Synthetic single-stranded nucleic acid compositions and methods thereof

Abstract

There are some major drawbacks and defects when using AAV particles as gene delivery vectors derived from traditional AAV production from host cells.Therefore, there is a strong need in the field of gene therapy for a technology that minimizes immunogenicity, can be re-administered, and allows the production of large amounts of recombinant vectors, and increases expression level, strand specificity, and purity, while also increasing the capacity of transgene size.The present application discloses a single-stranded deoxyribonucleic acid (DNA) molecule that comprises at least one nucleic acid sequence of interest adjacent to at least one first stem-loop structure, a method for making, and a method for using for the delivery and expression of transgenes in host cells.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 429,461, filed December 1, 2022, U.S. Provisional Patent Application No. 63 / 449,872, filed March 3, 2023, U.S. Provisional Patent Application No. 63 / 529,637, filed July 28, 2023, and U.S. Provisional Patent Application No. 63 / 544,571, filed October 17, 2023. The entire contents of each of the foregoing applications are expressly incorporated herein by reference. [Background technology]

[0002] Adeno-associated viruses (AAV) (e.g., recombinant AAV (rAAV) or AAV vectors) are attractive for delivering genetic material because (i) they can infect ("transduce") a wide variety of dividing and non-dividing cell types, such as muscle cells and neurons; (ii) they lack viral structural genes, thereby reducing the host cell response to viral infection, e.g., the interferon-mediated response; (iii) wild-type AAV is considered non-pathological in humans; and (iv) in contrast to wild-type AAV, they can integrate into the host cell genome; replication-deficient AAV vectors lack the rep gene and generally persist episomally, thus significantly limiting the risk of insertional mutagenesis or genotoxicity.

[0003] However, there are several major drawbacks and deficiencies in using AAV particles as gene delivery vectors derived from traditional AAV production from host cells (e.g., Sf9 insect cells under large-scale production conditions). One major drawback associated with rAAV is its limited viral packaging capacity of approximately 4.5 kb of heterologous DNA (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010). As a result, this limitation in viral packaging restricts the use of AAV vectors to protein-coding capacities of less than 150 kDa. A second drawback is related to capsid immunogenicity, which prevents re-administration to patients. The patient's immune system can respond to the vector, effectively acting as a booster to stimulate the immune system to generate high titers of anti-AAV antibodies that hinder future treatment. Some recent reports have raised concerns about immunogenicity in high-dose situations. Another significant drawback is that production of AAV in host cells (e.g., insect cells) during large-scale production of the viral genome results in a random mixture of plus (+) and minus (-) strand vectors, dramatically reducing strand specificity of the transgene for desired therapeutic expression of the sense strand.

[0004] In addition, conventional capsid-containing AAV virions are produced by introducing a plasmid containing the AAV genome, rep gene, and cap gene (Grimm et al., 1998). However, such encapsidated AAV viral vectors have been found to inefficiently transduce certain cell and tissue types, and the capsids have also been found to induce severe immune responses in the host. Therefore, the use of adeno-associated virus (AAV) vectors for gene therapy (including gene editing) is limited to a single administration to patients due to the patient's immune response, the limited range of transgene genetic material suitable for delivery in AAV vectors due to the minimal viral packaging capacity (approximately 4.5 kb), and slow AAV-mediated gene expression. Furthermore, methods for producing such AAV vectors rely heavily on traditional insect cell-dependent production methods. Such methods can be hindered by contaminants from the cells used to produce the vector, which are inconvenient or expensive to remove or purify, and may result in undesirable side effects if included in a therapeutic formulation.

[0005] Thus, there is a strong need in the gene therapy field for technologies that are minimally immunogenic, re-administrable, and allow for the generation of large quantities of recombinant vectors, increasing expression levels, strand specificity, and purity while at the same time increasing transgene size capacity. Summary of the Invention

[0006] According to some aspects, the present disclosure provides isolated linear single-stranded deoxyribonucleic acid (ssDNA) molecules comprising at least one nucleic acid sequence of interest adjacent to at least one stem-loop structure at the 3' end for priming replication and / or transcription. According to some embodiments, the ssDNA molecules of the present disclosure are characterized in that they do not comprise an inverted terminal repeat (ITR) structure.

[0007] An unexpected finding was that the tolerability and immune response of the ssDNA molecules described herein compared to double-stranded (ds) ceDNA in vivo was comparable to or better than that of ds ceDNA. As shown in the Examples, the cytokine response to the ssDNA molecules described herein was dramatically lower or undetectable compared to ds ceDNA when administered at equivalent doses. The reduced immunogenicity observed with the ssDNA molecules of the present disclosure is advantageous because it allows for repeated administration or higher dose concentrations.

[0008] According to a first aspect, the present disclosure provides a lipid nanoparticle (LNP) comprising (a) a linear single-stranded deoxyribonucleic acid (ssDNA) molecule comprising at least one nucleic acid sequence of interest, and (b) a lipid. In one embodiment, the ssDNA molecule is single-stranded along its entire length. In one embodiment of the aspects and embodiments herein, the ssDNA molecule does not contain any virus-derived sequence. In one embodiment of the aspects and embodiments herein, the ssDNA molecule is at least 200 nucleotides in length. In one embodiment of the aspects and embodiments herein, the ssDNA molecule is at least 300 nucleotides in length, at least 400 nucleotides in length, at least 500 nucleotides in length, at least 600 nucleotides in length, at least 700 nucleotides in length, at least 800 nucleotides in length, at least 900 nucleotides in length, at least 1000 nucleotides in length, at least 1500 nucleotides in length, at least 2000 nucleotides in length, at least 2500 nucleotides in length, at least 3000 nucleotides in length, at least 3500 nucleotides in length, at least 4000 nucleotides in length, at least 4500 nucleotides in length, at least 5000 nucleotides in length, at least 5500 nucleotides in length, at least 6000 nucleotides in length, at least 6500 nucleotides in length, at least 7000 nucleotides in length, at least 7500 nucleotides in length, at least 8000 nucleotides in length, at least 8500 nucleotides in length, at least 9000 nucleotides in length, at least 9500 nucleotides in length, or at least 10,000 nucleotides in length. In one embodiment of the aspects and embodiments herein, at least one nucleic acid sequence of interest is adjacent at its 3' end to at least one stem-loop structure, wherein the at least one stem-loop structure comprises at least one stem and at least one loop. In further embodiments, the at least one stem-loop structure at the 3' end is sufficient to prime replication and / or transcription. In some embodiments of the aspects and embodiments herein, the at least one stem at the 3' end comprises a partial DNA duplex of 4 to 500 nucleotides.In one embodiment of the aspects and embodiments herein, at least one stem at the 3'-end comprises a partial DNA duplex of 4 to 5 nucleotides. In one embodiment of the aspects and embodiments herein, at least one loop at the 3'-end comprises 3 to 500 unlinked nucleotides. In one embodiment of the aspects and embodiments herein, at least one loop at the 3'-end comprises a minimum of 3 unlinked nucleotides. In one embodiment of the aspects and embodiments herein, the ssDNA molecule comprises at least two stem-loop structures at the 3'-end. In one embodiment of the aspects and embodiments herein, the ssDNA molecule comprises at least three stem-loop structures at the 3'-end. In one embodiment of the aspects and embodiments herein, the ssDNA molecule comprises at least four or more stem-loop structures at the 3'-end. In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 3'-end comprises a hairpin DNA structure. In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 3'-end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadruplex DNA structure, a bulge-forming DNA structure, and a multi-branched loop structure. In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 3'-end does not comprise the A or A' region that would be present in a wild-type AAV ITR. In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 3'-end does not comprise the A, A', D, or D' region that would be present in a wild-type AAV ITR. In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 3'-end does not comprise the A, A', B, B', C, C', D, or D' region that would be present in a wild-type AAV ITR. In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 3'-end does not comprise the rep-binding element (RBE) that would be present in a wild-type AAV ITR. In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 3' end does not include a terminal resolving site (trs) present in the wild-type ITR.In one aspect and embodiment herein, the ssDNA molecule does not contain any virus-derived sequences. In one aspect and embodiment herein, the stem at the 3' end of the ssDNA molecule contains one or more nucleotides modified to be exonuclease-resistant. In one aspect and embodiment herein, the 3' end of the ssDNA molecule contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more nucleotides modified to be exonuclease-resistant. In one aspect and embodiment herein, the nucleotides modified to be exonuclease-resistant are phosphorothioate-modified (PS) nucleotides. In one aspect and embodiment herein, the ssDNA molecule contains at least one functional moiety. In one aspect and embodiment herein, at least one stem-loop structure at the 3' end further comprises at least one functional moiety. In one aspect and embodiment herein, the at least one functional moiety is an aptamer. In a further embodiment, the aptamer is capable of nuclear translocation in a cell. In one embodiment of the aspects and embodiments herein, the ssDNA molecule comprises at least one stem-loop structure at its 5'-end, wherein the at least one stem-loop structure at the 5'-end comprises at least one stem and at least one loop. In one embodiment of the aspects and embodiments herein, the ssDNA comprises at least two stem-loop structures at the 5'-end. In one embodiment of the aspects and embodiments herein, the ssDNA molecule comprises at least three stem-loop structures at the 5'-end. In one embodiment of the aspects and embodiments herein, the ssDNA molecule comprises at least four or more stem-loop structures at the 5'-end. In one embodiment of the aspects and embodiments herein, the at least one stem-loop structure at the 5'-end comprises a hairpin DNA structure. In one embodiment of the aspects and embodiments herein, the at least one stem-loop structure at the 5'-end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadruplex DNA structure, a bulge-forming DNA structure, and a multi-branched loop structure.In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 5'-end does not include the A or A' region that would be present in a wild-type AAV ITR. In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 5'-end does not include the A, A', D, or D' region that would be present in a wild-type AAV ITR. In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 5'-end does not include the A, A', B, B', C, C', D, or D' region that would be present in a wild-type AAV ITR. In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 5'-end does not include the rep binding element (RBE) that is present in a wild-type ITR. In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 5'-end does not include the terminal resolving site (trs) that is present in a wild-type ITR. In one aspect and embodiment herein, the stem at the 5' end of the ssDNA molecule comprises one or more nucleotides modified to be exonuclease resistant. In one aspect and embodiment herein, the 5' end of the ssDNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more nucleotides modified to be exonuclease resistant. In a further embodiment, the nucleotides modified to be exonuclease resistant are phosphorothioate-modified (PS) nucleotides. In one aspect and embodiment herein, the loop at the 5' end further comprises one or more nucleic acids (one or more, two or more, three or more, five or more, ten or more, twenty or more) for stabilizing the end. In one aspect and embodiment herein, the e-loop at the 5' end further comprises one or more chemically modified nucleic acids. In one aspect and embodiment herein, the stem-loop structure at the 5' end comprises at least one functional moiety. In a further embodiment, the at least one functional moiety is an aptamer. In another further embodiment, the aptamer is capable of nuclear translocation in a cell.In one embodiment of the aspects and embodiments herein, the functional moiety is a ribozyme.In one embodiment of the aspects and embodiments herein, the functional moiety is an antisense oligonucleotide (ASO). In one embodiment of the aspects and embodiments herein, the functional moiety is a small interfering RNA (siRNA). In one embodiment of the aspects and embodiments herein, the functional moiety is an antiviral nucleoside analog (ANA). In one embodiment of the aspects and embodiments herein, the 5'-end and / or 3'-end loop further comprises one or more triplex-forming oligonucleotides. In one embodiment of the aspects and embodiments herein, the 5'-end and / or 3'-end loop further comprises one or more gRNAs or gDNAs. In one embodiment of the aspects and embodiments herein, the 5'-end and / or 3'-end loop further comprises one or more molecular probes. In one embodiment of the aspects and embodiments herein, the ssDNA molecule lacks any viral capsid protein coding sequence.

[0009] In one embodiment of the aspects and embodiments herein, the ssDNA molecule is produced synthetically in vitro.

[0010] In one embodiment of the aspects and embodiments herein, the ssDNA molecules are synthetically produced in vitro in a cell-free environment.

[0011] In one embodiment of the aspects and embodiments herein, the ssDNA molecule does not activate or only minimally activates an immune pathway. In a further embodiment, the immune pathway is an innate immune pathway. In some embodiments, the innate immune pathway is selected from the group consisting of the cGAS / STING pathway, the TLR9 pathway, the inflammasome-mediated pathway, and combinations thereof.

[0012] In one embodiment of the aspects and embodiments herein, the ssDNA molecule further comprises at least one promoter. In one embodiment of the aspects and embodiments herein, the ssDNA molecule further comprises at least one enhancer. In one embodiment of the aspects and embodiments herein, the promoter is a hAAT promoter. In one embodiment of the aspects and embodiments herein, the promoter is a TTR promoter. In one embodiment of the aspects and embodiments herein, the enhancer is a serpin (SERP) enhancer. In one embodiment of the aspects and embodiments herein, the ssDNA molecule comprises a TTR promoter and a SERP enhancer. In one embodiment of the aspects and embodiments herein, the promoter comprises a transcription start site (TSS). In one embodiment of the aspects and embodiments herein, the promoter is double-stranded. In one embodiment of the aspects and embodiments herein, the TSS is double-stranded.

[0013] In one aspect and embodiment of the present specification, the ssDNA molecule is capable of expressing at least one therapeutic protein or therapeutic fragment thereof, hi one embodiment, the at least one therapeutic protein is selected from the group consisting of an antibody, an enzyme, a clotting factor, a transcription factor, a replication factor, a growth factor, a hormone, and a fusion protein. In one embodiment of the aspects and embodiments herein, the at least one therapeutic protein is useful for treating a genetic disorder selected from the group consisting of sickle cell anemia, melanoma, hemophilia A (factor VIII (FVIII) deficiency) and hemophilia B (factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited disorders of liver metabolism, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharide storage diseases (e.g., Hurler syndrome (MPS type I), Shelley syndrome (MPS type IS), Hurler-Scheie syndrome (MPS type IHS), Hunter syndrome (MPS type IHS), and the like. II), Sanfilippo types A, B, C, and D (MPS type III A, B, C, and D), Morquio types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS type VI), Sly syndrome (MPS type VII), hyaluronidase deficiency (MPSIX), Niemann-Pick disease types A / B, C1 and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III and IV, sialidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II and III, Fabry disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipids, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar degeneration, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), external nucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital maculopathy, Stargardt macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Uscher syndrome, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency.

[0014] According to another aspect, the present disclosure provides a pharmaceutical composition comprising the LNP of any one of the aspects and embodiments herein and a pharmaceutically acceptable excipient. In one embodiment of the aspects and embodiments herein, the ssDNA molecule is encapsulated in a lipid. In one embodiment of the aspects and embodiments herein, the LNP further comprises a sterol. In a further embodiment, the sterol is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, derivatives thereof, and combinations thereof. In one embodiment of the aspects and embodiments herein, the sterol is cholesterol. In one embodiment of the aspects and embodiments herein, the sterol is beta-sitosterol. In one embodiment of the aspects and embodiments herein, the LNP further comprises a non-cationic lipid. In a further embodiment, the non-cationic lipid is distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)ethanolamine 1-O-methyl-2-oleoyl-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine (such as 16-O-monomethyl PE), dimethylphosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS),Sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dicoil phosphatidylcholine (DEPC), palmitoyl methyl phosphatidylglycerol (POPG), diazidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-diphthaloyl In one aspect and embodiment herein, the non-cationic lipid is selected from the group consisting of di-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. In one aspect and embodiment herein, the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylethanolamine (DOPE). In one aspect and embodiment herein, the LNP further comprises at least one PEGylated lipid. In a further embodiment, the at least one PEGylated lipid is selected from the group consisting of PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, PEG-distearyloxypropyl, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (DMG-PEG), PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaotanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol) (PEG-cholesterol),The PEGylated lipid is selected from the group consisting of 3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether (PEG-DMB) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) (DSPE-PEG). In another further embodiment, the at least one PEGylated lipid is DMG-PEG, DSPE-PEG, or both. In one aspect and embodiment herein, the at least one PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, or both. In one aspect and embodiment herein, the LNP further comprises a tissue-specific and / or cell-type-specific targeting moiety. In a further embodiment, the tissue- and / or cell-type-specific targeting ligand is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. In another further embodiment, the tissue- and / or cell-type-specific targeting ligand is an antibody, an antibody fragment, or an antibody derivative. In yet another further embodiment, the antibody, antibody fragment, or antibody derivative is selected from the group consisting of a full-length antibody, Fab, Fab', single-domain antibody, and single-chain antibody (scFv). In one embodiment of the aspects and embodiments herein, the antibody, antibody fragment, or antibody derivative is an scFv. In one embodiment of the aspects and embodiments herein, the tissue-specific and / or cell-type-specific targeting group is covalently attached to at least one PEGylated lipid to form a PEGylated lipid conjugate. In one embodiment, the PEGylated lipid conjugate comprises a tetra-antennary GalNAc covalently attached to DSPE-PEG2000. In one embodiment of the aspects and embodiments herein, the LNP further comprises an ionizable lipid. In a further embodiment, the ionizable lipid is a cationic lipid. In another further embodiment, the ionizable lipid is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA),2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), DLin-MC3-DMA, N-[1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-sn-glycerol 1,2-Dilauroyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-Dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC), 1,2-Dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-Dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamidoethyl-3,4-di[oleyloxy]benzamide (MVL5), Dioctadecylamino-glycero-3-ethylphosphocholine (DOEPC), Silspermine (DOGS), 3b-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), dioctadecyldimethylammonium bromide (DDAB), Saint lipids (e.g., SAINT-2,N-methyl-4-(dioleyl)methylpyridinium), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 1,2-dioleoyl 1-3-dimethyl-hydroxyethylammonium bromide (DORIE), 1,2-dioleoyloxypropyl In some variations, the condensing agent is a cationic lipid, such as dioctadecyldimethylammonium bromide (DDAB), diisopropyl-3-dimethylhydroxyethylammonium chloride (DORI), dialkylated amino acids (DILA2) (e.g., C18:1-norArg-C16), dioleyldimethylammonium chloride (DODAC), 1-palmitoyl-2-oleyl-sn-glycero-3-ethylphosphocholine (POEPC), and 1,2-dimethylestreoyl-sn-glycero-3-ethylphosphocholine (MOEPC). ...1,2-Dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), Heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-Dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-Dioleoyloxy-3-dimethylaminopropane The lipids include dimethylaminopropane (DODMA), morpholinocholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-diyl 1-diolate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-diyl 1-diolate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminium chloride (DOTAPen), SS-cleavable lipids, and mixtures thereof. In one embodiment of the aspects and embodiments herein, the ionizable lipid is present in a molar proportion of about 30% to about 80%, e.g., about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 80%, about 60% to about 70%, or about 70% to about 80%. In one aspect and embodiment of the present specification, the sterol is present in a molar proportion of about 20% to about 50%, e.g., about 20% to about 50%, about 25% to about 50%, about 30% to about 40%, about 20% to about 40%, about 20% to about 30%, or about 25% to about 35%. In one aspect and embodiment of the present specification, the non-cationic lipid is present in a molar proportion of about 2% to about 20%, e.g., about 2% to about 20%, about 10% to about 20%, about 15% to about 20%, about 2% to about 15%, about 2% to about 10%, about 5% to about 10%, about 5% to about 15%, or about 2% to about 5%. In one embodiment of the aspects and embodiments herein, the at least one PEGylated lipid is present in an amount of from about 2.1% to about 10%, e.g., from about 2.1% to about 10%, from about 5% to about 10%, from about 2.1% to about 5%, from about 2.1% to about 8%,or about 5% to about 7% molar percentage. In one aspect and embodiment herein, the PEGylated lipid conjugate is present in a molar percentage of about 0.1% to about 10%, e.g., about 0.1% to about 10%, about 0.1% to about 1%, about 0.1% to about 2%, about 0.1% to about 4%, about 0.1% to about 6%, about 0.1% to about 8%, about 1% to about 10%, about 1% to about 5%, about 5% to about 10%, or about 1% to about 2%. In one aspect and embodiment herein, the LNP is a sterol, non-cationic, In one embodiment of the aspects and embodiments herein, the LNP further comprises dexamethasone palmitate.

[0015] In one embodiment of the aspects and embodiments herein, the LNP has a total lipid to ssDNA ratio of about 10:1 to about 40:1, e.g., about 10:1 to about 40:1, about 10:1 to about 30:1, or about 10:1 to about 20:1.

[0016] In one aspect and embodiment herein, the LNP has a diameter of about 40 nm to about 120 nm. In one aspect and embodiment herein, the LNP has a diameter of less than about 100 nm. In one aspect and embodiment herein, the LNP has a diameter of about 60 nm to about 80 nm. In one aspect and embodiment herein, the LNP is present in an LNP composition comprising a plurality of LNPs having an average diameter of about 40 nm to about 120 nm. In one aspect and embodiment herein, the LNP is present in an LNP composition comprising a plurality of LNPs having an average diameter of less than about 100 nm. In one aspect and embodiment herein, the LNP is present in an LNP composition comprising a plurality of LNPs having an average diameter of about 60 nm to about 80 nm (e.g., about 60, 65, 70, 75, or 80 nm).

[0017] In another aspect, the present disclosure provides a pharmaceutical composition comprising the LNP of any one of the aspects and embodiments herein and a pharmaceutically acceptable excipient.

[0018] In another aspect, the disclosure provides a method of treating a genetic disorder in a subject, the method comprising administering a therapeutically effective amount of the LNP of any one of the aspects and embodiments herein, or the pharmaceutical composition of any of the aspects or embodiments herein, to the subject. In a further embodiment of the method, the subject is human. In another further embodiment, the genetic disorder is sickle cell anemia, melanoma, hemophilia A (factor VIII (FVIII) deficiency) and hemophilia B (factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited disorders of liver metabolism, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharide storage diseases (e.g., Hurler syndrome (MPS type I), Shelley syndrome (MPS type I S), Hurler-Scheie syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippotypes A, B, C, and D (MPS type III), A, B, C, and D), Morquio syndrome types A and B (MPS IVA and IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPS IX), Niemann-Pick disease types A / B, C1 and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III, and IV, sialidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II, and III, cystinosis, Batten disease, aspartame disease, Glucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipid galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar degeneration, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD),The genetic disorder is selected from the group consisting of Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), external nucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital maculopathy, Stargardt macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, age-related macular degeneration (AMD), alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency. In one aspect and embodiment herein, the genetic disorder is hemophilia A. In one aspect and embodiment herein, the genetic disorder is hemophilia B. In one aspect and embodiment herein, the genetic disorder is phenylketonuria (PKU). In one embodiment of the aspects and embodiments herein, the genetic disorder is Wilson's disease. In one embodiment of the aspects and embodiments herein, the genetic disorder is Gaucher's disease type I, type II, or type III. In one embodiment of the aspects and embodiments herein, the genetic disorder is Stargardt's macular dystrophy. In one embodiment of the aspects and embodiments herein, the genetic disorder is LCA10. In one embodiment of the aspects and embodiments herein, the genetic disorder is Usher's syndrome. In one embodiment of the aspects and embodiments herein, the genetic disorder is wet AMD.

[0019] According to another aspect, the present disclosure provides a host cell comprising the LNP of any one of the aspects and embodiments herein. In one embodiment, the host cell is in vitro. In another embodiment, the host cell is in vivo.

[0020] In another aspect, the present disclosure provides a method for delivering a therapeutic gene and / or a therapeutic protein to a subject, the method comprising administering a therapeutically effective amount of the LNP of any one of the aspects and embodiments herein, or the pharmaceutical composition of the aspects and embodiments herein, to the subject. According to some embodiments, the subject is a human.

[0021] According to another aspect, the present disclosure provides a method of delivering a therapeutic gene and / or a therapeutic protein to a cell, the method comprising contacting the cell with an LNP of any one of the aspects and embodiments herein, or a pharmaceutical composition of the aspects and embodiments herein, thereby delivering the therapeutic gene and / or the therapeutic protein to the cell.

[0022] According to another aspect, the present disclosure provides a method for delivering a therapeutic gene to the nucleus of a cell, the method comprising contacting the cell with an LNP according to any one of the aspects and embodiments herein or a pharmaceutical composition according to the aspects and embodiments herein, thereby delivering the therapeutic gene and / or therapeutic protein to the nucleus of the cell. According to some embodiments, the cell is in vitro. According to other embodiments, the cell is in vivo.

[0023] In another aspect, the disclosure provides a method of minimizing an immune response in a subject, wherein the subject is being treated with a therapeutic gene or protein, comprising administering a therapeutically effective amount of an LNP according to any one of the aspects and embodiments herein, or a pharmaceutical composition according to the aspects and embodiments herein, wherein the nucleic acid of the subject encodes the therapeutic gene or protein. According to some embodiments, the subject is human.

[0024] In one aspect and embodiment of the present specification, the dosage of the ssDNA molecule administered to the subject is about 0.05 mg / kg to about 5.0 mg / kg. In one aspect and embodiment of the present specification, the dosage of the ssDNA molecule administered to the subject is about 0.05 mg / kg, about 0.1 mg / kg, about 0.15 mg / kg, about 0.2 mg / kg, about 0.25 mg / kg, about 0.3 mg / kg, about 0.35 mg / kg, about 0.4 mg / kg, about 0.45 mg / kg, about 0.5 mg / kg, about 0.55 mg / kg, about 0.6 mg / kg, about 0.65 mg / kg, about 0.7 mg / kg, or about 0.8 mg / kg. kg, about 0.75mg / kg, about 0.8mg / kg, about 0.85mg / kg, about 0.9mg / kg, about 0.95mg / kg, about 1.0mg / kg, about 1.1mg / kg, about 1.2mg / kg, about 1.25mg / kg , about 1.3mg / kg, about 1.4mg / kg, about 1.5mg / kg, about 1.6mg / kg, about 1.7mg / kg, about 1.75mg / kg, about 1.8mg / kg, about 1.9mg / kg, about 2.0mg / kg, about 2.1m g / kg, approx. 2.2 mg / kg, approx. 2.25 mg / kg, approx. 2.3 mg / kg, approx. 2.4 mg / kg, approx. 2.5 mg / kg, approx. 2.6 mg / kg, approx. 2.7 mg / kg, approx. 2.75 mg / kg, approx. 2.8 mg / kg , about 2.9mg / kg, about 3.0mg / kg, about 3.1mg / kg, about 3.2mg / kg, about 3.25mg / kg, about 3.3mg / kg, about 3.4mg / kg, about 3.5mg / kg, about 3.6mg / kg, about 3.7m

[0023] In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is less than about 4.0 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is less than about 3.0 mg / kg.In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is less than about 2.0 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is less than about 1.75 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is less than about 1.5 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is less than about 1.25 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is less than about 1.0 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is less than about 0.75 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is less than about 0.5 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is less than about 0.25 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is about 2.0 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is about 1.75 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is about 1.5 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is about 1.25 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is about 1.0 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is about 0.75 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is about 0.5 mg / kg. In one embodiment of the aspects and embodiments herein, the dosage of the ssDNA molecule administered to the subject is about 0.25 mg / kg.In one aspect and embodiment of the present specification, the dosage of the ssDNA molecule administered to a subject is about 0.075 mg / kg to about 4.0 mg / kg. In one aspect and embodiment of the present specification, the dosage of the ssDNA molecule administered to a subject is about 0.1 mg / kg to about 3.0 mg / kg. In one aspect and embodiment of the present specification, the dosage of the ssDNA molecule administered to a subject is about 0.125 mg / kg to about 2.0 mg / kg. In one aspect and embodiment of the present specification, the dosage of the ssDNA molecule administered to a subject is about 0.15 mg / kg to about 1.5 mg / kg. In one aspect and embodiment of the present specification, the dosage of the ssDNA molecule administered to a subject is about 0.175 mg / kg to about 1.25 mg / kg. In one aspect and embodiment of the present specification, the dosage of the ssDNA molecule administered to a subject is about 0.2 mg / kg to about 1.0 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is about 0.1 mg / kg to about 0.5 mg / kg. In one aspect and embodiment herein, the dosage of the ssDNA molecule administered to the subject is about 0.1 mg / kg to about 1.0 mg / kg. In one aspect and embodiment herein, the method further comprises administering at least two doses of the LNP or pharmaceutical composition. In one aspect and embodiment herein, the method further comprises administering at least three doses of the LNP or pharmaceutical composition. In one aspect and embodiment herein, the method further comprises administering four or more doses of the LNP or pharmaceutical composition.

[0025] In another aspect, the present disclosure provides an isolated linear single-stranded deoxyribonucleic acid (ssDNA) molecule comprising at least one nucleic acid sequence of interest, wherein the at least one nucleic acid sequence of interest is adjacent to at least one stem-loop structure at its 3'-end, the at least one stem-loop structure comprising at least one stem and at least one loop. In one embodiment, at least one stem-loop structure at the 3'-end is sufficient to prime replication and / or transcription. In another embodiment, at least one stem at the 3'-end comprises a partial DNA duplex of 4-500 nucleotides. In another further embodiment, at least one stem at the 3'-end comprises a partial DNA duplex of 4-5 nucleotides. In one aspect and embodiment herein, at least one loop at the 3'-end comprises 3-500 unlinked nucleotides. In one aspect and embodiment herein, at least one loop at the 3'-end comprises a minimum of 3 unlinked nucleotides. In one aspect and embodiment herein, the ssDNA molecule comprises at least two stem-loop structures at the 3'-end. In one aspect and embodiment herein, the ssDNA molecule comprises at least three stem-loop structures at the 3'-end. In one aspect and embodiment herein, the ssDNA molecule comprises at least four or more stem-loop structures at the 3'-end. In one aspect and embodiment herein, at least one stem-loop structure at the 3'-end comprises a hairpin DNA structure. In one aspect and embodiment herein, at least one stem-loop structure at the 3'-end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadruplex DNA structure, a bulge-forming DNA structure, and a multi-branched loop structure. In one aspect and embodiment herein, at least one stem-loop structure at the 3'-end does not comprise the A or A' region that would be present in a wild-type AAV ITR. In one aspect and embodiment herein, at least one stem-loop structure at the 3'-end does not comprise the A, A', D, or D' region that would be present in a wild-type AAV ITR.In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 3'-end does not contain the A, A', B, B', C, C', D, or D' regions that would be present in a wild-type AAV ITR. In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 3'-end does not contain the rep binding element (RBE) present in a wild-type AAV ITR. In one embodiment of the aspects and embodiments herein, at least one stem-loop structure at the 3'-end does not contain the terminal resolving site (trs) present in a wild-type ITR. In one embodiment of the aspects and embodiments herein, the ssDNA molecule does not contain any virus-derived sequences. In one embodiment of the aspects and embodiments herein, the stem at the 3'-end of the ssDNA molecule contains one or more nucleotides modified to be exonuclease resistant. In one aspect and embodiment of the present specification, the 3'-end of the ssDNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more nucleotides modified to be exonuclease-resistant. In one aspect and embodiment of the present specification, the nucleotides modified to be exonuclease-resistant are phosphorothioate-modified (PS) nucleotides. In one aspect and embodiment of the present specification, the ssDNA molecule comprises at least one functional moiety. In one aspect and embodiment of the present specification, at least one stem-loop structure at the 3'-end further comprises at least one functional moiety. In one aspect and embodiment of the present specification, the at least one functional moiety is an aptamer. In a further embodiment, the aptamer is capable of nuclear translocation in cells. In one aspect and embodiment of the present specification, the ssDNA molecule comprises at least one stem-loop structure at its 5'-end, and the at least one stem-loop structure at the 5'-end comprises at least one stem and at least one loop. In one embodiment of the aspects and embodiments herein, the ssDNA comprises at least two stem-loop structures at the 5' end. In one embodiment of the aspects and embodiments herein, the ssDNA molecule comprises at least three stem-loop structures at the 5' end.In one aspect and embodiment herein, the ssDNA molecule comprises at least four or more stem-loop structures at the 5'-end. In one aspect and embodiment herein, at least one stem-loop structure at the 5'-end comprises a hairpin DNA structure. In one aspect and embodiment herein, at least one stem-loop structure at the 5'-end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadruplex DNA structure, a bulge-forming DNA structure, and a multi-branched loop structure. In one aspect and embodiment herein, at least one stem-loop structure at the 5'-end does not comprise the A or A' region that would be present in a wild-type AAV ITR. In one aspect and embodiment herein, at least one stem-loop structure at the 5'-end does not comprise the A, A', D, or D' region that would be present in a wild-type AAV ITR. In one aspect and embodiment herein, at least one stem-loop structure at the 5'-end does not contain the A, A', B, B', C, C', D, or D' regions that would be present in a wild-type AAV ITR. In one aspect and embodiment herein, at least one stem-loop structure at the 5'-end does not contain the rep binding element (RBE) that is present in a wild-type ITR. In one aspect and embodiment herein, at least one stem-loop structure at the 5'-end does not contain the terminal resolving site (trs) that is present in a wild-type ITR. In one aspect and embodiment herein, the stem at the 5'-end of the ssDNA molecule contains one or more nucleotides modified to be exonuclease resistant. In one aspect and embodiment herein, the 5'-end of the ssDNA molecule contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more nucleotides modified to be exonuclease resistant. In a further embodiment, the nucleotides modified to be exonuclease resistant are phosphorothioate-modified (PS) nucleotides. In one embodiment of the aspects and embodiments herein, the 5'-terminal loop further comprises one or more nucleic acids to stabilize the end.In one embodiment of the aspects and embodiments herein, the 5'-terminal loop further comprises one or more chemically modified nucleic acids. In one embodiment of the aspects and embodiments herein, the 5'-terminal stem-loop structure comprises at least one functional moiety. In a further embodiment, the at least one functional moiety is an aptamer. In another further embodiment, the aptamer is capable of nuclear translocation within a cell. In one embodiment of the aspects and embodiments herein, the functional moiety is a ribozyme. In one embodiment of the aspects and embodiments herein, the functional moiety is an antisense oligonucleotide (ASO). In one embodiment of the aspects and embodiments herein, the functional moiety is a small interfering RNA (siRNA). In one embodiment of the aspects and embodiments herein, the functional moiety is an antiviral nucleoside analog (ANA). In one embodiment of the aspects and embodiments herein, the 5'-terminal and / or 3'-terminal loop further comprises one or more triplex-forming oligonucleotides. In one embodiment of the aspects and embodiments herein, the 5'-terminal and / or 3'-terminal loop further comprises one or more gRNAs or gDNAs. In one aspect and embodiment herein, the 5'-end and / or 3'-end loops further comprise one or more molecular probes. In one aspect and embodiment herein, the ssDNA molecule lacks any viral capsid protein coding sequence. In one aspect and embodiment herein, the ssDNA molecule is synthetically produced in vitro. In one aspect and embodiment herein, the ssDNA molecule is synthetically produced in vitro in a cell-free environment. In one aspect and embodiment herein, the ssDNA molecule does not activate or minimally activates an immune pathway. In one aspect and embodiment herein, the immune pathway is an innate immune pathway. In a further embodiment, the innate immune pathway is selected from the group consisting of the cGAS / STING pathway, the TLR9 pathway, the inflammasome-mediated pathway, and combinations thereof. In one aspect and embodiment herein, the ssDNA molecule further comprises at least one promoter.In one embodiment of the aspects and embodiments herein, the ssDNA molecule further comprises at least one enhancer.

[0026] In one embodiment of the aspects and embodiments herein, the promoter is a hAAT promoter. In one embodiment of the aspects and embodiments herein, the promoter is a TTR promoter. In one embodiment of the aspects and embodiments herein, the enhancer is a serpin (SERP) enhancer. In one embodiment of the aspects and embodiments herein, the ssDNA molecule comprises a TTR promoter and a SERP enhancer. In one embodiment of the aspects and embodiments herein, the promoter comprises a transcription start site (TSS). In one embodiment of the aspects and embodiments herein, the promoter is double-stranded. In one embodiment of the aspects and embodiments herein, the enhancer is double-stranded. In one embodiment of the aspects and embodiments herein, the TSS is double-stranded. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is at least 10 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 60 base pairs, at least 70 base pairs, at least 80 base pairs, at least 90 base pairs, at least 100 base pairs, at least 110 base pairs, at least 120 base pairs, at least 130 base pairs, at least 140 base pairs, at least 150 base pairs, at least 160 base pairs, at least 170 base pairs, at least 180 base pairs, at least 190 base pairs, at least 200 base pairs, at least 220 base pairs, at least 240 base pairs, at least 260 base pairs, at least 270 base pairs, at least 280 base pairs, at least 290 base pairs, at least 300 base pairs, at least 310 base pairs, at least 320 base pairs, at least 330 base pairs, at least 340 base pairs, at least 350 base pairs, at least 360 base pairs, at least 370 base pairs, at least 380 base pairs, at least 390 base pairs, at least 400 base pairs, at least 410 base pairs, at least 420 base pairs, at least 430 base pairs, at least 440 base pairs, at least 450 base pairs, at least 460 base pairs, at least 470 base pairs, at least 480 base pairs, at least 490 base pairs, at least 500 base pairs, at least 510 base pairs, at least 520 base pairs, at least 530 base pairs, at least 540 base pairs, at least 550 base pairs, at least 560 base pairs, at least 570 base pairs, at least 5 base pairs, at least 280 base pairs, at least 300 base pairs, at least 320 base pairs, at least 340 base pairs, at least 360 base pairs, at least 380 base pairs, at least 400 base pairs, at least 420 base pairs, at least 440 base pairs, at least 460 base pairs, at least 480 base pairs, at least 500 base pairs, at least 550 base pairs, at least 600 base pairs, at least 650 base pairs, at least 700 base pairs, at least 750 base pairs, at least 800 base pairs, at least 850 base pairs, at least 900 base pairs, at least 950 base pairs, at least 1000 base pairs, at least 1100 base pairs, at least 1200 base pairs, at least 1300 base pairs, at least 1400 base pairs,In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is less than 1500 base pairs, less than 1400 base pairs, less than 1300 base pairs, less than 1200 base pairs, less than 1100 base pairs, less than 1000 base pairs, less than 950 base pairs, less than 900 base pairs, less than 850 base pairs, less than 800 base pairs, less than 750 base pairs, less than 700 base pairs, less than 650 base pairs, less than 600 base pairs, less than 550 base pairs, less than 500 base pairs, less than 480 base pairs, less than 460 base pairs, less than 440 base pairs, less than 420 base pairs, less than 400 base pairs, less than 5 ...00 base pairs, less than 500 base pairs, less than 500 base pairs, less than 500 base pairs, less than 500 base pairs, less than 500 base pairs, less than 500 base pairs, less than 500 base pairs, less than 500 base pairs, less than 500 base pairs, less than 500 base pairs, less than 500 base pairs, less than 500 base In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is between about 30 and 1500 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is about 40 to 1400 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is about 50 to 1300 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is about 60 to 1200 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is about 70 to 1100 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is about 80 to 1000 base pairs in length. In one embodiment of the aspects and embodiments herein, the promoter,The double-stranded region comprising the enhancer and / or TSS is approximately 90 to 900 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 90 to 900 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 100 to 800 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 110 to 700 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 120 to 600 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 130 to 500 base pairs in length. In one aspect and embodiment of the present specification, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 140-400 base pairs in length. In one aspect and embodiment of the present specification, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 150-300 base pairs in length. In one aspect and embodiment of the present specification, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 160-200 base pairs in length. In one aspect and embodiment of the present specification, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 170-190 base pairs in length. In one aspect and embodiment of the present specification, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 1381 base pairs in length. In one aspect and embodiment of the present specification, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 499 base pairs in length. In one embodiment of the aspects and embodiments herein, the ssDNA molecule is capable of expressing at least one therapeutic protein or therapeutic fragment thereof. In one embodiment of the aspects and embodiments herein, the at least one therapeutic protein is an antibody, an enzyme, a clotting factor, a transcription factor, a replication factor, a growth factor,The compound is selected from the group consisting of a hormone, and a fusion protein. In one embodiment of the aspects and embodiments herein, the at least one therapeutic protein is selected from the group consisting of sickle cell anemia, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited disorders of liver metabolism, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharide storage diseases (e.g., Hurler syndrome (MPS type I), Shelley syndrome (MPS type IS), Hurler-Scheie syndrome (MPS type IHS), Hunter syndrome (MPS type II), Sanfilippotypes A, B, C, and D (MPS type III), and the like. A, B, C, and D), Morquio syndrome types A and B (MPS IVA and IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPS IX), Niemann-Pick disease types A / B, C1 and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III, and IV, sialidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II, and III, Fabry disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (L AL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipids, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar degeneration, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital maculopathy,It is useful for treating a genetic disorder selected from the group consisting of Stargardt macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency.

[0027] In one embodiment of the aspects and embodiments herein, the ssDNA molecule further comprises a lipid. In a further embodiment, the ssDNA molecule is encapsulated in a lipid. In one embodiment of the aspects and embodiments herein, the lipid is a lipid nanoparticle (LNP).

[0028] In one embodiment of the aspects and embodiments herein, the present disclosure provides a pharmaceutical composition comprising an ssDNA molecule according to any one of the aspects and embodiments herein and a pharmaceutically acceptable excipient.

[0029] In another aspect, the present disclosure provides a method of treating a genetic disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of an ssDNA molecule according to any one of the aspects and embodiments herein, or a pharmaceutical composition according to any one of the aspects and embodiments herein.

[0030] In another aspect, the present disclosure provides a host cell comprising an ssDNA molecule of any one of the aspects and embodiments herein.

[0031] In another aspect, the present disclosure provides a method for delivering a therapeutic gene and / or a therapeutic protein to a subject, the method comprising administering to the subject a therapeutically effective amount of an ssDNA molecule according to any one of the aspects and embodiments herein, or a pharmaceutical composition according to any one of the aspects and embodiments herein.

[0032] In another aspect, the present disclosure provides a method of delivering a therapeutic gene and / or a therapeutic protein to a cell, the method comprising contacting the cell with an ssDNA molecule of any one of the aspects and embodiments herein or a pharmaceutical composition of any one of the aspects and embodiments herein, thereby delivering the therapeutic gene and / or the therapeutic protein to the cell.

[0033] In another aspect, the present disclosure provides a method of delivering a therapeutic gene to the nucleus of a cell, the method comprising contacting the cell with an ssDNA molecule of any one of the aspects and embodiments herein or a pharmaceutical composition of any one of the aspects and embodiments herein, thereby delivering the therapeutic gene and / or therapeutic protein to the nucleus of the cell.

[0034] In another aspect, the present disclosure provides a method of minimizing an immune response in a subject, wherein the subject is treated with a therapeutic gene or therapeutic protein, comprising administering a therapeutically effective amount of an ssDNA molecule according to any one of the aspects and embodiments herein, or a pharmaceutical composition according to any one of the aspects and embodiments herein, wherein the nucleic acid of the subject encodes the therapeutic gene or therapeutic protein.

[0035] In one embodiment of the aspects and embodiments herein, the enhancer is double-stranded. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is at least 10 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 60 base pairs, at least 70 base pairs, at least 80 base pairs, at least 90 base pairs, at least 100 base pairs, at least 110 base pairs, at least 120 base pairs, at least 130 base pairs, at least 140 base pairs, at least 150 base pairs, at least 160 base pairs, at least 170 base pairs, at least 180 base pairs, at least 190 base pairs, at least 200 base pairs, at least 220 base pairs, at least 240 base pairs, at least 260 base pairs, at least 280 base pairs, at least 300 base pairs, at least 320 base pairs, at least 340 base pairs, at least 360 base pairs, at least 380 base pairs, at least 390 base pairs, at least 400 base pairs, at least 410 base pairs, at least 420 base pairs, at least 430 base pairs, at least 440 base pairs, at least 450 base pairs, at least 460 base pairs, at least 470 base pairs, at least 480 base pairs, at least 490 base pairs, at least 500 base pairs, at least 510 base pairs, at least 520 base pairs, at least 530 base pairs, at least 540 base pairs, at least 550 base pairs, at least 560 base pairs, at least 570 base pairs, at least 580 base pairs, at least 590 base pairs, at least 600 base pairs, at least 610 base pairs, at least 0 base pairs, at least 300 base pairs, at least 320 base pairs, at least 340 base pairs, at least 360 base pairs, at least 380 base pairs, at least 400 base pairs, at least 420 base pairs, at least 440 base pairs, at least 460 base pairs, at least 480 base pairs, at least 500 base pairs, at least 550 base pairs, at least 600 base pairs, at least 650 base pairs, at least 700 base pairs, at least 750 base pairs, at least 800 base pairs, at least 850 base pairs, at least 900 base pairs, at least 950 base pairs, at least 1000 base pairs, at least 1100 base pairs, at least 1200 base pairs, at least 1300 base pairs, at least 1400 base pairs, or at least 1500 base pairs in length.In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is less than 1500 base pairs, less than 1400 base pairs, less than 1300 base pairs, less than 1200 base pairs, less than 1100 base pairs, less than 1000 base pairs, less than 950 base pairs, less than 900 base pairs, less than 850 base pairs, less than 800 base pairs, less than 750 base pairs, less than 700 base pairs, less than 650 base pairs, less than 600 base pairs, less than 550 base pairs, less than 500 base pairs, less than 480 base pairs, less than 460 base pairs, less than 440 base pairs, less than 420 base pairs, less than 400 base pairs, less than 5 ... In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is between about 30 and 1500 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 40 to 1,400 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 50 to 1,300 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 60 to 1,200 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 70 to 1,100 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 80 to 1,000 base pairs in length.In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 90-900 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 90-900 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 100-800 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 110-700 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 120-600 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 130-500 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 140-400 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 150-300 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 160-200 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 1381 base pairs in length. In one embodiment of the aspects and embodiments herein, the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 499 base pairs in length. [Brief explanation of the drawings]

[0036] Embodiments of the present disclosure, briefly summarized above and discussed in more detail below, can be understood by reference to illustrative embodiments thereof, which are depicted in the accompanying drawings. However, because the present disclosure may admit of other equally effective embodiments, the accompanying drawings illustrate only typical embodiments of the present disclosure and therefore should not be considered limiting in scope.

[0037] [Figure 1A] Figure 1A shows that starting from the double-stranded ceDNA construct 10429, ssAAV vectors were obtained by ssDNA endonuclease treatment. In Figure 1A, a clear approximately 830 bp band is seen for the ssRES ss360 control, and there was clear degradation of the ΦX174 ssDNA control. There was loss of full-sized ssRES ss429 in the well, with the appearance of a band migrating at approximately 100 bp that was more prominent / clear in the 5U Mung Bean nuclease treatment. [Figure 1B] FIG. 1B shows a schematic diagram of the double-stranded ceDNA10429 construct (ds429) and the single-stranded 10429 and 10360. [Figure 2] FIG. 2 shows Klenow exonuclease filling of ss synthetic AAV vector molecules favoring the presence of a 3′-OH. [Figure 3] Figure 3 shows schematic diagrams of symmetric and asymmetric ITR oligos. The top diagram shows a symmetric overhang. The bottom diagram shows an asymmetric overhang, where the 3' end of the left ITR has a PS bond (closer to the 3' end of the molecule) and the right ITR has a PS bond shifted two bases to the right. [Figure 4] Figure 4 shows a schematic of the left and right ITR sequences of the single-stranded AAV synthetic vector hAAT luciferase with symmetric ITR oligos from constructs ss10429 and ss10483. As shown in Figure 4, a CpG-free spacer was included that altered several nucleotides in the RBE of the A / A' stem. A nick site, Nb.BbvCI, was engineered downstream of the end-resolving site (trs). [Figure 5]Figure 5 shows a schematic of the left and right ITR sequences of the single-stranded AAV synthetic vector hAAT luciferase with asymmetric ITR oligos from construct ss10485. As shown in Figure 5, a CpG-free spacer was included that changes several nucleotides in the RBE of the A / A' stem. A nick site, Nb.BbvCI, was engineered downstream of the cleavage. [Figure 6] Figure 6 shows a schematic diagram of the single-stranded AAV synthetic vector FVIII symmetric with the ITR oligos from construct ss10491. A nick site Nb.BbvCI was engineered downstream of the cleavage. [Figure 7] Figure 7 shows a schematic of the left and right ITR sequences of the single-stranded AAV synthetic vector FVIII from construct ss10484. A nick site Nb.BbvCI was engineered downstream of the cleavage. [Figure 8] Figure 8 is a graph showing that HepG2 cells transfected with single-stranded AAV synthetic vectors express luciferase. A clear dose response was observed with ceDNA and single-stranded AAV synthetic vectors. [Figure 9] Figure 9 is a graph showing that gel-extracted, Zymo column-purified single-stranded AAV synthetic vectors induced a lower innate immune response compared to ceDNA. Single-stranded AAV synthetic vectors induced lower Lucia IFN reporter activity than ceDNA in WT THP1 cells at matched molecular doses. [Figure 10] Figure 10 is a graph showing that gel-extracted and Zymo column-purified single-stranded AAV synthetic vector induced a lower innate immune response compared to ceDNA. In cGAS KO cells, an IFN response was present with the highest ceDNA dose and column-purified single-stranded AAV synthetic vector, but not otherwise, indicating cGAS-sensing single-stranded AAV synthetic vector and ceDNA. [Figure 11] FIG. 11 is a graph showing preliminary longitudinal body weights in animals treated with single-stranded synthetic DNA (ssDNA) “SSD” (40004) and double-stranded (ds) ceDNA (10541). [Figure 12]FIG. 12 is a panel of graphs showing interim longitudinal body weights in animals treated with single-stranded synthetic DNA (ssDNA) “SSD” (40004) and double-stranded (ds) ceDNA (10541) for each dose. [Figure 13] FIG. 13 shows the results of IVIS imaging performed on day 4. [Figure 14] Figure 14 shows the results of IVIS imaging performed on days 4 to 7. [Figure 15-1] Figure 15 shows the nucleic acid sequence of double-stranded (ds) ceDNA construct 10360 (SEQ ID NO: 1). As depicted in Figure 15, the Factor VIII ORF is located at nucleotides 828-5219 of SEQ ID NO: 1. [Figure 15-2] Same as above. [Figure 16-1] Figure 16 shows the nucleic acid sequence of ds ceDNA construct 10429 (SEQ ID NO:2). As depicted in Figure 16, the luciferase ORF is located at nucleotides 1449-3101 of SEQ ID NO:2. [Figure 16-2] Same as above. [Figure 17-1] Figure 17 shows the nucleic acid sequence of ds ceDNA construct 10483 (SEQ ID NO: 3). As depicted in Figure 17, the luciferase ORF is located at nucleotides 1449-3101 of SEQ ID NO: 3. [Figure 17-2] Same as above. [Figure 18-1] Figure 18 shows the nucleic acid sequence of ds ceDNA construct 10484 (SEQ ID NO: 4). As depicted in Figure 18, the Factor VIII ORF is located at nucleotides 835-5226 of SEQ ID NO: 4. [Figure 18-2] Same as above. [Figure 19-1] Figure 19 shows the nucleic acid sequence of ds ceDNA construct 10485 (SEQ ID NO: 5). As depicted in Figure 19, the luciferase ORF is located at nucleotides 1449-3101 of SEQ ID NO: 5. [Figure 19-2] Same as above. [Figure 20-1]Figure 20 shows the nucleic acid sequence of ds ceDNA construct 10491 (SEQ ID NO: 6). As depicted in Figure 20, the Factor VIII ORF is located at nucleotides 835-5226 of SEQ ID NO: 6. [Figure 20-2] Same as above. [Figure 21-1] Figure 21 shows the nucleic acid sequence of ds ceDNA construct 10376 (SEQ ID NO: 7). As depicted in Figure 21, the luciferase ORF is located at nucleotides 1443-3095 of SEQ ID NO: 7. [Figure 21-2] Same as above. [Figure 22-1] Figure 22 shows the nucleic acid sequence of ds ceDNA construct 10541 (SEQ ID NO: 8). As depicted in Figure 22, the luciferase ORF is located at nucleotides 1449-3101 of SEQ ID NO: 8. [Figure 22-2] Same as above. [Figure 23-1] Figure 23 shows the nucleic acid sequence of plasmid 210150 (SEQ ID NO: 9), which contains one of the exemplary ds ceDNA sequences. As depicted in Figure 23, the Factor VIII ORF is located at nucleotides 894-5285. [Figure 23-2] Same as above. [Figure 23-3] Same as above. [Figure 24-1] Figure 24 shows the nucleic acid sequence of single-stranded (ss) DNA construct 40004 (SEQ ID NO: 10). As depicted in Figure 24, the luciferase ORF reverse complement is located at nucleotides 503-2155 of SEQ ID NO: 10. [Figure 24-2] Same as above. [Figure 25] Figure 25 is a panel of graphs showing interim longitudinal body weights in single-stranded synthetic DNA (ssDNA) "SSD" (ssDNA construct 40004, referred to as "ss004")-treated animals and double-stranded (ds)-treated animals (ds ceDNA construct 10541, referred to as "ds541") at five different doses matched by molecular number. [Figure 26]Figure 26 is a panel of graphs showing weight loss over a 5-day period in animals treated with single-stranded synthetic DNA (ssDNA) "SSD" (40004) and double-stranded (ds) ceDNA (10541). As shown in Figure 26, the difference in BW reduction was most evident on day 2, when animals treated with ssDNA rapidly recovered from weight loss. [Figure 27] Figure 27 is a panel of graphs showing the effect of ssDNA (construct 40004, designated "ss004") and ds ceDNA (construct 10541, designated "ds541") on cytokine expression after 6 hours. ds ceDNA construct 10376 ("376") served as a positive control. The figure legend indicates whether PS binding is present (No PS) or absent (No PS) in the construct. [Figure 28] Figure 28 is a panel of graphs from a second experiment showing the effect of ssDNA (construct 40004, designated "ss004") and ds ceDNA (construct 10541, designated "ds541") on cytokine expression after 6 hours. ds ceDNA construct 10376 ("376") served as a positive control. The figure legend indicates whether PS linkages are present (No PS) or absent (No PS) in the construct. [Figure 29] FIG. 29 is a graph showing that ds ceDNA (construct 10541, designated ds541) and ss DNA (construct 40004, designated ss004) had similar levels of mRNA in the liver as determined by qtPCR. [Figure 30]Figure 30 is a panel of graphs showing the effect of loading LNP-formulated mRNA, ceDNA, and ssDNA on blood cytokine levels in mice. Mice (n=5 per group) were given intravenous injections of LNP-formulated mRNA, ceDNA, or ssDNA. Blood levels (pg / mL) of IFN-α, IL-18, TNF-α, IL-6, and IFN-γ were measured 6 hours after injection. Several batches of ssDNA produced at scale for subsequent use in non-human primates (NHPs) were also tested. Groups for each graph, left to right: PBS control, mRNA (2.0 mg / kg), ceDNA (2.0 mg / kg), ssDNA batch B1 (2.0 mg / kg), ssDNA batch B1 (NHP scale, 0.5 mg / kg), ssDNA batch B1 (NHP scale, 2.0 mg / kg), ssDNA batch C (NHP scale, 0.5 mg / kg), ssDNA batch C (NHP scale, 2.0 mg / kg). [Figure 31] Figure 31 is a panel of graphs showing the effect of loading 1.0 mg / kg of LNP-formulated ceDNA (DNA), ssDNA (squares), and mRNA (triangles) on blood cytokine levels (pg / mL) 6 and 24 hours after intravenous infusion in cynomolgus monkeys. [Figure 32] Figure 32 shows the levels of complement activation (left: C3a, right: C5b-9) measured in NHPs after administration of 1.0 mg / mL of LNP-formulated ceDNA (circles), ssDNA (squares), or mRNA (triangles). [Figure 33] Figure 33 shows the results of an in vitro PBMC assay. Human peripheral blood mononuclear cells (PBMCs) were contacted with LNP-formulated ceDNA or ssDNA, and TNF-α stimulation was measured. In both the first experiment (two left bars) and the second experiment (three right bars, including an untreated control), ssDNA induced significantly lower TNF-α stimulation compared to ceDNA (ssDNA-induced TNF-α stimulation was equivalent to that of the untreated control). [Figure 34]Figure 34 is a panel of graphs showing sustained and robust expression from LNP-formulated ssDNA. Mice were given an intravenous injection of 0.25 mg / kg of LNP-formulated ceDNA or ssDNA containing a luciferase reporter gene. Luciferase expression (IVIS) was measured 1 and 4 days (right) and at additional time points up to 30 days (left) after injection. [Figure 35] Figures 35A-35E show schematic diagrams of ssDNA constructs with different components. All constructs include a hAAT luciferase expression construct. Figure 35A: Construct ss004 (the left ITR is from AAV2, has a PS binding site on the bottom strand, and contains an Nb.BbvCI cleavage site; the right ITR is from AAV2, with most of the A region removed, a PS binding site at the 5' end, and the minus strand removed from the expression construct). Figure 35B: Construct ss020 (same as ss004, but without the PS binding site). Figure 35C: Construct ss021 (same as ss004, but with wild-type AAV2 ITRs, no PS binding site, and no Nb.BbvCI nick site). Figure 35D: Construct ss022 (similar to ss021, except for the + strand of the expression construct). Figure 35E: A hAAT luciferase expression construct without ITR sequences. [Figure 36] Figure 36 is a graph of in vitro luciferase expression by ssDNA constructs. HepG2 cells were transfected with 100 ng or 200 ng of ss004 or ss011, respectively, and luciferase expression (normalized to cell viability) was measured at 48 hours. [Figure 37] Figure 37 is a panel of graphs showing the results of in vivo luciferase expression by ssDNA constructs in mice after hydrodynamic injection (HDI). Mice were injected with either ceDNA541 (5 ng or 500 ng per animal), constructs ss004, ss020, ss021, ss022, or ss011 (500 ng per animal), or a mixture of ss021 + ss022 (250 ng per animal) or ss011 + s022 (250 ng per animal). IVIS luciferase expression is shown on days 1 (upper left) and 4 (lower left), as well as in a longitudinal plot (upper right). [Figure 38] Figure 38 is a graph showing the results of in vivo luciferase expression in mice injected with LNP-formulated ssDNA constructs. Results are shown, from left to right, for PBS control (upper and lower triangles), ss011 (diamonds, ionizable lipid MC3), ss011 (open circles, ionizable lipid Y), ss011 (squares, ionizable lipid Z), and ss004 (ionizable lipid Z). [Figure 39] Figures 39A-39D show schematic diagrams of ssDNA constructs with different PS binding configurations. All constructs contain a hAAT luciferase expression construct. The location of the PS binding is indicated by an arrow. Figure 39A: Construct ss004 (the left ITR is derived from AAV2 and has a PS binding on the bottom strand; the right ITR is derived from AAV2 with most of the A region removed and has a PS binding at the 5' end). Figure 39B: Construct 034 (same as ss004, but the PS binding in the left ITR is on the top strand near the 3' end). Figure 39C: Construct ss039 (same as ss034, but the right ITR has been truncated and has a PS binding on the bottom strand near the 5' end). Figure 39D: Construct ss040 (same as ss039, but without the PS binding). [Figure 40] Figure 40 is a panel of graphs showing the results of in vivo luciferase expression in mice injected with LNP-formulated ssDNA constructs. Mice were injected with ceDNA541, ceDNA654, ss004, ss034, ss039, or ss040 (0.25 mg / kg). IVIS luciferase expression is shown on days 1 (top left) and 4 (top right), as well as in longitudinal plots (bottom). [Figure 41]Figure 41 is a panel of graphs showing the effect of loading LNP-formulated mRNA, ceDNA, and ssDNA on blood cytokine levels in mice. Mice (n=5 per group) were intravenously injected with LNP-formulated ceDNA or ssDNA constructs. Six hours after injection, blood levels (pg / mL) of IFN-α (top left), IFN-γ (top right), IL-6 (center left), TNF-α (center right), and IL-18 (bottom left) were measured. Groups in each graph, from left to right: PBS control, ceDNA541, ceDNA654, ss004, ss034, ss039, and ss040. [Figure 42] Figures 42A-42D show schematic diagrams of ssDNA constructs with single- or double-stranded promoter regions. All constructs contain a luciferase reporter. Figure 43A: ss004, single-stranded hAAT enhancer / promoter set. Figure 43B: ss104, double-stranded hAAT enhancer / promoter set. Figure 43C: ss102, single-stranded 1xSERP / TTR enhancer / promoter set. Figure 43D: ss104, double-stranded 1xSERP / TTR enhancer / promoter set. [Figure 43] Figure 43 is a panel of graphs measuring luciferase expression in mice injected via HDI with ssDNA constructs having single-stranded promoter regions (ss004, ss102) or double-stranded promoter regions (ss104, ss103). Left: Luciferase expression at day 1. Middle: Luciferase expression at day 7. Right: Longitudinal expression from days 1 to 7. [Figure 44] Figure 44 is a panel of graphs showing the fold change in expression of ssDNA constructs with double-stranded promoter regions (ss104, ss103, respectively) compared to ssDNA constructs with single-stranded promoter regions (ss004, ss102, respectively). Left: Day 1. Middle: Day 7. Right: Vertical fold change from day 1 to day 7. [Figure 45]Figure 45 is a panel of graphs measuring luciferase expression in mice injected via HDI with ssDNA constructs having single-stranded (ss004, ss102) or double-stranded (ss104, ss103) promoter regions compared to expression from ceDNA541. Left: luciferase expression at day 1. Right: luciferase expression at day 7. DETAILED DESCRIPTION OF THE INVENTION

[0038] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art to which this disclosure belongs. It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein, and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the disclosure, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), Fields Virology, 6th Edition, published by Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013); Knipe, DM and Howley, PM (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN). 1936113414);Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, Inc., 2014 (ISBN047150338X, 9780471503385). 2005;およびCurrent Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated herein by reference in their entireties.

[0039] As used herein, the term "AAV" or "adeno-associated virus" refers to a single-stranded DNA parvovirus that grows only in cells. Certain functions of AAV can only be provided by co-infection with a helper virus. Thirteen serotypes of AAV have been identified. General information and reviews about AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York).

[0040] As used herein, the terms "single-stranded (ss) synthetic DNA molecule," "single-stranded (ss) synthetic AAV vector," "synthetic production of ss DNA molecules," and "synthetic production of ss AAV vectors" refer to single-stranded (ss) synthetic DNA molecules (ssDNA), single-stranded AAV vectors, and methods for their synthetic production in a completely cell-free environment. This production can involve one or more molecules in a manner that does not require replication or other propagation of the molecule by a cell or the interior of a cell or by the use of a cell extract. Synthetic production avoids contamination of the produced molecule with cellular contaminants, such as cellular proteins or nucleic acids, viral proteins or DNA, insect proteins or DNA, and further avoids undesired cell-specific modifications of the molecule during the production process, such as methylation or glycosylation, or other post-translational modifications.

[0041] As used herein, the terms "gap" and "nick" are used interchangeably and refer to an interrupted portion of the synthetic DNA vector of the present disclosure, creating a section of single-stranded DNA in the otherwise double-stranded ceDNA. Gaps can be from 1 base pair to 100 base pairs in length. Exemplary gaps designed and created by the methods described herein, and synthetic vectors generated by the methods, can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 base pairs (bp) in length. Exemplary gaps in the present disclosure can be 1 bp to 10 bp in length, 1 to 20 bp in length, 1 to 30 bp in length, or any length necessary to nick double-stranded DNA to enable or maintain efficient transcription of the expression cassette in a host cell. According to some embodiments, the gap can be located 5' upstream of the expression cassette. According to some embodiments, the gap can be located 3' downstream of the expression cassette. According to some embodiments, the gap can be located 5' upstream of the expression cassette and 3' downstream of the expression cassette.

[0042] As used herein, the term "nick" refers to a discontinuity in a double-stranded DNA molecule in which a phosphodiester bond is absent between adjacent nucleotides on one strand, typically due to damage or enzymatic action. It is understood that one or more nicks allow for the release of a twist within the strand during DNA replication, and that nicks are also thought to play a role in facilitating the binding of the transcription machinery.

[0043] As used herein, the term "ceDNA" refers to capsid-free, closed-end, linear, double-stranded (ds) double-stranded DNA for non-viral gene transfer, synthesis, or other methods. A detailed description of ceDNA is provided in the international application PCT / US2017 / 020828 (published internationally as WO 2017 / 152149A1), filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Specific methods for producing ceDNA containing various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Application PCT / US18 / 49996 (published as WO 2019 / 051255A1), filed September 7, 2018, and PCT / US2018 / 064242 (published as WO 2019 / 113310A1), filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Specific methods for producing synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Application PCT / US2019 / 14122 (published as WO 2019 / 143885A1), filed January 18, 2019, the entire contents of which are incorporated herein by reference. According to some embodiments, the ceDNA comprises one or more phosphorothioate modified nucleotides.

[0044] As used herein, the term "neDNA" or "nicked ceDNA" refers to closed-end DNA that has a nick or a gap of 1 to 100 base pairs in the stem or spacer region upstream of the open reading frame (e.g., the expressed promoter and transgene).

[0045] As used herein, the term "inverted terminal repeat" or "ITR" refers to a nucleic acid sequence located at the 5' and / or 3' end of an ssDNA vector disclosed herein, which comprises at least one stem-loop structure including a partial double strand and at least one loop. According to some embodiments, the ITR may be an artificial sequence (e.g., does not contain a sequence derived from a virus). The ITR may further comprise one stem-loop structure (e.g., a "hairpin"), or two or more stem-loop structures. For example, the ITR may comprise two stem-loop structures (e.g., a "hammerhead," "dogbone," or "dumbbell"), three stem-loop structures (e.g., a "cruciform"), or a more complex structure. The ITR may comprise an aptamer sequence or one or more chemical modifications.

[0046] According to some embodiments, "ITRs" can be artificially synthesized using a set of oligonucleotides containing one or more desired functional sequences (e.g., palindromic sequences). The ITR sequences can be artificial AAV ITRs, artificial non-AAV ITRs, or ITRs physically derived from viral AAV ITRs (e.g., ITR fragments removed from the viral genome). For example, ITRs can be derived from the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, and human parvovirus B-19). Alternatively, the SV40 hairpin, which serves as the SV40 origin of replication, can be used as an ITR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. Parvoviridae viruses are divided into two subfamilies: the Parvovirinae subfamily, which infect vertebrates, and the Densovirinae subfamily, which infect invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses that can replicate in vertebrate hosts, including, but not limited to, humans, primates, cattle, canines, equines, and bovine species. Typically, ITR sequences can be derived from parvoviruses, lentiviruses, goose viruses, and B19, in wild-type, "duckbone," and "dumbbell-shaped," symmetric, or asymmetric ITR orientations. ITRs are typically present at both the 5' and 3' ends of AAV vectors in single-stranded DNA (ssDNA) molecules, but ITRs can be present at only one end of a linear vector. For example, ITRs can be present only at the 5' end. In some other cases, ITRs can be present only at the 3' end in single-stranded DNA (ssDNA) molecules. For convenience herein, an ITR located 5' to (upstream of) an expression cassette in a single-stranded DNA (ssDNA) molecule will be referred to as the "5' ITR" or "left ITR," and an ITR located 3' to (downstream of) an expression cassette in a single-stranded DNA (ssDNA) molecule will be referred to as the "3' ITR" or "right ITR."

[0047] As used herein, "wild-type ITR" or "WT-ITR" refers to a native ITR sequence in an AAV or other dependant virus that retains, for example, Rep binding activity and Rep nicking ability. The nucleic acid sequence of a WT-ITR from any AAV serotype may vary slightly from the standard native sequence due to degeneracy or drift in the genetic code; therefore, WT-ITR sequences encompassed for use herein include WT-ITR sequences that result from natural variations (e.g., replication errors) that occur during the production process.

[0048] As used herein, the terms "substantially symmetric WT-ITR" or "substantially symmetric WT-ITR pair" refer to a pair of WT-ITRs in a single-stranded DNA (ssDNA) molecule (e.g., a synthetic AAV vector, e.g., a single-stranded (ss) synthetic AAV vector) that are wild-type ITRs with reverse-complementary sequences across their entire length. For example, an ITR can be considered a wild-type sequence even if it has one or more nucleotides that deviate from the standard, naturally occurring, canonical sequence, as long as the changes do not affect the physical or functional properties or overall three-dimensional structure (secondary and tertiary structure) of the sequence. In some embodiments, the deviating nucleotides represent conservative sequence changes. An example is a sequence that has 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical sequence (e.g., as determined using BLAST default settings) and a symmetrical three-dimensional spatial arrangement in which the three-dimensional structure has the same shape in geometric space as the other WT-ITR. A substantially symmetric WT ITR has the same A, C-C', and B-B' loops in 3D space. A substantially symmetric WT ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE') and terminal resolving region (trs) that pairs with an appropriate Rep protein. Optionally, other functions can be tested, including transgene expression under permissive conditions.

[0049] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutated ITR" are used interchangeably herein and refer to an ITR that has a mutation in at least one or more nucleotides compared to a wild-type ITR from the same serotype. The mutation can result in a change in one or more of the A, C, C', B, and B' regions in the ITR, and can result in a change in the three-dimensional spatial configuration (i.e., its three-dimensional structure in geometric space) compared to the three-dimensional spatial configuration of a wild-type ITR from the same serotype.

[0050] As used herein, the term "asymmetric ITR," also referred to as an "asymmetric ITR pair," refers to a pair of ITRs in a single-stranded ssDNA vector that are not reverse complements over their entire length. As one non-limiting example, an asymmetric ITR pair does not have a symmetrical three-dimensional spatial configuration relative to its cognate ITR, such that their 3D structures are different shapes in geometric space. Stated differently, an asymmetric ITR pair has a different overall geometric structure, i.e., they have different configurations of their A, C-C', and B-B' loops in 3D space (e.g., one ITR may have a shorter C-C' arm and / or a shorter B-B' arm compared to the cognate ITR). The difference in sequence between the two ITRs may be due to one or more nucleotide additions, deletions, truncations, or point mutations. In one embodiment, one ITR of the asymmetric ITR pair may be a wild-type AAV ITR sequence, and the other ITR may be a modified ITR (e.g., a non-wild-type or synthetic ITR sequence) as defined herein. In another embodiment, neither ITR of the asymmetric ITR pair is a wild-type AAV sequence, and the two ITRs are modified ITRs that have different shapes in geometric space (i.e., different overall geometric structures). In some embodiments, one mod-ITR of the asymmetric ITR pair can have a short C-C' arm, and the other ITR can have a different modification (e.g., a single arm or a short B-B' arm) such that it has a different three-dimensional spatial configuration compared to the cognate asymmetric mod-ITR.

[0051] As used herein, the term "symmetric ITRs" refers to a pair of ITRs in an ssDNA vector that are mutated or modified compared to the wild-type Depend virus ITR sequence and are reverse-complementary across their entire length. Neither ITR is the wild-type AAV2 ITR sequence (i.e., they are modified ITRs, also referred to as mutant ITRs), and may have sequence differences from the wild-type ITR due to nucleotide additions, deletions, substitutions, truncations, or point mutations. For convenience, the ITR located 5' (upstream) of the expression cassette in a single-stranded DNA (ssDNA) molecule is referred to herein as the "5' ITR" or "left ITR," and the ITR located 3' (downstream) of the expression cassette is referred to as the "right ITR." (In a single-stranded DNA, ssDNA) molecule, the ITR located 5' (upstream) of the expression cassette is referred to as the "5' ITR" or "left ITR," and the ITR located 3' (downstream) of the expression cassette is referred to as the "3' ITR" or "right ITR."

[0052] As used herein, the term "substantially symmetric modified ITR" or "substantially symmetric modified ITR pair" refers to a pair of modified ITRs in a single-stranded DNA (ssDNA) molecule (e.g., a synthetic AAV vector, e.g., a single-stranded (ss) synthetic AAV vector) that have reverse-complementary sequences throughout their entire length. For example, a modified ITR can be considered substantially symmetric even if it has a portion of its nucleotide sequence that deviates from its reverse-complementary sequence, as long as the change does not affect its properties and overall shape. As a non-limiting example, a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence (as measured using BLAST with default settings) and also has a symmetrical three-dimensional spatial configuration with respect to its cognate modified ITR, such that their three-dimensional structures have the same shape in geometric space. In other words, a substantially symmetric modified ITR pair has the same stem-loop structure configured in three-dimensional space. In some embodiments, the ITRs from a mod-ITR pair can have different reverse-complementary nucleotide sequences but still have the same symmetrical three-dimensional spatial organization, i.e., both ITRs have mutations that result in the same overall 3D shape. For example, in a virus-derived ITR, one ITR (e.g., the 5' ITR) of a mod-ITR pair can be from one serotype, and the other ITR (e.g., the 3' ITR) can be from a different serotype, but both can have the same corresponding mutations so that the modified ITR pair has the same symmetrical three-dimensional spatial organization (e.g., if the 5' ITR has a deletion in the C region, the cognate modified 3' ITR from the different serotype has a deletion at a corresponding position in the C' region). In such embodiments, each ITR of a modified ITR pair can be from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), e.g., a combination of AAV2 and AAV6, and the modification of one ITR is reflected at the corresponding position in the cognate ITR from the different serotype. In one embodiment, a substantially symmetric modified ITR pair refers to a pair of modified ITRs (mod-ITRs) so long as the nucleotide sequence differences between the ITRs do not affect the properties or overall shape and they have substantially the same shape in three-dimensional space.Non-limiting examples include mod-ITRs that share 95%, 96%, 97%, 98%, or 99% sequence identity with a standard mod-ITR, as determined by standard means (e.g., BLAST (Basic Local Alignment Search Tool) or BLASTN with default settings), and further have a symmetrical three-dimensional spatial organization such that their three-dimensional structures have the same shape in geometric space. A substantially symmetric mod-ITR pair will have the same A, C-C', and B-B' loops in 3D space; for example, if the modified ITR in a substantially symmetric mod-ITR pair has a deletion of the C-C' arm, then the cognate mod-ITR will have a corresponding deletion of the C-C' loop and a similar 3D structure of the remaining A and B-B' loops that are the same shape in geometric space as its cognate mod-ITR.

[0053] As used herein, the term "flanking" refers to the relative position of one nucleic acid sequence with respect to another. Generally, in the sequence ABC, B is flanked by A and C. The same is true for the sequence AxBxC. Thus, a flanking sequence precedes or follows the flanking sequence, but need not be contiguous with or immediately adjacent to the flanking sequence. In one embodiment, the term flanking refers to the terminal repeat sequences at each end of a linear single-stranded DNA (ssDNA) molecule.

[0054] As used herein, the term closed-end DNA or ceDNA refers to a synthetic, double-stranded, linear DNA construct with at least one covalently closed end that contains a gene of interest and other regulatory elements.

[0055] As used herein, the term "closed-end DNA vector" refers to a capsid-free DNA vector with at least one covalently closed end, at least a portion of the vector having an intramolecular double-stranded structure.

[0056] As defined herein, a "reporter" refers to a protein that can be used to provide a detectable readout. Reporters generally produce a measurable signal, such as fluorescence, color, or luminescence. A reporter protein coding sequence encodes a protein whose presence in a cell or organism is easily observed. For example, fluorescent proteins cause cells to fluoresce when excited with a particular wavelength of light, luciferases cause cells to catalyze light-producing reactions, and enzymes such as β-galactosidase convert a substrate into a colored product. Exemplary reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP) and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art.

[0057] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable readout, e.g., as a reporter polypeptide, or more appropriately, as a cell-killing polypeptide, e.g., a toxin or drug that sensitizes a cell to killing with, or lack thereof, a selected agent. Effector proteins include any protein or peptide that directly targets or damages host cell DNA and / or RNA. For example, effector proteins can include, but are not limited to, restriction endonucleases that target host cell DNA sequences (whether genomic or on extrachromosomal elements), proteases that degrade polypeptide targets necessary for cell survival, DNA gyrase inhibitors, and ribonuclease-type toxins. According to some embodiments, the expression of effector proteins controlled by the synthetic biological circuits described herein can participate as factors in another synthetic biological circuit, thereby expanding the range and complexity of the responsiveness of the biological circuit system.

[0058] Transcriptional regulators refer to transcriptional activators and repressors that either activate or repress the transcription of a gene of interest. A promoter is a region of nucleic acid that initiates transcription of a specific gene. Transcriptional activators typically bind near the transcriptional promoter and recruit RNA polymerase to directly initiate transcription. Repressors bind to the transcriptional promoter and sterically hinder transcription initiation by RNA polymerase. Other transcriptional regulators can act as either activators or repressors, depending on where they bind and on cellular and environmental conditions. Non-limiting examples of transcriptional regulator classes include, but are not limited to, homeodomain proteins, zinc finger proteins, winged helix (forkhead) proteins, and leucine zipper proteins.

[0059] As used herein, a "repressor protein" or "inducer protein" is a protein that binds to a regulatory sequence element and represses or activates, respectively, the transcription of a sequence operatively linked to the regulatory sequence element. Preferred repressor and inducer proteins described herein are sensitive to the presence or absence of at least one input or environmental input. Preferred proteins described herein are modular in nature, containing, for example, separable DNA-binding and input-binding or response elements or domains.

[0060] As used herein, "carrier" includes any and all solvents, dispersion media, excipients, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar adverse reactions when administered to a host.

[0061] As used herein, an "input agent response domain" is a domain of a transcription factor that binds to or otherwise responds to a condition or input agent in a manner that renders the linked DNA-binding fusion domain responsive to the presence of that condition or input. In certain embodiments, the presence of the condition or input results in a conformational change in the input agent response domain, or in the protein to which it is fused, that modifies the transcriptional regulatory activity of the transcription factor.

[0062] As used herein, the term "in vivo" refers to an assay or process performed on or within an organism, such as a multicellular animal. In some of the embodiments described herein, a method or use can be said to be performed "in vivo" when a unicellular organism, such as a bacterium, is used. The term "ex vivo" particularly refers to methods and uses performed using living cells with intact membranes that are outside the body of a multicellular animal or plant, such as, inter alia, explants, cultured cells including primary cells and cell lines, transformed cell lines, and extracted tissues or cells including blood cells. The term "in vitro" refers to assays and methods that do not require the presence of cells with intact membranes, such as cell extracts, and can refer to the introduction of programmable synthetic biological circuits in a non-cellular system, such as cell-free medium, or in a cellular system, such as a cell extract.

[0063] The term "promoter," as used herein, refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of that nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. Promoters can be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence that controls the initiation and rate of transcription of the remainder of the nucleic acid sequence. Promoters can also contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. Within the promoter sequence, a transcription initiation site and protein binding domains involved in the binding of RNA polymerase are found. Eukaryotic promoters often, but not necessarily, contain "TATA" and "CAT" boxes. A variety of promoters, including inducible promoters, can be used to drive the expression of transgenes in the single-stranded (ssDNA) molecules disclosed herein. The promoter sequence may be bounded at its 3' end by a transcription initiation site and may extend upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background.

[0064] As used herein, the terms "expression cassette" and "expression unit" are used interchangeably and refer to a heterologous DNA sequence operably linked to a promoter or other DNA regulatory sequence sufficient to direct the transcription of a transgene in a DNA vector, e.g., a single-stranded (ssDNA) molecule. Suitable promoters include, for example, tissue-specific promoters. The promoter can also be of AAV origin.

[0065] As used herein, the term renaturation, when referring to a renatured double-stranded expression cassette or renatured double-stranded transgene, refers to a double-stranded expression cassette or double-stranded transgene that is formed after an ssDNA molecule is transported into the nucleus of a host cell and is responsive to DNA polymerase activity that generates double-stranded DNA from the ssDNA by filling in the single-stranded portion of the ssDNA molecule.

[0066] "Operably linked" refers to a juxtaposition in which the components so described are in a relationship that allows them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. A promoter can be said to drive expression or drive transcription of the nucleic acid sequence it regulates. The terms "operably linked," "operably positioned," "operably linked," "under control," and "under transcriptional control" indicate that the promoter is in the correct functional location and / or orientation with respect to the nucleic acid sequence it regulates so as to control transcription initiation and / or expression of that sequence. "Reverse promoter," as used herein, refers to a promoter in which the nucleic acid sequence is in the reverse orientation, so that what was the coding strand is now the non-coding strand, or vice versa. Reverse promoter sequences can be used in various embodiments to regulate the state of a switch. Also, in various embodiments, a promoter can be used in conjunction with an enhancer.

[0067] The terms "DNA regulatory sequence," "control element," and "regulatory element" are used interchangeably herein and refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, proteolysis signals, and the like, that provide for and / or regulate the transcription of a non-coding sequence (e.g., a DNA-targeting RNA) or a coding sequence (e.g., a site-directed modifying polypeptide, or a Cas9 / Csn1 polypeptide) and / or regulate the translation of the encoded polypeptide.

[0068] As used herein, the term "enhancer" refers to a cis-acting regulatory sequence (e.g., 50 to 1,500 base pairs) that binds to one or more proteins (e.g., activator proteins or transcription factors) to increase transcriptional activation of a nucleic acid sequence. Naturally, enhancers can be located up to 1,000,000 base pairs upstream of the gene start site they regulate or downstream of the gene start site. Enhancers can be located within intronic regions or in the exon regions of unrelated genes. Typically, cis-acting enhancer sequences of 20 to 200 base pairs can be used to increase transgene expression.

[0069] A promoter can be a promoter naturally associated with a gene or sequence, such as can be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exons of a given gene or sequence. Such a promoter can be referred to as "endogenous." Similarly, according to some embodiments, an enhancer can be an enhancer naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. In some embodiments, a coding nucleic acid segment is positioned under the control of a "recombinant promoter" or "heterologous promoter," both of which refer to promoters that are not normally associated with the coding nucleic acid sequence to which it is operably linked in its natural environment. Similarly, a "recombinant or heterologous enhancer" refers to an enhancer that is not normally associated with a given nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes; promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell; and synthetic promoters or enhancers that are not "naturally occurring," i.e., contain different elements of different transcriptional regulatory regions and / or mutations that alter expression through methods of genetic engineering known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, promoter sequences can be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, in the context of the synthetic biology circuits and modules disclosed herein (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference). Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, and the like, can also be used.

[0070] As described herein, an "inducible promoter" is characterized by initiating or enhancing transcriptional activity when in the presence of, affected by, or contacted by an inducer or inducing agent. An "inducer" or "inducing agent," as defined herein, can be an endogenous or, usually exogenous, compound or protein administered in such a way that it is active in inducing transcriptional activity from an inducible promoter. In some embodiments, the inducer or inducing agent, i.e., a chemical, compound, or protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., an inducer can be an inducer protein expressed by another component or module), which can itself be under the control of an inducible promoter. In some embodiments, an inducible promoter is induced in the absence of certain agents, such as a repressor. Examples of inducible promoters include, but are not limited to, tetracycline, metallothionine, ecdysone, mammalian viruses (e.g., adenovirus late promoter; and mouse mammary tumor virus long terminal repeat (MMTV-LTR)), as well as other steroid-responsive promoters, rapamycin-responsive promoters, and the like.

[0071] As used herein, the term "subject" refers to a human or animal to which treatment, including prophylactic treatment, using a single-stranded (ssDNA) molecule according to the present disclosure is provided. Typically, the animal is a vertebrate, such as, but not limited to, a primate, a rodent, a domestic animal, or a game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, felines, such as domestic cats, canines, such as dogs, foxes, wolves, avian species, such as chickens, emus, and ostriches, and fish, such as trout, catfish, and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, such as a primate or a human. The subject can be male or female. Additionally, the subject can be an infant or child. In some embodiments, the subject can be a newborn or unborn subject, e.g., the subject is present in utero. Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects that represent animal models of diseases and disorders. Additionally, the methods and compositions described herein can be used with domestic animals and / or pets. Human subjects can be of any age, sex, race, or ethnic group, e.g., Caucasian (white), Asian, African, Black, African-American, African-European, Hispanic, Middle Eastern, etc. In some embodiments, the subject can be a patient or other subject in a clinical setting. In some embodiments, the subject has already received treatment. In some embodiments, the subject is an embryo, fetus, newborn, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, a human newborn, a human infant, a human child, a human adolescent, or a human adult.In some embodiments, the subject is an animal embryo, or a non-human embryo, or a non-human primate embryo, hi some embodiments, the subject is a human embryo.

[0072] As used herein, the term "host cell" includes any cell type that is amenable to transformation, transfection, transduction, etc. with the single-stranded (ssDNA) molecules described in this disclosure. By way of non-limiting example, host cells include isolated primary cells, pluripotent stem cells, CD34 + The host cell may be a human cell, an induced pluripotent stem cell, or any of a number of immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell may be an in situ or in vivo cell in a tissue, organ, or organism. Furthermore, the host cell may be a target cell, for example, in a mammalian subject (e.g., a human patient in need of gene therapy).

[0073] As used herein, the term "exogenous" refers to a substance present in a cell other than its natural source. As used herein, the term "exogenous" can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide that has been introduced, by a process involving the hand of man, into a biological system, such as a cell or organism in which it is not normally found, and in which it is desired to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, "exogenous" can refer to a nucleic acid or polypeptide that has been introduced, by a process involving the hand of man, into a biological system, such as a cell or organism in which it is found in relatively low abundance, and in which it is desired to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to a biological system or cell.

[0074] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. An "oligonucleotide" generally refers to a polynucleotide of between about 5 and about 100 nucleotides, either single- or double-stranded. However, for purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as "oligomers" or "oligos" and may be isolated from genes or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded polynucleotides (such as sense or antisense) and double-stranded polynucleotides, as applicable to the described embodiments. According to some embodiments, the nucleic acid is a single-stranded DNA (ssDNA) molecule as described in this disclosure. DNA can be in the form of, for example, antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations thereof. DNA can be in the form of minicircles, plasmids, bacmids, minigenes, ministring DNA (covalently closed linear DNA vectors), closed-end linear double-stranded DNA (CELiD or ceDNA), doggybone (dbDNA™) DNA, dumbbell-shaped DNA, minimal immunologically defined gene expression (MIDGE) vectors, viral vectors, or non-viral vectors. RNA can be in the form of small interfering RNA (siRNA), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof.Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which may be synthetic, naturally occurring, or non-naturally occurring, and have similar binding properties to the reference nucleic acid. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphorodiamidate morpholino oligomers (morpholinos), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNA™), and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequence.

[0075] As used herein, "inhibitory polynucleotide" refers to a DNA molecule or RNA molecule that reduces or prevents the expression (transcription or translation) of a second (target) polynucleotide. Inhibitory polynucleotides include antisense polynucleotides, ribozymes, and external guide sequences. The term "inhibitory polynucleotide" also includes DNA molecules and RNA molecules, such as RNAi that encode the actual inhibitory species, such as DNA molecules that encode ribozymes.

[0076] A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group.

[0077] "Base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues, as well as synthetic derivatives of purines and pyrimidines, including modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0078] By "hybridizable" or "complementary" or "substantially complementary," it is meant that a nucleic acid (e.g., RNA) contains a sequence of nucleotides that allows it to non-covalently bind, i.e., form Watson-Crick base pairs and / or G / U base pairs, to "anneal" or "hybridize" to another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid) under suitable in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base pairing includes adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C). It is also known in the art that guanine (G) base pairs with uracil (U) for hybridization between two RNA molecules (e.g., dsRNA). For example, G / U base pairing, in the context of tRNA anticodon base pairing with codons in mRNA, is partially responsible for the degeneracy (i.e., redundancy) of the genetic code. In the context of the present disclosure, a guanine (G) in the protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered complementary to a uracil (U), and vice versa. As such, if a G / U base pair can be made at a given nucleotide position in the protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule, that position is not considered non-complementary, but instead is considered complementary.

[0079] As used herein, the term "nucleic acid construct" refers to either a single-stranded or double-stranded nucleic acid molecule, which is isolated from a native gene or modified to contain a segment of nucleic acid in a manner that would not otherwise occur in nature or is synthetic. The term "nucleic acid construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present disclosure. An "expression cassette" contains a DNA coding sequence operably linked to a promoter.

[0080] As used herein, the terms "nucleic acid therapeutic," "therapeutic nucleic acid," and "TNA" are used interchangeably and refer to any therapeutic modality that uses nucleic acid as the active ingredient of the therapeutic agent for treating disease or disorder.As used herein, these terms refer to RNA-based therapeutic agents and DNA-based therapeutic agents.Non-limiting examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), and guide RNA (gRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigene, viral DNA (e.g., lentivirus or AAV genome) or non-viral synthetic DNA vector, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmid, bacmid, Doggybone (dbDNA™) DNA vector, minimal immunologically defined gene expression (MIDGE) vector, non-viral ministring DNA vector (linear covalently closed DNA vector), or dumbbell-shaped DNA minimal vector ("dumbbell DNA").

[0081] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0082] As used herein, the term "sequence identity" refers to the relationship between two nucleotide sequences. For purposes of this disclosure, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. Optional parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (the EMBOSS version in NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest homology" (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment - total number of gaps in the alignment). The length of the alignment is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[0083] As used herein, the term "homology" or "homology" is defined as the percentage of nucleotide residues in homologous arms that are identical to the nucleotide residues in the corresponding sequence on the target chromosome, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage.Alignment for determining the percentage of nucleotide sequence homology can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2, or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. In some embodiments, for example, a nucleic acid sequence (e.g., a DNA sequence) of a homologous arm of a repair template is considered to be "homologous" if the sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the corresponding native or unedited nucleic acid sequence (e.g., genomic sequence) of the host cell.

[0084] As used herein, homology arm refers to the polynucleotide that is suitable for targeting donor sequence to genome through homologous recombination.Typically, two homology arms flank donor sequence, and each homology arm comprises the genome sequence upstream and downstream of integration locus.

[0085] As used herein, a donor sequence refers to a polynucleotide that is inserted into a host cell genome or used as a repair template. The donor sequence may contain the desired modification during gene editing. The sequence to be integrated may be introduced into a target nucleic acid molecule through homology-directed repair at the target sequence, thereby changing the target sequence from the original target sequence to the sequence contained in the donor sequence. Thus, the sequence contained in the donor sequence may be an insertion, deletion, indel, point mutation, or repair of a mutation in the target sequence. The donor sequence may be, for example, a single-stranded DNA molecule, a double-stranded DNA molecule, a DNA / RNA hybrid molecule, or a DNA / modRNA (modified RNA) hybrid molecule. In one embodiment, the donor sequence is exogenous to the homologous arm. Editing may be RNA or DNA editing. The donor sequence may be endogenous or exogenous to the host cell genome, depending on the nature of the desired gene editing. As used herein, the term "heterologous" refers to a nucleotide sequence or polypeptide sequence that is not found in natural nucleic acids or proteins, respectively. A heterologous nucleic acid sequence can be linked (e.g., by genetic engineering) to a naturally occurring nucleic acid sequence (or a variant thereof) to generate a chimeric nucleotide sequence encoding a chimeric polypeptide. A heterologous nucleic acid sequence can be linked (e.g., by genetic engineering) to a variant polypeptide to generate a nucleic acid sequence encoding a fusion variant polypeptide.

[0086] As used herein, a "vector" or "expression vector" is a replicon, such as a plasmid, bacmid, phage, virus, virion, or cosmid, to which another DNA segment, i.e., an "insert," "transgene," or "expression cassette," can be attached so as to result in expression or replication of the attached segment ("expression cassette") in a cell. A vector can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be of viral or non-viral origin in its final form. However, for purposes of this disclosure, a vector generally refers to a synthetic AAV, e.g., a single-stranded (ss) synthetic AAV vector or a nicked ceDNA vector. Thus, the term "vector" encompasses any genetic element that, when associated with the appropriate control elements, is capable of replication and transfer of a gene sequence to a cell. In some embodiments, a vector can be a recombinant vector or an expression vector.

[0087] As used herein, the term "recombinant vector" refers to a vector containing a heterologous nucleic acid sequence or "transgene" that can be expressed in vivo. It should be understood that the vectors described herein can be combined with other suitable compositions and therapies in some embodiments. In some embodiments, the vector is episomal. The use of an appropriate episomal vector provides a means to maintain the target nucleotide in a subject in high copy number extrachromosomal DNA, thereby eliminating the potential effects of chromosomal integration.

[0088] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptides from sequences linked to transcriptional regulatory sequences on the vector. The expressed sequences are often, but not necessarily, heterologous to the host cell. Expression vectors may contain additional elements; for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example, in human cells for expression and in prokaryotic hosts for cloning and amplification. An expression vector may be a recombinant vector.

[0089] As used herein, the term "expression" refers to the cellular processes involved in the production of RNA and proteins, and optionally secreted proteins, including, for example, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, as applicable.

[0090] As used herein, the phrase "expression product" includes RNA transcribed from a gene (e.g., a transgene) and polypeptides obtained by translation of mRNA transcribed from a gene.

[0091] As used herein, the term "gene" refers to a nucleic acid sequence that is transcribed (from DNA) into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequence and 3'UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0092] As used herein, the term gene delivery refers to the process by which foreign DNA is transferred into host cells for gene therapy applications.

[0093] As used herein, the term gene editing molecule refers to one or more of a protein or a nucleic acid encoding a protein, wherein the protein is selected from the group including a transportase, a nuclease, an integrase, a guide RNA (gRNA), a guide DNA, a ribonucleoprotein (RNP), or an activator RNA. A nuclease gene editing molecule is a protein with nuclease activity, and non-limiting examples include CRISPR protein (Cas), CRISPR-associated protein 9 (Cas9), type IIS restriction enzymes, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs), meganucleases, engineered site-specific nucleases, or inactivated CAS for CRISPRi or CRISPRa systems. A gene editing molecule may also include a DNA binding domain and a nuclease. In certain embodiments, the gene editing molecule includes a DNA binding domain and a nuclease. In certain embodiments, the DNA binding domain includes a guide RNA. In certain embodiments, the DNA binding domain includes the DNA binding domain of a TALEN. In certain embodiments, at least one gene editing molecule comprises one or more transposable elements. In certain embodiments, the one or more transposable elements comprise circular DNA. In certain embodiments, the one or more transposable elements comprise a plasmid vector or a minicircle DNA vector. In certain embodiments, the DNA-binding domain comprises a DNA-binding domain of a zinc finger nuclease. In certain embodiments, at least one gene editing molecule comprises one or more transposable elements. In certain embodiments, the one or more transposable elements comprise linear DNA. Linear recombinant DNA sequences encoding transposons and non-native DNA sequences may be produced in vitro. The linear recombinant DNA sequences and non-native DNA sequences of the present disclosure may be the product of restriction digestion of circular DNA. In certain embodiments, the circular DNA is a plasmid vector or a minicircle DNA vector. The linear recombinant DNA sequences and non-native DNA sequences of the present disclosure may be the product of polymerase chain reaction (PCR).The linear recombinant DNA sequences and non-naturally occurring DNA sequences of the present disclosure may be double-stranded DOGGYBONE™ DNA sequences. The DOGGYBONE™ DNA sequences of the present disclosure may be generated by an enzymatic process that encodes only the antigen expression cassette, including the antigen, promoter, polyA tail, and telomere end.

[0094] As used herein, the term gene editing function refers to the insertion, deletion, or loss-of-function or gain-of-function replacement of DNA at a specific site in a genome. The insertion, deletion, or replacement of DNA at a specific site can be achieved, for example, by homology-directed repair (HDR) or non-homologous end joining (NHEJ), or single-base change editing. In some embodiments, a donor template is used, for example, for HDR, such that the desired sequence in the donor template is inserted into the genome by a homologous recombination event. In one embodiment, the donor template or repair template includes two homologous arms (e.g., a 5' homologous arm and a 3' homologous arm) flanking the donor sequence containing the desired mutation or insertion in the nucleic acid sequence to be introduced into the host genome. The 5' homologous arm and the 3' homologous arm are substantially homologous to the genomic sequence of the target gene at the site of endonuclease-mediated cleavage. The 3' homology arm is generally immediately downstream of a protospacer adjacent motif (PAM) site that undergoes endonuclease cleavage (eg, double-stranded DNA break), or in some embodiments, nicks the DNA.

[0095] As used herein, the term "gene editing system" refers to the minimum components required to cause genome editing in cells.For example, zinc finger nuclease or TALEN system may only require the expression of endonuclease fused to the nucleic acid complementary to the sequence of target gene, while in CRISPR / Cas gene editing system, the minimum components may require, for example, Cas endonuclease and guide RNA.Gene editing system can be coded on a single ceDNA vector or multiple vectors as desired.Those skilled in the art will easily understand the components required for gene editing system.

[0096] As used herein, the term base editing moiety refers to an enzyme or enzyme system that can change a single base in a sequence, for example, an enzyme or enzyme system that can change a cytosine / guanine nucleotide pair "G / C" to an adenine and thymine "T" / uridine "U" nucleotide pair (A / T,U) (e.g., as described in Shevidi et al. Dev Dyn 31 (2017) PMID:28857338, Kyoungmi et al. Nature Biotechnology 35:435-437 (2017), the contents of each of which are incorporated herein by reference in their entireties) or an adenine / thymine "A / T" nucleotide pair to a guanine / cytosine "G / C" nucleotide pair (e.g., as described in Gaudelli et al. Nature (2017), in press doi:10.1038 / nature24644, the contents of which are incorporated herein by reference in their entireties).

[0097] As used herein, the term "genomic safe harbor gene" or "safe harbor gene" refers to a gene or locus into which a nucleic acid sequence can be inserted such that the sequence can integrate and function in a predictable manner (e.g., express a protein of interest) without significantly adversely affecting endogenous gene activity or the promotion of cancer. In some embodiments, a safe harbor gene is also a locus or gene where the inserted nucleic acid sequence can be expressed more efficiently and at higher levels than at a non-safe harbor site.

[0098] As used herein, the term "gene delivery" refers to the process by which foreign DNA is transferred into host cells for gene therapy applications.

[0099] As used herein, the term "CRISPR" refers to Clustered Regularly Interspaced Short Palindromic Repeats, a feature of the bacterial defense system that forms the basis of CRISPR-Cas9 genome editing technology.

[0100] As used herein, the term "homologous recombination" refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. Homologous recombination also produces new combinations of DNA sequences. These new DNA combinations represent genetic variations. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of viruses.

[0101] As used herein, the terms "correction," "genome editing," and "restoration" refer to altering a mutant gene that encodes a truncated protein or no protein at all, resulting in full-length or partial full-length functional protein expression. Correcting or restoring a mutant gene can involve replacing the region of the gene that has the mutation, or replacing a copy of the gene that has the mutation with a repair mechanism such as homology-directed repair (HDR). Correcting or restoring a mutant gene can also include repairing a frameshift mutation that causes a premature stop codon, an aberrant splice acceptor site, or an aberrant splice donor site by generating a double-strand break in the gene that is repaired using non-homologous end joining (NHEJ). NHEJ may add or delete at least one base pair during repair, thereby restoring the proper reading frame and removing the premature stop codon. Correcting or restoring a mutant gene can also involve disrupting the aberrant splice acceptor site or splice donor sequence. Correcting or restoring a mutated gene can also involve deleting a non-essential gene segment by the simultaneous action of two nucleases on the same DNA strand to remove the DNA between two nuclease target sites and repair the DNA break by NHEJ, thereby restoring the proper reading frame.

[0102] As used herein, the phrase "nonhomologous end joining (NHEJ) pathway" refers to a pathway that repairs double-stranded DNA breaks by directly ligating the broken ends without the need for a homologous template. Template-independent religation of DNA ends by NHEJ is a stochastic, error-prone repair process that introduces random microinsertions and microdeletions (indels) at the DNA breakpoint. This method may be used to intentionally disrupt, delete, or alter the reading frame of a target gene sequence. NHEJ typically uses short homologous DNA sequences, called microhomologies, to guide repair. These microhomologies are often present in single-stranded overhangs at the end of the double-stranded break. If the overhangs are perfectly compatible, NHEJ typically repairs the break accurately; however, imprecise repair, resulting in nucleotide loss, can also occur. However, as used herein, the term "NHEJ pathway" refers to the more common scenario in which the overhangs are incompatible, in which NHEJ is initiated after the action of a nuclease, such as Cas9 or other nucleases, breaks the double-stranded DNA. In the CRISPR / CAS system, NHEJ can be targeted by using a single guide RNA sequence.

[0103] As used herein, the term " site-specific nuclease " or " sequence-specific nuclease " refers to an enzyme that can specifically recognize and cut DNA sequences. Site-specific nucleases can be engineered. Examples of engineered site-specific nucleases include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR / Cas-based systems that use various natural and non-natural Cas enzymes.

[0104] As used herein, the phrase "genetic disease" refers to a disease caused, in part or in whole, directly or indirectly, by one or more abnormalities in the genome, particularly a condition that is present from birth and can be treated by the single-stranded (ssDNA) molecules described herein. The abnormality can be a mutation, insertion, or deletion. The abnormality can affect the coding sequence of a gene or its regulatory sequence. Genetic diseases include phenylketonuria (PKU), sickle cell anemia, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis, Huntington's bile duct disease, familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited disorders of liver metabolism, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, and mucopolysaccharide storage diseases (e.g., Hurler syndrome (MPS type I), Scheie syndrome (MPS type IS), Hurler-Scheie syndrome (MPS type IHS), Hunter syndrome (MPS type II), Sanfilippo types A, B, C, and D (MPS type III)). A, B, C, and D), Morquio syndrome types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPS These may include, but are not limited to, Niemann-Pick disease types A / B, C1 and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III and IV, sialidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II and III, Fabry disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipid galactosialidosis.Genetic disorders also include amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital amaurosis (LCA, e.g., LCA10 [CEP290]), Stargardt macular dystrophy (ABCA4), or cathepsin A deficiency.

[0105] As used herein, the term "treatment" or "treatment" and / or "treatment" refers to suppressing, substantially inhibiting, slowing down, or reversing the progression of a condition, substantially improving the clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms of a condition, or obtaining beneficial or desired clinical results. Treating also refers to achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting the onset of symptoms characteristic of the disorder being treated; (c) limiting the worsening of symptoms characteristic of the disorder being treated; (d) limiting the recurrence of the disorder in patients who previously had the disorder; and (e) limiting the recurrence of symptoms in patients who did not previously exhibit symptoms of the disorder. In some embodiments, treatment involves gene editing. In some embodiments, treatment involves gene therapy.

[0106] Beneficial or desirable clinical results, such as pharmacological and / or physiological effects, include, but are not limited to, preventing a disease, disorder, or condition from occurring in a subject who would be susceptible to the disease, disorder, or condition, but who has not yet experienced or exhibited symptoms of the disease (prophylactic treatment), alleviating the disease, disorder, or condition, reducing the severity of the disease, disorder, or condition, stabilizing (i.e., not worsening) the disease, disorder, or condition, preventing the spread of the disease, disorder, or condition, delaying or slowing the progression of the disease, disorder, or condition, ameliorating or alleviating the disease, disorder, or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0107] As used herein, the terms "increase," "enhance," and "elevate" (and similar terms) generally refer to the act of increasing, either directly or indirectly, a concentration, level, function, activity, or behavior compared to natural, expected, or average conditions, or compared to a control condition.

[0108] As used herein, the terms "suppress," "reduce," "interfere," "inhibit," and / or "reduce" (and similar terms) generally refer to the act of decreasing, either directly or indirectly, a concentration, level, function, activity, or behavior compared to natural, expected, or average conditions, or compared to a control condition.

[0109] As used herein, the terms synthetic AAV vector, single-stranded (ss) synthetic AAV vector, and synthetic production of AAV vector refer to AAV vectors and methods for their synthetic production in a cell-free environment.

[0110] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, processes, and their respective components that are essential to the process, method, or composition, but which embrace the inclusion of unspecified elements, whether essential or not. The use of "comprising" indicates inclusion rather than limitation.

[0111] The term "consisting of" refers to compositions, methods, processes, and their respective components described herein, excluding any element not recited in the description of the embodiment.

[0112] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic novel or functional characteristics of that embodiment of the present disclosure.

[0113] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure, etc. Similarly, the term "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below.

[0114] The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to denote a non-limiting example. As such, the abbreviation "eg" is synonymous with the term "for example."

[0115] Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±1%. The present disclosure is further illustrated in detail by the following examples, but the scope of the present disclosure should not be limited thereto.

[0116] Grouping of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limiting. Each group member can be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification herein includes the modified group and, as such, is deemed to satisfy the written description of all Markush groups used in the appended claims.

[0117] In some embodiments of any of its aspects, the disclosure described herein does not relate to processes for cloning humans, processes for modifying the germline genetic identity of humans, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals that are likely to cause suffering to humans or animals without providing any substantial medical benefit to them, and similarly processes for modifying the genetic identity of animals resulting from such processes.

[0118] Other terms are defined herein within the description of various aspects of the disclosure.

[0119] All patents and other publications, including literature references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure or for any other reason. All statements as to dates or representations regarding the contents of these documents are based on information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0120] The description of embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples thereof, the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or may perform the functions substantially simultaneously. The teachings of the present disclosure provided herein can be applied to other procedures or methods where appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, where necessary, to employ compositions, functions, and concepts from the above references and applications to provide still further embodiments of the present disclosure. Furthermore, due to considerations of biological functional equivalence, some changes can be made in protein structure without affecting biological or chemical activity in kind or amount. These and other changes can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0121] Particular elements of any of the foregoing embodiments can be combined with or substituted for elements in other embodiments. Furthermore, while advantages associated with particular embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages, to fall within the scope of the present disclosure.

[0122] The techniques described herein are further illustrated by the following examples, which should not be construed as further limiting in any way. It should be understood that the present disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein, and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.

[0123] II. Single-stranded (ss) DNA molecule As described herein, the present disclosure relates to synthetic single-stranded (ssDNA) molecules. According to some aspects, the present disclosure provides single-stranded deoxyribonucleic acid (ssDNA) molecules comprising at least one nucleic acid sequence of interest adjacent to at least one stem-loop structure at the 3' end. According to some embodiments, the ssDNA molecule further comprises a 5' end comprising at least one stem-loop structure.

[0124] In some embodiments, the ssDNA molecules described herein are linear single-stranded DNA molecules that are completely single-stranded along their entire length (i.e., contain no double-stranded regions).

[0125] A.3' end As described herein, in some embodiments, the present disclosure provides ssDNA molecules comprising at least one nucleic acid sequence of interest adjacent to at least one stem-loop structure at the 3' end. As described herein, the stem structure may include a partial DNA duplex (e.g., with a free 3'-OH group) for priming replication or transcription. The partial DNA duplex functions, in part, to hold the stem-loop structure together.

[0126] According to some embodiments, the partial DNA double-stranded strand comprises 4 to 500 nucleotides, for example, 4 to 10 nucleotides, 4 to 25 nucleotides, 4 to 50 nucleotides, 4 to 100 nucleotides, 4 to 200 nucleotides, 4 to 300 nucleotides, 4 to 400 nucleotides, 20 to 25 nucleotides, 20 to 50 nucleotides, 20 to 100 nucleotides, 20 to 200 nucleotides, 20 to 300 nucleotides, 20 to 400 nucleotides, 20 to 500 nucleotides, 50 to 100 nucleotides, 50 to 200 nucleotides, 50 to 300 nucleotides, or 50 to 400 nucleotides. , 50 to 500 nucleotides, 150 to 200 nucleotides, 150 to 300 nucleotides, 150 to 400 nucleotides, 150 to 500 nucleotides, 200 to 300 nucleotides, 200 to 400 nucleotides, 200 to 500 nucleotides, 250 to 300 nucleotides, 250 to 400 nucleotides, 250 to 500 nucleotides, 300 to 400 nucleotides, 300 to 500 nucleotides, 350 to 400 nucleotides, 350 to 500 nucleotides, 400 to 500 nucleotides, or 450 to 500 nucleotides, and at least one loop on the 3' end. According to some embodiments, the DNA duplex comprises at least 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides and comprises at least one loop at the 3' end.

[0127] According to some embodiments, the loop structure at the 3' end has a minimum length of 3 to 500 nucleotides, for example, 3 to 450 nucleotides, 3 to 400 nucleotides, 3 to 350 nucleotides, 3 to 300 nucleotides, 3 to 250 nucleotides, 3 to 200 nucleotides, 3 to 150 nucleotides, 3 to 100 nucleotides, 3 to 90 nucleotides, 3 to 80 nucleotides, 3 to 70 nucleotides, 3 to 60 nucleotides, 3 to 50 nucleotides, 3 to 40 nucleotides, 3 to 30 nucleotides, 3 to 20 nucleotides, 3 to 10 nucleotides, 3 to 5 nucleotides, 10 to 450 nucleotides, 10 to 400 nucleotides, 10 to 350 nucleotides, 10 to 300 nucleotides, 10 to 250 nucleotides, 10 to 200 nucleotides, 10 to 150 nucleotides, 10 to 100 nucleotides, 10 to 90 nucleotides, 10 to 80 nucleotides, 10 to 70 nucleotides, 10 to 60 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 30 nucleotides, 10 to 20 nucleotides, 50 to 450 nucleotides, 50 to 400 nucleotides, 50 to 350 nucleotides, 50 to 300 nucleotides tide, 50-250 nucleotides, 50-200 nucleotides, 50-150 nucleotides, 50-100 nucleotides, 50-90 nucleotides, 50-80 nucleotides, 50-70 nucleotides, 50-60 nucleotides, 100-450 nucleotides, 100-400 nucleotides, 100-350 nucleotides, 100-300 nucleotides, 100-250 nucleotides, 100-200 nucleotides, 150-450 nucleotides, 150-400 nucleotides, 150-350 nucleotides, 150-300 nucleotides, 150-2 and 50 nucleotides, 150-200 nucleotides, 200-450 nucleotides, 200-400 nucleotides, 200-350 nucleotides, 200-300 nucleotides, 200-250 nucleotides, 250-450 nucleotides, 250-400 nucleotides, 250-350 nucleotides, 250-300 nucleotides, 300-450 nucleotides, 300-400 nucleotides, 300-350 nucleotides, 350-450 nucleotides, 350-400 nucleotides, or 400-450 nucleotides.

[0128] According to some embodiments, the stem portion of the stem-loop is 4 to 500 nucleotides in length, and the loop portion of the stem-loop is 3 to 500 nucleotides in length. According to some embodiments, the stem portion of the stem-loop is 4 to 50 nucleotides in length, and the loop portion of the stem-loop is 3 to 50 nucleotides in length. According to some embodiments, the stem portion of the stem-loop is 4 to 20 nucleotides in length, and the loop portion of the stem-loop is 3 to 20 nucleotides in length. According to some embodiments, the stem portion of the stem-loop is 4 to 10 nucleotides in length, and the loop portion of the stem-loop is 3 to 10 nucleotides in length.

[0129] In some embodiments, the loop further comprises one or more nucleic acids or is used to stabilize the termini. In other embodiments, the loop further comprises one or more nucleic acids that may be used in therapeutic methods. In other embodiments, the loop further comprises one or more nucleic acids that may be used in diagnostic methods. In other embodiments, the loop further comprises one or more nucleic acids that may be used for research purposes.

[0130] According to some embodiments, the minimum nucleic acid structure required at the 3' end of the ssDNA is any structure that loops back on itself, i.e., a hairpin structure. However, it should be understood that various structures are contemplated at the 3' end, as long as there is at least one stem and one loop. For example, in some embodiments, the ssDNA described herein may contain at least one stem-loop structure at the 3' end. In some embodiments, the ssDNA may contain at least two stem-loop structures at the 3' end. In some embodiments, the ssDNA may contain at least three stem-loop structures at the 3' end. In some embodiments, the ssDNA may contain at least four stem-loop structures at the 3' end. In some embodiments, the ssDNA may contain at least five stem-loop structures at the 3' end.

[0131] According to some embodiments, the 3'-terminal nucleotides form a cruciform DNA structure. A DNA cruciform structure can be formed when both strands form a stem-loop structure at the same position within the molecule and contain a four-way junction and two closed hairpin-shaped points.

[0132] According to some embodiments, the 3'-terminal nucleotides form a hairpin DNA structure, wherein the hairpin loop structure in the nucleic acid comprises a base-paired stem structure and a loop sequence having unpaired or non-Watson-Crick paired nucleotides.

[0133] According to some embodiments, the 3' terminal nucleotides form a hammerhead DNA structure composed of three base-paired helices separated by short linkers of conserved sequence.

[0134] According to some embodiments, the 3'-terminal nucleotides form a quadruplex DNA structure. A G-quadruplex is a four-stranded DNA secondary structure (G4) formed from certain guanine-rich sequences.

[0135] According to some embodiments, the 3' terminal nucleotides form a bulge DNA structure.

[0136] According to some embodiments, the 3' terminal nucleotides form a multi-branched loop.

[0137] According to some embodiments, the 3'-terminal nucleotides do not form two stem-loop structures. In one embodiment, the 3'-terminal nucleotides do not form an AAV ITR structure.

[0138] According to some embodiments, at least one stem-loop structure at the 3' end does not include the A, A', D, and D' regions that would be present in a wild-type AAV ITR.

[0139] According to some embodiments, at least one stem-loop structure at the 3' end does not include regions A, A', B, B', C, C', D, and D' that would be present in a wild-type AAV ITR.

[0140] According to some embodiments, at least one stem-loop structure at the 3' end does not contain a rep-binding element (RBE) that would be present in a wild-type ITR. According to some embodiments, at least one stem-loop structure at the 3' end does not contain a terminal resolving site (trs) that would be present in a wild-type ITR. According to some embodiments, at least one stem-loop structure at the 3' end lacks any viral capsid protein-encoding sequence. According to some embodiments, the ssDNA molecule does not contain any virus-derived sequences.

[0141] According to some embodiments, the stem structure at the 3' end comprises one or more nucleotides modified to be exonuclease resistant. According to some embodiments, the stem structure at the 3' end comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or twenty or more nucleotides modified to be exonuclease resistant.

[0142] According to some embodiments, the stem structure at the 3'-end comprises one or more phosphorothioate-modified nucleotides. According to some embodiments, the stem structure at the 3'-end comprises about 4 to about 10, e.g., about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 to about 10, or about 9 to about 10, phosphorothioate-modified nucleotides. According to some embodiments, the stem structure comprises more than 10 phosphorothioate-modified nucleotides.

[0143] According to some embodiments, the phosphorothioate modified nucleotides are located adjacent to one another.

[0144] According to some embodiments, one or more phosphorothioate-modified nucleotides at the 3'-end are resistant to exonuclease degradation. Boranophosphate-modified DNA is also resistant to nuclease degradation and can be considered an alternative to phosphorothioate modification.

[0145] According to a further embodiment, the stem structure may comprise at least one functional moiety. In one embodiment, the at least one functional moiety is an aptamer sequence. In a further embodiment, the aptamer sequence has high binding affinity to a nuclear-localized protein.

[0146] According to some embodiments, the nucleotides in the loop are chemically modified with functional groups to alter their properties.

[0147] According to some embodiments, the loop further comprises one or more aptamers, which according to some embodiments are identified from the publicly available Apta-index database of aptamers (aptagen.com / apta-index).

[0148] According to some embodiments, the loop further comprises one or more synthetic ribozymes.

[0149] According to some embodiments, the loop further comprises one or more antisense oligonucleotides (ASOs).

[0150] According to some embodiments, the loop further comprises one or more short interfering RNAs (siRNAs).

[0151] According to some embodiments, the loop further comprises one or more antiviral nucleoside analogs (ANAs).

[0152] According to some embodiments, the loop further comprises one or more triplex-forming oligonucleotides.

[0153] According to some embodiments, the loop further comprises one or more gRNAs or gDNAs.

[0154] According to some embodiments, the loop further comprises one or more molecular probes, for example, nucleic acid-based fluorescent probes.

[0155] According to some embodiments, "click" azide-alkyne cycloaddition (Kolb et al., Angew. Chem. Int. Ed. Engl. 2001, 40, 2004-2021) is used to modify the nucleotides in the loop. Click chemistry was developed to link organic molecules together under mild conditions in the presence of various functional groups. Most click-mediated modifications are performed on nitrogenous bases by introducing novel base analogs, attaching fluorophores or isotopic elements for molecular imaging, forming interstrand linkages between oligonucleotides, and for molecular bioconjugation. The best examples of click chemistry are the CuAAC reaction, independently discovered by Sharpless and Meldal (Angew. Chem., Int. Ed. 2002, 41, 2596-2599) and the Cu-based [3+2] azide-alkyne cycloaddition reaction by Huisgen (Angew. Chem., Int. Ed. 1963, 2, 633-645). I This is the catalytic version.

[0156] According to some embodiments, introduction of a reactive amino or thiol group into a synthetic oligonucleotide provides, for example, an acceptor for subsequent chemiluminescent labeling.

[0157] According to some embodiments, the stem-loop structure may comprise alternative or modified nucleotides, including, but not limited to, ribonucleic acid (RNA), peptide-nucleic acid (PNA), locked nucleic acid (LNA). According to some embodiments, the loop portion of the stem-loop structure may comprise a chemical structure that does not include a nucleic acid.

[0158] According to some embodiments, the 3' end of the ssDNA molecule is single-stranded and does not contain any double-stranded region. As described in Example 5 and Figures 35-38, the completely single-stranded ssDNA molecule can induce transgene expression. In some embodiments, the completely single-stranded ssDNA molecule without a double-stranded region is at least 200 nucleotides long, at least 300 nucleotides long, at least 400 nucleotides long, at least 500 nucleotides long, at least 600 nucleotides long, at least 700 nucleotides long, at least 800 nucleotides long, at least 900 nucleotides long, at least 1000 nucleotides long, at least 1500 nucleotides long, at least 2000 nucleotides long, at least 2500 nucleotides long, at least 30 ... It may be 3500 nucleotides in length, at least 4000 nucleotides in length, at least 4500 nucleotides in length, at least 5000 nucleotides in length, at least 5500 nucleotides in length, at least 6000 nucleotides in length, at least 6500 nucleotides in length, at least 7000 nucleotides in length, at least 7500 nucleotides in length, at least 8000 nucleotides in length, at least 8500 nucleotides in length, at least 9000 nucleotides in length, at least 9500 nucleotides in length, or at least 10,000 nucleotides in length.

[0159] B.5' end As described herein, according to some aspects, the present disclosure provides ssDNA molecules comprising at least one nucleic acid sequence of interest adjacent to at least one stem-loop structure at the 3' end, as described in detail above. According to some embodiments, the ssDNA molecule further comprises a 5' end comprising at least one stem-loop structure. According to some embodiments, the DNA structure at the 5' end is the same as the DNA structure at the 3' end. According to some embodiments, the DNA structure at the 5' end is different from the DNA structure at the 3' end.

[0160] For example, in some embodiments, the ssDNA described herein may comprise at least one stem-loop structure at its 5' end. According to some embodiments, the ssDNA may comprise at least two stem-loop structures at its 5' end. According to some embodiments, the ssDNA may comprise at least three stem-loop structures at its 5' end. According to some embodiments, the ssDNA may comprise at least four stem-loop structures at its 5' end. According to some embodiments, the ssDNA may comprise at least five stem-loop structures at its 5' end.

[0161] According to some embodiments, the 5' terminal nucleotides form a cruciform DNA structure.

[0162] According to some embodiments, the 5' terminal nucleotides form a hairpin structure.

[0163] According to some embodiments, the 5' terminal nucleotide forms a hammerhead structure.

[0164] According to some embodiments, the 5' terminal nucleotides form a quadruplex structure.

[0165] According to some embodiments, the 5' terminal nucleotide forms a bulge structure.

[0166] According to some embodiments, the 5' terminal nucleotides form a multi-branched loop.

[0167] According to some embodiments, the 5'-terminal nucleotides do not form two stem-loop structures. In one embodiment, the 5'-terminal nucleotides do not form an AAV ITR structure.

[0168] According to some embodiments, at least one stem-loop structure at the 5' end does not include the A, A', D, and D' regions that would be present in a wild-type AAV ITR.

[0169] According to some embodiments, at least one stem-loop structure at the 5' end does not include regions A, A', B, B', C, C', D, and D' that would be present in a wild-type AAV ITR.

[0170] According to some embodiments, at least one stem-loop structure at the 5'-end does not contain a rep-binding element (RBE) that would be present in a wild-type ITR. According to some embodiments, at least one stem-loop structure at the 5'-end does not contain a terminal resolving site (trs) that would be present in a wild-type ITR. According to some embodiments, at least one stem-loop structure at the 5'-end lacks any viral capsid protein-encoding sequence. According to some embodiments, the ssDNA molecule does not contain any virus-derived sequences.

[0171] According to some embodiments, the stem structure at the 5' end comprises one or more nucleotides modified to be exonuclease resistant. According to some embodiments, the stem structure at the 5' end comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or twenty or more nucleotides modified to be exonuclease resistant.

[0172] According to some embodiments, the stem structure comprises one or more phosphorothioate-modified nucleotides. According to some embodiments, the stem structure comprises about 4 to about 10, e.g., about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 to about 10, or about 9 to about 10 phosphorothioate-modified nucleotides. According to some embodiments, the stem structure comprises more than 10 phosphorothioate-modified nucleotides.

[0173] According to some embodiments, the phosphorothioate modified nucleotides are located adjacent to one another. According to some embodiments, the one or more phosphorothioate modified nucleotides are resistant to exonuclease degradation.

[0174] In some embodiments, the loop further comprises one or more nucleic acids or is used to stabilize the termini. In other embodiments, the loop further comprises one or more nucleic acids that may be used in therapeutic methods. In other embodiments, the loop further comprises one or more nucleic acids that may be used in diagnostic methods. In other embodiments, the loop further comprises one or more nucleic acids that may be used for research purposes.

[0175] According to some embodiments, the nucleotides in the loop are chemically modified with functional groups to alter their properties.

[0176] According to some embodiments, the loop further comprises one or more aptamers, which according to some embodiments are identified from the publicly available Apta-index database of aptamers (aptagen.com / apta-index).

[0177] According to some embodiments, the loop further comprises one or more synthetic ribozymes.

[0178] According to some embodiments, the loop further comprises one or more antisense oligonucleotides (ASOs).

[0179] According to some embodiments, the loop further comprises one or more short interfering RNAs (siRNAs).

[0180] According to some embodiments, the loop further comprises one or more antiviral nucleoside analogs (ANAs).

[0181] According to some embodiments, the loop further comprises one or more triplex-forming oligonucleotides.

[0182] According to some embodiments, the loop further comprises one or more gRNAs or gDNAs.

[0183] According to some embodiments, the loop further comprises one or more molecular probes, for example, nucleic acid-based fluorescent probes.

[0184] According to some embodiments, "click" azide-alkyne cycloaddition (Kolb et al., Angew. Chem. Int. Ed. Engl. 2001, 40, 2004-2021) is used to modify the nucleotides in the loop. Click chemistry was developed to link organic molecules together under mild conditions in the presence of various functional groups. Most click-mediated modifications are performed on nitrogenous bases by introducing novel base analogs, attaching fluorophores or isotopic elements for molecular imaging, forming interstrand linkages between oligonucleotides, and for molecular bioconjugation. The best examples of click chemistry are the CuAAC reaction, independently discovered by Sharpless and Meldal (Angew. Chem., Int. Ed. 2002, 41, 2596-2599) and the Cu-based [3+2] azide-alkyne cycloaddition reaction by Huisgen (Angew. Chem., Int. Ed. 1963, 2, 633-645). I This is the catalytic version.

[0185] According to some embodiments, introduction of a reactive amino or thiol group into a synthetic oligonucleotide provides, for example, an acceptor for subsequent chemiluminescent labeling.

[0186] According to some embodiments, the stem-loop structure may comprise alternative or modified nucleotides, including, but not limited to, ribonucleic acid (RNA), peptide-nucleic acid (PNA), locked nucleic acid (LNA). According to some embodiments, the loop portion of the stem-loop structure may comprise a chemical structure that does not include a nucleic acid.

[0187] According to some embodiments, the 5' end of the ssDNA molecule is single-stranded and does not contain any double-stranded regions. As described in Example 5 and Figures 35-38, the completely single-stranded ssDNA molecule can induce transgene expression.

[0188] C. Nucleic Acid Sequence of Interest The single-stranded DNA (ssDNA) molecules described herein do not have the packaging constraints imposed by the limited space within the viral capsid, allowing for the insertion of one or more genetic elements, such as single-stranded enhancers, single-stranded introns, single-stranded posttranscriptional regulatory elements, single-stranded polyadenylation signals, and single-stranded regulatory switches, large transgenes, multiple transgenes, etc.

[0189] According to some embodiments, the nucleic acid sequence of interest further comprises at least one single-stranded promoter linked to the at least one nucleic acid sequence of interest.

[0190] In other aspects of the present disclosure, single-stranded transgene cassettes are used in gene editing applications, as described in more detail herein.

[0191] According to some embodiments, the nucleic acid sequence of interest (also referred to herein as a transgene) encodes a gene encoding a protein that is absent, inactive, or insufficiently active in a recipient subject, or a protein that has a desired biological or therapeutic effect. The transgene can encode a gene product that can function to correct the expression of a defective gene or transcript. In principle, the expression cassette can include any gene that encodes a protein, polypeptide, or RNA that is reduced or absent due to a mutation, or that provides a therapeutic effect if overexpression is considered within the scope of the present disclosure.

[0192] The nucleic acid sequence of interest can include any sequence useful for treating a disease or disorder in a subject. ssDNA molecules can be used to deliver and express any gene of interest to a subject, including, but not limited to, exogenous genes and nucleotide sequences, including polypeptide-encoding or non-coding nucleic acids (e.g., RNAi, miR, etc.), as well as viral sequences in the subject's genome, such as HIV viral sequences. In some embodiments, the ssDNA molecules disclosed herein are used for therapeutic purposes (e.g., for medical, diagnostic, or veterinary use). In certain embodiments, the ssDNA molecules are useful for expressing any gene of interest in a subject, including one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, or RNAs (encoding or non-encoding, e.g., siRNAs, shRNAs, microRNAs, mRNAs, or gRNAs, and their antisense counterparts (e.g., antagomirs)), antibodies, antigen-binding fragments, or any combination thereof.

[0193] The sequence can be codon-optimized for the target host cell. As used herein, the term "codon-optimized" or "codon optimization" refers to the process of modifying a nucleic acid sequence for enhanced expression in the cells of a target vertebrate, such as a mouse or human, by replacing at least one, two, or a significant number of codons in a native sequence (e.g., a prokaryotic sequence) with codons more frequently or most frequently used in the genes of that vertebrate. Various species exhibit specific biases for specific codons of specific amino acids. Typically, codon optimization does not alter the amino acid sequence of the original translated protein. Optimized codons can be determined, for example, using Aptagen's GENEFORGE® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or another publicly available database.

[0194] In some embodiments, the transgene expressed by the ssDNA molecule is a therapeutic gene, hi some embodiments, the therapeutic gene is an antibody, or antibody fragment, or antigen-binding fragment thereof, such as a neutralizing antibody or antibody fragment.

[0195] Specifically, a therapeutic gene is one or more therapeutic agents, including, but not limited to, proteins, polypeptides, peptides, enzymes, antibodies, antigen-binding fragments, and variants and / or active fragments thereof, for use in treating, preventing, and / or ameliorating one or more symptoms of a disease, dysfunction, injury, and / or disorder. Exemplary therapeutic genes are described herein in the section entitled "Methods of Treatment."

[0196] According to any of the above aspects and embodiments, the ssDNA molecule is synthetically produced.

[0197] According to any of the above aspects and embodiments, the ssDNA molecule is devoid of any viral capsid protein coding sequence.

[0198] According to any of the above embodiments, the DNA is a peptide nucleic acid (PNA), which is a synthetic mimic of DNA.

[0199] III. Single-stranded synthetic AAV vector As described herein, the present disclosure relates to a single-stranded (ssDNA) molecule. In some embodiments, the ssDNA molecule is, for example, a synthetic AAV vector, for example, a single-stranded (ss) synthetic AAV vector generated from double-stranded closed-end DNA containing phosphorothioate (PS) bonds. PS bonds replace non-bridging oxygen in the phosphate backbone of oligonucleotides with sulfur atoms. Advantageously, this modification makes internucleotide bonds resistant to nuclease degradation and provides precision for exonuclease targeting.

[0200] In some aspects, the present disclosure provides a single-stranded transgene cassette comprising at least one single-stranded transgene and at least one inverted terminal repeat (ITR) comprising one or more phosphorothioate-modified nucleotides. According to some embodiments, the ssDNA molecule comprises a first ITR and an optional second ITR, wherein at least one of the first ITR and the optional second ITR comprises one or more phosphorothioate-modified nucleotides. In further embodiments, the ssDNA molecule comprises a 3'-end fragment comprising a terminal resolving site (trs) sequence.

[0201] According to some aspects, the present disclosure provides an isolated linear single-stranded DNA (ssDNA) molecule comprising a single-stranded transgene cassette comprising at least one single-stranded transgene and a first inverted terminal repeat (ITR) and a second ITR each flanking the at least one single-stranded transgene cassette, wherein at least one of the first ITR and the second ITR comprises one or more phosphorothioate-modified nucleotides.

[0202] As described in more detail herein, ssDNA molecules are synthetically generated in vitro from dsDNA containing phosphorothioate (PS) linkages ("starting material") by removing one DNA strand from specific nicking sites at the PS linkage sites of the dsDNA. According to further embodiments, the ssDNA molecules are synthetically generated in vitro in a cell-free environment.

[0203] According to some embodiments, it is a feature of the present disclosure that the 3'-terminal portion of the double-stranded DNA molecule ("starting material") comprises a nickase recognition sequence. In one embodiment, the 3'-terminal portion of the dsDNA molecule comprises the sequence 5'-CCAA-3'. In some embodiments, the 3'-terminal portion of the dsDNA molecule comprises any one or more of the sequences shown in Table 1 below. Furthermore, because these are unique sequences after double-stranded ceDNA with the specific engineered nick sites shown in the table is nicked by a nicking endonuclease, the resulting ssDNA molecule also comprises any one or more of the sequences shown in the table below in its 3'-terminal fragment. [Table 1]

[0204] According to some embodiments, the 3'-terminal fragment of the ssDNA molecule comprises a terminal residue that is hydroxylated (-OH) to allow polymerase activity upon transport of the ssDNA to the nucleus of a host cell where it is converted into expressible renatured dsDNA.

[0205] According to some embodiments, the ssDNA molecule comprises a 3' end fragment that includes a terminal resolving site (trs) sequence.

[0206] A key finding of the present disclosure is that the ssDNA molecules described herein can be transported across the nuclear membrane from the cytoplasm into the nucleus of a host cell and accessed by a host cell DNA polymerase to generate double-stranded DNA (regenerated dsDNA) for expression of a transgene in the host cell. Thus, in some embodiments, it is important that the hydroxylated (-OH) terminal residue in the ssDNA molecule is responsive to DNA polymerase activity in the nucleus of the host cell. According to further embodiments, the DNA polymerase generates the dsDNA molecule.

[0207] Importantly, ssDNA molecules do not activate or only minimally activate innate immune pathways in host cells. As used herein, the term "innate immune response" refers to a cellular pathway that responds to pathogen-associated molecular patterns and activates a defensive response via RIG-I-like receptors, Toll-like receptors, or other pathogen-associated molecular pattern receptors to activate interferon, NF-kappa-B, STATs, IRFs, and other response pathways that protect against pathogen infection. According to some embodiments, the innate immune pathway may be the cGAS / STING pathway, the TLR9 pathway, the inflammasome-mediated pathway, or a combination thereof. Indicators of innate immune response activation include increased expression and / or phosphorylation of IRF family members, increased expression of RIG-I-like receptors, and increased expression of interferons and / or chemokines.

[0208] According to some embodiments, the single-stranded transgene cassette further comprises at least one single-stranded promoter operably linked to the at least one single-stranded transgene, and the dsDNA molecule comprises a regenerated double-stranded expression cassette comprising at least one regenerated double-stranded transgene and at least one double-stranded promoter operably linked to the regenerated double-stranded transgene, controlling the expression of the at least one regenerated double-stranded transgene. The double-stranded expression cassette can be expressed in a host cell, for example, in a host cell in vivo. In some embodiments, the double-stranded expression cassette can express at least one therapeutic protein or fragment thereof.

[0209] In a further embodiment, the single-stranded transgene cassette further comprises one or more genetic elements selected from the group consisting of a single-stranded enhancer, a single-stranded intron, a single-stranded post-transcriptional regulatory element, a single-stranded polyadenylation signal, and a single-stranded regulatory switch.

[0210] In other aspects of the present disclosure, single-stranded transgene cassettes are used in gene editing applications.

[0211] Thus, in some embodiments, the at least one single-stranded transgene cassette is a promoterless transgene cassette, and the dsDNA molecule comprises at least one regenerated promoterless double-stranded transgene. In some embodiments, the at least one regenerated promoterless double-stranded transgene can be inserted into a target locus in the genome of a host cell. In further embodiments, the at least one regenerated promoterless double-stranded transgene can be inserted into a target locus in the genome of a host cell in vivo. In some embodiments, the at least one regenerated promoterless double-stranded transgene can be inserted into a target locus to replace or supplement at least one target gene. In other embodiments, the at least one regenerated promoterless double-stranded transgene can be inserted into a target locus via homologous recombination (HDR) or microhomology-mediated end joining (MMEJ). In other further embodiments, the at least one single-stranded transgene is a single-stranded donor sequence, and the single-stranded transgene cassette further comprises a single-stranded 5' homologous arm and a single-stranded 3' homologous arm adjacent to the single-stranded donor sequence. The single-stranded 5' homologous arm and the single-stranded 3' homologous arm are each about 10 to 2,000 nt in length, e.g., about 100 to 2,000 nt in length, or about 1,000 to 2,000 nt in length, or about 10 to 1,000 nt in length, e.g., about 100 to 1,000 nt in length, or about 10 to 500 nt in length, about 50 to 500 nt in length, or about 100 to 500 nt in length, about 10 to 50 nt in length, about 50 to 500 nt in length, or about 500 to 1,000 nt in length, about 500 to 1,500 nt in length, about 1,500 to 2,000 nt in length, about 2 to 1,000 nt in length, about 2 to 500 nt in length, about 2 to 100 nt in length, or about 2 to 50 nt in length. In some embodiments, at least one regenerating promoterless double-stranded transgene can be inserted into the target locus via non-homologous end joining (NHEJ). In some embodiments, at least one single-stranded transgene is a single-stranded donor sequence, and the single-stranded transgene cassette lacks a single-stranded 5' homology arm and a single-stranded 3' homology arm.In other embodiments, the single-stranded transgene cassette is cleavable and further comprises at least a first single-stranded guide RNA (gRNA) target sequence (TS), at least a first single-stranded protospacer adjacent motif (PAM), at least a second single-stranded gRNA TS, and at least a second single-stranded PAM.

[0212] As described in more detail herein, in some embodiments, the ssDNA molecules described herein are synthetically produced from a dsDNA construct by a method comprising: a) contacting the dsDNA construct with one or more nicking endonucleases that nick one of the single strands of the dsDNA construct at one or more nick sites; and b) contacting the dsDNA construct with an exonuclease capable of removing nucleotides from the nicked strand of the dsDNA construct, thereby producing the ssDNA molecule. A. Double-stranded (ds) terminated DNA (ceDNA)

[0213] In some embodiments, the present disclosure provides double-stranded closed-end DNA (ceDNA) containing phosphorothioate (PS) bonds. As described herein, this modification is advantageously located in the ITR region of the exonuclease-active space, and functions as a lock on the 5' and / or 3' ends, making the internucleotide bond resistant to nuclease degradation and ensuring the accuracy of exonuclease activity. The double-stranded ceDNA described herein is used to produce the ssDNA molecules described herein.

[0214] In one aspect, the present disclosure provides an isolated double-stranded DNA (dsDNA) construct comprising a double-stranded transgene cassette comprising at least one double-stranded transgene, and a first inverted terminal repeat (ITR) and an optional second ITR, each flanking the at least one double-stranded transgene, wherein at least one of the first ITR and the optional second ITR comprises one or more phosphorothioate-modified nucleotides. According to some embodiments, the dsDNA construct comprises a nickase recognition sequence (nick site).

[0215] In one embodiment, the dsDNA construct comprises an end-resolving region (trs) sequence of an AAV ITR containing a nick site. According to some embodiments, the dsDNA construct comprises one or more recognition nucleotide sequences for one or more nicking endonucleases, each independently selected from Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BssSI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, and an isoschizomer of any of the foregoing. According to further embodiments, the one or more recognition nucleotide sequences comprise any one or more of the following sequences shown in Table 2 below: [Table 2]

[0216] According to some embodiments, each of the one or more recognition nucleotide sequences is an engineered sequence. According to further embodiments, each of the one or more recognition nucleotide sequences comprises one or more nick sites for one or more nicking endonucleases.

[0217] According to some embodiments, the one or more nick sites are about 0 to about 20 nucleotides downstream of the end resolving region (trs), e.g., about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream of the end resolving region (trs), or e.g., about 0 to about 15, about 0-10, about 0-5, about 5-15, about 10-20, about 15-20, about 10-20, or about 5-20 nucleotides downstream of the end resolving region (trs). According to some embodiments, only one nick site functions as an exonuclease entry site.

[0218] According to some embodiments, the dsDNA construct comprises one or more recognition nucleotide sequences for Nb.BbvCI or its isoschizomers.

[0219] According to some embodiments, the dsDNA construct comprises a single recognition nucleotide sequence for Nb.BbvCI or an isoschizomer thereof.

[0220] According to some embodiments, the dsDNA construct comprises one or more recognition nucleotide sequences for Nb.BtsI or its isoschizomers.

[0221] According to some embodiments, the dsDNA construct comprises a single recognition nucleotide sequence for Nb.BtsI or an isoschizomer thereof.

[0222] According to embodiments of the present disclosure, the double-stranded transgene cassette further comprises at least one double-stranded promoter operably linked to the at least one double-stranded transgene for controlling expression of the at least one double-stranded transgene. In further embodiments, the double-stranded transgene cassette further comprises one or more genetic elements selected from the group consisting of a double-stranded enhancer, a double-stranded intron, a double-stranded post-transcriptional regulatory element, a double-stranded polyadenylation signal, and a double-stranded regulatory switch. According to other further embodiments, the at least one double-stranded transgene is a promoterless double-stranded transgene. As described herein, the at least one double-stranded transgene is a double-stranded donor sequence, and the double-stranded transgene cassette further comprises a double-stranded 5' homologous arm and a double-stranded 3' homologous arm adjacent to the double-stranded donor sequence. According to some embodiments, the double-stranded 5' homologous arm and the double-stranded 3' homologous arm are each about 10 to 2000 nt in length, e.g., about 100 to 2000 nt in length, or about 1000 to 2000 nt in length, or about 10 to 1000 nt in length, e.g., about 100 to 1000 nt in length, or about 10 to 500 nt in length, about 50 to 500 nt in length, or about 100 to 500 nt in length, about 10 to 50 nt in length, about 50 to 500 nt in length, or about 500 to 1000 nt in length, about 500 to 1500 nt in length, about 1500 to 2000 nt in length, about 2 to 1000 nt in length, about 2 to 500 nt in length, about 2 to 100 nt in length, or about 2 to 50 nt in length.

[0223] According to some embodiments, the at least one double-stranded transgene is a double-stranded donor sequence, and the double-stranded transgene cassette lacks a single-stranded 5' homology arm and a single-stranded 3' homology arm. Further, in some embodiments, the double-stranded transgene cassette is cleavable and further comprises at least a first double-stranded guide RNA (gRNA) target sequence (TS), at least a first double-stranded protospacer adjacent motif (PAM), at least a second double-stranded gRNA TS, and at least a second double-stranded PAM.

[0224] As described in more detail herein, in some embodiments, the dsDNA construct is synthetically produced by a method comprising: a) contacting a dsDNA template with at least one restriction endonuclease; the template comprising the double-stranded transgene cassette comprising the at least one double-stranded transgene, wherein the template comprises a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the double-stranded transgene cassette, a second non-palindromic restriction endonuclease recognition and a corresponding second cleavage site downstream of the double-stranded transgene cassette, wherein the at least one restriction endonuclease is capable of cleaving the template at the first and second cleavage sites to release an insert having single-stranded overhangs at the 5' and 3' ends of the insert; and b) ligating the 5' and 3' ends of the insert to a first inverted terminal repeat (ITR) oligonucleotide and an optional second ITR oligonucleotide to form the dsDNA construct. According to some embodiments, at least one of the first ITR oligonucleotide and the optional second ITR oligonucleotide comprises one or more phosphorothioate-modified nucleotides. According to some other embodiments, at least one of the first ITR oligonucleotide and the optional second ITR oligonucleotide comprises one or more phosphorothioate-modified nucleotides and at least one functional moiety. In one embodiment, at least one functional moiety is an aptamer sequence, and optionally, the aptamer sequence has high binding affinity for a nuclear-localized protein. In another embodiment, at least one functional moiety is a nuclear-localization peptide conjugated to at least one of the ITR oligonucleotides. In another embodiment, at least one functional moiety is a fluorophore chemically conjugated to the ITR oligonucleotide.

[0225] B. A single-stranded DNA molecule or vector derived from double-stranded DNA Single-stranded DNA (ssDNA) molecules (e.g., synthetic AAV vectors, e.g., single-stranded (ss) synthetic AAV vectors according to embodiments of the present disclosure) are derived from double-stranded DNA (dsDNA) constructs, particularly double-stranded ceDNA (ds ceDNA) with phosphorothioate-modified nucleotides, and the physical attributes of ds ceDNA vectors are also present in single-stranded DNA (ssDNA) molecules, including the presence of at least one functional moiety, such as an aptamer sequence, e.g., having high binding affinity for a nuclear-localized protein or a fluorophore chemically conjugated to an ITR oligonucleotide. In another embodiment, the at least one functional moiety is a fluorophore chemically conjugated to an ITR oligonucleotide.

[0226] Single-stranded DNA (ssDNA) molecules (e.g., synthetic AAV vectors, e.g., single-stranded (ss) synthetic AAV vectors) and ds DNA constructs (e.g., ds ceDNA) produced using the synthetic processes described herein do not have the packaging constraints imposed by the limited space within the viral capsid, allowing for the insertion of control elements, such as regulatory switches disclosed herein, large transgenes, multiple transgenes, etc.

[0227] According to some embodiments, the one or more phosphorothioate modified nucleotides of the ss DNA molecule are each independently located in any region selected from A, A', B, B', C, C', and D' of at least one of the first and second ITRs. According to some embodiments, the one or more phosphorothioate modified nucleotides of the dsDNA construct are each independently located in any region selected from A, A', B, B', C, C', D, and D' of at least one of the first and optional second ITRs.

[0228] According to some embodiments, the one or more phosphorothioate modified nucleotides of the ssDNA molecule are each independently located in any region selected from A, A', and D of at least one of the first ITR and the optional second ITR. According to some embodiments, the one or more phosphorothioate modified nucleotides of the dsDNA construct are each independently located in any region selected from A, A', and D of at least one of the first ITR and the optional second ITR.

[0229] According to some embodiments, the one or more phosphorothioate-modified nucleotides of the ssDNA molecule are each independently located in any region selected from A and A' of at least one of the first ITR and the optional second ITR. According to some embodiments, the one or more phosphorothioate-modified nucleotides of the dsDNA construct are each independently located in any region selected from A and A' of at least one of the first ITR and the optional second ITR.

[0230] According to some embodiments, all of the one or more phosphorothioate modified nucleotides of the ssDNA molecule in the first ITR are located in the A' and / or D regions of the first ITR. According to some embodiments, all of the one or more phosphorothioate modified nucleotides of the dsDNA construct in the first ITR are located in the A' and / or D regions of the first ITR.

[0231] According to some embodiments, all of the one or more phosphorothioate-modified nucleotides in the first ITR of the ssDNA molecule are located in the A region of the first ITR. According to some embodiments, all of the one or more phosphorothioate-modified nucleotides in the first ITR of the dsDNA construct are located in the A region of the first ITR.

[0232] According to some embodiments, all of the one or more phosphorothioate modified nucleotides in the second ITR of the ssDNA molecule, if present, are located in the A' and / or D regions of the second ITR. According to some embodiments, all of the one or more phosphorothioate modified nucleotides in the second ITR of the dsDNA construct, if present, are located in the A' and / or D regions of the second ITR.

[0233] According to some embodiments, all of the one or more phosphorothioate-modified nucleotides in the second ITR of the ssDNA molecule, if present, are located in the A region of the second ITR. According to some embodiments, all of the one or more phosphorothioate-modified nucleotide dsDNA constructs in the second ITR, if present, are located in the A region of the second ITR.

[0234] According to some embodiments, one or more phosphorothioate-modified nucleotides of the ssDNA molecule are adjacent to one another. According to some embodiments, one or more phosphorothioate-modified nucleotides of the dsDNA construct are adjacent to one another.

[0235] According to some embodiments, the one or more phosphorothioate-modified nucleotides of the ssDNA molecule, if present, are about 1-15 nucleotides from the B-B' and C-C' arms of the first ITR or optional second ITR. According to some embodiments, the one or more phosphorothioate-modified nucleotide dsDNA construct, if present, are about 1-15 nucleotides from the B-B' and C-C' arms of the first ITR or optional second ITR.

[0236] According to some embodiments, the one or more phosphorothioate-modified nucleotides of the ssDNA molecule, if present, are about 1-10 nucleotides from the B-B' and C-C' arms of the first ITR or optional second ITR. According to some embodiments, the one or more phosphorothioate-modified nucleotide dsDNA construct, if present, are about 1-10 nucleotides from the B-B' and C-C' arms of the first ITR or optional second ITR.

[0237] According to some embodiments, the one or more phosphorothioate-modified nucleotides of the ssDNA molecule, if present, are about 1-5 nucleotides from the B-B' and C-C' arms of the first ITR or optional second ITR. According to some embodiments, the one or more phosphorothioate-modified nucleotide dsDNA construct, if present, are about 1-5 nucleotides from the B-B' and C-C' arms of the first ITR or optional second ITR.

[0238] According to some embodiments, one or more phosphorothioate-modified nucleotides of the ssDNA molecule are resistant to exonuclease degradation. According to some embodiments, one or more phosphorothioate-modified nucleotides comprising the dsDNA construct are resistant to exonuclease degradation at the PS-binding sequence.

[0239] According to some embodiments, at least one of the first and optional second ITRs of the ssDNA molecule each comprises from about 1 to about 60, e.g., from about 1 to about 3, from about 1 to about 5, from about 1 to about 7, from about 1 to about 10, from about 1 to about 20, from about 1 to about 30, from about 1 to about 40, from about 1 to about 50, from about 10 to about 20, from about 10 to about 30, from about 10 to about 40, from about 10 to about 50, from about 20 to about 30, from about 20 to about 40, from about 20 to about 50, from about 30 to about 40, from about 30 to about 50, from about 40 to about 50, from about 25 to about 50, from about 5 to about 10, from about 5 to about 15, from about 5 to about 20, or from about 5 to about 25, phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and optional second ITR dsDNA constructs each comprises about 1 to about 60 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and optional second ITR of the ssDNA molecule each comprises about 1 to about 5 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and optional second ITR of the ssDNA molecule each comprises about 1 to about 10 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and optional second ITR of the ssDNA molecule each comprises about 1 to about 15 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and optional second ITR dsDNA constructs each comprises about 1 to about 20 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first and optional second ITRs of the ssDNA molecule each comprises from about 1 to about 25 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first and optional second ITR dsDNA constructs each comprises from about 1 to about 30 phosphorothioate-modified nucleotides.

[0240] According to some embodiments, one or more phosphorothioate-modified nucleotides are located at the 5'-end of the ssDNA molecule. According to some embodiments, one or more phosphorothioate-modified nucleotides are located at the 3'-end of the ssDNA molecule. According to some embodiments, one or more phosphorothioate-modified nucleotides are located at the 3'-end of the ssDNA molecule, the 5'-end of the ssDNA molecule, or both.

[0241] According to some embodiments, one or more phosphorothioate modified nucleotides are located upstream of each of the one or more nicking endonuclease recognition sequences.

[0242] According to some embodiments, the one or more phosphorothioate modified nucleotides are located at the 5' end of the first ITR and / or the optional second ITR.

[0243] According to some embodiments, the ssDNA molecule comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more phosphorothioate-modified nucleotides. According to some embodiments, the ssDNA molecule comprises at least 1, 2, 3, 4, 5, or more phosphorothioate-modified nucleotides at the 3' end of the ssDNA molecule, the 5' end of the ssDNA molecule, or both. According to some embodiments, the ssDNA molecule comprises at least 1, 2, 3, 4, 5, or more phosphorothioate-modified nucleotides upstream of each of one or more nicking endonuclease recognition sequences. According to some embodiments, the ssDNA molecule comprises at least 1, 2, 3, 4, 5, or more phosphorothioate-modified nucleotides at the 5' end of the first ITR and / or at least 1, 2, 3, 4, 5, or more phosphorothioate-modified nucleotides at the 5' end of the optional second ITR.

[0244] According to some embodiments, at least one of the first ITR and the optional second ITR of the ssDNA molecule each contains about 6 or fewer phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR of the ssDNA molecule each contains about 5 or fewer phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR of the ssDNA molecule each contains about 4 or fewer phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR of the ssDNA molecule each contains about 3 or fewer phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR of the ssDNA molecule each contains about 2 or fewer phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR of the ssDNA molecule each contains about 1 or fewer phosphorothioate-modified nucleotide. According to some embodiments, at least one of the first ITR and the optional second ITR dsDNA constructs each contains about 6 or less phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR dsDNA constructs each contains about 5 or less phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR dsDNA constructs each contains about 4 or less phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR dsDNA constructs each contains about 3 or less phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR dsDNA constructs each contains about 2 or less phosphorothioate-modified nucleotides.According to some embodiments, at least one of the first ITR and optional second ITR dsDNA constructs each comprises no more than about one phosphorothioate modified nucleotide.

[0245] According to some embodiments, at least one of the first ITR and the optional second ITR of the ssDNA molecule each comprises about 3, about 4, or about 5 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR dsDNA construct each comprises about 3, about 4, or about 5 phosphorothioate-modified nucleotides.

[0246] According to some embodiments, the first and second ITRs of the ssDNA molecule are symmetrical or substantially symmetrical to each other. According to some embodiments, the first and second ITR dsDNA constructs are symmetrical or substantially symmetrical to each other.

[0247] According to some embodiments, the first and second ITRs of the ssDNA molecule are asymmetric with respect to each other. According to some embodiments, the first and second ITRs of the dsDNA construct are asymmetric with respect to each other.

[0248] According to some embodiments, at least one of both the first and second ITRs of the ssDNA molecule is a wild-type ITR. According to some embodiments, at least one of both the first and second ITRs of the dsDNA construct is a wild-type ITR.

[0249] According to some embodiments, at least one of the first and second ITRs of the ssDNA molecule is modified by deletion, insertion, and / or base substitution in at least one region selected from A, A', B, B', C, C', D, and D'. According to some embodiments, at least one of both the first and second ITR dsDNA constructs is modified by deletion, insertion, and / or base substitution in at least one region selected from A, A', B, B', C, C', D, and D'.

[0250] According to some embodiments, the first and second ITRs of the ssDNA molecule are each AAV ITRs, each independently an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV 12. According to some embodiments, the first and second ITR dsDNA constructs are each AAV ITRs, each independently an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0251] According to some embodiments, the ssDNA molecule lacks any viral capsid protein coding sequence. According to some embodiments, the dsDNA construct lacks any viral capsid protein coding sequence.

[0252] According to some embodiments, the ssDNA molecule comprises one or more viral capsid protein coding sequences. According to some embodiments, the dsDNA construct comprises one or more viral capsid protein coding sequences.

[0253] C. Expression Cassettes, Transgenes, and Nucleic Acid Sequences of Interest An expression cassette can include a transgene (nucleic acid sequence of interest) and one or more regulatory sequences that permit and / or control expression of the transgene. For example, an expression cassette can include one or more of the following, in order: an enhancer / promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA). An expression cassette can also include an internal ribosome entry site (IRES) and / or a 2A element. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir-regulatable elements, post-transcriptional regulatory elements, tissue- and cell-type-specific promoters, and enhancers. In some embodiments, an ITR can act as a promoter for the transgene. In some embodiments, the ssDNA molecules or dsDNA constructs described in Part II or Part III comprise additional components for regulating expression of a transgene or nucleic acid sequence of interest, e.g., a regulatory switch as described herein in the section entitled "Regulatory Switch" for controlling and regulating expression of the transgene, and optionally a regulatory switch that is a kill switch to allow controlled cell death of cells containing the ssDNA molecule.

[0254] The expression cassette or nucleic acid sequence of interest in the ssDNA construct can comprise more than 4,000 nucleotides, more than 5,000 nucleotides, more than 10,000 nucleotides, or more than 20,000 nucleotides, or more than 30,000 nucleotides, or more than 40,000 nucleotides, or more than 50,000 nucleotides, or any range of about 4,000 to 10,000 nucleotides, or 10,000 to 50,000 nucleotides, or more than 50,000 nucleotides. According to some embodiments, the expression cassette can comprise a transgene in the length range of 500 to 50,000 nucleotides. According to some embodiments, the expression cassette can comprise a transgene in the length range of 500 to 75,000 nucleotides. According to some embodiments, the expression cassette can comprise a transgene in the length range of 500 to 10,000 nucleotides. According to some embodiments, the expression cassette can comprise a transgene in the length range of 1,000 to 10,000 nucleotides. According to some embodiments, the expression cassette can contain a transgene ranging from 500 to 5,000 nucleotides in length. The ssDNA molecules and dsDNA constructs described herein are free of the size limitations of encapsidated AAV vectors, allowing for the delivery of large expression cassettes into hosts to provide efficient transgenes. In some embodiments, the ssDNA molecules and dsDNA constructs described in Section II or Section III lack prokaryote-specific methylation.

[0255] An expression cassette can include, for example, an expressible exogenous sequence (e.g., an open reading frame) or transgene or nucleic acid sequence of interest that encodes a protein that is either absent, inactive, or insufficiently active in the recipient subject, or a gene that encodes a protein with a desired biological or therapeutic effect. The transgene or nucleic acid sequence of interest can encode a gene product that can function to correct expression of a defective gene or transcript. In principle, an expression cassette can include any gene that encodes a protein, polypeptide, or RNA that is reduced or absent due to a mutation, or that provides a therapeutic effect if overexpression is considered within the scope of the present disclosure.

[0256] The expression cassette can include any transgene or nucleic acid sequence of interest useful for treating a disease or disorder in a subject. The ssDNA molecules or dsDNA constructs described in Part II or Part III, produced using the synthetic processes described herein, can be used to deliver and express any gene of interest in a subject, including, but not limited to, exogenous genes and nucleotide sequences, including polypeptide-encoding or non-coding nucleic acids (e.g., RNAi, miR, etc.), and viral sequences in the subject's genome, such as HIV viral sequences. In some embodiments, the ssDNA molecules and dsDNA constructs described in Part II or Part III are used for therapeutic purposes (e.g., medical, diagnostic, or veterinary uses). In certain embodiments, the ssDNA molecules and dsDNA constructs described in Part II or Part III are useful for expressing any gene of interest in a subject and include one or more polypeptides, be they peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, or RNA (coding or non-coding; e.g., siRNA, shRNA, microRNA, mRNA, or gRNA, and their antisense counterparts (e.g., antagoMiRs)), antibodies, antigen-binding fragments, or any combination thereof.

[0257] The expression cassette can also encode a polypeptide, a sense or antisense oligonucleotide, or RNA (e.g., coding or non-coding, e.g., siRNA, shRNA, microRNA, and their antisense counterparts (e.g., antagomirs)). The expression cassette can include exogenous sequences encoding reporter proteins used for experimental or diagnostic purposes, such as β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art.

[0258] The sequences provided in the expression cassettes, expression constructs for the ssDNA molecules described in Part II or Part III, and dsDNA constructs described herein can be codon-optimized for the target host cell. As used herein, the term "codon-optimized" or "codon optimization" refers to the process of modifying a nucleic acid sequence for enhanced expression in the cells of a target vertebrate, such as a mouse or human, by replacing at least one, two, or a significant number of codons in the native sequence (e.g., a prokaryotic sequence) with codons more frequently or most frequently used in the genes of that vertebrate. Various species exhibit specific biases for particular codons for particular amino acids. Typically, codon optimization does not alter the amino acid sequence of the original translated protein. Optimized codons can be determined, for example, using Aptagen's GENEFORGE® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or another publicly available database.

[0259] In some embodiments, the transgene or nucleic acid sequence of interest expressed by the ssDNA molecules and dsDNA constructs described herein in Section II or Section III is a therapeutic gene. In some embodiments, the therapeutic gene is an antibody, or antibody fragment, or an antigen-binding fragment thereof, such as a neutralizing antibody or antibody fragment.

[0260] Specifically, a therapeutic gene is one or more therapeutic agents, including, but not limited to, proteins, polypeptides, peptides, enzymes, antibodies, antigen-binding fragments, and variants and / or active fragments thereof, for use in treating, preventing, and / or ameliorating one or more symptoms of a disease, dysfunction, injury, and / or disorder. Exemplary therapeutic genes are described herein in the section entitled "Methods of Treatment."

[0261] The ssDNA molecules and dsDNA constructs described in Parts II and III of this document have several structural features that distinguish them from plasmid-based expression vectors. The ssDNA molecules described in Parts II and III of this document and the dsDNA constructs produced by the synthetic methods of this document may possess one or more of the following characteristics: lack of native (i.e., uninserted) bacterial DNA; lack of a prokaryotic origin of replication; self-sufficiency, i.e., no need for sequences other than the two ITRs (containing the Rep binding site and terminal resolution site); lack of exogenous sequences between the ITRs; hairpin formation of ITR sequences; and lack of bacterial-type DNA methylation or other methylation associated with production in specific cell types and considered aberrant by mammalian hosts. Generally, the vectors are preferably free of any prokaryotic DNA; however, it is contemplated that some prokaryotic DNA may be inserted as exogenous sequences, as a non-limiting example in promoter or enhancer regions.

[0262] There are several advantages to using the ssDNA molecules and dsDNA constructs described in Part II or Part III of this specification over plasmid-based expression vectors. These advantages include, but are not limited to: 1) while plasmids contain bacterial DNA sequences and are subject to prokaryote-specific methylation, such as 6-methyladenosine and 5-methylcytosine methylation, capsid-free AAV vector sequences are of eukaryotic origin and are not subject to prokaryote-specific methylation, resulting in capsid-free AAV vectors being less likely to induce inflammatory and immune responses compared to plasmids; 2) while plasmids require the presence of resistance genes during the production process, the ssDNA molecules of the present disclosure do not require such presence; and 3) while circular plasmids are not delivered to the nucleus upon introduction into cells and require overloading to bypass degradation by cellular nucleases, ssDNA molecules or dsDNA constructs contain viral cis-elements, i.e., ITRs, that confer resistance to nucleases and can be designed to be targeted and delivered to the nucleus. The minimal defining elements essential for ITR function include a Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' for AAV2) and a terminal resolving site (TRS; 5'-AGTTGG-3' for AAV2), plus a variable palindromic sequence that allows hairpin formation, and 4) the ssDNA molecule and dsDNA construct vector do not have an overrepresentation of CpG dinucleotides often found in prokaryotic-derived plasmids that bind members of the Toll-like family of receptors and elicit T cell-mediated immune responses.

[0263] D. Inverted Terminal Repeats (ITRs) As described herein, according to some aspects, the present disclosure provides single-stranded deoxyribonucleic acid (ssDNA) molecules comprising at least one nucleic acid sequence of interest adjacent to at least one stem-loop structure comprising a partial DNA duplex and at least one loop on the 3' end. In some embodiments, the ssDNA molecule comprises at least one stem-loop structure comprising a partial DNA duplex and at least one loop on the 5' end.

[0264] According to some embodiments, ssDNA molecules and dsDNA constructs comprise a transgene or heterologous nucleic acid sequence located between two inverted terminal repeat (ITR) sequences, which can be an asymmetric ITR pair or a symmetric or substantially symmetric ITR pair, as these terms are defined herein. The ssDNA molecules and dsDNA constructs disclosed herein can comprise ITR sequences selected from: (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (ITR) (e.g., an asymmetric modified ITR), (ii) two modified ITRs, where the modified ITR pair has a different three-dimensional spatial arrangement relative to each other (e.g., an asymmetric modified ITR), or (iii) a symmetric or substantially symmetric WT-WT ITR pair, where each WT-ITR has the same three-dimensional spatial arrangement, or (iv) a symmetric or substantially symmetric modified ITR pair, where each mod-ITR has the same three-dimensional spatial arrangement, wherein the methods of the present disclosure can include a delivery system, including, but not limited to, a liposomal nanoparticle delivery system.

[0265] In some embodiments, the ITR sequences can be derived from viruses of the Parvoviridae family, which is divided into two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects. The Parvovirinae subfamily (also referred to as parvoviruses) includes the Dependovirus genus, whose members, under most conditions, require co-infection with a helper virus, such as an adenovirus or a herpesvirus, for productive infection. The Dependovirus genus includes adeno-associated viruses (AAVs), which typically infect humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4), as well as related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses). Parvoviruses and other members of the family Parvoviridae are generally described in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996).

[0266] While the ITRs exemplified herein and in the Examples herein are AAV2 WT-ITRs, one of skill in the art can use ITRs from any known parvovirus, e.g., dependovirus, such as AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes; e.g., NCBI: NC 002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261), chimeric ITRs, or ITRs from any synthetic AAV, as described above. In some embodiments, the AAV can infect warm-blooded animals, such as avian (AAAV), bovine (BAAV), canine, equine, and ovine adeno-associated viruses. In some embodiments, the ITRs are derived from B19 parvovirus (GenBank Accession No. NC000883), minute virus of mice (MVM) (GenBank Accession No. NC001510), goose parvovirus (GenBank Accession No. NC001701), or snake parvovirus 1 (GenBank Accession No. NC006148). In some embodiments, the 5'WT-ITR can be derived from one serotype and the 3'WT-ITR can be derived from a different serotype, as discussed herein.

[0267] Those skilled in the art recognize that ITR sequences have a common structure of a double-stranded Holliday junction, typically a T-shaped or Y-shaped hairpin structure, where each WT-ITR has two palindromic arms or loops (B-B' and C-C') embedded in a larger palindromic arm (A-A') and a single-stranded D sequence (the order of these palindromic sequences determines the flip or flop configuration of the ITR). For example, see the structural analysis and sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6). Grimm et al., J. Virology, 2006; 80(1); 426-439; Yan et al., J. Virology, 2005; 364-379; Duan et al., Virology 1999; 261; 8-14. Those skilled in the art can easily determine the WT-ITR sequence from any AAV serotype for use in ssDNA molecules and dsDNA constructs based on the exemplary AAV2 ITR sequences provided herein.See, for example, the sequence comparison of ITRs from different AAV serotypes (AAV1 to AAV6, and avian AAV (AAAV) and bovine AAV (BAAV)) described in Grimm et al., J. Virology, 2006; 80(1); 426-439; which shows the percent identity of the left ITR of AAV2 to the left ITRs from other serotypes: AAV-1 (84%), AAV-3 (86%), AAV-4 (79%), AAV-5 (58%), AAV-6 (left ITR) (100%), and AAV-6 (right ITR) (82%).

[0268] According to some other embodiments, at least one of the first ITR oligonucleotide and the optional second ITR oligonucleotide comprising one or more phosphorothioate-modified nucleotides of the present invention can further comprise one or more functional moieties. In one embodiment, at least one functional moiety is an aptamer sequence, and optionally, the aptamer sequence has high binding affinity for a nuclear-localized protein. In another embodiment, at least one functional moiety is a nuclear-localization peptide conjugated to at least one of the ITR oligonucleotides. In another embodiment, at least one functional moiety is a fluorophore chemically conjugated to the ITR oligonucleotide.

[0269] In some embodiments, the ITRs used herein may be entirely synthetic, e.g., they may contain no viral-derived sequences.

[0270] E. Regulatory Elements The single-stranded DNA (ssDNA) molecules described in Part II or Part III may further comprise a specific combination of cis-regulatory elements. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir-regulatory elements, post-transcriptional regulatory elements, tissue- and cell-type-specific promoters, and enhancers. In some embodiments, the single-stranded DNA (ssDNA) molecules described in Part II or Part III include additional components for regulating the expression of a transgene or nucleic acid of interest, such as a control switch described herein, or a death switch that can kill cells containing the single-stranded DNA (ssDNA) molecules described in Part II or Part III. Regulatory elements, including regulatory switches, that can be used in the present disclosure are fully discussed in International Patent Application No. PCT / US18 / 49996 (published as International Patent Publication No. 2019 / 051255A1), which is incorporated herein by reference in its entirety.

[0271] According to some embodiments, the second nucleic acid sequence comprises a regulatory sequence and a nucleic acid sequence encoding a nuclease. In certain embodiments, a gene regulatory sequence is operably linked to the nucleic acid sequence encoding the nuclease. In certain embodiments, the regulatory sequence is suitable for controlling the expression of the nuclease in the host cell. In certain embodiments, the regulatory sequence comprises a suitable promoter sequence capable of directing the transcription of a gene operably linked to the promoter sequence, such as the nucleic acid sequence encoding the nuclease of the present disclosure. In certain embodiments, the second nucleic acid sequence comprises an intron sequence linked to the 5' end of the nucleic acid sequence encoding the nuclease. In certain embodiments, an enhancer sequence is provided upstream of the promoter to increase the effectiveness of the promoter. In certain embodiments, the regulatory sequence comprises an enhancer and a promoter, and the second nucleic acid sequence comprises an intron sequence upstream of the nucleic acid sequence encoding the nuclease, the intron comprising one or more nuclease cleavage sites, and the promoter is operably linked to the nucleic acid sequence encoding the nuclease.

[0272] The single-stranded DNA (ssDNA) and dsDNA molecules described in Part II or Part III produced using the synthetic processes described herein may further comprise specific combinations of cis-regulatory elements, such as a WHP post-transcriptional regulatory element (WPRE) and a BGH polyA. Expression cassettes suitable for use in expression constructs are not limited by packaging constraints imposed by viral capsids.

[0273] (i) Promoter Those skilled in the art will understand that promoters used in the synthetically produced single-stranded DNA (ssDNA) and dsDNA molecules described in Part II or Part III of this disclosure should be adjusted as necessary for the specific sequences they are promoting. For example, guide RNAs may not require a promoter at all, since their function is to form duplexes with specific target sequences on natural DNA to generate recombination events. In contrast, nucleases encoded by ssDNA molecules or dsDNA construct vectors will benefit from a promoter so that they can be expressed efficiently and, optionally, in a regulatable manner from the vector.

[0274] The expression cassettes of the present disclosure contain promoters that can affect overall expression levels and cell specificity. In the case of transgene expression, they can include highly active viral immediate-early promoters. The expression cassette can also contain tissue-specific eukaryotic promoters to restrict transgene expression to specific cell types and reduce toxic effects and immune responses resulting from unregulated ectopic expression. In a preferred embodiment, the expression cassette can contain synthetic regulatory elements such as the CAG promoter. The CAG promoter contains (i) a cytomegalovirus (CMV) early enhancer element, (ii) the promoter, first exon, and first intron of the chicken beta-actin gene, and (iii) the splice acceptor of the rabbit beta-globin gene. Alternatively, the expression cassette can contain the alpha-1-antitrypsin (AAT) promoter, the liver-specific (LP1) promoter, the human elongation factor-1 alpha (EF1a) promoter, or the human transthyretin (TTR) promoter. In some embodiments, the expression cassette comprises one or more constitutive promoters, such as the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), or the cytomegalovirus (CMV) immediate early promoter (optionally with a CMV enhancer). Alternatively, an inducible promoter, the native promoter for the transgene, a tissue-specific promoter, or various promoters known in the art can be used.

[0275] Suitable promoters, including those described above, can be derived from viruses and therefore can be referred to as viral promoters, or they can be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, e.g., the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the human U6 micronucleus promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31 (17)), the human H1 promoter (H1), the CAG promoter, the human alpha 1-antitypsin (HAAT) promoter, and the like. In certain embodiments, these promoters are modified at their downstream intron-containing ends to include one or more nuclease cleavage sites. In certain embodiments, the DNA containing the nuclease cleavage site is exogenous to the promoter DNA.

[0276] In one embodiment, the promoter used is the native promoter of the gene encoding the therapeutic protein. The promoter and other regulatory sequences of each gene encoding the therapeutic protein are known and characterized. The promoter region used may further comprise one or more additional regulatory sequences (e.g., native enhancers). Preferably, the gap is located 5' upstream of the promoter.

[0277] In some embodiments, the region of the ssDNA molecule comprising the promoter is double-stranded. In some embodiments, the transcription start site (TSS) of the promoter is double-stranded. In some embodiments, the ssDNA molecule further comprises a regulatory element, such as an enhancer. In some embodiments, the enhancer may be a serpin enhancer (e.g., 1xSERP, 2xSERP, or 3xSERP). As described in Example 7 and Figures 42-45, the inclusion of a double-stranded promoter, enhancer, and / or TSS can substantially increase expression from the ssDNA molecules described herein.

[0278] Thus, in some embodiments, the double-stranded region comprising a promoter, enhancer, and / or TSS is at least 10 base pairs in length, at least 20 base pairs in length, at least 30 base pairs in length, at least 40 base pairs in length, at least 50 base pairs in length, at least 60 base pairs in length, at least 70 base pairs in length, at least 80 base pairs in length, at least 90 base pairs in length, at least 100 base pairs in length, at least 110 base pairs in length, at least 120 base pairs in length, at least 130 base pairs in length, at least 140 base pairs in length, at least 150 base pairs in length, at least 160 base pairs in length, at least 170 base pairs in length, at least 180 base pairs in length, at least 190 base pairs in length, at least 200 base pairs in length, at least 220 base pairs in length, at least 240 base pairs in length, at least 260 base pairs in length, at least 280 base pairs in length, at least 300 base pairs in length, at least 320 base pairs in length, at least 340 base pairs in length, at least 360 base pairs in length, at least 380 base pairs in length, at least 390 base pairs in length, at least 400 base pairs in length, at least 410 base pairs in length, at least 420 base pairs in length, at least 430 base pairs in length, at least 440 base pairs in length, at least 450 base pairs in length, at least 460 base pairs in length, at least 470 base pairs in length, at least 480 base pairs in length, at least 490 base pairs in length, at least 500 base pairs in length, at least 510 base pairs in length, at least 520 base pairs in length, at least 530 base pairs in length, at least 540 base pairs in length pairs in length, at least 300 base pairs in length, at least 320 base pairs in length, at least 340 base pairs in length, at least 360 base pairs in length, at least 380 base pairs in length, at least 400 base pairs in length, at least 420 base pairs in length, at least 440 base pairs in length, at least 460 base pairs in length, at least 480 base pairs in length, at least 500 base pairs in length, at least 550 base pairs in length, at least 600 base pairs in length, at least 650 base pairs in length, at least 700 base pairs in length, at least 750 base pairs in length, at least 800 base pairs in length, at least 850 base pairs in length, at least 900 base pairs in length, at least 950 base pairs in length, at least 1000 base pairs in length, at least 1100 base pairs in length, at least 1200 base pairs in length, at least 1300 base pairs in length, at least 1400 base pairs in length, or at least 1500 base pairs in length.

[0279] In some embodiments, the double-stranded region comprising the promoter, enhancer, and / or TSS is less than 1500 base pairs in length, less than 1400 base pairs in length, less than 1300 base pairs in length, less than 1200 base pairs in length, less than 1100 base pairs in length, less than 1000 base pairs in length, less than 950 base pairs in length, less than 900 base pairs in length, less than 850 base pairs in length, less than 800 base pairs in length, less than 750 base pairs in length, less than 700 base pairs in length, less than 650 base pairs in length, less than 600 base pairs in length, less than 550 base pairs in length, less than 500 base pairs in length, less than 480 base pairs in length, less than 460 base pairs in length, less than 440 base pairs in length, less than 420 base pairs in length, less than 400 base pairs in length, less than 380 base pairs in length, less than 4 ...400 base pairs in length, less than 400 base pairs in length, less than 400 base pairs in length, less than 400 base pairs in length, less than 400 base pairs in length, less than 400 base pairs in length, less than 400 base pairs in length, less than 400 base pairs in length, less than 400 base pairs in length, less than 400 base pairs in length, less than 400 base pairs in length, less than 400 base pairs less than 360 base pairs, less than 340 base pairs, less than 320 base pairs, less than 300 base pairs, less than 280 base pairs, less than 260 base pairs, less than 240 base pairs, less than 220 base pairs, less than 200 base pairs, less than 190 base pairs, less than 180 base pairs, less than 170 base pairs, less than 160 base pairs, less than 150 base pairs, less than 140 base pairs, less than 130 base pairs, less than 120 base pairs, less than 110 base pairs, less than 100 base pairs, less than 90 base pairs, less than 80 base pairs, less than 70 base pairs, less than 60 base pairs, less than 50 base pairs, less than 40 base pairs, or less than 30 base pairs in length.

[0280] In some embodiments, the double-stranded region comprising the promoter, enhancer, and / or TSS is about 30 to 1500 base pairs in length, about 40 to 1400 base pairs in length, about 50 to 1300 base pairs in length, about 60 to 1200 base pairs in length, about 70 to 1100 base pairs in length, about 80 to 1000 base pairs in length, about 90 to 900 base pairs in length, about 90 to 900 base pairs in length, about 100 to 800 base pairs in length, about 110 to 700 base pairs in length, about 120 to 600 base pairs in length, about 130 to 500 base pairs in length, about 140 to 400 base pairs in length, about 150 to 300 base pairs in length, about 160 to 200 base pairs in length, about 1381 base pairs in length, or about 499 base pairs in length.

[0281] (ii) Polyadenylation sequence: A sequence encoding a polyadenylation sequence can be included in a synthetically produced AAV vector to stabilize mRNA expressed from a single-stranded DNA (ssDNA) molecule (e.g., a synthetic AAV vector, e.g., a single-stranded (ss) synthetic AAV vector) and to aid in nuclear transport and translation. In one embodiment, the synthetically produced AAV vector does not contain a polyadenylation sequence. In other embodiments, the vector contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, or more adenine dinucleotides. In some embodiments, the polyadenylation sequence contains about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range therebetween.

[0282] The expression cassette can include a polyadenylation sequence known in the art or a variant thereof, such as a naturally occurring sequence isolated from bovine BGHpA or viral SV40pA, or a synthetic sequence. Some expression cassettes also include an SV40 late polyA signal upstream enhancer (USE) sequence. In some embodiments, the USE sequence can be used in combination with SV40pA or a heterologous polyA signal.

[0283] The expression cassette can also include post-transcriptional elements to increase transgene expression. In some embodiments, the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) is used to increase transgene expression. Other post-transcriptional processing elements can be used, such as the post-transcriptional elements from herpes simplex virus or the thymidine kinase gene of hepatitis B virus (HBV). A secretory sequence can be linked to the transgene, for example, the VH-02 sequence and the VK-A26 sequence.

[0284] (iii) Nuclear localization sequence In some embodiments, the vector encoding the RNA-guided endonuclease contains one or more nuclear localization sequences (NLSs), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the one or more NLSs are located at or near the amino terminus, at or near the carboxy terminus, or a combination thereof (e.g., one or more NLSs at the amino terminus and / or one or more NLSs at the carboxy terminus). When two or more NLSs are present, each can be selected independently from the others, such that a single NLS is present in two or more copies and / or in combination with one or more other NLSs present in one or more copies. Non-limiting examples of NLSs are shown in Table 3 below. [Table 3]

[0285] F. Additional Components The single-stranded DNA (ssDNA) and dsDNA molecules described in Section II or Section III produced using the synthetic processes described herein may contain nucleotides encoding other components for gene expression. For example, to select specific gene targeting events, a protective shRNA may be embedded in a microRNA and inserted into the recombinant single-stranded DNA (ssDNA) molecules described herein in Section II or Section III, designed to site-specifically integrate into a highly active locus, such as the albumin locus. Such embodiments may provide a system for in vivo selection and expansion of genetically modified hepatocytes in any genetic background, as described in Nygaard et al., "A universal system to select gene-modified hepatocytes in vivo," Gene Therapy, June 8, 2016. The single-stranded DNA (ssDNA) molecules described in Section II or Section III of the present disclosure may contain one or more selectable markers that allow for the selection of transformed, transfected, transduced, or similar cells. A selectable marker is a gene the product of which provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, NeoR, etc. In certain embodiments, a positive selection marker is incorporated into the donor sequence, such as NeoR. A negative selection marker may be incorporated downstream of the donor sequence, for example, a nucleic acid sequence encoding the negative selection marker, HSV-tk, may be incorporated into the nucleic acid construct downstream of the donor sequence.

[0286] The single-stranded DNA (ssDNA) molecules described in Part II or Part III of this specification and double-stranded DNA (dsDNA) molecules produced using the synthesis processes described herein can be used for gene editing, for example, as disclosed in International Application PCT / US2018 / 064242 filed December 6, 2018 (published as International Patent Publication No. 2019 / 113310), and may include one or more of a 5' homology arm, a 3' homology arm, or a polyadenylation site located upstream and adjacent to the homology arm. Exemplary homology arms are 5' and 3' albumin homology arms or CCR5 5' and 3' homology arms.

[0287] G. Switch A molecular regulatory switch is one that generates a measurable change in state in response to a signal. Such a regulatory switch can be usefully combined with the single-stranded DNA (ssDNA) molecules described in Section II or Section III and the dsDNA molecules produced using the synthetic processes described herein to control the output of transgene expression from the single-stranded DNA (ssDNA) molecules described in Section II or Section III. In some embodiments, the single-stranded DNA (ssDNA) molecules described in Section II or Section III contain a regulatory switch that serves to fine-tune transgene expression. For example, this can function as a biological containment function for the single-stranded DNA (ssDNA) molecules described herein in Section II or Section III. In some embodiments, the switch is an "on / off" switch designed to start or stop (i.e., shut off) expression of a gene of interest in the synthetic AAV in a controllable and tunable manner. In some embodiments, the switch can include a "kill switch" that can instruct a cell containing a single-stranded DNA (ssDNA) molecule described herein, in Section II or Section III, to undergo programmed cell death when the switch is activated. Exemplary regulatory switches encompassed for use in the single-stranded DNA (ssDNA) molecules described herein in Part II or Part III can be used to regulate expression of a transgene and are more fully discussed in International Application No. PCT / US18 / 49996 (published as International Patent Publication No. WO 2019 / 051255A1), which is incorporated herein by reference in its entirety.

[0288] (i) Binary Adjustment Switch In some embodiments, the single-stranded DNA (ssDNA) molecules described in Section II or Section III produced using the synthetic processes described herein contain a regulatory switch that can function to controllably regulate the expression of a transgene. For example, the expression cassette located between the ITRs of the single-stranded DNA (ssDNA) molecules described herein in Section II or Section III may additionally contain regulatory regions, such as promoters, cis-elements, repressors, enhancers, etc., that are operably linked to a gene of interest, and the regulatory region is controlled by one or more cofactors or exogenous agents. By way of example only, the regulatory region can be regulated by a small molecule switch or an inducible or repressible promoter. Non-limiting examples of inducible promoters are hormone-inducible promoters or metal-inducible promoters. Other exemplary inducible promoter / enhancer elements include, but are not limited to, the RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, and metallothionein promoter.

[0289] (ii) small molecule regulatory switches A variety of art-known small molecule-based control switches are known in the art and can be combined with synthetically produced single-stranded DNA (ssDNA) molecules described in Part II or Part III disclosed herein to form a control switch-controlled single-stranded DNA (ssDNA) molecule described in Part II or Part III. In some embodiments, the regulatory switch is an orthogonal ligand / nuclear receptor pair (e.g., retinoid receptor variants / LG335 and GRQCIMFI) plus an artificial promoter controlling expression of an operably linked transgene (such as that disclosed in Taylor, et al. BMC Biotechnology 10 (2010): 15), an engineered steroid receptor, for example, a modified progesterone receptor with a C-terminal truncation that cannot bind progesterone but binds RU486 (mifepristone) (U.S. Pat. No. 5,364,791), the ecdysone receptor from Drosophila and its ecdysteroid ligands (Saez, et al., PNAS, 97(26)(2000), 14512-14517, or Sando R 3 rd or a switch controlled by the antibiotic trimethoprim (TMP) disclosed in Nat Methods. 2013, 10(11):1085-8, or a combination thereof. In some embodiments, the controlled switch controlling the transgene or expressed by a single-stranded DNA (ssDNA) molecule (e.g., a synthetic AAV vector, e.g., a single-stranded (ss) synthetic AAV vector) is a prodrug activation switch, such as those disclosed in U.S. Patent Nos. 8,771,679 and 6,339,070.

[0290] (iii) "Password" restriction switch In some embodiments, the control switch can be a "passcode switch" or "passcode circuit." The passcode switch allows for fine-tuning of the control of transgene expression from a synthetically produced single-stranded DNA (ssDNA) molecule described in Section II or Section III when certain conditions occur. That is, a combination of conditions must be present for transgene expression and / or repression to occur. For example, at least condition A and condition B must occur for transgene expression to occur. The passcode regulatory switch can have any number of conditions present for transgene expression to occur, e.g., at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or more. In some embodiments, at least two conditions must occur (e.g., condition A, condition B), and in some embodiments, at least three conditions must occur (e.g., A, B, and C, or A, B, and D). By way of example only, conditions A, B, and C must be present for gene expression to occur from a synthetic AAV having a passcode "ABC" regulatory switch. Conditions A, B, and C can be as follows: Condition A is the presence of a condition or disease, Condition B is a hormonal response, and Condition C is a response to transgene expression. For example, if the transgene edits a defective EPO gene, Condition A is the presence of chronic kidney disease (CKD), Condition B occurs when the subject has hypoxia in the kidney, and Condition C occurs when erythropoietin-producing cell (EPC) recruitment in the kidney is impaired or HIF-2 activation is impaired. When oxygen levels increase or a desired level of EPO is reached, the transgene will be turned off and back on again until one of the three conditions occurs.

[0291] In some embodiments, part II or part III of the passcode-controlled switch or "passcode circuit" encompassed for use in the synthetically produced single-stranded DNA (ssDNA) molecules described herein includes hybrid transcription factors (TFs) to expand the range and complexity of environmental signals used to define biocontainment conditions. In contrast to dead-man switches that induce cell death in the presence of predetermined conditions, "passcode circuits" allow cell survival or transgene expression in the presence of specific "passcodes" and can be easily reprogrammed to allow transgene expression and / or cell survival only when the predetermined environmental conditions or passcodes are present.

[0292] Any and all combinations of the regulatory switches disclosed herein, e.g., small molecule switches, nucleic acid-based switches, small molecule-nucleic acid hybrid switches, post-transcriptional transgene regulatory switches, post-translational regulatory switches, radiation-controlled switches, hypoxia-mediated switches, and other regulatory switches disclosed herein and known to those of skill in the art, can be used in the passcode regulatory switches disclosed herein. Regulatory switches encompassed for use are also discussed in the review Kis et al., JR Soc Interface. 12:20141000 (2015) and summarized in Table 1 of Kis et al. In some embodiments, regulatory switches for use in the passcode system can be selected from any or combination of the switches in Table 4 below.

[0293] (iv) a nucleic acid-based regulatory switch for controlling transgene expression In some embodiments, the control switch for controlling the transgene expressed by the synthetically produced single-stranded DNA (ssDNA) molecule described herein to part II or part III is based on a nucleic acid-based control mechanism.Exemplary nucleic acid control mechanisms are known in the art and are envisioned for use.For example, such mechanisms include riboswitches, such as those disclosed in, for example, US Patent Application Publication No. 2009 / 0305253, US Patent Application Publication No. 2008 / 0269258, US Patent Application Publication No. 2017 / 0204477, International Publication No. 2018026762A1, US Patent No. 9,222,093, and European Patent Application No. 288071, and also disclosed in the review article by Prain JK et al., Microbiol Spectr. 2018 May;6(3). Also included are metabolite-responsive transcriptional biosensors such as those disclosed in International Publication Nos. WO 2018 / 075486 and WO 2017 / 147585. Other art-known mechanisms envisioned for use include silencing transgenes using siRNA or RNAi molecules (e.g., miR, shRNA). For example, the single-stranded DNA (ssDNA) molecule described in Section II or Section III may contain a control switch encoding an RNAi molecule complementary to the transgene expressed by the single-stranded DNA (ssDNA) molecule described in Section II or Section III. When such an RNAi is expressed, the transgene is silenced by the complementary RNAi molecule, even when expressed by the single-stranded DNA (ssDNA) molecule described herein in Section II or Section III; when the transgene is expressed by the single-stranded DNA (ssDNA) molecule described herein in Section II or Section III, the transgene is not silenced by the RNAi.

[0294] In some embodiments, the control switch is a tissue-specific self-inactivating control switch, e.g., as disclosed in U.S. Patent Application Publication No. 2002 / 0022018, whereby the control switch purposefully switches off transgene expression at sites where transgene expression would otherwise be disadvantageous. In some embodiments, the control switch is a recombinase reversible gene expression system, e.g., as disclosed in U.S. Patent Application Publication No. 2014 / 0127162 and U.S. Patent No. 8,324,436.

[0295] (v) Post-transcriptional and post-translational control switches. In some embodiments, the regulatory switch for controlling the transgene or gene of interest expressed by the synthetically produced single-stranded DNA (ssDNA) molecule described herein in part II or part III is a post-transcriptional modification system. For example, such regulatory switches are described in US2018 / 0119156, GB201107768, WO2001 / 064956A3, EP2707487, and Beilstein et al., ACS Synth. Biol., 2015, 4(5), pp 526-534; Zhong et al., Elife. 2016 Nov 2;5.pii:e18858. In some embodiments, a person skilled in the art can encode both the transgene and the inhibitory siRNA containing the ligand-sensitive (off-switch) aptamer, and the end result is expected to be a ligand-sensitive on-switch.

[0296] (vi) Other exemplary control switches Any known regulatory switch in a synthetically produced ssDNA molecule can be used to control gene expression of a transgene expressed by a single-stranded DNA (ssDNA) molecule described in Section II or Section III, including those triggered by environmental changes. Additional examples include, but are not limited to, Suzuki et al., Scientific Reports 8; 10051 (2018), genetic code expansion and non-physiological amino acids, radiation-regulated or ultrasound-regulated on / off switches (see, e.g., Scott S et al., Gene Ther. 2000). Jul;7(13):1121-5, U.S. Patent Nos. 5,612,318, 5,571,797, 5,770,581, 5,817,636, and WO 1999 / 025385A1. In some embodiments, the control switch is controlled by an implantable system, e.g., as disclosed in U.S. Patent No. 7,840,263, U.S. Patent Application Publication No. 2007 / 0190028, and gene expression is controlled by one or more forms of energy, including electromagnetic energy, that activates a promoter operably linked to a transgene in a single-stranded DNA (ssDNA) molecule described in Part II or Part III.

[0297] In some embodiments, regulatory switches contemplated for use in the synthetically generated single-stranded DNA (ssDNA) molecules described in Part II or III herein are hypoxia-dependent or stress-activated switches, including, for example, those disclosed in WO 1999 / 060142A2, U.S. Patent Nos. 5,834,306, 6,218,179, and 6,709,858, U.S. Patent Application No. 2015 / 0322410, and Greco et al. (2004) Targeted Cancer Therapies 9, S368. Also included are FROG, TOAD, NRSE elements, and conditionally inducible silence elements (e.g., hypoxia response elements (HREs), inflammatory response elements (IREs), and shear-responsive activation elements (SSAEs)), including, for example, those disclosed in U.S. Patent No. 9,394,526. Such embodiments are useful for turning on transgene expression from single-stranded DNA (ssDNA) molecules described herein after ischemia or in ischemic tissues and / or tumors.

[0298] H. Kill Switch Other embodiments of the present disclosure relate to synthetically produced single-stranded DNA (ssDNA) molecules described in Part II or Part III and dsDNA molecules comprising a kill switch. The kill switch disclosed herein allows cells containing the single-stranded DNA (ssDNA) molecules described in Part II or Part III to be killed or undergo programmed cell death as a means of permanently removing the introduced single-stranded DNA (ssDNA) molecules described in Part II or Part III from a subject's system. Those skilled in the art will understand that the use of a kill switch in a synthetically produced single-stranded DNA (ssDNA) molecule described herein in Part II or Part III of the present disclosure is typically coupled with targeting of the single-stranded DNA (ssDNA) molecules described herein in Part II or Part III to a limited number of cells that a subject can acceptably lose, or to a cell type (e.g., cancer cells) in which apoptosis is desired. In all aspects, the "kill switches" disclosed herein are designed to provide rapid and robust cell killing of cells containing a single-stranded DNA (ssDNA) molecule described in Part II or Part III in the absence of an input survival signal or other specific condition. Stated another way, a kill switch encoded by a single-stranded DNA (ssDNA) molecule described in Part II or Part III herein can restrict cell survival of cells containing a single-stranded DNA (ssDNA) molecule described in Part II or Part III herein to an environment defined by a specific input signal. Such a kill switch functions as a biological containment function when it is desirable to remove a synthetically produced single-stranded DNA (ssDNA) molecule described herein in Part II or Part III from a subject or when it is desirable to ensure that the encoded transgene is not expressed.

[0299] Thus, a kill switch is a synthetic biological circuit within a ssDNA molecule or dsDNA construct that links an environmental signal to the conditional survival of a cell containing the ssDNA molecule or dsDNA construct. In some embodiments, different ssDNA molecules or dsDNA constructs can be designed with different kill switches.

[0300] In some embodiments, the single-stranded DNA (ssDNA) molecules described in Part II or Part III can include a kill switch, a modular biological containment circuit. In some embodiments, a kill switch incorporated for use in an ssDNA molecule or dsDNA construct is disclosed in WO 2017 / 059245, which describes a switch called a "dead man kill switch" that includes a mutually inhibitory arrangement of at least two repressible sequences such that an environmental signal suppresses the activity of a second molecule in the construct (e.g., using a small molecule-binding transcription factor to produce a "survival" state due to the suppression of toxin production). In a cell containing a single-stranded DNA (ssDNA) molecule described in Part II or Part III herein, upon loss of the environmental signal, the circuit permanently switches to a "dead" state, where the toxin is now suppressed, resulting in toxin production that kills the cell. In another embodiment, a synthetic biological circuit called a "code circuit" or "code kill switch" is provided that uses hybrid transcription factors (TFs) to construct complex environmental requirements for cell survival. The deadman and passcode kill switches described in WO2017 / 059245 are particularly useful for use with single-stranded DNA (ssDNA) molecules described in Part II or Part III because they are modular and customizable, both with respect to the environmental conditions that control circuit activation and the output modules that control cell fate. By appropriate selection of toxins, including but not limited to endonucleases such as EcoRI, the passcode circuits present in the ssDNA molecule or dsDNA construct can be used not only to kill host cells containing the ssDNA molecule or dsDNA construct, but also to degrade its genome and associated plasmids.

[0301] Other kill switches known to those of skill in the art are encompassed for use with the single-stranded DNA (ssDNA) molecules described in Part II or Part III disclosed herein, and are disclosed, for example, in U.S. Patent Application Publication Nos. 2010 / 0175141, 2013 / 0009799, 2011 / 0172826, 2013 / 0109568, and reviewed in Jusiak et al., Reviews in Cell Biology and molecular Medicine, 2014, 1-56; Kobayashi et al., PNAS, 2004; 101; 8419-9; Marchisio et al., Int. Journal of Biochem and Cell Biol., 2011; 43; 310-319; and Reinshagen et al., Science Translational Medicine, 2018, 11.

[0302] Thus, in some embodiments, a single-stranded DNA (ssDNA) molecule described in Part II or Part III can comprise a kill-switch nucleic acid construct comprising a nucleic acid encoding an effector toxin or reporter protein, wherein expression of the effector toxin (e.g., death protein) or reporter protein is controlled by a predetermined condition. For example, the predetermined condition can be the presence of an environmental factor, e.g., an exogenous factor, without which the cell will not express the effector toxin (e.g., death protein) and will die. In an alternative embodiment, the predetermined condition is the presence of two or more environmental agents, e.g., the cell will survive only when two or more necessary exogenous agents are provided, and cells containing a single-stranded DNA (ssDNA) molecule described herein, but not Part II or Part III, will die.

[0303] In some embodiments, the single-stranded DNA (ssDNA) molecules described in Section II or Section III are modified to incorporate a kill switch for destroying cells containing the single-stranded DNA (ssDNA) molecules described in Section II or Section III, effectively terminating in vivo expression of a transgene expressed by the ssDNA molecule or dsDNA construct (e.g., a therapeutic gene, protein, or peptide, etc.). Specifically, the single-stranded DNA (ssDNA) molecules described in Section II or Section III are further engineered to express a switch protein that does not function in mammalian cells under normal physiological conditions. Only upon administration of a drug or environmental condition that specifically targets this switch protein are cells expressing the switch protein destroyed, thereby terminating expression of the therapeutic protein or peptide. For example, it has been reported that cells expressing HSV thymidine kinase can be killed upon administration of drugs such as ganciclovir and cytosine deaminase. See, for example, Day and Evans, Suicide Gene Therapy by Herpes Simplex Virus-1 Thymidine Kinase (HSV-TK), in Targets in Gene Therapy, edited by You (2011), and Bertinger et al., Proc. Natl. Acad. Scientific USA 96(15):8699-8704 (1999). In some embodiments, the ssDNA molecule or dsDNA construct can contain an siRNA kill switch called DISE (Death Induced by Survival Gene Elimination) (Murmann et al., Oncotarget. 2017;8:84643-84658. Induction of DISE in ovarian cancer cells in vivo).

[0304] In some embodiments, a deadman kill switch is a biological circuit or system that sensitizes a cellular response to a predetermined condition, such as a factor in a cellular growth environment, e.g., the absence of an exogenous factor. Such a circuit or system can comprise a nucleic acid construct comprising an expression module forming a deadman control circuit sensitive to the predetermined condition, the construct comprising an expression module forming a control circuit, the construct comprising a first repressor protein expression module, the first repressor protein binding element binding to a first repressor protein nucleic acid binding element and repressing transcription from a coding sequence containing the first repressor protein binding element, the repression activity of the first repressor protein being sensitive to inhibition by a first exogenous agent, the presence or absence of the first exogenous agent establishing the predetermined condition;

[0305] ii) a second repressor protein expression module, wherein the second repressor protein binds to a second repressor protein nucleic acid binding element and represses transcription from a coding sequence that includes the second repressor protein binding element, and the second repressor protein is different from the first repressor protein; and

[0306] iii) an effector expression module, comprising a nucleic acid sequence encoding an effector protein, operably linked to a genetic element comprising a binding element for said second repressor protein, said second expression module comprising a first repressor protein nucleic acid binding element that enables repression of transcription of said second repressor protein when said element is bound by said first repressor protein, such that expression of said second repressor protein causes repression of effector expression from said effector expression module, said respective modules comprising a first repressor protein nucleic acid binding element that enables repression of transcription of said second repressor protein when said element is bound by said first repressor protein, said first repressor protein being capable of inhibiting expression of said first repressor protein in the absence of said first exogenous agent; a first exogenous agent produced from the current module and repressing transcription from the second repressor protein expression module, resulting in expression of an effector protein such that repression of effector expression by the second repressor protein is relieved, but in the presence of a first exogenous agent, the activity of the first repressor protein is inhibited, allowing expression of the second repressor protein; and maintaining expression of effector protein expression in an "off" state, wherein removal or absence of the first exogenous agent defaults to expression of the effector protein such that the first exogenous agent is required by the circuit to maintain effector protein expression in an "off" state.

[0307] In some embodiments, the effector is a toxin or protein that induces a cell death program. Any protein toxic to host cells can be used. In some embodiments, the toxin kills only the cell in which it is expressed. In other embodiments, the toxin kills other cells in the same host organism. Any of a number of products that lead to cell death can be used in the dead-man kill switch. Agents that inhibit DNA replication, protein translation, or other processes, or agents that degrade host cell nucleic acids, for example, are particularly useful. To identify an efficient mechanism for killing host cells upon circuit activation, several toxin genes that directly damage host cell DNA or RNA were tested. The endonuclease ecoRI, the DNA zyrase inhibitor ccdB, and the ribonuclease-type toxin mazF were tested because they are well characterized, native to E. coli, and offer a broad killing mechanism. To increase circuit robustness and provide an independent method of circuit-dependent cell death, the system can be further adapted to express, for example, targeted proteases or nucleases that further interfere with repressors that maintain death genes in an "off" state. Upon loss or withdrawal of the survival signal, death gene repression is removed even more efficiently, for example, by active degradation of the repressor protein or its message. As a non-limiting example, mf-Lon protease was used to not only degrade LacI but also target proteins essential for degradation. The mf-Lon degradation tag pdt#1 can be attached to the 3' ends of five essential genes whose protein products are particularly sensitive to mf-Lon degradation, and cell viability was measured after removal of aTc. Of the essential gene targets tested, the peptidoglycan biosynthesis gene murC provided the strongest and fastest cell death phenotype (1 x 10 viability within 6 hours). -4 less than).

[0308] As used herein, the term "predetermined input" refers to an agent or condition that affects the activity of a transcription factor polypeptide in a known manner. Generally, such factors can bind to and / or change the conformation of a transcription factor polypeptide, thereby modifying the activity of the transcription factor polypeptide. Examples of predetermined inputs include, but are not limited to, environmental input factors that are not required for the survival of a given host organism (i.e., in the absence of a synthetic biological circuit described herein). Conditions under which a predetermined input can be provided include, for example, temperature, e.g., a temperature at which the activity of one or more factors is temperature-sensitive, the presence or absence of light, including light of a given wavelength spectrum, and concentrations of gases, salts, metals, or minerals. Environmental input factors include, for example, concentrations of small molecules, biological agents, e.g., pheromones, hormones, growth factors, metabolites, nutrients, etc., and their analogs, chemicals, environmental by-products, metal ions, and other such molecules or agents, light levels, temperature, mechanical stress or pressure, or electrical signals, e.g., current and voltage.

[0309] In some embodiments, reporters are used to quantify the strength or activity of signals received by modules or programmable synthetic biological circuits of the present disclosure. In some embodiments, reporters can be fused in-frame to other protein-coding sequences to identify where the proteins are located in a cell or organism. Luciferase can be used as an effector protein in various embodiments described herein, for example, to measure low levels of gene expression, because in the absence of luciferase, cells tend to have little or no background luminescence. In other embodiments, enzymes that produce colored substrates can be quantified using a spectrophotometer or other instrument capable of measuring absorbance, including a plate reader. Similar to luciferase, enzymes such as β-galactosidase tend to amplify low signals and can therefore be used to measure low levels of gene expression. In some embodiments, the effector protein can be an enzyme that can degrade or otherwise destroy a given toxin. In some embodiments, the effector protein can be an odorant enzyme that converts a substrate into an odorant product. In some embodiments, the effector protein can be an enzyme that phosphorylates or dephosphorylates either small molecules or other proteins, or that methylates or demethylates other proteins or DNA.

[0310] In some embodiments, the effector protein can be a receptor, ligand, or lytic protein. Receptors tend to have three domains: an extracellular domain for binding ligands such as proteins, peptides, or small molecules; a transmembrane domain; and an intracellular or cytoplasmic domain, which may frequently be involved in some signaling event, such as phosphorylation. In some embodiments, transporter, channel, or pump gene sequences are used as effector proteins. Non-limiting examples and sequences of effector proteins for use in the kill switches described herein can be found at the Registry of Standard Biological Parts on the World Wide Web at parts.igem.org.

[0311] As used herein, a "regulator protein" is a protein that regulates expression from a target nucleic acid sequence. Modulator proteins include, for example, transcription factors, including transcription activators and repressors, as well as proteins that bind to or modify transcription factors to affect their activity. In some embodiments, modulator proteins include, for example, proteases that degrade protein factors involved in regulating expression from a target nucleic acid sequence. Preferred modulator proteins include, for example, modular proteins in which the DNA-binding and input factor binding or response elements or domains are separable and transportable, such that, for example, fusing the DNA-binding domain of a first modulator protein to the input factor response domain of a second modulator protein results in a new protein that binds to the DNA sequence recognized by the first protein but is sensitive to the input factor to which the second protein normally responds. Thus, as used herein, the terms "regulator polypeptide," and more specifically "repressor polypeptide," include not only the particular polypeptide, but also "LacI (repressor) polypeptides, variants, or derivatives of such polypeptides that respond to different or variant input agents. Thus, in the case of LacI polypeptides, LacI mutants or variants that bind agents other than lactose or IPTG are included. A wide variety of such agents are known in the art.

[0312] Table 4. Exemplary regulatory switches: b Switching to the ON state is accomplished by an effector, and by any means other than removing the effector that confers the OFF state. c The switch to the OFF state is effected by an effector and by any means other than removing the effector that confers the ON state. d Ligands or other physical stimuli (e.g., temperature, electromagnetic waves, electricity) stabilize the switch in either the ON or OFF state. eSee reference numbers cited in Kis et al., JR Soc Interface. 12:20141000 (2015), which article and all references cited therein are incorporated herein in their entirety. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0313] IV. Synthesis Preparation Method The methods and compositions provided herein are based in part on the discovery of synthetic and cell-free production processes and methods useful for producing single-stranded (ssDNA) molecules as described herein in Part II or Part III. According to some embodiments, in the method for producing ssDNA molecules, PS bond replaces the sulfur atom of the non-bridging oxygen in the phosphate backbone of oligonucleotide. Advantageously, this modification stabilizes nucleic acid and makes internucleotide bond resistant to nuclease degradation.

[0314] The present disclosure provides methods for the synthetic production of single-stranded DNA (ssDNA) molecules described in Part II or Part III, and double-stranded closed-ended DNA (ceDNA) molecules described herein.

[0315] According to some embodiments, the disclosed methods and / or production steps are performed entirely in a cell-free environment. According to some embodiments, the disclosed methods and / or production steps are performed partially in a cell-free environment. According to some embodiments, the dsDNA constructs (e.g., linear, double-stranded ceDNA) are synthetically produced in vitro. According to some embodiments, the dsDNA constructs (e.g., double-stranded ceDNA) are synthetically produced in vitro in a cell-free environment.

[0316] According to some embodiments, the ssDNA molecule is synthetically generated in vitro. According to further embodiments, the ssDNA molecule is synthetically generated in vitro in a cell-free environment. According to some embodiments, the ssDNA molecule is synthetically produced from a double-stranded DNA (dsDNA) construct. According to further embodiments, the dsDNA construct comprises a double-stranded transgene cassette comprising at least one double-stranded transgene, a first ITR, and an optional second ITR. According to other further embodiments, the dsDNA construct comprises a double-stranded transgene cassette comprising at least one double-stranded transgene, a first ITR, and a second ITR.

[0317] According to one aspect, the present disclosure provides a method for synthetically producing a ssDNA molecule, e.g., a synthetic AAV vector, e.g., a single-stranded (ss) synthetic AAV vector described herein, from a double-stranded DNA (dsDNA) construct, the method comprising: a) contacting the dsDNA construct with one or more nick endonucleases that nick one of the single strands of the dsDNA construct at one or more nick sites; and b) contacting the dsDNA construct with an exonuclease capable of removing nucleotides from the nicked strand of the dsDNA construct, thereby producing a ssDNA molecule. In some embodiments, the method does not include a purification step between steps a) and b). In other embodiments, the method includes a purification step between steps a) and b). An advantage of the present disclosure is that the method for synthetically producing a ssDNA molecule does not include a purification step after step a), which reduces or minimizes DNA loss.

[0318] According to some embodiments, step a) comprises contacting the dsDNA construct with a single nick endonuclease. According to further embodiments, the single nick endonuclease generates a single nick on one of the single strands of the dsDNA construct. In other further embodiments, the single nick endonuclease is Nb.BbvCI or an isoschizomer thereof, or the single nick endonuclease is Nb.BtsI or an isoschizomer thereof.

[0319] According to some embodiments, the exonuclease can remove the nicked strand of the dsDNA construct, starting at one or more nick sites and ending with one or more phosphorothioate-modified nucleotides. The exonuclease can be selected from, but not limited to, T7 exonuclease, lambda exonuclease, T5 exonuclease, and exonuclease V. According to some embodiments, the exonuclease is T7 exonuclease.

[0320] According to some embodiments, steps a) and b) are carried out simultaneously or sequentially in a single reaction vessel. According to some embodiments, when T7 exonuclease is used, steps a) and b) are carried out sequentially due to the short digestion reaction of T7 exonuclease.

[0321] According to some embodiments, the one or more nick endonucleases are selected from Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BssSI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII, and isoschizomers of any of the foregoing. In some embodiments, the one or more nick endonucleases comprise Nb.BbvCI or an isoschizomer thereof. In some embodiments, the one or more nick endonucleases comprise Nb.BtsI or an isoschizomer thereof.

[0322] According to some embodiments, one or more nick sites are about 0 to about 20 nucleotides downstream of the end resolving region (trs), e.g., about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream of the end resolving region (trs), or, e.g., about 0 to about 15, about 0-10, about 0-5, about 5-15, about 10-20, about 15-20, about 10-20, or about 5-20 nucleotides downstream of the end resolving region (trs). According to some embodiments, there is only one nick site.

[0323] According to another aspect, the present disclosure provides a method for synthetically producing a dsDNA construct (e.g., a ds closed-end DNA), the method comprising: a) contacting a dsDNA template with at least one restriction endonuclease, wherein the template comprises a double-stranded transgene cassette comprising at least a double-stranded transgene, wherein the template comprises a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the double-stranded transgene cassette, a second non-palindromic restriction endonuclease recognition and a corresponding second cleavage site downstream of the double-stranded transgene cassette, wherein the at least one restriction endonuclease is capable of cleaving the template at the first and second cleavage sites to release an insert having single-stranded overhangs at the 5' and 3' ends of the insert; and b) ligating the 5' and 3' ends of the insert to a first inverted terminal repeat (ITR) oligonucleotide and an optional second ITR oligonucleotide to form a dsDNA construct.

[0324] The single-stranded DNA (ssDNA) molecules described in Sections II or III of the present specification have an advantage over other vectors in that they can be used more safely to express transgenes in cells, tissues, or subjects compared to DNA vectors produced in a cell culture environment (e.g., insect cell lines such as Sf9 cell lines, yeast cells, or mammalian cell lines such as HEK293). That is, producing linear vectors by such cell-free methods can minimize potentially undesirable side effects because the resulting vectors are free of bacterial or insect cell contaminants. This synthetic production method may also result in a desired vector of higher purity. This synthetic production method may also be more efficient and / or cost-effective than traditional cell-based production methods for such vectors. Vectors synthesized as described herein can express any desired transgene, for example, a transgene for treating or curing a given disease. Those skilled in the art will readily recognize that any transgene used in conventional gene therapy methods using conventional recombinant vectors can be adapted for expression by single-stranded DNA (ssDNA) molecules and dsDNA constructs (e.g., ds ceDNA) generated by the methods described herein, for example, without the limitations of size capabilities of the transgene insert.

[0325] It should be understood that the production process of the present disclosure can potentially be carried out in a completely cell-free environment if desired. However, depending on the starting material, some DNA components can be obtained from nucleotide fragments originally prepared in cells (e.g., plasmid-ceDNA, AAV vectors produced from insect cells). In some embodiments, non-viral ssDNA can be prepared by introducing nicks at desired positions and lengths into existing double-stranded ceDNA vectors produced by cell replication (e.g., insect or mammalian cell lines), which have a designed sequence of nicking endonuclease binding sites in the stems of the ITRs and contain one or more phosphorothioate-modified nucleotides that form PS bonds in one or both ITRs. In other embodiments, single-stranded DNA (ssDNA) molecules (e.g., synthetic AAV vectors, e.g., single-stranded (ss) synthetic AAV vectors) are synthesized from double-stranded ceDNA with PS bonds in a cell-free manner. As described herein, one result of adding phosphorothioate linkages is the stabilization of the ITRs, ensuring precision in the location where the exonuclease functions to produce ssDNA.

[0326] It will be understood by those skilled in the art that one or more enzymes or one or more oligonucleotide components for the present synthetic production methods can be produced from cells and used in the disclosed methods in purified form. Thus, in some embodiments, the present synthetic production methods are cell-free methods, but the restriction enzymes and / or ligase enzymes can be produced from cells.

[0327] In one embodiment, the restriction endonuclease and / or ligatable protein may be expressed or provided from an expression vector in a cell, e.g., a bacterial cell. In one embodiment, a cell, such as a bacterial cell, may be present that contains an expression vector expressing one or more of the restriction endonuclease or ligase enzymes. Thus, while the methods disclosed herein are primarily directed to cell-free synthetic methods for producing the ssDNA molecules disclosed herein, in some embodiments, synthetic production methods are also encompassed in which cells, e.g., bacterial cells, but not insect cells, are present and can be used to express one or more of the enzymes required for the method. In such embodiments, the cell expressing the restriction endonuclease and / or ligatable protein is not an insect cell. In all embodiments in which a cell is present and expresses one or more restriction endonucleases or proteins with ligation capabilities, the cell does not replicate single-stranded DNA (ssDNA) molecules (e.g., synthetic AAV vectors, e.g., single-stranded (ss) synthetic AAV vectors). In other words, the cell's intracellular machinery does not replicate or participate in replicating single-stranded DNA (ssDNA) molecules (e.g., synthetic AAV vectors, e.g., single-stranded (ss) synthetic AAV vectors).

[0328] A. Isolation and Purification Described herein are methods for generating and isolating single-stranded DNA (ssDNA) molecules (e.g., synthetic AAV vectors, e.g., single-stranded (ss) synthetic AAV vectors) and dsDNA constructs (e.g., ds ceDNA). For example, the single-stranded DNA (ssDNA) molecules and dsDNA constructs (e.g., ds ceDNA) described in Section II or Section III produced by the synthesis methods described herein can be harvested or collected at an appropriate time point after the final ligation reaction and optimized to achieve high-yield vector production. The ssDNA molecules and dsDNA constructs (e.g., ds ceDNA) can be purified by any means known to those skilled in the art for purifying DNA. In one embodiment, the ssDNA molecules or dsDNA constructs (e.g., ds ceDNA) are purified as DNA molecules. Generally, any nucleic acid purification method known in the art can be employed, as well as commercially available DNA extraction kits.

[0329] Purification can be carried out by subjecting the reaction mixture to chromatographic separation. As a non-limiting example, this process can be carried out by loading the reaction mixture onto an ion exchange column (e.g., SARTOBIND Q®) that retains nucleic acids, followed by elution (e.g., with a 1.2 M NaCl solution), and further chromatographic purification on a gel filtration column (e.g., 6 Fast Flow GE). The DNA vector is then recovered, for example, by precipitation.

[0330] The presence of ssDNA molecules or dsDNA constructs can be confirmed by digesting the vector DNA with a restriction enzyme that has a single recognition site on the DNA vector and analyzing both the digested and undigested DNA material using gel electrophoresis to confirm the presence of the characteristic band of linear, continuous DNA compared to linear, non-contiguous, single-stranded DNA known in the art.

[0331] In some embodiments, ssDNA molecules or dsDNA constructs can be delivered to target cells in vitro or in vivo by various suitable methods discussed herein. The vector alone can be applied or injected. The vector can be delivered to cells without the aid of transfection reagents or other physical means. Alternatively, the vector can be delivered using transfection reagents or other physical means that facilitate DNA entry into cells, such as liposomes, alcohol, polylysine-rich compounds, arginine-rich compounds, calcium phosphate, microvesicles, and microinjection.

[0332] V. Gene Editing Applications In some aspects, the present disclosure provides a gene editing system comprising an ssDNA molecule described herein, at least one guide RNA (gRNA), and at least one site-specific nuclease or a messenger RNA (mRNA) encoding at least one site-specific nuclease.

[0333] A. Nucleic acid-guided endonucleases Different types of nucleic acid-guided endonucleases can be used in the compositions and methods of the present invention to facilitate gene editing. Exemplary, non-limiting types of nucleic acid-guided endonucleases suitable for the compositions and methods of the present invention include RNA-guided endonucleases, DNA-guided endonucleases, and single-base editors.

[0334] In some embodiments, the nuclease can be an RNA-guided endonuclease.As used herein, the term RNA-guided endonuclease refers to an endonuclease that forms a complex with an RNA molecule that comprises a region that is complementary to a selected target DNA sequence, so that the RNA molecule binds to the selected sequence and directs endonuclease activity to the selected target DNA sequence.

[0335] In one embodiment, the RNA-guided endonuclease is a CRISPR enzyme. In some embodiments, the RNA-guided endonuclease comprises nickase activity. In some embodiments, the RNA-guided endonuclease induces cleavage of one or both strands at the location of the target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the RNA-guided endonuclease induces cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence. In other embodiments, the nickase activity targets one or more sequences on the ceDNA vector itself, for example, to relax sequence constraints so that the HDR template is exposed to HDR interaction with the genomic sequence of the target gene.

[0336] In certain embodiments, the nicase is intended to cleave at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more sites in a desired nucleic acid sequence (e.g., one or more regions of an ssDNA molecule or dsDNA construct). In another embodiment, the nicase is intended to cleave at one and / or two sites via transnicking. Transnicking can enhance genome editing by HDR, which is high-fidelity and produces fewer errors, thus reducing undesirable off-target effects.

[0337] In some embodiments, the expression construct or vector encodes an RNA-guided endonuclease that is mutated relative to the corresponding wild-type enzyme such that the mutant endonuclease lacks the ability to cleave a single strand of a target polynucleotide containing the target sequence.

[0338] In some embodiments, the nucleic acid sequence encoding the RNA-guided endonuclease is codon-optimized for expression in a specific cell, such as a eukaryotic cell. The eukaryotic cell may be derived from a specific organism, such as a mammal. Non-limiting examples of mammals include humans, mice, rats, rabbits, dogs, or non-human primates. Generally, codon optimization refers to the process of modifying a nucleic acid sequence for enhanced expression in a target host cell by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence.

[0339] In some embodiments, the RNA-guided endonuclease is part of a fusion protein that includes one or more heterologous protein domains (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the endonuclease). The RNA-guided endonuclease fusion protein can include any additional protein sequences and, optionally, a linker sequence between any two domains. Examples of protein domains that can be fused to the RNA-guided endonuclease include, but are not limited to, epitope tags, reporter gene sequences, purification tags, fluorescent proteins, and protein domains with one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, SV-G tag, glutathione-S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein (MBP), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, nus, Softag1, Softag3, Strp, SBP, Glu-Glu, HSV, KT3, S, SI, T7, biotin carboxyl carrier protein (BCCP), calmodulin, and thioredoxin (Trx) tags.Examples of reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, emerald, thistle green, monomeric thistle green, CopGFP, AceGFP, ZsGreen1), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (e.g., YFP, EYFP, citrine, Venus yPet, PhiYFP, ZsYellow1), cyan fluorescent protein (e.g., ECFP, cerulean, CyPet Autofluorescent proteins include, but are not limited to, red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRaspberry, mStrawberry JRed), orange fluorescent proteins (e.g., mOrange, mKO, Kusabira Orange Monomer, Kusabira Orange, mTangin, tdTomato), and blue fluorescent proteins (BFP). RNA-guided endonucleases can be fused to gene sequences encoding proteins or protein fragments that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA-binding domain (DBD) fusions, GAL4 DNA-binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. In some embodiments, a tagged endonuclease is used to identify the location of the target sequence.

[0340] It is contemplated herein that at least two (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, or more) different Cas enzymes are administered or contacted with cells substantially simultaneously. Any combination of double-strand break-inducing Cas enzymes, Cas nickases, catalytically inactive Cas enzymes (e.g., dCas9), modified Cas enzymes, truncated Cas9, etc. is contemplated for use in combination with the methods and compositions described herein.

[0341] In some embodiments, nucleic acid-guided endonuclease is DNA-guided endonuclease.See, for example, Varshney and Burgess Genome Biol.17:187 (2016).In one embodiment, the enzyme involved in DNA repair and / or replication can be fused to endonuclease to form DNA-guided nuclease. One non-limiting example is the fusion of Flap Endonuclease 1 (FEN-1) to Fok1 endonuclease (Xu et al., Genome Biol. 17:186 (2016)). In another embodiment, a naturally occurring DNA-guided nuclease may be used. A non-limiting example of such a natural nuclease is a prokaryotic endonuclease from the Argonaute protein family (see Kropocheva et al., FEBS Open Bio. 8(S1):P01-074(2018)). In some embodiments, the nucleic acid-guided endonuclease is a "single base editor," a chimeric protein composed of a DNA-targeting module and a catalytic domain that can modify a single type of nucleotide base (Rusk, N, Nature Methods 15:763 (2018); Eid et al., Biochem J. 475(11): 1955-64 (2018)). Such single-base editors cannot generate double-strand breaks in the target DNA, resulting in DNA base editing, thereby limiting the generation of insertions and deletions (e.g., indels), thus improving the fidelity of the editing process. Different types of single-base editors are known. For example, cytidine deaminase (an enzyme that catalyzes the conversion of cytosine to uracil) can be coupled to a nuclease such as APOBEC-dCas9, where APOBEC contributes the cytidine deaminase functional group and, guided by dCas9, deamidates the specific cytidine ...

Claims

1. A lipid nanoparticle (LNP), (a) a linear single-stranded deoxyribonucleic acid (ssDNA) molecule comprising at least one nucleic acid sequence of interest; and (b) A lipid, comprising:

2. 2. The LNP of claim 1, wherein the ssDNA molecule is single-stranded along its entire length.

3. The LNP of claim 1 or 2, wherein the ssDNA molecule does not contain any viral-derived sequences.

4. The LNP of any one of claims 1 to 3, wherein the ssDNA molecule is at least 200 nucleotides in length.

5. 5. The LNP of any one of claims 1-4, wherein the ssDNA molecule is at least 300 nucleotides in length, at least 400 nucleotides in length, at least 500 nucleotides in length, at least 600 nucleotides in length, at least 700 nucleotides in length, at least 800 nucleotides in length, at least 900 nucleotides in length, at least 1000 nucleotides in length, at least 1500 nucleotides in length, at least 2000 nucleotides in length, at least 2500 nucleotides in length, at least 3000 nucleotides in length, at least 3500 nucleotides in length, at least 4000 nucleotides in length, at least 4500 nucleotides in length, at least 5000 nucleotides in length, at least 5500 nucleotides in length, at least 6000 nucleotides in length, at least 6500 nucleotides in length, at least 7000 nucleotides in length, at least 7500 nucleotides in length, at least 8000 nucleotides in length, at least 8500 nucleotides in length, at least 9000 nucleotides in length, at least 9500 nucleotides in length, or at least 10000 nucleotides in length.

6. The LNP of any one of claims 1 to 5, wherein the at least one nucleic acid sequence of interest is adjacent to at least one stem-loop structure at its 3' end, and the at least one stem-loop structure comprises at least one stem and at least one loop.

7. The LNP of claim 6, wherein the at least one stem-loop structure at the 3' end is sufficient to prime replication and / or transcription.

8. The LNP of claim 6 or 7, wherein the at least one stem at the 3' end comprises a partial DNA duplex of 4 to 500 nucleotides.

9. The LNP of any one of claims 6 to 8, wherein the at least one stem at the 3' end comprises a partial DNA duplex of 4 to 5 nucleotides.

10. The LNP of any one of claims 6 to 9, wherein the at least one loop at the 3' end comprises 3 to 500 unlinked nucleotides.

11. The LNP of any one of claims 6 to 10, wherein the at least one loop at the 3' end comprises at least three unlinked nucleotides.

12. The LNP of any one of claims 6 to 11, wherein the ssDNA molecule comprises at least two stem-loop structures at the 3' end.

13. The LNP of any one of claims 6 to 12, wherein the ssDNA molecule comprises at least three stem-loop structures at the 3' end.

14. The LNP of any one of claims 6 to 13, wherein the ssDNA molecule comprises at least four or more stem-loop structures at the 3' end.

15. The LNP of any one of claims 6 to 14, wherein the at least one stem-loop structure at the 3' end comprises a hairpin DNA structure.

16. The LNP of any one of claims 6 to 15, wherein the at least one stem-loop structure at the 3' end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadruplex DNA structure, a bulge-forming DNA structure, and a multi-branched loop structure.

17. The LNP of any one of claims 6 to 16, wherein the at least one stem-loop structure at the 3' end does not include the A region or the A' region that would be present in a wild-type AAV ITR.

18. The LNP of any one of claims 6 to 17, wherein the at least one stem-loop structure at the 3' end does not contain the A, A', D, or D' region that would be present in a wild-type AAV ITR.

19. The LNP of any one of claims 6 to 18, wherein the at least one stem-loop structure at the 3' end does not contain the A, A', B, B', C, C', D, or D' regions that would be present in a wild-type AAV ITR.

20. The LNP of any one of claims 6 to 19, wherein the at least one stem-loop structure at the 3' end does not include a rep binding element (RBE) that would be present in a wild-type AAV ITR, and / or the at least one stem-loop structure at the 3' end does not include a terminal resolving site (trs) that would be present in a wild-type ITR.

21. The LNP of any one of claims 6 to 20, wherein the ssDNA molecule does not contain any virus-derived sequences.

22. The LNP of any one of claims 6 to 21, wherein the stem at the 3' end of the ssDNA molecule comprises one or more nucleotides modified to be exonuclease resistant.

23. The LNP of any one of claims 6 to 22, wherein the 3' end of the ssDNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more nucleotides modified to be exonuclease resistant.

24. 24. The LNP of claim 23, wherein the nucleotide modified to be exonuclease resistant is a phosphorothioate-modified (PS) nucleotide.

25. The LNP of any one of claims 6 to 24, wherein the ssDNA molecule comprises at least one functional moiety.

26. The LNP of any one of claims 6 to 25, wherein the at least one stem-loop structure at the 3' end further comprises at least one functional moiety.

27. 27. The LNP of claim 25 or 26, wherein the at least one functional moiety is an aptamer.

28. 28. The LNP of claim 27, wherein the aptamer has the ability to translocate to the nucleus in a cell.

29. The LNP of any one of claims 6 to 28, wherein the ssDNA molecule comprises at least one stem-loop structure at its 5' end, and the at least one stem-loop structure at the 5' end comprises at least one stem and at least one loop.

30. 30. The LNP of claim 29, wherein the ssDNA comprises at least two stem-loop structures at the 5' end.

31. The LNP of any one of claims 29 or 30, wherein the ssDNA molecule comprises at least three stem-loop structures at the 5' end.

32. The LNP of any one of claims 29 to 31, wherein the ssDNA molecule comprises at least four or more stem-loop structures at the 5' end.

33. The LNP of any one of claims 29 to 32, wherein the at least one stem-loop structure at the 5' end comprises a hairpin DNA structure.

34. The LNP of any one of claims 29 to 33, wherein the at least one stem-loop structure at the 5' end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadruplex DNA structure, a bulge-forming DNA structure, and a multi-branched loop structure.

35. The LNP of any one of claims 29 to 34, wherein the at least one stem-loop structure at the 5' end does not include the A region or the A' region that would be present in a wild-type AAV ITR.

36. The LNP of any one of claims 29 to 35, wherein the at least one stem-loop structure at the 5' end does not contain the A, A', D, or D' region that would be present in a wild-type AAV ITR.

37. The LNP of any one of claims 29 to 36, wherein the at least one stem-loop structure at the 5' end does not contain the A, A', B, B', C, C', D, or D' regions that would be present in a wild-type AAV ITR.

38. The LNP of any one of claims 29 to 37, wherein the at least one stem-loop structure at the 5' end does not contain a rep binding element (RBE) that would be present in a wild-type ITR.

39. The LNP of any one of claims 29 to 38, wherein the at least one stem-loop structure at the 5' end does not include a terminal resolving site (trs) present in a wild-type ITR.

40. The LNP of any one of claims 29 to 39, wherein the stem at the 5' end of the ssDNA molecule comprises one or more nucleotides modified to be exonuclease resistant.

41. The LNP of any one of claims 29 to 40, wherein the 5' end of the ssDNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more nucleotides modified to be exonuclease resistant.

42. 30. The LNP of claim 29, wherein the nucleotide modified to be exonuclease resistant is a phosphorothioate-modified (PS) nucleotide.

43. The LNP of any one of claims 29 to 42, wherein the loop at the 5' end further comprises one or more nucleic acids to stabilize the end.

44. The LNP of any one of claims 29 to 43, wherein the loop at the 5' end further comprises one or more chemically modified nucleic acids.

45. The LNP of any one of claims 29 to 44, wherein the stem-loop structure at the 5' end comprises at least one functional moiety.

46. 46. ​​The LNP of claim 45, wherein the at least one functional moiety is an aptamer.

47. 47. The LNP of claim 46, wherein the aptamer has the ability to translocate to the nucleus in a cell.

48. The LNP of any one of claims 27 to 47, wherein the functional moiety is a ribozyme.

49. The LNP of any one of claims 27 to 48, wherein the functional moiety is an antisense oligonucleotide (ASO).

50. 46. ​​The LNP of any one of claims 27 or 45, wherein the functional moiety is a small interfering RNA (siRNA).

51. 46. ​​The LNP of any one of claims 27 or 45, wherein the functional moiety is an antiviral nucleoside analog (ANA).

52. The LNP of any one of claims 6 to 51, wherein the loop at the 5' end and / or the 3' end further comprises one or more triplex-forming oligonucleotides.

53. The LNP of any one of claims 6 to 52, wherein the loop at the 5' end and / or 3' end further comprises one or more gRNAs or gDNAs.

54. The LNP of any one of claims 6 to 53, wherein the loop at the 5' end and / or the 3' end further comprises one or more molecular probes.

55. The LNP of any one of claims 1 to 54, wherein the ssDNA molecule lacks any viral capsid protein coding sequence.

56. The LNP of any one of claims 1 to 55, wherein the ssDNA molecule is synthetically produced in vitro.

57. The LNP of any one of claims 1 to 56, wherein the ssDNA molecule is synthetically produced in vitro in a cell-free environment.

58. 58. The LNP of any one of claims 1-57, wherein the ssDNA molecule does not activate or minimally activates immune pathways.

59. 59. The LNP of claim 58, wherein the immune pathway is an innate immune pathway.

60. 60. The LNP of claim 59, wherein the innate immune pathway is selected from the group consisting of the cGAS / STING pathway, the TLR9 pathway, the inflammasome-mediated pathway, and combinations thereof.

61. The LNP of any one of claims 1 to 60, wherein the ssDNA molecule further comprises at least one promoter.

62. The LNP of any one of claims 1 to 61, wherein the ssDNA molecule further comprises at least one enhancer.

63. The LNP of any one of claims 61 to 62, wherein the promoter is a hAAT promoter.

64. The LNP of any one of claims 61 to 62, wherein the promoter is a TTR promoter.

65. The LNP of any one of claims 62 to 64, wherein the enhancer is a serpin (SERP) enhancer.

66. The LNP of any one of claims 61-62 or 64-65, wherein the ssDNA molecule comprises a TTR promoter and a SERP enhancer.

67. The LNP of any one of claims 61 to 66, wherein the promoter comprises a transcription start site (TSS).

68. The LNP of any one of claims 61 to 67, wherein the promoter is double-stranded.

69. The LNP of any one of claims 61 to 68, wherein the TSS is double-stranded.

70. The LNP of any one of claims 1 to 69, wherein the ss DNA molecule is capable of expressing at least one therapeutic protein or therapeutic fragment thereof.

71. 71. The LNP of claim 70, wherein the at least one therapeutic protein is selected from the group consisting of an antibody, an enzyme, a clotting factor, a transcription factor, a replication factor, a growth factor, a hormone, and a fusion protein.

72. The at least one therapeutic protein is selected from the group consisting of sickle cell anemia, melanoma, hemophilia A (factor VIII (FVIII) deficiency) and hemophilia B (factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited disorders of liver metabolism, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharide storage diseases (e.g., Hurler syndrome (MPS type I), Shelley syndrome (MPS type I-S), Hurler-Scheie syndrome (MPS type I-H-S), Hunter syndrome (MPS type II), Sanfilippo syndromes A, B, C, and D (MPS type III), A, B, C, and D), Morquio syndrome types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPSIX), Niemann-Pick disease types A / B, C1 and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III and IV, sialidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II and I II, Fabry disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipid galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, 72. The LNPs of any one of claims 70 or 71, which are useful for treating a genetic disorder selected from the group consisting of spinocerebellar degeneration, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital maculopathy, Stargardt macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency.

73. 73. A pharmaceutical composition comprising the LNP of any one of claims 1 to 72 and a pharmaceutically acceptable excipient.

74. The LNP of any one of claims 1 to 72, wherein the ssDNA molecule is encapsulated in the lipid.

75. 75. The LNP of any one of claims 1 to 72 or 74, further comprising a sterol.

76. 76. The LNP of claim 75, wherein the sterol is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brass asterol, derivatives thereof, and combinations thereof.

77. 77. The LNP of any one of claims 75 or 76, wherein the sterol is cholesterol.

78. 77. The LNP of any one of claims 75 or 76, wherein the sterol is beta-sitosterol.

79. The LNP of any one of claims 1 to 72 or 74 to 78, further comprising a non-cationic lipid.

80. The non-cationic lipid may be distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), Monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), 16-O-monomethyl, dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dicoil phosphatidylcholine (DEPC), palmitoyl erythritol phosphatidylglycerol (POPG), diazidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,80. The LNP of claim 79, selected from the group consisting of 2-diphthanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecytin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

81. 81. The LNP of claim 79 or claim 80, wherein the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylethanolamine (DOPE).

82. The LNP of any one of claims 1 to 72 or 74 to 81, further comprising at least one PEGylated lipid.

83. The at least one PEGylated lipid is selected from the group consisting of PEG-dilauryloxypropyl, PEG-dimyrityloxypropyl, PEG-dipalmityloxypropyl, PEG-distearyloxypropyl, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (DMG-PEG), PEG-diaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-diaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disteryl glycamide, (l-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaotanyl]carbamoyl-ω-methyl-poly(ethylene glycol) (PEG-cholesterol), 3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether (PEG-DMB), and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) (DSPE-PEG).

84. The LNP of any one of claims 82 or 83, wherein the at least one PEGylated lipid is DMG-PEG, DSPE-PEG, or both.

85. The LNP described in any one of 82 to 84, wherein the at least one PEGylated lipid is DMG-PEG2000, DSPE-PEG2000.

86. 86. The LNP of any one of claims 1-72 or 74-85, further comprising a tissue and / or cell type specific targeting moiety.

87. 87. The LNP of claim 86, wherein the tissue and / or cell type specific targeting ligand is N-acetylgalactosamine (GalNAc) or a GalNAc derivative.

88. 87. The LNP of claim 86, wherein the tissue and / or cell type-specific targeting ligand is an antibody, an antibody fragment, or an antibody derivative.

89. The lipid nanoparticle of claim 88, wherein the antibody, antibody fragment, or antibody derivative is selected from the group consisting of a full-length antibody, a Fab, a Fab', a single domain antibody, and a single-chain antibody (scFv).

90. 90. The lipid nanoparticle of claim 88 or 89, wherein the antibody, antibody fragment, or antibody derivative is an scFv.

91. The LNP of any one of claims 86 to 90, wherein the tissue- and / or cell-type-specific targeting moiety is covalently attached to the at least one PEGylated lipid to form a PEGylated lipid conjugate.

92. The LNP of claim 91, wherein the PEGylated lipid conjugate comprises a tetra-antennary GalNAc covalently attached to DSPE-PEG2000.

93. The LNP of any one of claims 1 to 72 or 74 to 92, further comprising an ionizable lipid.

94. 94. The LNP of claim 93, wherein the ionizable lipid is a cationic lipid.

95. The ionizable lipid is 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-C2-DMA), Solan (DLin-K-DMA), DLin-MC3-DMA, N-[1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLE PC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)aminolbutylquarboxamidoethyl-3,4-di[oleyloxy]-benzamide (MVL5), dioctadecylamido-glycylspermine (DOGS), 3b-[N-( N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), dioctadecyldimethylammonium bromide (DDAB), Saint lipids (e.g., SAINT-2,N-methyl-4-(dioleoyl)methylpyridinium), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 1,2-dioleoyl-1-3-dimethyl-hydroxyethylammonium bromide (DORIE), 1,2-dioleoyloxypropyl-3-dimethylhydroxyethyl ammonium chloride (DORI), dialkylated amino acids (DILA2) (e.g., C18:1-norArg-C16), dioleyldimethylammonium chloride (DODAC), 1-palmitoyl 1-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), and 1,2-dimethylistoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC). In some variations, the condensing agent is combined with a cationic lipid, such as, for example, a lipid, such as, for example, dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-Dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-di ...

95. The LNP of claim 93 or 94, wherein the lipid is selected from the group consisting of N,N,N-trimethyl-4,5-bis(oleoyloxy)pentane-1-aminium chloride (DOTAPen), N-yloxy-3-dimethylaminopropane (DODMA), morpholinocholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-diyl 1-diolate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-diyl 1-diolate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentane-1-aminium chloride (DOTAPen), SS-cleavable lipids, and mixtures thereof.

96. The LNP of any one of claims 93 to 95, wherein the ionizable lipid is present in a molar proportion of about 30% to about 80%.

97. The LNP of any one of claims 75 to 96, wherein the sterol is present in a molar proportion of about 20% to about 50%.

98. The LNP of any one of claims 79 to 97, wherein the non-cationic lipid is present in a molar proportion of about 2% to about 20%.

99. The LNP of any one of claims 82 to 98, wherein the at least one PEGylated lipid is present in a molar proportion of about 2.1% to about 10%.

100. The LNP of any one of claims 91 to 99, wherein the PEGylated lipid conjugate is present in a molar proportion of about 0.1% to about 10%.

101. The LNP of any one of claims 93 to 100, further comprising a sterol, a non-cationic lipid, a PEGylated lipid, and a PEGylated lipid conjugate.

102. The LNP of any one of claims 1 to 101, further comprising dexamethasone palmitate.

103. The LNP of any one of claims 1 to 102, wherein the LNP has a total lipid to ssDNA ratio of about 10:1 to about 40:

1.

104. The LNP of any one of claims 1 to 103, wherein the LNP has a diameter of about 40 nm to about 120 nm.

105. The LNP of any one of claims 1 to 104, wherein the LNP has a diameter of less than about 100 nm.

106. The LNP of any one of claims 1 to 105, wherein the LNP has a diameter of about 60 nm to about 80 nm.

107. The LNP of any one of claims 1 to 106, wherein the LNP is present in an LNP composition comprising a plurality of LNPs having an average diameter of about 40 nm to about 120 nm.

108. The LNP of any one of claims 1 to 107, wherein the LNP is present in an LNP composition comprising a plurality of LNPs having an average diameter of less than about 100 nm.

109. The LNP of any one of claims 1 to 108, wherein the LNP is present in an LNP composition comprising a plurality of LNPs having an average diameter of about 60 nm to about 80 nm.

110. A pharmaceutical composition comprising the LNP of any one of claims 1 to 72 or 74 to 109 and a pharmaceutically acceptable excipient.

111. A method for treating a genetic disorder in a subject, comprising administering to the subject a therapeutically effective amount of the LNP of any one of claims 1 to 72 or 74 to 109, or the pharmaceutical composition of claim 73 or claim 110.

112. 112. The method of claim 111, wherein the subject is a human.

113. The genetic disorder may be sickle cell anemia, melanoma, hemophilia A (factor VIII (FVIII) deficiency) and hemophilia B (factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited disorders of liver metabolism, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharide storage diseases (e.g., Hurler syndrome (MPS type I), Shelley syndrome (MPS type I S), Hurler-Scheie syndrome (MPS type I H-S), Hunter syndrome (MPS type II), Sanfilipotypes A, B, C, and D (MPS type III), A, B, C, and D), Morquio syndrome types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPSIX), Niemann-Pick disease types A / B, C1 and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III and IV, sialidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II and III, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipids, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar 113. The method of any one of claims 111 or 112, wherein the inflammatory bowel disease is selected from the group consisting of: degeneration, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital maculopathy, Stargardt macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, age-related macular degeneration (AMD), alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency.

114. 114. The method of any one of claims 111 to 113, wherein the genetic disorder is hemophilia A.

115. 114. The method of any one of claims 111 to 113, wherein the genetic disorder is hemophilia B.

116. 114. The method of any one of claims 111 to 113, wherein the genetic disorder is phenylketonuria (PKU).

117. 114. The method of any one of claims 111 to 113, wherein the genetic disorder is Wilson's disease.

118. 114. The method of any one of claims 111 to 113, wherein the genetic disorder is Gaucher disease type I, type II, or type III.

119. 114. The method of any one of claims 111 to 113, wherein the genetic disorder is Stargardt's macular dystrophy.

120. 114. The method of any one of claims 111 to 113, wherein the genetic disorder is LCA10.

121. 114. The method of any one of claims 111 to 113, wherein the genetic disorder is Usher syndrome.

122. 114. The method of any one of claims 111 to 113, wherein the genetic disorder is wet AMD.

123. A host cell comprising the LNP of any one of claims 1 to 72 or 74 to 109.

124. The host cell of claim 123, wherein the host cell is in vitro.

125. The host cell of claim 123, wherein the host cell is in vivo.

126. A method for delivering a therapeutic gene and / or a therapeutic protein to a subject, comprising administering to the subject a therapeutically effective amount of the LNP described in any one of claims 1 to 72 or 74 to 109, or the pharmaceutical composition described in claim 73 or claim 110.

127. 127. The method of claim 126, wherein the subject is a human.

128. A method for delivering a therapeutic gene and / or a therapeutic protein to a cell, comprising contacting the cell with an LNP described in any one of claims 1 to 72 or 74 to 109 or the pharmaceutical composition described in claim 73 or 110, thereby delivering the therapeutic gene and / or therapeutic protein to the cell.

129. A method for delivering a therapeutic gene to the nucleus of a cell, comprising contacting the cell with an LNP described in any one of claims 1 to 72 or 74 to 109 or the pharmaceutical composition described in claim 73 or 110, thereby delivering the therapeutic gene and / or therapeutic protein to the nucleus of the cell.

130. 130. The method of any one of claims 128 or 129, wherein the cell is in vitro.

131. 130. The method of any one of claims 128 or 129, wherein the cell is in vivo.

132. A method for minimizing an immune response in a subject, wherein the subject is being treated with a therapeutic gene or therapeutic protein, the method comprising administering a therapeutically effective amount of the LNP of any one of claims 1 to 72 or 74 to 109, or the pharmaceutical composition of claim 73 or claim 110, wherein the subject's nucleic acid encodes the therapeutic gene or therapeutic protein.

133. 133. The method of claim 132, wherein the subject is a human.

134. 134. The method of any one of claims 111-122, 126-129, or 131-133, wherein the dosage of the ssDNA molecule administered to the subject is from about 0.05 mg / kg to about 5.0 mg / kg.

135. The method of any one of claims 111 to 122, 126 to 129, or 131 to 134, wherein the dosage of ssDNA molecule administered to the subject is about 0.05 mg / kg, about 0.1 mg / kg, about 0.15 mg / kg, about 0.2 mg / kg, about 0.25 mg / kg, about 0.3 mg / kg, about 0.35 mg / kg, about 0.4 mg / kg, about 0.45 mg / kg, about 0.5 mg / kg, about 0.55 mg / kg, about 0.6 mg / kg, or about 0. 65mg / kg, about 0.7mg / kg, about 0.75mg / kg, about 0.8mg / kg, about 0.85mg / kg, about 0.9mg / kg, about 0.95mg / kg, about 1.0mg / kg, about 1.1mg / kg, about 1.2mg / kg g, about 1.25 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.75 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 m g / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.25 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.75 mg / kg, about 2 .8mg / kg, about 2.9mg / kg, about 3.0mg / kg, about 3.1mg / kg, about 3.2mg / kg, about 3.25mg / kg, about 3.4mg / kg, about 3.5mg / kg, about 3.6mg / kg, Selected from the group consisting of about 3.7 mg / kg, about 3.75 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.25 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.75 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg, and about 5.0 mg / kg.

136. 136. The method of any one of claims 111-122, 126-129, or 131-135, wherein the dose of ssDNA molecule administered to the subject is less than about 4.0 mg / kg.

137. 137. The method of any one of claims 111-122, 126-129, or 131-136, wherein the dosage of ssDNA molecule administered to the subject is less than about 3.0 mg / kg.

138. 138. The method of any one of claims 111-122, 126-129, or 131-137, wherein the dose of ssDNA molecule administered to the subject is less than about 2.0 mg / kg.

139. 139. The method of any one of claims 111-122, 126-129, or 131-138, wherein the dosage of ssDNA molecule administered to the subject is less than about 1.75 mg / kg.

140. 140. The method of any one of claims 111-122, 126-129, or 131-139, wherein the dose of ssDNA molecule administered to the subject is less than about 1.5 mg / kg.

141. 141. The method of any one of claims 111-122, 126-129, or 131-140, wherein the dose of ssDNA molecule administered to the subject is less than about 1.25 mg / kg.

142. 142. The method of any one of claims 111-122, 126-129, or 131-141, wherein the dosage of ssDNA molecule administered to the subject is less than about 1.0 mg / kg.

143. 143. The method of any one of claims 111-122, 126-129, or 131-142, wherein the dose of ssDNA molecule administered to the subject is less than about 0.75 mg / kg.

144. 144. The method of any one of claims 111-122, 126-129, or 131-143, wherein the dose of ssDNA molecule administered to the subject is less than about 0.5 mg / kg.

145. 145. The method of any one of claims 111-122, 126-129, or 131-144, wherein the dosage of ssDNA molecule administered to the subject is less than about 0.25 mg / kg.

146. 138. The method of any one of claims 111-122, 126-129, or 131-137, wherein the dose of ssDNA molecule administered to the subject is about 2.0 mg / kg.

147. 139. The method of any one of claims 111-122, 126-129, or 131-138, wherein the dose of ssDNA molecule administered to the subject is about 1.75 mg / kg.

148. 140. The method of any one of claims 111-122, 126-129, or 131-139, wherein the dose of ssDNA molecule administered to the subject is about 1.5 mg / kg.

149. 141. The method of any one of claims 111-122, 126-129, or 131-140, wherein the dose of ssDNA molecule administered to the subject is about 1.25 mg / kg.

150. 142. The method of any one of claims 111-122, 126-129, or 131-141, wherein the dose of ssDNA molecule administered to the subject is about 1.0 mg / kg.

151. 143. The method of any one of claims 111-122, 126-129, or 131-142, wherein the dose of ssDNA molecule administered to the subject is about 0.75 mg / kg.

152. 144. The method of any one of claims 111-122, 126-129, or 131-143, wherein the dose of ssDNA molecule administered to the subject is about 0.5 mg / kg.

153. 145. The method of any one of claims 111-122, 126-129, or 131-144, wherein the dose of ssDNA molecule administered to the subject is about 0.25 mg / kg.

154. 154. The method of any one of claims 111-122, 126-129, or 131-153, wherein the dose of ssDNA molecule administered to the subject is from about 0.075 mg / kg to about 4.0 mg / kg.

155. 154. The method of any one of claims 111-122, 126-129, or 131-153, wherein the dose of ssDNA molecule administered to the subject is from about 0.1 mg / kg to about 3.0 mg / kg.

156. 154. The method of any one of claims 111-122, 126-129, or 131-153, wherein the dose of ssDNA molecule administered to the subject is from about 0.125 mg / kg to about 2.0 mg / kg.

157. 154. The method of any one of claims 111-122, 126-129, or 131-153, wherein the dose of ssDNA molecule administered to the subject is from about 0.15 mg / kg to about 1.5 mg / kg.

158. 154. The method of any one of claims 111-122, 126-129, or 131-153, wherein the dose of ssDNA molecule administered to the subject is from about 0.175 mg / kg to about 1.25 mg / kg.

159. 154. The method of any one of claims 111-122, 126-129, or 131-153, wherein the dose of ssDNA molecule administered to the subject is from about 0.2 mg / kg to about 1.0 mg / kg.

160. 154. The method of any one of claims 111-122, 126-129, or 131-153, wherein the dose of ssDNA molecule administered to the subject is from about 0.1 mg / kg to about 0.5 mg / kg.

161. 154. The method of any one of claims 111-122, 126-129, or 131-153, wherein the dose of ssDNA molecule administered to the subject is from about 0.1 mg / kg to about 1.0 mg / kg.

162. 162. The method of any one of claims 111-122, 126-129, or 131-161, further comprising administering at least two doses of said LNP or pharmaceutical composition.

163. 163. The method of any one of claims 111-122, 126-129, or 131-162, further comprising administering at least three doses of said LNP or pharmaceutical composition.

164. 164. The method of any one of claims 111-122, 126-129, or 131-163, further comprising administering four or more doses of said LNP or pharmaceutical composition.

165. An isolated linear single-stranded deoxyribonucleic acid (ssDNA) molecule comprising at least one nucleic acid sequence of interest, wherein the at least one nucleic acid sequence of interest is adjacent at its 3' end to at least one stem-loop structure, the at least one stem-loop structure comprising at least one stem and at least one loop.

166. 166. The ssDNA molecule of claim 165, wherein said at least one stem-loop structure at said 3' end is sufficient to prime replication and / or transcription.

167. 167. The ssDNA molecule of any one of claims 165 or 166, wherein the at least one stem at the 3' end comprises a partial DNA duplex of 4 to 500 nucleotides.

168. 168. The ssDNA molecule of any one of claims 165 to 167, wherein the at least one stem at the 3' end comprises a partial DNA duplex of 4 to 5 nucleotides.

169. 169. The ssDNA molecule of any one of claims 165 to 168, wherein the at least one loop at the 3' end comprises 3 to 500 unlinked nucleotides.

170. 170. The ssDNA molecule of any one of claims 165 to 169, wherein the at least one loop at the 3' end comprises a minimum of three unlinked nucleotides.

171. 171. The ssDNA molecule of any one of claims 165 to 170, wherein the ssDNA molecule comprises at least two stem-loop structures at the 3' end.

172. 172. The ssDNA molecule of any one of claims 165 to 171, wherein the ssDNA molecule comprises at least three stem-loop structures at the 3' end.

173. 173. The ssDNA molecule of any one of claims 165 to 172, wherein the ssDNA molecule comprises at least four or more stem-loop structures at the 3' end.

174. 174. The ssDNA molecule of any one of claims 165 to 173, wherein the at least one stem-loop structure at the 3' end comprises a hairpin DNA structure.

175. 175. The ssDNA molecule of any one of claims 165 to 174, wherein the at least one stem-loop structure at the 3' end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadruplex DNA structure, a bulge-forming DNA structure, and a multi-branched loop structure.

176. 176. The ssDNA molecule of any one of claims 165 to 175, wherein the at least one stem-loop structure at the 3' end does not contain the A region or the A' region that would be present in a wild-type AAV ITR.

177. 177. The ssDNA molecule of any one of claims 165 to 176, wherein the at least one stem-loop structure at the 3' end does not contain an A, A', D, or D' region that would be present in a wild-type AAV ITR.

178. 178. The ssDNA molecule of any one of claims 165-177, wherein the at least one stem-loop structure at the 3' end does not contain an A, A', B, B', C, C', D, or D' region that would be present in a wild-type AAV ITR.

179. 179. The ssDNA molecule of any one of claims 165 to 178, wherein said at least one stem-loop structure at said 3' end does not contain a rep binding element (RBE) that would be present in a wild-type AAV ITR.

180. 180. The ssDNA molecule of any one of claims 165 to 179, wherein said at least one stem-loop structure at said 3' end does not contain a terminal resolving site (trs) present in a wild-type ITR.

181. 181. The ssDNA molecule of any one of claims 165 to 180, wherein the ssDNA molecule does not contain any viral-derived sequences.

182. 182. The ssDNA molecule of any one of claims 165 to 181, wherein the stem at the 3' end of the ssDNA molecule comprises one or more nucleotides modified to be exonuclease resistant.

183. 183. The ssDNA molecule of any one of claims 165-182, wherein the 3' end of the ssDNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more nucleotides modified to be exonuclease resistant.

184. 184. The ssDNA molecule of claim 183, wherein the nucleotides modified to be exonuclease resistant are phosphorothioate-modified (PS) nucleotides.

185. 185. The ssDNA molecule of any one of claims 165 to 184, wherein the ssDNA molecule comprises at least one functional moiety.

186. 186. The ssDNA molecule of any one of claims 165 to 185, wherein the at least one stem-loop structure at the 3' end further comprises at least one functional moiety.

187. 187. The ssDNA molecule of any one of claims 185 to 186, wherein said at least one functional moiety is an aptamer.

188. 188. The ssDNA molecule of claim 187, wherein the aptamer is capable of nuclear translocation in a cell.

189. 189. The ssDNA molecule of any one of claims 165 to 188, wherein the ssDNA molecule comprises at least one stem-loop structure at its 5' end, the at least one stem-loop structure at the 5' end comprising at least one stem and at least one loop.

190. 190. The ssDNA molecule of claim 189, wherein said ssDNA comprises at least two stem-loop structures at said 5' end.

191. 191. The ssDNA molecule of any one of claims 189 or 190, wherein the ssDNA molecule comprises at least three stem-loop structures at the 5' end.

192. 192. The ssDNA molecule of any one of claims 189 to 191, wherein the ssDNA molecule comprises at least four or more stem-loop structures at the 5' end.

193. 193. The ssDNA molecule of any one of claims 189 to 192, wherein the at least one stem-loop structure at the 5' end comprises a hairpin DNA structure.

194. 194. The ssDNA molecule of any one of claims 189 to 193, wherein the at least one stem-loop structure at the 5' end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadruplex DNA structure, a bulge-forming DNA structure, and a multi-branched loop structure.

195. 195. The ssDNA molecule of any one of claims 189 to 194, wherein the at least one stem-loop structure at the 5' end does not contain an A or A' region that would be present in a wild-type AAV ITR.

196. 196. The ssDNA molecule of any one of claims 189 to 195, wherein the at least one stem-loop structure at the 5' end does not contain an A, A', D, or D' region that would be present in a wild-type AAV ITR.

197. 197. The ssDNA molecule of any one of claims 189-196, wherein the at least one stem-loop structure at the 5' end does not contain an A, A', B, B', C, C', D, or D' region that would be present in a wild-type AAV ITR.

198. 198. The ssDNA molecule of any one of claims 189 to 197, wherein the at least one stem-loop structure at the 5' end does not contain a rep binding element (RBE) that would be present in a wild-type ITR.

199. 200. The ssDNA molecule of any one of claims 189 to 198, wherein said at least one stem-loop structure at said 5' end does not comprise a terminal resolving site (trs) present in a wild-type ITR.

200. 200. The ssDNA molecule of any one of claims 189 to 199, wherein the stem at the 5' end of the ssDNA molecule comprises one or more nucleotides modified to be exonuclease resistant.

201. 201. The ssDNA molecule of any one of claims 189-200, wherein the 5' end of the ssDNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more nucleotides modified to be exonuclease resistant.

202. 202. The ssDNA molecule of claim 201, wherein the nucleotides modified to be exonuclease resistant are phosphorothioate-modified (PS) nucleotides.

203. 203. The ssDNA molecule of any one of claims 189 to 202, wherein the loop at the 5' end further comprises one or more nucleic acids to stabilize the end.

204. 204. The ssDNA molecule of any one of claims 189 to 203, wherein the loop at the 5' end further comprises one or more chemically modified nucleic acids.

205. 205. The ssDNA molecule of any one of claims 189 to 204, wherein the stem-loop structure at the 5' end comprises at least one functional moiety.

206. 206. The ssDNA molecule of claim 205, wherein said at least one functional moiety is an aptamer.

207. 207. The ssDNA molecule of claim 206, wherein the aptamer is capable of nuclear translocation in a cell.

208. 205. The ssDNA molecule of any one of claims 186 to 204, wherein the functional moiety is a ribozyme.

209. 205. The ssDNA molecule of any one of claims 186 to 204, wherein the functional moiety is an antisense oligonucleotide (ASO).

210. 205. The ssDNA molecule of any one of claims 186 to 204, wherein the functional moiety is a small interfering RNA (siRNA).

211. 205. The ssDNA molecule of any one of claims 186 to 204, wherein the functional moiety is an antiviral nucleoside analog (ANA).

212. 212. The ssDNA molecule of any one of claims 165 to 211, wherein the loop at the 5' end and / or the 3' end further comprises one or more triplex-forming oligonucleotides.

213. 213. The ssDNA molecule of any one of claims 165 to 212, wherein the loop at the 5' end and / or 3' end further comprises one or more gRNAs or gDNAs.

214. 214. The ssDNA molecule of any one of claims 165 to 213, wherein the loop at the 5' end and / or the 3' end further comprises one or more molecular probes.

215. 215. The ssDNA molecule of any one of claims 165 to 214, wherein the ssDNA molecule lacks any viral capsid protein coding sequence.

216. 216. The ssDNA molecule of any one of claims 165 to 215, wherein the ssDNA molecule is synthetically produced in vitro.

217. 217. The ssDNA molecule of any one of claims 165 to 216, wherein the ssDNA molecule is synthetically produced in vitro in a cell-free environment.

218. 218. The ssDNA molecule of any one of claims 165-217, wherein the ssDNA molecule does not activate or minimally activates immune pathways.

219. 219. The ssDNA molecule of claim 218, wherein the immune pathway is the innate immune pathway.

220. 220. The ssDNA molecule of claim 219, wherein the innate immune pathway is selected from the group consisting of the cGAS / STING pathway, the TLR9 pathway, the inflammasome-mediated pathway, and combinations thereof.

221. 221. The ssDNA molecule of any one of claims 165 to 220, wherein the ssDNA molecule further comprises at least one promoter.

222. 222. The ssDNA molecule of any one of claims 165 to 221, wherein the ssDNA molecule further comprises at least one enhancer.

223. 223. The ssDNA molecule of any one of claims 221 to 222, wherein the promoter is a hAAT promoter.

224. 223. The sDNA molecule of any one of claims 221 to 222, wherein the promoter is a TTR promoter.

225. 225. The ssDNA molecule of any one of claims 222 to 224, wherein the enhancer is a serpin (SERP) enhancer.

226. 226. The ssDNA molecule of any one of claims 221-222 or 224-225, wherein the ssDNA molecule comprises a TTR promoter and a SERP enhancer.

227. 227. The ssDNA molecule of any one of claims 221 to 226, wherein the promoter comprises a transcription start site (TSS).

228. 228. The sDNA molecule of any one of claims 221 to 227, wherein the promoter is double-stranded.

229. 229. The ssDNA molecule of any one of claims 222 to 228, wherein the enhancer is double-stranded.

230. 230. The ssDNA molecule of any one of claims 227 to 229, wherein the TSS is double-stranded.

231. The double-stranded region comprising the promoter, enhancer, and / or TSS may be at least 10 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 60 base pairs, at least 70 base pairs, at least 80 base pairs, at least 90 base pairs, at least 100 base pairs, at least 110 base pairs, at least 120 base pairs, at least 130 base pairs, at least 140 base pairs, at least 150 base pairs, at least 160 base pairs, at least 170 base pairs, at least 180 base pairs, at least 190 base pairs, at least 200 base pairs, at least 220 base pairs, at least 240 base pairs, at least 260 base pairs, at least 280 base pairs, at least 300 base pairs, at least 32 231. The ssDNA molecule of any one of claims 228-230, wherein the ssDNA molecule is at least 0 base pairs, at least 340 base pairs, at least 360 base pairs, at least 380 base pairs, at least 400 base pairs, at least 420 base pairs, at least 440 base pairs, at least 460 base pairs, at least 480 base pairs, at least 500 base pairs, at least 550 base pairs, at least 600 base pairs, at least 650 base pairs, at least 700 base pairs, at least 750 base pairs, at least 800 base pairs, at least 850 base pairs, at least 900 base pairs, at least 950 base pairs, at least 1000 base pairs, at least 1100 base pairs, at least 1200 base pairs, at least 1300 base pairs, at least 1400 base pairs, or at least 1500 base pairs in length.

232. The double-stranded region comprising the promoter, enhancer, and / or TSS is less than 1500 base pairs, less than 1400 base pairs, less than 1300 base pairs, less than 1200 base pairs, less than 1100 base pairs, less than 1000 base pairs, less than 950 base pairs, less than 900 base pairs, less than 850 base pairs, less than 800 base pairs, less than 750 base pairs, less than 700 base pairs, less than 650 base pairs, less than 600 base pairs, less than 550 base pairs, less than 500 base pairs, less than 480 base pairs, less than 460 base pairs, less than 440 base pairs, less than 420 base pairs, less than 400 base pairs, less than 380 base pairs, less than 360 base pairs, less than 34 232. The ssDNA molecule of any one of claims 228-231, wherein the ssDNA molecule is less than 0 base pairs, less than 320 base pairs, less than 300 base pairs, less than 280 base pairs, less than 260 base pairs, less than 240 base pairs, less than 220 base pairs, less than 200 base pairs, less than 190 base pairs, less than 180 base pairs, less than 170 base pairs, less than 160 base pairs, less than 150 base pairs, less than 140 base pairs, less than 130 base pairs, less than 120 base pairs, less than 110 base pairs, less than 100 base pairs, less than 90 base pairs, less than 80 base pairs, less than 70 base pairs, less than 60 base pairs, less than 50 base pairs, less than 40 base pairs, or less than 30 base pairs in length.

233. 233. The sDNA molecule of any one of claims 228 to 232, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 30 to 1500 base pairs in length.

234. 234. The ssDNA molecule of any one of claims 228 to 233, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 40 to 1400 base pairs in length.

235. 235. The ssDNA molecule of any one of claims 228 to 234, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 50 to 1300 base pairs in length.

236. 236. The ssDNA molecule of any one of claims 228 to 235, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 60 to 1200 base pairs in length.

237. 237. The ssDNA molecule of any one of claims 228 to 236, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 70 to 1100 base pairs in length.

238. 238. The ssDNA molecule of any one of claims 228 to 237, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 80 to 1000 base pairs in length.

239. 239. The ssDNA molecule of any one of claims 228-238, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 90-900 base pairs in length.

240. 240. The ssDNA molecule of any one of claims 228-239, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 90-900 base pairs in length.

241. 241. The sDNA molecule of any one of claims 228 to 240, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 100 to 800 base pairs in length.

242. 242. The ssDNA molecule of any one of claims 228-241, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 110-700 base pairs in length.

243. 243. The sDNA molecule of any one of claims 228 to 242, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 120 to 600 base pairs in length.

244. 244. The ssDNA molecule of any one of claims 228 to 243, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 130 to 500 base pairs in length.

245. 245. The sDNA molecule of any one of claims 228 to 244, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 140 to 400 base pairs in length.

246. 246. The ssDNA molecule of any one of claims 228-245, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 150-300 base pairs in length.

247. 247. The ssDNA molecule of any one of claims 228 to 246, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 160 to 200 base pairs in length.

248. 248. The ssDNA molecule of any one of claims 228-247, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 170-190 base pairs in length.

249. 235. The ssDNA molecule of any one of claims 228 to 234, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 1381 base pairs in length.

250. 245. The ssDNA molecule of any one of claims 228 to 244, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 499 base pairs in length.

251. 251. The ssDNA molecule of any one of claims 165 to 250, wherein the ssDNA molecule is capable of expressing at least one therapeutic protein or a therapeutic fragment thereof.

252. 252. The ssDNA molecule of claim 251, wherein the at least one therapeutic protein is selected from the group consisting of an antibody, an enzyme, a clotting factor, a transcription factor, a replication factor, a growth factor, a hormone, and a fusion protein.

253. The at least one therapeutic protein is selected from the group consisting of sickle cell anemia, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited disorders of liver metabolism, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharide storage diseases (e.g., Hurler syndrome (MPS type I), Shelley syndrome (MPS type I-S), Hurler-Scheie syndrome (MPS type I-H-S), Hunter syndrome (MPS type I-H-S), and others. MPS type II), Sanfilippotypes A, B, C, and D (MPS type III A, B, C, and D), Morquio types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS type VI), Sly syndrome (MPS type VII), hyaluronidase deficiency (MPSIX), Niemann-Pick disease types A / B, C1 and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III and IV, sialidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II and I II, Fabry disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipids, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinal cord injury 253. The sDNA molecule of any one of claims 251 or 252, useful for treating a genetic disorder selected from the group consisting of cerebral degeneration, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital maculopathy, Stargardt macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency.

254. 254. The ssDNA molecule of any one of claims 165 to 253, further comprising a lipid.

255. 255. The ssDNA molecule of claim 254, wherein the ssDNA molecule is encapsulated in the lipid.

256. 256. The ssDNA molecule of any one of claims 254 to 255, wherein the lipid is in a lipid nanoparticle (LNP).

257. 257. A pharmaceutical composition comprising the ssDNA molecule of any one of claims 165 to 256 and a pharmaceutically acceptable excipient.

258. A host cell comprising the ssDNA molecule of any one of claims 165 to 256.

259. 257. A method of treating a genetic disorder in a subject, comprising administering to the subject a therapeutically effective amount of the ssDNA molecule of any one of claims 165-256 or the pharmaceutical composition of claim 257.

260. 257. A method for delivering a therapeutic gene and / or a therapeutic protein to a subject, comprising administering to the subject a therapeutically effective amount of the ssDNA molecule of any one of claims 165-256 or the pharmaceutical composition of claim 257.

261. 257. A method for delivering a therapeutic gene and / or a therapeutic protein to a cell, comprising contacting said cell with an ssDNA molecule of any one of claims 165-256 or the pharmaceutical composition of claim 257, thereby delivering said therapeutic gene and / or therapeutic protein to said cell.

262. 257. A method of delivering a therapeutic gene to the nucleus of a cell, comprising contacting said cell with an ssDNA molecule of any one of claims 165-256 or the pharmaceutical composition of claim 257, thereby delivering said therapeutic gene and / or therapeutic protein to the nucleus of said cell.

263. 257. A method of minimizing an immune response in a subject, wherein the subject is being treated with a therapeutic gene or therapeutic protein, comprising administering a therapeutically effective amount of a ssDNA molecule of any one of claims 165-256 or the pharmaceutical composition of claim 257, wherein the subject's nucleic acid encodes the therapeutic gene or therapeutic protein.

264. The LNP of any one of claims 68 to 109, wherein the enhancer is double-stranded.

265. The double-stranded region comprising the promoter, enhancer, and / or TSS may be at least 10 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 60 base pairs, at least 70 base pairs, at least 80 base pairs, at least 90 base pairs, at least 100 base pairs, at least 110 base pairs, at least 120 base pairs, at least 130 base pairs, at least 140 base pairs, at least 150 base pairs, at least 160 base pairs, at least 170 base pairs, at least 180 base pairs, at least 190 base pairs, at least 200 base pairs, at least 220 base pairs, at least 240 base pairs, at least 260 base pairs, at least 280 base pairs, at least 300 base pairs, at least 320 base pairs, at least 340 base pairs, at least 360 base pairs, at least 380 base pairs, at least 390 base pairs, at least 400 base pairs, at least 410 base pairs, at least 420 base pairs, at least 430 base pairs, at least 440 base pairs, at least 450 base pairs, at least 460 base pairs, at least 470 base pairs, at least 480 base pairs, at least 490 base pairs, at least 500 base pairs, at least 510 base pairs, at least 520 base pairs, at least 530 base pairs, at least 540 base pairs, at least 550 base pairs, at least 560 base pairs, at least 570 base pairs, at least 580 base pairs, at least 590 base pairs, at least 600 base pairs, at least 610 base pairs, at least 620 base pairs, at least 630 base pairs, at least 640 base pairs, at least 6 base pairs, at least 340 base pairs, at least 360 base pairs, at least 380 base pairs, at least 400 base pairs, at least 420 base pairs, at least 440 base pairs, at least 460 base pairs, at least 480 base pairs, at least 500 base pairs, at least 550 base pairs, at least 600 base pairs, at least 650 base pairs, at least 700 base pairs, at least 750 base pairs, at least 800 base pairs, at least 850 base pairs, at least 900 base pairs, at least 950 base pairs, at least 1000 base pairs, at least 1100 base pairs, at least 1200 base pairs, at least 1300 base pairs, at least 1400 base pairs, or at least 1500 base pairs in length.

266. The double-stranded region comprising the promoter, enhancer, and / or TSS is less than 1500 base pairs, less than 1400 base pairs, less than 1300 base pairs, less than 1200 base pairs, less than 1100 base pairs, less than 1000 base pairs, less than 950 base pairs, less than 900 base pairs, less than 850 base pairs, less than 800 base pairs, less than 750 base pairs, less than 700 base pairs, less than 650 base pairs, less than 600 base pairs, less than 550 base pairs, less than 500 base pairs, less than 480 base pairs, less than 460 base pairs, less than 440 base pairs, less than 420 base pairs, less than 400 base pairs, less than 380 base pairs, less than 360 base pairs, less than 340 base pairs. The LNP of any one of claims 68-72, 74-109, or 263-265, having a length of less than 320 base pairs, less than 300 base pairs, less than 280 base pairs, less than 260 base pairs, less than 240 base pairs, less than 220 base pairs, less than 200 base pairs, less than 190 base pairs, less than 180 base pairs, less than 170 base pairs, less than 160 base pairs, less than 150 base pairs, less than 140 base pairs, less than 130 base pairs, less than 120 base pairs, less than 110 base pairs, less than 100 base pairs, less than 90 base pairs, less than 80 base pairs, less than 70 base pairs, less than 60 base pairs, less than 50 base pairs, less than 40 base pairs, or less than 30 base pairs.

267. The LNP of any one of claims 68-72, 74-109, or 263-266, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 30-1500 base pairs in length.

268. The LNP of any one of claims 68-72, 74-109, or 263-267, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 40-1400 base pairs in length.

269. The LNP of any one of claims 68-72, 74-109, or 263-268, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 50-1300 base pairs in length.

270. The LNP of any one of claims 68-72, 74-109, or 263-269, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 60-1200 base pairs in length.

271. The LNP of any one of claims 68-72, 74-109, or 263-270, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 70-1100 base pairs in length.

272. The LNP of any one of claims 68-72, 74-109, or 263-271, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 80-1000 base pairs in length.

273. The LNP of any one of claims 68-72, 74-109, or 263-272, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 90-900 base pairs in length.

274. The LNP of any one of claims 68-72, 74-109, or 263-273, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 90-900 base pairs in length.

275. The LNP of any one of claims 68-72, 74-109, or 263-274, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 100-800 base pairs in length.

276. The LNP of any one of claims 68-72, 74-109, or 263-275, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 110-700 base pairs in length.

277. The LNP of any one of claims 68-72, 74-109, or 263-276, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 120-600 base pairs in length.

278. The LNP of any one of claims 68-72, 74-109, or 263-277, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 130-500 base pairs in length.

279. The LNP of any one of claims 68-72, 74-109, or 263-278, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 140-400 base pairs in length.

280. The LNP of any one of claims 68-72, 74-109, or 263-279, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 150-300 base pairs in length.

281. The LNP of any one of claims 68-72, 74-109, or 263-280, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is about 160-200 base pairs in length.

282. The LNP of any one of claims 68-72, 74-109, or 263-268, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 1381 base pairs in length.

283. The LNP of any one of claims 68-72, 74-109, or 263-277, wherein the double-stranded region comprising the promoter, enhancer, and / or TSS is approximately 499 base pairs in length.

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