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 the United States. Provisional Patent Application No. 63 / 490,978, filed on March 17, 2023, is incorporated herein by reference in its entirety.

[0002] Background Single-stranded nucleic acid molecules (such as antisense oligonucleotides (ASOs)) are a versatile class of oligonucleotide therapeutics designed to target nucleic acid molecules such as messenger ribonucleic acid (mRNA), premRNA, and / or non-coding (intron or regulatory) regions of RNA via Watson-Crick base pairs. Broadly speaking, the mechanisms of action of these therapeutics fall into two distinct categories: stereoblocking ASOs, RNA processing, and mRNA translation, which are single-stranded oligonucleotides that are complementary to the target sequence and can modulate splicing (exon inclusion or skipping) solely through occupation. The second class of ASOs acts as guides for enzymes that can degrade mRNA (such as RNase H and Ago2) or edit target mRNA (such as ADAR) or DNA sequences (such as Cas9). The latter two are often classified as guide RNAs rather than ASOs due to their longer size. The occupation-only class of ASOs can result in the upregulation or downregulation of target mRNA through a wide range of mechanisms, including the modulation of splicing, nonsense-mediated disintegration (NMD) alteration, inhibition of miRNA function, and modulation of upstream ORFs (uORFs) or translation inhibitory elements (TIEs) (all activations), or it can lead to the downregulation of targets by inhibiting the translation mechanism or altering polyadenylation. Splice-switching oligonucleotides (SSOs) can also modulate premRNA splicing (exon inclusion or exclusion / skipping) by binding to exons, introns, or exon / intron junctions. Steriblocky by ASOs can also modulate gene expression by binding to non-protein-coding and regulatory regions of genes (such as UTR regions), thereby regulating the expression of gene expression products from target nucleic acid molecules. Single-stranded nucleotides have numerous applications, including but not limited to their use as therapeutic agents to treat a variety of diseases and disorders. These single-stranded nucleic acid ASOs can be DNA, RNA, chimeric gamma (RNA:DNA hybrid) based molecules and / or chemically modified variants thereof.

[0003] overview The embodiments disclosed herein provide a synthetic single-stranded nucleic acid molecule comprising: a single-stranded non-coding nucleic acid molecule complementary to a 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 joined to produce 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) a target binding sequence complementary to the coding or regulatory region of a target nucleic acid sequence; (b) a single-stranded nucleic acid with 5' and 3' ends reversibly or irreversibly bound, resulting in a single-stranded nucleic acid molecule structure with no free ends; and (c) one or more adapters configured to enhance the specificity of target binding between the target binding sequence and the target nucleic acid sequence of the single-stranded non-coding ribonucleic acid molecule compared to a single-stranded non-coding ribonucleic acid molecule without 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 codes for 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 comprises 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 bond, intron / exon bond, 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 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 chemical linker. In some embodiments, the chemical linker is substantially cleavable under intracellular conditions.In some embodiments, the chemical linker is 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 tosylated linker containing a phosphorotoate, 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 substantially incleavable under intracellular conditions. In some embodiments, the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer. In some embodiments, a single-stranded non-coding nucleic acid molecule includes a targeting moiety, the targeting moiety being specific to a target cell or target tissue. In some embodiments, the target cells are hepatocytes, cardiac cells, neurons, muscle cells, hematopoiesis, 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 target moiety comprises polypeptides, macrosilicul peptides, RNA molecules, lipophilic moieties, nanoparticles, or small molecules. In some embodiments, the polypeptide comprises antibodies, single-domain antibodies, miniproteins, or their antigen-binding fragments. In some embodiments, the polypeptide is glucagon-like peptide 1 receptor (GLP1R), asialoglicoprotein 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-0(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, vomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In some embodiments, the small molecule includes a sugar moiety. In some embodiments, the sugar moiety includes an amino sugar. In some embodiments, the amino sugar is N-acetylgalactosamine (GalNAc). In some embodiments, the target moiety is specific to an antigen or receptor in a target cell or target tissue. In some embodiments, the receptor comprises an Asialoglicoprotein receptor (ASGPR). In some embodiments, the single-stranded non-coding nucleic acid molecule comprises 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, the nucleotide modifications include 2'-O-Me, 2'F, 2'-MOE, N(6)-methyladenosine, 5-methylcytidine, 5-methyluridine (ribothymidine), ribose modification by crosslinked nucleic acids, or nucleotides with alternative chemical properties. In some embodiments, the ribose modification by crosslinked nucleic acids is loc nucleic acid (LNA), ethylene-crosslinked nucleic acid (ENA), or constrained ethyl-crosslinked nucleic acid (cEt). In some embodiments, nucleotides with alternative chemical properties are phosphorodiamide morpholinonucleotides (PMO), thiophosphoamides, peptide nucleic acids (PNA), tricycloDNA (tcDNA), unlocked nucleic acids (UNA), or glycol nucleic acids (GNA).In some embodiments, the modification of the phosphate backbone bond is a phosphodiester, a phosphorothioate isomer (Sp or Rp), a phosphoryl DMI amidine diester isomer, a phosphorodithioate, a methylphosphonate, a 5'-phosphothioate, a peptide nucleic acid, a 5'-(E)-vinylphosphonate, or a 5'-methylphosphonate. In some embodiments, the binding of the 5' and 3' ends of a single-stranded nucleic acid allows for a lower content of chemically modified nucleotides associated with adverse medical side effects compared to an unbound single-stranded nucleic acid of control. In some embodiments, the adverse medical side effects are selected from the group consisting of thrombocytopenia, thrombocytopenia, heart rate perturbation, increased blood pressure, or increased cardiac output. In some embodiments, one or more adapters include a peptide or polypeptide adapter, or a nucleotide or oligonucleotide adapter. In some embodiments, the peptide 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 the synthetic nucleic acid molecule and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical formulation is formulated for subcutaneous administration. In some embodiments, the cell contains the synthetic nucleic acid molecule.

[0005] The embodiments disclosed herein provide synthetic nucleic acid molecules comprising: a single-stranded non-coding ribonucleic acid molecule complementary to a 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 joined 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 a chemically modified variant thereof. In some embodiments, the target nucleic acid sequence is a gene expression product of a gene from 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 an exon, intron, exon / intron linkage, intron / exon linkage, untranslated region, or regulatory region of the target nucleic acid sequence. In some embodiments, a single-stranded non-coding nucleic acid molecule is configured to regulate the outcome of a gene product expressed from a target nucleic acid sequence. In some embodiments, the single-stranded non-coding nucleic acid molecule is 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 joined 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 is 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 tosylated linker containing a phosphorotoate, 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 substantially incleavable under intracellular conditions. In some embodiments, the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer. In some embodiments, a single-stranded non-coding nucleic acid molecule includes a targeting moiety, the targeting moiety being specific to a target cell or target tissue. In some embodiments, the target cells are hepatocytes, cardiac cells, neurons, muscle cells, hematopoiesis, 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 target moiety comprises polypeptides, macrosilicul peptides, RNA molecules, lipophilic moieties, nanoparticles, or small molecules. In some embodiments, the polypeptide comprises antibodies, single-domain antibodies, miniproteins, or their antigen-binding fragments. In some embodiments, the polypeptide is glucagon-like peptide 1 receptor (GLP1R), asialoglicoprotein 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-0(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, vomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In some embodiments, the small molecule includes a sugar moiety. In some embodiments, the sugar moiety includes an amino sugar. In some embodiments, the amino sugar is N-acetylgalactosamine (GalNAc). In some embodiments, the target moiety is specific to an antigen or receptor in a target cell or target tissue. In some embodiments, the receptor comprises an Asialoglicoprotein receptor (ASGPR). In some embodiments, the single-stranded non-coding nucleic acid molecule comprises 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, the nucleotide modifications include 2'-O-Me, 2'-F, 2'-MOE, N(6)-methyladenosine, 5-methylcytidine, 5-methyluridine (ribothymidine), ribose modifications by crosslinked nucleic acids, or nucleotides with alternative chemistry properties. In some embodiments, the ribose modifications by crosslinked nucleic acids are loc nucleic acids (LNA), ethylene-crosslinked nucleic acids (ENA), or constrained ethyl-crosslinked nucleic acids (cEt). In some embodiments, nucleotides with alternative chemistry properties are phosphorodiamide 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 bond 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, bonding of the 5' and 3' ends of single-stranded nucleic acids allows for a lower content of chemically modified nucleotides associated with adverse medical side effects compared to unbonded single-stranded nucleic acids of control. In some embodiments, adverse medical side effects are selected from the group consisting of thrombocytopenia, thrombocytopenia, heart rate perturbation, 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 as disclosed herein, and pharmaceutically acceptable excipients, carriers, or diluents. In some embodiments, the pharmaceutical formulation is formulated for subcutaneous administration. In some embodiments, the cells include synthetic nucleic acid molecules such as those disclosed herein.

[0006] Embodiments disclosed herein provide a method for delivering synthetic nucleic acid molecules to a subject, the method of targeting a single-stranded non-coding nucleic acid molecule complementary to the coding and / or regulatory regions of a target gene for the purpose of regulating the expression from the gene, generating a structure with no free ends, where the 5' and 3' ends of the single-stranded non-coding nucleic acid molecule are ligated, 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 synthetic nucleic acid molecules to a subject, the method comprising: (a) a target binding sequence complementary to the coding or regulatory region of a target nucleic acid sequence; and (b) generating a structure of a single-stranded nucleic acid molecule with no free ends, where the 5' and 3' ends of the single-stranded nucleic acid are reversibly or irreversibly bound. (c) One or more adapters configured to enhance the specificity of target binding between the target binding sequence of a single-stranded non-coding ribonucleic acid molecule and the target nucleic acid sequence, compared to the same single-stranded non-coding ribonucleic acid molecule without the adapters of 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, administration includes subcutaneous, intravenous, intravitreous, or intrathecal administration of the single-stranded non-coding ribonucleic acid molecule to a subject. In some embodiments, administration is performed less than once a month.

[0007] Aspects disclosed herein provide a method for activating the transcription of a gene of interest, the method comprising: (a) providing a single-stranded non-coding nucleic acid molecule complementary to the coding, intronic, or regulatory region of the 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 with no 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. Aspects disclosed herein provide a method for activating the transcription of a gene of interest, comprising: (a) a target binding sequence complementary to the coding or regulatory region of the target nucleic acid sequence; and (b) a single-stranded nucleic acid structure with reversibly or irreversibly bound 5' and 3' ends, thereby producing a single-stranded nucleic acid molecule structure with no free ends. (c) One or more adapters configured to enhance the specificity of target binding between the target binding sequence of a single-stranded non-coding ribonucleic acid molecule and the target nucleic acid sequence introduce the single-stranded non-coding ribonucleic acid molecule into a sample containing the gene of interest under conditions sufficient to activate the transcription of the gene of interest, compared to the same single-stranded non-coding ribonucleic acid molecule without the adapters of one or more adapters. Provides a single-stranded non-coding ribonucleic acid molecule: In some embodiments, the single-stranded non-coding ribonucleic acid molecule includes an antisense strand comprising the 5' end of the antisense strand, which is fused 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, where 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 an untranslated region, intron, exon, intron / exon bond, exon / intron bond, 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 joined 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 phosphorotoate-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 substantially incleavable 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 a modification including (E) vinyl phosphonate (5'-VP), 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), or 6'(phosphonooxybutyl-sulfide)purine (6-PBuS-purine). In some embodiments, the single-stranded non-coding nucleic acid molecule comprises a targeting moiety, the targeting moiety being 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 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 glucagon-like peptide 1 receptor (GLP1R), asialoglicoprotein receptor (ASGPR), prostate-specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), epidermal 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-0(hexadecyl)glycerol, geranyloxyhexianol, hexadecylglycerol, vomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In some embodiments, the small molecule includes a sugar moiety. In some embodiments, the sugar moiety includes an amino sugar. In some embodiments, the amino sugar is N-acetylgalactosamine (GalNAc). In some embodiments, the target moiety is specific to an antigen or receptor in a target cell or target tissue. In some embodiments, the receptor includes the Asialoglicoprotein 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 modifications include 2'-O-Me, 2'-F, 2'-MOE, N(6)-methyladenosine, 5-methylcytidine, 5-methyluridine (ribothymidine), ribose modification by crosslinked nucleic acids, or nucleotides with alternative chemistry properties. In some embodiments, ribose modification by crosslinked nucleic acids is located nucleic acid (LNA), ethylene-crosslinked nucleic acid (ENA), or constrained ethyl-crosslinked nucleic acid (cEt). In some embodiments, nucleotides with alternative chemistry properties are phosphorodiamide morpholinonucleotides (PMO), thiophosphoamide, peptide nucleic acid (PNA), tricycloDNA (tcDNA), unlocated nucleic acid (UNA), or glycol nucleic acid (GNA). In some embodiments, single-stranded non-coding nucleic acid molecules exhibit less 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 less toxicity in subjects 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 off-target effects in subjects compared to otherwise identical linear non-coding nucleic acid molecules. In some embodiments, off-target effects are measured by gene expression analysis. In some embodiments, single-stranded non-coding nucleic acid molecules exhibit improved durability in vivo compared to otherwise identical linear non-coding nucleic acid molecules. In some embodiments, durability in vivo is measured using nucleic acid detection techniques. In some embodiments, single-stranded non-coding nucleic acid molecules without free ends include cyclic oligonucleotides. In some embodiments, the cyclized oligonucleotides constitute monovalent or polyvalent ASOs that may or may not be separated by linker sequences of different lengths. In some embodiments, the polyvalent ASOs include two or more ASOs.In some embodiments, two or more ASOs in a polyvalent ASO target the same sequence and / or gene. In some embodiments, two or more ASOs in a polyvalent ASO target different sequences and / or genes. In some embodiments, two or more ASOs in a polyvalent ASO have the same mechanism of action. In some embodiments, two or more ASOs in 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; (a) a functionally active oligonucleotide targeting a congeneral mRNA; (b) an adapter element for enhancing functionality; and (c) a portion that reversibly or irreversibly binds the ends of (a) and (b). In some embodiments, the synthetic nucleic acid molecule further comprises a second portion (d) that reversibly or irreversibly binds the ends of (a) and (b).

[0009] Embedding 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 specifically and individually indicated as being incorporated by reference. To the extent that any publication, patent, or patent application incorporated by reference conflicts with any disclosure contained herein, this specification is intended to take precedence and / or supersede such conflicting material.

[0010] Novel features of the present invention are specifically described in the appended claims. A deeper understanding of the features and advantages of the present invention can be obtained by referring to the following detailed description, and its accompanying drawings, illustrating exemplary embodiments in which the principles of the present invention are utilized. [Brief explanation of the drawing]

[0011] [Figure 1A]Figure 1A shows non-limiting examples of loop-shaped antisense oligonucleotides (ASOs) according to some embodiments of this specification.

[0012] [Figure 1B] Figure 1B shows an exemplary ASO that forms a loop structure designed via an adapter element (A1) that connects the ends of the loop via J1 and J2 junction sites, facilitating irreversible and / or reversible bonding between A1 and L1.

[0013] [Figure 1C] Figure 1C shows non-restrictive examples of nucleotide backbone, ribose ring, and base modifications that can improve the stability, immunogenicity, and potency of loop-shaped ASO constructs.

[0014] [Figure 2A] Figure 2A shows non-limiting examples of various monovalent or polyvalent rounding therapeutic ASOs according to some embodiments of this specification.

[0015] [Figure 2B] Figure 2B illustrates the versatility and modularity of the designed loop ASO construct (top panel). Multiple L modules (L1-L3 in this case) can be joined via adapters (A1-A3) to form a multivalent functional loop unit capable of acting on one or more target genes.

[0016] [Figure 2C] Figure 2C shows non-restrictive examples of divalent (left) and trivalent (right) ASOs. Each ASO unit (L) of a polyvalent ASO can target an independent gene or RNA, thereby increasing the valence of the loop construct.

[0017] [Figure 2D]FIG. 2D shows a non-limiting example of a portion embedded in the adapter section of a looped construct that can act as either a linker or a cell-specific targeting moiety (e.g., an antibody, aptamer, small molecule, and lipophilic moiety) to improve looped ASO delivery, according to some embodiments of the present specification.

[0018] [Figure 3] FIG. 3 shows non-limiting examples of locked and open conformations of an ASO, according to some embodiments of the present specification.

[0019] [Figure 4A] FIG. 4A depicts a linear ASO structure with a phosphorothioate nucleotide backbone.

[0020] [Figure 4B] FIG. 4B depicts a circular ASO structure with a phosphodiester backbone.

[0021] [Figure 4C] FIG. 4C shows the chemical structures of phosphodiester bonds and phosphorothioate bonds.

[0022] [Figure 5A] FIG. 5A shows a diagram of the enzymatic cyclization of an ASO, its separation and purification on a denaturing urea PAGE gel. Note that the migration pattern of the circular ASO in PAGE (right lane) is slow. GelRed was used for visualization of open ASO and loop ASO.

[0023] [Figure 5B] FIG. 5B shows a flowchart depicting a non-limiting example of steps involved in the generation of cyclized ASO.

[0024] [Figure 6A]Figure 6A shows that the accessible open end of ASO is vulnerable to degradation by exonucleases (Pacman figure).

[0025] [Figure 6B] Figure 6B illustrates the resistance of loop / cyclized ASO to degradation by exonucleases due to the absence of open ends.

[0026] [Figure 7A] Figure 7A shows the results of experiments using linear and cyclic ASOs targeting CFB and ApoB. mRNA was treated with exonuclease T, a 3'-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-shaped ASOs and the resistance of loop-shaped ASO constructs to digestion by exonuclease T. ASOs were separated on a 12% urea PAGE gel and subsequently stained with GelRed.

[0027] [Figure 7B] Figure 7B shows a graph illustrating the resistance of loop-shaped CFB ASO after treatment with exonuclease T at the indicated time. The intensity of the intact ASO band was quantified in ImageJ and expressed as a fraction at 0 minutes. Error bars indicate the standard deviation of the three independent experiments.

[0028] [Figure 8A] Figure 8A shows that open, loop-shaped chemically modified single-strand oligonucleotides were digested by rattlesnake phosphodiesterase I (PDI), a nuclease that primarily exhibits exonuclease activity towards nucleic acids. The loop-shaped constructs showed higher resistance to digestion by PDI.

[0029] [Figure 8B]Figure 8B shows gel images of a portion of an intact open (left) or loop-shaped (right) construct after digestion with the indicated PDI time.

[0030] [Figure 9A] Figure 9A shows the absence of RNase H-mediated CFB degradation by the standard loop structure after treating human hepatoblastoma HepG2 cells with 50 nM standard CFB ASO or standard loop-shaped ASO for 24 hours. Isolated RNA was reverse transcribed, and cellular levels of CFB mRNA were quantified by probe-based RT qPCR. Rpb1 was used as a housekeeping gene for normalization of CFB transcripts.

[0031] [Figure 9B] Figure 9B shows an open canonical ASO (top) and a loop canonical (adapterless) ASO (bottom).

[0032] [Figure 10] Figure 10 shows that a manipulated loop ASO (L1-A1 loop construct) targeting CFB is functional and can target and degrade congeneral CFB mRNA. Human hepatoblastoma HepG2 cells were transfected with lipofectamine for 24 hours with 50 nM open or loop-manipulated ASO (L1-A1 containing the ASO), and the effect on mRNA knockdown was measured by probe-based RT-q-PCR.

[0033] [Figure 11A] Figure 11A shows that loop formation by standard ApoB ASO (adapterless, L1 only) deactivates the standard ApoB ASO. The panel shows the loss of RNase H-mediated ApoB degradation activity by the loop-shaped standard structure (adapterless).

[0034] [Figure 11B]Figure 11B shows that adding A1 to the L1 ApoB standard structure (operational loop ASO) restores the function of ASO, and that target ApoB mRNA in HepG2 cells is degraded 24 hours after treatment with 50 nM lipofectamine reagent (error bars indicate standard deviation).

[0035] [Figure 11C] Figure 11C summarizes the activity (function) of open normalized ASO (top), loop normalized (adapterless) ASO (center), and engineered loop ASO (L1-A1) (bottom). Experiments were performed in human hepatoblastoma HepG2 cells, and it was demonstrated that removing the A1 adapter from the loop-shaped L1 ASO structure eliminated the depletion of targeted ApoB mRNA degradation. Cells were treated with 50 nM of each ApoB ASO for 24 hours. Isolated RNA was reverse transcribed, and cellular levels of ApoB mRNA were quantified by SYBR-based RT qPCR. RPL13A was used as a housekeeping gene for normalization of ApoB transcripts.

[0036] [Figure 12] Figure 12 shows that the addition of adapter A1 restores function to the engineered loop ASO (L1-A1 loop structure) targeting ApoB, highlighting the importance of incorporating and optimizing the adapter for loop ASO function. The figure also shows 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 openly designed ApoB ASO. Primer 1 and Primer 2 are two separate amplicons of ApoB measured by SYBR green q RT-PCR. RPL13A was used as a housekeeping gene for normalization.

[0037] [Figure 13A]Figure 13A shows the activity of the multivalent engineered loop CFB ASO. Two modified loop dimer ASOs (#1 and #2) and a single trivalent ASO were tested for RNase-H-mediated depletion activity against CFB mRNA. All constructs showed activity against CFB mRNA at the tested concentration (37.5 nM ASO, 24-hour treatment in HepG2 cells).

[0038] [Figure 13B] Figure 13B shows the structural features of divalent and trivalent loop ASOs, including the L1 ASO and A1 adapter that facilitate loop formation.

[0039] [Figure 13C] Figure 13C shows the structural features of divalent and trivalent loop ASOs, including L1, L2 ASOs and A1, A2 adapters that facilitate loop formation.

[0040] [Figure 13D] Figure 13D shows the structural features of divalent and trivalent loop ASOs, including L1, L2, L3 ASOs and A1-A3 adapters that facilitate loop formation. [Modes for carrying out the invention]

[0041] Detailed explanation Drug design is a very difficult problem, especially with regard to genetic diseases. Existing single-stranded nucleic acid molecules used in gene therapy (such as antisense oligonucleotides) have limited clinical efficacy, at least in part, due to their instability in vivo and / or toxicity or off-target effects. Continuing research and advancements in oligonucleotide technologies for use in gene therapy, as described in the US App (No. 20230257745), are incorporated here as a whole, but at least in part, their instability limits clinical efficacy in vivo and / or toxicity or off-target effects. For example, antisense oligonucleotides are vulnerable to degradation by nucleases and other cellular enzymes, reducing their efficacy and potentially requiring high doses to achieve therapeutic effects. However, such existing single-stranded nucleic acid molecules (e.g., antisense oligonucleotides) lack adapters and other features, resulting in low specificity and reduced target binding efficiency.

[0042] Disclosed herein are compositions, methods, and kits comprising single-stranded non-coding nucleic acid molecules that, in some embodiments, modulate (e.g., increase or decrease) the transcription of target genes (e.g., disease-related genes) exhibiting superior durability (e.g., less instability) in vivo, lower toxicity, lower immunogenicity, or any combination thereof. In some embodiments, the single-stranded non-coding nucleic acid molecules disclosed herein include an antisense strand having bound 5' and 3' ends, 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 bound so that the single-stranded non-coding nucleic acid molecule can be linearized in vivo to induce modulation of the transcription of target genes at the site of action. Additional moieties (e.g., antibodies, RNA aptamers, small molecules, lipophilic moieties, etc.) can be conjugated to the single-stranded non-coding nucleic acid molecule to increase stability, direct targeting, reduce off-target immunogenicity, or improve the pharmacological quality of the single-stranded nucleic acid molecule.

[0043] Disclosed herein are methods for delivering single-stranded non-coding nucleic acid molecules to a subject's cells in vivo or ex vivo. In some embodiments, such delivery includes administering a double-stranded non-coding nucleic acid molecule to a subject, such as by subcutaneous administration. In some embodiments, such delivery includes administering a single-stranded non-coding nucleic acid molecule to a subject, such as by subcutaneous administration. In some embodiments, the method further includes treating a disease or condition in a subject by regulating the transcription of a target gene (e.g., a disease-related gene) with a single-stranded non-coding nucleic acid molecule.

[0044] Also disclosed herein are kits comprising the compositions and systems disclosed herein, and instructions for methods of regulating the transcription of a target gene using the single-stranded non-coding nucleic acid molecules disclosed herein. Such kits may include containers for storing the system components and instructions.

[0045] The section headings used herein are for organizational purposes only and should not be interpreted as limiting the subject matter described.

[0046] I. Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or vocabulary used herein are intended to have the same meaning as that commonly understood by an ordinary skilled worker in the art relating to the claimed subject matter. In some cases, terms that have a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from that commonly understood in the art.

[0047] Throughout this application, various embodiments may be presented in range form. It is important to understand that range form descriptions are merely for convenience and brevity and should not be interpreted as inflexible limitations on the scope of disclosure. Therefore, a range description should be considered to specifically disclose not only the individual numbers within that range, but also all possible subranges. For example, a range description of 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numbers within those ranges: 1, 2, 3, 4, 5, 6. This applies regardless of the breadth of the range.

[0048] As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context explicitly indicates otherwise. For example, the term “sample” includes multiple samples, including mixtures thereof.

[0049] The terms “determination,” “measurement,” “evaluation,” “assessment,” and “analysis” are often used interchangeably in this specification to refer to forms of measurement. These terms include determining whether an element is present or absent (e.g., detection). These terms include quantitative, qualitative, or both quantitative and qualitative determinations. Evaluations can be relative or absolute. “Detection of presence” includes determining the quantity of something present, in addition to determining whether it is present or absent depending on the context.

[0050] In this specification, the term “circular” as used in relation to single-stranded non-coding nucleic acid molecules means that the 5' and 3' ends of a single-stranded non-coding nucleic acid molecule are directly or indirectly linked to each other, regardless of the shape or conformation of the single-stranded non-coding nucleic acid molecule, such that the single-stranded non-coding nucleic acid molecule has no free ends.

[0051] The term "in vivo" is used to describe events that occur inside a subject's body.

[0052] The term "ex vivo" is used to describe events that occur outside the subject's body. An ex vivo assay is not performed on the subject; rather, it is performed on a sample separate from the subject. An example of an ex vivo assay is an assay performed on a sample, which is called an "in vitro" assay.

[0053] The term "in vitro" is used to describe the event of separation of a material from its biological source, where it is contained in a container for holding experimental reagents. In vitro assays include cell-based assays that use live or dead cells. In vitro assays may also include cell-free assays that do not use intact cells.

[0054] The term "approximately" is used herein to refer to a number that represents plus or minus 10% of that number. The term "approximately" for a range refers to the range minus 10% of the minimum value plus 10% of the maximum value.

[0055] The terms “polynucleotide,” “nucleotide,” or “nucleic acid” are used herein interchangeably to refer to polymers of nucleotides of any length, including DNA and RNA or hybrids thereof. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase. Polynucleotides may, but are not limited to, modified nucleotides such as methylated nucleotides and their analogs or non-nucleotide components. Modifications to the nucleotide structure may be given before or after the assembly of polynucleotides. Polynucleotides may be further modified after polymerization, such as by binding with labeling components.

[0056] As used herein, unless otherwise specified, “polynucleotide,” “nucleotide,” or “nucleic acid” includes double-stranded or triple-stranded nucleic acids, as well as single-stranded molecules. In double-stranded or triple-stranded nucleic acids, the nucleic acid strands do not need to be cobroad (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 5' to 3' in orientation unless otherwise specified. The methods described herein provide the production of isolated nucleic acids. The methods described herein further define the production of isolated and purified nucleic acids. “Nucleic acids” as referred herein may include 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, and at least about 125. At least approximately 150, at least approximately 175, at least approximately 200, at least approximately 225, at least approximately 250, at least approximately 275, at least approximately 300, at least approximately 325, at least approximately 350, at least approximately 375, at least approximately 400, at least approximately 425, at least approximately 450, at least approximately 475, at least approximately 500 people, at least approximately 600 people, at least approximately 700 people, at least approximately 800 people, at least approximately 900 people, at least approximately 1000 people, at least approximately 1100 people, at least approximately 1200 people, at least approximately 1300 people, at least approximately 1400 people, at least approximately 1500 people, at least approximately 1600 people, at least approximately 1700 people, at least approximately 1800 people, at least approximately 1900, at least approximately 2000, or more bases of a certain length.Furthermore, this specification includes methods for synthesizing any number of polypeptide segments encoding nucleotide sequences, including sequences encoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomal peptide synthetase (NRPS) modules and synthetic variants, polypeptide segments of other modular proteins such as antibodies, polypeptide segments from other protein families including non-coding DNA or RNA, regulatory sequences such as promoters, transcription factors, enhancers, siRNAs, shRNAs, RNAi, miRNAs, micronucleolar RNAs derived from microRNAs, or functional or structural DNA or RNA units of interest. The following are non-restrictive examples of polynucleotides: coding or non-coding regions of genes or gene fragments, intergenetic DNA, loci (gene loci) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), micronucleolar RNA, ribozymes, complementary DNA (cDNA), which is the DNA representation of mRNA, typically obtained by reverse transcription or amplification of messenger RNA (mRNA); 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.

[0057] In this specification, the term "synthesis" as used with respect to nucleic acid molecules refers to production by chemical and / or enzymatic synthesis in vitro.

[0058] As used herein, the term "cell" generally refers to a biological cell.

[0059] 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”), and optionally includes any associated regulatory regions that may be located upstream or downstream of the coding sequence, such as promoters, operators, and terminators. As used herein, a “genetic locus” is a specific location within a gene.

[0060] The terms "increase" or "growth" are used herein to generally mean an increase by a statically significant amount.

[0061] The terms "decrease" or "decrease" are used herein to generally mean a decrease by a statistically significant amount.

[0062] The terms “polypeptide,” “peptide,” and “protein” may be used interchangeably herein in relation to polymers of amino acid residues. While a protein may refer to a full-length polypeptide translated from a coding open reading frame or processed into its mature form, a polypeptide or peptide may nevertheless refer to a protein degradation or processing fragment that is uniquely or identifiablely mapped to a particular protein. A polypeptide may be a single linear polymer chain of amino acids linked by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Polypeptides may be modified, for example, by the addition of carbohydrates or phosphorylation.

[0063] In this specification, the terms “homology,” “homology,” or “homology percentage” used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence are as defined by Karlin and Altschul (Proc. Natl. Acad. Scient. USA 87: 2264-2268, 1990, modified as follows: Proc. Natl. Acad. Scient. USA 90: 5873-5877, 1993). Such formulas are incorporated into Altschul et al.’s basic local location search tool (BLAST) program. (J Mol Biol. 1990 Oct 5; 215(3):403-10; Nucleic Acid Resistance. 1997 Sep 1; 25(17):3389-402). The percentage of sequence homology can be determined using the latest version of BLAST as of the filing date of this application. The percentage of sequence identity can be determined using the latest version of BLAST as of the filing date of this application.

[0064] The term “percent (%) identity” or “percent sequence identity” in reference polypeptide sequences is the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve maximum percentage sequence identity, without considering conservative substitutions as part of sequence identity. As used herein, the term “percent (%) identity” or “percent sequence identity” in reference nucleic acid sequences is the percentage of nucleotides in a candidate sequence that are identical to nucleotides in a reference nucleic acid sequence after aligning the sequences and introducing gaps as necessary to achieve maximum percentage sequence identity. Alignment for the purpose of determining sequence identity can be achieved in various known ways using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the % amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code has been submitted with user documentation in the United States. It is registered with the Copyright Office, Washington, DC, 20559, USA. Copyright registration number TXU510087. The ALIGN-2 program is released by Genentech, Inc. in South San Francisco, California, or can be compiled from source code. The ALIGN-2 program must be compiled for use with UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0065] In this specification, the terms “target gene” or “GOI” as used interchangeably refer to a gene that encodes a gene expression product that can be detected directly or indirectly.

[0066] II. Composition Antisense oligonucleotides Disclosed herein are, in some embodiments, 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 comprising 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), which generally comprises a single-stranded nucleic acid sequence that can bind to a target nucleic acid sequence, such as another RNA (e.g., mRNA), via a Watson-Crick base pair. Single-stranded nucleic acid molecules can be synthesized. Alternatively, or in addition to the above, a single-stranded nucleic acid molecule may be a chemically modified nucleic acid molecule. A single-stranded nucleic acid molecule may contain an antisense strand that may be complementary or substantially complementary to the target nucleic acid sequence.

[0067] The target nucleic acid sequence may be a single-stranded nucleic acid such as single-stranded DNA or RNA. The target nucleic acid sequence may be coding RNA (e.g., mRNA). For example, the target nucleic acid sequence may be a gene expression product from a gene encoding complement factor B (CFB), complement factor C5 (C5), or ApoB. Alternatively, the target nucleic acid sequence may be non-coding RNA (e.g., tRNA, rRNA, microRNA, non-coding RNA, small non-coding RNA, intron, exon, intron / exon or exon / intron linkage, long non-coding RNA, small interfering RNA, or piwi-interacting RNA). The target nucleic acid sequence may be a synthetic nucleic acid sequence. Alternatively, the target nucleic acid sequence may be specific 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 the 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, regulation of the expression of a target nucleic acid sequence (e.g., in the case of an RNA target) may be therapeutically effective in treating a disease or condition. In some embodiments, the regulation of target nucleic acid sequence expression includes post-transcriptional modifications such as capping, splicing, and polyadenylation of RNA targets. In some embodiments, single-stranded nucleic acids (e.g., ASOs) regulate premRNA splicing (e.g., exon inclusion or exon exclusion) by binding to exons, introns, exon / intron bonds, or intron / exon bonds, thereby promoting or repressing splicing events. In some embodiments, steric blockage by single-stranded nucleic acids (e.g., ASOs) can regulate gene expression through binding to non-protein-coding and regulatory regions of genes, thereby modulating expression.

[0068] Oligo length A single-stranded nucleic acid (e.g., ASO) is a nucleotide with at least approximately 5 nucleotides, at least approximately 6 nucleotides, at least approximately 7 nucleotides, at least approximately 8 nucleotides, at least approximately 9 nucleotides, at least approximately 10 nucleotides, at least approximately 11 nucleotides, at least approximately 12 nucleotides, at least approximately 13 nucleotides, at least approximately 14 nucleotides, at least approximately 15 nucleotides, at least approximately 16 nucleotides, at least approximately 17 nucleotides, at least approximately 18 nucleotides, at least approximately 19 nucleotides, at least approximately 20 nucleotides, at least approximately 21 nucleotides, at least approximately 22 nucleotides, at least approximately 23 nucleotides, at least approximately 24 nucleotides, at least approximately 25 nucleotides, at least approximately 26 nucleotides, at least approximately 27 nucleotides, at least approximately 28 nucleotides, at least approximately 29 nucleotides, at least approximately 30 nucleotides, at least approximately 31 nucleotides, at least approximately 32 nucleotides, at least approximately 33 nucleotides, at least approximately 34 nucleotides, at least approximately 34 nucleotides, at least approximately 35 nucleotides, at least approximately 36 nucleotides, at least approximately 37 nucleotides, at least approximately 38 nucleotides, at least approximately 39 nucleotides, at least approximately 40 nucleotides, or more nucleotides of that length.

[0069] Single-stranded nucleic acids (such as ASOs) have at most 40 nucleotides, a maximum of approximately 39 nucleotides, a maximum of approximately 38 nucleotides, a maximum of approximately 37 nucleotides, a maximum of approximately 36 nucleotides, a maximum of approximately 35 nucleotides, a maximum of approximately 34 nucleotides, a maximum of approximately 33 nucleotides, a maximum of approximately 32 nucleotides, a maximum of approximately 31 nucleotides, a maximum of approximately 30 nucleotides, a maximum of approximately 29 nucleotides, a maximum of approximately 28 nucleotides, a maximum of approximately 27 nucleotides, a maximum of approximately 26 nucleotides, a maximum of approximately 25 nucleotides, a maximum of approximately 24 nucleotides, a maximum of approximately 23 nucleotides, a maximum of approximately 22 nucleotides, a maximum of approximately 21 nucleotides, a maximum of approximately 20 nucleotides, a maximum of approximately 19 nucleotides, a maximum of approximately 18 nucleotides, and a maximum of approximately 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 the length of the nucleotides is small.

[0070] Single-stranded nucleic acids (such as ASO) can have a length of approximately 5 to 40 nucleotides. Single-stranded nucleic acids (e.g., ASO) can have a length of approximately 5 to 35 nucleotides. Single-stranded nucleic acids (such as ASO) can have a length of approximately 5 to 30 nucleotides. Single-stranded nucleic acids (such as ASO) can have a length of approximately 5 to 25 nucleotides. Single-stranded nucleic acids (such as ASO) can have a length of approximately 5 to 20 nucleotides. Single-stranded nucleic acids (such as ASO) can have a length of approximately 5 to 15 nucleotides. Single-stranded nucleic acids (such as ASO) can have a length of approximately 5 to 10 nucleotides. Single-stranded nucleic acids (such as ASO) can have a length of approximately 10 to 40 nucleotides. Single-stranded nucleic acids (such as ASO) can have a length of approximately 15 to 40 nucleotides. Single-stranded nucleic acids (such as ASO) can have a length of approximately 20 to 40 nucleotides. Single-stranded nucleic acids (such as ASOs) can be approximately 25 to 40 nucleotides long. Single-stranded nucleic acids (such as ASOs) can be approximately 30 to 40 nucleotides long. Single-stranded nucleic acids (such as ASOs) may be approximately 35 to 40 nucleotides long.

[0071] Complementary combination Antisense oligonucleotides can form a bond complementary to the entire target strand, or they can form a bond complementary to a portion of the target nucleic acid sequence. The antisense strand can form a bond complementary to one or more portions of the target nucleic acid sequence (e.g., mRNA strand) that include the 5'UTR, 3'UTR, regulatory regions, coding sequences, introns, exons, intron / exon bonds, and / or exon / intron bonds. 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.

[0072] ASOs can form a binding complementary to part or all of the mRNA target chain. Antisense oligonucleotides may be perfectly complementary (e.g., 100% complementary) to their target chain counterparts, or they may have incomplete complementarity with the ASO to their target chain counterparts. ASO has 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 to the corresponding target chain. ASOs have at best approximately 99% complementarity, at best approximately 98% complementarity, at best approximately 97% complementarity, at best approximately 96% complementarity, at best approximately 95% complementarity, at best approximately 94% complementarity, at best approximately 93% complementarity, at best approximately 92% complementarity, at best approximately 91% complementarity, at best approximately 90% complementarity, at best approximately 85% complementarity, at best approximately 80% complementarity, at best approximately 75% complementarity, at best approximately 70% complementarity, at best approximately 65% ​​complementarity, at best approximately 60% complementarity, at best approximately 50% complementarity, or less than or equal to the corresponding target chain.

[0073] Oligoformation and linkers 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 (e.g., hairpin) conformation. Alternatively, an ASO can exist in a circular conformation (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 joined such that the antisense stand does not have free ends.

[0074] An ASO can exist as a single ASO or as an ASO composed of multiple units or modules (for example, an ASO composed of multiple ASOs) (Figures 13A-D). An ASO composed of multiple ASOs can 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 up to about 10 ASOs, up to about 9 ASOs, up to about 8 ASOs, up to about 7 ASOs, up to about 6 ASOs, up to about 5 ASOs, up to about 4 ASOs, up to about 3 ASOs, up to about 2 ASOs, or fewer. Other examples of monovalent or polyvalent ASOs are shown in Figure 2.

[0075] Polyvalent ASOs can be circularized (Figures 13A-D). In some embodiments, a circularized polyvalent ASO containing two or more ASOs can refer to a polyvalent loop ASO. In some embodiments, a cyclic polyvalent ASO containing two ASOs is an ASO loop dimer (Figures 13A-D). In some embodiments, a cyclic polyvalent ASO containing three ASOs is an ASO loop trimer (Figures 13A-D). In some embodiments, a polyvalent ASO can be linked by at least about two linkers, at least about three linkers, at least about four linkers, at least about five linkers, at least about six linkers, at least about seven linkers, at least about eight linkers, at least about nine linkers, at least about ten linkers, or more linkers. In some embodiments, the polyvalent ASO can be linked by up to about 10 linkers, up to about 9 linkers, up to about 8 linkers, up to about 7 linkers, up to about 6 linkers, up to about 5 linkers, up to about 4 linkers, up to about 3 linkers, or up to about 2 linkers.

[0076] Circular ASOs can be fabricated from linear ASOs through enzymatic or chemical reactions, by joining the 5' and 3' ends of the linear chain (Figure 1). Circular ASOs can be formed by the formation of phosphodiester bonds, joining the 5' and 3' ends of a linear antisense chain. Alternatively, circular ASOs can be formed by adding linker elements that connect the 5' and 3' ends of a linear antisense chain. The linker elements can be chemical linkers. Chemical linkers act as junctions for joining the 5' and 3' ends of a linear antisense chain to form a circular ASO. Alternatively, linkers can be used to span exon / intron junctions. Linkers may 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, phosphorotoates, tosylated linkers, phosphate-activator-mediated phosphate-hydroxyl bonds, or metal chelate ligation linkers. Nucleotide linkers may be oligonucleotide linkers, oligoaptamer linkers, RNA linkers, or DNA linkers. Peptide linkers may be polypeptide linkers (e.g., antibody linkers). Linkers may contain phosphodiester bonds, alkyl groups, sulfhydryl groups, amine groups, or polymers.

[0077] 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 by, for example, enzymes or catalysts. Enzymes with cleavage properties include, but are not limited to, proteases and endonucleases. Catalysts can be chemical catalysts (e.g., acid-based or metal catalysts) or non-chemical catalysts (e.g., light, heat, or pH). Alternatively, the linker can be cleaved using a reversible click reaction. Alternatively, the linker can be cleaved independently.

[0078] Click reactions are reactions used to combine two specific molecular entities in the absence of water. Examples of click chemistry 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, alkene-tetrazine reverse demand Diels-Alder reaction, and alkene-tetrazole photoclick reaction. Amines and thiols can be used to facilitate reversible click reactions.

[0079] Linkers can be cleaved 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 not cleavable or substantially cleavable under intracellular conditions, but cleavable or substantially cleavable under extracellular conditions. Selective cleavage may be related to conditions such as pH and ion concentration, but is not limited to these.

[0080] adapter In some embodiments, single-stranded non-coding nucleic acid molecules (e.g., ASOs) can constitute 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).

[0081] 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. The ASO may include one or more adapters (Figures 13A-D). In some embodiments, the 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 more. In some embodiments, the ASO may include up to about 10 adapters, up to about 9 adapters, up to about 8 adapters, up to about 7 adapters, up to about 6 adapters, up to about 5 adapters, up to about 4 adapters, up to about 3 adapters, up to about 2 adapters, or fewer adapters.

[0082] A single ASO (e.g., a monovalent ASO) can be circularized by comprising adapters. A monovalent ASO including adapters can be circularized by reversibly or irreversibly coupling the free end of the adapter to the free end of the ASO via linkers. A multivalent ASO, comprising two or more adapters, can be circularized (Figure 13A-D). An ASO can consist of a single adapter and one or more linkers. For example, the first free end of an adapter can be coupled to the first free end of an ASO via a first linker, and the second free end of an adapter can be coupled to the second free end of an ASO via a second linker, forming a loop ASO (Figures 13A-D). A multivalent ASO can have two or more adapters and two or more linkers (Figures 13A-D). In some embodiments, a multivalent ASO may be linked by 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 more adapters. In some embodiments, a multivalent ASO may be linked by up to about ten adapters, up to about nine adapters, up to about eight adapters, up to about seven adapters, up to about six adapters, up to about five adapters, up to about four adapters, up to about three adapters, or up to about two adapters.

[0083] The adapter is a single-stranded non-coding nucleic acid molecule (e.g., ASO). In some cases, the adapter can extend the 5' and 3' ends of a single-stranded non-coding nucleic acid molecule (e.g., ASO). The adapter is the gene of interest and / or the target gene (e.g., the adapter may contain a hybridizing region and / or sticky ends). Alternatively, the adapter may contain a non-hybridizing region (e.g., non-lateral region, blunt end, etc.). The adapter is a single-stranded non-coding nucleic acid molecule (e.g., ASO). For example, a single-stranded non-coding nucleic acid molecule (e.g., ASO) interacts with another single-stranded non-coding nucleic acid molecule (e.g., ASO) to prevent degradation by a downstream event (e.g., RNase H). Alternatively, or additionally, the adapter is a single-stranded non-coding nucleic acid molecule (e.g., ASO). The length of the adapter varies. For example, a glycan adapter may be about 5-10 glycan residues long to form a glycan. Alternatively, the antibody adapter may contain a single antibody.

[0084] In some embodiments, the adapter may be a nucleotide adapter. Nucleotide adapters (e.g., DNA, RNA, etc.) of at least approximately 5 nucleotides, at least approximately 6 nucleotides, at least approximately 7 nucleotides, at least approximately 8 nucleotides, at least approximately 9 nucleotides, at least approximately 10 nucleotides, at least approximately 11 nucleotides, at least approximately 12 nucleotides, at least approximately 13 nucleotides, at least approximately 14 nucleotides, at least approximately 15 nucleotides, at least approximately 16 nucleotides, at least approximately 17 nucleotides, at least approximately 18 nucleotides, at least approximately 19 nucleotides, at least approximately 20 nucleotides, at least approximately 21 nucleotides, at least approximately 22 nucleotides, at least approximately 23 nucleotides, at least approximately 24 nucleotides, at least approximately 25 nucleotides, at least approximately 26 nucleotides, at least approximately 27 nucleotides, at least approximately 28 nucleotides, at least approximately 29 nucleotides, at least approximately 30 nucleotides, at least approximately 31 nucleotides, at least approximately 32 nucleotides, at least approximately 33 nucleotides, at least approximately 34 nucleotides, at least approximately 34 nucleotides, at least approximately 35 nucleotides, at least approximately 36 nucleotides, at least approximately 37 nucleotides, at least approximately 38 nucleotides, at least approximately 39 nucleotides, at least approximately 40 nucleotides, or nucleotides of a length greater than or equal to 30 nucleotides.

[0085] Nucleotide adapters (e.g., DNA, RNA, etc.) up to approximately 40 nucleotides, up to approximately 39 nucleotides, up to approximately 38 nucleotides, up to approximately 37 nucleotides, up to approximately 36 nucleotides, up to approximately 35 nucleotides, up to approximately 34 nucleotides, up to approximately 33 nucleotides, up to approximately 32 nucleotides, up to approximately 31 nucleotides, up to approximately 30 nucleotides, up to approximately 29 nucleotides, up to approximately 28 nucleotides, up to approximately 27 nucleotides, up to approximately 26 nucleotides, up to approximately 25 nucleotides, up to approximately 24 nucleotides, up to approximately 23 nucleotides, up to approximately 22 nucleotides, up to approximately 21 nucleotides, up to approximately 20 nucleotides, up to approximately 19 nucleotides, up to approximately 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 less than a nucleotide of that length.

[0086] Nucleotide adapters (e.g., DNA, RNA, etc.) have a length of approximately 5 to 40 nucleotides. Nucleotide adapters (e.g., DNA, RNA, etc.) have a length of approximately 5 to 35 nucleotides. Nucleotide adapters (e.g., DNA, RNA, etc.) have a length of approximately 5 to 30 nucleotides. Nucleotide adapters (e.g., DNA, RNA, etc.) have a length of approximately 5 to 25 nucleotides. Nucleotide adapters (e.g., DNA, RNA, etc.) have a length of approximately 5 to 20 nucleotides. Nucleotide adapters (e.g., DNA, RNA, etc.) have a length of approximately 5 to 15 nucleotides. Nucleotide adapters (e.g., DNA, RNA, etc.) have a length of approximately 5 to 10 nucleotides. Nucleotide adapters (e.g., DNA, RNA, etc.) have a length of approximately 10 to 40 nucleotides. Nucleotide adapters (e.g., DNA, RNA, etc.) have a length of approximately 15 to 40 nucleotides. Nucleotide adapters (e.g., DNA, RNA, etc.) are approximately 15 to 20 nucleotides long. Nucleotide adapters (e.g., DNA, RNA, etc.) are approximately 20 to 40 nucleotides long. Nucleotide adapters (e.g., DNA, RNA, etc.) are approximately 25 to 40 nucleotides long. Nucleotide adapters (e.g., DNA, RNA, etc.) are between approximately 30 and 40 nucleotides long. Nucleotide adapters (e.g., DNA, RNA, etc.) are approximately 35 to 40 nucleotides long.

[0087] Up to a certain point, the adapter may be a peptide adapter. A peptide adapter (e.g., polypeptide) is a peptide 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 longer.

[0088] A peptide adapter (e.g., polypeptide) can contain at most about 20 peptides, a maximum of about 19 peptides, a maximum of about 18 peptides, a maximum of about 17 peptides, a maximum of about 16 peptides, a maximum of about 15 peptides, a maximum of about 14 peptides, a maximum of about 13 peptides, a maximum of about 12 peptides, a maximum of about 11 peptides, a maximum of about 10 peptides, a maximum of about 9 peptides, a maximum of about 8 peptides, a maximum of about 7 peptides, a maximum of about 6 peptides, a maximum of about 5 peptides, a maximum of about 4 peptides, a maximum of about 3 peptides, a maximum of about 2 peptides, or less than the length of a peptide.

[0089] A peptide adapter (e.g., polypeptide) can have a length of approximately 2 to approximately 20 peptides. A peptide adapter (e.g., polypeptide) can have a length of approximately 2 to approximately 18 peptides. A peptide adapter (e.g., polypeptide) can have a length between approximately 2 and approximately 16 peptides. A peptide adapter (e.g., polypeptide) can have a length of approximately 2 to approximately 14 peptides. A peptide adapter (e.g., polypeptide) can have a length between approximately 2 and approximately 12 peptides. A peptide adapter (e.g., polypeptide) can have a length between approximately 2 and approximately 10 peptides. A peptide adapter (e.g., polypeptide) can have a length between approximately 2 and approximately 8 peptides. A peptide adapter (e.g., polypeptide) can have a length between approximately 2 and approximately 6 peptides. A peptide adapter (e.g., polypeptide) can have a length between approximately 2 and approximately 4 peptides. A peptide adapter (e.g., polypeptide) can have a length between approximately 4 and approximately 20 peptides. A peptide adapter (e.g., polypeptide) can have a length between approximately 6 and approximately 20 peptides. The peptide adapter (e.g., polypeptide) may have a length of approximately 8 to approximately 20 peptides. The peptide adapter (e.g., polypeptide) may have a length of approximately 10 to approximately 20 peptides. The peptide adapter (e.g., polypeptide) may have a length of approximately 12 to approximately 20 peptides. The peptide adapter (e.g., polypeptide) may have a length of approximately 14 to approximately 20 peptides. The peptide adapter (e.g., polypeptide) may have a length of approximately 16 to approximately 20 peptides. The peptide adapter (e.g., polypeptide) may have a length of approximately 18 to approximately 20 peptides.

[0090] Adapters can enhance the functionality of ASOs. For example, the enhancement of ASO functionality by adapters is due to an increase in the steric degrees of freedom of the modified ASO, allowing it to interact unrestrictedly with proteins involved in downstream events (such as degradation by RNAse). In some embodiments, ASOs with adapters may have increased steric degrees of freedom compared to ASOs without adapters. In some embodiments, ASOs with adapters had increased steric degrees of freedom by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, and at least about 500% or more compared to ASOs without adapters. In some embodiments, ASOs with adapters showed little increase in three-dimensional degrees of freedom compared to ASOs without adapters, with increases of at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less.

[0091] In some embodiments, ASOs with adapters can enhance target specificity compared to ASOs without adapters. In some embodiments, ASOs with adapters can enhance target specificity by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, at least about 800%, at least about 900%, at least about 1,000%, or more compared to ASOs without adapters. In some embodiments, the ASO having an adapter is 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 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%. Compared to ASO without an adapter, the increase in target specificity is at most about 15%, at most about 10%, at most about 5%, or less.

[0092] In some embodiments, an ASO with an adapter may increase target binding compared to an ASO without an adapter. In some embodiments, an ASO with an adapter may increase target binding by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, at least about 800%, at least about 900%, at least about 1,000%, or more compared to an ASO without an adapter. In some embodiments, the ASO having an adapter is 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 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%. Target binding increased by up to approximately 15%, up to approximately 10%, up to approximately 5%, or less, compared to ASO without an adapter.

[0093] change 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 can be modified by adding a moiety to the molecule. The modification may be chemical, synthetic, or native.

[0094] Nucleic acid molecules can be modified with nucleic acid bases. Nucleobase modifications include 2'-O-methylation (2'-OMe), uridine to pudouridine conversion, N(6)-methyladenosine, 5-methylcytidine, 5-methyluridine (ribothymidine), 2'-fluoro (2'F), and 2'-O-methoxyethyl (2'-MOE), ribose modification and cross-linked nucleic acids (locked nucleic acid (LNA), ethylene-cross-linked nucleic acid (ENA), or restricted ethyl-cross-linked nucleic acid (cEt) modification), or nucleotides with alternative chemical properties (e.g., phosphorodiamide morpholino oligonucleotide (PMO), peptide nucleic acid (PNA), tricycloDNA (tcDNA), unlocked nucleic acid (UNA), or glycol nucleic acid (GNA)).

[0095] Nucleic acid molecules have a phosphate backbone (Figure 4C). The phosphate backbone can be a phosphodiester, a phosphorothioate isomer (e.g., stereoisomers Sp and / or Rp), a phosphoryl DMI amide diester isomer, a phosphorodithioate, a methylphosphonate, a 5'-phosphorothioate, a thiophosphoamide, a peptide nucleic acid, a 5'-(E)-vinylphosphonate, or a 5'-methylphosphonate.

[0096] Additional parts can be added to or attached to single-stranded nucleic acids (such as ASOs). Additional parts may include, but are not limited to, antibodies, lipophilic moieties, small molecules, and RNA aptamers (e.g., ribozymes). Adding additional parts to single-stranded nucleic acids can alter their pharmacological characteristics (such as structural or chemical parameters). Single-stranded nucleic acids can be modified with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 or more parts. Additional parts can be added to the 5' end of a single-stranded nucleic acid. Alternatively, additional parts can be added to the 3' end of a single-stranded nucleic acid. Alternatively, additional parts can be added to the middle of a single-stranded nucleic acid (e.g., neither the 3' nor the 5' end).

[0097] To enhance stability, single-stranded nucleic acids (such as ASOs) can be modified. The single-stranded nucleic acids described herein contain 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 95%, at least about 100%, or more compared to the control single-stranded nucleic acid.

[0098] The stability of nucleic acids can be measured by analyzing the half-life of single-stranded non-coding nucleic acid molecules. The single-stranded nucleic acid molecules described herein have a half-life of at least about 30 minutes, at least about 1 hour, at least about 90 minutes, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours or more compared to control single-stranded nucleic acids.

[0099] To reduce off-target effects, single-stranded non-coding nucleic acid molecules (such as ASOs) can be modified. The number of off-target effects measured by sequencing is 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.

[0100] To reduce the adverse effects on immunogenicity, single-stranded non-coding nucleic acid molecules (such as ASOs) can be modified. The number of immunogenic adverse events measured by immunogenicity assays is 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 90%, at least approximately 95%, at least approximately 100%, or more compared to control single-stranded nucleic acids.

[0101] To reduce toxicity, single-stranded non-coding nucleic acid molecules (such as ASOs) can be modified. The toxicity measured by toxicity assays is 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 may be an unmodified, otherwise identical single-stranded non-coding nucleic acid molecule.

[0102] To enhance stability and / or durability, single-stranded non-coding nucleic acid molecules (such as ASOs) can be modified. Durability and / or stability, as measured by nucleic acid detection, are 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 may be an unmodified, otherwise identical single-stranded non-coding nucleic acid molecule.

[0103] Modifications such as cyclization can be performed on single-stranded non-coding nucleic acid molecules (such as ASOs). Cyclized non-coding nucleic acid molecules can be single-stranded non-coding nucleic acid molecules without free ends. Single-stranded non-coding nucleic acid molecules without free ends can refer to cyclic oligonucleotides. Cyclized oligonucleotides are single (e.g., monovalent) ASOs or polyvalent ASOs. A polyvalent ASO can consist of two or more ASOs. The two or more ASOs can be separated by linker sequences, and the lengths of the linker sequences may differ. The two or more ASOs in a polyvalent ASO may target the same sequence and / or gene. Alternatively, the two or more ASOs in a polyvalent ASO may target different sequences and / or genes. The two or more ASOs in a polyvalent ASO may have the same mechanism of action and / or different mechanisms of action. Cyclization and / or circularization of single-stranded non-coding nucleic acid molecules can reduce the inclusion of chemically modified nucleotides (such as nucleotides containing phosphorothioates) associated with adverse medical side effects. Cyclization and / or cyclicization may involve 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 chemically modified nucleotides. Adverse medical side effects include, but are not limited to, a decrease in platelets, thrombocytopenia, perturbations of heart rate, increased blood pressure, or increased cardiac output due to activation of the complement cascade.

[0104] Target modulation Single-stranded non-coding nucleic acid molecules can be used to influence transcriptional regulation. 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, an ASO can bind to a specific region of the target gene or to a regulatory region of the target mRNA (e.g., UTR, intron, exon, intron / exon junction, exon / intron junction) to regulate the target gene. Or, in addition to the above, 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.

[0105] By regulating a target gene, its expression may increase. Alternatively, by regulating a target gene, its expression may decrease. Or, by regulating a target gene, its expression may be maintained.

[0106] In some cases, single-stranded non-coding nucleic acid molecules are present in amounts of 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 more compared to control expression levels.

[0107] In some cases, single-stranded non-coding nucleic acid molecules reduce the expression of target genes by at most approximately 500%, at most approximately 400%, at most approximately 300%, at most approximately 200%, at most approximately 100%, 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%, at most approximately 10%, at most approximately 9%, at most approximately 8%, at most approximately 7%, at most approximately 6%, at most approximately 5%, at most approximately 4%, at most approximately 3%, at most approximately 2%, at most approximately 1%, and at most approximately 0.9%. Compared to the control expression level, the levels are at most around 0.8%, at most around 0.7%, at most around 0.6%, at most around 0.5%, at most around 0.4%, at most around 0.3%, at most around 0.2%, and at most around 0.1% or less.

[0108] In some cases, single-stranded non-coding nucleic acid molecules fold the expression of target genes by at least or up to approximately 0.1 times, at least up to approximately 0.2 times, at least 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 approximately 0.6 times, at least up to approximately 0.7 times, at least up to approximately 0.8 times, at least up to approximately 0.9 times, at least up to approximately 1 time, at least up to approximately 2 times, at least up to approximately 3 times, at least 4 times or more, at least 5 times or more, at least approximately 6 times, at least approximately 7 times, at least up to approximately 8 times, at least up to approximately 9 times, at least up to approximately 10 times, at least up to approximately 20 times, at least up to approximately 30 times, at least up to approximately 40 times, At least 50 times, at least up to approximately 60 times, at least up to approximately 70 times, at least up to approximately 80 times, at least up to approximately 90 times, at least up to approximately 100 times, at least up to approximately 500 times, at least or up to approximately 1,000 times, at least up to approximately 5,000 times, or at least or up to approximately 10,000 times compared to the control expression level.

[0109] In some cases, single-stranded non-coding nucleic acid molecules fold the expression of target genes by up to approximately 10,000 times, up to approximately 5,000 times, up to approximately 1,000 times, up to approximately 500 times, up to approximately 100 times, up to approximately 90 times, up to approximately 80 times, up to approximately 70 times, up to approximately 60 times, up to approximately 50 times, up to approximately 40 times, up to approximately 30 times, up to approximately 20 times, up to approximately 10 times, up to approximately 9 times, up to approximately 8 times, up to approximately 7 times, up to approximately 6 times, up to approximately 5 times, up to approximately 4 times, up to approximately 3 times, up to approximately 2 times, up to approximately 1 time, up to approximately 0.9 times. Up to approximately 0.8 times or less, up to approximately 0.7 times or less, up to approximately 0.6 times or less compared to the control expression level, up to approximately 0.5 times or less, up to approximately 0.4 times or less, up to approximately 0.3 times or less, up to approximately 0.2 times or less, up to approximately 0.1 times or less.

[0110] In some cases, single-stranded non-coding nucleic acid molecules increase the expression of target genes 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 more compared to control expression levels.

[0111] In some cases, single-stranded non-coding nucleic acid molecules reduce the expression of target genes by at most approximately 500%, at most approximately 400%, at most approximately 300%, at most approximately 200%, at most approximately 100%, 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%, at most approximately 10%, at most approximately 9%, at most approximately 8%, at most approximately 7%, at most approximately 6%, at most approximately 5%, at most approximately 4%, at most approximately 3%, at most approximately 2%, at most approximately 1%, and at most approximately 0.9%. Compared to the control expression level, the levels are at most around 0.8%, at most around 0.7%, at most around 0.6%, at most around 0.5%, at most around 0.4%, at most around 0.3%, at most around 0.2%, and at most around 0.1% or less.

[0112] In some cases, single-stranded non-coding nucleic acid molecules fold 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 approximately 0.6 times, at least up to approximately 0.7 times, at least up to approximately 0.8 times, at least up to approximately 0.9 times, at least up to approximately 1 time, at least up to approximately 2 times, at least up to approximately 3 times, at least 4 times or more, at least 5 times or more, at least approximately 6 times, at least approximately 7 times, at least up to approximately 8 times, at least up to approximately 9 times, at least up to approximately 10 times, at least up to approximately 20 times, at least up to approximately 30 times, at least up to or up to approximately 40 times, at least 50 times or more, at least up to approximately 60 times, at least up to approximately 70 times, at least up to approximately 80 times, at least up to approximately 90 times, at least up to approximately 100 times, at least up to approximately 500 times, Compared to control expression levels, the levels were 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.

[0113] In some cases, single-stranded non-coding nucleic acid molecules fold the expression of target genes by up to approximately 10,000 times, up to approximately 5,000 times, up to approximately 1,000 times, up to approximately 500 times, up to approximately 100 times, up to approximately 90 times, up to approximately 80 times, up to approximately 70 times, up to approximately 60 times, up to approximately 50 times, up to approximately 40 times, up to approximately 30 times, up to approximately 20 times, up to approximately 10 times, up to approximately 9 times, up to approximately 8 times, up to approximately 7 times, up to approximately 6 times, up to approximately 5 times, up to approximately 4 times, up to approximately 3 times, up to approximately 2 times, up to approximately 1 time, up to approximately 0.9 times. Up to approximately 0.8 times or less, up to approximately 0.7 times or less, up to approximately 0.6 times or less compared to the control expression level, up to approximately 0.5 times or less, up to approximately 0.4 times or less, up to approximately 0.3 times or less, up to approximately 0.2 times or less, up to approximately 0.1 times or less.

[0114] Regulation of target mRNA may increase the expression of the gene product expressed from that target mRNA. Alternatively, regulation of target mRNA may decrease the expression of the gene product expressed from that target mRNA. Alternatively, regulation of target mRNA may maintain the expression of the gene product expressed from that target mRNA.

[0115] In some cases, single-stranded non-coding nucleic acid molecules increase the expression of target mRNA 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 more compared to control expression levels.

[0116] In some cases, single-stranded non-coding nucleic acid molecules can reduce the expression of target mRNA by 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%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%. Compared to the control expression level, the levels are at most around 0.8%, at most around 0.7%, at most around 0.6%, at most around 0.5%, at most around 0.4%, at most around 0.3%, at most around 0.2%, and at most around 0.1% or less.

[0117] In some cases, single-stranded non-coding nucleic acid molecules fold the expression of target mRNA by at least or up to approximately 0.1 times, at least up to approximately 0.2 times, at least 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 approximately 0.6 times, at least up to approximately 0.7 times, at least up to approximately 0.8 times, at least up to approximately 0.9 times, at least up to approximately 1 time, at least up to approximately 2 times, at least up to approximately 3 times, at least 4 times or more, at least 5 times or more, at least approximately 6 times, at least approximately 7 times, at least up to approximately 8 times, at least up to approximately 9 times, at least up to approximately 10 times, at least up to approximately 20 times, at least up to approximately 30 times, at least up to or up to approximately 40 times, at least 50 times or more, at least up to approximately 60 times, at least up to approximately 70 times, at least up to approximately 80 times, at least up to approximately 90 times, at least up to approximately 100 times, at least up to approximately 500 times, Compared to control expression levels, the levels were 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.

[0118] In some cases, single-stranded non-coding nucleic acid molecules fold the expression of target mRNA by up to approximately 10,000 times, up to approximately 5,000 times, up to approximately 1,000 times, up to approximately 500 times, up to approximately 100 times, up to approximately 90 times, up to approximately 80 times, up to approximately 70 times, up to approximately 60 times, up to approximately 50 times, up to approximately 40 times, up to approximately 30 times, up to approximately 20 times, up to approximately 10 times, up to approximately 9 times, up to approximately 8 times, up to approximately 7 times, up to approximately 6 times, up to approximately 5 times, up to approximately 4 times, up to approximately 3 times, up to approximately 2 times, up to approximately 1 time, up to approximately 0.9 times. Up to approximately 0.8 times or less, up to approximately 0.7 times or less, up to approximately 0.6 times or less compared to the control expression level, up to approximately 0.5 times or less, up to approximately 0.4 times or less, up to approximately 0.3 times or less, up to approximately 0.2 times or less, up to approximately 0.1 times or less.

[0119] In some cases, single-stranded non-coding nucleic acid molecules increase the expression of target mRNA 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 more compared to control expression levels.

[0120] In some cases, single-stranded non-coding nucleic acid molecules can reduce the expression of target mRNA by 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%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%. Compared to the control expression level, the levels are at most around 0.8%, at most around 0.7%, at most around 0.6%, at most around 0.5%, at most around 0.4%, at most around 0.3%, at most around 0.2%, and at most around 0.1% or less.

[0121] In some cases, single-stranded non-coding nucleic acid molecules fold 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 approximately 0.6 times, at least up to approximately 0.7 times, at least up to approximately 0.8 times, at least up to approximately 0.9 times, at least up to approximately 1 time, at least up to approximately 2 times, at least up to approximately 3 times, at least 4 times or more, at least 5 times or more, at least approximately 6 times, at least approximately 7 times, at least up to approximately 8 times, at least up to approximately 9 times, at least up to approximately 10 times, at least up to approximately 20 times, at least up to approximately 30 times, at least up to or up to approximately 40 times, at least up to 50 times, at least up to approximately 60 times, at least up to approximately 70 times, at least up to approximately 80 times, at least up to approximately 90 times, at least up to approximately 100 times, at least up to approximately 500 times. Compared to control expression levels, the levels were 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.

[0122] In some cases, single-stranded non-coding nucleic acid molecules fold the expression of target mRNA by up to approximately 10,000 times, up to approximately 5,000 times, up to approximately 1,000 times, up to approximately 500 times, up to approximately 100 times, up to approximately 90 times, up to approximately 80 times, up to approximately 70 times, up to approximately 60 times, up to approximately 50 times, up to approximately 40 times, up to approximately 30 times, up to approximately 20 times, up to approximately 10 times, up to approximately 9 times, up to approximately 8 times, up to approximately 7 times, up to approximately 6 times, up to approximately 5 times, up to approximately 4 times, up to approximately 3 times, up to approximately 2 times, up to approximately 1 time, up to approximately 0.9 times. Up to approximately 0.8 times or less, up to approximately 0.7 times or less, up to approximately 0.6 times or less compared to the control expression level, up to approximately 0.5 times or less, up to approximately 0.4 times or less, up to approximately 0.3 times or less, up to approximately 0.2 times or less, up to approximately 0.1 times or less.

[0123] Target part Disclosed herein are single-stranded non-coding nucleic acid molecules comprising one or more target moieties. The targeting moieties can be used to guide the single-stranded non-coding nucleic acid molecules to target cells or tissues. The targeting moieties may be lipophilic moieties, small molecules, peptides (e.g., polypeptides, macrosilicul peptides, etc.), RNA molecules, nanoparticles, antibodies, single-domain antibodies, miniproteins, or antigen-binding fragments thereof. The targeting moieties may be specific to an antigen or receptor on the target cell or tissue (e.g., Asian lycoprotein receptor (ASGPR)).

[0124] The target moiety may be a lipophilic moiety. The lipophilic moiety may contain one or more fatty acid groups or salts thereof. Lipids are lipids. Lipids are fatty acids and their derivatives, which are insoluble in water but soluble in organic solvents. In some embodiments, the lipophilic moiety may be unsaturated. Alternatively, or in addition, the lipophilic moiety may be monosaturated. Alternatively, or in addition, the lipophilic moiety may be polysaturated. In some embodiments, the double bond of the unsaturated lipophilic moiety may be in a cis conformation. Alternatively, or in addition, the double bond of the unsaturated lipophilic moiety may be in a transconformation. 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, geranyloxyhexianol, hexadecylglycerol, vomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0125] The target part can be a small molecule. Small molecules 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 to that, small molecules are molecules with a size on the order of 1 nm.

[0126] The target portion can be the sugar portion or the sugar portion. The sugar can be a monosaccharide. Alternatively, the sugar may be a disaccharide. Alternatively, the sugar may be a polysaccharide. Non-limiting examples of sugars include glucose, dextrose, fructose, galactose, sugar alcohols, pentose, xylose, ribose, sucrose, cellulose, starch, lactose, maltose, trehalose, lactulose, cellobiose, chitobiose, glycogen, or chitin. Low molecular weight sugars may be amino sugars such as acetylgalactosamine (GalNAc), N-acetylglucosamine, or sialic acid, but are not limited to these.

[0127] The target region 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 they can be polyclonal antibodies. Antibodies can be single-domain antibodies. Antibodies can be antibody fragments. Antibodies can be agonists, or they can be antagonists. Alternatively, antibodies can be allosteric modulators (e.g., positive or negative allosteric modulators).

[0128] The target moiety can be a polypeptide. Non-limiting examples of polypeptides include macrocillin peptides, glucagon-like peptide 1 receptor (GLP1R) agonists, asialoglicoprotein receptor (ASGPR), prostate-specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), epidermal 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).

[0129] The target portion can be an RNA molecule. This RNA molecule can include aptamers, ribozymes, hairpin RNA, siRNA, or miRNA.

[0130] Single-stranded nucleic acids can target target genes. Alternatively, single-stranded nucleic acids can target RNA molecules that encode target genes. Non-limiting 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, CTNS, 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, JAG1, 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, Orai1, ORF1Ab / N-protein, OTC, p21 (CDKN1A), P27Kip1, PAH, PAX2, Pax6, PC, PCCa, PCCb, PCSK9, PD-L1, PER1, PIK3R1, PKD2, PKK, PNPLA3, POGLUT1, P PARD, 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.

[0131] 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 may be involved in inflammation, homeostasis, and defense against pathogens. The C5 protein consists of C5 alpha and beta chains linked by disulfide crosslinks. Mutations in the C5 gene can cause complement component 5 deficiency, a disease characterized by recurrent bacterial infections.

[0132] Single-stranded non-coding nucleic acid molecules can manipulate target cells at their target regions. Target cells include stem cells (e.g., induced pluripotent stem cells, embryonic stem cells), osteocytes, blood cells, erythrocytes, leukocytes, platelets, dermal 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, chondrocytes, odontocytes, cementoblasts, chondrocytes, mesenchymal cells, epithelial cells, secretory cells, germ cells, nurse cells, storage cells, pituitary cells, glial cells, stromal cells, lymphocytes, 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.

[0133] Single-stranded non-coding nucleic acid molecules can be manipulated at target regions of target 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.

[0134] Illness and Disability Disclosed herein are single-stranded non-coding nucleic acid molecules and their pharmaceutical formulations that are therapeutically effective in several embodiments for treating diseases or disorders disclosed herein. In several embodiments, the single-stranded nucleic acids disclosed herein can be used to treat diseases or disorders in subjects. Limited examples of diseases or disorders include cancer, inflammatory diseases or disorders, metabolic diseases or disorders, cardiovascular diseases or disorders, immunodeficiency diseases or disorders, respiratory diseases or disorders, pain, digestive diseases or disorders, reproductive diseases or disorders, endocrine diseases or disorders, immune diseases or disorders, autoimmune diseases or disorders, or neurological diseases or disorders. Table 1 describes non-restrictive target genes, non-restrictive target tissues, and their relationships to non-restrictive 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 the gene of interest provided in Table 1. In some embodiments, the modulation of the expression of the gene of interest is therapeutically effective for treating the indications provided in Table 1. It corresponds to the gene of interest. In some embodiments, the modulation may be activation or inhibition. In some embodiments, the single-stranded non-coding nucleic acid molecule further includes a target region specific to the target tissue provided, corresponding to the genes of interest and indications shown in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0135] A subject may be a biological entity containing expressed genetic material. A biological entity may be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject may be a tissue, cell, or their offspring of the obtained biological entity in vivo or culture in vitro. A 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. A mammal may be a human. A subject may be diagnosed or suspected of being at high risk of disease. In some cases, a subject may not necessarily be diagnosed or suspected of being at high risk of disease.

[0136] Pharmaceutical preparations A pharmaceutical formulation may include the compositions disclosed herein. A pharmaceutical formulation may further include excipients. Excipients may be buffers, carriers, stabilizers, solubilizers, fillers, preservatives, diluents, vehicles, surfactants, salts, peptides, oligosaccharides, amino acids, adjuvants, carbohydrates, and / or bulking agents.

[0137] Pharmaceutical preparations require 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, at least approximately 22 hours, 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 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, or longer.

[0138] The preparation lasts for at most about 4 weeks, at most about 3 weeks, at most about 2 weeks, at most about 13 days, at most about 12 days, at most about 11 days, at most about 10 days, at most about 9 days, at most about 8 days, at most about 7 days, mostly about 6 days, mostly about 5 days, mostly about 4 days, mostly about 3 days, mostly about 2 days, mostly about 44 hours, mostly about 40 hours, mostly about 36 hours, mostly about 32 hours, mostly about 28 hours, mostly about 24 hours, mostly about 23 hours, at most about 22 hours, mostly about 21 hours, mostly about 20 hours, mostly about 19 hours, mostly about 18 hours, mostly about 17 hours, mostly about 17 hours, mostly about 16 hours, mostly about 15 hours, mostly about 14 hours, mostly about 13 hours, mostly about 12 hours. Mostly around 11 hours, mostly around 10 hours, mostly around 9 hours, mostly around 8 hours, mostly around 7 hours, mostly around 6 hours, mostly around 5 hours, mostly around 4 hours, mostly around 3 hours, mostly around 2 hours, mostly around 1 hour, mostly around 30 minutes, or less.

[0139] Pharmaceutical formulations may have a half-life measured by transcription inhibition assays. The half-lives of formulations can range from at least approximately 15 minutes, 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, at least approximately 22 hours, 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 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, or longer.

[0140] The half-lives of the formulations are approximately 4 weeks, 3 weeks, 2 weeks, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, and 7 days. Mostly around 6 days, mostly around 5 days, mostly around 4 days, mostly around 3 days, mostly around 2 days, mostly around 44 hours, mostly around 40 hours, mostly around 36 hours, mostly around 32 hours, mostly around 28 hours, mostly around 24 hours, mostly around 23 hours, at most around 22 hours, mostly around 21 hours, mostly around 20 hours, mostly around 19 hours, mostly around 18 hours, mostly around 17 hours, mostly around 17 hours, mostly around 16 hours, mostly around 15 hours, mostly around 14 hours, mostly around 13 hours, mostly around 12 hours, mostly around 11 hours, mostly around 10 hours, mostly around 9 hours, mostly around 8 hours, mostly around 7 hours, mostly around 6 hours, mostly around 5 hours, mostly around 4 hours, mostly around 3 hours, mostly around 2 hours, mostly around 1 hour, mostly around 30 minutes, It takes about 15 minutes at most, or even less.

[0141] cell Provided herein are cells that may be manipulated to contain or express one or more systems disclosed herein. In some embodiments, the cells contain single-stranded nucleic acids 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 cells may have a purity of at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The cells may be formulated in pharmaceutical compositions or formulations, such as for the treatment of diseases or conditions defined below. Table 1. The cells may be cell lines or multiple cells that contain or express one or more systems of this disclosure.

[0142] III. Method Disclosed herein, in several embodiments, are methods for preparing, isolating, and / or purifying the single-stranded nucleic acid compositions disclosed herein. Also disclosed herein, in several embodiments, are methods for utilizing the compositions or pharmaceutical formulations disclosed herein to treat diseases and disorders in subjects. The methods disclosed herein may be modified by applying molecular barcodes to nucleic acid molecules.

[0143] In some embodiments, the methods of the present disclosure include the purification or isolation of single-stranded non-coding nucleic acid molecules. Single-stranded non-coding nucleic acid molecules can be purified and / or isolated using several processes, including phenol-chloroform extraction, DNA filtration columns, salt and proteinase K treatment, and the use of silica gel membranes.

[0144] 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 several means, such as viral vector particles (e.g., retroviruses, adenoviruses, adeno-associated viruses (AAVs), or herpes simplex viruses), cationic nanoparticles, lipid nanoparticles, cationic polymers, plasmids, cells, or physical methods (e.g., sonication, electroporation, lipofection).

[0145] In some embodiments, these methods disclose a single-stranded non-coding nucleic acid molecule (e.g., ASO) to 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 transcription enhancer, transcription silencer, or promoter of transcription 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 (lncRNA) or microRNA (miRNA) to influence 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 the intron region (pre-splicing) of pre-mRNA to influence 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 a complementary region of mRNA (after splicing) to affect mRNA translation. For example, the antisense strand may silence or enhance the expression or translation of a gene of interest.

[0146] In some embodiments, these methods involve enhancing or silencing the expression and / or translation of a gene of interest by introducing a single-stranded non-coding nucleic acid molecule (e.g., ASO) of the Disclosed into a target cell or tissue. In some embodiments, introducing a single-stranded non-coding nucleic acid molecule of the Disclosed into a target cell or tissue enhances or silences the expression and / or translation of the gene of interest compared to introducing a linear, identical synthetic nucleic acid molecule into the target cell or tissue. In some embodiments, introducing a single-stranded non-coding nucleic acid molecule into target cells or tissues results in an increase of 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 the same linear synthetic nucleic acid molecule into target cells or tissues. In some embodiments, introducing a single-stranded non-coding nucleic acid molecule into target cells or tissues increases 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 the same synthetic nucleic acid molecule that is linear to the target cells or tissues.In some embodiments, introducing a single-stranded non-coding nucleic acid molecule into target cells or tissues can increase the expression and / or translation of a target gene by up to approximately 1,000%, up to approximately 900%, up to approximately 800%, up to approximately 700%, up to approximately 600%, up to approximately 500%, up to approximately 400%, at most approximately 300%, at most approximately 200%, at most approximately 150%, at most approximately 100%, 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%, at most approximately 10%, or less.

[0147] In some embodiments, introducing a single-stranded non-coding nucleic acid molecule into target cells or tissues can increase the expression and / or translation of a target gene by up to approximately 100 times, up to approximately 90 times, up to approximately 80 times, up to approximately 70 times, up to approximately 60 times, up to approximately 50 times, at most about 40 times, mostly about 30 times, mostly about 20 times, mostly about 10 times, mostly about 90 times, mostly about 8 times, mostly about 7 times, mostly about 6 times, mostly about 5 times, mostly about 4 times, mostly about 3 times, mostly about 2 times, or less than when introducing a linear, identical synthetic nucleic acid molecule into target cells or tissues.

[0148] In some embodiments, these methods involve enhancing or silencing the expression and / or translation of a gene of interest by introducing an adapter into a loop-shaped and / or circularized single-stranded non-coding nucleic acid molecule (e.g., ASO) of the present disclosure (Figure 11A-C). In some embodiments, the introduction of a loop-shaped single-stranded non-coding nucleic acid molecule including an adapter into a target cell or tissue enhances or silences the expression and / or translation of the gene of interest compared to introducing the same synthetic nucleic acid molecule into the target cell or tissue by other methods that do not include the adapter. In some embodiments, introducing a loop-shaped single-stranded non-coding nucleic acid molecule containing an adapter to target cells or tissues increases the expression and / or translation of the gene of interest by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more compared to introducing the same synthetic nucleic acid molecule without an adapter to target cells or tissues. In some embodiments, introducing a loop-shaped single-stranded non-coding nucleic acid molecule containing an adapter to target cells or tissues increases 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, or at least about 100 times or more compared to introducing the same synthetic nucleic acid molecule without the adapter to target cells or tissues.In some embodiments, introducing a loop-shaped single-stranded non-coding nucleic acid molecule containing an adapter to target cells or tissues can increase the expression and / or translation of the gene of interest by up to approximately 1,000%, up to approximately 900%, up to approximately 800%, up to approximately 700%, up to approximately 600%, up to approximately 500%, at most approximately 400%, at most approximately 300%, at most approximately 200%, at most approximately 150%, at most approximately 100%, 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%, at most approximately 10%, or less than when introducing an otherwise identical synthetic nucleic acid molecule without an adapter to target cells or tissues. In some embodiments, introducing a loop-shaped single-stranded non-coding nucleic acid molecule containing an adapter to target cells or tissues can increase the expression and / or translation of the target gene by up to approximately 100 times, up to approximately 90 times, up to approximately 80 times, up to approximately 70 times, up to approximately 60 times, at most about 50 times, mostly about 40 times, mostly about 30 times, mostly about 20 times, mostly about 10 times, mostly about 90 times, mostly about 8 times, mostly about 7 times, mostly about 6 times, mostly about 5 times, mostly about 4 times, mostly about 3 times, and at most about 2 times or less compared to introducing the same synthetic nucleic acid molecule by other methods without an adapter to target cells or tissues.

[0149] In some embodiments, these methods involve delivering (administering) a single-stranded non-coding nucleic acid molecule to a subject, where the single-stranded non-coding nucleic acid molecule exhibits fewer off-target effects in target cells or tissues compared to the same synthetic nucleic acid molecule by other linear methods. In some embodiments, the off-target effect is 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 in target cells or tissues having the single-stranded non-coding nucleic acid molecule. In some embodiments, the off-target effect is at least about 2x, at least about 3x, at least about 4x, at least about 5x, at least about 6x, at least about 7x, at least about 8x, at least about 9x, at least about 10x, at least about 20x, at least about 30x, at least about 40x, at least about 50x, at least about 60x, at least about 70x, at least about 80x, at least about 90x, at least about 100x, or more in target cells or tissues having a single-stranded non-coding nucleic acid molecule. In some embodiments, off-target effects in target cells or tissues having single-stranded non-coding nucleic acid molecules are 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 40%, at most about 30%, at most about 20%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less.In some embodiments, the off-target effect is up to about 100 times, up to about 90 times, up to about 80 times, up to about 70 times, up to about 60 times, up to about 50 times, up to 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 less, when compared to the same linear non-coding nucleic acid molecule in target cells or tissues having a single-stranded non-coding nucleic acid molecule.

[0150] In some embodiments, single-stranded non-coding nucleic acid molecules are less toxic in target cells or tissues compared to linear, identical synthetic nucleic acid molecules. In some embodiments, toxicity in target cells or tissues having a single-stranded non-coding nucleic acid molecule is 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. In some embodiments, the toxicity is 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 in target cells or tissues having a single-stranded non-coding nucleic acid molecule. In some embodiments, toxicity in target cells or tissues having single-stranded non-coding nucleic acid molecules is up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less.In some embodiments, toxicity is up to approximately 100 times, up to approximately 90 times, up to approximately 80 times, up to approximately 70 times, up to approximately 60 times, up to approximately 50 times, up to approximately 40 times, up to approximately 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 less, in target cells or tissues having a single-stranded non-coding nucleic acid molecule, otherwise compared to the same linear non-coding nucleic acid molecule.

[0151] In some embodiments, single-stranded non-coding nucleic acid molecules exhibit greater target nucleic acid sequence specificity in target cells or tissues compared to otherwise identical linear non-coding nucleic acid molecules. In some embodiments, single-stranded non-coding nucleic acid molecules exhibit target nucleic acid sequence specificity in target cells or tissues of 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. In some embodiments, a single-stranded non-coding nucleic acid molecule increases the target nucleic acid sequence specificity in target cells or tissues 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. In some embodiments, single-stranded non-coding nucleic acid molecules exhibit target nucleic acid sequence specificity in target cells or tissues of up to approximately 1,000%, up to approximately 900%, up to approximately 800%, up to approximately 700%, up to approximately 600%, up to approximately 500%, up to approximately 400%, up to approximately 300%, up to approximately 200%, at most approximately 150%, at most approximately 100%, 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%, at most approximately 10%, or less.In some embodiments, single-stranded non-coding nucleic acid molecules increase the target nucleic acid sequence specificity in target cells or tissues by up to approximately 100 times, up to approximately 90 times, up to approximately 80 times, up to approximately 70 times, up to approximately 60 times, up to approximately 50 times, and up to approximately 40 times, compared to the same linear non-coding nucleic acid molecule by at most approximately 30 times, at most approximately 20 times, at most approximately 10 times, at most approximately 90 times, at most approximately 8 times, at most approximately 7 times, at most approximately 6 times, at most approximately 5 times, at most approximately 4 times, at most approximately 3 times, at most approximately 2 times, or less.

[0152] In some embodiments, single-stranded non-coding nucleic acid molecules exhibit greater stability in vivo compared to otherwise identical linear non-coding nucleic acid molecules (Figure 9A). In some embodiments, single-stranded non-coding nucleic acid molecules show increased stability in vivo by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more, compared to otherwise identical linear non-coding nucleic acid molecules. In some embodiments, single-stranded non-coding nucleic acid molecules exhibit increased stability in vivo by 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, at least approximately 100 times, or more. In some embodiments, single-stranded non-coding nucleic acid molecules show increased stability in vivo by at most 1,000%, at most 900%, at most 800%, at most 700%, at most 600%, at most 500%, at most 400%, at most 300%, at most 200%, at most 150%, at most 100%, at most 90%, at most 80%, at most 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less, compared to the same linear non-coding nucleic acid molecule by other means.In some embodiments, single-stranded non-coding nucleic acid molecules exhibit increased stability in vivo, 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 less, otherwise compared to identical linear non-coding nucleic acid molecules.

[0153] In some embodiments, single-stranded non-coding nucleic acid molecules exhibit greater stability ex vivo compared to otherwise identical linear non-coding nucleic acid molecules (Figures 7A-8B). In some embodiments, single-stranded non-coding nucleic acid molecules show increased stability ex vivo by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more, compared to otherwise identical linear non-coding nucleic acid molecules. In some embodiments, single-stranded non-coding nucleic acid molecules exhibit increased stability ex vivo by 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, at least approximately 100 times, or more. In some embodiments, single-stranded non-coding nucleic acid molecules show increased stability ex vivo of at most 1,000%, at most 900%, at most 800%, at most 700%, at most 600%, at most 500%, at most 400%, at most 300%, at most 200%, at most 150%, at most 100%, at most 90%, at most 80%, at most 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less, compared to the same linear non-coding nucleic acid molecule by other means.In some embodiments, single-stranded non-coding nucleic acid molecules show increased stability ex vivo of 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 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 less, otherwise compared to the same linear non-coding nucleic acid molecule.

[0154] 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 associated with the disease or condition (e.g., Table 1). 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 associated with the disease or condition (e.g., Table 1). In some embodiments, the administration may be orally, intraarachnoid, transdermally, rectally, sublingually, intranasally, intravitreously, subcutaneously, intramuscularly, transdermally, or intravenously.

[0155] The compositions disclosed herein may be made into dose formulations that can be varied depending on the target being treated and the method of administration. The single-stranded nucleic acid active ingredient may be present in formulations containing 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. A formulation in which the single-stranded nucleic acid active ingredient is at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less.

[0156] The dosage formulations for intravenous administration are 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, 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.

[0157] The dosage formulations for intravenous administration are approximately 350 mg at most, 340 mg at most, 330 mg at most, 320 mg at most, 310 mg at most, 300 mg at most, 290 mg at most, 280 mg at most, 270 mg at most, 260 mg at most, approximately 250 mg at most, approximately 240 mg at most, approximately 230 mg at most, approximately 220 mg at most, approximately 210 mg at most, approximately 200 mg at most, approximately 190 mg at most, approximately 180 mg at most, approximately 170 mg at most, approximately 160 mg at most, approximately 150 mg at most, and approximately 140 mg at most. At most around 130mg, at most around 120mg, at most around 110mg, at most around 100mg, at most around 90mg, at most around 80mg, at most around 70mg, at most around 60mg, at most around 50mg, at most around 45mg, at most around 40mg, at most around 35mg, at most around 30mg, at most around 25mg, at most around 20mg, at most around 15mg, at most around 10mg, or less.

[0158] The dosage formulations for intravenous administration may range from 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, and 30-150 mg. 30mg~200mg, 30mg~250mg, 30mg~300mg, 50mg~100mg, 50mg~150mg, 50mg~200mg, 50mg~250mg, 50mg~300mg, 50mg~300mg, 100mg~150mg, 100mg~200mg, 100mg~250mg, 100 mg~300 mg, 100 mg~250 mg, 150 mg~200 mg, 150 mg~250 mg, 150 mg~300 mg, 150 mg~350 mg, 200~250 mg, 200~300 mg, 200~350 mg, 250 mg~300 mg, 250 mg~350 mg, or 300 mg~350 mg.

[0159] The dosage formulations for subcutaneous administration are 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, 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.

[0160] The dosage formulations for subcutaneous administration are at most about 350 mg, at most about 340 mg, at most about 330 mg, at most about 320 mg, at most about 310 mg, at most about 300 mg, at most about 290 mg, at most about 280 mg, at most about 270 mg, at most about 260 mg, at most about 250 mg, at most about 240 mg, at most about 230 mg, at most about 220 mg, at most about 210 mg, at most about 200 mg, at most about 190 mg, at most about 180 mg, at most about 170 mg, at most about 160 mg, at most about 150 mg, and at most about 140 mg. At most around 130mg, at most around 120mg, at most around 110mg, at most around 100mg, at most around 90mg, at most around 80mg, at most around 70mg, at most around 60mg, at most around 50mg, at most around 45mg, at most around 40mg, at most around 35mg, at most around 30mg, at most around 25mg, at most around 20mg, at most around 15mg, at most around 10mg, or less.

[0161] The dosage formulations for subcutaneous administration may range from 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, and 30-150 mg. 30mg~200mg, 30mg~250mg, 30mg~300mg, 50mg~100mg, 50mg~150mg, 50mg~200mg, 50mg~250mg, 50mg~300mg, 50mg~300mg, 100mg~150mg, 100mg~200mg, 100mg~250mg, 100 mg~300 mg, 100 mg~250 mg, 150 mg~200 mg, 150 mg~250 mg, 150 mg~300 mg, 150 mg~350 mg, 200~250 mg, 200~300 mg, 200~350 mg, 250 mg~300 mg, 250 mg~350 mg, or 300 mg~350 mg.

[0162] The compositions disclosed herein may be administered to a patient at a frequency of at least three times a day, at least twice a day, at least once a day, at least once a day, at least once every three days, at least once every four days, at least once every five days, or at least once every six days. Alternatively, they may be administered 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.

[0163] The compositions disclosed herein may be administered to patients 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. Alternatively, they may be administered once every 5 months, once every 4 months, once every 3 months, once every 2 months, once every 4 weeks, once every 3 weeks, once every 2 weeks, up to once every 7 days, up to once every 6 days, up to once every 5 days, up to once every 4 days, up to once every 3 days, up to once every 2 days, up to once a day, up to twice a day, or up to three times a day.

[0164] IV. Kit Provided herein are kits comprising, in several embodiments, one or more compositions disclosed herein. In some embodiments, the kit comprises one or more single-stranded nucleic acids described herein. In some embodiments, the kit further comprises cells or a plurality of 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 mevalonic acid and antibiotics. The exact properties of the components comprising the kit of the present invention depend on its intended purpose.

[0165] Instructions for use may be included in the kit. These instructions typically include specific descriptions of the techniques employed to influence the desired outcome using the kit's components, such as the production of single-stranded nucleic acids, the isolation of single-stranded nucleic acids, or the investigation of the therapeutic potential of one or more single-stranded nucleic acids. Optionally, the kit may also include other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measurement tools, or other useful instruments readily recognizable to those skilled in the art.

[0166] The materials or components assembled in the kit can be provided to the user in a convenient and appropriate storage manner to maintain operability and practicality. For example, components can be in a dissolved, dehydrated, or freeze-dried form. They can be provided at room temperature, refrigerated, or frozen temperature. Components are usually contained in appropriate packaging materials. As used herein, the term “packaging material” refers to one or more physical structures used to contain the contents of the kit, such as an inventive composition. The packaging material is constructed by a well-known method and preferably provides a sterile and contaminant-free environment. The packaging material used in the kit is one that is conventionally used for gene expression assays or the administration of therapeutics. As used herein, the term “package” refers to an appropriate solid matrix or material, such as glass, plastic, paper, or foil, that can hold individual kit components. Thus, for example, the package could be a plastic vial or tube used to contain an appropriate amount of genetically encoded system and / or cells. The packaging material usually has an external label indicating the contents and / or purpose of the kit and / or its components.

[0167] V. Example The following examples are included for illustrative purposes only and are not intended to limit the scope of the concept of the present invention.

[0168] Example 1: Method for forming circular ASO / RNA Circular ASOs exert their biological functions by acting as transcription regulators.

[0169] Formation of circular RNA and circular ASO gamma by T4 ligase 1 or 2r

[0170] A typical reaction involves preparing 10-50 μM Linear ASO, 0.5-2 U RNA ligase 2 or RNA ligase 1 and 20 U RiboLock RNase 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), and 400-1000 μM adenosine triphosphate (ATP), 10% PEG 8000, 1-3 M betaine). RNA samples are pretreated at 80°C for 3 minutes and cooled to reaction temperature at a rate of 6°C / min. The reaction is then carried out at 25°C for 2 hours, and the mixture is heated at 75°C for 10 minutes to terminate. The cyclic structure of the reaction product is confirmed by treating with exonuclease T(5U) at 25°C for 6 hours.

[0171] General methods for cyclic ASO synthesis

[0172] Standard universal control pore glass (CPG) is used for RNA oligonucleotide synthesis. All oligonucleotides are synthesized according to standard RNA synthesis on ABI394 using 2-tert-butyldimethylsilyl (TBDMS) RNA monomers. The concentration of all oligonucleotides is measured at 260 nm using a Thermo Scientific NanoDrop 2000 spectrophotometer. High-performance liquid chromatography (HPLC) is performed on a Waters Alliance e2695 system equipped with a Waters Xbridge OST C18 column (2.5 μm, 10.0 × 50 mm). 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.

[0173] Synthesis and Purification of Cyclic ASOs

[0174] Dissolve the RNA in water to prepare a final 100 μM RNA solution. The final composition of the reaction mixture for cycling the RNA is 7 μL RNA solution, 1 μL 10 mM ATP, 1 μL 10 × reaction buffer, and 1 μL T4 RNA ligase (10 U / μL). Place 10 μL / tube of the solution into the PCR chamber at 4°C for 12 hours. After mixing these liquids, mix the crude product with 6 × RNA loading buffer (0.25% bromophenol blue and 30% glycerol in DEPC-treated water). Load the solution (18 μL / well) onto a 20% natural polyacrylamide PAGE (1 mm thick) gel. Then, electrophoresis the gel at 220 V for 50 minutes using 1 × trisborate EDTA (TBE) buffer (pH 8.2). For each preparative gel, the sample lanes on both sides of the gel were cut, stained with 1 × SYBR Gold (Invitrogen), and then imaged. Since the images were printed according to the same size of the gel, it was possible to mark the gel positions without staining with SYBR Gold. The gel zones at the marked positions were cut and crushed into small particles, and immersed overnight at 37°C in 1 × TBE buffer at 1°C. After filtering from the solid particles, the RNA solution was desalted and concentrated using a Millipore-Amicon Ultra-0.5 mL centrifuge filter (cutoff = 3,000). The collected product was lyophilized to remove moisture to obtain the final circular single-stranded RNA.

[0175] RNA oligonucleotides are characterized using ESI-MS, where oligonucleotides (~0.2 nmol) are dissolved in water / acetonitrile (50:50, 20 μL) containing 1% triethylamine to a final concentration of 10 μM. The solution is then analyzed in negative ion mode of ESI using a Waters Xevo G2 Q-Tof spectrometer. The molecular weight of cyclic ASO / RNA is 18 smaller than that of linear RNA due to the condensation reaction.

[0176] Dissolve the circular ASO / RNA in 1×PBS buffer to prepare a 6 μmol stock solution. Add 10 μL of the stock solution and heat to 85°C for 5 minutes to anneal the phosphate-modified RNA to form the 5' circular ASO / RNA, then let it cool to room temperature for at least 1 hour before further use.

[0177] Enzymatic stability of cyclic ASO

[0178] Cyclic ASO or control linear ASO (3 μM, 5 μL) is incubated in enzyme solution at 37°C to a final concentration of 1 μmol / L (15 μL). In this study, RNases are used for enzymatic stability, respectively. 3 μL aliquots (containing 3 pmol siRNA) are dispensed at different time points (2, 4, 6, and 8 hours), immediately frozen in liquid nitrogen, and stored at -80°C until assay. 1 μL 6 × RNA loading buffer is added to the aliquots. Samples are run on a 10% natural polyacrylamide gel in TBE buffer according to the procedure described above.

[0179] chemical synthesis

[0180] Cyclic ASOs containing disulfide bonds are chemically synthesized as follows: A passenger chain containing disulfide bonds with protecting groups at both ends is obtained. The thiol modifier C6 SS amidite and the 3'-thiol modifier C3 SS CPG are used at the 5' and 3' ends, respectively, to synthesize a passenger chain containing disulfide bonds at both ends. 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. Circular passenger strands are prepared by adding 5 equivalents of 2-(methoxythio)-3-nitropyridine (Npys-OMe) and acetonitrile (MeCN / water = 1 / 3). Subsequently, the solution is 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 for 0–22.0 minutes at 60°C at a flow rate of 1 mL / min using a linear gradient of 5%–50% buffer B. The purified oligonucleotides are recovered, desalted on a NAP-10 column (GE Healthcare), and concentrated by centrifugation. The aqueous solution of the resulting circular passenger chain with disulfide bonds is mixed equimolarly with an aqueous solution of the guide chain purchased from GeneDesign, Inc., 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.

[0181] The chemical synthesis of non-cleavable cyclic ASOs is carried out as follows: Passenger strands containing a 5'-hexynyl phosphoramidite (Glen Research) at the 5' end and an azide-modified CPG (PRIMETECH ALC) at the 3' end were purchased from GeneDesign, Inc. Non-cleavable circular passenger strands are synthesized. Briefly, 10-20 Cu wires (Fujifilm Wako Pure Chemical Co., Ltd.) are added to a solution of passenger strands (50 μM) containing NaCl (200 mM). The solution is heated to 80°C for 3 minutes and 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 an NAP-10 column (GE Healthcare) and subjected to reverse-phase preparative HPLC as described above. Next, the purified oligonucleotides are recovered, desalted using an NAP-10 column, and concentrated by centrifugation. Annealing is carried out as described above.

[0182] Liquid chromatography-mass spectrometry

[0183] The purity and structure of the synthesized oligonucleotides are measured by liquid chromatography-mass spectrometry (LC-MS) using an Agilent 6120 series single quadrupole LC / MS system (Agilent Technologies). Liquid chromatography is performed using an ACQUITY BEH C18 column (1.7 μm, 2.1 × 50 mm; water) with buffers A (8.6 mM trimethylamine and 100 mM hexafluoroisopropanol in water) and B (methanol). Oligonucleotides are separated at 60°C at a flow rate of 0.3 mL / min with a linear gradient of 0 to 18.0 mins from 10% to 90% buffer B. 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 incubated with DPBS(-) at a flow rate of 1 mL / min at 25°C for 20 minutes.

[0184] 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) containing buffer A (8.6 mM trimethylamine and 100 mM hexafluoroisopropanol aqueous solution) and buffer B (methanol). Oligonucleotides are separated at 50°C at a flow rate of 0.3 mL / min for 0–10 minutes with a linear gradient of 10%–90% of buffer B. MaxEnt-1 software is used for mass deconvolution of MS data. Collision activation at the selected m / z is performed with a collision energy of 25 eV.

[0185] cell culture

[0186] HeLa cells (ATCC) and RAW264.7 cells (ATCC) were supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin in RPMI 1640 medium (Life Technologies; A10491-01) at 37°C in a humidified atmosphere of 5% CO2. Similarly, HepG2 cells (ATCC), Huh-7 cells (JCRB), and L929 cells (RCB) were maintained in MEM (11095-080; Gibco), Dulbecco's Modified Eagle Medium (DMEM), and Minimum Essential Medium (MEM) (11095-080; Gibco), each at 37°C in a humidified atmosphere of 5% CO2. Primary mouse hepatocytes (MSCP10; Life Technologies) were thawed and plated in William's E medium (A1217601; Life Technologies) with primary hepatocyte supplement (CM3000; Thermo Fisher Scientific).

[0187] In vitro knockdown assay

[0188] The transfection-based in vitro knockdown assay is performed in cultured cells as follows: Oligonucleotides and RNAiMAX (13778-075; Life Technologies) (fin. 0.2%) are mixed with Opti-MEM (31985-070; Life Technologies), and the oligonucleotide / RNAiMAX solution is prepared according to the manufacturer's protocol. Next, 20 μL of the solution is added to a 96-well plate (167008; Nunc), followed by 80 μL of cell suspension (10,000 cells / well). After gentle shaking, the plate is maintained in a humidified atmosphere of 5% CO2 at 37°C for 24 hours.

[0189] The in vitro knockdown assay by free uptake (nasal rhesus) in cultured cells is performed as follows: Add 20 microliters of oligonucleotide solution diluted with Opti-MEM (Life Technologies; 31985-070) to a 96-well plate (167008; Nunc), followed by 80 μL of cell suspension (2,000-3,000 cells / well). After gentle shaking, maintain the plate in a humidified atmosphere of 5% CO2 at 37°C for 96 hours. For primary mouse hepatocytes, add 20 μL of diluted oligonucleotide solution to a 96-well collagen I multi-well microplate (#356702; Corning), followed by 80 μL of cell suspension, and incubate for 24 hours in a humidified atmosphere of 5% CO2 at 37°C.

[0190] After culturing oligonucleotide-treated cells, total RNA is extracted and converted to cDNA using the SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo) according to the manufacturer's protocol. Reaction conditions are 15 minutes at 37°C, 5 minutes at 50°C, 5 minutes at 98°C, and 5 minutes at 4°C. mRNA levels are evaluated by quantitative RT-PCR using TaqMan Gene Expression Master Mix (Life Technologies), the TaqMan probes listed above (Table 2), and QuantStudio 12K flex (Thermo Fisher Scientific) for 40 cycles × reaction conditions of 2 minutes at 50°C, 10 minutes at 95°C, and (15 seconds at 95°C, 1 minute at 60°C). Relative mRNA expression is quantified using the comparative Ct method.

[0191] Example 2: Method for synthesizing oligonucleotides Oligonucleotides are synthesized using the MerMade-12 DNA / RNA synthesizer. Glen Research's Stirling solvents / reagents, Prime Synthesis's 500-A controlled-pore glass (CPG) solid supports, Thermo's 2'-deoxy 3'-phosphoramidites, and Hongene's 2'-OMe and 2'-F nucleoside 3'-phosphoramidites 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 15% dimethylformamide (DMF), and conjugated using standard conditions on the synthesizer. GalNAc CPG compatible versions are available. 5-bromohexyl phosphoramidite (Glen Research, Cat# 10-1946) is dissolved in 0.15 M acetonitrile and bound using standard synthesizer conditions. Alkyne CPG support and alkyne hydroxyprolinol phosphoramidite (Y) are prepared. Low-water-content acetonitrile is purchased from EMD Chemicals. As an activator, a solution of 0.6 M 5-(S-ethylthio)-1H-tetrazole in acetonitrile is used. The phosphoramidite solution is 0.15 M in anhydrous acetonitrile containing 15% DMF as a cosolvent for 2'-OMe uridine and cytidine. The oxidizing reagent 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 sulfidation agent. The detritylation agent is 3% dichloroacetic acid (DCA) in dichloromethane (DCM).

[0192] Example 3: Analysis of oligonucleotide stability in plasma and liver homogenates Rat plasma (BioIVT, Cat#RAT00PL38NCXNN) and liver homogenate (BioIVT, custom order) are diluted in 10× cofactor solution to achieve final concentrations of 1 mM MgCl2, 1 mM MnCl2, and 2 mM CaCl2. SciRNA is added to 50 μl of plasma or liver homogenate to achieve a final cohesiveness of 20 μg / ml. The reaction mixture is incubated at 37°C with gentle shaking. At each predetermined time point (0, 1, 4, 8, and 24 hours), the reaction is stopped by adding 450 μl of Clarity OTX lysis loading buffer (Phenomenex, Cat#AL0-8579) containing an internal standard (oligonucleotide U21 at a final concentration of 1 μg / ml), and the mixture is frozen at -80°C until analysis. The experiment is performed in three separate steps.

[0193] Oligonucleotide concentration for LC-MS analysis is performed using a Clarity OTX 96-well solid column (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 and 2 mM sodium azide in HPLC-grade water. The sample is loaded onto the SPE column under positive pressure. The column is then washed five times with 1 ml of 50 mM ammonium acetate in 50 / 50 (v / v) water and acetonitrile (pH 5.5). Finally, the oligonucleotide is eluted in 40 / 10 / 50 (v / v / v) acetonitrile / tetrahydrofuran / water (pH 8.8) using an elution buffer containing 10 mM EDTA and 100 mM ammonium bicarbonate. The eluent is dried under nitrogen and resuspended in 120 μl of LC-MS-grade water for LC-MS analysis.

[0194] Relative quantification and metabolite identification of modified oligonucleotides are performed using high-resolution mass spectrometry on a Thermo Scientific Q Exactive coupled with ion-pair reversed-phase liquid chromatography (Dionex Ultimate 3000) (LC-HRMS). For chromatographic separation, a Waters X-BridgeBEH C8 XP column (Cat# 176002554, 130 A, 2.5 μm, 2.1 mm × 30 mm, 80°C) is used. The injection volume and flow rate are 30 μl and 1 ml / min, respectively. Mobile phase A consisted of 16 mM triethylamine (Sigma, Cat# 471283) and 200 mM 1,1,1,3,3,3-hexafluoro-2-propanol (Fisher, Cat# 67-56-1) in LC-MS grade water (Fisher, Cat# 7732-18-5). Mobile phase B is 100% methanol (Fisher, Cat# 67-56-1). The gradient starts with 1% mobile phase B and progresses to 35% B in 4.3 minutes, after which the column is equilibrated with 1% mobile phase B for 1 minute. Mass spectrometer data acquisition is performed in full scan mode with a scan range of 500-3000 m / z and a resolution setting of 35,000. The spray voltage is 2.8kV. The auxiliary gas temperature and capillary temperature are set to 300°C.

[0195] 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 Deconvolution software (Novatia, LLC) to identify the linearization and major metabolic pathways of the modified oligonucleotide.

[0196] The half-life is calculated by monitoring the loss of the full-length test oligonucleotide over 24 hours. The amounts of the test oligonucleotide and the internal standard are normalized to time 0 at each point in time for each oligonucleotide. The percentage of remaining full length and the natural logarithm of the slope are calculated using linear regression. The half-life is calculated using the following formula: t12 = -Ln(2)k.

[0197] Thermal fusion research

[0198] Melting studies are performed using a Beckman DU800 spectrophotometer with a thermoprogrammer in a quartz cell with a 1 cm path length. Samples are diluted to obtain a final concentration of approximately 1 μM of oligonucleotide chains in 0.1 × PBS buffer (pH 7.4). The melting curve is monitored at 260 nm with a heating rate of 1 °C / min at 10–90 °C. The melting temperature (Tm) is calculated from the first derivative of the heating curve, and the reported value is the result of two independent measurements.

[0199] NMR research

[0200] Lyophilized RNA is dissolved in a mixture of 10% 2H2O / 90% H2O and 20 mM NaCl and 10 mM sodium phosphate buffer (pH 7). The final concentration of double-stranded RNA in 600 μl ranges from 20 to 60 μM. All spectra are acquired at 25°C using an Agilent VNMRS 800 MHz NMR spectrometer with a cold probe.

[0201] Circular dichroism spectroscopy

[0202] Circular dichroism (CD) spectra are acquired using a Jasco J-815 spectropolarimeter with a Julaba F25 circulating bath. Samples are equilibrated in 1×PBS at 10°C for 5 minutes, with a final double-stranded concentration of 1.57 μM. Spectra are averaged 5 scans. Spectra are collected using a fused silica cell (Starna 29-Q-10) at a rate of 50 nm / min with a bandwidth of 1 nm and a sampling wavelength of 0.2 nm. CD spectra are recorded at 350–200 nm at 10°C. Molar ellipticity is calculated from equation [0] = 0 / 10Cl, where 0 is the ellipticity (mdeg), C is the molar concentration of the oligonucleotide (M), and l is the cell path length (cm).

[0203] Example 4: Chemical synthesis oligonucleotide synthesis

[0204] Oligonucleotides (1 μmol scale) are synthesized in an ABI 381A or 394 DNA synthesizer using a cycle that includes phosphoramidite chemistry. 2.5% DCA is detritylated in CH2Cl2 for 60 seconds. Coupling step: BMT (0.3 M in dry acetonitrile) is used as the activator. Propynyl and bromohexyl phosphoramidites (0.09 M in CH3CN) are introduced with a coupling time of 45 seconds. Commercial phosphoramidites (0.09 M in CH3CN) are introduced with a binding time of 30 seconds. The capping step is performed in acetic anhydride for 15 seconds using commercial solutions (Cap A: Ac2O, pyridine, THF 10 / 10 / 80 and Cap B: 10% N-methylimidazole in THF). Oxidation is performed for 10 seconds with a commercially available iodide solution (0.1 M I2, THF, pyridine / water 90 / 5 / 5).

[0205] General procedure for azidization

[0206] Azidation of 5-hydroxyl oligonucleotides is carried out according to known methods. Azidation from bromohexyl oligonucleotides is performed as follows: A solution of NaN3 (13 mg) and NaI (30 mg) in dry DMF (1.5 mL) is inoculated onto solid-supported bromohexyl oligonucleotides at 65°C for 1 hour and 15 minutes. Then, CPG beads containing the oligonucleotides are washed with DMF (2.1 mL) and CH2-Cl2 (5 mL) and dried in a desiccator under reduced pressure for 30 minutes.

[0207] General steps for removing protection

[0208] Place the beads in a sealed vial and treat with concentrated ammonia aqueous solution (1 mL) for 24 hours at room temperature if the oligonucleotide contains phosphotrister function, or for 2 hours at room temperature, followed by 5 hours at 55°C if the oligonucleotide is otherwise. Filter the beads and evaporate the solution. Dissolve the residue in water for further analysis.

[0209] General procedure for 1,3-dipole cyclic addition using a Cu(I) catalyst.

[0210] Azide alkyne oligonucleotide (1 μmol) is added to CuSO4 (0.4 equivalents, 0.4 μmol, in 13.2 μL of 20 mM H2O solution), freshly prepared sodium ascorbate (2 equivalents, 2 μmol, in 13.2 μL of 100 mM H2O solution) (from degassed water), methanol (100 μL), and water (23.6 μL). The tube containing the resulting preparation is washed with argon and sealed. The reaction is carried out in a Biotage microwave synthesizer initiator set to 100 W with a pre-mixing time of 30 seconds for 1 to 1 hour 30 minutes at 60°C. The temperature is monitored with an internal infrared probe. The solution is then desalted with NAP10.

[0211] Example 6: Administration of antisense oligonucleotides to rats Twenty-four passive metastatic myasthenia gravis (PTMG) model rats were randomly assigned by subcutaneous injection to three groups: ASO-C5 (2.5 mg / kg), ASO-C5 (5 mg / kg), and a saline control. Treatment was administered 10, 7, and 3 days prior to PTMG induction. Blood was collected before siRNA administration and immediately prior to 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. Debilitation was graded on a standard disease severity scale of 1–4, with higher scores indicating more severe debilitation. Blood, liver, diaphragm, and anterior tibialis muscle were collected for analysis.

[0212] C5 mRNA expression is quantified by qRT-PCR. Flash-frozen livers are ground with a 2000 Geno / Grinder (Spex SamplePrep), and RNA is extracted using the RNeasy Mini Kit according to the manufacturer's protocol (QIAGEN). Reverse transcription is performed to generate cDNA according to the manufacturer's protocol (Life Technologies). qPCR is performed against cDNA using a Roche LightCycler 480 instrument with rat C5 Taqman FAM probe (Life Technologies) and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Taqman VIC probe (Life Technologies) as controls. C5 levels are normalized to GAPDH, and the percentage of remaining C5 mRNA is calculated relative to the mean of untreated or saline-treated rats.

[0213] Rat serum samples are analyzed by semi-quantitative Western blotting. Serum is rediluted 1:20 in 50 mM Tris (pH 7) with 1% SDS. Samples are run on 10% Bis-Tris protein gel (Life Technologies) and transferred to a PVDF membrane (Bio-Rad). Goat anti-human C5 antibody (complement technology) used to breed rats is diluted 1:1,000 with secondary fluorescent dye-conjugated donkey anti-goat antibody (LI-COR) and subsequently imaged using the LI-COR Odyssey imaging system. Western blotting analysis and quantification are performed using LI-COR Image Studio software. The percentage of C5 remaining in the PTMG test is calculated by normalizing rat samples at day 8 to individual pre-bred samples.

[0214] Example 7: Administration of antisense oligonucleotides to humans The ASO-C5 formulation is administered subcutaneously to human subjects with complement system disorders. One month later, samples are taken from the subjects to measure C5 levels. Additional samples are taken to measure toxicity, ASO-C5 half-life, and immunogenicity.

[0215] Example 8: Formation of circular ASO / RNA Circular ASOs exert their biological functions by acting as transcription regulators.

[0216] Formation of loop-shaped / circular RNA by ligase enzymes

[0217] A typical reaction involves 10-50 μM linear ASO, 0.5-2 U RNA ligase 2, RNA ligase 1 or a heat-stable ligase, and 20 U RiboLock RNase 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), and 400-1000 μM adenosine triphosphate (ATP), 10% PEG 8000, 1-3 M betaine). RNA samples are pretreated at 80°C for 3 minutes and cooled to reaction temperature at a rate of 6°C / min. The ligation reaction is then heated at 25°C-60°C for 2-4 hours, and the mixture is finished by heating at 75°C for 10 minutes (Figure 5). The cyclic structure of the reaction product was confirmed by treatment with exonuclease T(5U) at 25°C for 6 hours (Figures 7A-7B).

[0218] Synthesis and Purification of Cyclic ASOs

[0219] Loop RNA was mixed with 2× RNA loading buffer (0.25% bromophenol blue and 95% formamide in DEPC-treated water). The samples were loaded onto a 12% urea polyacrylamide PAGE (1 mm thick) gel and electrophoresed with 1× trisborate 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, a nucleic acid dye, to preferentially stain the double-stranded structures. After staining for 30 minutes, images were acquired using the iBright imaging system (Thermo Fisher Scientific) (Figure 5A).

[0220] For large-scale purification of ligation products, UV shadowing was used for PAGE purification of loop sense strands by exposing the gel placed on a fluorescent TLC plate to short-wave UV light (254 nm). The cyclized bands were excised from the gel, pulverized, and incubated at 15°C with 5–10 mL of nuclease-free water O / N on a tumbler. oC. Diffused ASO was concentrated using a Millipore-Amicon Ultra-15 mL centrifugal filter (cutoff = 3,000 kDa). The quality and quantity of the collected loop-shaped ASO product were determined and frozen.

[0221] Enzymatic stability of circular / loop-shaped ASO

[0222] The cyclic nature of the reaction product was determined by exonuclease T (NEB) and / or phosphodiesterase I (PDI) (Crotalus atrox phosphodiesterase I; Sigma: P4506-100MG). Linear and loop-shaped ASO (10-50 pmol) were digested. The reaction was terminated by adding EDTA pH 8 to 10-50 mM, 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.

[0223] cell culture

[0224] HepG2 cells (ATCC) were maintained in EMEM (30-2003, ATCC) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin in a humidified incubator (37°C with 5% CO2). Primary mouse hepatocytes (B129-7224F; Cell Biologics) were cultured in a complete hepatocyte medium kit (M1265, Cell Biologics).

[0225] In vitro knockdown assay

[0226] siRNA and lipofectamine RNAiMAX (13778-075; Life Technologies) (0.3 μL / well for 96-well plates) were mixed separately with 25 μL of Opti-MEM (31985-070; Life Technologies) 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 quantification of total 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 either a single-step or two-step RT-qPCR assay. Reverse transcription using the TOYOBO kit was performed at 37°C for 15 minutes, 50°C for 5 minutes, 98°C for 5 minutes, and 4°C for 5 minutes. CFB and ApoB mRNA levels were quantitatively measured by RT-PCR, and detection with TaqMan Gene Expression Master Mix (IDT) or SYBR green dye was quantitatively measured for 40 cycles × on a QuantStudio 6-7 real-time PCR instrument. Relative mRNA expression was quantified using the comparative Ct method and / or serial dilution and standard curve. RPL13A and / or RPB1 mRNA served as housekeeping genes for quantifying target mRNA (Figure 9-13).

[0227] Example 9: Synthesis of oligonucleotides Unmodified and chemically modified oligonucleotides of different lengths were purchased from IDT (Coralville, Iowa). Example 4: Analysis of oligonucleotide stability in plasma

[0228] Rat serum (10–50%) (Sigma R9759) was diluted in 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 by incubating with 5–10 mM EDTA and / or 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 gels as described above.

Claims

1. Synthetic nucleic acid molecules including the following, including: single-stranded non-coding ribonucleic acid molecules, 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 single-stranded nucleic acids, which are reversibly or irreversibly bound, create a structure of a single-stranded nucleic acid molecule with no free ends. c) One or more adapters configured to enhance the specificity of target binding between a single-stranded non-coding ribonucleic acid molecule and a target nucleic acid sequence are otherwise identical to a single-stranded non-coding ribonucleic acid molecule without one or more adapters.

2. A 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, or 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 the gene shown in Table 1.

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

5. The synthetic nucleic acid molecule described in claim 2 has an RNA sequence which is a messenger RNA (mRNA) sequence.

6. A 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 bond, intron / exon bond, untranslated region, or regulatory region of the target nucleic acid sequence.

7. A 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. A synthetic nucleic acid molecule according to claim 2, wherein the single-stranded non-coding nucleic acid molecule is configured to regulate the expression level of a splicing event from the target nucleic acid sequence.

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

10. A synthetic nucleic acid molecule according to claim 7 or claim 8, wherein the regulation is a reduction 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. A 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. A 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. A synthetic nucleic acid molecule according to claim 13, wherein the linker is a nucleotide linker.

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

16. A 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. A synthetic nucleic acid molecule according to claim 17, 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 azide-alkyne linker, an aldehyde-oxamine linker, a phosphorotoate-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. A synthetic nucleic acid molecule according to claim 16, wherein the chemical linker is substantially incapable of cleavage 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. A synthetic nucleic acid molecule according to claim 1, wherein the single-stranded non-coding nucleic acid molecule includes the target portion, and the target portion is specific to the 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. A synthetic nucleic acid molecule according to claim 23, wherein the target portion comprises a polypeptide, a macrosilicic peptide, an RNA molecule, a lipophilic portion, nanoparticles, or a small molecule.

27. A 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. A synthetic nucleic acid molecule according to claim 27, wherein the polypeptide is glucagon-like peptide 1 receptor (GLP1R), asialoglycoprotein receptor (ASGPR), prostate-specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), epidermal 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, geranyloxyhexianol, 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 small molecule contains a sugar moiety.

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

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

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

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

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

39. The synthetic nucleic acid molecule according to claim 38 is a nucleotide whose modification is 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 with alternative chemical properties.

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

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

42. A 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 binding of the 5' and 3' ends of the single-stranded nucleic acid reduces the content of chemically modified nucleotides associated with harmful medical side effects compared to a control unbound single-stranded nucleic acid.

44. The synthetic nucleic acid molecule according to claim 43, wherein the adverse medical side effect is selected from the group consisting of thrombocytopenia, hypocardic heart rate, heart rate perturbation, increased blood pressure, or increased cardiac output.

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

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

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

48. A synthetic nucleic acid molecule according to claim 47, wherein the length comprises about 15 to about 20 consecutive nucleotides.

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

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

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

52. A pharmaceutical formulation comprising the following: a synthetic nucleic acid molecule according to any one of claims 1 to 51; a pharmaceutically acceptable excipient, carrier, or diluent.

53. A pharmaceutical preparation according to claim 52, wherein the pharmaceutical preparation 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 subject, including: administering a single-stranded non-coding nucleic acid molecule containing the following to a subject, 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 single-stranded nucleic acids, which are reversibly or irreversibly bound, create a structure of a single-stranded nucleic acid molecule with no free ends. c) One or more adapters configured to enhance the specificity of target binding between a single-stranded non-coding ribonucleic acid molecule and a target nucleic acid molecule, compared to a single-stranded non-coding ribonucleic acid molecule without one or more adapters, and Here, single-stranded non-coding nucleic acid molecules have 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 a single-stranded non-coding nucleic acid molecule to a 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, the method provides a single-stranded non-coding nucleic acid molecule comprising the following: 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 single-stranded nucleic acids, which are reversibly or irreversibly bound, create a structure of a single-stranded nucleic acid molecule with no free ends. c) One or more adapters configured to enhance the specificity of target binding between the target binding sequence and the target nucleic acid sequence of a single-stranded non-coding ribonucleic acid molecule compared to the same single-stranded non-coding ribonucleic acid molecule without the adapters of one or more adapters; and Under conditions sufficient to activate the transcription of the target gene, a single-stranded non-coding nucleic acid molecule is introduced into a sample containing the target gene.

59. The method according to claim 58, wherein the single-stranded non-coding nucleic acid molecule comprises an antisense strand including the 5' end of the antisense strand, which is attached to the 3' end of the antisense strand.

60. The method according to claim 59, wherein the antisense strand comprises 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 an untranslated region, intron, exon, intron / exon bond, exon / intron bond, 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 azide-alkyne linker, an aldehyde-oxamine linker, a phosphorotoate-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 substantially incapable of cleavage 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 includes a modification comprising 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 comprises the target portion, and the target 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, a macrocyclic peptide, an RNA molecule, a lipophilic portion, or a small molecule.

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 is glucagon-like peptide 1 receptor (GLP1R), asialoglycoprotein receptor (ASGPR), prostate-specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), epidermal 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 a lipid, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-0(hexadecyl)glycerol, geranyloxyhexianol, 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 small molecule includes a sugar portion.

86. The method according to claim 85, wherein the sugar portion comprises 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 target 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 the Asialoglicoprotein 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 chemical modification.

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

93. The method according to claim 91, wherein the chemical modification of the nucleotide is 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 with alternative chemical properties.

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

95. The synthetic nucleic acid molecule according to claim 93, wherein the nucleotide having alternative chemical properties is a phosphorodiamic acid morpholino oligonucleotide (PMO), thiophosphoamide, 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 has lower immunogenicity in vivo compared to a linear non-coding nucleic acid molecule otherwise identical.

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

98. The method according to claim 58, wherein the single-stranded non-coding nucleic acid molecule is less toxic to the target compared to the same linear non-coding nucleic acid molecule by other methods.

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 fewer off-target effects in the target compared to the same linear non-coding nucleic acid molecule by other methods.

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 improved durability in vivo compared to an otherwise identical linear non-coding nucleic acid molecule.

103. The method according to claim 102, which is measured using a durable in vivo nucleic acid detection technique.

104. The method according to claim 58, wherein the single-stranded non-coding nucleic acid molecule that does not have a free end is a cyclized oligonucleotide.

105. The method according to claim 104, wherein the cyclic 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 is composed of two or more ASOs.

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

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

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

112. Synthetic nucleic acid molecules, including: Single-stranded nucleic acid molecules without free ends, including: a) Functionally active oligonucleotides that target homologous mRNA. b) Adapter elements to enhance functionality. c) The portion that reversibly or irreversibly joins the ends of (a) and (b).

113. The synthetic nucleic acid molecule according to claim 112 further comprises a second portion which reversibly or irreversibly binds the ends of (a) and (b).

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