASO. The engineered loop ASO depleted ApoB mRNA to the same extent as the open engineered ApoB ASO. Primers 1 and 2 are two separate amplicons of ApoB when measured by SYBR Green qRT-PCR. RPL13A was used as a housekeeping gene for normalization. [Figure 13A] Show the activity of multivalent engineered loop CFB ASOs. Two engineered loop dimer ASOs (#1 and #2) and a single trivalent ASO were examined for RNase H-mediated depletion activity against CFB mRNA. All constructs showed activity against CFB mRNA at the concentrations examined (37.5 nM ASO, 24-hour treatment in HepG2 cells). [Figure 13B] Illustrate the structural features of divalent and trivalent loop ASOs containing the L1 ASO and A1 adapter that promote loop formation. [Figure 13C] Illustrate the structural features of divalent and trivalent loop ASOs containing the L1, and L2 ASOs and A1, and A2 adapters that promote loop formation. [Figure 13D] The structural characteristics of divalent and trivalent loop-type ASOs, including L1, L2, and L3 ASOs and A1-A34 adapters, which promote loop formation, are illustrated. [Modes for carrying out the invention]

[0012] Drug discovery is an extremely difficult problem, particularly in the field related to genetic diseases. Existing single-stranded nucleic acid molecules used in gene therapy (e.g., antisense oligonucleotides) have limited clinical efficacy, at least in part, due to their instability and / or toxicity or mistargeting effects in vivo. Oligonucleotide technologies for use in gene therapy, such as those described in U.S. Patent Application No. 20230257745 (which is incorporated herein by reference in its entirety), also have limited clinical efficacy, at least in part, due to instability and / or toxicity or mistargeting effects in vivo. For example, antisense oligonucleotides are susceptible to degradation by nucleases and other intracellular enzymes, reducing their efficacy and requiring higher doses to achieve therapeutic effects. However, such existing single-stranded nucleic acid molecules (e.g., antisense oligonucleotides) lack adapters and other features, resulting in problems with low specificity and reduced target binding efficiency.

[0013] Disclosed herein are compositions, methods, and kits comprising single-stranded non-coding nucleic acid molecules that, in several embodiments, modulate (e.g., increase or decrease) the transcription of target genes (e.g., disease-related genes) exhibiting greater durability (e.g., reduced instability), reduced toxicity, reduced immunogenicity, or any combination thereof in vivo. In some embodiments, the single-stranded non-coding nucleic acid molecules disclosed herein include an antisense strand with 5' and 3' ends ligated, thereby protecting the antisense (e.g., guide) strand from degradation in vivo. In some embodiments, the 5' and 3' ends of the antisense strand are reversibly ligated so that the single-stranded non-coding nucleic acid molecule can linearize in vivo at the site of action to induce modulation of target gene transcription. Additional moieties (e.g., antibodies, RNA aptamers, small molecules, lipophilic moieties, etc.) can be ligated to the single-stranded non-coding nucleic acid molecule to increase stability, direct targeting, reduce off-target immunogenic effects, or otherwise improve the pharmacological quality of the single-stranded nucleic acid molecule.

[0014] Disclosed herein are methods for delivering single-stranded non-coding nucleic acid molecules to target cells in vivo or in vitro. In some embodiments, such delivery includes administering double-stranded non-coding nucleic acid molecules to the target, such as by subcutaneous administration. In some embodiments, such administration includes administering single-stranded non-coding nucleic acid molecules to the target, for example by subcutaneous administration. In some embodiments, the method further includes treating a disease or condition in the target by regulating the transcription of a target gene (e.g., a disease-related gene) with the single-stranded non-coding nucleic acid molecule.

[0015] Also disclosed herein are kits comprising the compositions and systems disclosed herein and instructions for using the single-stranded non-coding nucleic acid molecules disclosed herein to modulate the transcription of a gene of interest. Such kits may also include a container for storing the system components and instructions.

[0016] The section headings used in this specification are for organizational purposes only and should not be construed as limiting the subjects described herein.

[0017] I. Definition Unless otherwise defined, all technical terms, notations, and other technical or scientific terms or glossaries used herein are to have the same meaning as those generally understood by those skilled in the art in the field relating to the claimed subject matter. In some cases, terms that have a commonly understood meaning are defined herein for clarity and / or for immediate reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from those generally understood in the art.

[0018] Throughout this application, various embodiments may be presented in range form. It should be understood that range form is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of this disclosure. Therefore, range descriptions should be considered to have all possible partial ranges specifically disclosed, as well as the individual numerical values ​​within that range. For example, a range of 1 to 6 should be considered to have the individual numerical values ​​within that range, e.g., 1, 2, 3, 4, 5, 6, as well as the specifically disclosed partial ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, and 3 to 6. This applies regardless of the breadth of the range.

[0019] When used herein and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. For example, the term "a sample" includes multiple samples, including mixtures thereof.

[0020] The terms “determining,” “measuring,” “evaluating,” “assessing,” and “analyzing” are often used interchangeably in this specification to refer to forms of measurement. These terms include determining whether an element is present or not (e.g., detection). These terms may include quantitative, qualitative, or both quantitative and qualitative determinations. Evaluations may be relative or absolute. “Detecting the presence of ~” may, depending on the context, include determining the quantity of something that is present, in addition to determining whether it is present or not.

[0021] As used herein with respect to single-stranded non-coding nucleic acid molecules, the term "cyclic" means that the 5' and 3' ends of a single-stranded non-coding nucleic acid molecule are directly or indirectly linked to each other such that the single-stranded non-coding nucleic acid molecule has no free ends, regardless of the shape or structure of the single-stranded non-coding nucleic acid molecule.

[0022] The term "in vivo" is used to describe events that occur within the body of a subject.

[0023] The term "in vitro" is used to describe events that occur outside the body of the subject. In vitro assays are not performed on the subject; rather, they are performed on a sample separate from the subject. An example of an in vitro assay performed on a sample is an "in vitro" assay.

[0024] The term "in vitro" is used to describe events that occur within a container used to hold experimental reagents so that they can be separated from the biological source from which the material is obtained. In vitro assays can include cell-based assays in which live or dead cells are employed. In vitro assays can also include cell-free assays that do not use untreated cells.

[0025] When used herein in relation to a number, the term "approximately" refers to plus or minus 10% of that number. The term "approximately" in the context of a range refers to the range obtained by subtracting 10% of its lowest value and the range obtained by adding 10% of its highest value.

[0026] As used interchangeably herein, the terms “polynucleotide,” “nucleotide,” or “nucleic acid” refer to polymers of nucleotides of any length, including DNA and RNA or hybrids thereof. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or analogues thereof, or any substrate that can be incorporated into the polymer by DNA polymerase or RNA polymerase. A polynucleotide may contain modified nucleotides, such as, but not limited to, methylated nucleotides and their analogues, or non-nucleotide components. Modifications to the nucleotide structure may be introduced before or after the assembly of the polynucleotide. Polynucleotides may further be modified after polymerization, for example, by binding with a labeling component.

[0027] Unless otherwise specified, the terms “polynucleotide,” “nucleotide,” or “nucleic acid” as used herein include single-stranded molecules as well as double-stranded or triple-stranded nucleic acids. In double-stranded or triple-stranded nucleic acids, the nucleic acid strands do not need to be identically elongated (i.e., double-stranded nucleic acids do not need to be double-stranded along the entire length of both strands). Nucleic acid sequences, if provided, are enumerated in the 5' to 3' direction unless otherwise stated. The methods described herein provide the production of isolated nucleic acids. The methods described herein further provide the production of isolated and purified nucleic acids. The term "nucleic acid" as used herein refers to a group of 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, It may include base lengths of at least approximately 400, at least approximately 425, at least approximately 450, at least approximately 475, at least approximately 500, at least approximately 600, at least approximately 700, at least approximately 800, at least approximately 900, at least approximately 1000, at least approximately 1100, at least approximately 1200, at least approximately 1300, at least approximately 1400, at least approximately 1500, at least approximately 1600, at least approximately 1700, at least approximately 1800, at least approximately 1900, and at least approximately 2000 or more.Furthermore, provided herein are methods for synthesizing any number of polypeptide fragments encoding nucleotide sequences, including sequences encoding non-ribosomal peptides (NRPs), non-ribosomal peptide synthase (NRPS) modules and sequences encoding synthetic variants, polypeptide fragments of other modular proteins such as antibodies, regulatory sequences, such as promoters, transcription factors, enhancers, non-coding DNA or RNA such as micronucleolar RNA derived from siRNA, shRNA, RNAi, miRNA, or microRNA, or polypeptide fragments derived from other protein families, including any functional or structural DNA or RNA units of interest. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, intergenetic DNA, loci (plural) (singular) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), small RNA, ribozymes, complementary DNA (cDNA) (usually the DNA representation of mRNA obtained by reverse transcription or amplification of messenger RNA); DNA molecules produced by synthesis or 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 a gene or gene fragment as referred to herein may include at least one region encoding an exon sequence without intervening intron sequences in a genomic equivalent sequence.

[0028] As used herein with respect to nucleic acid molecules, the term "synthesis" refers to production by chemical synthesis and / or enzymatic synthesis in vitro.

[0029] As used herein, the term “cell” generally refers to a biological cell.

[0030] As used herein, the term “gene” refers to a segment of nucleic acid that codes for an individual protein or RNA (also called a “coding sequence” or “coding region”), along with any associated regulatory regions such as promoters, operators, or terminators, which may optionally be located upstream or downstream of the coding sequence. As used herein, a “locus” refers to a specific location within a gene.

[0031] The terms "increased" or "growth" are used herein to generally mean an increase of a statistically significant amount.

[0032] The terms “declined” or “declined” are used herein to generally mean a statistically significant decrease.

[0033] The terms “polypeptide,” “peptide,” and “protein” may be used interchangeably in relation to polymers of amino acid residues. A protein may refer to a full-length polypeptide translated from an encoding open reading frame or processed into its mature form, while a polypeptide or peptide may refer to a protein degradation or processing fragment that is still independently or identifiablely mapped to a specific protein. A polypeptide may be a single polymer chain of amino acids linked to one another by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Polypeptides may be modified, for example, by carbohydrate addition, phosphorylation, etc.

[0034] When used herein to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, the terms “homologous,” “homonymy,” or “homonymity percentage” are defined as Karlin The sequence homology percentage can be determined using the formula described by Altschul (Proc.Natl.Acad.Sci.USA,87:2264-2268,1990, modified as in Proc.Natl.Acad.Sci.USA,90:5873-5877,1993). Such a formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program by Altschul et al. (J.Mol.Biol.1990,Oct.5;215(3):403-10;Nucleic Acids Res.1997,Sep.1;25(17):3389-402). The sequence homology percentage can be determined using the latest version of BLAST as of the filing date of this application. The sequence identity percentage can be determined using the latest version of BLAST as of the filing date of this application.

[0035] The term “identity percentage (%)” or “sequence identity percentage” with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps where necessary to obtain the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. As used herein, the term “identity percentage (%)” or “sequence identity percentage” with respect to a reference nucleic acid sequence is the percentage of nucleotides in a candidate sequence that are identical to nucleotides in the reference nucleic acid sequence, after aligning the sequences and introducing gaps where necessary to achieve maximum sequence identity. Alignment for the purpose of determining the sequence identity percentage can be achieved using various known methods, such as publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. It is possible to determine appropriate parameters for aligning sequences, including algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes herein, the amino acid sequence identity % values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, along with the user documentation, is protected by the U.S. Copyright Office. The ALIGN-2 program is filed with the Office, Washington DC, 20559 and registered under U.S. copyright number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from source code. The ALIGN-2 program should be edited when used with UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and should not be changed.

[0036] When used interchangeably in this specification, the terms “Gene of Interest” or “GOI” refer to a gene that encodes a directly or indirectly detectable gene expression product.

[0037] II. Composition Antisense oligonucleotides Disclosed herein, in some embodiments, are single-stranded non-coding nucleic acid molecules. In some embodiments, a single-stranded non-coding nucleic acid molecule is any type of nucleic acid containing a single strand (e.g., ribonucleic acid (RNA), deoxyribonucleic acid (DNA), etc.). In some embodiments, a single-stranded non-coding nucleic acid molecule is a single-stranded oligonucleotide containing a short fragment of a nucleotide sequence (e.g., DNA, RNA, or a DNA-RNA hybrid). In some embodiments, a single-stranded non-coding nucleic acid molecule includes an antisense strand configured to silence a target single-stranded nucleic acid sequence, such as messenger RNA (mRNA). In some embodiments, a single-stranded non-coding nucleic acid molecule includes an antisense strand configured to enhance the expression of a target single-stranded nucleic acid sequence. In some embodiments, a single-stranded non-coding nucleic acid molecule is an antisense oligonucleotide (ASO) and generally includes a single-stranded nucleic acid sequence that can bind to a target nucleic acid sequence, e.g., another RNA (e.g., mRNA), via Watson-Crick base pairing. Single-stranded nucleic acid molecules can be synthetic. Alternatively, or in addition to the above, single-stranded nucleic acid molecules can be chemically modified nucleic acid molecules. Single-stranded nucleic acid molecules may contain an antisense strand that is complementary, or substantially complementary, to the target nucleic acid sequence.

[0038] The target nucleic acid sequence can be a single-stranded nucleic acid such as single-stranded DNA or RNA. The target nucleic acid sequence can be coding RNA (e.g., mRNA). For example, the target nucleic acid sequence can be a gene expression product derived from a gene encoding complement factor B (CFB), complement factor C5 (C5), or ApoB. Alternatively, the target nucleic acid sequence can be non-coding RNA (e.g., tRNA, rRNA, microRNA, non-coding RNA, small non-coding RNA, intron, exon, intron / exon or exon / intron junction, long non-coding RNA, small interfering RNA, or piwi-interacting RNA). The target nucleic acid sequence can be a synthetic nucleic acid sequence. Alternatively, the target nucleic acid sequence can be native to a cell or organism. In some embodiments, the target nucleic acid sequence is associated with a disease or condition disclosed herein. For example, regulation of transcription of a target nucleic acid sequence (e.g., in the case of a DNA target) may be therapeutically effective in treating a disease or condition. In another example, the regulation of the expression of a target nucleic acid sequence (e.g., an RNA target) may be therapeutically effective in treating a disease or condition. In some embodiments, the regulation of the expression of a target nucleic acid sequence includes post-transcriptional modifications such as capping, splicing, and polyadenylation of the RNA target. In some embodiments, single-stranded nucleic acids (e.g., ASOs) regulate the splicing of precursor mRNA (e.g., exon inclusion or exon exclusion) by binding to exons, introns, exon / intron junctions, or intron / exon junctions, thereby promoting or inhibiting splicing events. In some embodiments, steric blocking by single-stranded nucleic acids (e.g., ASOs) can regulate gene expression by binding to non-protein-coding and regulatory regions of a gene, thereby modulating expression.

[0039] Oligo length A single-stranded nucleic acid (e.g., ASO) 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. A nucleotide can be 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, at least about 28 nucleotides, at least about 29 nucleotides, at least about 30 nucleotides, at least about 31 nucleotides, at least about 32 nucleotides, at least about 33 nucleotides, at least about 34 nucleotides, at least about 34 nucleotides, at least about 35 nucleotides, at least about 36 nucleotides, at least about 37 nucleotides, at least about 38 nucleotides, at least about 39 nucleotides, at least about 40 nucleotides, or longer.

[0040] Single-stranded nucleic acids (e.g., ASO) have 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, and many It can be at least 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 10 nucleotides, at most about 9 nucleotides, at most about 8 nucleotides, at most about 7 nucleotides, at most about 6 nucleotides, at most about 5 nucleotides, or even shorter.

[0041] Single-stranded nucleic acids (e.g., ASO) can be approximately 5 to 40 nucleotides long. Single-stranded nucleic acids (e.g., ASO) can be approximately 5 to 35 nucleotides long. Single-stranded nucleic acids (e.g., ASO) can be approximately 5 to 30 nucleotides long. Single-stranded nucleic acids (e.g., ASO) can be approximately 5 to 25 nucleotides long. Single-stranded nucleic acids (e.g., ASO) can be approximately 5 to 20 nucleotides long. Single-stranded nucleic acids (e.g., ASO) can be approximately 5 to 15 nucleotides long. Single-stranded nucleic acids (e.g., ASO) can be approximately 5 to 10 nucleotides long. Single-stranded nucleic acids (e.g., ASO) can be approximately 10 to 40 nucleotides long. Single-stranded nucleic acids (e.g., ASO) can be approximately 15 to 40 nucleotides long. Single-stranded nucleic acids (e.g., ASO) can be approximately 20 to 40 nucleotides long. Single-stranded nucleic acids (e.g., ASO) can be approximately 25 to 40 nucleotides long. Single-stranded nucleic acids (e.g., ASO) can be approximately 30 to 40 nucleotides long. Single-stranded nucleic acids (e.g., ASO) can be approximately 35 to 40 nucleotides long.

[0042] Complementary bond An antisense oligonucleotide can form a complementary bond with the entire target strand. Alternatively, an antisense oligonucleotide can form a complementary bond with a portion of the target nucleic acid sequence. The antisense strand can form a complementary bond with one or more portions of the target nucleic acid sequence (e.g., an mRNA strand) including the 5'UTR, 3'UTR, regulatory regions, coding sequences, introns, exons, intron / exon junctions, and / or exon / intron junctions. The regulatory region of the target nucleic acid sequence (e.g., mRNA) may include a promoter region, an enhancer region, an operator region, or a repressor region.

[0043] ASOs can form complementary binding to part or all of the mRNA target chain. Antisense oligonucleotides can be perfectly complementary (e.g., 100% complementary) to their target chain counterparts. Alternatively, ASOs can have incomplete complementarity to their target chain counterparts. ASOs can have at least approximately 50% complementarity, at least approximately 55% complementarity, at least approximately 60% complementarity, at least approximately 65% ​​complementarity, at least approximately 70% complementarity, at least approximately 75% complementarity, at least approximately 80% complementarity, at least approximately 85% complementarity, at least approximately 90% complementarity, at least approximately 91% complementarity, at least approximately 92% complementarity, at least approximately 93% complementarity, at least approximately 94% complementarity, at least approximately 95% complementarity, at least approximately 96% complementarity, at least approximately 97% complementarity, at least approximately 98% complementarity, at least approximately 99% complementarity, or more, with respect to their corresponding target chain. ASOs can have at most approximately 99% complementarity with their corresponding target chain, at most approximately 98% complementarity, at most approximately 97% complementarity, at most approximately 96% complementarity, at most approximately 95% complementarity, at most approximately 94% complementarity, at most approximately 93% complementarity, at most approximately 92% complementarity, at most approximately 91% complementarity, at most approximately 90% complementarity, at most approximately 85% complementarity, at most approximately 80% complementarity, at most approximately 75% complementarity, at most approximately 70% complementarity, at most approximately 65% ​​complementarity, at most approximately 60% complementarity, at most approximately 55% complementarity, at most approximately 50% complementarity, or even lower.

[0044] Oligoformation and linker A single-stranded non-coding nucleic acid molecule can be a DNA oligonucleotide. Alternatively, an ASO can be an RNA oligonucleotide. Alternatively, an ASO can be a chimeric oligonucleotide containing both RNA and DNA. An ASO can exist as a single unfolded strand (e.g., a linear strand) (Figure 4A). Alternatively, an ASO can exist in a folded structure (e.g., a hairpin). Alternatively, an ASO can exist in a cyclized structure (e.g., without free ends) (Figure 4B). For example, the 5' and 3' ends of the antisense strand of a single-stranded non-coding nucleic acid molecule can be ligated so that the antisense strand does not have free ends.

[0045] An ASO can exist as a single ASO or as an ASO composed of multiple units or modules (for example, an ASO composed of more than one ASO) (Figures 13A-D). An ASO composed of more than one ASO may be called an ASO module and / or a multivalent ASO. In some embodiments, a multivalent ASO may contain at least about 2 ASOs, at least about 3 ASOs, at least about 4 ASOs, at least about 5 ASOs, at least about 6 ASOs, at least about 7 ASOs, at least about 8 ASOs, at least about 9 ASOs, at least about 10 ASOs, or more. In some embodiments, a multivalent ASO may contain at most about 10 ASOs, at most about 9 ASOs, at most about 8 ASOs, at most about 7 ASOs, at most about 6 ASOs, at most about 5 ASOs, at most about 4 ASOs, at most about 3 ASOs, at most about 2 ASOs, or even fewer. Further examples of monovalent or polyvalent ASOs can be found in Figure 2.

[0046] Polyvalent ASOs can be cyclized (Figures 13A-D). In some embodiments, a cyclized polyvalent ASO containing two or more ASOs may refer to a polyvalent loop-type ASO. In some embodiments, a cyclized polyvalent ASO containing two or more ASOs may refer to an ASO loop-type dimer (Figures 13A-D). In some embodiments, a cyclized polyvalent ASO containing three or more ASOs may refer to an ASO loop-type trimer (Figures 13A-D). In some embodiments, a polyvalent ASO may 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. In some embodiments, the polyvalent ASO may 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.

[0047] Cyclic ASOs can be prepared from their linear counterparts by enzymatic or chemical reactions to link the 5' and 3' ends of a linear chain (Figure 1). Cyclic ASOs can be formed via the formation of phosphodiester bonds linking the 5' and 3' ends of a linear antisense chain. Alternatively, cyclic ASOs can be formed via the addition of linker elements to connect the 5' and 3' ends of a linear antisense chain. The linker elements can be chemical linkers. Chemical linkers can act as junctions connecting the 5' and 3' ends of a linear antisense chain to form a cyclic ASO. Alternatively, linkers can be used to span exon / intron junctions. Chemical linkers include, but are not limited to, disulfide bonds, photocleavable linkers, diazo linkers, acid-unstable linkers, peptide linkers, nucleotide linkers, glucuronide groups, azido-alkyne linkers, aldehyde-oxamine linkers, phosphorothioate-tosylated linkers, phosphate-activator-mediated phosphate-hydroxyl bonds, or metal chelate ligation linkers. Nucleotide linkers can be oligonucleotide linkers, oligoaptamer linkers, RNA linkers, or DNA linkers. Peptide linkers can be polypeptide linkers (e.g., antibody linkers). Linkers can contain phosphodiester bonds, alkyl groups, sulfhydryl groups, amine groups, or polymers.

[0048] The linker can be permanent (e.g., indestructible) (Figure 3). Alternatively, the linker can be reversible. For example, the linker can be cleavable (Figure 3). The linker can be cleaved, for example, by an enzyme or catalyst. Enzymes with cleavage properties include, but are not limited to, proteases and endonucleases. The catalyst can be a chemical catalyst (e.g., an acidic catalyst or a metal catalyst) or a non-chemical catalyst (e.g., light, heat, or pH). Alternatively, the linker can be cleaved via a reversible click reaction. Alternatively, the linker can be cleaved independently.

[0049] Click reactions are reactions used to link two specific molecular entities in the absence of water. Examples of click reactions include, but are not limited to, copper(I) catalyzed azide-alkyne cycloaddition, strain-promoted alkyne-nitrone cycloaddition, strain-promoted azide-alkyne cycloaddition, alkene-azide cycloaddition, reverse electron-demanded Deal-Alder reactions between alkenes and tetrazines, and photoclick reactions between alkenes and tetrazoles. Amines and thiols can be used to facilitate reversible click reactions.

[0050] Linkers can be cleavable under specific conditions. For example, a linker may be cleavable or substantially cleavable under intracellular conditions, but not cleavable or substantially cleavable under extracellular conditions. Alternatively, a linker may be incapable or substantially incapable under intracellular conditions, but cleavable or substantially cleavable under extracellular conditions. Selective cleavage can be related to conditions such as, but not limited to, pH or ion concentration.

[0051] adapter In some embodiments, single-stranded non-coding nucleic acid molecules (e.g., ASOs) may include adapters. Non-limiting examples of adapters include peptide adapters, nucleotide adapters, antibody adapters, targeting moiety adapters (e.g., glycans), sugar adapters, lipid adapters, DNA aptamers, or RNA aptamers (Figure 2D). Nucleotide adapters can be oligonucleotide adapters, oligo aptamer adapters, RNA adapters, or DNA adapters. Peptide adapters can be polypeptide adapters (e.g., protein adapters).

[0052] In some embodiments, the antisense strand includes an adapter. In some embodiments, the adapter may be adjacent to a linker. Alternatively, in some embodiments, the adapter may not be adjacent to a linker. In some embodiments, a single-stranded non-coding nucleic acid molecule includes one adapter. Alternatively, in some embodiments, a single-stranded non-coding nucleic acid molecule includes two, three, four, five, six, seven, eight, nine, ten or more adapters. An ASO may include one or more adapters (Figures 13A-D). In some embodiments, an ASO may include at least about two adapters, at least about three adapters, at least about four adapters, at least about five adapters, at least about six adapters, at least about seven adapters, at least about eight adapters, at least about nine adapters, at least about ten adapters, or even more adapters. In some embodiments, the ASO may contain 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 even fewer adapters.

[0053] A single ASO containing an adapter (e.g., a monovalent ASO) can be cyclic. A monovalent ASO containing an adapter can be cyclic by reversibly or irreversibly linking the free end of the adapter to the free end of the ASO via a linker. A multivalent ASO containing two or more adapters can be cyclic (Figures 13A-D). An ASO may contain a single adapter and one or more linkers. For example, a first free end of the adapter may be linked to a first free end of the ASO via a first linker, and a second free end of the adapter may be linked to a second free end of the ASO via a second linker, thus forming a loop-type ASO (Figures 13A-D). A multivalent ASO may contain two or more adapters and two or more linkers (Figures 13A-D). In some embodiments, a polyvalent ASO can be connected 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 10 adapters, or more adapters. In some embodiments, a polyvalent ASO can be connected 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, at most about 2 adapters, or fewer adapters.

[0054] Adapters may include sequences that provide additional functionality to single-stranded non-coding nucleic acid molecules (e.g., ASOs). In some cases, adapters can extend the 5' and 3' ends of a single-stranded non-coding nucleic acid molecule (e.g., ASO). Adapters may have complete or partial complementarity to the adjacent regions of the gene of interest and / or target gene (e.g., adapters may include hybridization regions and / or adherent ends). Alternatively, adapters may include non-hybridization regions (e.g., non-adjacent regions, blunt ends, etc.). Adapters can add flexibility to single-stranded non-coding nucleic acid molecules (e.g., ASOs). For example, an adapter connecting the 5' and 3' ends of a single-stranded non-coding nucleic acid molecule (e.g., ASO) can improve the "steric freedom" of the single-stranded non-coding nucleic acid molecule (e.g., ASO), allowing it to interact unrestricted with proteins involved in downstream events (e.g., degradation by RNA-degrading enzyme H). Alternatively, or further, adapters can alter the shape of a single-stranded non-coding nucleic acid molecule (e.g., ASO). The adapter can vary in length. For example, a glycan adapter can be a glycan residue approximately 5-10 in length to form a glycan chain. Alternatively, an antibody adapter can contain a single antibody.

[0055] In some cases, the adapter can be a nucleotide adapter. A nucleotide adapter (e.g., DNA, RNA, etc.) has 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, and at least about 19 nucleotides. The length of a nucleotide can be 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, at least about 28 nucleotides, at least about 29 nucleotides, at least about 30 nucleotides, at least about 31 nucleotides, at least about 32 nucleotides, at least about 33 nucleotides, at least about 34 nucleotides, at least about 34 nucleotides, at least about 35 nucleotides, at least about 36 nucleotides, at least about 37 nucleotides, at least about 38 nucleotides, at least about 39 nucleotides, at least about 40 nucleotides, or more.

[0056] Nucleotide adapters (e.g., DNA, RNA, etc.) have 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, and at most about 23 nucleos. A nucleotide can have a length of at most approximately 22 nucleotides, at most approximately 21 nucleotides, at most approximately 20 nucleotides, at most approximately 19 nucleotides, at most approximately 18 nucleotides, at most approximately 17 nucleotides, at most approximately 16 nucleotides, at most approximately 15 nucleotides, at most approximately 14 nucleotides, at most approximately 13 nucleotides, at most approximately 12 nucleotides, at most approximately 11 nucleotides, at most approximately 10 nucleotides, at most approximately 9 nucleotides, at most approximately 8 nucleotides, at most approximately 7 nucleotides, at most approximately 6 nucleotides, at most approximately 5 nucleotides, or even shorter.

[0057] Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 5 and 40. Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 5 and 35. Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 5 and 30. Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 5 and 25. Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 5 and 20. Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 5 and 15. Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 5 and 10. Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 10 and 40. Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 15 and 40. Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 15 and 20. Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 20 and 40. Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 25 and 40. Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 30 and 40. Nucleotide adapters (e.g., DNA, RNA, etc.) can have a nucleotide length between approximately 35 and 40.

[0058] In some embodiments, the adapter can be a peptide adapter. The peptide adapter (e.g., polypeptide) can have a peptide length of at least about 2 peptides, at least about 3 peptides, at least about 4 peptides, at least about 5 peptides, at least about 6 peptides, at least about 7 peptides, at least about 8 peptides, at least about 9 peptides, at least about 10 peptides, at least about 11 peptides, at least about 12 peptides, at least about 13 peptides, at least about 14 peptides, at least about 15 peptides, at least about 16 peptides, at least about 17 peptides, at least about 18 peptides, at least about 19 peptides, at least about 20 peptides, or a longer peptide length.

[0059] The peptide adapter (e.g., polypeptide) can be at most about 20 peptides, at most about 19 peptides, at most about 18 peptides, at most about 17 peptides, at most about 16 peptides, at most about 15 peptides, at most about 14 peptides, at most about 13 peptides, at most about 12 peptides, at most about 11 peptides, at most about 10 peptides, at most about 9 peptides, at most about 8 peptides, at most about 7 peptides, at most about 6 peptides, at most about 5 peptides, at most about 4 peptides, at most about 3 peptides, at most about 2 peptides, or even shorter peptide lengths.

[0060] A peptide adapter (e.g., polypeptide) can have a peptide length between approximately 2 and approximately 20. A peptide adapter (e.g., polypeptide) can have a peptide length between approximately 2 and approximately 18. A peptide adapter (e.g., polypeptide) can have a peptide length between approximately 2 and approximately 16. A peptide adapter (e.g., polypeptide) can have a peptide length between approximately 2 and approximately 14. A peptide adapter (e.g., polypeptide) can have a peptide length between approximately 2 and approximately 12. A peptide adapter (e.g., polypeptide) can have a peptide length between approximately 2 and approximately 10. A peptide adapter (e.g., polypeptide) can have a peptide length between approximately 2 and approximately 8. A peptide adapter (e.g., polypeptide) can have a peptide length between approximately 2 and approximately 6. A peptide adapter (e.g., polypeptide) can have a peptide length between approximately 2 and approximately 4. A peptide adapter (e.g., polypeptide) can have a peptide length between approximately 4 and approximately 20. A peptide adapter (e.g., polypeptide) can have a peptide length between approximately 6 and approximately 20. A peptide adapter (e.g., polypeptide) can have a peptide length between approximately 8 and approximately 20. The peptide adapter (e.g., polypeptide) can have a peptide length between approximately 10 and approximately 20. The peptide adapter (e.g., polypeptide) can have a peptide length between approximately 12 and approximately 20. The peptide adapter (e.g., polypeptide) can have a peptide length between approximately 14 and approximately 20. The peptide adapter (e.g., polypeptide) can have a peptide length between approximately 16 and approximately 20. The peptide adapter (e.g., polypeptide) can have a peptide length between approximately 18 and approximately 20.

[0061] The use of adapters can enhance the function of ASOs. For example, the enhanced function of an ASO with an adapter may be due to an increased degree of steric freedom of the modified ASO, allowing it to interact unrestricted with proteins involved in downstream events (e.g., degradation by RNA-degrading enzymes). In some embodiments, an ASO with an adapter can have a higher degree of steric freedom than an ASO without an adapter. In some embodiments, an ASO 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%, or more degrees of steric freedom than an ASO without an adapter. In some embodiments, an ASO with an adapter can have a higher or less but still high degree of three-dimensional freedom compared to an ASO without an adapter, with a maximum of approximately 500%, 400%, 300%, 200%, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% higher.

[0062] In some embodiments, ASOs with adapters can have higher target specificity than ASOs without adapters. In some embodiments, ASOs with adapters can have target specificity that is 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 500%, at least about 600%, at least about 700%, at least about 80%, at least about 80%, at least about 80%, 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 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, and at least about 1,000% higher or higher than ASOs without adapters. In some embodiments, an ASO with an adapter can have higher target specificity than an ASO without an adapter, 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 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%, or at most about 5%, or less.

[0063] In some embodiments, ASOs with adapters can have higher target binding affinity than ASOs without adapters. In some embodiments, ASOs with adapters can have target binding affinity that is 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 500%, at least about 600%, at least about 700%, at least about 800%, at least about 800%, at least about 800%, 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 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, and at least about 1,000% higher or higher than ASOs without adapters. In some embodiments, an ASO with an adapter can have higher target binding affinity than an ASO without an adapter, 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 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%, or at most about 5%, or less.

[0064] qualification The single-stranded non-coding nucleic acid molecules disclosed herein may include modifications of sugars, phosphate backbones, or nucleic acid bases. For example, non-coding nucleic acid molecules may be modified by the addition of a moiety to the molecule. The modification may be chemical, synthetic, or native.

[0065] Nucleic acid molecules can be modified with nucleic acid bases. Nucleic acid base modifications include, but are not limited to, 2'-O-methylation (2'-OMe), uridine to pseudouridine conversion, N(6)-methyladenosine, 5-methylcytidine, 5-methyluridine (ribothymidine), 2'-fluoro (2'F), 2'-O-methoxyethyl (2'-MOE), ribose modification, and cross-linked nucleic acids (e.g., locked nucleic acid (LNA), ethylene-cross-linked nucleic acid (ENA), or restricted ethyl-cross-linked nucleic acid (cEt) modification), or nucleotides with alternative chemical structures (e.g., phosphorodiamidate morpholinonucleotides (PMO), peptide nucleic acid (PNA), tricycloDNA (tcDNA), unlocked nucleic acid (UNA), or glycol nucleic acid (GNA)).

[0066] Nucleic acid molecules can be modified with a phosphate backbone (Figure 4C). The phosphate backbone can be modified to include phosphate diesters, phosphorothioate isomers (e.g., stereoisomers Sp and / or Rp), phosphoryl DMI amide diester isomers, phosphorodithioates, methylphosphonates, 5'-phosphorothioates, thiophosphoamides, peptide nucleic acids, 5'-(E)-vinylphosphonates, or 5'-methylphosphonates.

[0067] Additional portions can be added to or bound to single-stranded nucleic acids (e.g., ASOs). These additional portions may include, but are not limited to, antibodies, lipophilic moieties, small molecules, and RNA aptamers (e.g., ribozymes). Adding additional portions to single-stranded nucleic acids can alter their pharmacological characteristics (e.g., structural or chemical parameters). Single-stranded nucleic acids can be modified by 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, or at least 10 or more portions. Additional portions can be added to the 5' end of the single-stranded nucleic acid, or to the 3' end, or to the middle of the single-stranded nucleic acid (neither the 3' nor the 5' end).

[0068] To enhance stability, single-stranded nucleic acids (e.g., ASOs) can be modified. Single-stranded nucleic acids as described herein 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%, and at least about 100% higher or greater stability compared to control single-stranded nucleic acids.

[0069] The stability of nucleic acids can be measured by analyzing the half-life of single-stranded non-coding nucleic acid molecules. Single-stranded nucleic acid molecules as described herein may have a half-life that is 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 longer than control single-stranded nucleic acids.

[0070] To reduce out-of-target effects, single-stranded non-coding nucleic acid molecules (e.g., ASOs) can be modified. When measured by sequencing, the number of out-of-target effects can be reduced by at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 100%, or more, compared to the control single-stranded nucleic acid.

[0071] To reduce harmful immunogenic effects, single-stranded non-coding nucleic acid molecules (e.g., ASOs) can be modified. When measured by immunogenicity assays, the number of harmful immunogenic effects can be reduced by at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 100%, or more, compared to control single-stranded nucleic acids.

[0072] To reduce toxicity, single-stranded non-coding nucleic acid molecules (e.g., ASOs) can be modified. When measured by toxicity assays, toxicity can be reduced by at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 100%, or more, compared to a control single-stranded nucleic acid. The control single-stranded nucleic acid molecule can be a single-stranded non-coding nucleic acid molecule that is identical except for the lack of modification.

[0073] To enhance stability and durability, single-stranded non-coding nucleic acid molecules (e.g., ASOs) can be modified. Durability and / or stability, as measured by nucleic acid detection, can be increased by at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 100%, or more, compared to a control single-stranded nucleic acid. The control single-stranded nucleic acid molecule can be a single-stranded non-coding nucleic acid molecule that is identical except for the lack of modification.

[0074] Modifications such as cyclization can be performed on single-stranded non-coding nucleic acid molecules (e.g., ASOs). A cyclized non-coding nucleic acid molecule can be a single-stranded non-coding nucleic acid molecule without free ends. A single-stranded non-coding nucleic acid molecule without free ends can refer to a cyclized oligonucleotide. A cyclized oligonucleotide can be a single (e.g., monovalent) ASO or a polyvalent ASO. A polyvalent ASO contains two or more ASOs. The two or more ASOs can be separated by a linker sequence, which can be of different lengths. The two or more ASOs in a polyvalent ASO can target the same sequence and / or gene. Alternatively, the two or more ASOs in a polyvalent ASO can target different sequences and / or genes. The two or more ASOs in a polyvalent ASO can have the same and / or different mechanisms of action. Cyclization and / or circularization of single-stranded non-coding nucleic acid molecules may allow for even fewer chemically modified nucleotides (e.g., nucleotides containing phosphorothioates) associated with adverse medical side effects. Cyclization and / or cyclicization may 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 even fewer chemically modified nucleotides associated with adverse medical side effects. Adverse medical side effects may include, but are not limited to, a decrease in platelets, thrombocytopenia, heart rate disturbances, increased blood pressure, or increased cardiac output due to activation of the complement cascade.

[0075] Target adjustment Transcriptional regulation can be induced using single-stranded non-coding nucleic acid molecules. For example, an ASO can bind to a target gene or the mRNA of a target gene (e.g., target RNA) to regulate the expression of the target gene. Alternatively, to regulate a target gene, an ASO can bind to a specific region of the target gene or target mRNA, such as a regulatory region (e.g., UTR, intron, exon, intron / exon junction, exon / intron junction). Alternatively, or in addition, an ASO can bind to a specific region of the target gene or target mRNA to regulate the splicing of the target gene or target mRNA.

[0076] Regulation of a target gene can lead to an increase in its expression, a decrease in its expression, or even maintenance of its expression.

[0077] In some cases, single-stranded non-coding nucleic acid molecules can increase target gene expression by at least approximately 0.1%, at least approximately 0.2%, at least approximately 0.3%, at least approximately 0.4%, at least approximately 0.5%, at least approximately 0.6%, at least approximately 0.7%, at least approximately 0.8%, at least approximately 0.9%, at least approximately 1%, at least approximately 2%, at least approximately 3%, at least approximately 4%, at least approximately 5%, at least approximately 6%, at least approximately 7%, at least approximately 8%, at least approximately 9%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 100%, at least approximately 200%, at least approximately 300%, at least approximately 400%, at least approximately 500%, or even more compared to control expression levels.

[0078] In some cases, single-stranded non-coding nucleic acid molecules may be expressed at up to approximately 500%, up to approximately 400%, up to approximately 300%, up to approximately 200%, up to approximately 100%, up to approximately 90%, up to approximately 80%, up to approximately 70%, up to approximately 60%, up to approximately 50%, up to approximately 40%, up to approximately 30%, up to approximately 20%, up to approximately 10%, up to approximately 9%, and up to approximately The expression of the target gene can be increased by 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 even less.

[0079] In some cases, single-stranded non-coding nucleic acid molecules can reduce the expression of target genes by at least or up to approximately 0.1 times, at least or up to approximately 0.2 times, at least or up to approximately 0.3 times, at least or up to approximately 0.4 times, at least or up to approximately 0.5 times, at least or up to approximately 0.6 times, at least or up to approximately 0.7 times, at least or up to approximately 0.8 times, at least or up to approximately 0.9 times, at least or up to approximately 1 time, at least or up to approximately 2 times, at least or up to approximately 3 times, at least or up to approximately 4 times, at least or up to approximately 5 times, at least or up to approximately 6 times, or less. It shows an increase of at least approximately 7 times, at least approximately 8 times, at least approximately 9 times, at least approximately 10 times, at least approximately 20 times, at least approximately 30 times, at least approximately 40 times, at least approximately 50 times, at least approximately 60 times, at least approximately 70 times, at least approximately 80 times, at least approximately 90 times, at least approximately 100 times, at least approximately 500 times, at least approximately 1,000 times, at least approximately 5,000 times, or at least approximately 10,000 times.

[0080] In some cases, single-stranded non-coding nucleic acid molecules increase the expression of target genes by at most about 10,000 times or less than about 10,000 times, at most about 5,000 times or less than about 5,000 times, at most about 1,000 times or less than about 1,000 times, at most about 500 times or less than about 500 times, at most about 100 times or less than about 100 times, at most about 90 times or less than about 90 times, at most about 80 times or less than about 80 times, at most about 70 times or less than about 70 times, at most about 60 times or less, at most about 50 times or less than about 50 times, at most about 40 times or less than about 40 times, at most about 30 times or less than about 30 times, at most about 20 times or less than about 20 times, at most about 10 times or less than about 10 times, at most about 9 times Alternatively, it can be increased by approximately 9 times or less, at most approximately 8 times or less, at most approximately 7 times or less, at most approximately 6 times or less, at most approximately 5 times or less, at most approximately 4 times or less, at most approximately 3 times or less, at most approximately 2 times or less, at most approximately 1 time or less, at most approximately 0.9 times or less, at most approximately 0.8 times or less, at most approximately 0.7 times or less, at most approximately 0.6 times or less, at most approximately 0.5 times or less, at most approximately 0.4 times or less, at most approximately 0.3 times or less, at most approximately 0.2 times or less, and at most approximately 0.1 times or less.

[0081] In some cases, single-stranded non-coding nucleic acid molecules can reduce target gene expression by at least approximately 0.1%, at least approximately 0.2%, at least approximately 0.3%, at least approximately 0.4%, at least approximately 0.5%, at least approximately 0.6%, at least approximately 0.7%, at least approximately 0.8%, at least approximately 0.9%, at least approximately 1%, at least approximately 2%, at least approximately 3%, at least approximately 4%, at least approximately 5%, at least approximately 6%, at least approximately 7%, at least approximately 8%, at least approximately 9%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 100%, at least approximately 200%, at least approximately 300%, at least approximately 400%, at least approximately 500%, or even more compared to control expression levels.

[0082] In some cases, single-stranded non-coding nucleic acid molecules may be expressed at up to approximately 500%, up to approximately 400%, up to approximately 300%, up to approximately 200%, up to approximately 100%, up to approximately 90%, up to approximately 80%, up to approximately 70%, up to approximately 60%, up to approximately 50%, up to approximately 40%, up to approximately 30%, up to approximately 20%, up to approximately 10%, up to approximately 9%, and up to approximately The expression of the target gene can be reduced by 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 even less.

[0083] In some cases, single-stranded non-coding nucleic acid molecules can reduce the expression of target genes by at least or up to approximately 0.1 times, at least or up to approximately 0.2 times, at least or up to approximately 0.3 times, at least or up to approximately 0.4 times, at least or up to approximately 0.5 times, at least or up to approximately 0.6 times, at least or up to approximately 0.7 times, at least or up to approximately 0.8 times, at least or up to approximately 0.9 times, at least or up to approximately 1 time, at least or up to approximately 2 times, at least or up to approximately 3 times, at least or up to approximately 4 times, at least or up to approximately 5 times, at least or up to approximately 6 times, or less. It can be reduced by or up to approximately 7 times, at least or up to approximately 8 times, at least or up to approximately 9 times, at least or up to approximately 10 times, at least or up to approximately 20 times, at least or up to approximately 30 times, at least or up to approximately 40 times, at least or up to approximately 50 times, at least or up to approximately 60 times, at least or up to approximately 70 times, at least or up to approximately 80 times, at least or up to approximately 90 times, at least or up to approximately 100 times, at least or up to approximately 500 times, at least or up to approximately 1,000 times, at least or up to approximately 5,000 times, or at least or up to approximately 10,000 times.

[0084] In some cases, single-stranded non-coding nucleic acid molecules increase the expression of target genes by at most about 10,000 times or less than about 10,000 times, at most about 5,000 times or less than about 5,000 times, at most about 1,000 times or less than about 1,000 times, at most about 500 times or less than about 500 times, at most about 100 times or less than about 100 times, at most about 90 times or less than about 90 times, at most about 80 times or less than about 80 times, at most about 70 times or less than about 70 times, at most about 60 times or less, at most about 50 times or less than about 50 times, at most about 40 times or less than about 40 times, at most about 30 times or less than about 30 times, at most about 20 times or less than about 20 times, at most about 10 times or less than about 10 times, at most about 9 times Alternatively, it can be reduced by less than approximately 9 times, at most about 8 times or less than 8 times, at most about 7 times or less than 7 times, at most about 6 times or less than 6 times, at most about 5 times or less than 5 times, at most about 4 times or less than 4 times, at most about 3 times or less than 3 times, at most about 2 times or less than 2 times, at most about 1 time or less than 1 time, at most about 0.9 times or less than 0.9 times, at most about 0.8 times or less than 0.8 times, at most about 0.7 times or less than 0.7 times, at most about 0.6 times or less than 0.6 times, at most about 0.5 times or less than 0.5 times, at most about 0.4 times or less than 0.4 times, at most about 0.3 times or less than 0.3 times, at most about 0.2 times or less than 0.2 times, and at most about 0.1 times or less than 0.1 times.

[0085] Regulation of target mRNA can lead to an increase in the expression of the gene product expressed from the target mRNA. Alternatively, regulation of target mRNA can lead to a decrease in the expression of the gene product expressed from the target mRNA. Alternatively, regulation of target mRNA can maintain the expression of the gene product expressed from the target mRNA.

[0086] In some cases, single-stranded non-coding nucleic acid molecules can increase target mRNA expression by at least approximately 0.1%, at least approximately 0.2%, at least approximately 0.3%, at least approximately 0.4%, at least approximately 0.5%, at least approximately 0.6%, at least approximately 0.7%, at least approximately 0.8%, at least approximately 0.9%, at least approximately 1%, at least approximately 2%, at least approximately 3%, at least approximately 4%, at least approximately 5%, at least approximately 6%, at least approximately 7%, at least approximately 8%, at least approximately 9%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 100%, at least approximately 200%, at least approximately 300%, at least approximately 400%, at least approximately 500%, or even more compared to control expression levels.

[0087] In some cases, single-stranded non-coding nucleic acid molecules may be expressed at up to approximately 500%, up to approximately 400%, up to approximately 300%, up to approximately 200%, up to approximately 100%, up to approximately 90%, up to approximately 80%, up to approximately 70%, up to approximately 60%, up to approximately 50%, up to approximately 40%, up to approximately 30%, up to approximately 20%, up to approximately 10%, up to approximately 9%, and up to approximately It is possible to increase the expression of target mRNA by 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 even less.

[0088] In some cases, single-stranded non-coding nucleic acid molecules can reduce the expression of target mRNA by at least or up to approximately 0.1 times, at least or up to approximately 0.2 times, at least or up to approximately 0.3 times, at least or up to approximately 0.4 times, at least or up to approximately 0.5 times, at least or up to approximately 0.6 times, at least or up to approximately 0.7 times, at least or up to approximately 0.8 times, at least or up to approximately 0.9 times, at least or up to approximately 1 time, at least or up to approximately 2 times, at least or up to approximately 3 times, at least or up to approximately 4 times, at least or up to approximately 5 times, at least or up to approximately 6 times, or less. It shows an increase of at least approximately 7 times, at least approximately 8 times, at least approximately 9 times, at least approximately 10 times, at least approximately 20 times, at least approximately 30 times, at least approximately 40 times, at least approximately 50 times, at least approximately 60 times, at least approximately 70 times, at least approximately 80 times, at least approximately 90 times, at least approximately 100 times, at least approximately 500 times, at least approximately 1,000 times, at least approximately 5,000 times, or at least approximately 10,000 times.

[0089] In some cases, single-stranded non-coding nucleic acid molecules increase the expression of target mRNA by at most about 10,000 times or less than about 10,000 times, at most about 5,000 times or less than about 5,000 times, at most about 1,000 times or less than about 1,000 times, at most about 500 times or less than about 500 times, at most about 100 times or less than about 100 times, at most about 90 times or less than about 90 times, at most about 80 times or less than about 80 times, at most about 70 times or less than about 70 times, at most about 60 times or less than about 60 times, at most about 50 times or less than about 50 times, at most about 40 times or less than about 40 times, at most about 30 times or less than about 30 times, at most about 20 times or less than about 20 times, at most about 10 times or less than about 10 times, at most about 9 This indicates an increase of less than two times or approximately nine times, at most about eight times or less than eight times, at most about seven times or less than seven times, at most about six times or less than six times, at most about five times or less than five times, at most about four times or less than four times, at most about three times or less than three times, at most about two times or less than two times, at most about one time or less than one time, at most about 0.9 times or less than 0.9 times, at most about 0.8 times or less than 0.8 times, at most about 0.7 times or less than 0.7 times, at most about 0.6 times or less than 0.6 times, at most about 0.5 times or less than 0.5 times, at most about 0.4 times or less than 0.4 times, at most about 0.3 times or less than 0.3 times, at most about 0.2 times or less than 0.2 times, and at most about 0.1 times or less than 0.1 times.

[0090] In some cases, single-stranded non-coding nucleic acid molecules can reduce target mRNA expression by at least approximately 0.1%, at least approximately 0.2%, at least approximately 0.3%, at least approximately 0.4%, at least approximately 0.5%, at least approximately 0.6%, at least approximately 0.7%, at least approximately 0.8%, at least approximately 0.9%, at least approximately 1%, at least approximately 2%, at least approximately 3%, at least approximately 4%, at least approximately 5%, at least approximately 6%, at least approximately 7%, at least approximately 8%, at least approximately 9%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 100%, at least approximately 200%, at least approximately 300%, at least approximately 400%, at least approximately 500%, or even more compared to control expression levels.

[0091] In some cases, single-stranded non-coding nucleic acid molecules may be expressed at up to approximately 500%, up to approximately 400%, up to approximately 300%, up to approximately 200%, up to approximately 100%, up to approximately 90%, up to approximately 80%, up to approximately 70%, up to approximately 60%, up to approximately 50%, up to approximately 40%, up to approximately 30%, up to approximately 20%, up to approximately 10%, up to approximately 9%, and up to approximately The expression of the target mRNA can be reduced by 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 even less.

[0092] In some cases, single-stranded non-coding nucleic acid molecules can reduce the expression of target mRNA by at least or up to approximately 0.1 times, at least or up to approximately 0.2 times, at least or up to approximately 0.3 times, at least or up to approximately 0.4 times, at least or up to approximately 0.5 times, at least or up to approximately 0.6 times, at least or up to approximately 0.7 times, at least or up to approximately 0.8 times, at least or up to approximately 0.9 times, at least or up to approximately 1 time, at least or up to approximately 2 times, at least or up to approximately 3 times, at least or up to approximately 4 times, at least or up to approximately 5 times, at least or up to approximately 6 times, or less. It can be reduced by or up to approximately 7 times, at least or up to approximately 8 times, at least or up to approximately 9 times, at least or up to approximately 10 times, at least or up to approximately 20 times, at least or up to approximately 30 times, at least or up to approximately 40 times, at least or up to approximately 50 times, at least or up to approximately 60 times, at least or up to approximately 70 times, at least or up to approximately 80 times, at least or up to approximately 90 times, at least or up to approximately 100 times, at least or up to approximately 500 times, at least or up to approximately 1,000 times, at least or up to approximately 5,000 times, or at least or up to approximately 10,000 times.

[0093] In some cases, single-stranded non-coding nucleic acid molecules increase the expression of target mRNA by at most about 10,000 times or less than about 10,000 times, at most about 5,000 times or less than about 5,000 times, at most about 1,000 times or less than about 1,000 times, at most about 500 times or less than about 500 times, at most about 100 times or less than about 100 times, at most about 90 times or less than about 90 times, at most about 80 times or less than about 80 times, at most about 70 times or less than about 70 times, at most about 60 times or less than about 60 times, at most about 50 times or less than about 50 times, at most about 40 times or less than about 40 times, at most about 30 times or less than about 30 times, at most about 20 times or less than about 20 times, at most about 10 times or less than about 10 times, at most about 9 It can be reduced by more than two times or about nine times, at most about eight times or less than eight times, at most about seven times or less than seven times, at most about six times or less than six times, at most about five times or less than five times, at most about four times or less than four times, at most about three times or less than three times, at most about two times or less than two times, at most about one time or less than one time, at most about 0.9 times or less than 0.9 times, at most about 0.8 times or less than 0.8 times, at most about 0.7 times or less than 0.7 times, at most about 0.6 times or less than 0.6 times, at most about 0.5 times or less than 0.5 times, at most about 0.4 times or less than 0.4 times, at most about 0.3 times or less than 0.3 times, at most about 0.2 times or less than 0.2 times, and at most about 0.1 times or less than 0.1 times.

[0094] Targeting section Disclosed herein are single-stranded non-coding nucleic acid molecules comprising one or more targeting moieties. The targeting moieties can be used to direct the single-stranded non-coding nucleic acid molecules toward target cells or target tissues. The targeting moieties may, but are not limited to, lipophilic moieties, small molecules, peptides (e.g., polypeptides, macrocyclic peptides, etc.), RNA molecules, nanoparticles, antibodies, single-domain antibodies, miniproteins, or antigen-binding fragments thereof. The targeting moieties may be specific to antigens or receptors on target cells or tissues (e.g., Asialoglycoprotein receptor (ASGPR)).

[0095] The targeting moiety can be a lipophilic moiety. The lipophilic moiety can contain one or more fatty acid groups or salts thereof. Lipids are lipids. Lipids are fatty acids and their derivatives that are insoluble in water but soluble in organic solvents. In some embodiments, the lipophilic moiety can be unsaturated. Alternatively, or in addition, the lipophilic moiety can be monosaturated. Alternatively, or in addition, the lipophilic moiety can be polysaturated. In some embodiments, the double bond of the unsaturated lipophilic moiety can be in a cis conformation. In some embodiments, the double bond of the unsaturated lipophilic moiety can be in a trans conformation. Non-limiting examples of lipophilic moieties include triglycerides, phospholipids, sterols, oils, waxes, hormones, vitamins, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-0(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, vomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0096] The targeting portion can be a low molecular weight. Low molecular weights can be sugars, amino acids, phenolic compounds, alkaloids, sterols, lipids, fatty acids, or other small compounds. Compounds can be molecules with a molecular weight of less than 1000 daltons. Alternatively, or in addition, low molecular weights are molecules with a size in the order of 1 nm.

[0097] The targeting portion can be a sugar or a sugar moiety. The sugar can be a monosaccharide, or a disaccharide, or a polysaccharide. Non-limiting examples of sugars include glucose, dextrose, fructose, galactose, sugar alcohols, pentoses, xylose, ribose, sucrose, cellulose, starch, lactose, maltose, trehalose, lactulose, cerbiose, chitobiose, glycogen, or chitin. Low molecular weight sugars can be amino sugars such as, but not limited to, acetylgalactosamine (GalNAc), N-acetylglucosamine, or sialic acid.

[0098] The targeting portion can be an antibody or its antigen-binding fragment. Antibodies, also known as immunoglobulins, are blood proteins produced to counteract specific antigens. Antibodies can be Y-shaped proteins containing variable binding sites specific to a particular epitope. Antibodies can be monoclonal antibodies, or polyclonal antibodies. Antibodies can be single-domain antibodies. Antibodies can be antibody fragments. Antibodies can be agonists, or antagonists. Alternatively, antibodies can be allosteric modulators (e.g., positive or negative allosteric modulators).

[0099] The targeting portion can be a polypeptide. Non-limiting examples of polypeptides include macrocyclic peptides, glucagon-like peptide 1 receptor (GLP1R) agonists, 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).

[0100] The targeting portion can be an RNA molecule. This RNA molecule may include an aptamer, ribozyme, hairpin RNA, siRNA, or miRNA.

[0101] Single-stranded nucleic acids can be directed to target genes, or to RNA molecules encoding target genes. Non-specific examples of 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, C1-INH (SERPING1), C3, C5, CEBPA, CERS2, CFB, Clcn7, CNGA3, CPS1, C TNS, CYP24A1, DGAT2, DMPK, DNM2, DUX4, Dystrophin, Dystrophin (Exon 44), Dystrophin (Exon 45), Dystrophin (Exon 51), Dystrophin (Exon 53), EPO, FAH, FGFR3, FOXP3, FUS, FXI, FXII, G6P, GALT, GCGR, GCK, GFAP, GHR, GOX (HAO1), GPLD1, GRANULIN, GYS1, HAMP, HBV, HBV (X ORF), hepatitis D virus protein, HFE, HMBS, HNF1A, HNF4A, HSD17B13, HSD3B7, HTT, IDS, IDUA, IL6, INHBE, insulin, IRF4, IVD, J AG1, LDHA, LDLR, LDLRAP1, Lp(a), LRAT, LRRK2, M1 / PA, MAPT, MAPT(TAU), MARC1, MBOAT7, MECP2, MFSD8, MMP7, MMUT, MUC5AC, MuRF1(TRIM63), MYOC, OPTN, NCOA5, NF1, NRARP, NRF2(NFE2L2), NXNL1, OPA1, OPTN, Ora1, ORF1Ab / N-protein , OTC, p21(CDKN1A), P27Kip1, PAH, PAX2, Pax6, PC, PCCa, PCCb, PCSK9, PD-L1, PER1, PIK3R1, PKD2, PKK, PNPLA3, POGLExamples include UT1, PPARD, PPOX, PRPF3, PRPF8, PSD3, RAGE, RDH12, RGR, RLBP1, RPE65, SARS-CoV-2 viral protein, SCN1A, SCNN1A(ENaC2), SERPINA1, SMN2, SNCA, SOD1, SOD2, STAT3, SYNGAP1, TCF4, TFEB, TGFB1, COX-2, VEGFR2, THPO, TMPRSS6, ApoC3, TRIB1, TRPV1, TSC2, TTR, UBE3A-ATS(SNHG14), UROD, VEGF, Wnt16, XDH, and YAP1.

[0102] C5, also known as complement 5, is a gene that codes for a component of the complement system, which is part of the innate immune system. C5 can be involved in inflammation, homeostasis, and defense against pathogens. The C5 protein consists of C5 alpha and beta chains, which are linked by disulfide crosslinks. Mutations in the C5 gene can cause complement component 5 deficiency, a disease characterized by recurrent bacterial infections.

[0103] Single-stranded non-coding nucleic acid molecules can be manipulated by a targeting portion that targets target cells. Target cells can be, but are not limited to, stem cells (e.g., induced pluripotent stem cells, embryonic stem cells), osteocytes, blood cells, erythrocytes, leukocytes, platelets, skin cells, fibroblasts, hepatocytes, lymphocytes, bone marrow cells, glandular cells, hepatocytes, cardiac cells, pancreatic cells, gallbladder cells, muscle cells, sperm cells, egg cells, adipocytes, nerve cells, neurons, Schwann cells, interneurons, immune cells, osteoblasts, chondroblasts, odontoblasts, cementoblasts, chondrocytes, mesenchymal cells, epithelial cells, secretory cells, germ cells, nurse cells, storage cells, pituitary cells, glial cells, stromal cells, lymphoid cells, B cells, T cells, natural killer cells, bone marrow cells, enterocrine cells, thyroid cells, parathyroid cells, sweat gland cells, mammary gland cells, pituitary cells, melanocytes, ductal cells, and photoreceptors.

[0104] The targeting portion can be used to manipulate single-stranded non-coding nucleic acid molecules and target specific tissues. Target tissues may include, but are not limited to, nerve tissue, brain tissue, spinal cord tissue, epithelial tissue, epidermal tissue, intestinal tissue, cardiac tissue, liver tissue, eye tissue, pancreatic tissue, lung tissue, bladder tissue, muscle tissue, cardiomyocyte tissue, smooth muscle tissue, skeletal muscle tissue, connective tissue, adipose tissue, bone tissue, and tendon tissue.

[0105] Diseases and Disabilities Disclosed herein are single-stranded non-coding nucleic acid molecules and their pharmaceutical formulations that, in some embodiments, are therapeutically effective in treating the diseases or disorders disclosed herein. In some embodiments, the single-stranded nucleic acids disclosed herein can be used to treat diseases or disorders in a subject. Non-limiting examples of diseases or disorders may be cancer, inflammatory diseases or disorders, metabolic diseases or disorders, cardiovascular diseases or disorders, immunodeficiency diseases or disorders, respiratory diseases or disorders, pain, gastrointestinal diseases or disorders, reproductive diseases or disorders, endocrine diseases or disorders, immune diseases or disorders, autoimmune diseases or disorders, or neurological diseases or disorders. Table 1 illustrates the relationships between non-limiting examples of target genes, non-limiting examples of target tissues, and non-limiting examples of diseases and disorders. In some embodiments, the single-stranded non-coding nucleic acid molecule includes an antisense strand configured to modulate the expression of a gene expression product expressed from a gene of interest presented in Table 1. In some embodiments, the modulation of the expression of a gene of interest is therapeutically effective in treating the indications presented in Table 1 corresponding to the gene of interest. In some embodiments, regulation can be activation or inhibition. In some embodiments, the single-stranded non-coding nucleic acid molecule further includes a targeting moiety specific to the target tissue presented in Table 1, corresponding to the gene of interest and the indication. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0106] The subject may be a biological entity containing expressed genetic material. Biological entities may be plants, animals, or microorganisms, including, for example, bacteria, viruses, fungi, and protozoa. The subject may be tissues, cells, and their offspring of a biological entity obtained in vivo or cultured in a test tube. The subject may be an animal such as a fish, bird, reptile, insect, amphibian, or mammal. Mammals may be cats, dogs, primates, apes, rodents, camelids, pigs, sheep, cattle, horses, goats, or rabbits. Mammals may be humans. The subject may be diagnosed as being at high risk of disease, or suspected of being at high risk of disease. In some cases, the subject may not necessarily be diagnosed as being at high risk of disease, or suspected of being at high risk of disease.

[0107] Pharmaceutical preparations Pharmaceutical formulations may include compositions disclosed herein. Pharmaceutical formulations may further include excipients. Excipients may be buffers, carriers, stabilizers, solubilizers, fillers, preservatives, diluents, vehicles, detergents, salts, peptides, surfactants, oligosaccharides, amino acids, adjuvants, carbohydrates, and / or bulking agents.

[0108] The pharmaceutical preparation will be exposed to the target for at least approximately 30 minutes, at least approximately 1 hour, at least approximately 2 hours, at least approximately 3 hours, at least approximately 4 hours, at least approximately 5 hours, at least approximately 6 hours, at least approximately 7 hours, at least approximately 8 hours, at least approximately 9 hours, at least approximately 10 hours, at least approximately 11 hours, at least approximately 12 hours, at least approximately 13 hours, at least approximately 14 hours, at least approximately 15 hours, at least approximately 16 hours, at least approximately 17 hours, at least approximately 18 hours, at least approximately 19 hours, at least approximately 20 hours, at least approximately 21 hours, and at least approximately 22 hours. During this time, you may stay for at least approximately 23 hours, at least approximately 24 hours, at least approximately 28 hours, at least approximately 32 hours, at least approximately 36 hours, at least approximately 40 hours, at least approximately 44 hours, at least approximately 2 days, at least approximately 3 days, at least approximately 4 days, at least approximately 5 days, at least approximately 6 days, at least approximately 7 days, at least approximately 8 days, at least approximately 9 days, at least approximately 10 days, at least approximately 11 days, at least approximately 12 days, at least approximately 13 days, at least approximately 2 weeks, at least approximately 3 weeks, at least approximately 4 weeks, or longer.

[0109] For pharmaceutical preparations, the time required for treatment is approximately 4 weeks at most, approximately 3 weeks at most, approximately 2 weeks at most, approximately 13 days at most, approximately 12 days at most, approximately 11 days at most, approximately 10 days at most, approximately 9 days at most, approximately 8 days at most, approximately 7 days at most, approximately 6 days at most, approximately 5 days at most, approximately 4 days at most, approximately 3 days at most, approximately 2 days at most, approximately 44 hours at most, approximately 40 hours at most, approximately 36 hours at most, approximately 32 hours at most, approximately 28 hours at most, approximately 24 hours at most, approximately 23 hours at most, approximately 22 hours at most, and approximately They can stay for 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 hours, at most about 4 hours, at most about 3 hours, at most about 2 hours, at most about 1 hour, at most about 30 minutes, or even shorter.

[0110] Pharmaceutical formulations may have a half-life when measured by a transcription inhibition assay. Pharmaceutical formulations may have a half-life of at least about 15 minutes, 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. During this time, the half-life may be at least approximately 23 hours, at least approximately 24 hours, at least approximately 28 hours, at least approximately 32 hours, at least approximately 36 hours, at least approximately 40 hours, at least approximately 44 hours, at least approximately 2 days, at least approximately 3 days, at least approximately 4 days, at least approximately 5 days, at least approximately 6 days, at least approximately 7 days, at least approximately 8 days, at least approximately 9 days, at least approximately 10 days, at least approximately 11 days, at least approximately 12 days, at least approximately 13 days, at least approximately 2 weeks, at least approximately 3 weeks, at least approximately 4 weeks, or even longer.

[0111] Pharmaceutical preparations are available for a maximum of approximately 4 weeks, a maximum of approximately 3 weeks, a maximum of approximately 2 weeks, a maximum of approximately 13 days, a maximum of approximately 12 days, a maximum of approximately 11 days, a maximum of approximately 10 days, a maximum of approximately 9 days, a maximum of approximately 8 days, a maximum of approximately 7 days, a maximum of approximately 6 days, a maximum of approximately 5 days, a maximum of approximately 4 days, a maximum of approximately 3 days, a maximum of approximately 2 days, a maximum of approximately 44 hours, a maximum of approximately 40 hours, a maximum of approximately 36 hours, a maximum of approximately 32 hours, a maximum of approximately 28 hours, a maximum of approximately 24 hours, a maximum of approximately 23 hours, a maximum of approximately 22 hours, a maximum of approximately 21 hours, and a maximum of approximately 2 It can have a half-life of 0 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 hours, at most about 4 hours, at most about 3 hours, at most about 2 hours, at most about 1 hour, at most about 30 minutes, at most about 15 minutes, or an even shorter half-life.

[0112] cell Provided herein are cells that contain or may be manipulated to express one or more systems disclosed herein. In some embodiments, the cells contain single-stranded nucleic acids as disclosed herein. The cells may be isolated from other sample components or reaction components. The cells may be purified. For example, a cell sample containing the cells 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 cells may be formulated into pharmaceutical compositions or formulations for the treatment of diseases or conditions presented in Table 1. The cells may be cell lines or a number of cells that contain or express one or more systems of this disclosure.

[0113] III. Method Disclosed herein, in some embodiments, are methods for producing, isolating, and / or purifying the single-stranded nucleic acid compositions disclosed herein. Also disclosed herein, in some embodiments, are methods for utilizing the compositions or pharmaceutical formulations disclosed herein to treat the diseases and disorders of interest. The methods disclosed herein may be modified by applying molecular barcodes to nucleic acid molecules.

[0114] In some embodiments, the methods of the present disclosure include purifying or isolating single-stranded non-coding nucleic acid molecules. Single-stranded non-coding nucleic acid molecules may be purified and / or isolated by the use of several processes, including, but not limited to, phenol-chloroform extraction, DNA filtration columns, salt and proteinase K treatment, and the use of silica gel membranes.

[0115] In some embodiments, the methods of the Disclosure involve delivering single-stranded non-coding nucleic acid molecules to target cells or tissues. The compositions disclosed herein can be delivered to targets by using viral vector particles (e.g., retroviruses, adenoviruses, adeno-associated viruses (AAVs), or herpes simplex viruses), cationic nanoparticles, lipid nanoparticles, cationic polymers, plasmids, cells, or by several means such as physical methods (e.g., sonication, electroporation, lipofection).

[0116] In some embodiments, the method involves enhancing or halting the expression or translation of a gene of interest by introducing a single-stranded non-coding nucleic acid molecule (e.g., ASO) of the Disclosure into target cells or tissues. In some embodiments, the antisense strand of the single-stranded non-coding nucleic acid molecule (e.g., ASO) is complementary to a target nucleic acid sequence that codes for the gene of interest or modulates the expression of the gene of interest. In some embodiments, the antisense strand of the single-stranded non-coding nucleic acid molecule (e.g., ASO) is complementary to a transcriptional enhancer, transcriptional silencer, or transcriptional promoter of the gene of interest. In some embodiments, the antisense strand of the single-stranded non-coding nucleic acid molecule (e.g., ASO) is complementary to a long non-coding RNA (IncRNA) or microRNA (miRNA) and influences the regulation of the expression of the gene of interest. In some embodiments, the antisense strand of the single-stranded non-coding nucleic acid molecule (e.g., ASO) is complementary to an intron region (before splicing) of the precursor mRNA and influences the translation of the resulting mRNA isoform. In some embodiments, the antisense strand of a single-stranded non-coding nucleic acid molecule (e.g., ASO) is complementary to the mRNA region (after splicing) and affects mRNA translation. For example, the antisense strand may halt the expression or translation of the gene of interest, or it may enhance the expression or translation of the gene of interest.

[0117] In some embodiments, the method involves enhancing or halting the expression or translation of a gene of interest by introducing a single-stranded non-coding nucleic acid molecule (e.g., ASO) of the Disclosure into target cells or tissue. In some embodiments, introducing a single-stranded non-coding nucleic acid molecule of the Disclosure into target cells or tissue enhances or halts the expression and / or translation of a gene of interest compared to introducing a synthetic nucleic acid molecule that is identical to the target cells or tissue except for being linear. In some embodiments, introducing a single-stranded non-coding nucleic acid molecule into a target cell or tissue enhances or inhibits the expression and / or translation of a target gene 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, compared to introducing a synthetic nucleic acid molecule that is otherwise identical except for being linear into the target cell or tissue. In some embodiments, introducing a single-stranded non-coding nucleic acid molecule into a target cell or tissue enhances or halts the expression and / or translation of a target gene by 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, or more, compared to introducing a synthetic nucleic acid molecule that is otherwise identical except for being linear into the target cell or tissue.In some embodiments, introducing a single-stranded non-coding nucleic acid molecule into a target cell or tissue enhances or halts the expression and / or translation of the target gene 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 even less, compared to introducing a synthetic nucleic acid molecule that is otherwise identical except for being linear.

[0118] In some embodiments, introducing a single-stranded non-coding nucleic acid molecule into a target cell or tissue enhances or halts the expression and / or translation of the target gene by at most about 100 times, at most about 90 times, at most about 80 times, at most about 70 times, at most about 60 times, at most about 50 times, at most about 40 times, at most about 30 times, at most about 20 times, at most about 10 times, at most about 90 times, at most about 8 times, at most about 7 times, at most about 6 times, at most about 5 times, at most about 4 times, at most about 3 times, at most about 2 times, or even less, compared to introducing a synthetic nucleic acid molecule that is identical to the target cell or tissue except for being linear.

[0119] In some embodiments, the method involves enhancing or halting the expression or translation of a gene of interest by introducing an adapter into a loop-type and / or cyclic single-stranded non-coding nucleic acid molecule (e.g., ASO) of the present disclosure. In some embodiments, introducing a loop-type single-stranded non-coding nucleic acid molecule containing an adapter into a target cell or tissue enhances or halts the expression and / or translation of a gene of interest compared to introducing the same synthetic nucleic acid molecule, except that it does not contain an adapter, into the target cell or tissue. In some embodiments, introducing a loop-type single-stranded non-coding nucleic acid molecule containing an adapter into target cells or tissues enhances or halts the expression and / or translation of a target gene 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, compared to introducing an identical synthetic nucleic acid molecule, except that it does not contain an adapter. In some embodiments, introducing a loop-type single-stranded non-coding nucleic acid molecule containing an adapter into a target cell or tissue enhances or halts the expression and / or translation of the gene of interest by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, or more, compared to introducing an identical synthetic nucleic acid molecule, except without the adapter, into the target cell or tissue.In some embodiments, introducing a loop-type single-stranded non-coding nucleic acid molecule containing an adapter into target cells or tissues enhances or halts the expression and / or translation of the target gene 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 even less. In some embodiments, introducing a loop-type single-stranded non-coding nucleic acid molecule containing an adapter into a target cell or tissue enhances or halts the expression and / or translation of the target gene by at most about 100 times, at most about 90 times, at most about 80 times, at most about 70 times, at most about 60 times, at most about 50 times, at most about 40 times, at most about 30 times, at most about 20 times, at most about 10 times, at most about 90 times, at most about 8 times, at most about 7 times, at most about 6 times, at most about 5 times, at most about 4 times, at most about 3 times, at most about 2 times, or even less, compared to introducing the same synthetic nucleic acid molecule, except without the adapter, into the target cell or tissue.

[0120] In some embodiments, the method involves delivering (administering) a single-stranded non-coding nucleic acid molecule to a target, where the single-stranded non-coding nucleic acid molecule exhibits less mistargeting effect in the target cells or tissues compared to a synthetic nucleic acid molecule that is otherwise identical except for being linear. In some embodiments, the mistargeting effect of the single-stranded non-coding nucleic acid molecule is reduced by 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, compared to a non-coding nucleic acid molecule that is otherwise identical except for being linear. In some embodiments, the mistargeting effect of single-stranded non-coding nucleic acid molecules is reduced by at least approximately 2, at least approximately 3, at least approximately 4, at least approximately 5, at least approximately 6, at least approximately 7, at least approximately 8, at least approximately 9, at least approximately 10, at least approximately 20, at least approximately 30, at least approximately 40, at least approximately 50, at least approximately 60, at least approximately 70, at least approximately 80, at least approximately 90, at least approximately 100, or more, in target cells or tissues compared to non-coding nucleic acid molecules that are otherwise identical except for being linear. In some embodiments, the mistargeting effect of single-stranded non-coding nucleic acid molecules is reduced 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%, and at most about 10% in target cells or tissues, or even less, compared to non-coding nucleic acid molecules that are otherwise identical except for being linear.In some embodiments, the mistargeting effect of single-stranded non-coding nucleic acid molecules is reduced by at most 100 times, at most 90 times, at most 80 times, at most 70 times, at most 60 times, at most 50 times, at most 40 times, at most 30 times, at most 20 times, at most 10 times, at most 90 times, at most 8 times, at most 7 times, at most 6 times, at most 5 times, at most 4 times, at most 3 times, at most 2 times, or even less, in target cells or tissues compared to non-coding nucleic acid molecules that are otherwise identical except for being linear.

[0121] In some embodiments, single-stranded non-coding nucleic acid molecules exhibit lower toxicity in target cells or tissues compared to synthetic nucleic acid molecules that are otherwise identical except for their linear structure. In some embodiments, the toxicity of single-stranded non-coding nucleic acid molecules is reduced by 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, compared to non-coding nucleic acid molecules that are otherwise identical. In some embodiments, the toxicity of single-stranded non-coding nucleic acid molecules is reduced by at least approximately 2, at least approximately 3, at least approximately 4, at least approximately 5, at least approximately 6, at least approximately 7, at least approximately 8, at least approximately 9, at least approximately 10, at least approximately 20, at least approximately 30, at least approximately 40, at least approximately 50, at least approximately 60, at least approximately 70, at least approximately 80, at least approximately 90, at least approximately 100, or more, in target cells or tissues compared to linear non-coding nucleic acid molecules that are otherwise identical. In some embodiments, the toxicity of single-stranded non-coding nucleic acid molecules is reduced 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%, and at most about 10% in target cells or tissues, or even less, compared to linear non-coding nucleic acid molecules that are otherwise identical.In some embodiments, the toxicity of single-stranded non-coding nucleic acid molecules is reduced to at most 100 times, at most 90 times, at most 80 times, at most 70 times, at most 60 times, at most 50 times, at most 40 times, at most 30 times, at most 20 times, at most 10 times, at most 90 times, at most 8 times, at most 7 times, at most 6 times, at most 5 times, at most 4 times, at most 3 times, at most 2 times, or even less, in target cells or tissues compared to linear non-coding nucleic acid molecules that are otherwise identical.

[0122] In some embodiments, single-stranded non-coding nucleic acid molecules exhibit even higher target nucleic acid sequence specificity in target cells or tissues compared with linear non-coding nucleic acid molecules that are otherwise identical. In some embodiments, single-stranded non-coding nucleic acid molecules have 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 even higher target nucleic acid sequence specificity in target cells or tissues compared with linear non-coding nucleic acid molecules that are otherwise identical. In some embodiments, single-stranded non-coding nucleic acid molecules have at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, and at least about 100 times higher, or even higher, target nucleic acid sequence specificity in target cells or tissues compared to linear non-coding nucleic acid molecules that are otherwise identical. In some embodiments, single-stranded non-coding nucleic acid molecules exhibit at least approximately 1,000%, at most approximately 900%, at most approximately 800%, at most approximately 700%, at most approximately 600%, at most approximately 500%, at most approximately 400%, at most approximately 300%, at most approximately 200%, at most approximately 150%, at most approximately 10%, at most approximately 90%, at most approximately 80%, at most approximately 70%, at most approximately 60%, at most approximately 50%, at most approximately 40%, at most approximately 30%, at most approximately 20%, and at most approximately 10% higher, or even higher, target nucleic acid sequence specificity in target cells or tissues compared to linear non-coding nucleic acid molecules that are otherwise identical.In some embodiments, single-stranded non-coding nucleic acid molecules have a target nucleic acid sequence specificity that is at most about 100 times, at most about 90 times, at most about 80 times, at most about 70 times, at most about 60 times, at most about 50 times, at most about 40 times, at most about 30 times, at most about 20 times, at most about 10 times, at most about 90 times, at most about 8 times, at most about 7 times, at most about 6 times, at most about 5 times, at most about 4 times, at most about 3 times, and at most about 2 times higher in target cells or tissues compared to linear non-coding nucleic acid molecules that are otherwise identical.

[0123] In some embodiments, single-stranded non-coding nucleic acid molecules exhibit higher stability in vivo compared to linear non-coding nucleic acid molecules that are identical except for their linear shape (Figure 9A). In some embodiments, single-stranded non-coding nucleic acid molecules have approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 100%, at least approximately 150%, at least approximately 200%, at least approximately 300%, at least approximately 400%, at least approximately 500%, at least approximately 600%, at least approximately 700%, at least approximately 800%, at least approximately 900%, and at least approximately 1000% higher or even higher stability in vivo compared to linear non-coding nucleic acid molecules that are identical except for their linear shape. In some embodiments, single-stranded non-coding nucleic acid molecules have at least approximately 2 times, at least approximately 3 times, at least approximately 4 times, at least approximately 5 times, at least approximately 6 times, at least approximately 7 times, at least approximately 8 times, at least approximately 9 times, at least approximately 10 times, at least approximately 20 times, at least approximately 30 times, at least approximately 40 times, at least approximately 50 times, at least approximately 60 times, at least approximately 70 times, at least approximately 80 times, at least approximately 90 times, and at least approximately 100 times higher stability in vivo compared to linear non-coding nucleic acid molecules that are otherwise identical. In some embodiments, single-stranded non-coding nucleic acid molecules 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 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%, and at most about 10% higher stability in vivo compared to linear non-coding nucleic acid molecules that are otherwise identical.In some embodiments, the toxicity of single-stranded non-coding nucleic acid molecules is at most about 100 times, at most about 90 times, at most about 80 times, at most about 70 times, at most about 60 times, at most about 50 times, at most about 40 times, at most about 30 times, at most about 20 times, at most about 10 times, at most about 90 times, at most about 8 times, at most about 7 times, at most about 6 times, at most about 5 times, at most about 4 times, at most about 3 times, and at most about 2 times higher in vivo compared to linear non-coding nucleic acid molecules that are identical except for their linear structure, or they have even less but higher stability.

[0124] In some embodiments, single-stranded non-coding nucleic acid molecules exhibit greater in vitro stability compared to linear non-coding nucleic acid molecules that are otherwise identical (Figures 7A-8B). In some embodiments, single-stranded non-coding nucleic acid molecules have approximately 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, and at least 1000% higher or even greater stability compared to linear non-coding nucleic acid molecules that are otherwise identical. In some embodiments, single-stranded non-coding nucleic acid molecules have at least approximately 2 times, at least approximately 3 times, at least approximately 4 times, at least approximately 5 times, at least approximately 6 times, at least approximately 7 times, at least approximately 8 times, at least approximately 9 times, at least approximately 10 times, at least approximately 20 times, at least approximately 30 times, at least approximately 40 times, at least approximately 50 times, at least approximately 60 times, at least approximately 70 times, at least approximately 80 times, at least approximately 90 times, and at least approximately 100 times higher stability in vitro compared to linear non-coding nucleic acid molecules that are otherwise identical. In some embodiments, single-stranded non-coding nucleic acid molecules 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 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%, and at most about 10% higher stability in vitro compared to linear non-coding nucleic acid molecules that are otherwise identical.In some embodiments, single-stranded non-coding nucleic acid molecules have at most about 100 times, at most about 90 times, at most about 80 times, at most about 70 times, at most about 60 times, at most about 50 times, at most about 40 times, at most about 30 times, at most about 20 times, at most about 10 times, at most about 90 times, at most about 8 times, at most about 7 times, at most about 6 times, at most about 5 times, at most about 4 times, at most about 3 times, and at most about 2 times higher stability in vitro compared to linear non-coding nucleic acid molecules that are identical except for their linear shape.

[0125] In some embodiments, the methods of the present disclosure include treating a disease or condition in a subject by administering a double-stranded non-coding nucleic acid molecule to the subject under conditions sufficient to modulate the expression of a target gene (e.g., Table 1) associated with the disease or condition. In some embodiments, the methods of the present disclosure include treating a disease or condition in a subject by administering a single-stranded non-coding nucleic acid molecule to the subject under conditions sufficient to modulate the expression of a target gene (e.g., Table 1) associated with the disease or condition. In some embodiments, the administration may be orally, intrathecal, percutaneously, rectally, sublingually, intranasally, intravitreously, subcutaneously, intramuscularly, percutaneously, or intravenously.

[0126] The compositions disclosed herein can be prepared into drug formulations that can be modified based on the target being treated and the mode of administration. Single-stranded nucleic acid active ingredients may constitute 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 the drug formulation. Single-stranded nucleic acid active ingredients can constitute at most approximately 95%, at most approximately 90%, at most approximately 85%, at most approximately 80%, at most approximately 75%, at most approximately 70%, at most approximately 65%, at most approximately 60%, at most approximately 55%, at most approximately 50%, at most approximately 45%, at most approximately 40%, at most approximately 35%, at most approximately 30%, at most approximately 25%, at most approximately 20%, at most approximately 15%, at most approximately 10%, at most approximately 5%, or less of these components in a drug formulation.

[0127] The intravenous administration formulations contain at least approximately 10 mg, at least approximately 15 mg, at least approximately 20 mg, at least approximately 25 mg, at least approximately 30 mg, at least approximately 35 mg, at least approximately 40 mg, at least approximately 45 mg, at least approximately 50 mg, at least approximately 60 mg, at least approximately 70 mg, at least approximately 80 mg, at least approximately 90 mg, at least approximately 100 mg, at least approximately 110 mg, at least approximately 120 mg, at least approximately 130 mg, at least approximately 140 mg, at least approximately 150 mg, at least approximately 160 mg, and less than It may be at least approximately 170 mg, at least approximately 180 mg, at least approximately 190 mg, at least approximately 200 mg, at least approximately 210 mg, at least approximately 220 mg, at least approximately 230 mg, at least approximately 240 mg, at least approximately 250 mg, at least approximately 260 mg, at least approximately 270 mg, at least approximately 280 mg, at least approximately 290 mg, at least approximately 300 mg, at least approximately 310 mg, at least approximately 320 mg, at least approximately 330 mg, at least approximately 340 mg, at least approximately 350 mg, or more.

[0128] Intravenous drug formulations are available in doses of approximately 350 mg, 340 mg, 330 mg, 320 mg, 310 mg, 300 mg, 290 mg, 280 mg, 270 mg, 260 mg, 250 mg, 240 mg, 230 mg, 220 mg, 210 mg, 200 mg, 190 mg, 180 mg, 170 mg, and many others. It can be as low as approximately 160 mg, as high as approximately 150 mg, as high as approximately 140 mg, as high as approximately 130 mg, as high as approximately 120 mg, as high as approximately 110 mg, as high as approximately 100 mg, as high as approximately 90 mg, as high as approximately 80 mg, as high as approximately 70 mg, as high as approximately 60 mg, as high as approximately 50 mg, as high as approximately 45 mg, as high as approximately 40 mg, as high as approximately 35 mg, as high as approximately 30 mg, as high as approximately 25 mg, as high as approximately 20 mg, as high as approximately 15 mg, as high as approximately 10 mg, or less.

[0129] The intravenous dosage formulations are available in the following doses: 25-50 mg, 25-100 mg, 25-150 mg, 25-200 mg, 25-250 mg, 25-300 mg, 25-300 mg, 30-50 mg, 30-100 mg, 30-150 mg, 30-200 mg, 30-250 mg, 30-300 mg, 50-100 mg, 50-150 mg, 50-200 mg, 50-250 mg, and 50-300 mg. The dosage can be in the range of 50mg-300mg, 100mg-150mg, 100mg-200mg, 100mg-250mg, 100mg-300mg, 100mg-250mg, 150mg-200mg, 150mg-250mg, 150mg-300mg, 150mg-350mg, 200-250mg, 200-300mg, 200-350mg, 250mg-300mg, 250mg-350mg, or 300mg-350mg.

[0130] Subcutaneously administered drug formulations contain at least approximately 10 mg, at least approximately 15 mg, at least approximately 20 mg, at least approximately 25 mg, at least approximately 30 mg, at least approximately 35 mg, at least approximately 40 mg, at least approximately 45 mg, at least approximately 50 mg, at least approximately 60 mg, at least approximately 70 mg, at least approximately 80 mg, at least approximately 90 mg, at least approximately 100 mg, at least approximately 110 mg, at least approximately 120 mg, at least approximately 130 mg, at least approximately 140 mg, at least approximately 150 mg, at least approximately 160 mg, and less Each may be approximately 170 mg, at least approximately 180 mg, at least approximately 190 mg, at least approximately 200 mg, at least approximately 210 mg, at least approximately 220 mg, at least approximately 230 mg, at least approximately 240 mg, at least approximately 250 mg, at least approximately 260 mg, at least approximately 270 mg, at least approximately 280 mg, at least approximately 290 mg, at least approximately 300 mg, at least approximately 310 mg, at least approximately 320 mg, at least approximately 330 mg, at least approximately 340 mg, at least approximately 350 mg, or more.

[0131] Subcutaneously administered drug formulations are available in doses of approximately 350 mg, 340 mg, 330 mg, 320 mg, 310 mg, 300 mg, 290 mg, 280 mg, 270 mg, 260 mg, 250 mg, 240 mg, 230 mg, 220 mg, 210 mg, 200 mg, 190 mg, 180 mg, 170 mg, and many others. It can be as low as approximately 160 mg, as high as approximately 150 mg, as high as approximately 140 mg, as high as approximately 130 mg, as high as approximately 120 mg, as high as approximately 110 mg, as high as approximately 100 mg, as high as approximately 90 mg, as high as approximately 80 mg, as high as approximately 70 mg, as high as approximately 60 mg, as high as approximately 50 mg, as high as approximately 45 mg, as high as approximately 40 mg, as high as approximately 35 mg, as high as approximately 30 mg, as high as approximately 25 mg, as high as approximately 20 mg, as high as approximately 15 mg, as high as approximately 10 mg, or less.

[0132] Subcutaneously administered drug formulations are available in the following dosages: 25-50 mg, 25-100 mg, 25-150 mg, 25-200 mg, 25-250 mg, 25-300 mg, 25-300 mg, 30-50 mg, 30-100 mg, 30-150 mg, 30-200 mg, 30-250 mg, 30-300 mg, 50-100 mg, 50-150 mg, 50-200 mg, 50-250 mg, 50-300 mg, The dosage can be in the range of 50mg-300mg, 100mg-150mg, 100mg-200mg, 100mg-250mg, 100mg-300mg, 100mg-250mg, 150mg-200mg, 150mg-250mg, 150mg-300mg, 150mg-350mg, 200-250mg, 200-300mg, 200-350mg, 250mg-300mg, 250mg-350mg, or 300mg-350mg.

[0133] The 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 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.

[0134] The compositions disclosed herein can be administered to a patient at a frequency of once a year or less, once every 11 months or less, once every 10 months or less, once every 9 months or less, once every 8 months or less, once every 7 months or less, once every 6 months or less, once every 5 months or less, once every 4 months or less, once every 3 months or less, once every 2 months or less, once every 4 weeks or less, once every 3 weeks or less, once every 2 weeks or less, once every 7 days or less, once every 6 days or less, once every 5 days or less, once every 4 days or less, once every 3 days or less, once every 2 days or less, once a day or less, twice a day or less, or three times a day or less.

[0135] IV. Kit Provided herein are kits comprising, in some embodiments, one or more compositions disclosed herein. In some embodiments, the kit comprises one or more single-stranded nucleic acids as disclosed herein. In some embodiments, the kit further comprises one or more cells. In some embodiments, the kit further comprises a cell medium, such as a growth medium. In some embodiments, the kit further comprises additional components of the cell medium, such as mevalonate or antibiotics. The exact properties of the components comprising the kit of the present invention depend on its intended purpose.

[0136] Instructions for use may be included in the kit. These instructions typically include tangible representations describing the techniques employed in using the kit's components to influence desired outcomes, such as generating single-stranded nucleic acids, isolating single-stranded nucleic acids, or investigating the therapeutic potential of single-stranded nucleic acids. Optionally, the kit also contains other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measurement tools, or other useful instruments, as readily recognizable to those skilled in the art.

[0137] The materials or components assembled in the kit may be provided to the user in a convenient and suitable storage manner that maintains their functionality and usefulness. For example, components may be in a dissolved, dehydrated, or lyophilized form; they may be provided at room temperature, refrigerated, or frozen temperature. Components are usually contained in suitable packaging material(s). As used herein, the term “packaging material” refers to one or more physical structures used to contain the contents of the kit, such as the compositions of the present invention. The packaging material is preferably constructed by a well-known method to provide a sterile and contaminant-free environment. The packaging material employed in the kit is one that is commonly used in gene expression assays and the administration of therapies. As used herein, the term “package” refers to a suitable solid matrix or material, such as glass, plastic, paper, or foil, that can hold individual kit components. Thus, for example, a package may be a plastic vial or tube used to contain a suitable amount of genetically encoded systems and / or cells. The packaging material generally has an external label indicating the contents and / or purpose of the kit and / or its components. [Examples]

[0138] V. Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the concepts of the present invention.

[0139] Example 1: Method for forming circular ASO / RNA Cyclic ASOs exert their biological functions by acting as transcription regulators.

[0140] Formation of circular RNA and circular ASO gapmers by T4 ligase 1 or 2r

[0141] A typical reaction consists of 10–50 μM linear ASO, 0.5–2 U of RNA ligase 2 or RNA ligase 1, and 20 U of RiboLock RNA-degrading enzyme inhibitor in 1×T4 ligase 1 or 2 buffer (50 mM Tris-HCl (pH 7.5), 2–10 mM MgCl2, 1 mM DL-dithiothreitol (DTT), 400–1000 μM adenosine triphosphate (ATP), 10% PEG8000, 1–3 M betaine). The RNA sample is pretreated at 80°C for 3 minutes and cooled to the reaction temperature at a rate of 6°C / min. The reaction is then carried out at 25°C for 2 hours, and the reaction is terminated by heating the mixture at 75°C for 10 minutes. The cyclic structure of the reaction product is confirmed by treating them with exonuclease T (5 U) at 25°C for 6 hours.

[0142] General method for cyclic ASO synthesis

[0143] Standard universal CPGs (CPGs) are used for RNA oligonucleotide synthesis. All oligonucleotides are synthesized using 2-tert-butyldimethylsilyl (TBDMS) RNA monomers, following standard RNA synthesis procedures in ABI394. The concentrations of all oligonucleotides are measured at 260 nm using a Thermo Scientific NanoDrop 2000 spectrophotometer. A Waters Xbridge OST C18 column (2.5 μm, 10.0 × 50 mm) is used. High-performance liquid chromatography (HPLC) is performed using an Alliance e2695 system. MS data is acquired using a Waters Xevo G2 Q-TOF spectrometer with ESI. Diethyl pyrocarbonate (DEPC) treated water is used for all solutions and HPLC purification.

[0144] Synthesis and Purification of Cyclic ASOs

[0145] The RNA is dissolved in water to prepare a final 100 μM RNA solution. The final composition of the reaction mixture for RNA cyclization is as follows: 7 μL RNA solution, 1 μL 10 mM ATP, 1 μL 10× reaction buffer, and 1 μL T4 RNA ligase (10 U / μL). 10 μL / tube of the solution is placed in PCR at 4°C for 12 hours. After mixing these liquids together, the crude product is mixed with 6× RNA loading buffer (0.25% bromophenol blue and 30% glycerol in DEPC-treated water). The solution (18 μL / well) is loaded onto a 20% natural polyacrylamide PAGE (1 mm thick) gel. The gel is then electrophoresed for 50 minutes at 220 V using 1× Tris-boric acid-EDTA (TBE) buffer (pH 8.2). For each preparative gel, the sample lanes on both sides of the gel are cut, stained with 1× SYBR gold (Invitrogen), and then imaged. The image was printed to the same size as the gel, which allowed marking the gel location without SYBR gold staining. The gel strip at the marked location was cut out, crushed into small particles, and immersed overnight in 1×TBE buffer at 37°C. After filtering off the solid particles, the RNA solution was desalted and concentrated using a Millipore-Amicon Ultra-0.5 mL centrifuge filter (cutoff = 3,000). The recovered product was freeze-dried to remove water and obtain the final circular single-stranded RNA.

[0146] RNA oligonucleotides were characterized using ESI-MS, and the oligonucleotide (approximately 0.2 nmol) was dissolved in water / acetonitrile (50:50, 20 μL) containing 1% triethylamine to obtain a final concentration of 10 μM. Next, Waters Xevo The solution was analyzed using ESI in negative ion mode with a G2 Q-Tof spectrometer. The molecular weight of cyclic ASO / RNA is 18 lower than that of linear RNA due to the condensation reaction.

[0147] Dissolve circular ASO / RNA in 1×PBS buffer to prepare a 6 μmol stock solution. Mix 10 μL of the stock solution with an equal amount of complementary RNA having 5'-phosphate modification to form circular ASO / RNA. Anneal the RNA by heating at 85°C for 5 minutes, then cool to room temperature for at least 1 hour before use.

[0148] Enzyme stability of cyclic ASO

[0149] Cyclic ASO or control linear ASO (3 μM, 5 μL) is incubated in enzyme solution at 37°C to obtain a final concentration of 1 μmol / L (15 μL). RNA-degrading enzymes are used for enzyme stability in this test. 3 μL aliquots (containing 3 pmol of siRNA) are divided equally at different time points (2, 4, 6, and 8 hours), immediately frozen in liquid nitrogen, and then stored at -80°C until assay. 1 μL of 6× RNA loading buffer is added to the aliquots. The samples are electrophoresed on a 10% natural polyacrylamide gel in TBE buffer according to the procedure described above.

[0150] chemical synthesis

[0151] A cyclic ASO containing disulfide bonds is chemically synthesized as follows. A passenger chain with disulfide bonds to protecting groups at both ends is obtained. A passenger chain with disulfide bonds at both ends is synthesized using the thiol modifier C6 SS amidite and the 3'-thiol modifier C3 SS CPG at the 5' and 3' ends, respectively. To deprotect both end protecting groups, 50 mM dithiothreitol (DTT) in Tris buffer is added to the passenger chain solution, and the mixture is incubated at room temperature for 18 hours. The reaction solution is purified using a NAP-10 column (GE Healthcare) to obtain a passenger chain with thiol groups at both ends in aqueous solution. A cyclic passenger chain is prepared by adding 5 equivalents of 2-(methoxythio)-3-nitropyridine (Npys-OMe) and acetonitrile (MeCN / water = 1 / 3). The mixture is then incubated at room temperature for 48 hours, concentrated by centrifugation, and purified using a Shimadzu reverse-phase preparative high-performance liquid chromatography (HPLC) system. Liquid chromatography is performed using an XBridge C18 column (Waters; 5 μm, 4.6 × 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% to 50% buffer B at a flow rate of 1 mL / min at 60°C for 0 to 22.0 minutes. The purified oligonucleotides are collected, desalted through a NAP-10 column (GE Healthcare), and then concentrated by centrifugation. The aqueous solution of the resulting disulfide-bonded cyclic passenger chain and the aqueous solution of the guide chain purchased from GeneDesign, Inc. are mixed in equimolar amounts, and the required amount of D-PBS(-) (Nacalai Tesque) is added. The mixture is placed in a heating block preheated to 85°C and incubated for 5 minutes. The solution is then gently cooled to room temperature.

[0152] The chemical synthesis of an incleavable cyclic ASO is carried out as follows: A passenger chain containing a 5'-hexynyl phosphoramidite (Glen Research) at the 5' end and an azide-modified CPG (PRMETECH ALC) at the 3' end is purchased from GeneDesign, Inc. An incleavable cyclic passenger chain is synthesized. Briefly, 10 to 20 Cu wires (GIFIFILMWako Pure Chemical Corporation) are added to a solution of the passenger chain (50 μM) with NaCl (200 mM). This solution is heated to 80 °C for 3 minutes and then gently cooled to room temperature. Prior to these reactions, copper-catalyzed azide-alkyne cycloaddition (CuAAC) occurs to form a triazole. The solution is purified using a NAP-10 column (GE Healthcare) and reverse-phase preparative HPLC is performed as previously described. The purified oligonucleotide is then recovered, desalted through a NAP-10 column, and concentrated by centrifugation. Annealing should be performed as described above.

[0153] Analysis of liquid chromatography-mass spectroscopy

[0154] The purity and structure of the synthesized oligonucleotides are determined by liquid chromatography-mass spectrometry (LC-MS) using an Agilent 6120 series quadrupole LC / MS system (Agilent Technologies). Liquid chromatography is performed using an ACQUITY BEH C18 column (1.7 μm, 2.1 × 50 mm, Waters) with buffers A (8.6 mM trimethylamine and 100 mM hexafluoroisopropanol aqueous solution) and B (methanol). Oligonucleotides are separated using a linear gradient of 10% to 90% buffer B at a flow rate of 0.3 mL / min at 60°C for 0 to 18.0 minutes. To further confirm the purity and molecular size of the synthesized oligonucleotides, size exclusion chromatography (SEC) (Shimadzu High-Performance Liquid Chromatography System) is also performed. Liquid chromatography is performed using a G2000SWXL column (TOSOH; 5 μm, 7.8 × 300 mm). Oligonucleotides are separated using D-PBS(-) at a flow rate of 1 mL / min at 25°C for 20 minutes.

[0155] LC-MS / MS analysis is performed using 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 C18 column (1.7 μm, 2.1 × 50 mm) with buffers A (8.6 mM trimethylamine and 100 mM hexafluoroisopropanol aqueous solution) and B (methanol). Oligonucleotides are separated using a linear gradient of 10% to 90% buffer B at a flow rate of 0.3 mL / min for 0 to 10 minutes at 50°C. MaxEnt-1 software is used for mass spectrometry of MS data. Collision activation of selected m / z is performed with a collision energy of 25 eV.

[0156] cell culture

[0157] HeLa cells (ATCC) and RAW264.7 cells (ATCC) are maintained at 37°C in a humidified atmosphere of 5% CO2 in RPMI1640 medium (Life Technologies; A10491-01) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Similarly, HepG2 cells (ATCC), Huh-7 cells (JCRB), and L929 cells (RCB) are maintained at 37°C in a humidified atmosphere of 5% CO2 in MEM (11095-080; Gibco), Dulbecco's Modified Eagle Medium (DMEM), and Minimum Essential Medium (MEM) (11095-080; Gibco), respectively. Primary mouse hepatocytes (MSCP10, Life Technologies) were cultured in William's E medium (A1217601; Life Technologies) supplemented with a primary hepatocyte thawing and plating aid (CM3000, ThermoFisher Scientific).

[0158] In vitro knockdown assay

[0159] The in vitro knockdown assay by translocation is performed in cultured cells as follows: Prepare the oligonucleotide / RNAiMAX solution according to the manufacturer's protocol by diluting oligonucleotides and RNAiMAX (13778-075; Life Technologies) (fin. 0.2%) with Opti-MEM (31985-070; Life Technologies). Next, treat a 96-well plate (167008; Nunc) with 20 μL of the solution, then add 80 μL of cell suspension (10,000 cells / well). After gentle shaking, maintain the plate at 37°C for 24 hours in a humidified atmosphere of 5% CO2.

[0160] The in vitro knockdown assay by free uptake (gymnosis) in cultured cells is performed as follows: A 20 microliter oligonucleotide solution diluted with Opti-MEM (Life Technologies; 31985-070) is treated in a 96-well plate (167008; Nunc), followed by the addition of 80 μL of cell suspension (2,000-3,000 cells / well). After gentle shaking, the plate is maintained at 37°C for 96 hours in a humidified atmosphere of 5% CO2. For primary mouse hepatocytes, a 20 μL diluted oligonucleotide solution is treated in a 96-well collagen I multi-well microplate (#356702; Corning), followed by the addition of 80 μL of cell suspension, and incubated at 37°C for 24 hours in a humidified atmosphere of 5% CO2.

[0161] After culturing cells treated with oligonucleotides, total RNA was extracted and converted to cDNA according to the manufacturer's protocol using the SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo). The reaction conditions were as follows: 15 minutes at 37°C, 5 minutes at 50°C, 5 minutes at 98°C, and 5 minutes at 4°C. mRNA levels were evaluated by quantitative RT-PCR using the TaqMan gene expression master mix (Life Technologies), the above TaqMan probes (Table 2), and QuantStudio 12K Flex (Thermo Fisher Scientific) under reaction conditions of 2 minutes at 50°C, 10 minutes at 95°C, and 40 cycles of (15 seconds at 95°C, 1 minute at 60°C). Relative mRNA expression was quantified using the comparative Ct method.

[0162] Example 2: Method for synthesizing oligonucleotides Oligonucleotides are synthesized using a MerMed-12 DNA / RNA synthesizer. Sterile solvents / reagents from Glen Research, a 500-Å CPG solid support from Prime Synthesis, 2'-deoxy-3'-phosphoramidite from Thermo, and 2'-OMe and 2'-F nucleoside-3'-phosphoramidites from Hongene are all used as received. 2'-OMe-uridine-5'-bis-POM-(E) vinylphosphonate (VP)3'-phosphoramidite is synthesized, dissolved in 0.15 M 85% acetonitrile and dimethylformamide (DMF), and ligated in the synthesizer using standard conditions. GalNAc CPG supports are prepared. 5-bromohexyl phosphoramidite (Glen Research, catalog no. 10-1946) is dissolved in 0.15 M acetonitrile and ligated in the synthesizer using standard conditions. Alkyne CPG supports and alkyne hydroxyprolinol phosphoramidite (Y) are prepared. Low-water content acetonitrile is purchased from EMD Chemicals. A 0.6 M solution of 5-(S-ethylthio)-1H-tetrazole in acetonitrile is used as the activator. The phosphoramidite solution is 0.15 M in anhydrous acetonitrile containing 15% DMF as a cosolvent for 2'-OMe uridine and cytidine. The oxidizing agent is 0.02 M I2 in THF / pyridine / water. N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)methaneimidamide (DDTT) (0.09 M in pyridine) is used as the sulfidating agent. The detritylation agent is a 3% dichloroacetic acid (DCA) solution in dichloromethane (DCM).

[0163] Example 3: Analysis of oligonucleotide stability in plasma and liver homogenates Rat plasma (BioIVT, catalog number RAT00PL38NCXNN) and liver homogenate (BioIVT, custom-made) were diluted with a 10-fold cofactor solution to achieve final concentrations of 1 mM MgCl2, 1 mM MnCl2, and 2 mM CaCl2. SciRNA was added to 50 μl of plasma or liver homogenate to achieve a final concentration of 20 μg / ml. The reaction mixture was incubated at 37°C with gentle shaking. At each predetermined time point (0, 1, 4, 8, and 24 hours), the reaction was stopped by adding 450 μl of Clarity OTX lysis-load buffer (Phenomenex, catalog number AL0-8579) containing an internal standard (oligonucleotide U21 at a final concentration of 1 μg / ml), and the mixture was frozen at -80°C until analysis. The experiment was performed in sets of three.

[0164] Oligonucleotide concentration for LC-MS analysis is performed using a Clarity OTX 96-well solid-phase extraction plate as described by Liu et al. (49). The SPE column is first prepared with 1 ml of methanol, followed by equilibration with 2 ml of 50 mM ammonium acetate (containing 2 mM sodium azide) dissolved in HPLC-grade water. The sample is loaded onto the SPE column by applying positive pressure. The column is then washed five times with 1 ml of 50 mM ammonium acetate solution in a mixture of 50 / 50 (v / v) water and acetonitrile (pH 5.5). Finally, the oligonucleotides are eluted using an elution buffer containing 10 mM EDTA and 100 mM ammonium bicarbonate in 40 / 10 / 50 (v / v / v) acetonitrile / tetrahydrofuran / water (pH 8.8). The eluent is dried under nitrogen and resuspended in 120 μl of LC-MS-grade water for LC-MS analysis.

[0165] High-resolution mass spectrometry using Thermo Scientific Q Exactive coupled to ion-pair reverse-phase liquid chromatography (Dionex timtim 3000) (LC-HRMS) is used to perform relative quantification of modified oligonucleotides and identification of metabolites. A Waters X-BridgeBEH C8 is used for chromatographic separation. An XP column (catalog number 176002554, 130 Å, 2.5 μm, 2.1 mm × 30 mm, 80°C) was used. The injection volume and flow rate were 30 μl and 1 ml / min, respectively. Mobile phase A consisted of 16 mM triethylamine (Sigma, catalog number 471283) and 200 mM 1,1,1,3,3,3-hexafluoro-2-propanol (Fisher, catalog number 67-56-1) in LC-MS grade water (Fisher, catalog number 7732-18-5); mobile phase B was 100% methanol (Fisher, catalog number 67-56-1). The gradient was started with 1% mobile phase B and advanced to 35% B over 4.3 minutes, after which the column was equilibrated with 1% mobile phase B for 1 minute. Mass spectrometer data acquisition is performed in full scan mode with a resolution setting of 35000 and a scan range of 500-3000 m / z. The spray voltage is 2.8 kV. The auxiliary gas temperature and capillary temperature are set to 300°C.

[0166] Using the Thermo Quan browser, the area ratio of the extracted ion chromatogram (XIC) of the test oligonucleotide to the internal standard is calculated with a mass precision of 10 ppm. After LC-HRMS analysis, the data is processed using ProMass HR Deconvoulation software (Novatia, LLC) to identify the linearization and major metabolism of the modified oligonucleotide.

[0167] The half-life is calculated by monitoring the depletion of the full-length test oligonucleotide over 24 hours. The amounts of the test oligonucleotide and the internal standard are normalized to time 0 for each time point of each oligonucleotide. Linear regression is used to calculate the natural logarithm and slope of the percentage of remaining full length. The half-life is calculated using the equation: t12 = -Ln(2)k.

[0168] Thermal fusion test

[0169] Melting tests are performed using a Beckman DU800 spectrophotometer with a thermoprogrammer and a 1 cm path length quartz cell. The sample is diluted in 0.1 × PBS buffer (pH 7.4) to obtain a final oligonucleotide chain concentration of approximately 1 μM. The melting curve is monitored at 260 nm at a heating rate of 1 °C / min from 10 to 90 °C. The melting temperature (Tm) is calculated from the first derivative of the heating curve, and the reported value represents the result of two independent measurements.

[0170] NMR test

[0171] Lyophilized RNA is dissolved in a 10% 2H2O / 90% H2O mixture of 20 mM NaCl and 10 mM sodium phosphate buffer (pH 7). The final concentration of double-stranded RNA in 600 μl is in the range of 20–60 μM. All spectra are acquired at 25°C using an Agilent VNMRS 800 MHz NMR spectrometer equipped with a cold probe.

[0172] Circular dichroism spectroscopy

[0173] Circular dichroism (CD) spectra were obtained using a Jasco J-815 spectropolarimeter equipped with a Julaba F25 circulation cell. The sample was equilibrated in 1×PBS at 10°C for 5 minutes at a final double-strand concentration of 1.57 μM. This spectrum is the average of 5 scans. Spectra were collected using a fused silica cell (StaRNA 29-Q-10) at a speed of 50 nm / min with a bandwidth of 1 nm and a sampling wavelength of 0.2 nm. CD spectra were recorded at 350–200 nm at 10°C. Molar ellipticity was calculated from the equation [0] = 0 / 10Cl, where 0 is the ellipticity (mdeg), C is the molar concentration of the oligonucleotide (M), and l is the optical path length of the cell (cm).

[0174] Example 4: Chemical Synthesis Synthesis of oligonucleotides

[0175] Oligonucleotides (1 μmol scale) are synthesized in an ABI 381A or 394 DNA synthesizer using a phosphoramidite chemistry-involved cycle. Detritylation is performed with CH2Cl2 containing 2.5% DCA for 60 seconds. Coupling step: BMT (0.3 M in anhydrous acetonitrile) is used as an activator; propynyl and bromohexyl phosphoramidites (0.09 M in CH3CN) are introduced with a coupling time of 45 seconds; commercially available phosphoramidites (0.09 M in CH3CN) are introduced with a coupling time of 30 seconds. Capping is performed with acetic anhydride for 15 seconds using commercially available solutions (Cap A: Ac2O, pyridine, THF10 / 10 / 80, and Cap B: 10% N-methylimidazole in THF). Oxidation is performed for 10 seconds using a commercially available iodide solution (0.1 M I2, THF, pyridine / water 90 / 5 / 5).

[0176] General procedures for azinization

[0177] Azation of 5-hydroxyl oligonucleotides is carried out according to known methods. Azation from bromohexyl oligonucleotides is performed as follows: A solution of NaN3 (13 mg) and NaI (30 mg) in anhydrous DMF (1.5 mL) is added to the solid-supported bromohexyl oligonucleotide at 65°C for 1 hour and 15 minutes. Then, the CPG beads supporting the oligonucleotide are washed with DMF (2.1 mL) and CH2-Cl2 (5 mL) and dried under reduced pressure in a desiccator for 30 minutes.

[0178] General procedure for deprotection

[0179] Place the beads in a sealed vial and treat with concentrated aqueous ammonia (1 mL) for 24 hours at room temperature if the oligonucleotide contains a phosphate triester group, or for 2 hours at room temperature, followed by 5 hours at 55°C if the oligonucleotide contains one group. Remove the beads by filtration and evaporate the solution. Dissolve the residue in water for subsequent analysis.

[0180] General procedure for Cu(I)-catalyzed 1,3-dipolar cycloaddition

[0181] Add azide-alkyne oligonucleotide (1 μmol) to CuSO4 (0.4 equivalents, 0.4 μmol, 13.2 μL of 20 mM H2O solution), freshly prepared sodium ascorbate (2 equivalents, 2 μmol, 13.2 μL of 100 mM H2O solution) (from degassed water), methanol (100 μL), and water (23.6 μL). Wash the tube containing the resulting preparation with argon and seal it. Place the reaction mixture into a Biotage microwave synthesizer initiator, set to 100 W, with a premixing time of 30 seconds, and react at 60°C for 1 to 1.5 hours. Monitor the temperature with an internal infrared probe. Next, desalt the solution with NAP10.

[0182] Example 6: Administration of antisense oligonucleotides to rats Twenty-four passive metastatic myasthenia gravis (PTMG) model rats were randomly assigned to three groups: ASO-C5 (2.5 mg / kg) by subcutaneous injection, ASO-C5 (5 mg / kg), and a saline control. Treatment was performed 10 days, 7 days, 3 days, and on the day of PTMG induction. Blood was collected before siRNA administration and immediately before PTMG induction. PTMG was induced by administration of the rat anti-mouse muscle AChR monoclonal antibody (mAb) McAb3. McAb3 is an IgG2b isotype known to activate complement. Animals were euthanized 48 hours after PTMG induction. Muscle weakness was assessed on a standard 1-4 severity scale, with higher scores indicating more severe muscle weakness. Blood, liver, diaphragm, and anterior tibia were collected for analysis.

[0183] C5 mRNA expression is quantified by qRT-PCR. Rapidly frozen liver is ground using a 2000 Geno / Grinder (SPEx sample prep), and RNA is extracted using the RNeasy Mini kit according to the manufacturer's protocol (QIAGEN). Reverse transcription is performed according to the manufacturer's protocol (Life Technologies) to generate cDNA. Using a Roche LightCycler 480 instrument, qPCR is performed on cDNA using a rat C5 Taqman FAM probe (Life Technologies) and, as a control, a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Taqman VIC probe (Life Technologies). C5 levels are normalized against GAPDH, and the percentage of remaining C5 mRNA is calculated compared to the mean of untreated or saline-treated rats.

[0184] Rat serum samples are analyzed by semi-quantitative Western blotting. Serum is diluted 20:1 in 50 mM Tris (pH 7) with 1% SDS. Samples are electrophoresed on a 10% Bis-Tris protein gel (Life Technologies) and transferred to a PVDF membrane (Bio-Rad). A goat anti-human C5 antibody (Complement Technology) that cross-reacts with rats is used at a 1:1,000 dilution, and a fluorescently labeled donkey anti-goat antibody (LI-COR) is used as the secondary antibody. The samples are then imaged using the LI-COR Odyssey imaging system. Western blotting analysis and quantification are performed using LI-COR ImageStudio software. The percentage of residual C5 in the PTMG test is calculated by normalizing rat samples from day 8 onward against their individual pre-blood collection samples.

[0185] Example 7: Administration of antisense oligonucleotides to humans Human subjects with complement system disorders will be administered an ASO-C5 preparation subcutaneously. One month later, samples will be collected from the subjects to measure C5 levels. Additional samples will be collected to measure toxicity, ASO-C5 half-life, and immunogenicity.

[0186] Example 8: Formation of circular ASO / RNA Cyclic ASOs exert their biological functions by acting as transcription regulators.

[0187] Formation of loop-type / circular RNA by ligase enzymes

[0188] A typical reaction consists of 10–50 μM linear ASO in 1×T4 ligase 1 or 2 buffer (50 mM Tris-HCl (pH 7.5), 2–10 mM MgCl2, 1 mM DL-dithiothreitol (DTT), and 400–1000 μM adenosine triphosphate (ATP), 10% PEG8000, and 1–3 M betaine), 0.5–2 U of RNA ligase 2, RNA ligase 1, or a heat-stable ligase, and 20 U of RiboLock RNA-degrading enzyme inhibitor. The RNA sample was pretreated at 80°C for 3 minutes and cooled to the reaction temperature at a rate of 6°C / min. The ligation reaction was then carried out at 25°C–60°C for 2–4 hours, and the reaction was terminated by heating the mixture at 75°C for 10 minutes (Figure 5). The cyclic structure of the reaction products was confirmed by treating them with exonuclease T(5U) at 25°C for 6 hours (Figures 7A-7B).

[0189] Synthesis and Purification of Cyclic ASOs

[0190] Loop RNA was mixed with 2× RNA loading buffer (0.25% bromophenol blue and 95% formamide in DEPC-treated water). The sample was loaded onto a 12% urea polyacrylamide PAGE (1 mm thick) gel and electrophoresis was performed in 1× Tris-boric acid-EDTA (TBE) buffer (pH 8.2) at 100-200 V for 50 minutes. For visualization purposes, the gel was immersed in a 1× GelRED (Biotium) solution of nucleic acid dye to preferentially stain the double-stranded structures for 30 minutes, and then images were acquired using the iBright imaging system (Thermo Fisher Scientific) (Figure 5A).

[0191] For the large-scale purification of ligation products, PAGE purification of loop-type sense strands was performed using UV shadowing by exposing the gel placed on a fluorescent TLC plate to short-wavelength UV light (254 nm). The cyclized bands were excised from the gel, lysed, and incubated overnight in a 15°C tumbler with 5-10 mL of nuclease-free water. The diffused ASO was concentrated using a Millipore-Amicon Multi-15 mL centrifugal filter (cutoff = 3,000 kDa). The quality and quantity of the recovered loop-type ASO product were determined and frozen.

[0192] Enzyme stability of cyclic / loop-type ASOs

[0193] The cyclic nature of the reaction products was confirmed by exonuclease T (NEB) and / or phosphodiesterase I (PDI) (Crotalus atrox phosphodiesterase I, Sigma: P4506-100MG), and linear and loop-type ASO (10-50 pmol) were digested. The reaction was terminated by adding EDTA pH 8 to a concentration of 10-50 mM, and the products were separated on a 12% denatured urea PAGE gel and visualized by GelRED (Biotium) staining (Figures 7 and 8), a nucleic acid fluorescent dye that preferentially stains double-stranded nucleic acids.

[0194] cell culture

[0195] HepG2 cells (ATCC) were maintained in EMEM (30-2003, ATCC) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin in a humidified incubator (5% CO2, 37°C). Primary mouse hepatocytes (B129-7224F, Cell) were also used. Cells were cultured in a complete hepatocyte culture medium kit (M1265, Cell Biologics).

[0196] In vitro knockdown assay

[0197] siRNA and lipofectamine RNAiMAX (13778-075; Life Technologies) (0.3 μL / well for a 96-well plate) were added separately to 25 μL of Opti-MEM (31985-070; Life Technologies) and mixed according to the manufacturer's protocol. 50 μL of this mixture was added to a 96-well plate (167008; Nunc), and 50 μL of cell suspension (5-10,000 cells / well) was added to the lipofectamine siRNA mixture. After gentle shaking, the plates were incubated at 37°C for 24 hours in a humidified atmosphere of 5% CO2. For the quantification of target messenger RNA, total RNA was extracted and converted to cDNA using the Luna Cell Ready Lysis module (NEB E3023S) or SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo) according to the manufacturer's protocol. RNA levels were determined by single-step or two-step RT-qPCR assays. Reverse transcription using the TOYOBO kit was performed as follows: 15 minutes at 37°C, 5 minutes at 50°C, 5 minutes at 98°C, and 5 minutes at 4°C. mRNA levels of CFB and ApoB were measured by quantitative RT-PCR and / or detection by SYBR green staining using TaqMan Gene Expression Master Mix (IDT) after 40 cycles on a real-time PCR instrument, QuantStudio6 or 7. Relative mRNA expression was quantified using the comparative Ct method and / or serial dilutions and standard curves. RPL13A and / or RPB1 mRNA served as housekeeping genes for the quantification of target mRNA (Figures 9-13).

[0198] Example 9: Synthesis of oligonucleotides Unmodified and chemically modified oligonucleotides of different lengths were purchased from IDT (Coralvia, IA). Example 4: Analysis of oligonucleotide stability in plasma.

[0199] Rat serum (10–50%) (Sigma R9759) was diluted with PBS containing 1 mM MgCl2, 1 mM MnCl2, and 2 mM CaCl2. ASO was added to a final concentration of 5–50 pmol. The reaction mixture was incubated at 37°C with gentle shaking. At each time point (0, 1 / 2, 1, 2, 4, 6 hours), the reaction was stopped with 5–10 mM EDTA and / or incubated at 95°C for 5–10 minutes, followed by treatment with proteinase K at 50°C for 30 minutes, and then loaded onto urea PAGE gel as described above.

Claims

1. A synthetic nucleic acid molecule comprising a single-stranded non-coding ribonucleic acid molecule, wherein the single-stranded non-coding ribonucleic acid molecule a) A target binding sequence that is complementary to the coding or regulatory region of the target nucleic acid sequence; b) The 5' and 3' ends of the single-stranded nucleic acid, which are reversibly or irreversibly linked to form a structure of the single-stranded nucleic acid molecule without free ends; c) The synthetic nucleic acid molecule comprising one or more adapters configured to enhance the specificity of target binding between the target binding sequence of the single-stranded non-coding ribonucleic acid molecule and the target nucleic acid sequence, compared to a single-stranded non-coding ribonucleic acid molecule that is identical except for lacking one of the one or more adapters.

2. The synthetic nucleic acid molecule according to claim 1, wherein the target nucleic acid sequence is a ribonucleic acid (RNA) sequence, a deoxyribonucleic acid (DNA) sequence, a DNA / RNA hybrid sequence, or a chemically modified variant thereof.

3. The synthetic nucleic acid molecule according to claim 2, wherein the target nucleic acid sequence is the gene expression product of a gene from Table 1.

4. The synthetic nucleic acid molecule according to claim 1, wherein the target nucleic acid sequence is a gene expression product derived from a gene encoding complement factor B (CFB), complement factor C5 (C5), or ApoB.

5. The synthetic nucleic acid molecule according to claim 2, wherein the RNA sequence is a messenger RNA (mRNA) sequence.

6. The synthetic nucleic acid molecule according to claim 2, wherein the target binding sequence of the single-stranded non-coding nucleic acid molecule is complementary to an exon, intron, exon / intron junction, intron / exon junction, untranslated region, or regulatory region of the target nucleic acid sequence.

7. The synthetic nucleic acid molecule according to claim 2, wherein the single-stranded non-coding nucleic acid molecule is configured to regulate the outcome of a gene product expressed from the target nucleic acid sequence.

8. The synthetic nucleic acid molecule according to claim 2, wherein the single-stranded non-coding nucleic acid molecule is configured to regulate the level of expression of splicing events from the target nucleic acid sequence.

9. The synthetic nucleic acid molecule according to claim 7 or claim 8, wherein the regulation is an increase in the expression level.

10. The synthetic nucleic acid molecule according to claim 7 or 8, wherein the regulation is a decrease in the expression level.

11. The synthetic nucleic acid molecule according to claim 1, wherein the single-stranded non-coding nucleic acid molecule is a chemically modified nucleic acid molecule.

12. The synthetic nucleic acid molecule according to claim 1, wherein the single-stranded non-coding nucleic acid molecule contains about 16 to about 100 consecutive nucleotides.

13. The synthetic nucleic acid molecule according to claim 1, wherein the 5' end of the single-stranded non-coding nucleic acid molecule is linked to the 3' end of the single-stranded non-coding nucleic acid molecule by a linker.

14. The synthetic nucleic acid molecule according to claim 13, wherein the linker is a nucleotide linker.

15. The synthetic nucleic acid molecule according to claim 13, wherein the linker is a peptide linker.

16. The synthetic nucleic acid molecule according to claim 13, wherein the linker is a chemical linker.

17. The synthetic nucleic acid molecule according to claim 16, wherein the chemical linker is substantially cleavable under intracellular conditions.

18. The synthetic nucleic acid molecule according to claim 17, wherein the chemical linker comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-unstable linker, a peptide linker, a nucleotide linker, a glucuronide group, an azido-alkyne linker, an aldehyde-oxamine linker, a phosphorothioate-tosylated linker, a phosphate activator-mediated phosphate-hydroxyl bond, or a metal chelate ligation linker.

19. The synthetic nucleic acid molecule according to claim 17, wherein the chemical linker is substantially cleavable by an enzyme under intracellular conditions.

20. The synthetic nucleic acid molecule according to claim 17, wherein the chemical linker is substantially cleavable independently under intracellular conditions.

21. The synthetic nucleic acid molecule according to claim 16, wherein the chemical linker is substantially not cleavable under intracellular conditions.

22. The synthetic nucleic acid molecule according to claim 21, wherein the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.

23. The synthetic nucleic acid molecule according to claim 1, wherein the single-stranded non-coding nucleic acid molecule includes a targeting portion, and the targeting portion is specific to a target cell or target tissue.

24. The synthetic nucleic acid molecule according to claim 23, wherein the target cell is a hepatocyte, cardiac cell, neuron, muscle cell, blood cell, photoreceptor cell, pancreatic cell, or stem cell.

25. The synthetic nucleic acid molecule according to claim 23, wherein the target tissue is liver tissue, heart tissue, brain tissue, muscle tissue, nerve tissue, epithelial tissue, connective tissue, eye, or pancreatic tissue.

26. The synthetic nucleic acid molecule according to claim 23, wherein the targeting portion comprises a polypeptide, a macrocyclic peptide, an RNA molecule, a lipophilic portion, nanoparticles, or a small molecule.

27. The synthetic nucleic acid molecule according to claim 26, wherein the polypeptide comprises an antibody, a single-domain antibody, a miniprotein, or an antigen-binding fragment thereof.

28. The synthetic nucleic acid molecule according to claim 27, wherein the polypeptide comprises a glucagon-like peptide-1 receptor (GLP1R) agonist, 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).

29. The synthetic nucleic acid molecule according to claim 26, wherein the RNA molecule comprises an aptamer, a ribozyme, hairpin RNA, siRNA, or miRNA.

30. The synthetic nucleic acid molecule according to claim 26, wherein the lipophilic portion comprises a lipid, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-0(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, vomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)colenic acid, dimethoxytrityl, or phenoxazine.

31. The synthetic nucleic acid molecule according to claim 26, wherein the low molecular weight includes a sugar portion.

32. The synthetic nucleic acid molecule according to claim 31, wherein the sugar portion contains an amino sugar.

33. The synthetic nucleic acid molecule according to claim 32, wherein the amino sugar is N-acetylgalactosamine (GalNAc).

34. The synthetic nucleic acid molecule according to claim 23, wherein the targeting portion is specific to the antigen or receptor of the target cell or target tissue.

35. The synthetic nucleic acid molecule according to claim 34, wherein the receptor comprises an asialoclycoprotein receptor (ASGPR).

36. The synthetic nucleic acid molecule according to claim 1, wherein the single-stranded non-coding nucleic acid molecule comprises one or more nucleotides including modifications.

37. The synthetic nucleic acid molecule according to claim 36, wherein the modification includes chemical modification.

38. The synthetic nucleic acid molecule according to claim 37, wherein the chemical modification includes modification of a sugar, a phosphate backbone, or a nucleic acid base.

39. The synthetic nucleic acid molecule according to claim 38, wherein the modification of the nucleotide includes 2'-O-Me, 2'-F, 2'-MOE, N(6)-methyladenosine, 5-methylcytidine, 5-methyluridine (ribothymidine), ribose modification by a cross-linked nucleic acid, or a nucleotide having an alternative chemical structure.

40. The synthetic nucleic acid molecule according to claim 39, wherein the ribose modification by the cross-linked nucleic acid is locked nucleic acid (LNA), ethylene-crosslinked nucleic acid (ENA), or restricted ethyl-crosslinked nucleic acid (cEt).

41. The synthetic nucleic acid molecule according to claim 39, wherein the nucleotide having an alternative chemical structure is a phosphorodiamidate morpholino oligonucleotide (PMO), a thiophosphoamide, a peptide nucleic acid (PNA), tricycloDNA (tcDNA), unlocked nucleic acid (UNA), or glycol nucleic acid (GNA).

42. The synthetic nucleic acid molecule according to claim 38, wherein the modification of the phosphate backbone bond comprises a phosphodiester, a phosphorothioate isomer (Sp or Rp), a phosphoryl DMI amide diester isomer, a phosphorodithioate, a methylphosphonate, a 5'-phosphorothioate, a peptide nucleic acid, a 5'-(E)-vinylphosphonate, or a 5'-methylphosphonate.

43. The synthetic nucleic acid molecule according to claim 1, wherein the ligation of the 5' and 3' ends of the single-stranded nucleic acid allows for the inclusion of even fewer chemically modified nucleotides associated with harmful medical side effects compared to an unligated control single-stranded nucleic acid.

44. The synthetic nucleic acid molecule according to claim 43, wherein the adverse medical side effects are selected from the group consisting of a decrease in platelets, thrombocytopenia, heart rate disturbance, increased blood pressure, or increased cardiac output.

45. The synthetic nucleic acid molecule according to claim 1, wherein the one or more adapters include a peptide adapter or a polypeptide adapter, or a nucleotide adapter or an oligonucleotide adapter.

46. The synthetic nucleic acid molecule according to claim 45, wherein the peptide or polypeptide adapter is an antibody adapter.

47. The synthetic nucleic acid molecule according to claim 45, wherein the oligonucleotide adapter has a length comprising about 10 to about 25 consecutive nucleotides.

48. The synthetic nucleic acid molecule according to claim 47, comprising a sequence of nucleotides having a length of approximately 15 to approximately 20.

49. The synthetic nucleic acid molecule according to claim 1, wherein the synthetic nucleic acid molecule is isolated.

50. The synthetic nucleic acid molecule according to claim 1, wherein the synthetic nucleic acid molecule is purified and isolated.

51. The synthetic nucleic acid molecule according to claim 1, wherein the one or more adapters include sugars, lipids, peptides, antibodies, nucleotide sequences, aptamers, or combinations thereof.

52. A pharmaceutical preparation comprising a synthetic nucleic acid molecule according to any one of claims 1 to 51 and a pharmaceutically acceptable excipient, carrier, or diluent.

53. The pharmaceutical formulation according to claim 52, wherein the pharmaceutical formulation is formulated for subcutaneous administration.

54. A cell comprising a synthetic nucleic acid molecule according to any one of claims 1 to 53.

55. A method for delivering synthetic nucleic acid molecules to a target, wherein the method includes administering a single-stranded non-coding nucleic acid molecule to the target, and the single-stranded non-coding nucleic acid molecule is a) A target binding sequence that is complementary to the coding or regulatory region of the target nucleic acid sequence, b) The 5' and 3' ends of the single-stranded nucleic acid, which are reversibly or irreversibly linked to form a structure of the single-stranded nucleic acid molecule without free ends; c) comprising one or more adapters configured to enhance the specificity of target binding between the target binding sequence of the single-stranded non-coding ribonucleic acid molecule and the target nucleic acid sequence, compared to a single-stranded non-coding ribonucleic acid molecule that is identical except for lacking one of the one or more adapters; The method wherein the single-stranded non-coding nucleic acid molecule has an in vivo half-life of approximately 10 hours or more when measured using a quantitative nucleic acid detection assay.

56. The method according to claim 55, wherein the administration includes subcutaneous, intravenous, intravitreous, or intrathecal administration of the single-stranded non-coding nucleic acid molecule to the subject.

57. The method according to claim 55, wherein the administration is performed at a frequency of less than once a month.

58. A method for activating the transcription of a target gene, wherein the method includes providing a single-stranded non-coding nucleic acid molecule, a) A target binding sequence that is complementary to the coding or regulatory region of the target nucleic acid sequence, b) The 5' and 3' ends of the single-stranded nucleic acid, which are reversibly or irreversibly linked to form a structure of the single-stranded nucleic acid molecule without free ends; c) comprising one or more adapters configured to enhance the specificity of target binding between the target binding sequence of the single-stranded non-coding ribonucleic acid molecule and the target nucleic acid sequence, compared to a single-stranded non-coding ribonucleic acid molecule that is identical except for lacking one of the one or more adapters; The method comprises introducing the single-stranded non-coding nucleic acid molecule into a sample containing the target gene under conditions sufficient to activate the transcription of the target gene.

59. The method according to claim 58, wherein the single-stranded non-coding nucleic acid molecule includes an antisense strand, and includes the 5' end of the antisense strand ligated to the 3' end of the antisense strand.

60. The method according to claim 59, wherein the antisense strand includes a nucleic acid sequence complementary to the nucleic acid sequence of the target RNA, and the target RNA encodes one of the targets in Table 1.

61. The method according to claim 59, wherein the target RNA includes mRNA.

62. The method according to claim 60, wherein the nucleic acid sequence of the antisense strand is complementary to the untranslated region, intron, exon, intron / exon junction, exon / intron junction, promoter, enhancer, or regulatory element of the target RNA.

63. The method according to claim 58, wherein the single-stranded non-coding nucleic acid molecule comprises about 19 to about 27 consecutive nucleotides.

64. The method according to claim 58, wherein the single-stranded non-coding nucleic acid molecule is an RNA molecule or a chemically modified nucleic acid.

65. The method according to claim 64, wherein the single-stranded non-coding nucleic acid molecule comprises about 16 to about 100 consecutive nucleotides.

66. The method according to claim 64, wherein the 5' end of the single-stranded non-coding nucleic acid molecule is linked to the 3' end of the single-stranded non-coding nucleic acid molecule by a linker.

67. The method according to claim 66, wherein the linker is a nucleotide linker.

68. The method according to claim 66, wherein the linker is a peptide linker.

69. The method according to claim 66, wherein the linker is a chemical linker.

70. The method according to claim 69, wherein the chemical linker is substantially cleavable under intracellular conditions.

71. The method according to claim 69, wherein the chemical linker includes a disulfide bond, a photocleavable linker, a diazo linker, an acid-unstable linker, a peptide linker, a nucleotide linker, a glucuronide group, an azido-alkyne linker, an aldehyde-oxamine linker, a phosphorothioate-tosylated linker, a phosphate activator-mediated phosphate-hydroxyl bond, or a metal chelate ligation linker.

72. The method according to claim 69, wherein the chemical linker is substantially cleavable by an enzyme under intracellular conditions.

73. The method according to claim 69, wherein the chemical linker is substantially cleavable independently under intracellular conditions.

74. The method according to claim 69, wherein the chemical linker is not substantially cleavable under intracellular conditions.

75. The method according to claim 74, wherein the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer.

76. The method according to claim 58, wherein the 5' end of the single-stranded non-coding nucleic acid molecule comprises a modification including 5'-(E)-vinylphosphonate (5'-VP), 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), or 6'(phosphonoxy-butyl-sulfide)purine (6-PBuS-purine).

77. The method according to claim 58, wherein the single-stranded non-coding nucleic acid molecule includes a targeting portion, and the targeting portion is specific to a target cell or target tissue.

78. The method according to claim 77, wherein the target cell is a hepatocyte, cardiac cell, neuron, muscle cell, blood cell, photoreceptor cell, pancreatic cell, or stem cell.

79. The method according to claim 77, wherein the target tissue is liver tissue, heart tissue, brain tissue, muscle tissue, nerve tissue, epithelial tissue, connective tissue, eye, or pancreatic tissue.

80. The method according to claim 77, wherein the targeting portion comprises a polypeptide, a macrocyclic peptide, an RNA molecule, a lipophilic portion, or a low molecular weight.

81. The method according to claim 80, wherein the polypeptide comprises an antibody, a single-domain antibody, a miniprotein, or an antigen-binding fragment thereof.

82. The method according to claim 80, wherein the polypeptide comprises a glucagon-like peptide-1 receptor (GLP1R) agonist, 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).

83. The method according to claim 80, wherein the RNA molecule comprises an aptamer, a ribozyme, hairpin RNA, siRNA, or miRNA.

84. The method according to claim 80, wherein the lipophilic portion comprises lipids, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-0(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, vomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

85. The method according to claim 80, wherein the low molecular weight includes a sugar portion.

86. The method according to claim 85, wherein the sugar portion includes an amino sugar.

87. The method according to claim 86, wherein the amino sugar is N-acetylgalactosamine (GalNAc).

88. The method according to claim 77, wherein the targeting portion is specific to the antigen or receptor of the target cell or target tissue.

89. The method according to claim 88, wherein the receptor comprises an asialoglycoprotein receptor (ASGPR).

90. The method according to claim 58, wherein the single-stranded non-coding nucleic acid molecule comprises one or more nucleotides including modifications.

91. The method according to claim 90, wherein the modification includes a chemical modification.

92. The method according to claim 91, wherein the chemical modification includes modification of a sugar, a phosphate backbone, or a nucleic acid base.

93. The method according to claim 91, wherein the chemical modification of the nucleotide includes 2'-O-Me, 2'-F, 2'-MOE, N(6)-methyladenosine, 5-methylcytidine, 5-methyluridine (ribothymidine), ribose modification by cross-linked nucleic acids, or a nucleotide having an alternative chemical structure.

94. The synthetic nucleic acid molecule according to claim 93, wherein the ribose modification by the cross-linked nucleic acid is locked nucleic acid (LNA), ethylene-crosslinked nucleic acid (ENA), or restricted ethyl-crosslinked nucleic acid (cEt).

95. The synthetic nucleic acid molecule according to claim 93, wherein the nucleotide having an alternative chemical structure is a phosphorodiamidate morpholino oligonucleotide (PMO), a thiophosphoamide, a peptide nucleic acid (PNA), tricycloDNA (tcDNA), unlocked nucleic acid (UNA), or glycol nucleic acid (GNA).

96. The method according to claim 58, wherein the single-stranded non-coding nucleic acid molecule exhibits lower immunogenicity in vivo compared to a linear non-coding nucleic acid molecule that is identical to it except for its linear shape.

97. The method according to claim 96, wherein the immunogenicity in vivo is measured by an immunogenicity assay.

98. The method according to claim 58, wherein the single-stranded non-coding nucleic acid molecule exhibits lower toxicity in the subject compared to a linear non-coding nucleic acid molecule that is identical except for its linear shape.

99. The method according to claim 98, wherein toxicity is measured by subchronic or chronic toxicity testing.

100. The method according to claim 58, wherein the single-stranded non-coding nucleic acid molecule exhibits less of a mistargeting effect in the subject compared to a linear non-coding nucleic acid molecule that is identical except for its linear shape.

101. The method according to claim 100, wherein the off-target effect is measured by gene expression analysis.

102. The method according to claim 58, wherein the single-stranded non-coding nucleic acid molecule exhibits higher durability in vivo compared to a linear non-coding nucleic acid molecule that is identical to it except for its linear shape.

103. The method according to claim 102, wherein the durability in vivo is measured using a nucleic acid detection technique.

104. The method according to claim 58, wherein the single-stranded non-coding nucleic acid molecule without free ends includes a cyclized oligonucleotide.

105. The method according to claim 104, wherein the cyclized oligonucleotide constitutes a monovalent or polyvalent ASO that may or may not be separated by linker sequences of different lengths.

106. The method according to claim 105, wherein the polyvalent ASO contains two or more ASOs.

107. The method according to claim 106, wherein two or more of the polyvalent ASOs target the same sequence and / or gene.

108. The method according to claim 106, wherein two or more ASOs of the polyvalent ASO target different sequences and / or genes.

109. The method according to claim 106, wherein the two or more ASOs of the polyvalent ASO have the same mechanism of action.

110. The method according to claim 102, wherein the two or more ASOs of the polyvalent ASO have different mechanisms of action.

111. The method according to claim 58, wherein the one or more adapters include sugars, lipids, peptides, antibodies, nucleotide sequences, aptamers, or combinations thereof.

112. A synthetic nucleic acid molecule comprising a single-stranded nucleic acid molecule without free ends, wherein the single-stranded nucleic acid molecule comprises a) a functionally active oligonucleotide that targets homologous mRNA; b) Adapter elements to enhance functionality; c) The synthetic nucleic acid molecule comprising a portion reversibly or irreversibly attached to the ends of (a) and (b).

113. Furthermore, the synthetic nucleic acid molecule according to claim 112, comprising a second portion that is reversibly or irreversibly bound to the ends of (d), (a) and (b).

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