WSGR Docket No.65107-701.602 CHEMICALLY MODIFIED NUCLEOTIDES CROSS-REFERENCE [0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/490,978 filed on March 17, 2023, which is incorporated by reference in its entirety. BACKGROUND [0002] Single-stranded nucleic acid molecules (e.g., antisense oligonucleotides (ASOs)) are a versatile class of oligonucleotide therapeutics designed to target nucleic acid molecules, such as a messenger ribonucleic acid (mRNA), pre-mRNA, and/or non-coding (e.g., intronic or regulatory) regions of the RNA through Watson-Crick base pairing. Broadly speaking, the mechanism of action of these therapeutics falls under two different categories: Steric blocking ASOs, which are occupancy-only mediated single-stranded oligonucleotides that are complementary to the target sequence and can modulate splicing (exon inclusion or skipping), RNA processing and mRNA translation. The second class of ASOs act as guides for enzymes that can degrade mRNA (e.g., RNase H or Ago2), edit a target mRNA (e.g., ADAR) or DNA sequence (e.g., Cas9). The latter two are often classified as guide RNAs rather than ASOs, because of their longer size. The occupancy-only class of ASOs can result in upregulation or downregulation of the target mRNA through a wide range of mechanisms such as modulation of splicing, nonsense- mediated decay (NMD) alteration, miRNA function inhibition, modulation of upstream ORF (uORF) or translation inhibitory elements (TIE) utilization (all activation) or lead to downregulation of targets by inhibiting the translation machinery or altering polyadenylation. Splice switching oligonucleotides (SSOs) can also modulate splicing (exon inclusion or exclusion/skipping) of the pre-mRNA by binding to the exon, intron, or exon/intron junction. Steric blockade by ASOs can also modulate gene expression by binding to the non-protein coding and regulatory regions of genes (e.g., UTR regions) thereby modulating expression of gene expression products from the target nucleic acid molecule. There are numerous applications of single-stranded nucleotides, including but not limited to use as therapeutics to treat a variety of diseases or disorders. These single stranded nucleic acid ASO could be DNA, RNA, chimeric gapmer (RNA:DNA hybrid) based molecules and/or chemically-modified variations thereof. SUMMARY [0003] Aspects disclosed herein provide synthetic single-stranded nucleic acid molecules comprising: a single-stranded noncoding nucleic acid molecule complementary to the coding or
WSGR Docket No.65107-701.602 regulatory regions of the target gene (RNA or DNA) for modulation of expression from the said gene, wherein the 5’ and 3’ ends of the single-stranded nucleic acid are reversibly or irreversibly linked, generating a structure of the single-stranded nucleic acid molecule with no free ends. In some embodiments, the single-stranded noncoding nucleic acid molecule comprises a nucleic acid sequence complementary to a target nucleic acid sequence. [0004] Aspects disclosed herein provide synthetic nucleic acid molecule, comprising: a single-stranded noncoding ribonucleic acid molecule comprising: (a) a target-binding sequence that is complementary to the coding or regulatory regions of a target nucleic acid sequence; (b) a 5’ end and a 3’ end of the single-stranded nucleic acid that are reversibly or irreversibly linked, generating a structure of the single-stranded nucleic acid molecule with no free ends; and (c) one or more adapters configured to enhance specificity of target binding between the target-binding sequence of the single-stranded noncoding ribonucleic acid molecule and the target nucleic acid sequence, as compared with an otherwise identical single-stranded noncoding ribonucleic acid molecule that does not have an adapter of the one or more adapters. In some embodiments, the target nucleic acid sequence is a ribonucleic acid (RNA) sequence, a deoxyribonucleic acid (DNA) sequence, or a DNA/RNA hybrid sequence. In some embodiments, the target nucleic acid sequence encodes any one of the targets of 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 noncoding nucleic acid molecule is complementary to an exon, an intron, an exon/intron junction, an intron/exon junction, an untranslated region, or a regulatory region of the target nucleic acid sequence. In some embodiments, the single-stranded noncoding nucleic acid molecule is configured to modulate expression levels of a gene product expressed from the target nucleic acid sequence. In some embodiments, the single-stranded noncoding nucleic acid molecule is configured to modulate outcome of a splicing event from the target nucleic acid sequence. In some embodiments, the modulation is an increase in expression levels. In some embodiments, the modulation is a decrease in expression levels. In some embodiments, the single-stranded noncoding nucleic acid molecule is a chemically modified nucleic acid molecule. In some embodiments, the single-stranded noncoding nucleic acid molecule comprises about 16 to about 100 contiguous nucleotides. In some embodiments, the 5’ end of the single-stranded noncoding nucleic acid molecule is coupled to the 3’ end of the single-stranded noncoding 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 comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a
WSGR Docket No.65107-701.602 nucleotide linker, a glucuronide group, an azide-alkyne linker, an aldehyde-oxamine linker, a phosphorothoates-tosylated linker, a phosphate activation agent mediated phosphate-hydroxyl linkage, or a metal chelation ligation linker. In some embodiments, the chemical linker is substantially cleavable under intracellular conditions by an enzyme. In some embodiments, the chemical linker is independently substantially cleavable under intracellular conditions. In some embodiments, the chemical linker is substantially non-cleavable under intracellular conditions. In some embodiments, the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer. In some embodiments, the single-stranded noncoding nucleic acid molecule comprises a targeting moiety, wherein the targeting moiety is specific to a target cell or target tissue. In some embodiments, the target cell is a hepatic cell, a cardiac cell, a neuron, a muscle cell, a blood cell, a photoreceptor cell, a pancreatic cell, or a stem cell. In some embodiments, the target tissue is liver tissue, heart tissue, brain tissue, muscle tissue, nervous tissue, epithelial tissue, connective tissue, eye, or pancreatic tissue. In some embodiments, the targeting moiety comprises a polypeptide, a macrocylic peptide, an RNA molecule, a lipophilic moiety, a nanoparticle, 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 an agonist of Glucagon Like Peptide 1 Receptor (GLP1R), Asialoglycoprotein receptor (ASGPR), prostate specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), Epithelial cell adhesion molecule (EpCam), AXL receptor tyrosine kinase (AXL), protein tyrosine kinase 7 (PTK7), Programmed death-ligand 1 (PD-L1), T-cell immunoglobulin and mucin domain-3 (Tim-3). In some embodiments, the RNA molecule comprises an aptamer, a ribozyme, a hairpin RNA, a siRNA, or miRNA. In some embodiments, the lipophilic moiety comprises lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-0(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, bomeol, 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 comprises a sugar moiety. In some embodiments, the sugar moiety comprises an amino sugar. In some embodiments, the amino sugar is N-acetyl Galactosamine (GalNAc). In some embodiments, the targeting moiety is specific to an antigen or receptor of the target cell or target tissue. In some embodiments, the receptor comprises Asialoglycoprotein receptor (ASGPR). In some embodiments, the single- stranded noncoding nucleic acid molecule comprises one or more nucleotides comprising a modification. In some embodiments, the modification comprises a chemical modification. In some embodiments, the chemical modification comprises a modification of the sugar, the phosphate
WSGR Docket No.65107-701.602 backbone, or the nucleobase. In some embodiments, the modification of the nucleotide comprises a 2’-O-Me, a 2’-F, 2’-MOE, a N(6)-methyladenosine, a 5-methylcytidine, a 5-Methyluridine (ribothymidine), a ribose modification with bridged nucleic acids, or a nucleotide with an alternative chemistry. In some embodiments, the ribose modification with bridged nucleic acids is a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), or a constrained ethyl bridged nucleic acid (cEt). In some embodiments, the nucleotide with an alternative chemistry is a phosphorodiamidate morpholino oligonucleotide (PMO), a thiophosphoroamidate, a peptide nucleic acid (PNA), a tricyclo DNA (tcDNA), an unlocked nucleic acid (UNA), or a glycol nucleic acid (GNA). In some embodiments, the modification of the phosphate backbone linkage comprises a phosphodiester, phosphorothioate isomers (Sp or Rp), phosphoryl DMI amidate diester isomers, phosphorodithioate, methylphosphonate, 5’-phosphorothioate, peptide nucleic acid, 5’-(E)-vinylphosphonate, or 5’-methyl phosphonate. In some embodiments, the linkage of the 5’ and 3’ ends of the single-stranded nucleic acid allows for the inclusion of fewer chemically- modified nucleotides linked to adverse medical side-effects as compared to a control unlinked single-stranded nucleic acid. In some embodiments, the adverse medical side effects are selected from the group consisting of reduced platelets, thrombocytopenia, perturbation of heart rate, increased blood pressure, or increased cardiac output. In some embodiments, the one or more adapters comprises 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 comprising about 10 to about 25 contiguous nucleotides. In some embodiments, the length comprises about 15 to about 20 contiguous 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, the one or more adapters comprise a sugar, a lipid, a peptide, an antibody, a nucleotide sequence, an aptamer, or a combination thereof. In some embodiments, a pharmaceutical formulation is comprised 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, a cell comprises the synthetic nucleic acid molecule. [0005] Aspects disclosed here in provide synthetic nucleic acid molecules, comprising: a single-stranded noncoding ribonucleic acid molecule complementary to the coding or regulatory regions of a target nucleic acid sequence, wherein a 5’ end and a 3’ end of the single-stranded nucleic acid are reversibly or irreversibly linked, generating a structure of the single-stranded nucleic acid molecule with no 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
WSGR Docket No.65107-701.602 hybrid sequence or chemically-modified variations 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 noncoding nucleic acid molecule is complementary to an exon, an intron, an exon/intron junction, an intron/exon junction, an untranslated region or a regulatory region of the target nucleic acid sequence. In some embodiments, the single-stranded noncoding nucleic acid molecule is configured to modulate the outcome of a gene product expressed from the target nucleic acid sequence. In some embodiments, the single-stranded noncoding nucleic acid molecule is configured to modulate expression levels of a splicing event from the target nucleic acid sequence. In some embodiments, the modulation is an increase in expression levels. In some embodiments, the modulation is a decrease in expression levels. In some embodiments, the single- stranded noncoding nucleic acid molecule is a chemically modified nucleic acid molecule. In some embodiments, the single-stranded noncoding nucleic acid molecule comprises about 16 to about 100 contiguous nucleotides. In some embodiments, the 5’ end of the single-stranded noncoding nucleic acid molecule is coupled to the 3’ end of the single-stranded noncoding 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 comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, a glucuronide group, an azide-alkyne linker, an aldehyde-oxamine linker, a phosphorothoates-tosylated linker, a phosphate activation agent mediated phosphate-hydroxyl linkage, or a metal chelation ligation linker. In some embodiments, the chemical linker is substantially cleavable under intracellular conditions by an enzyme. In some embodiments, the chemical linker is independently substantially cleavable under intracellular conditions. In some embodiments, the chemical linker is substantially non-cleavable under intracellular conditions. In some embodiments, the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer. In some embodiments, the single-stranded noncoding nucleic acid molecule comprises a targeting moiety, wherein the targeting moiety is specific to a target cell or target tissue. In some embodiments, the target cell is a hepatic cell, a cardiac cell, a neuron, a muscle cell, a blood cell, a photoreceptor cell, a pancreatic cell, or a stem cell. In some embodiments, the target tissue is liver tissue, heart tissue, brain tissue, muscle tissue, nervous tissue, epithelial tissue, connective tissue, eye, or pancreatic tissue. In some embodiments,
WSGR Docket No.65107-701.602 the targeting moiety comprises a polypeptide, a macrocylic peptide, an RNA molecule, a lipophilic moiety, a nanoparticle, 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 an agonist of Glucagon Like Peptide 1 Receptor (GLP1R), Asialoglycoprotein receptor (ASGPR), prostate specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), Epithelial cell adhesion molecule (EpCam), AXL receptor tyrosine kinase (AXL), protein tyrosine kinase 7 (PTK7), Programmed death-ligand 1 (PD-L1), T-cell immunoglobulin and mucin domain-3 (Tim-3). In some embodiments, the RNA molecule comprises an aptamer, a ribozyme, a hairpin RNA, a siRNA, or miRNA. In some embodiments, the lipophilic moiety comprises lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-0(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, bomeol, 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 comprises a sugar moiety. In some embodiments, the sugar moiety comprises an amino sugar. In some embodiments, the amino sugar is N-acetyl Galactosamine (GalNAc). In some embodiments, the targeting moiety is specific to an antigen or receptor of the target cell or target tissue. In some embodiments, the receptor comprises Asialoglycoprotein receptor (ASGPR). In some embodiments, the single- stranded noncoding nucleic acid molecule comprises one or more nucleotides comprising a modification. In some embodiments, the modification comprises a chemical modification. In some embodiments, the chemical modification comprises a modification of the sugar, the phosphate backbone, or the nucleobase. In some embodiments, the modification of the nucleotide comprises a 2’-O-Me, a 2’-F, 2’-MOE, a N(6)-methyladenosine, a 5-methylcytidine, a 5-Methyluridine (ribothymidine), a ribose modification with bridged nucleic acids, or a nucleotide with an alternative chemistry. In some embodiments, the ribose modification with bridged nucleic acids is a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), or a constrained ethyl bridged nucleic acid (cEt). In some embodiments, the nucleotide with an alternative chemistry is a phosphorodiamidate morpholino oligonucleotide (PMO), a thiophosphoroamidate, a peptide nucleic acid (PNA), a tricyclo DNA (tcDNA), an unlocked nucleic acid (UNA), or a glycol nucleic acid (GNA). In some embodiments, the modification of the phosphate backbone linkage comprises a phosphodiester, phosphorothioate isomers (Sp or Rp), phosphoryl DMI amidate diester isomers, phosphorodithioate, methylphosphonate, 5’-phosphorothioate, peptide nucleic acid, 5’-(E)-vinylphosphonate, or 5’-methyl phosphonate. In some embodiments, the linkage of the 5’ and 3’ ends of the single-stranded nucleic acid allows for the inclusion of fewer chemically-
WSGR Docket No.65107-701.602 modified nucleotides linked to adverse medical side-effects as compared to a control unlinked single-stranded nucleic acid. In some embodiments, the adverse medical side effects are selected from the group consisting of reduced platelets, thrombocytopenia, perturbation of heart rate, increased blood pressure, or increased cardiac output. 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, a pharmaceutical formulation comprises a synthetic nucleic acid molecule as disclosed herein, and a pharmaceutically acceptable: excipient, carrier, or diluent. In some embodiments, the pharmaceutical formulation is formulated for subcutaneous administration. In some embodiments, a cell comprises the synthetic nucleic acid molecules as disclosed herein. [0006] Aspects disclosed herein provide methods for delivering a synthetic nucleic acid molecule to a subject, the method comprising: administering to the subject a single-stranded noncoding nucleic acid molecule complementary to the coding and/or regulatory regions of the target gene for modulation of expression from the said gene, wherein the 5’ and 3’ ends of the single-stranded noncoding nucleic acid molecule are linked, generating a structure with no free ends, and wherein the single-stranded noncoding nucleic acid molecule has a half-life in vivo that is greater than or equal to about 10 hours when measured using a quantitative nucleic acid detection assay. Aspects disclosed herein provide methods of delivering a synthetic nucleic acid molecule to a subject, the methods comprising: administering to the subject a single-stranded noncoding nucleic acid molecule comprising: (a) a target-binding sequence that is complementary to the coding or regulatory regions of a target nucleic acid sequence; (b) a 5’ end and a 3’ end of the single-stranded nucleic acid that are reversibly or irreversibly linked, generating a structure of the single-stranded nucleic acid molecule with no free ends; and (c) one or more adapters configured to enhance specificity of target binding between the target-binding sequence of the single-stranded noncoding ribonucleic acid molecule and the target nucleic acid sequence, as compared with an otherwise identical single-stranded noncoding ribonucleic acid molecule that does not have an adapter of the one or more adapters; and wherein the single-stranded noncoding nucleic acid molecule has a half-life in vivo that is greater than or equal to about 10 hours when measured using a quantitative nucleic acid detection assay. In some embodiments, the administering comprises subcutaneous, intravenous, intravitreal, or intrathecal administration of the single-stranded noncoding nucleic acid molecule to the subject. In some embodiments, the administering is performed at a frequency of less than once per month. [0007] Aspects disclosed herein provide methods for activating transcription of a gene of interest, the method comprising: (a) providing a single-stranded noncoding nucleic acid molecule complementary to the coding, intronic, or regulatory regions of a targeted gene, wherein a 5’ end
WSGR Docket No.65107-701.602 and a 3’ end of the single-stranded noncoding nucleic acid molecule are linked, generating a structure of the single-stranded nucleic acid molecule with no free ends; and (b) introducing the single-stranded noncoding nucleic acid molecule to a sample comprising the gene of interest under conditions sufficient to activate transcription of the gene of interest. Aspects disclosed herein provide methods for activating transcription of a gene of interest, the methods (a) a target-binding sequence that is complementary to the coding or regulatory regions of a target nucleic acid sequence; (b) a 5’ end and a 3’ end of the single-stranded nucleic acid that are reversibly or irreversibly linked, generating a structure of the single-stranded nucleic acid molecule with no free ends; and (c) one or more adapters configured to enhance specificity of target binding between the target-binding sequence of the single-stranded noncoding ribonucleic acid molecule and the target nucleic acid sequence, as compared with an otherwise identical single-stranded noncoding ribonucleic acid molecule that does not have an adapter of the one or more adapters; and introducing the single-stranded noncoding nucleic acid molecule to a sample comprising the gene of interest under conditions sufficient to activate transcription of the gene of interest. comprising: providing a single-stranded noncoding nucleic acid molecule comprising: In some embodiments, the single-stranded noncoding nucleic acid molecule comprises an antisense strand comprising a 5’ end of the antisense strand coupled to the 3’ end of the antisense strand. In some embodiments, the antisense strand comprises a nucleic acid sequence complementary to a nucleic acid sequence of a target RNA, wherein the target RNA encodes any one of the targets of Table 1. In some embodiments, the target RNA comprises a mRNA. In some embodiments, the nucleic acid sequence of the antisense strand is complementary to an untranslated region, an intron, an exon, an intron/exon junction, an exon/intron junction, a promoter, an enhancer, or a regulatory element of the target RNA. In some embodiments, the single-stranded noncoding nucleic acid molecule comprises about 19 to about 27 contiguous nucleotides. In some embodiments, the single-stranded noncoding nucleic acid molecule is an RNA molecule or a chemically modified nucleic acid. In some embodiments, the single-stranded noncoding nucleic acid molecule comprises about 16 to about 100 contiguous nucleotides. In some embodiments, the 5’ end of the single-stranded noncoding nucleic acid molecule is coupled to the 3’ end of the single-stranded noncoding 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 comprises a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, a glucuronide group, an azide-alkyne linker, an aldehyde-oxamine linker, a phosphorothoates- tosylated linker, a phosphate activation agent mediated phosphate-hydroxyl linkage, or a metal
WSGR Docket No.65107-701.602 chelation ligation linker. In some embodiments, the chemical linker is substantially cleavable under intracellular conditions by an enzyme. In some embodiments, the chemical linker is independently substantially cleavable under intracellular conditions. In some embodiments, the chemical linker is substantially non-cleavable under intracellular conditions. In some embodiments, the chemical linker comprises a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer. In some embodiments, the 5’ end of the antisense strand comprises a modification comprising 5’-(E)-Vinylphosphonate (5’-VP), 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), or 6’(phosphonooxy-Butyl-Sulfide)Purine (6-PBuS-purine). In some embodiments, the single- stranded noncoding nucleic acid molecule comprises a targeting moiety, wherein the targeting moiety is specific to a target cell or target tissue. In some embodiments, the target cell is a hepatic cell, a cardiac cell, a neuron, a muscle cell, a blood cell, a photoreceptor cell, a pancreatic cell, or a stem cell. In some embodiments, the target tissue is liver tissue, heart tissue, brain tissue, muscle tissue, nervous 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 an agonist of Glucagon Like Peptide 1 Receptor (GLP1R), Asialoglycoprotein receptor (ASGPR), prostate specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), Epithelial cell adhesion molecule (EpCam), AXL receptor tyrosine kinase (AXL), protein tyrosine kinase 7 (PTK7), Programmed death-ligand 1 (PD- L1), T-cell immunoglobulin and mucin domain-3 (Tim-3). In some embodiments, the RNA molecule comprises an aptamer, a ribozyme, a hairpin RNA, a siRNA, or miRNA. In some embodiments, the lipophilic moiety comprises lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-0(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, bomeol, 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 comprises a sugar moiety. In some embodiments, the sugar moiety comprises an amino sugar. In some embodiments, the amino sugar is N-acetyl Galactosamine (GalNAc). In some embodiments, the targeting moiety is specific to an antigen or receptor of the target cell or target tissue. In some embodiments, the receptor comprises Asialoglycoprotein receptor (ASGPR). In some embodiments, the single- stranded noncoding nucleic acid molecule comprises one or more nucleotides comprising a modification. In some embodiments, the modification comprises a chemical modification. In some
WSGR Docket No.65107-701.602 embodiments, the chemical modification comprises a modification of the sugar, the phosphate backbone, or the nucleobase. In some embodiments, the modification of the nucleotide comprises a 2’-O-Me, a 2’-F, 2’-MOE, a N(6)-methyladenosine, a 5-methylcytidine, a 5-Methyluridine (ribothymidine), a ribose modification with bridged nucleic acids, or a nucleotide with an alternative chemistry. In some embodiments, the ribose modification with bridged nucleic acids is a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), or a constrained ethyl bridged nucleic acid (cEt). In some embodiments, the nucleotide with an alternative chemistry is a phosphorodiamidate morpholino oligonucleotide (PMO), a thiophosphoroamidate, a peptide nucleic acid (PNA), a tricyclo DNA (tcDNA), an unlocked nucleic acid (UNA), or a glycol nucleic acid (GNA). In some embodiments, the single-stranded noncoding nucleic acid molecule exhibits less immunogenicity in vivo as compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, immunogenicity in vivo is measured by an immunogenicity assay. In some embodiments, the single-stranded noncoding nucleic acid molecule exhibits less toxicity in the subject as compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, toxicity is measured by subchronic or chronic toxicity tests. In some embodiments, the single-stranded noncoding nucleic acid molecule exhibits fewer off-target effects in the subject as compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, off-target effects are measured by gene expression analysis. In some embodiments, the single-stranded noncoding nucleic acid molecule exhibits increased durability in vivo as compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, durability in vivo is measured using nucleic acid detection techniques. In some embodiments, the single-stranded noncoding nucleic acid molecule with no free ends comprises a cyclized oligonucleotide. In some embodiments, the cyclized oligonucleotide constitutes a single or multivalent ASO that may or may not be separated by linker sequences of different length. In some embodiments, the multivalent ASO comprises two or more ASOs. In some embodiments, the two or more ASOs of the multivalent ASO target the same sequence and/or gene. In some embodiments, the two or more ASOs of the multivalent ASO target different sequences and/or genes. In some embodiments, the two or more ASOs of the multivalent ASO have the same mechanism of action. In some embodiments, the two or more ASOs of the multivalent ASO have different mechanisms of action. In some embodiments, the one or more adapters comprise a sugar, a lipid, a peptide, an antibody, a nucleotide sequence, an aptamer, or a combination thereof. [0008] Aspects disclosed herein provide synthetic nucleic acid molecules, comprising: a single-stranded nucleic acid molecule with no free ends, comprising: (a) a functionally active oligonucleotide targeting a cognate mRNA; (b) an adapter element that enhances functionality;
WSGR Docket No.65107-701.602 and (c) a moiety that reversibly or irreversibly joins the ends of (a) and (b). In some embodiments, the synthetic nucleic acid molecules further comprise (d) second moiety that reversibly or irreversibly joins the ends of (a) and (b). INCORPORATION BY REFERENCE [0009] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS [0010] The novel features of the inventive concepts are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present inventive concepts will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concepts are utilized, and the accompanying drawings of which: [0011] FIG.1A shows a non-limiting example of a looped antisense oligonucleotide (ASO), according to some embodiments herein. [0012] FIG. 1B depicts an exemplary ASO forming an engineered looped structure via an adapter element (A1) that conjoins the terminal ends of the loop through J1 and J2 junction sites, which facilitate irreversible and/or reversible binding of A1 and L1. [0013] FIG.1C shows non-limiting examples of backbone, ribose ring and base modifications of nucleotides, which can improve stability, immunogenicity, and potency of the looped ASO constructs. [0014] FIG. 2A shows non-limiting examples of various monovalent or multivalent circularized therapeutic ASOs, according to some embodiments herein. [0015] FIG. 2B illustrates the versatility and modularity of the engineered looped ASO constructs (top row). Multiple L modules (here L1-L3) can join via adapters (A1-A3) to form multivalent functional looped units that can act upon one or more target genes.
WSGR Docket No.65107-701.602 [0016] FIG.2C shows non-limiting exemplary demonstration of a bivalent (left) and trivalent (right) ASOs. Each ASO unit (L) of the multivalent ASOs could target an independent gene or RNA, thereby enhancing the valency of the looped construct. [0017] FIG. 2D shows non-limiting examples of moieties embedded in the Adapter section of the looped construct, which can act as either linkers or cell-specific targeting moieties (e.g., antibodies, aptamers, small molecules, and lipophilic moieties to improve looped ASO delivery, according to some embodiments herein. [0018] FIG. 3 shows non-limiting examples of locked and open conformations of ASOs, according to some embodiments herein. [0019] FIG.4A depicts linear ASO structures with phosphorothioate nucleotide backbones. [0020] FIG.4B depicts circular ASO structures with phosphodiester backbones. [0021] FIG.4C shows the chemical structure of phosphodiester and phosphorothioate bonds. [0022] FIG. 5A shows a diagram of enzymatic cyclization of an ASO, its separation and purification on a denaturing urea PAGE gel. Note the slower migration pattern of the cyclized ASO on PAGE (right lane). GelRed was used for visualization of open and looped ASOs. [0023] FIG. 5B shows a flowchart depicting a non-limiting example of steps involved in generation of cyclized ASO. [0024] FIG.6A shows accessible open ends of ASOs make them vulnerable to degradation by exonucleases (Pacman figures). [0025] FIG. 6B depicts an illustration showing resistance of looped/cyclized ASO to degradation by exonucleases due to the absence of open termini. [0026] FIG. 7A shows results from an experiment with linear and cyclized ASOs targeting CFB and ApoB. mRNAs were treated with Exonuclease T, a 3’to 5’ nuclease, for the indicated period of time. Cyclized oligonucleotides remained predominantly intact up to 2 hours after digestion with 0.16 U/µL exonuclease T, confirming formation of looped ASO and resistance of looped ASO construct against digestion by Exonuclease T. ASOs were separated on a 12% urea PAGE gel and stained with GelRed afterwards. [0027] FIG.7B shows a graph showing resistance of looped CFB ASO after treatment with Exonuclease T for the indicated time. Intensity of intact ASO bands was quantified with ImageJ and expressed as a fraction of the 0 minute time point. Error bars denote a standard deviation of 3 independent experiments. [0028] FIG.8A shows open and looped chemically-modified single stranded oligonucleotides were digested with rattlesnake phosphodiesterase I (PDI), a nuclease with primarily exonuclease activity towards nucleic acids. The looped construct displayed higher resistance to digestion by PDI.
WSGR Docket No.65107-701.602 [0029] FIG. 8B shows a gel image of the fraction of intact open (left) or looped (right) constructs after digestion with PDI for the indicated time. [0030] FIG.9A shows a lack of RNase H-mediated CFB degradation by the looped canonical structure after treating human hepatablastoma HepG2 cells with 50 nM of a canonical CFB ASO, or a canonical looped 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] FIG.9B illustrates the open canonical ASO (top) and looped canonical (adapter-less) ASO (bottom). [0032] FIG.10 shows that a CFB-targeting engineered looped ASO (L1-A1 looped construct) is functional and capable of targeting and degrading cognate CFB mRNA. Human hepatoblastoma HepG2 cells were Lipofectamine transfected with 50 nM open or looped engineered ASOs (L1- A1 containing ASOs) for 24 hours and the effect on mRNA knock-down was measured by probe- based RT q-PCR. [0033] FIG.11A shows loop formation by the canonical ApoB ASO (adapter-less, L1 only) abolishes the activity of canonical ApoB ASO. Panel depicts loss of RNase H-mediated ApoB degradation activity by the looped canonical structure (adapter-less). [0034] FIG.11B shows that the addition of A1 to L1 ApoB canonical structure (engineered looped ASO) restores the function of ASO and results in degradation of target ApoB mRNA in HepG2 cells 24 hours after treatment with 50nM lipofectamine reagent (error bars denote standard deviation). [0035] FIG. 11C summarizes the activity (functionality) of the open canonical ASO (top), looped canonical (adapter-less) ASO (center), and engineered looped ASO (L1-A1) (bottom). Experiments were carried out in human hepatoblastoma HepG2 cells and demonstrates that removal of A1 adapter from the looped L1 ASO structure abolishes depletion of target ApoB mRNA degradation. Cells were treated with 50 nM of a respective ApoB ASOs for 24 hrs. 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] FIG. 12 shows that the addition of Adapter A1 restores function to ApoB-targeting engineered looped ASO (L1-A1 looped construct), demonstrating the importance of adapter inclusion and optimization for the function of looped ASO. Figure also demonstrates generalizability of RNase H-mediated KD-effect by another looped ASO. Engineered looped ASO depleted ApoB mRNA to the same extent as open engineered ApoB ASOs. Primer 1 and primer 2 are two separate amplicons of ApoB as measured by SYBR green q RT-PCR. RPL13A was used as a housekeeping gene for normalization.
WSGR Docket No.65107-701.602 [0037] FIG. 13A shows the activity of multivalent engineered looped CFB ASOs. Two engineered looped 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] FIG. 13B depicts structural features of the bivalent and trivalent looped ASOs including the L1 ASOs and A1 adapters, which facilitate loop formation. [0039] FIG. 13C depicts structural features of the bivalent and trivalent looped ASOs including the L1, and L2 ASOs and A1, and A2 adapters, which facilitate loop formation. [0040] FIG. 13D shows structural features of the bivalent and trivalent looped ASOs including the L1, L2 and L3 ASOs and A1-A3 adapters, which facilitate loop formation. DETAILED DESCRIPTION [0041] Drug design is an exceedingly difficult problem, especially relating to genetic diseases. Existing single-stranded nucleic acid molecules (e.g., antisense oligonucleotides) used in gene therapies have limited clinical efficacy due, at least in part, to their instability in vivo and/or toxicity or off-target effects. Ongoing research and advancements in oligonucleotide technologies for use in gene therapies, such as those described in US App. No. 20230257745, hereby incorporated in its entirety, have limited clinical efficacy due, at least in part, to their instability in vivo and/or toxicity or off-target effects. For example, antisense oligonucleotides can be vulnerable to degradation by nucleases and other cellular enzymes, reducing their effectiveness and requiring higher doses for therapeutic efficacy. However, such existing single-stranded nucleic acid molecules (e.g., antisense oligonucleotides) lack adapters and other features, and thus suffer from less specificity and reduced target binding efficiency. [0042] Disclosed herein, in some embodiments, are compositions, methods, and kits comprising single-stranded noncoding nucleic acid molecules that modulate (e.g., increase or decrease) transcription of a target gene (e.g., a disease-associated gene) that exhibits greater durability (e.g., less instability) in vivo, less toxicity, or less immunogenicity, or any combination thereof. In some embodiments, the single-stranded noncoding nucleic acid molecules disclosed herein comprise an antisense strand with 5’ and 3’ ends that are coupled, 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 coupled reversibly, such that the single-stranded noncoding nucleic acid molecule can linearize in vivo at the site of action to induce modulation of transcription of the target gene. Additional moieties (e.g., antibodies, RNA aptamers, small molecules, lipophilic moieties, etc.) can be attached to the single-stranded noncoding nucleic acid molecule in order to
WSGR Docket No.65107-701.602 increase stability, direct targeting, lower off-target immunogenic effects, or otherwise improve pharmacological qualities of the single-stranded nucleic acid molecule. [0043] Disclosed herein are methods of delivering the single-stranded noncoding nucleic acid molecules of the present disclosure to a cell of a subject in vivo or ex vivo. In some embodiments, such delivery comprises administering a double-stranded noncoding nucleic acid molecule to the subject, such as by subcutaneous administration. In some embodiments, such delivery comprises administering a single-stranded noncoding nucleic acid molecule to the subject, such as by subcutaneous administration. In some embodiments, methods further comprise treating a disease or a condition in the subject by modulating the transcription of the target gene (e.g., disease- associated gene) with the single-stranded noncoding nucleic acid molecule. [0044] Also disclosed here are kits comprising the compositions and systems disclosed herein, and instructions for how to use the single-stranded noncoding nucleic acid molecules disclosed herein to modulate the transcription of a gene of interest. Such kits may comprise a container to store the system components and instructions. [0045] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. DEFINITIONS [0046] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. [0047] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
WSGR Docket No.65107-701.602 [0048] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof. [0049] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative, or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of” can include determining the amount of something present in addition to determining whether it is present or absent depending on the context. [0050] The term “circular” as used herein in reference to a single-stranded noncoding nucleic acid molecule, means that the 5’ and 3’ end of the single-stranded noncoding nucleic acid molecule are coupled to each other directly or indirectly such that the single-stranded noncoding nucleic acid molecule has no free ends, irrespective of the shape or conformation of the single- stranded noncoding nucleic acid molecule. [0051] The term “in vivo” is used to describe an event that takes place in a subject’s body. [0052] The term “ex vivo” is used to describe an event that takes place outside of a subject’s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an “in vitro” assay. [0053] The term “in vitro” is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed. [0054] The term “about” is used herein with reference to a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value. [0055] The terms, “polynucleotide,” “nucleotide” or “nucleic acid,” are used interchangeably herein to refer to polymers of nucleotides of any length and include DNA and RNA or hybrids thereof. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as, but not limited to methylated nucleotides and their analogs or non-nucleotide components. Modifications to the nucleotide structure may be imparted before or after assembly of the polynucleotide. A
WSGR Docket No.65107-701.602 polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. [0056] Unless specifically stated, as used herein, the terms “polynucleotide,” “nucleotide” or “nucleic acid,” encompass double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands). Nucleic acid sequences, when provided, are listed in the 5’ to 3’ direction, unless stated otherwise. Methods described herein provide for the generation of isolated nucleic acids. Methods described herein additionally provide for the generation of isolated and purified nucleic acids. A “nucleic acid” as referred to herein can comprise at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 375, at least about 400, at least about 425, at least about 450, at least about 475, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, or more bases in length. Moreover, provided herein are methods for the synthesis of 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, such as regulatory sequences e.g. promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived from microRNA, or any functional or structural DNA or RNA unit of interest. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. cDNA encoding for a gene or gene fragment referred herein
WSGR Docket No.65107-701.602 may comprise at least one region encoding for exon sequences without an intervening intron sequence in the genomic equivalent sequence. [0057] The term “synthetic,” as used herein with reference to a nucleic acid molecule, refers to production by in vitro chemical and/or enzymatic synthesis. [0058] The term “cell,” as used herein, generally refers to a biological cell. [0059] The term “gene,” as used herein, refers to a segment of nucleic acid that encodes an individual protein or RNA (also referred to as a “coding sequence” or “coding region”), optionally together with associated regulatory region such as promoter, operator, terminator and the like, which may be located upstream or downstream of the coding sequence. A “genetic locus” referred to herein, is a particular location within a gene. [0060] The terms “increased” or “increase” are used herein to generally mean an increase by a statically significant amount. [0061] The terms “decreased” or “decrease” are used herein generally to mean a decrease by a statistically significant amount. [0062] The terms “polypeptide,” “peptide” and “protein” may be used interchangeably herein in reference to a polymer of amino acid residues. A protein may refer to a full-length polypeptide as translated from a coding open reading frame, or as processed to its mature form, while a polypeptide or peptide may refer to a degradation fragment or a processing fragment of a protein that nonetheless uniquely or identifiably maps to a particular protein. A polypeptide may be a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Polypeptides may be modified, for example, by the addition of carbohydrate, phosphorylation, etc. [0063] The terms “homologous,” “homology,” or “percent homology” when used herein to describe to an amino acid sequence or a nucleic acid sequence, relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J Mol Biol.1990 Oct 5;215(3):403-10; Nucleic Acids Res. 1997 Sep 1;25(17):3389-402). Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application. Percent identity of sequences can be determined using the most recent version of BLAST, as of the filing date of this application. [0064] The term “percent (%) identity”, or “percent sequence identity,” with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence
WSGR Docket No.65107-701.602 identity, and not considering any conservative substitutions as part of the sequence identity. As used herein, the term “percent (%) identity”, or “percent sequence identity,” with respect to a reference nucleic acid sequence is the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. [0065] The terms “gene of interest,” or “GOI,” as used interchangeably herein, refer to a gene encoding a gene expression product that is detectable directly or indirectly. II. COMPOSITIONS Antisense Oligonucleotides [0066] Disclosed herein, in some embodiments, are single-stranded noncoding nucleic acid molecules. In some embodiments, the single-stranded noncoding nucleic acid molecules are any type of nucleic acid (e.g., ribonucleic acid (RNA), deoxyribonucleic acid (DNA), etc.) that contains one strand. In some embodiments, the single-stranded noncoding nucleic acid molecules are single-stranded oligonucleotides, which comprise short pieces of nucleotide sequence (e.g., DNA, RNA, or DNA and RNA hybrid). In some embodiments, the single-stranded noncoding nucleic acid molecule comprises an antisense strand configured to silence a target single-stranded nucleic acid sequence, such as a messenger RNA (mRNA). In some embodiments, the single- stranded noncoding nucleic acid molecule comprises an antisense strand configured to enhance expression of a target single-stranded nucleic acid sequence. In some embodiments, the single- stranded noncoding nucleic acid molecule is an antisense oligonucleotide (ASO), which generally comprise a single-stranded nucleic acid sequence that can bind to the target nucleic acid sequence,
WSGR Docket No.65107-701.602 such as another RNA (e.g., mRNA) through Watson-Crick base pairing. The single-stranded nucleic acid molecule can be synthetic. Alternatively, or in addition to, the single-stranded nucleic acid molecule can be a chemically modified nucleic acid molecule. The single-stranded nucleic acid molecule can comprise an antisense strand, which can be complementary or substantially complementary to a target nucleic acid sequence. [0067] A target nucleic acid sequence can be a single-stranded nucleic acid such as a single- stranded DNA or an RNA. A target nucleic acid sequence can be a coding RNA (e.g., an mRNA). For example, a target nucleic acid sequence can be a gene expression product from a gene encoding complement factor B(CFB), complement factor C5 (C5) or ApoB. Alternatively, a target nucleic acid sequence can be a non-coding RNA (e.g., a tRNA, an rRNA, a microRNA, a noncoding RNA, a small noncoding RNA, an intron, an exon, an intron/exon or exon/intron junction, a long noncoding RNA, a small interfering RNA, or a piwi-interacting RNA). Target nucleic acid sequences can be synthetic nucleic acid sequences. Alternatively, target nucleic acid sequences can be native to a cell or organism. In some embodiments, the target nucleic acid sequence is associated with a disease or a condition disclosed herein. For example, modulation of transcription of the target nucleic acid sequence (e.g., in the case of a DNA target) may be therapeutically effective to treat the disease or the condition. In another example, modulation of expression of the target nucleic acid sequence (e.g., in the case of an RNA target) may be therapeutically effective to treat the disease or the condition. In some embodiments, the modulation of the expression of the target nucleic acid sequence comprises post-transcriptional modifications, such as capping, splicing and polyadenylation of the RNA target. In some embodiments, the single-stranded nucleic acid (e.g., ASO) modulates splicing (e.g., exon inclusion or exon exclusion) of the pre-mRNA by binding to the exon, the intron, the exon/intron junction, or the intron/exon junction and promoting or suppressing splicing events. In some embodiment, steric blocking by the single-stranded nucleic acid (e.g., ASO) can modulate gene expression through binding to the non-protein coding and regulatory regions of genes, thereby modulating expression. Oligo Length [0068] Single-stranded nucleic acids (e.g., ASOs) can be at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24
WSGR Docket No.65107-701.602 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides, at least about 28 nucleotides, at least about 29 nucleotides, at least about 30 nucleotides, at least about 31 nucleotides, at least about 32 nucleotides, at least about 33 nucleotides, at least about 34 nucleotides, at least about 34 nucleotides, at least about 35 nucleotides, at least about 36 nucleotides, at least about 37 nucleotides, at least about 38 nucleotides, at least about 39 nucleotides, at least about 40 nucleotides, or more nucleotides in length. [0069] Single-stranded nucleic acids (e.g., ASOs) can be at most about 40 nucleotides, at most about 39 nucleotides, at most about 38 nucleotides, at most about 37 nucleotides, at most about 36 nucleotides, at most about 35 nucleotides, at most about 34 nucleotides, at most about 33 nucleotides, at most about 32 nucleotides, at most about 31 nucleotides, at most about 30 nucleotides, at most about 29 nucleotides, at most about 28 nucleotides, at most about 27 nucleotides, at most about 26 nucleotides, at most about 25 nucleotides, at most about 24 nucleotides, at most about 23 nucleotides, at most about 22 nucleotides, at most about 21 nucleotides, at most about 20 nucleotides, at most about 19 nucleotides, at most about 18 nucleotides, 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 fewer nucleotides in length. [0070] Single-stranded nucleic acids (e.g., ASOs) can be between about 5 to about 40 nucleotides in length. Single-stranded nucleic acids (e.g., ASOs) can be between about 5 to about 35 nucleotides in length. Single-stranded nucleic acids (e.g., ASOs) can be between about 5 to about 30 nucleotides in length. Single-stranded nucleic acids (e.g., ASOs) can be between about 5 to about 25 nucleotides in length. Single-stranded nucleic acids (e.g., ASOs) can be between about 5 to about 20 nucleotides in length. Single-stranded nucleic acids (e.g., ASOs) can be between about 5 to about 15 nucleotides in length. Single-stranded nucleic acids (e.g., ASOs) can be between about 5 to about 10 nucleotides in length. Single-stranded nucleic acids (e.g., ASOs) can be between about 10 to about 40 nucleotides in length. Single-stranded nucleic acids (e.g., ASOs) can be between about 15 to about 40 nucleotides in length. Single-stranded nucleic acids (e.g., ASOs) can be between about 20 to about 40 nucleotides in length. Single-stranded nucleic acids (e.g., ASOs) can be between about 25 to about 40 nucleotides in length. Single-stranded nucleic acids (e.g., ASOs) can be between about 30 to about 40 nucleotides in length. Single- stranded nucleic acids (e.g., ASOs) can be between about 35 to about 40 nucleotides in length. Complementary Binding
WSGR Docket No.65107-701.602 [0071] Antisense oligonucleotides can form complementary binding with an entire target strand. Alternatively, antisense oligonucleotides can form complementary binding with a part of a target nucleic acid sequence. The antisense strand can form complementary binding to one or more portions of a target nucleic acid sequence (e.g., an mRNA strand) including a 5’ UTR, a 3’UTR, a regulatory region, a coding sequence, an intron, an exon, an intron/exon junction, and/or exon/intron junction. A regulatory region of a target nucleic acid sequence (e.g., an mRNA) can comprise a promoter region, an enhancer region, an operator region, or a repressor region. [0072] ASOs can form complementary binding to part or all of a portion of an mRNA target strand. Antisense oligonucleotides can be completely complementary (e.g., 100% complementary) to their target strand counterparts. Alternatively, ASOs and can have imperfect complementarity to their target strand counterparts. An ASO can have at least about 50% complementarity, at least about 55% complementarity, at least about 60% complementarity, at least about 65% complementarity, at least about 70% complementarity, at least about 75% complementarity, at least about 80% complementarity, at least about 85% complementarity, at least about 90% complementarity, at least about 91% complementarity, at least about 92% complementarity, at least about 93% complementarity, at least about 94% complementarity, at least about 95% complementarity, at least about 96% complementarity, at least about 97% complementarity, at least about 98% complementarity, at least about 99% complementarity, or more to its corresponding target strand. An ASO can have at most about 99% complementarity, at most about 98% complementarity, at most about 97% complementarity, at most about 96% complementarity, at most about 95% complementarity, at most about 94% complementarity, at most about 93% complementarity, at most about 92% complementarity, at most about 91% complementarity, at most about 90% complementarity, at most about 85% complementarity, at most about 80% complementarity, at most about 75% complementarity, at most about 70% complementarity, at most about 65% complementarity, at most about 60% complementarity, at most about 55% complementarity, at most about 50% complementarity, or less to its corresponding target strand. Oligo Formations and Linkers [0073] A single-stranded noncoding nucleic acid molecule can be a DNA oligonucleotide. Alternatively, an ASO can be an RNA oligonucleotide. Alternatively, an ASO can be a chimeric oligonucleotide, which comprises RNA and DNA. ASOs can exist as a single, unfolded strand (e.g., a linear strand) (FIG. 4A). Alternatively, ASOs can exist in a folded (e.g., a hairpin) conformation. Alternatively, ASOs can exist in a circularized conformation (e.g., having no free ends) (FIG. 4B). For example, the 5’ and 3’ end of the antisense strand of the single-stranded noncoding nucleic acid molecule can be coupled such that the antisense stand has no free ends.
WSGR Docket No.65107-701.602 [0074] ASOs can exist as a single ASO or as an ASO comprised of multiple units or modules (e.g., an ASO comprised of more than one ASO) (FIG.13A-D). An ASO comprised of more than one ASO can be referred to as an ASO module and/or a multivalent ASO. In some embodiments, a multivalent ASO can comprise 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 ASOs. In some embodiments, a multivalent ASO can comprise at most about 10 ASOs, at most about 9 ASOs, at most about 8 ASOs, at most about 7 ASOs, at most about 6 ASOs, at most about 5 ASOs, at most about 4 ASOs, at most about 3 ASOs, at most about 2 ASOs, or fewer ASOs. Additional examples of monovalent or multivalent ASOs can be found in FIG.2. [0075] Multivalent ASOs can be circularized (FIG. 13A-D). In some embodiments, a circularized multivalent ASO comprising two or more ASOs can refer to a multivalent looped ASO. In some embodiments, a circularized multivalent ASO comprising two ASOs can refer to an ASO looped dimer (FIG. 13A-D). In some embodiments, a circularized multivalent ASO comprising three ASOs can refer to an ASO looped trimer (FIG.13A-D). In some embodiments, a multivalent ASO can be linked by at least about 2 linkers, at least about 3 linkers, at least about 4 linkers, at least about 5 linkers, at least about 6 linkers, at least about 7 linkers, at least about 8 linkers, at least about 9 linkers, at least about 10 linkers, or more linkers. In some embodiments, a multivalent ASO can be linked by at most about 10 linkers, at most about 9 linkers, at most about 8 linkers, at most about 7 linkers, at most about 6 linkers, at most about 5 linkers, at most about 4 linkers, at most about 3 linkers, or at most about 2 linkers. [0076] Circular ASOs can be made from their linear counterparts through enzymatic or chemical reactions to link the 5’ and 3’ ends of the linear strand (FIG.1). A circular ASO can be formed through the formation of a phosphodiester bond to link the 5’ and 3’ ends of a linear antisense strand. Alternatively, a circular ASO can be formed through the addition of a linker element to connect the 5’ and 3’ ends of the linear antisense strand. The linker element can be a chemical linker. The chemical linker can act as a junction to connect the 5’ and 3’ ends of the linear antisense strand to form a circular ASO. Alternatively, a linker can be used to span an exon/intron junction. A linker can include, but is not limited to a disulfide bond, a photocleavable linker, a diazo linker, an acid-labile linker, a peptide linker, a nucleotide linker, a glucuronide group, an azide-alkyne linker, an aldehyde-oxamine linker, a phosphorothoates-tosylated linker, a phosphate activation agent mediated phosphate-hydroxyl linkage, or a metal chelation ligation linker. A nucleotide linker can be an oligonucleotide linker, an oligo aptamer linker, an RNA linker, a DNA linker. A peptide linker can be a polypeptide linker (e.g., an antibody linker). A
WSGR Docket No.65107-701.602 linker can comprise a phosphodiester bond, an alkyl group, a sulfhydryl group, an amine group, or a polymer. [0077] A linker can be permanent (e.g., non-cleavable) (FIG.3). Alternatively, a linker can be reversible. For example, a linker can be cleavable (FIG. 3). A linker can be cleaved by, for example, an enzyme or a catalyst. Enzymes which have cleaving properties include but are not limited to proteases and endonucleases. A catalyst can be a chemical catalyst (e.g., an acid-based catalyst or a metal catalyst) or a non-chemical catalyst (e.g., light, heat, or pH). Alternatively, a linker can be cleaved through the use of a reversible click chemistry reaction. Alternatively, a linker can be independently cleaved. [0078] A click chemistry reaction is a reaction used to joining two specific molecular entities in the absence of water. Examples of click-chemistry reactions can include but are not limited to copper(I)-catalyzed azide-alkyne cycloadditions, strain promoted alkyne-nitrone cycloadditions, strain promoted azide-alkyne cycloadditions, alkene and azide cycloadditions, alkene and tetrazine invers-demand Diels-Alder reactions, and alkene and tetrazole photoclick reactions. Amines and thiols can be used to facilitate reversible click chemistry reactions. [0079] A linker can be cleavable under certain conditions. For example, a linker can be cleavable or substantially cleavable in intracellular conditions but non-cleavable or substantially non-cleavable in extracellular conditions. Alternatively, a linker can be non-cleavable or substantially non-cleavable in intracellular conditions but cleavable or substantially cleavable in extracellular conditions. Selective cleavability can be related to conditions such as but not limited to pH or ionic concentration. Adapters [0080] In some embodiments, a single-stranded noncoding nucleic acid molecule (e.g., ASO) can comprise an adapter. Non-limiting examples of adapters are peptide adapters, nucleotide adapters, antibody adapters, targeting moiety adapters (e.g., glycans), sugar adapters, lipid adapters, DNA aptamers, or RNA aptamers (FIG. 2D). A nucleotide adapter can be an oligonucleotide adapter, an oligo aptamer adapter, an RNA adapter, a DNA adapter. A peptide adapter can be a polypeptide adapter (e.g., a protein adapter). [0081] In some embodiments, the antisense strand comprises an adapter. In some embodiments, an adapter can be contiguous with a linker. Alternatively, in some embodiments, an adapter is not adjacent to a linker. In some embodiments, the single-stranded noncoding nucleic acid molecule comprises one adapter. Alternatively, in some embodiments, the single-stranded noncoding nucleic acid molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more adapters. ASOs can comprise a single adapter or multiple adapters (FIG.13A-D). In some embodiments, an ASO can comprise at least about 2 adapters, at least about 3 adapters, at least about 4 adapters, at least about
WSGR Docket No.65107-701.602 5 adapters, at least about 6 adapters, at least about 7 adapters, at least about 8 adapters, at least about 9 adapters, at least about 10 adapters, or more adapters. In some embodiments, an ASO can comprise at most about 10 adapters, at most about 9 adapters, at most about 8 adapters, at most about 7 adapters, at most about 6 adapters, at most about 5 adapters, at most about 4 adapters, at most about 3 adapters, at most about 2 adapters, or fewer adapters. [0082] A single ASO (e.g., monovalent ASO) comprising an adapter can be circularized. Monovalent ASOs comprising an adapter can be circularized by reversibly or irreversibly joining a free end of the adapter to a free end of the ASO through a linker. Multivalent ASOs comprising two or more adapters can be circularized (FIG.13A-D). ASOs can comprise a single adapter and one or more linkers. For example, a first free end of an adapter can be joined to a first free end of an ASO through a first linker, and a second free end of the adapter can be joined to a second free end of the ASO through a second linker, thus forming a looped ASO (FIG.13A-D). Multivalent ASOs can comprise two or more adapters and two or more linkers (FIG. 13A-D). In some embodiments, a multivalent ASO can be linked by at least about 2 adapters, at least about 3 adapters, at least about 4 adapters, at least about 5 adapters, at least about 6 adapters, at least about 7 adapters, at least about 8 adapters, at least about 9 adapters, at least about 10 adapters, or more adapters. In some embodiments, a multivalent ASO can be linked by at most about 10 adapters, at most about 9 adapters, at most about 8 adapters, at most about 7 adapters, at most about 6 adapters, at most about 5 adapters, at most about 4 adapters, at most about 3 adapters, or at most about 2 adapters. [0083] Adapters can comprise sequences that provide additional functionality for the single- stranded noncoding nucleic acid molecule (e.g., ASO). In some cases, an adapter can extend the 5’ and 3’ ends of the single-stranded noncoding nucleic acid molecule (e.g., ASO). Adapters can have full or partial complementarity to a flanking region of a gene of interest and/or a target gene (e.g., adapters can comprise hybridizing regions and/or sticky ends). Alternatively, adapters can comprise non-hybridizing regions (e.g., non-flanking regions, blunt ends, etc.). Adapters can add flexibility to a single-stranded noncoding nucleic acid molecule (e.g., ASO). For example, an adapter connecting the 5’ and 3’ ends of the single-stranded noncoding nucleic acid molecule (e.g., ASO) can improve the “steric freedom” of the single-stranded noncoding nucleic acid molecule (e.g., ASO) and allow it to interact with protein(s) involved in downstream events (e.g., degradation by RNase H) in an unrestricted manner. Alternatively, or additionally, an adapter can change the shape of a single-stranded noncoding nucleic acid molecule (e.g., ASO). An adapter can vary in length. For example, a glycan adapter can be between about 5 to 10 glycan residues in length to form a glycan chain. Alternatively, an antibody adapter can comprise a single antibody.
WSGR Docket No.65107-701.602 [0084] In some embodiments, an adapter can be a nucleotide adapter. A nucleotide adapter (e.g., DNA, RNA, etc.) can be at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides, at least about 28 nucleotides, at least about 29 nucleotides, at least about 30 nucleotides, at least about 31 nucleotides, at least about 32 nucleotides, at least about 33 nucleotides, at least about 34 nucleotides, at least about 34 nucleotides, at least about 35 nucleotides, at least about 36 nucleotides, at least about 37 nucleotides, at least about 38 nucleotides, at least about 39 nucleotides, at least about 40 nucleotides, or more nucleotides in length. [0085] A nucleotide adapter (e.g., DNA, RNA, etc.) can be at most about 40 nucleotides, at most about 39 nucleotides, at most about 38 nucleotides, at most about 37 nucleotides, at most about 36 nucleotides, at most about 35 nucleotides, at most about 34 nucleotides, at most about 33 nucleotides, at most about 32 nucleotides, at most about 31 nucleotides, at most about 30 nucleotides, at most about 29 nucleotides, at most about 28 nucleotides, at most about 27 nucleotides, at most about 26 nucleotides, at most about 25 nucleotides, at most about 24 nucleotides, at most about 23 nucleotides, at most about 22 nucleotides, at most about 21 nucleotides, at most about 20 nucleotides, at most about 19 nucleotides, at most about 18 nucleotides, 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 fewer nucleotides in length. [0086] A nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 5 to about 40 nucleotides in length. A nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 5 to about 35 nucleotides in length. A nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 5 to about 30 nucleotides in length. A nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 5 to about 25 nucleotides in length. A nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 5 to about 20 nucleotides in length. A nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 5 to about 15 nucleotides in length. A nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 5 to about 10 nucleotides in length. A nucleotide adapter (e.g., DNA,
WSGR Docket No.65107-701.602 RNA, etc.) can be between about 10 to about 40 nucleotides in length. A nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 15 to about 40 nucleotides in length. A nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 15 to about 20 nucleotides in length. A nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 20 to about 40 nucleotides in length. A nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 25 to about 40 nucleotides in length. A nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 30 to about 40 nucleotides in length. A nucleotide adapter (e.g., DNA, RNA, etc.) can be between about 35 to about 40 nucleotides in length. [0087] In some embodiments, an adapter can be a peptide adapter. A peptide adapter (e.g., polypeptide) can be 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 more peptides in length. [0088] A peptide adapter (e.g., polypeptide) can be at most about 20 peptides, at most about 19 peptides, at most about 18 peptides, at most about 17 peptides, at most about 16 peptides, at most about 15 peptides, at most about 14 peptides, at most about 13 peptides, at most about 12 peptides, at most about 11 peptides, at most about 10 peptides, at most about 9 peptides, at most about 8 peptides, at most about 7 peptides, at most about 6 peptides, at most about 5 peptides, at most about 4 peptides, at most about 3 peptides, at most about 2 peptides, or fewer peptides in length. [0089] A peptide adapter (e.g., polypeptide) can be between about 2 to about 20 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 2 to about 18 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 2 to about 16 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 2 to about 14 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 2 to about 12 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 2 to about 10 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 2 to about 8 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 2 to about 6 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 2 to about 4 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 4 to about 20 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 6 to about 20 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 8 to about 20 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 10 to about 20 peptides in length. A peptide adapter (e.g.,
WSGR Docket No.65107-701.602 polypeptide) can be between about 12 to about 20 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 14 to about 20 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 16 to about 20 peptides in length. A peptide adapter (e.g., polypeptide) can be between about 18 to about 20 peptides in length. [0090] The use of an adapter can enhance the function of an ASO. For example, the enhanced function of the ASO with an adapter can be due to increased steric freedom of the modified ASO, allowing it to interact with proteins involved in downstream events (e.g., degradation by RNAse) in an unrestricted manner. In some embodiments, an ASO with an adapter can have increased steric freedom as compared to an ASO without an adapter. In some embodiments, an ASO with an adapter can have at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500% or more increased steric freedom as compared to an ASO without an adapter. In some embodiments, an ASO with an adapter can have at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less increased steric freedom as compared to an ASO without an adapter. [0091] In some embodiments, an ASO with an adapter can have increased target specificity as compared to an ASO without an adapter. In some embodiments, an ASO with an adapter can have at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, or more increased target specificity as compared to an ASO without an adapter. In some embodiments, an ASO with an adapter can have at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at
WSGR Docket No.65107-701.602 most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less increased target specificity as compared to an ASO without an adapter. [0092] In some embodiments, an ASO with an adapter can have increased target binding as compared to an ASO without an adapter. In some embodiments, an ASO with an adapter can have at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, or more increased target binding as compared to an ASO without an adapter. In some embodiments, an ASO with an adapter can have at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less increased target binding as compared to an ASO without an adapter. Modifications [0093] Single-stranded noncoding nucleic acid molecules disclosed herein can comprise a modification of the sugar, the phosphate backbone, or the nucleobase. For example, noncoding nucleic acid molecules can be modified through the addition of moieties onto the molecule. A modification can be a chemical modification, a synthetic modification, or a natural modification. [0094] Nucleic acid molecules can be modified at the nucleobase. Nucleobase modifications include but are not limited to 2’-O-methylation (2’-OMe), conversion of uridine to pseudouridine, N(6)-methyladenosine, 5-methylcytidine, 5-methyluridine (ribothymidine), 2’-fluoro (2’F), and 2’-O-methoxyethyl (2’-MOE), ribose modification and bridged nucleic acids (e.g., locked nucleic acid (LNA) , ethylene-bridged nucleic acids (ENA), or constrained ethyl bridged nucleic acid (cEt) modifications), or nucleotides with alternative chemistries (e.g., phosphorodiamidate morpholino oligonucleotides (PMO), peptide nucleic acids (PNA), tricyclo DNA (tcDNA), unlocked nucleic acids (UNA), or glycol nucleic acids (GNA)).
WSGR Docket No.65107-701.602 [0095] Nucleic acid molecules can be modified at the phosphate backbone (FIG. 4C). A phosphate backbone can be modified to comprise a phosphodiester, phosphorothioate isomers (e.g., stereoisomers Sp and/or Rp), phosphoryl DMI amidate diester isomers, a phosphorodithioate, a methylphosphonate, a 5’-phosphorothioate, a thiophosphoroamidate, a peptide nucleic acid, a 5’-(E)-vinylphosphonate, or a 5’-methyl phosphonate. [0096] Additional moieties can be added or attached onto a single-stranded nucleic acid (e.g., an ASO). Additional moieties can include, but are not limited to antibodies, lipophilic moieties, small molecules, and RNA aptamers (e.g., ribozymes). Additional moieties can be added to single- stranded nucleic acids to alter the pharmacological features (e.g., structural or chemical parameters) of the single-stranded nucleic acid. A single-stranded nucleic acid can be modified with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more moieties. An additional moiety can be added at the 5’ end of a single-stranded nucleic acid. Alternatively, an additional moiety can be added at the 3’ end of a single-stranded nucleic acid. Alternatively, an additional moiety can be added in the middle of a single-stranded nucleic acid (e.g., neither at the 3’ end nor the 5’ end). [0097] Modification can be made to single-stranded nucleic acids (e.g., ASOs) in order to increase stability. The single-stranded nucleic acids as described herein can have an increased stability of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more as compared to a control single-stranded nucleic acid. [0098] Nucleic acid stability can be measured by analyzing the half-life of the single-stranded noncoding nucleic acid molecule. The single-stranded nucleic acid molecule as described herein can have an increased half-life of at least about 30 minutes, at least about 1 hour, at least about 90 minutes, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, or more as compared to a control single-stranded nucleic acid. [0099] Modifications can be made to single-stranded noncoding nucleic acid molecules (e.g., ASOs) in order to lower off-target effects. The number of off-target effects as measured by
WSGR Docket No.65107-701.602 sequencing can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more as compared to a control single-stranded nucleic acid. [0100] Modifications can be made to single-stranded noncoding nucleic acid molecules (e.g., ASOs) in order to lower adverse immunogenic effects. The number of adverse immunogenic effects as measured by an immunogenicity assay can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more as compared to a control single-stranded nucleic acid. [0101] Modifications can be made to single-stranded noncoding nucleic acid molecules (e.g., ASOs) in order to lower toxicity. Toxicity as measured by an toxicity assay can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more as compared to a control single-stranded nucleic acid. The control single-stranded nucleic acid molecule can be an otherwise identical single-stranded noncoding nucleic acid molecule without the modification. [0102] Modifications can be made to single-stranded noncoding nucleic acid molecules (e.g., ASOs) in order to increase stability and/or durability. Durability and/or stability as measured by nucleic acid detection can be increased by least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more as compared to a control single-stranded nucleic acid. The control single-stranded nucleic acid molecule can be an otherwise identical single- stranded noncoding nucleic acid molecule without the modification. [0103] Modifications, such as cyclization, can be made to single-stranded noncoding nucleic acid molecules (e.g., ASOs). A cyclized noncoding nucleic acid molecule can be a single-stranded noncoding nucleic acid molecule with no free ends. The single-stranded noncoding nucleic acid molecule with no free ends can refer to a cyclized oligonucleotide. A cyclized oligonucleotide can
WSGR Docket No.65107-701.602 be a single (e.g., monovalent) ASO or a multivalent ASO. The multivalent ASO can comprise two or more ASOs. The two or more ASOs can be separated by linker sequences, where the linker sequences can be different lengths. The two or more ASOs of the multivalent ASO can target the same sequence and/or gene. Alternatively, the two or more ASOs of the multivalent ASO can target different sequences and/or genes. The two or more ASOs of the multivalent ASO can have the same mechanism of action and/or a different mechanism of action. Cyclization and/or circularization of the single-stranded noncoding nucleic acid molecules can allow for the inclusion of fewer chemically-modified nucleotides (e.g., nucleotides containing phosphorothioates) which are linked to adverse medical side effects. Cyclization and/or circularization can allow for the inclusion of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 fewer chemically-modified nucleotides which are linked to adverse medical side effects. Adverse medical side effects can include but are not limited to reduced platelets, thrombocytopenia, perturbation of heart rate, raised blood pressure, or raised cardiac output through the activation of the complement cascade. Target Modulation [0104] Single-stranded noncoding nucleic acid molecules can be used to effect transcriptional regulation. For example, an ASO can bind to a target gene or an mRNA of a target gene (e.g., a target RNA) in order to modulate the expression of the target gene. Alternatively, an ASO can bind to a specific region of a target gene or a target mRNA such as a regulatory region (e.g., a UTR, an intron, an exon, an intron/exon junction, an exon/intron junction) in order to modulate the target gene. Alternatively, or in addition to, an ASO can bind to a specific region of a target gene or a target mRNA to modulate the splicing of the target gene or target mRNA. [0105] Modulation of a target gene can result in increased expression of the target gene. Alternatively, modulation of a target gene can result in decreased expression of the target gene. Alternatively, modulation of a target gene can maintain the expression of the target gene. [0106] In some cases, a single-stranded noncoding nucleic acid molecule can increase expression of a target gene by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more as compared to a control expression level.
WSGR Docket No.65107-701.602 [0107] In some cases, a single-stranded noncoding nucleic acid molecule can increase expression of a target gene by at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0.1%, or less as compared to a control expression level. [0108] In some cases, a single-stranded noncoding nucleic acid molecule can increase expression of a target gene by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9- fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control expression level. [0109] In some cases, a single-stranded noncoding nucleic acid molecule can increase expression of a target gene by at most or less than about 10,000-fold, at most or less than about 5,000-fold, at most or less than about 1,000-fold, at most or less than about 500-fold, at most or less than about 100-fold, at most or less than about 90-fold, at most or less than about 80-fold, at most or less than about 70-fold, at most or less than about 60-fold, at most or less than about 50-fold, at most or less than about 40-fold, at most or less than about 30-fold, at most or less than about 20-fold, at most or less than about 10-fold, at most or less than about 9-fold, at most or less than about 8-fold, at most or less than about 7-fold, at most or less than about 6-fold, at most or less than about 5-fold, at most or less than about 4-fold, at most or less than about 3-fold, at most or less than about 2-fold, at most or less than about 1-fold, at most or less than about 0.9-fold, at most or less than about 0.8-fold, at most or less than about 0.7-fold, at most or less than about 0.6-fold, at most or less than about 0.5-fold, at most or less than about 0.4-fold, at most or less
WSGR Docket No.65107-701.602 than about 0.3-fold, at most or less than about 0.2-fold, at most or less than about 0.1-fold, as compared to a control expression level. [0110] In some cases, a single-stranded noncoding nucleic acid molecule can decrease expression of a target gene by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more as compared to a control expression level. [0111] In some cases, a single-stranded noncoding nucleic acid molecule can decrease expression of a target gene by at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0.1%, or less as compared to a control expression level. [0112] In some cases, a single-stranded noncoding nucleic acid molecule can decrease expression of a target gene by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9- fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control expression level. [0113] In some cases, a single-stranded noncoding nucleic acid molecule can decrease expression of a target gene by at most or less than about 10,000-fold, at most or less than about
WSGR Docket No.65107-701.602 5,000-fold, at most or less than about 1,000-fold, at most or less than about 500-fold, at most or less than about 100-fold, at most or less than about 90-fold, at most or less than about 80-fold, at most or less than about 70-fold, at most or less than about 60-fold, at most or less than about 50-fold, at most or less than about 40-fold, at most or less than about 30-fold, at most or less than about 20-fold, at most or less than about 10-fold, at most or less than about 9-fold, at most or less than about 8-fold, at most or less than about 7-fold, at most or less than about 6-fold, at most or less than about 5-fold, at most or less than about 4-fold, at most or less than about 3-fold, at most or less than about 2-fold, at most or less than about 1-fold, at most or less than about 0.9-fold, at most or less than about 0.8-fold, at most or less than about 0.7-fold, at most or less than about 0.6-fold, at most or less than about 0.5-fold, at most or less than about 0.4-fold, at most or less than about 0.3-fold, at most or less than about 0.2-fold, at most or less than about 0.1-fold, as compared to a control expression level. [0114] Modulation of a target mRNA can result in increased expression of the gene product expressed from the target mRNA. Alternatively, modulation of a target mRNA can result in decreased expression of the gene product expressed from the target mRNA. Alternatively, modulation of a target mRNA can maintain expression of the gene product expressed from the target mRNA. [0115] In some cases, a single-stranded noncoding nucleic acid molecule can increase expression of a target mRNA by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more as compared to a control expression level. [0116] In some cases, a single-stranded noncoding nucleic acid molecule can increase expression of a target mRNA by at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0.1%, or less as compared to a control expression level.
WSGR Docket No.65107-701.602 [0117] In some cases, a single-stranded noncoding nucleic acid molecule can increase expression of a target mRNA by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9- fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control expression level. [0118] In some cases, a single-stranded noncoding nucleic acid molecule can increase expression of a target mRNA by at most or less than about 10,000-fold, at most or less than about 5,000-fold, at most or less than about 1,000-fold, at most or less than about 500-fold, at most or less than about 100-fold, at most or less than about 90-fold, at most or less than about 80-fold, at most or less than about 70-fold, at most or less than about 60-fold, at most or less than about 50-fold, at most or less than about 40-fold, at most or less than about 30-fold, at most or less than about 20-fold, at most or less than about 10-fold, at most or less than about 9-fold, at most or less than about 8-fold, at most or less than about 7-fold, at most or less than about 6-fold, at most or less than about 5-fold, at most or less than about 4-fold, at most or less than about 3-fold, at most or less than about 2-fold, at most or less than about 1-fold, at most or less than about 0.9-fold, at most or less than about 0.8-fold, at most or less than about 0.7-fold, at most or less than about 0.6-fold, at most or less than about 0.5-fold, at most or less than about 0.4-fold, at most or less than about 0.3-fold, at most or less than about 0.2-fold, at most or less than about 0.1-fold, as compared to a control expression level. [0119] In some cases, a single-stranded noncoding nucleic acid molecule can decrease expression of a target mRNA by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at
WSGR Docket No.65107-701.602 least about 200%, at least about 300%, at least about 400%, at least about 500%, or more as compared to a control expression level. [0120] In some cases, a single-stranded noncoding nucleic acid molecule can decrease expression of a target mRNA by at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0.1%, or less as compared to a control expression level. [0121] In some cases, a single-stranded noncoding nucleic acid molecule can decrease expression of a target mRNA by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9- fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control expression level. [0122] In some cases, a single-stranded noncoding nucleic acid molecule can decrease expression of a target mRNA by at most or less than about 10,000-fold, at most or less than about 5,000-fold, at most or less than about 1,000-fold, at most or less than about 500-fold, at most or less than about 100-fold, at most or less than about 90-fold, at most or less than about 80-fold, at most or less than about 70-fold, at most or less than about 60-fold, at most or less than about 50-fold, at most or less than about 40-fold, at most or less than about 30-fold, at most or less than about 20-fold, at most or less than about 10-fold, at most or less than about 9-fold, at most or less than about 8-fold, at most or less than about 7-fold, at most or less than about 6-fold, at most or less than about 5-fold, at most or less than about 4-fold, at most or less than about 3-fold, at most or less than about 2-fold, at most or less than about 1-fold, at most or less than about 0.9-fold, at most or less than about 0.8-fold, at most or less than about 0.7-fold, at most or less than about
WSGR Docket No.65107-701.602 0.6-fold, at most or less than about 0.5-fold, at most or less than about 0.4-fold, at most or less than about 0.3-fold, at most or less than about 0.2-fold, at most or less than about 0.1-fold, as compared to a control expression level. Targeting Moieties [0123] Disclosed herein are single stranded noncoding nucleic acid molecules comprising one or more targeting moieties. A targeting moiety can be used to direct a single-stranded noncoding nucleic acid molecule to a target cell or tissue. A targeting moiety can be but is not limited to a lipophilic moiety, a small molecule, a peptide (e.g., a polypeptide, a macrocylic peptide, etc.), an RNA molecule, a nanoparticle, an antibody, a single-domain antibody, a miniprotein, or an antigen binding fragment thereof. A targeting moiety can be specific to an antigen or receptor on the target cell or tissue (e.g., Asialoglycoprotein receptor (ASGPR)). [0124] A targeting moiety can be a lipophilic moiety. lipophilic moiety can comprise one or more fatty acid groups or salts thereof. A lipophilic be a lipid. Lipids are fatty acids and their derivatives which are insoluble in water but soluble in organic solvents. In some embodiments, a lipophilic moiety can be unsaturated. Alternatively, or in addition to, a lipophilic moiety can be monosaturated. Alternatively, or in addition to, a lipophilic moiety can be polysaturated. In some embodiments, a double bond of an unsaturated lipophilic moiety can be in a cis conformation. Alternatively, or in addition to, a double bond of an unsaturated lipophilic moiety can be in a trans conformation. Non-limiting examples of lipophilic moieties can be a triglyceride, a phospholipid, a sterol, an oil, a wax, a hormone, a vitamin, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis- 0(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, bomeol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03- (oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. [0125] A targeting moiety can be a small molecule. A small molecule can be a sugar, an amino acid, a phenolic compound, an alkaloid, a sterol, a lipid, a fatty acid, or other small chemical compound. A chemical compound can be a molecule that has a molecular weight of less than 1000 Daltons. Alternatively, or in addition to, a small molecule is a molecule with a size on the order of 1 nm. [0126] A targeting moiety can be a sugar or sugar moiety. A sugar can be a monosaccharide. Alternatively, a sugar can be a disaccharide. Alternatively, a sugar can be a polysaccharide. Non- limiting examples of sugars include glucose, dextrose, fructose, galactose, a sugar alcohol, a pentose, xylose, ribose, sucrose, cellulose, starch, lactose, maltose, trehalose, lactulose, cellobiose, chitobiose, glycogen, or chitin. A small molecule can be an amino sugar such as but not limited to -acetyl Galactosamine (GalNAc), N-acetylglucosamine or sialic acid.
WSGR Docket No.65107-701.602 [0127] A targeting moiety can be an antibody or an antigen-binding fragment thereof. An antibody, also known as an immunoglobulin, is a blood protein produced to counteract a specific antigen. Antibodies can be Y-shaped proteins which comprise variable binding sites that are specific to particular epitopes. An antibody can be a monoclonal antibody. Alternatively, an antibody can be a polyclonal antibody. An antibody can be a single-domain antibody. An antibody can be an antibody fragment. An antibody can be an agonist. Alternatively, an antibody can be an antagonist. Alternatively, an antibody can be an allosteric modulator (e.g., a positive allosteric modulator or a negative allosteric modulator). [0128] A targeting moiety can be a polypeptide. Non-limiting examples of polypeptides can be a macrocylic peptide, an agonist of Glucagon Like Peptide 1 Receptor (GLP1R), Asialoglycoprotein receptor (ASGPR), prostate specific membrane antigen (PSMA), human transferrin receptor (hTfR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), Epithelial cell adhesion molecule (EpCam), AXL receptor tyrosine kinase (AXL), protein tyrosine kinase 7 (PTK7), Programmed death-ligand 1 (PD-L1), and T-cell immunoglobulin and mucin domain-3 (Tim-3). [0129] A targeting moiety can be an RNA molecule. An RNA molecule can comprise an aptamer, a ribozyme, a hairpin RNA, a siRNA, or a miRNA. [0130] A single-stranded nucleic acid can be targeted to a target gene. Alternatively, a single- stranded nucleic acid can be targeted to an RNA molecule that encodes a target gene. 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, PPARD, PPOX, PRPF3, PRPF8, PSD3, RAGE, RDH12, RGR, RLBP1, RPE65, SARS-CoV-2 virus protein, SCN1A, SCNN1A (ENaC2), SERPINA1, SMN2, SNCA, SOD1, SOD2, STAT3,
WSGR Docket No.65107-701.602 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 which encode a component of the complement system, a part of the innate immune system. C5 can be involved in inflammation, homeostasis, and defense against pathogens. The C5 protein is comprised of the C5 alpha and beta chains, which are linked by a disulfide bridge. Mutations in the C5 gene can cause complement component 5 deficiency, a disease characterized by recurrent bacterial infections. [0132] A single-stranded noncoding nucleic acid molecule can be engineered with a targeting moiety to target a target cell. A target cell can be but is not limited to a stem cell (e.g., an induced pluripotent stem cell, an embryonic stem cell), a bone cell, a blood cell, a red blood cell, a white blood cell, a platelet, a dermal cell, a fibroblast, a hepatocyte, a lymphocyte, a bone marrow cell, a glandular cell, a hepatic cell, a cardiac cell, a pancreatic cell, a gall bladder cell, a muscle cell, a sperm cell, an egg cell, a fat cell, a nerve cell, a neuron, a Schwann cell, an interneuron, an immune cell, an osteoblast, a chondrogenic cell, an odontoblast, a cementoblast, a chondrocyte, a mesenchymal cell, an epithelial cell, a secretory cell, a germ cell, a nurse cell, a storage cell, a pituitary cell, a glial cell, an interstitial cell, an lymphoid cell, a B cell, a T cell, a natural killer cell, a myeloid cell, a enteroendocrine cell, a thyroid gland cell, a parathyroid gland cell, a sweat gland cell, a mammary gland cell, a pituitary cell, a melanocyte, a duct cell, and a photoreceptor. [0133] A single-stranded noncoding nucleic acid molecule can be engineered with a targeting moiety to target a target tissue. A target tissue can be but is not limited to nervous tissue, brain tissue, spinal cord tissue, epithelial tissue, epidermal tissue, intestinal tissue, heart tissue, liver tissue, eye tissue, pancreatic tissue, lung tissue, bladder tissue, muscle tissue, cardiac muscle tissue, smooth muscle tissue, skeletal muscle tissue, connective tissue fat tissue, bone tissue, and tendon tissue. Diseases and Disorders [0134] Disclosed herein, in some embodiments, are single-stranded noncoding nucleic acid molecules and pharmaceutical formulations thereof that are therapeutically effective to treat a disease or disorder disclosed herein. In some embodiments, the single-stranded nucleic acids disclosed herein can be used to treat a disease or disorder in a subject. Non-limiting examples of diseases or disorders can be cancer, inflammatory diseases or disorder, metabolic diseases or disorders, cardiovascular diseases or disorders, immunodeficiency diseases or disorders, respiratory diseases or disorders, pain, digestive diseases or disorders, reproductive diseases or disorders, endocrine diseases or disorders, immune diseases or disorders, autoimmune diseases or disorders, or neurological diseases or disorders. Table 1 describes the relationship between non- limiting examples target genes, non-limiting examples of target tissues, and non-limiting
WSGR Docket No.65107-701.602 examples of diseases and disorders. In some embodiments, the single-stranded noncoding nucleic acid molecule comprises an antisense strand configured to modulate expression of a gene expression product expressed from a gene of interest provided in Table 1. In some embodiments, the modulation of the expressing of the gene of interest is therapeutically effective to treat the indication provided in Table 1 that corresponds with the gene of interest. In some embodiments, the modulation can be activation or inhibition. In some embodiments, the single-stranded noncoding nucleic acid molecule further comprises a targeting moiety specific to the target tissue provided in Table 1 corresponding with the gene of interest and indication. Table 1
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[0135] A subject can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be an animal such as a fish, a bird, a reptile, an insect, an amphibian, or a mammal. The mammal can be a feline, a canine, a primate, an ape, a rodent, a camelid, a pig, a sheep, a cow, a horse, a goat, or a rabbit. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease. Pharmaceutical Formulations [0136] A pharmaceutical formulation can comprise a composition disclosed herein. A pharmaceutical formulation can further comprise an excipient. An excipient can be a buffer, a carrier, a stabilizer, a solubilizer, a filler, a preservative, a dilutant, a vehicle, a detergent, a salt, a
WSGR Docket No.65107-701.602 peptide, a surfactant, an oligosaccharide, an amino acid, an adjuvant, a carbohydrate, and/or a bulking agent. [0137] A pharmaceutical formulation can remain in the subject for at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 28 hours, at least about 32 hours, at least about 36 hours, at least about 40 hours, at least about 44 hours, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, or more. [0138] A pharmaceutical formulation can remain in the subject for at most about 4 weeks, at most about 3 weeks, at most about 2 weeks, at most about 13 days, at most about 12 days, at most about 11 days, at most about 10 days, at most about 9 days, at most about 8 days, at most about 7 days, at most about 6 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, at most about 44 hours, at most about 40 hours, at most about 36 hours, at most about 32 hours, at most about 28 hours, at most about 24 hours, at most about 23 hours, at most about 22 hours, at most about 21 hours, at most about 20 hours, at most about 19 hours, at most about 18 hours, at most about 17 hours, at most about 17 hours, at most about 16 hours, at most about 15 hours, at most about 14 hours, at most about 13 hours, at most about 12 hours, at most about 11 hours, at most about 10 hours, at most about 9 hours, at most about 8 hours, at most about 7 hours, at most about 6 hours, at most about 5 hours, at most about 4 hours, at most about 3 hours, at most about 2 hours, at most about 1 hour, at most about 30 minutes, or less. [0139] A pharmaceutical formulation can have a half-life as measured by a transcription inhibition assay. A pharmaceutical formulation can have a half-life of at least about 15 minutes, 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 28 hours, at least about 32 hours, at least about 36 hours, at least about 40 hours, at
WSGR Docket No.65107-701.602 least about 44 hours, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, or more. [0140] A pharmaceutical formulation can have a half-life of at most about 4 weeks, at most about 3 weeks, at most about 2 weeks, at most about 13 days, at most about 12 days, at most about 11 days, at most about 10 days, at most about 9 days, at most about 8 days, at most about 7 days, at most about 6 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, at most about 44 hours, at most about 40 hours, at most about 36 hours, at most about 32 hours, at most about 28 hours, at most about 24 hours, at most about 23 hours, at most about 22 hours, at most about 21 hours, at most about 20 hours, at most about 19 hours, at most about 18 hours, at most about 17 hours, at most about 17 hours, at most about 16 hours, at most about 15 hours, at most about 14 hours, at most about 13 hours, at most about 12 hours, at most about 11 hours, at most about 10 hours, at most about 9 hours, at most about 8 hours, at most about 7 hours, at most about 6 hours, at most about 5 hours, at most about 4 hours, at most about 3 hours, at most about 2 hours, at most about 1 hour, at most about 30 minutes, at most about 15 minutes, or less. Cells [0141] Provided herein are cells that may be engineered to contain or express one or more systems disclosed herein. In some embodiments, the cell comprises the single-stranded nucleic acids as disclosed herein. The cell(s) may be isolated from other sample components, or reaction components. The cell(s) may be purified. For example, a cell sample comprising the cell(s) may have a purity of at least or about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The cell(s) may be formulated in a pharmaceutical composition or formulation, such as for treatment of a disease or a condition provided in Table 1. The cell(s) may be a cell line, or a plurality of cells containing or expressing the one or more systems of the present disclosure. III. METHODS [0142] Disclosed herein, in some embodiments, are methods of creating, isolating, and/or purifying the single-stranded nucleic acid compositions disclosed herein. Also disclosed herein, in some embodiments, are methods of utilizing the compositions or pharmaceutical formulations disclosed herein to treat diseases and disorders of a subject. The methods disclosed herein may be modified by applying molecular barcodes to the nucleic acid molecules.
WSGR Docket No.65107-701.602 [0143] In some embodiments, the methods of the present disclosure comprise purifying or isolating the single-stranded noncoding nucleic acid molecules. Single-stranded noncoding nucleic acid molecules can be purified and/or isolated through the use of several processes such as but not limited to phenol-chloroform extraction, a DNA filtration column, salt and proteinase K treatments, and the use of silica-gel membranes. [0144] In some embodiments, the methods of the present disclosure comprise delivering the single-stranded noncoding nucleic acid molecule to a target cell or tissue. The compositions disclosed herein can be delivered to a target through the use of several means such as viral vector particles (e.g., retrovirus, adenovirus, adeno-associated virus (AAV), or herpes simplex virus), cationic nanoparticles, lipid nanoparticles, cationic polymers, plasmids, cells, or physical methods (e.g., sonication, electroporation, lipofection). [0145] In some embodiments, the methods comprise enhancing or silencing expression or translation of a gene of interest by introducing the single-stranded noncoding nucleic acid molecule (e.g., ASO) of the present disclosure to a target cell or tissue. In some embodiments, the antisense strand of the single-stranded noncoding nucleic acid molecule (e.g., ASO) is complementary to a target nucleic acid sequence that either encodes the gene of interest or modulates expression of the gene of interest. In some embodiments, the antisense strand of the single-stranded noncoding nucleic acid molecule (e.g., ASO) is complementary to a transcriptional enhancer, transcriptional silencer, or a promoter of transcription of a gene of interest. In some embodiments, the antisense strand of the single-stranded noncoding nucleic acid molecule (e.g., ASO) is complementary to long noncoding RNAs (lncRNAs) or microRNAs (miRNAs) to affect regulation of expression of the gene of interest. In some embodiments, the antisense strand of the single-stranded noncoding nucleic acid molecule (e.g., ASO) is complementary to intronic regions of pre-mRNA (prior to splicing) to affect translation of the resulting mRNA isoforms. In some embodiments, the antisense strand of the single-stranded noncoding nucleic acid molecule (e.g., ASO) is complementary regions of mRNA (after splicing) to affect translation of the mRNA. For example, the antisense strand may silence expression or translation of the gene of interest or enhance expression or translation of the gene of interest. [0146] In some embodiments, the methods comprise enhancing or silencing expression and/or translation of a gene of interest by introducing the single-stranded noncoding nucleic acid molecules (e.g., ASOs) of the present disclosure to a target cell or tissue. In some embodiments, introducing the single-stranded noncoding nucleic acid molecule of the present disclosure to a target cell or tissue enhances or silences expression and/or translation of a gene of interest as compared with introducing an otherwise identical synthetic nucleic acid molecule that is linear to the target cell or tissue. In some embodiments, introducing the single-stranded noncoding nucleic
WSGR Docket No.65107-701.602 acid molecule to a target cell or tissue enhances or silences expression and/or translation of a gene of interest by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more as compared with introducing an otherwise identical synthetic nucleic acid molecule that is linear to the target cell or tissue. In some embodiments, introducing the single-stranded noncoding nucleic acid molecule to a target cell or tissue enhances or silences expression and/or translation of a gene of interest by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more as compared with introducing an otherwise identical synthetic nucleic acid molecule that is linear to the target cell or tissue. In some embodiments, introducing the single-stranded noncoding nucleic acid molecule to a target cell or tissue enhances or silences expression and/or translation of a gene of interest by at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less as compared with introducing an otherwise identical synthetic nucleic acid molecule that is linear to the target cell or tissue. [0147] In some embodiments, introducing the single-stranded noncoding nucleic acid molecule to a target cell or tissue enhances or silences expression and/or translation of a gene of interest by at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70- fold, at most about 60-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3- fold, at most about 2-fold, or less as compared with introducing an otherwise identical synthetic nucleic acid molecule that is linear to the target cell or tissue. [0148] In some embodiments, the methods comprise enhancing or silencing expression and/or translation of a gene of interest by introducing an adapter to a looped and/or circularized single- stranded noncoding nucleic acid molecules (e.g., ASOs) of the present disclosure (FIG.11A-C). In some embodiments, introducing the looped single-stranded noncoding nucleic acid molecule comprising an adapter to a target cell or tissue enhances or silences expression and/or translation
WSGR Docket No.65107-701.602 of a gene of interest when compared with introducing an otherwise identical synthetic nucleic acid molecule that does not comprise an adapter to the target cell or tissue. In some embodiments, introducing the looped single-stranded noncoding nucleic acid molecule comprising an adapter to a target cell or tissue enhances or silences expression and/or translation of a gene of interest by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more as compared with introducing an otherwise identical synthetic nucleic acid molecule that does not comprise an adapter to the target cell or tissue. In some embodiments, introducing the looped single-stranded noncoding nucleic acid molecule comprising an adapter to a target cell or tissue enhances or silences expression and/or translation of a gene of interest by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more as compared with introducing an otherwise identical synthetic nucleic acid molecule that does not comprise an adapter to the target cell or tissue. In some embodiments, introducing the looped single-stranded noncoding nucleic acid molecule comprising an adapter to a target cell or tissue enhances or silences expression and/or translation of a gene of interest by at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less as compared with introducing an otherwise identical synthetic nucleic acid molecule that does not comprise an adapter to the target cell or tissue. In some embodiments, introducing the looped single-stranded noncoding nucleic acid molecule comprising an adapter to a target cell or tissue enhances or silences expression and/or translation of a gene of interest by at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70-fold, at most about 60-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, or less as compared with introducing an otherwise identical synthetic nucleic acid molecule that does not comprise an adapter to the target cell or tissue.
WSGR Docket No.65107-701.602 [0149] In some embodiments, the methods comprise delivering (administering) the single- stranded noncoding nucleic acid molecule to a subject, wherein the single-stranded noncoding nucleic acid molecule exhibits less off-target effects in the target cell or tissue as compared with an otherwise identical synthetic nucleic acid molecule that is linear. In some embodiments, off- target effects are reduced in a target cell or tissue with the single-stranded noncoding nucleic acid molecule by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, off-target effects are reduced in a target cell or tissue with the single-stranded noncoding nucleic acid molecule by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, off-target effects are reduced in a target cell or tissue with the single-stranded noncoding nucleic acid molecule by at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, off-target effects are reduced in a target cell or tissue with the single-stranded noncoding nucleic acid molecule by at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70-fold, at most about 60-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, or less when compared with an otherwise identical linear noncoding nucleic acid molecule. [0150] In some embodiments, the single-stranded noncoding nucleic acid molecule exhibits less toxicity in the target cell or tissue as compared with an otherwise identical synthetic nucleic acid molecule that is linear. In some embodiments, toxicity is reduced in a target cell or tissue with the single-stranded noncoding nucleic acid molecule by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about
WSGR Docket No.65107-701.602 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, toxicity is reduced in a target cell or tissue with the single-stranded noncoding nucleic acid molecule by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, toxicity is reduced in a target cell or tissue with the single-stranded noncoding nucleic acid molecule by at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, toxicity is reduced in a target cell or tissue with the single-stranded noncoding nucleic acid molecule by at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70-fold, at most about 60-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, or less when compared with an otherwise identical linear noncoding nucleic acid molecule. [0151] In some embodiments, the single-stranded noncoding nucleic acid molecule exhibits more target nucleic acid sequence specificity in the target cell or tissue as compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, the single- stranded noncoding nucleic acid molecule has increased target nucleic acid sequence specificity in a target cell or tissue by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, the single-stranded noncoding nucleic acid molecule has increased target nucleic acid sequence specificity in a target cell or
WSGR Docket No.65107-701.602 tissue by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, the single-stranded noncoding nucleic acid molecule has increased target nucleic acid sequence specificity in a target cell or tissue by at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, the single-stranded noncoding nucleic acid molecule has increased target nucleic acid sequence specificity in a target cell or tissue by at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70-fold, at most about 60-fold, at most about 50- fold, at most about 40-fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, or less when compared with an otherwise identical linear noncoding nucleic acid molecule. [0152] In some embodiments, the single-stranded noncoding nucleic acid molecule exhibits more stability in vivo as compared with an otherwise identical linear noncoding nucleic acid molecule (FIG.9A). In some embodiments, the single-stranded noncoding nucleic acid molecule has increased stability in vivo by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, the single-stranded noncoding nucleic acid molecule has increased stability in vivo by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, the single-stranded noncoding nucleic acid molecule has increased stability in vivo
WSGR Docket No.65107-701.602 by at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, the single-stranded noncoding nucleic acid molecule has increased stability in vivo by at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70-fold, at most about 60-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90- fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, or less when compared with an otherwise identical linear noncoding nucleic acid molecule. [0153] In some embodiments, the single-stranded noncoding nucleic acid molecule exhibits more stability ex vivo as compared with an otherwise identical linear noncoding nucleic acid molecule (FIG. 7A-8B). In some embodiments, the single-stranded noncoding nucleic acid molecule has increased stability ex vivo by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, or more when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, the single- stranded noncoding nucleic acid molecule has increased stability ex vivo by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, the single-stranded noncoding nucleic acid molecule has increased stability ex vivo by at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less when compared with an otherwise identical linear noncoding nucleic acid molecule. In some embodiments, the single-stranded noncoding nucleic acid molecule has increased stability ex vivo by at most about 100-fold, at most about 90-fold, at most
WSGR Docket No.65107-701.602 about 80-fold, at most about 70-fold, at most about 60-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 90- fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, or less when compared with an otherwise identical linear noncoding nucleic acid molecule. [0154] In some embodiments, the methods of the present disclosure comprise treating a disease or a condition in a subject by administering the double-stranded noncoding nucleic acid molecule to the subject under conditions sufficient to modulate expression of a gene of interest associated with the disease or condition (e.g., Table 1). In some embodiments, the methods of the present disclosure comprise treating a disease or a condition in a subject by administering the single-stranded noncoding nucleic acid molecule to the subject under conditions sufficient to modulate expression of a gene of interest associated with the disease or condition (e.g., Table 1). In some embodiments, the administering can be administered orally, intrathecally, percutaneously, rectally, sublingually, intranasally, intravitreally, subcutaneously, intramuscularly, transdermally, or intravenously. [0155] The compositions disclosed herein can be made into a dosage formulation that can vary based on the subject being treated and the mode of administration. The single-stranded nucleic acid active ingredient can comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of a dosage formulation. The single-stranded nucleic acid active ingredient can comprise at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less of a dosage formulation. [0156] A dosage formulation for intravenous administration can be at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 110 mg, at least about 120 mg, at least about 130 mg, at least about 140 mg, at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, at least about 200 mg, at least about 210 mg, at least about 220 mg, at least about 230 mg, at least about 240 mg, at least about 250 mg, at least about 260 mg, at least about 270 mg, at least about
WSGR Docket No.65107-701.602 280 mg, at least about 290 mg, at least about 300 mg, at least about 310 mg, at least about 320 mg, at least about 330 mg, at least about 340 mg, at least about 350 mg, or more. [0157] A dosage formulation for intravenous administration can be at most about 350 mg, at most about 340 mg, at most about 330 mg, at most about 320 mg, at most about 310 mg, at most about 300 mg, at most about 290 mg, at most about 280 mg, at most about 270 mg, at most about 260 mg, at most about 250 mg, at most about 240 mg, at most about 230 mg, at most about 220 mg, at most about 210 mg, at most about 200 mg, at most about 190 mg, at most about 180 mg, at most about 170 mg, at most about 160 mg, at most about 150 mg, at most about 140 mg, at most about 130 mg, at most about 120 mg, at most about 110 mg, at most about 100 mg, at most about 90 mg, at most about 80 mg, at most about 70 mg, at most about 60 mg, at most about 50 mg, at most about 45 mg, at most about 40 mg, at most about 35 mg, at most about 30 mg, at most about 25 mg, at most about 20 mg, at most about 15 mg, at most about 10 mg, or less. [0158] A dosage formulation for intravenous administration can be in a range from 25 to 50 mg, 25 to 100 mg, 25 mg to 150 mg, 25 mg to 200 mg, 25 mg to 250 mg, 25 mg to 300 mg, 25 mg to 300 mg, 30 to 50 mg, 30 to 100 mg, 30 mg to 150 mg, 30 mg to 200 mg, 30 mg to 250 mg, 30 mg to 300 mg, 50 mg to 100 mg, 50 mg to 150 mg, 50 mg to 200 mg, 50 mg to 250 mg, 50 mg to 300 mg, 50 mg to 300 mg, 100 mg to 150 mg, 100 mg to 200 mg, 100 mg to 250 mg, 100 mg to 300 mg, 100 mg to 250 mg, 150 mg to 200 mg, 150 mg to 250 mg, 150 mg to 300 mg, 150 mg to 350 mg, 200 to 250 mg, 200 to 300 mg, 200 to 350 mg, 250 mg to 300 mg, 250 mg to 350 mg, or 300 mg to 350 mg. [0159] A dosage formulation for subcutaneous administration can be at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 110 mg, at least about 120 mg, at least about 130 mg, at least about 140 mg, at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, at least about 200 mg, at least about 210 mg, at least about 220 mg, at least about 230 mg, at least about 240 mg, at least about 250 mg, at least about 260 mg, at least about 270 mg, at least about 280 mg, at least about 290 mg, at least about 300 mg, at least about 310 mg, at least about 320 mg, at least about 330 mg, at least about 340 mg, at least about 350 mg, or more. [0160] A dosage formulation for subcutaneous administration can be at most about 350 mg, at most about 340 mg, at most about 330 mg, at most about 320 mg, at most about 310 mg, at most about 300 mg, at most about 290 mg, at most about 280 mg, at most about 270 mg, at most about 260 mg, at most about 250 mg, at most about 240 mg, at most about 230 mg, at most about 220 mg, at most about 210 mg, at most about 200 mg, at most about 190 mg, at most about 180
WSGR Docket No.65107-701.602 mg, at most about 170 mg, at most about 160 mg, at most about 150 mg, at most about 140 mg, at most about 130 mg, at most about 120 mg, at most about 110 mg, at most about 100 mg, at most about 90 mg, at most about 80 mg, at most about 70 mg, at most about 60 mg, at most about 50 mg, at most about 45 mg, at most about 40 mg, at most about 35 mg, at most about 30 mg, at most about 25 mg, at most about 20 mg, at most about 15 mg, at most about 10 mg, or less. [0161] A dosage formulation for subcutaneous administration can be in a range from 25 to 50 mg, 25 to 100 mg, 25 mg to 150 mg, 25 mg to 200 mg, 25 mg to 250 mg, 25 mg to 300 mg, 25 mg to 300 mg, 30 to 50 mg, 30 to 100 mg, 30 mg to 150 mg, 30 mg to 200 mg, 30 mg to 250 mg, 30 mg to 300 mg,50 mg to 100 mg, 50 mg to 150 mg, 50 mg to 200 mg, 50 mg to 250 mg, 50 mg to 300 mg, 50 mg to 300 mg, 100 mg to 150 mg, 100 mg to 200 mg, 100 mg to 250 mg, 100 mg to 300 mg, 100 mg to 250 mg, 150 mg to 200 mg, 150 mg to 250 mg, 150 mg to 300 mg, 150 mg to 350 mg, 200 to 250 mg, 200 to 300 mg, 200 to 350 mg, 250 mg to 300 mg, 250 mg to 350 mg, or 300 mg to 350 mg. [0162] The compositions disclosed herein can be administered to a patient at a frequency of at least three times a day, at least twice a day, at least once a day, at least once every other day, at least once every three days, at least once every four days, at least once every five days, at least once every six days, at least once a week, at least once every two weeks, at least once every three weeks, at least once every four weeks, at least once every two months, at least once every three months, at least once every four months, at least once every five months, at least once every six months, at least once every seven months, at least once every eight months, at least once every nine months, at least once every ten months, at least once every eleven months, or at least once a year. [0163] The compositions disclosed herein can be administered to a patient at a frequency of no more than once a year, no more than once every eleven months, no more than once every ten months, no more than once every nine months, no more than once every eight months, no more than once every seven months, no more than once every six months, no more than once every five months, no more than once every four months, no more than once every three months, no more than once every two months, no more than once every four weeks, no more than once every three weeks, no more than once every two weeks, no more than once every seven days, no more than once every six days, no more than once every five days, no more than once every four days, no more than once every three days, no more than once every two days, no more than once a day, no more than twice a day, or no more than three times a day.
WSGR Docket No.65107-701.602 IV. KITS [0164] Provided herein, in some aspects, are kits comprising the one or more compositions disclosed herein. In some embodiments, the kits comprise one or more single-stranded nucleic acids described herein. In some embodiments, the kits further comprise a cell, or a plurality of cells. In some embodiments, the kits further comprise cell media, such as growth media. In some embodiments, the kits further comprise additional constituents of the cell media, such as mevalonate, antibiotics, and so forth. The exact nature of the components configured in the inventive kit depends on its intended purpose. [0165] Instructions for use may be included in the kit. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to affect a desired outcome, e.g., producing a single-stranded nucleic acid, isolating a single- stranded nucleic acid, or investigating therapeutic potential of the one or more single-stranded nucleic acids. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, or other useful paraphernalia as will be readily recognized by those of skill in the art. [0166] The materials or components assembled in the kit can be provided to the user stored in any convenient and suitable ways that preserve their operability and utility. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging materials employed in the kit are those customarily utilized in gene expression assays and in the administration of treatments. As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a plastic vial or tube used to contain suitable quantities of the genetically-encoded system, and/or cells. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components. V. EXAMPLES [0167] The following examples are included for illustrative purposes only and are not intended to limit the scope of the inventive concepts.
WSGR Docket No.65107-701.602 Example 1: Methods for the Formation of Circular ASOs/RNAs [0168] Circular ASOs (Circular ASOs) exert biological functions by acting as transcriptional regulators. [0169] Formation of Circular RNA, circular ASO Gapmer by T4 ligase 1 or 2r [0170] A typical reaction is composed of 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, 1-3M betaine). RNA samples are pre-treated at 80°C for 3 minutes, and cooled to the reaction temperature at a rate of 6°C/min. Then, the reaction is carried out at 25°C for 2 hours and terminated by heating the mixture at 75°C for 10 minutes. The cyclic structures of reaction products are confirmed by treating them with Exonuclease T (5 U) at 25°C for 6 hours. [0171] General Methods for Circular ASO Synthesis [0172] A normal universal controlled pore glass (CPG) is used for RNA oligonucleotide synthesis. All the oligonucleotides are synthesized according to the standard RNA synthesis on ABI394 with 2-tert-butyldimethylsilyl (TBDMS) RNA monomers. The concentrations of all oligonucleotides are measured at 260 nm using a Thermo Scientific NanoDrop 2000 Spectrophotometer. High-performance liquid chromatography (HPLC) is performed on a Waters Alliance e2695 system equipped with a Waters Xbridge OST C18 column (2.5 μm, 10.0 × 50 mm). MS data are obtained with a Waters Xevo G2 Q-TOF spectrometer using ESI. Diethyl pyrocarbonate (DEPC)-treated water is used for all solutions and HPLC purification. [0173] Synthesis and Purification of Circular ASO [0174] RNA is dissolved in water to make the final 100 μM RNA solution. The final composition of the reaction mixture to circularize the RNA is as follows: 7 μL RNA solution, 1 μL 10 mM ATP, 1 μL 10 × reaction buffer, and 1 μL T4 RNA ligase (10 U/μL). The solution 10 μL/tube is placed in PCR at 4°C for 12 hr. After mixing these liquids together, the crude products are mixed with 6 × RNA loading buffer (0.25% bromophenol blue and 30% glycerol in DEPC- treated water). The solution (18 μL/well) is loaded into 20% native polyacrylamide PAGE (1 mm thick) gels. The gels are then electrophoresed at 220 V for 50 minutes using 1 × Tris-borate-EDTA (TBE) buffer (pH 8.2). For each preparative gel, the two-side sample lanes of the gel are cut and stained with 1 × SYBR Gold (Invitrogen) and then imaged. The images are printed out according to the same size of the gel, which made it possible to mark the location of the gels without SYBR Gold stain. The gel zones at the marked location are cut, crumbled into tiny particles, and immersed into a 1 × TBE buffer at 37°C overnight. After filtration out of the solid particles, the solutions of the RNA are desalted and concentrated using Millipore-Amicon Ultra-0.5 mL
WSGR Docket No.65107-701.602 Centrifugal Filters (cutoff = 3,000). The collected products are frozen dried to remove water and gave the final circular single-stranded RNAs. [0175] RNA oligonucleotides are characterized using ESI-MS, and oligonucleotides (∼0.2 nmol) are dissolved in water/acetonitrile (50:50, 20 μL) containing 1% triethylamine to make a final concentration of 10 μM. The solutions are then analyzed with a Waters Xevo G2 Q-Tof spectrometer with ESI in the negative ion mode. The molecular weight of the circular ASO/RNA is 18 less than the linear RNA due to the condensation reaction. [0176] The circular ASO/RNAs are dissolved in 1 × PBS buffer to make the 6-μmol stock solution. A 10-μL stock solution is mixed with an equal amount of the complementary RNA with 5′-phosphate modification to form the circular ASO/RNA. The RNA is annealed by heating to 85°C for 5 minutes and is subsequently cooled to room temperature for at least 1 hour for further use. [0177] Enzymatic Stability of Circular ASO [0178] Circular ASO or control linear ASO (3 μM, 5 μL) is incubated at 37°C in an enzyme solution to make a final concentration of 1 μmol/L (15 μL). RNase is used for enzymatic stability in this study, respectively. Aliquots of 3 μL (containing 3 pmol siRNA) are aliquoted at different time points (2, 4, 6, and 8 hours) and immediately frozen in liquid nitrogen and then stored at −80°C until assayed.1 μL 6 × RNA-loading buffer is added to the aliquots. The samples are run on 10% native polyacrylamide gels in TBE buffer according to the procedure mentioned before. [0179] Chemical Synthesis [0180] Circular ASOs containing a disulfide bond are chemically synthesized as follows. Passenger strands including a disulfide bond with protective groups at both ends are obtained. Thiol-Modifier C6 S-S Amidite and 3′-Thiol-Modifier C3 S-S CPG are used at the 5′ end and at the 3′ end, respectively, to synthesize passenger strands containing a disulfide bond at both ends. To deprotect both terminal protecting groups, 50 mM dithiothreitol (DTT) in Tris-buffer is added to the solution of the passenger strand, and the mixture is incubated at room temperature for 18 hours. The reaction solution is purified using a NAP-10 column (GE Healthcare) to obtain a passenger strand with a thiol group at both terminals in an aqueous solution. Circular passenger strands are prepared by adding five equivalents of 2-(methoxythio)-3-nitropyridine (Npys-OMe) and acetonitrile (MeCN/water = 1/3). Thereafter, the mixture is incubated at room temperature for 48 hours, concentrated by centrifugation, and purified using Shimadzu reversed-phase preparative high performance liquid chromatography (HPLC) system. Liquid chromatography is performed utilizing a XBridge C18 column (Waters; 5 μm, 4.6 × 250 mm) with buffers A (100 mM triethylammonium acetate [TEAA] in distilled water) and B (acetonitrile). Oligonucleotides are separated using a linear gradient of 5%–50% buffer B from 0 to 22.0 min at a flow rate of 1
WSGR Docket No.65107-701.602 mL/min at 60°C. Purified oligonucleotides are collected, desalted through an NAP-10 column (GE Healthcare), and then concentrated by centrifugation. An aqueous solution of the obtained circular passenger strand with a disulfide bond and an aqueous solution of the guide strand purchased from GeneDesign, Inc., are mixed in equimolar amounts, and the necessary amount of D-PBS (−) (Nacalai Tesque) is added. The mixture is placed in a heating block previously heated to 85°C and incubated for 5 minutes. Thereafter, the solution is gently cooled to room temperature. [0181] Chemical synthesis of non-cleavable circular ASO is performed as follows. A passenger strand containing 5′-hexynyl phosphoramidite (Glen Research) at the 5′ end and azide- modified CPG (PRIMETECH ALC) at the 3′ end are purchased from GeneDesign, Inc. Non- cleavable circular passenger strands are synthesized. In brief, 10–20 Cu wires (FUJIFILM Wako Pure Chemical Corporation) are added to the solution of the passenger strand (50 μM) with NaCl (200 mM). The solution is heated to 80°C for 3 minutes and gently cooled to room temperature. These reactions are preceded by a copper-catalyzed azide-alkyne cycloaddition (CuAAC) to form a triazole. The solution is purified using an NAP-10 column (GE Healthcare), and reversed-phase preparative HPLC is carried out as described previously. Then, purified oligonucleotides are collected, desalted through an NAP-10 column, and concentrated by centrifugation. The annealing is conducted as described previously. [0182] Liquid Chromatography-Mass Spectrometry Analysis [0183] The purity and structure of the synthesized oligonucleotides are determined by liquid chromatography–mass spectrometry (LC-MS) on 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; Waters) with buffers A (8.6 mM trimethylamine and 100 mM hexafluoroisopropanol in water) and B (methanol). The oligonucleotides are separated with a linear gradient of 10%–90% buffer B from 0 to 18.0 min at a flow rate of 0.3 mL/min at 60°C. To further confirm the purity and molecular size of the synthesized oligonucleotides, size- exclusion chromatography (SEC) (Shimadzu high-performance liquid chromatography systems) is also conducted. Liquid chromatography is performed using a G2000SWXL column (TOSOH; 5 μm, 7.8 × 300 mm). The oligonucleotides are separated with D-PBS(−) at a flow rate of 1 mL/min for 20 minutes at 25°C. [0184] LC-MS/MS analysis is conducted by ACQUITY UPLC H-class for UPLC, Synapt G2 HDMS (Waters) for MS, and MassLynx V4.1 for data processing. Liquid chromatography is performed using an ACQUITY BEH C18 column (1.7 μm, 2.1 × 50 mm) with buffer A (8.6 mM trimethylamine and 100 mM hexafluoroisopropanol in water) and buffer B (methanol). The oligonucleotides are separated with a linear gradient of 10%–90% of buffer B from 0 to 10 minutes
WSGR Docket No.65107-701.602 at a flow rate of 0.3 mL/min at 50°C. MaxEnt-1 software is used for mass deconvolution of the MS data. Collisional activation of the selected m/z is performed with a collision energy of 25 eV. [0185] Cell Culture [0186] HeLa cells (ATCC) and RAW264.7 cells (ATCC) are maintained in RPMI 1640 medium (Life Technologies; A10491-01) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Similarly, HepG2 cells (ATCC), Huh-7 cells (JCRB), and L929 cells (RCB) cells are maintained in MEM (11095-080; Gibco), Dulbecco's modified Eagle's medium (DMEM), and Minimum Essential Medium (MEM) (11095-080; Gibco), respectively, in a humidified atmosphere of 5% CO2 at 37°C. Primary mouse hepatocytes (MSCP10; Life Technologies) are cultured in William's E Medium (A1217601; Life Technologies) supplemented with Primary Hepatocyte Thawing and Plating Supplements (CM3000; ThermoFisher Scientific). [0187] In Vitro Knockdown Assay [0188] An in vitro knockdown assay by transfection is conducted in cultured cells as follows. Oligonucleotide and RNAiMAX (13778-075; Life Technologies) (fin. 0.2%) are diluted with Opti-MEM (31985-070; Life Technologies), and oligonucleotides/RNAiMAX solutions are prepared according to the manufacturer's protocol. Next, 20 μL solution is treated in a 96-well plate (167008; Nunc), and then 80 μL cell suspension (10,000 cells/well) is added. After gentle shaking, the plate is maintained in a humidified atmosphere of 5% CO2 at 37°C for 24 hours. [0189] In vitro knockdown assay by free uptake (gymnosis) in cultured cells is conducted as follows. Twenty microliters oligonucleotide solution diluted with Opti-MEM (Life Technologies; 31985- 070) is treated in a 96-well plate (167008; Nunc), and then 80 μL cell suspension (2,000– 3,000 cells/well) is added. After gentle shaking, the plate is maintained in a humidified atmosphere of 5% CO2 at 37°C for 96 hours. As for mouse primary hepatocytes, 20 μL diluted oligonucleotide solution is treated in a 96-well Collagen I Multiwell Microplate (#356702; Corning), followed by the addition of 80 μL cell suspension and incubation in a humidified atmosphere of 5% CO2 at 37°C for 24 hours. [0190] After culturing oligonucleotide-treated cells, total RNA is extracted and converted to cDNA using SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo) according to the manufacturer's protocol. The reaction conditions are as follows: 37°C for 15 min, 50°C for 5 min, 98°C for 5 min, and 4°C for 5 minutes. mRNA levels are evaluated by quantitative RT-PCR using TaqMan Gene Expression Master Mix (Life Technologies), the TaqMan probes listed above (Table 2), and QuantStudio 12K flex (Thermo Fisher Scientific) with the reaction conditions of 50°C for 2 min, 95°C for 10 min, and (95°C for 15 s and 60°C for 1 min) × 40 cycles. The relative mRNA expression is quantified using the comparative Ct method.
WSGR Docket No.65107-701.602 Example 2: Methods for Synthesis of Oligonucleotides [0191] Oligonucleotides are synthesized on a MerMade-12 DNA/RNA synthesizer. Sterling solvents/reagents from Glen Research, 500-Å controlled pore glass (CPG) solid supports from Prime Synthesis, 2′-deoxy 3′-phosphoramidites from Thermo, and 2′-OMe and 2′-F nucleoside 3′- phosphoramidites from Hongene are all used as received. The 2′-OMe-uridine-5′-bis-POM-(E) vinylphosphonate (VP) 3′-phosphoramidite is synthesized, dissolved to 0.15 M in 85% acetonitrile 15% dimethylformamide (DMF), and coupled using standard conditions on the synthesizer. GalNAc CPG support is prepared. 5-Bromohexyl phosphoramidite (Glen Research, Cat# 10- 1946) is dissolved to 0.15 M in acetonitrile and coupled using standard conditions on the synthesizer. Alkyne CPG support and alkyne hydroxyprolinol phosphoramidite (Y) are prepared. Low-water content acetonitrile is purchased from EMD Chemicals. A solution of 0.6 M 5-(S- ethylthio)-1H-tetrazole in acetonitrile is used as the activator. The phosphoramidite solutions are 0.15 M in anhydrous acetonitrile with 15% DMF as a co-solvent for 2′-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)methanimidamide (DDTT), 0.09 M in pyridine, is used as the sulfurizing reagent. The detritylation reagent is 3% dichloroacetic acid (DCA) in dichloromethane (DCM). Example 3: Analysis of Oligonucleotide Stability in Plasma and Liver Homogenates [0192] Rat plasma (BioIVT, Cat# RAT00PL38NCXNN) and liver homogenate (BioIVT, custom order) are diluted with a 10× cofactor solution to achieve a final concentration of 1 mM MgCl2, 1 mM MnCl2 and 2 mM CaCl2. The sciRNA is added to 50 μl of the plasma or the liver homogenate to achieve the final concertation of 20 μg/ml. The reaction mixture is incubated with gently shaking at 37°C. 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 internal standard (oligonucleotide U21 at 1 μg/ml final concentration) and frozen at −80°C until analysis. Experiments are performed in triplicate. [0193] Oligonucleotide enrichment for LC–MS analysis is performed using Clarity OTX 96- well solid-phase extraction plates as described by Liu et al. (49). The SPE columns are conditioned initially with 1 ml of methanol followed by equilibration with 2 ml of 50 mM ammonium acetate with 2 mM sodium azide in HPLC-grade water. The samples are loaded on to SPE column by applying positive pressure. The columns are then washed five times with 1 ml of 50 mM ammonium acetate in 50/50 (v/v) water and acetonitrile (pH 5.5). Finally, the oligonucleotides are eluted using elution buffer containing 10 mM EDTA, 100 mM ammonium bicarbonate in 40/10/50 (v/v/v) acetonitrile/tetrahydrofuran/water (pH 8.8). The eluant is dried under nitrogen and resuspended in 120 μl of LC–MS grade water for LC–MS analysis.
WSGR Docket No.65107-701.602 [0194] Relative quantitation and metabolite identification of modified oligonucleotides is performed using high-resolution mass spectrometry on a Thermo Scientific Q Exactive coupled to ion-pairing reverse-phase liquid chromatography (Dionex Ultimate 3000) (LC-HRMS). A Waters X-BridgeBEH C8 XP Column (Cat# 176002554,130 Å, 2.5 μm, 2.1 mm × 30 mm, 80°C) is used for the chromatographic separation. The injection volume and flow rates are 30 μl and 1 ml/min, respectively. Mobile phase A consisted of 16 mM triethylamine (Sigma, Cat# 471283), 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 started with 1% mobile phase B and progressed to 35% B over 4.3 min, then the column is equilibrated with 1% mobile phase B for 1 min. The mass spectrometer data acquisition is performed in full scan mode with a scan range of 500–3000 m/z at a resolution setting of 35000. Spray voltage is 2.8 kV. The auxiliary gas temperature and the capillary temperature are set to 300°C. [0195] The Thermo Quan browser is used calculate the area ratio of extracted ion chromatograms (XIC) of test oligonucleotide to internal standard with 10 ppm mass accuracy. After LC-HRMS analysis, data are processed using ProMass HR Deconvolution software (Novatia, LLC) to identify linearization and major metabolism of the modified oligonucleotides. [0196] The half-lives are calculated by monitoring loss of full-length test oligonucleotide for 24 hours. The amounts of test oligonucleotide and internal standard are normalized to time 0 hours for each time-point for respective oligonucleotides. The natural log of percentage full length remaining and the slope are calculated using linear regression. The half-life is calculated using the equation: t12=−Ln(2)k. [0197] Thermal Melting Studies [0198] Melting studies are performed in 1-cm path length quartz cells on a Beckman DU800 spectrophotometer equipped with a thermoprogrammer. Samples are diluted to obtain a final concentration of oligonucleotide strand of approximately 1 μM in 0.1× PBS buffer (pH 7.4). Melting curves are monitored at 260 nm with a heating rate of 1°C/min from 10 to 90°C. Melting temperatures (Tm) are calculated from the first derivatives of the heating curves, and the reported values are the result of two independent measurements. [0199] NMR Studies [0200] Lyophilized RNA is dissolved in a mixture of 10% 2H2O/90% H2O with 20 mM NaCl and 10 mM sodium phosphate buffer (pH 7). Final concentrations of duplexes in 600 μl are in the range from 20 to 60 μM. All spectra are acquired at 25°C on an Agilent VNMRS 800 MHz NMR spectrometer equipped with a cold probe. [0201] Circular Dichroism Spectroscopy
WSGR Docket No.65107-701.602 [0202] The circular dichroism (CD) spectra are obtained on a Jasco J-815 spectropolarimeter equipped with a Julaba F25 circulating bath. The sample is allowed to equilibrate for 5 minutes at 10 ºC in 1× PBS at a final duplex concentration of 1.57 μM. The spectrum is an average of 5 scans. Spectra are collected at a rate of 50 nm/min, with a bandwidth of 1 nm and sampling wavelength of 0.2 nm using fused quartz cells (Starna 29-Q-10). The CD spectra are recorded from 350 to 200 nm at 10 ºC. The molar ellipticity is calculated from the equation [0] = 0/10Cl, where 0 is the ellipticity (mdeg), C is the molar concentration of oligonucleotide (M), and l is the path length of the cell (cm). Example 4: Chemical Synthesis [0203] Oligonucleotide Synthesis [0204] Oligonucleotides (1 μmol scale) are synthesized on an ABI 381A or 394 DNA synthesizer using a cycle involving phosphoramidite chemistry. Detritylation is performed with 2.5% DCA in CH2Cl2 for 60 seconds. Coupling step: BMT (0.3 M in dry acetonitrile) is used as activator; propynyl and bromohexyl phosphoramidites (0.09 M in CH3CN) are introduced with a 45 second coupling time; commercially available phosphoramidites (0.09M in CH3CN) are introduced with a 30 second coupling time. The capping step is performed with acetic anhydride using commercial solution (Cap A: Ac2O, pyridine, THF 10/10/80 and Cap B: 10% N- methylimidazole in THF) for 15 seconds. Oxidation is performed with commercial solution of iodide (0.1 M I2, THF, pyridine/water 90/ 5/5) for 10 seconds. [0205] General Procedure for Azidation [0206] Azidation of 5-hydroxyl oligonucleotides is performed according to known methods. Azidation from bromohexyl oligonucleotides is performed as follows: a solution of NaN3 (13 mg) and NaI (30 mg) in dry DMF (1.5 mL) is applied on the solid-supported bromohexyl oligonucleotide for 1 hour and 15 minutes at 65 °C. Then the CPG beads bearing the oligonucleotide are washed with DMF (2 _ 1 mL) and CH2-Cl2 (5 mL) and dried in a desiccator under reduced pressure for 30 minutes. [0207] General Procedure for Deprotection [0208] The beads are placed into a sealed vial and treated with concentrated aqueous ammonia (1 mL) for 24 hours at room temperature for oligonucleotides containing phosphotriester functions or 2 hours at room temperature and then 5 hours at 55 °C for the others. The beads are filtered off, and the solution is evaporated. The residue is dissolved in water for subsequent analyses. [0209] General procedure for Cu(I)-Catalyzed 1,3-Dipolar Cycloaddition [0210] Azido-alkyne oligonucleotide (1 μmol) is added to CuSO4 (0.4 equiv, 0.4 μmol, 13.2 μL of a 20 mM solution in H2O), freshly prepared (from degassed water) sodium ascorbate (2
WSGR Docket No.65107-701.602 equiv, 2 μmol, 13.2 μL of a 100 mM solution in H2O), methanol (100 μL), and water (23.6μL). The tube containing the resulting preparation is flushed with argon and sealed. The reaction is placed in a microwave synthesizer Initiator from Biotage set at 100 W with a 30 second premixing time for 1 hour to 1 hour and 30 minutes at 60 °C. Temperature is monitored with an internal infrared probe. The solution is then desalted on NAP10. Example 6: Administration of an Antisense Oligonucleotide to Rats [0211] Twenty-four passive transfer myasthenia gravis (PTMG) model rats are randomly assigned to three groups: ASO-C5 (2.5 mg/kg), ASO-C5 (5 mg/kg), and saline control, by subcutaneous injection. Treatment is administered ten, seven, and three days before and on the day of PTMG induction. Blood is obtained prior to the siRNA dose and immediately prior to PTMG induction. PTMG is induced by administration of the rat anti-mouse muscle AChR monoclonal antibody (mAb) McAb3. McAb3 is an IgG2b isotype known to activate complement. Animals undergo euthanasia 48 hours after PTMG induction. Weakness is graded on a standard 1–4 disease severity scale, with a higher score indicating more severe weakness. Blood, liver, diaphragm, and tibialis anterior are harvested for analysis. [0212] C5 mRNA expression is quantified by qRT-PCR. Flash-frozen livers are pulverized by a 2000 Geno/Grinder (Spex SamplePrep), and RNA is extracted using the RNeasy Mini Kit according to the manufacturer’s protocol (QIAGEN). Reverse transcription is performed to generate cDNA according to the manufacturer’s protocol (Life Technologies). qPCR is performed on cDNA using a Roche LightCycler 480 instrument with a rat C5 Taqman FAM probe (Life Technologies) and a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Taqman VIC probe (Life Technologies) as the control. The C5 level is normalized to GAPDH, and the percentage of C5 mRNA remaining is calculated relative to the average of untreated or saline- treated rats. [0213] Rat serum samples are analyzed by semiquantitative western blot. Sera re diluted 1:20 into 50 mM Tris (pH 7) with 1% SDS. Samples are run on 10% Bis-Tris protein gels (Life Technologies) and transferred onto a PVDF membrane (Bio-Rad). A goat anti-human C5 antibody (Complement Technology) which crosses to rat, is used at a 1:1,000 dilution with a secondary fluorophore-conjugated donkey anti-goat antibody (LI-COR) followed by imaging with the LI- COR Odyssey imaging system. Analysis and quantification of the western blot is done using LI- COR’s Image Studio software. The percent of C5 remaining in the PTMG study is calculated by normalizing rat samples from day 8 to their individual pre-bleed sample.
WSGR Docket No.65107-701.602 Example 7: Administration of an Antisense Oligonucleotide to Humans [0214] ASO-C5 formulations are administered subcutaneously to human subjects with complement system disorders. After one month, samples are taken from the subjects to measure C5 levels. Additional samples are taken to measure toxicity, ASO-C5 half-life, and immunogenicity. Example 8: Formation of Circular ASOs/RNAs [0215] Circular ASOs (Circular ASOs) exert biological functions by acting as transcriptional regulators. [0216] Formation of looped/Circular RNA, by ligase enzymes [0217] A typical reaction is composed of 10-50 μM Linear ASO, 0.5-2 U RNA ligase 2, RNA ligase 1 or thermostable ligases 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, 1-3M betaine). RNA samples were pre-treated at 80°C for 3 minutes, and cooled to the reaction temperature at a rate of 6°C/min. Then, the ligation reaction was carried out between 25°C-60°C for 2-4 hours and terminated by heating the mixture at 75°C for 10 minutes (Fig. 5). The cyclic structures of reaction products were confirmed by treating them with Exonuclease T (5 U) at 25°C for 6 hours (FIG.7A-7B). [0218] Synthesis and Purification of Circular ASO [0219] Looped RNA was mixed with 2 × RNA loading buffer (0.25% bromophenol blue and 95 % formamide in DEPC-treated water). Samples were loaded on a 12% urea polyacrylamide PAGE (1 mm thick) gel and electrophoresed at 100-200 V for 50 minutes in 1 × Tris-borate- EDTA (TBE) buffer (pH 8.2). For visualization purposes gels were soaked in 1 × GelRed (Biotium) solution, a nucleic acid dye, which preferentially stains double stranded structures for 30 min, followed by image acquisition using iBright imaging system (Thermo Fisher Scientific) (FIG.5A). [0220] For large scale purification of ligated products, UV shadowing was used for PAGE purification of the looped sense strands by exposing the gels placed on top of a fluorescent TLC plate to shortwave UV light (254 nm). Cyclized bands were excised from the gel, crushed and incubated with 5-10 mL of nuclease free water O/N on a tumbler at 15
oC. diffused ASOs were concentrated using Millipore-Amicon Ultra-15 mL Centrifugal Filters (cutoff = 3,000 kDa). The collected looped ASO products quality and quantity was determined and frozen. [0221] Enzymatic Stability of Circular/looped ASO [0222] The cyclic nature of the reaction products were confirmed by Exonuclease T (NEB) and or Phosphodiesterase I (PDI) (Crotalus atrox Phosphodiesterase I; Sigma: P4506-100MG). to
WSGR Docket No.65107-701.602 digest linear and looped ASOs (10-50pmol). Reactions were terminated by addition of EDTA pH 8 to 10-50 mM and separated on a 12% denaturing urea PAGE gel and visualized by GelRed (Biotium) stain (Fig. 7, Fig. 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 (5% CO2 at 37°C). Primary mouse hepatocytes (B129-7224F; Cell Biologics) were cultured in 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 added to 25 μL of Opti-MEM (31985-070; Life Technologies) separately and mixed according to the manufacturer's protocol.50 μL of this mixture was added to a 96-well plate (167008; Nunc), and 50 μL cell suspension (5-10,000 cells/well) was added to the lipofectamine siRNA mixture. After gentle shaking, the plate was incubated in a humidified atmosphere of 5% CO2 at 37°C for 24 hours.totalFor quantification of target messenger RNA, total RNA was extracted and converted to cDNA using 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 2-step RT qPCR assay. Reverse transcription with the TOYOBO kit was carried out as follows: 37°C for 15 min, 50°C for 5 min, 98°C for 5 min, and 4°C for 5 minutes, and CFB, ApoB mRNA levels were measured by quantitative RT-PCR using TaqMan Gene Expression Master Mix (IDT) and or SYBR green dye detection on a QuantStudio 6-7 real time PCR machine for × 40 cycles. The relative mRNA expression was quantified using the comparative Ct method and or serial dilution and standard curve. RPL13A and or RPB1 mRNA served as housekeeping genes for quantification of target mRNAs (FIG.9-13). Example 9: Synthesis of Oligonucleotides [0227] Non-modified and chemically-modified oligonucleotides of different length were purchased from IDT (Coralville, IA). Example 4: Analysis of Oligonucleotide Stability in Plasma [0228] Rat serum (10-50%) (Sigma R9759) was diluted with PBS containing 1 mM MgCl2, 1 mM MnCl2 and 2 mM CaCl2. The ASOs were added to a final concentration of 5-50 pmol. The reaction mixture was incubated with gently shaking at 37°C. At each time point (0, ½ , 1, 2, 4, 6 hours).Reactions were stopped by EDTA 5-10mM and/or incubation at 95°C for 5-10 min
WSGR Docket No.65107-701.602 followed by treatment with proteinase K at 50°C for 30 min and loaded on urea PAGE gel as described above.
WSGR Docket No.65107-701.602
WSGR Docket No.65107-701.602
WSGR Docket No.65107-701.602