Synthetic single-stranded nucleic acid compositions and methods thereof

Abstract

Disclosed are single-stranded deoxyribonucleic acid (DNA) molecules comprising at least one nucleic acid sequence of interest flanked with a first at least one stem-loop structure, methods of making and methods of use for delivery and expression of a transgene in a host cell.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 429,461, filed December 1, 2022, U.S. Provisional Application No. 63 / 449,872, filed March 3, 2023, U.S. Provisional Application No. 63 / 529,637, filed July 28, 2023, and U.S. Provisional 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 Art

[0003] Vectors derived from the adeno-associated virus (AAV) (e.g., recombinant AAV (rAAV) or AAV vectors) are attractive for delivering genetic material because (i) the vectors are able to infect ("transduce") a variety of dividing and non-dividing cell types, such as myocytes and neurons; (ii) the vectors lack viral structural genes, thereby reducing host cell responses to viral infection, e.g., interferon-mediated responses; (iii) wild-type AAV is considered non-pathological in humans; and (iv) in contrast to wild-type AAV, the vectors are able to integrate into the host cell genome, replication-defective AAV vectors lack the rep gene and typically persist as episomes, thereby greatly limiting the risk of insertional mutagenesis or genotoxicity.

[0004] However, there are several major drawbacks and deficiencies in the use of AAV particles as gene delivery vectors derived from traditional AAV production by host cells (e.g., Sf9 insect cells in a high-scale production environment). A 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). Therefore, due to this limitation in viral packaging, the use of AAV vectors has been limited 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 may respond to a vector that effectively acts as a booster to stimulate the immune system, thereby producing high titers of anti-AAV antibodies, which hinder further treatment. Recent reports have pointed out concerns about immunogenicity in high-dose situations. Another significant drawback is that producing AAV on a large scale in host cells (e.g., insect cells) to prepare viral genomes produces a random mixture of positive (+) and negative (-) strand vectors. This greatly reduces the strand specificity of the transgene for the much-needed therapeutic expression of the sense strand.

[0005] In addition, conventional AAV virus particles with capsids are produced by introducing one or more plasmids containing the AAV genome, rep gene and cap gene (Grimm et al., 1998). However, it was found that such encapsulated AAV viral vectors inefficiently transduce certain cell and tissue types, and it was also found that capsids induce severe immune responses in the host. Therefore, due to the patient's immune response, the use of adeno-associated virus (AAV) vectors for gene therapy (including gene editing) is limited to a single administration to the patient, and due to the minimum viral packaging capacity (about 4.5 kb), the scope of transgenic genetic materials suitable for delivery in AAV vectors is limited, and AAV-mediated gene expression is slow. In addition, the method for producing such AAV vectors is greatly dependent on traditional insect cell-dependent production methods. Such methods can be hindered by pollutants from cells used to produce vectors, which are inconvenient or costly when removed or purified, and may produce undesirable side effects if included in therapeutic formulations.

[0006] Therefore, there is a strong need in the field of gene therapy for a technology that minimizes immunogenicity, is re-administrable, and allows for the large-scale production of recombinant vectors, and that increases expression levels, chain specificity, and purity while increasing transgene size capacity. Summary of the Invention

[0007] According to some aspects, the present disclosure provides an isolated linear single-stranded deoxyribonucleic acid (ssDNA) molecule comprising at least one nucleic acid sequence of interest flanked at its 3' end by at least one stem-loop structure to initiate replication and / or transcription. According to some embodiments, the ssDNA molecule of the present disclosure is characterized in that the ssDNA molecule does not comprise an inverted terminal repeat (ITR) structure.

[0008] Unexpectedly, the ssDNA molecules described herein were found to have tolerability and immune responses equivalent to or superior to double-stranded (ds)ceDNA in vivo. As shown in the Examples, when administered in equivalent amounts, the cytokine response to the ssDNA molecules described herein was significantly lower to undetectable compared to dsceDNA. The reduced immunogenicity observed with the ssDNA molecules of the present disclosure is advantageous because it allows for repeated dosing or higher dose concentrations.

[0009] According to a first aspect, the present disclosure provides a lipid nanoparticle (LNP) comprising: (a) a linear single-stranded deoxyribonucleic acid (ssDNA) molecule, the linear 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 various aspects and embodiments herein, the ssDNA molecule does not comprise any viral-derived sequence. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule is at least 200 nucleotides in length. In one embodiment of the various 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 50 ...0 nucleotides in length, at least 7000 nucleotides in length, at least 8000 nucleotides in length, at least 900 nucleotides in length At least 3500 nucleotides, 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 various aspects and embodiments herein, the at least one nucleic acid sequence of interest is flanked by at least one stem-loop structure at its 3' end, wherein the at least one stem-loop structure comprises at least one stem and at least one loop. In another embodiment, the at least one stem-loop structure at the 3' end is sufficient to induce replication and / or transcription. In some embodiments of the various aspects and embodiments herein, the at least one stem at the 3' end comprises a partial DNA duplex of 4-500 nucleotides. In one embodiment of the various aspects and embodiments herein, the at least one stem at the 3' end comprises a partial DNA duplex of 4-5 nucleotides. In one embodiment of the various aspects and embodiments herein, the at least one loop at the 3' end comprises 3-500 unbound nucleotides. In one embodiment of the various aspects and embodiments herein, the at least one loop at the 3' end comprises a minimum of 3 unbound nucleotides. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule comprises at least two stem-loop structures at the 3' end.In one embodiment of the various aspects and embodiments herein, the ssDNA molecule comprises at least three stem-loop structures at the 3' end. In one embodiment of the various 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 various aspects and embodiments herein, the at least one stem-loop structure at the 3' end comprises a hairpin DNA structure. In one embodiment of the various aspects and embodiments herein, 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 protruding DNA structure, and a multi-branched loop structure. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 3' end does not comprise an A or A' region that would be present in a wild-type AAV ITR. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 3' end does not comprise an A, A', D, or D' region that would be present in a wild-type AAV ITR. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 3' end does not comprise an 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 various aspects and embodiments herein, the at least one stem-loop structure at the 3' end does not comprise a rep binding element (RBE) that would be present in a wild-type AAV ITR. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 3' end does not comprise a terminal melting site (trs) that would be present in a wild-type ITR. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule does not comprise any viral-derived sequence. In one embodiment of the various aspects and embodiments herein, the stem at the 3' end of the ssDNA molecule comprises one or more nucleotides modified to confer exonuclease resistance. In one embodiment of the various aspects and embodiments herein, the 3' end of the ssDNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more nucleotides modified to confer exonuclease resistance. In one embodiment of the various aspects and embodiments herein, the nucleotides modified to have exonuclease resistance are phosphorothioate-modified (PS) nucleotides. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule comprises at least one functional moiety. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 3' end further comprises at least one functional moiety. In one embodiment of the various aspects and embodiments herein, the at least one functional moiety is an aptamer.In another embodiment, the aptamer is capable of nuclear translocation in a cell. In one embodiment of the various 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 various aspects and embodiments herein, the ssDNA molecule comprises at least two stem-loop structures at the 5' end. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule comprises at least three stem-loop structures at the 5' end. In one embodiment of the various 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 various 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 various 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 protruding DNA structure, and a multi-branched loop structure. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 5' end does not comprise an A or A' region that would be present in a wild-type AAV ITR. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 5' end does not comprise an A, A', D, or D' region that would be present in a wild-type AAV ITR. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 5' end does not comprise an 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 various aspects and embodiments herein, the at least one stem-loop structure at the 5' end does not comprise a rep binding element (RBE) that would be present in a wild-type ITR. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 5' end does not comprise a terminal melting site (trs) that would be present in a wild-type ITR. In one embodiment of the various aspects and embodiments herein, the stem at the 5' end of the ssDNA molecule comprises one or more nucleotides modified to confer exonuclease resistance. In one embodiment of the various aspects and embodiments 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 confer exonuclease resistance. In further embodiments, the nucleotides modified to confer exonuclease resistance are phosphorothioate-modified (PS) nucleotides.In one embodiment of the various aspects and embodiments herein, the loop at the 5' end further comprises one or more nucleic acids to stabilize the end (one or more, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more). In one embodiment of the various aspects and embodiments herein, the loop at the 5' end further comprises one or more chemically modified nucleic acids. In one embodiment of the various aspects and embodiments herein, the stem-loop structure at the 5' end comprises at least one functional moiety. In another 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 various aspects and embodiments herein, the functional moiety is a ribozyme. In one embodiment of the various aspects and embodiments herein, the functional moiety is an antisense oligonucleotide (ASO). In one embodiment of the various aspects and embodiments herein, the functional moiety is a short interfering RNA (siRNA). In one embodiment of the various aspects and embodiments herein, the functional moiety is an antiviral nucleoside analog (ANA). In one embodiment of the various aspects and embodiments herein, the loop at the 5' end and / or the 3' end further comprises one or more triplex-forming oligonucleotides. In one embodiment of the various aspects and embodiments herein, the loop at the 5' end and / or the 3' end further comprises one or more gRNAs or gDNAs. In one embodiment of the various aspects and embodiments herein, the loop at the 5' end and / or the 3' end further comprises one or more molecular probes. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule lacks any viral capsid protein coding sequence.

[0010] In one embodiment of the various aspects and embodiments herein, the ssDNA molecule is produced synthetically in vitro.

[0011] In one embodiment of the various aspects and embodiments herein, the ssDNA molecule is synthetically produced in vitro in a cell-free environment.

[0012] In one embodiment of the various aspects and embodiments herein, the ssDNA molecule does not activate or minimally activates an immune pathway. In further embodiments, the immune pathway is an innate immune pathway. In some embodiments, the innate immune pathway is selected from the group consisting of a cGAS / STING pathway, a TLR9 pathway, an inflammasome-mediated pathway, and combinations thereof.

[0013] In one embodiment of the various aspects and embodiments herein, the ssDNA molecule further comprises at least one promoter. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule further comprises at least one enhancer. In one embodiment of the various aspects and embodiments herein, the promoter is the hAAT promoter. In one embodiment of the various aspects and embodiments herein, the promoter is the TTR promoter. In one embodiment of the various aspects and embodiments herein, the enhancer is a serpin (SERP) enhancer. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule comprises a TTR promoter and a SERP enhancer. In one embodiment of the various aspects and embodiments herein, the promoter comprises a transcription start site (TSS). In one embodiment of the various aspects and embodiments herein, the promoter is double-stranded. In one embodiment of the various aspects and embodiments herein, the TSS is double-stranded.

[0014] In one embodiment of the various aspects and embodiments herein, the ssDNA molecule is capable of expressing at least one therapeutic protein or a therapeutic fragment thereof. In one embodiment, the at least one therapeutic protein is selected from the group consisting of an antibody, an enzyme, a coagulation factor, a transcription factor, a replication factor, a growth factor, a hormone, and a fusion protein. In one embodiment of the various aspects and embodiments herein, the at least one therapeutic protein can be used to treat a genetic disease 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 disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi's anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis (e.g.,Hurler syndrome (MPS type I), Scheie syndrome (MPS type I S), Hurler-Scheie syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo types A, B, C, and D (MPS types 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 (MPS type IX), Niemann-Pick disease types A / B, C1, and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), 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, aspartate glucosamineuria, sialidosis (Salla disease), Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease 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 congenital amaurosis, 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.

[0015] According to other aspects, the present disclosure provides a pharmaceutical composition comprising the LNP of any one of the various aspects and embodiments herein, and a pharmaceutically acceptable excipient. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule is encapsulated in the lipid. In one embodiment of the various aspects and embodiments herein, the LNP further comprises a sterol. In another embodiment, the sterol is selected from the group consisting of cholesterol, β-sitosterol, stigmasterol, β-sitostanol, campesterol, rapeseed sterol, derivatives thereof, and combinations thereof. In one embodiment of the various aspects and embodiments herein, the sterol is cholesterol. In one embodiment of the various aspects and embodiments herein, the sterol is β-sitosterol. In one embodiment of the various aspects and embodiments herein, the LNP further comprises a non-cationic lipid. In further embodiments, the non-cationic lipid is selected from the group consisting of: distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmito ... dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DPPG), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DPPG), dioleoylphosphatidylcholine (DP oleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE , 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dieucoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dioleoyl-phosphatidyl Phytosterylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine and mixtures thereof.In one embodiment of the various aspects and embodiments herein, the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC) and dioleoyl-phosphatidylethanolamine (DOPE). In one embodiment of the various aspects and embodiments herein, the LNP further comprises at least one PEGylated lipid. In another embodiment, the at least one PEGylated lipid is selected from the group consisting of: PEG-dilauryloxypropyl; PEG-dimyristoyloxypropyl; PEG-dipalmitoyloxypropyl, PEG-distearoyloxypropyl; 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (DMG-PEG); PEG-dilaurylglycerol; PEG-dipalmitoylglycerol; PEG-distearylglycerol; PEG-dilaurylglycolamide; PEG-dimyristylglycolamide; PE G-dipalmitoyl glycylamide; PEG-distearyl glycylamide; (1-[8'-(cholest-5-ene-3[β]-oxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol))(PEG-cholesterol); 3,4-ditetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether (PEG-DMB) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG). In another additional embodiment, the at least one PEGylated lipid is DMG-PEG, DSPE-PEG, or both. In one embodiment of the various aspects and embodiments herein, the at least one PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, or both. In one embodiment of the various aspects and embodiments herein, the LNP further comprises a tissue and / or cell type specific targeting moiety. In another embodiment, the tissue and / or cell type specific targeting ligand is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. In another another embodiment, the tissue and / or cell type specific targeting ligand is an antibody, an antibody fragment or an antibody derivative. In still another another embodiment, the antibody, the antibody fragment or the antibody derivative is selected from the group consisting of: full-length antibody, Fab, Fab', single domain antibody and single-chain antibody (scFv). In one embodiment of the various aspects and embodiments herein, the antibody, the antibody fragment or the antibody derivative is a scFv. In one embodiment of the various aspects and embodiments herein, 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. In one embodiment, the PEGylated lipid conjugate comprises a tetraantennary GalNAc covalently attached to DSPE-PEG2000.In one embodiment of the various 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 selected from the group consisting of:.

[0016] 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Di-γ-linoleyloxy-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-Dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP); 1,2-Dioleoyl-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-[bis(3-amino-propyl)amino]butylcarboxamidoethyl]-3,4-di[oleyloxy]-benzamide (MVL5); dioctadecylamido-glycyl spermine (DOGS); 3b-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol); dioctadecyldimethylammonium bromide (DDAB); Saint lipids (e.g., SAINT-2,N-methyl-4-(dioleyl)methylpyridinium ion); 1,2-dimyristoyl Oxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORIE); 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI); dialkylated amino acids (DILA2) (e.g., C18:1-norArg-C16); dioleyldimethylammonium chloride (DODAC); 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC); and 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC).In some variations, the condensing agent, e.g., cationic lipid, is a lipid such as dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,31]-dioxolane (DLin-KC2-DMA), heptathriacontane-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), 1,2-dioleo ...2,2-dioleoyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,31]-dioxolane (DLin- propane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), morpholino cholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G) and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentane-1-ammonium chloride (DOTAPen), SS-cleavable lipids and mixtures thereof. In one embodiment of the various aspects and embodiments herein, the ionizable lipid is present in a molar percentage of about 30% to about 80%, for example, 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 embodiment of the various aspects and embodiments herein, the sterol is present in a molar percentage of about 20% to about 50%, for example, 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 embodiment of the various aspects and embodiments herein, the non-cationic lipid is present in a molar percentage of about 2% to about 20%, for example, 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 various aspects and embodiments herein, the at least one PEGylated lipid is present in a molar percentage of about 2.1% to about 10%, for example, about 2.1% to about 10%, about 5% to about 10%, about 2.1% to about 5%, about 2.1% to about 8% or about 5% to about 7%.In one embodiment of the various aspects and embodiments herein, the PEGylated lipid conjugate is present in a molar percentage of about 0.1% to about 10%, for example, 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 embodiment of the various aspects and embodiments herein, the LNP comprises a sterol, a non-cationic lipid, a PEGylated lipid and a PEGylated lipid conjugate. In one embodiment of the various aspects and embodiments herein, the LNP further comprises dexamethasone palmitate.

[0017] In one embodiment of the various 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.

[0018] In one embodiment in the various aspects and embodiments herein, the diameter of the LNP is about 40nm to about 120nm. In one embodiment in the various aspects and embodiments herein, the diameter of the LNP is less than about 100nm. In one embodiment in the various aspects and embodiments herein, the diameter of the LNP is about 60nm to about 80nm. In one embodiment in the various aspects and embodiments herein, the LNP is present in an LNP composition, and the LNP composition comprises multiple average diameters and is about 40nm to about 120nm LNP. In one embodiment in the various aspects and embodiments herein, the LNP is present in an LNP composition, and the LNP composition comprises multiple average diameters and is less than about 100nm LNP. In one embodiment in the various aspects and embodiments herein, the LNP is present in an LNP composition, and the LNP composition comprises multiple average diameters and is about 60nm to about 80nm (for example, about 60, 65, 70, 75 or 80nm) LNP.

[0019] In another aspect, the present disclosure provides a pharmaceutical composition comprising the LNP according to any one of the aspects and embodiments herein, and a pharmaceutically acceptable excipient.

[0020] In another aspect, the present disclosure provides a method of treating a genetic disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the LNP of any one of the various aspects and embodiments herein or the pharmaceutical composition of any one of the various aspects and embodiments herein. In further embodiments of the method, the subject is a human.In another further embodiment, the genetic disease 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 disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Leschnehan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharidoses (e.g., Hurler syndrome (MPS type I), Shay syndrome (MPS type I S), Hurler-Shay syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo A, B, C, and D (MPS MPS III A, B, C, and D), Morquer A and B (MPS IVA and MPS IVB), Mallory-Lami syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPS Type IX), Niemann-Pick disease A / B, C1, and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff 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, aspartate glucosamineuria, sialidosis, Dannon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, 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), epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber congenital amaurosis, 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 embodiment of the various aspects and embodiments herein, the genetic disease is hemophilia A. In one embodiment of the various aspects and embodiments herein, the genetic disease is hemophilia B.In one embodiment of the various aspects and embodiments herein, the genetic disease is phenylketonuria (PKU). In one embodiment of the various aspects and embodiments herein, the genetic disease is Wilson's disease. In one embodiment of the various aspects and embodiments herein, the genetic disease is Gaucher disease type I, type II, or type III. In one embodiment of the various aspects and embodiments herein, the genetic disease is Stargardt macular dystrophy. In one embodiment of the various aspects and embodiments herein, the genetic disease is LCA10. In one embodiment of the various aspects and embodiments herein, the genetic disease is Usher syndrome. In one embodiment of the various aspects and embodiments herein, the genetic disease is wet AMD.

[0021] According to another aspect, the present disclosure provides a host cell comprising the LNPs of any one of the various aspects and embodiments herein. In one embodiment, the host cell is in vitro. In another embodiment, the host cell is in vivo.

[0022] In another aspect, the present disclosure provides a method for delivering a therapeutic gene and / or therapeutic protein to a subject, the method comprising administering to the subject a therapeutically effective amount of the LNP of any one of the various aspects and embodiments herein or the pharmaceutical composition of the various aspects and embodiments herein. According to some embodiments, the subject is a human.

[0023] According to other aspects, the present disclosure provides a method for delivering a therapeutic gene and / or a therapeutic protein to a cell, the method comprising contacting the cell with the LNP of any one of the various aspects and embodiments herein or the pharmaceutical composition of the various aspects and embodiments herein, thereby delivering the therapeutic gene and / or the therapeutic protein to the cell.

[0024] According to other aspects, 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 as described in any one of the various aspects and embodiments herein or a pharmaceutical composition as described in any one of the various aspects and embodiments herein, thereby delivering the therapeutic gene and / or the therapeutic protein to the nucleus of the cell. According to some embodiments, the cell is in vitro. According to some embodiments, the cell is in vivo.

[0025] In another aspect, the present disclosure provides a method of 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 the various aspects and embodiments herein, or the pharmaceutical composition of the various aspects and embodiments herein, wherein the nucleic acid of interest encodes the therapeutic gene or the therapeutic protein. According to some embodiments, the subject is a human.

[0026] In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 0.05 mg / kg to about 5.0 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is selected from the group consisting of 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, about 0.75 mg / kg, about 0.8 mg / kg, about 0.85 mg / kg, about 0.9 mg / kg, about 0.95 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, 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 mg / 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 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.25 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3. In one embodiment of the present invention, the dose of the ssDNA molecule administered to the subject is less than about 4.0 mg / kg. In one embodiment of the present invention, the dose of the ssDNA molecule administered to the subject is less than about 3.0 mg / kg. In one embodiment of the present invention, the dose of the ssDNA molecule administered to the subject is less than about 2.0 mg / kg.In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is less than about 1.75 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is less than about 1.5 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is less than about 1.25 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is less than about 1.0 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is less than about 0.75 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is less than about 0.5 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is less than about 0.25 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 2.0 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 1.75 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 1.5 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 1.25 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 1.0 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 0.75 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 0.5 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 0.25 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 0.075 mg / kg to about 4.0 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 0.1 mg / kg to about 3.0 mg / kg.In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 0.125 mg / kg to about 2.0 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 0.15 mg / kg to about 1.5 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 0.175 mg / kg to about 1.25 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 0.2 mg / kg to about 1.0 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 0.1 mg / kg to about 0.5 mg / kg. In one embodiment of the various aspects and embodiments herein, the dose of the ssDNA molecule administered to the subject is about 0.1 mg / kg to about 1.0 mg / kg. In one embodiment of the various aspects and embodiments herein, the method further comprises administering at least two doses of the LNP or the pharmaceutical composition. In one embodiment of the various aspects and embodiments herein, the method further comprises administering at least three doses of the LNP or the pharmaceutical composition. In one embodiment of the various aspects and embodiments herein, the method further comprises administering four or more doses of the LNP or the pharmaceutical composition.

[0027] 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 flanked at its 3' end by 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 one embodiment, the at least one stem-loop structure at the 3' end is sufficient to initiate replication and / or transcription. In another embodiment, the at least one stem at the 3' end comprises a partial DNA duplex of 4-500 nucleotides. In another additional embodiment, the at least one stem at the 3' end comprises a partial DNA duplex of 4-5 nucleotides. In one embodiment of the various aspects and embodiments herein, the at least one loop at the 3' end comprises 3-500 unbound nucleotides. In one embodiment of the various aspects and embodiments herein, the at least one loop at the 3' end comprises a minimum of 3 unbound nucleotides. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule comprises at least two stem-loop structures at the 3' end. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule comprises at least three stem-loop structures at the 3' end. In one embodiment of the various 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 various aspects and embodiments herein, the at least one stem-loop structure at the 3' end comprises a hairpin DNA structure. In one embodiment of the various aspects and embodiments herein, 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 protruding DNA structure, and a multi-branched loop structure. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 3' end does not comprise an A or A' region that would be present in a wild-type AAV ITR. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 3' end does not comprise an A, A', D, or D' region that would be present in a wild-type AAV ITR. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 3' end does not comprise an 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 various aspects and embodiments herein, the at least one stem-loop structure at the 3' end does not comprise a rep binding element (RBE) that would be present in a wild-type AAV ITR. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 3' end does not comprise a terminal melting site (trs) that would be present in a wild-type ITR.In one embodiment of the various aspects and embodiments herein, the ssDNA molecule does not contain any viral-derived sequences. In one embodiment of the various aspects and embodiments herein, the stem at the 3' end of the ssDNA molecule comprises one or more nucleotides modified to have exonuclease resistance. In one embodiment of the various aspects and embodiments herein, the 3' end of the ssDNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more nucleotides modified to have exonuclease resistance. In one embodiment of the various aspects and embodiments herein, the nucleotides modified to have exonuclease resistance are phosphorothioate-modified (PS) nucleotides. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule comprises at least one functional moiety. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 3' end further comprises at least one functional moiety. In one embodiment of the various aspects and embodiments herein, the at least one functional moiety is an aptamer. In another embodiment, the aptamer is capable of nuclear translocation in a cell. In one embodiment of the various 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 various aspects and embodiments herein, the ssDNA molecule comprises at least two stem-loop structures at the 5' end. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule comprises at least three stem-loop structures at the 5' end. In one embodiment of the various 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 various 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 various 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 DNA structure, and a multi-branched loop structure. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 5' end does not comprise an A or A' region that would be present in a wild-type AAV ITR. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 5' end does not comprise an A, A', D, or D' region as would be present in a wild-type AAV ITR. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 5' end does not comprise an A, A', B, B', C, C', D, or D' region as would be present in a wild-type AAV ITR.In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 5' end does not comprise a rep binding element (RBE) that would be present in a wild-type ITR. In one embodiment of the various aspects and embodiments herein, the at least one stem-loop structure at the 5' end does not comprise a terminal melting site (trs) that would be present in a wild-type ITR. In one embodiment of the various aspects and embodiments herein, the stem at the 5' end of the ssDNA molecule comprises one or more nucleotides modified to confer exonuclease resistance. In one embodiment of the various aspects and embodiments 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 confer exonuclease resistance. In another embodiment, the nucleotides modified to confer exonuclease resistance are phosphorothioate-modified (PS) nucleotides. In one embodiment of the various aspects and embodiments herein, the loop at the 5' end further comprises one or more nucleic acids to stabilize the end. In one embodiment of the various aspects and embodiments herein, the loop at the 5' end further comprises one or more chemically modified nucleic acids. In one embodiment of the various aspects and embodiments herein, the stem-loop structure at the 5' end includes at least one functional portion. In another embodiment, the at least one functional portion is an aptamer. In another further embodiment, the aptamer is capable of performing nuclear translocation in a cell. In one embodiment of the various aspects and embodiments herein, the functional portion is a ribozyme. In one embodiment of the various aspects and embodiments herein, the functional portion is an antisense oligonucleotide (ASO). In one embodiment of the various aspects and embodiments herein, the functional portion is a short interfering RNA (siRNA). In one embodiment of the various aspects and embodiments herein, the functional portion is an antiviral nucleoside analog (ANA). In one embodiment of the various aspects and embodiments herein, the loop at the 5' end and / or the 3' end further includes one or more triplex-forming oligonucleotides. In one embodiment of the various aspects and embodiments herein, the loop at the 5' end and / or the 3' end further includes one or more gRNAs or gDNAs. In one embodiment of the various aspects and embodiments herein, the loop at the 5' end and / or the 3' end further includes one or more molecular probes. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule lacks any viral capsid protein coding sequence. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule is synthetically produced in vitro. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule is synthetically produced in vitro in a cell-free environment.In one embodiment of the various aspects and embodiments herein, the ssDNA molecule does not activate or minimally activates an immune pathway. In one embodiment of the various aspects and embodiments herein, the immune pathway is an innate immune pathway. In further embodiments, the innate immune pathway is selected from the group consisting of a cGAS / STING pathway, a TLR9 pathway, an inflammasome-mediated pathway, and combinations thereof. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule further comprises at least one promoter. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule further comprises at least one enhancer.

[0028] In one embodiment of the various aspects and embodiments herein, the promoter is a hAAT promoter. In one embodiment of the various aspects and embodiments herein, the promoter is a TTR promoter. In one embodiment of the various aspects and embodiments herein, the enhancer is a serpin (SERP) enhancer. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule comprises a TTR promoter and a SERP enhancer. In one embodiment of the various aspects and embodiments herein, the promoter comprises a transcription start site (TSS). In one embodiment of the various aspects and embodiments herein, the promoter is double-stranded. In one embodiment of the various aspects and embodiments herein, the enhancer is double-stranded. In one embodiment of the various aspects and embodiments herein, the TSS is double-stranded. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the 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 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 one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the 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, or less than 500 base pairs. base pairs, less than 380 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 one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 30-1500 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 40-1400 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 50-1300 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 60-1200 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 70-1100 base pairs. In one embodiment of the various aspects and embodiments herein, the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 80-1000 base pairs in length. In one embodiment of the various aspects and embodiments herein, the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 90-900 base pairs in length.In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 90-900 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 100-800 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 110-700 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 120-600 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 130-500 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 140-400 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 150-300 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 160-200 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 170-190 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 1381 base pairs. In one embodiment of the various aspects and embodiments herein, the double-stranded region comprising the promoter, the enhancer, and / or the TSS is approximately 499 base pairs in length. In one embodiment of the various aspects and embodiments herein, the ssDNA molecule is capable of expressing at least one therapeutic protein or a therapeutic fragment thereof. In one embodiment of the various aspects and embodiments herein, the at least one therapeutic protein is selected from the group consisting of an antibody, an enzyme, a coagulation factor, a transcription factor, a replication factor, a growth factor, a hormone, and a fusion protein.In one embodiment of the various aspects and embodiments herein, the at least one therapeutic protein can be used to treat a genetic disease 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 disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Leschnehan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharidoses (e.g., Hurler syndrome (MPS type I), Shay syndrome (MPS type I S), Hurler-Shay syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo A, B, C, and D (MPS MPS III A, B, C, and D), Morquer A and B (MPS IVA and MPS IVB), Mallory-Lami 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 (Sandhoff 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, aspartate glucosamineuria, sialidosis, Dannon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), LS), 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 congenital amaurosis, 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.

[0029] In one embodiment of the various aspects and embodiments herein, the ssDNA molecule further comprises a lipid. In another embodiment, the ssDNA molecule is encapsulated in a lipid. In one embodiment of the various aspects and embodiments herein, the lipid is a lipid nanoparticle (LNP).

[0030] In one embodiment of the various aspects and embodiments herein, the present disclosure provides a pharmaceutical composition comprising the ssDNA molecule of any one of the various aspects and embodiments herein, and a pharmaceutically acceptable excipient.

[0031] In another aspect, the present disclosure provides a method of treating a genetic disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the ssDNA molecule of any one of the various aspects and embodiments herein or the pharmaceutical composition of any one of the various aspects and embodiments herein.

[0032] In another aspect, the present disclosure provides a host cell comprising the ssDNA molecule of any of the aspects and embodiments herein.

[0033] In another aspect, the present disclosure provides a method of delivering a therapeutic gene and / or therapeutic protein to a subject, the method comprising administering to the subject a therapeutically effective amount of the ssDNA molecule of any one of the various aspects and embodiments herein or the pharmaceutical composition of any one of the various aspects and embodiments herein.

[0034] In another aspect, the present disclosure provides a method for delivering a therapeutic gene and / or a therapeutic protein to a cell, the method comprising contacting the cell with the ssDNA molecule of any one of the various aspects and embodiments herein or the pharmaceutical composition of any one of the various aspects and embodiments herein, thereby delivering the therapeutic gene and / or the therapeutic protein to the cell.

[0035] In 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 the ssDNA molecule of any one of the various aspects and embodiments herein or the pharmaceutical composition of any one of the various aspects and embodiments herein, thereby delivering the therapeutic gene and / or the therapeutic protein to the nucleus of the cell.

[0036] In another aspect, the present disclosure provides a method of 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 ssDNA molecule of any one of the various aspects and embodiments herein, or the pharmaceutical composition of any one of the various aspects and embodiments herein, wherein the nucleic acid of interest encodes the therapeutic gene or the therapeutic protein.

[0037] In one embodiment of the various aspects and embodiments herein, the enhancer is double-stranded. In one embodiment of the various aspects and embodiments herein, the double-stranded region comprising the promoter, the enhancer and / or the 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 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 one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the 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, or less than 500 base pairs. base pairs, less than 380 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 one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 30-1500 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 40-1400 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 50-1300 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 60-1200 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 70-1100 base pairs. In one embodiment of the various aspects and embodiments herein, the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 80-1000 base pairs in length. In one embodiment of the various aspects and embodiments herein, the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 90-900 base pairs in length.In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 90-900 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 100-800 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 110-700 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 120-600 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 130-500 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 140-400 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 150-300 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 160-200 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 1381 base pairs. In one embodiment of the various aspects and embodiments herein, the length of the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 499 base pairs. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The embodiments of the present disclosure, briefly summarized above and discussed in more detail below, may be understood by reference to the illustrative embodiments of the disclosure depicted in the accompanying drawings. However, the drawings illustrate only typical embodiments of the disclosure and are therefore not to be considered limiting of its scope, as the disclosure may admit to other equally effective embodiments.

[0039] Figure 1A It is shown that ssAAV vectors were obtained by ssDNA endonuclease treatment starting from double-stranded ceDNA construct 10429. Figure 1A As can be seen, a clear band of approximately 830 bp was observed for the ssRES ss360 control, and there was clear degradation of the ΦX174 ssDNA control. The loss of the full-size ssRES ss429 in the wells and the appearance of a band migrating at approximately 100 bp were more pronounced / clearer in the 5U mung bean nuclease treatment.

[0040] Figure 1B Schematic diagrams of the double-stranded ceDNA 10429 construct ("ds429") and single-stranded 10429 and 10360 are depicted.

[0041] Figure 2 KLENOW exonuclease filling of ss synthetic AAV vector molecules supporting the presence of 3'-OH is shown.

[0042] Figure 3 Schematic diagrams of symmetric and asymmetric ITR oligomers are depicted. The top diagram shows symmetric overhangs. The bottom diagram shows asymmetric overhangs, where the 3' end of the left ITR has a PS bond (closer to the 3' end of the molecule) and the PS bond of the right ITR is shifted two bases to the right.

[0043] Figure 4 Schematic diagram depicting the left and right ITR sequences of the single-stranded AAV synthetic vector hAAT-luciferase with symmetrical ITR oligomers from constructs ss10429 and ss10483. Figure 4 As shown in , a CpG-free spacer was included that changed some nucleotides in the RBE of the A / A' stem. The nicking site Nb.BbvCI was engineered downstream of the terminal melting site (trs).

[0044] Figure 5 Schematic diagram of the left and right ITR sequences of the single-stranded AAV synthetic vector hAAT-luciferase with asymmetric ITR oligomers from construct ss10485 is depicted. Figure 5 As shown in , a CpG-free spacer was constructed by changing some nucleotides in the RBE of the A / A' stem. The nicking site Nb.BbvCI was engineered downstream of trs.

[0045] Figure 6 Schematic diagram depicting the symmetrical ITR oligomers of the single-stranded AAV synthetic vector FVIII left and right ITR sequences from construct ss10491. The nicking site Nb.BbvCI was engineered downstream of trs.

[0046] Figure 7 Schematic diagram depicting the asymmetric ITR oligomer of the single-stranded AAV synthetic vector FVIII left and right ITR sequences from construct ss10484. The nicking site Nb.BbvCI was engineered downstream of trs.

[0047] Figure 8 Graph showing luciferase expression in HepG2 cells transfected with single-stranded AAV synthetic vectors. A clear dose response was observed for both ceDNA and single-stranded AAV synthetic vectors.

[0048] Figure 9 Figure 2 shows that single-stranded AAV synthetic vectors induced lower innate immune responses compared to ceDNA, which was purified using a Zymo column. At matched molecular doses, single-stranded AAV synthetic vectors induced less Lucia IFN reporter than ceDNA in WT THP1 cells.

[0049] Figure 10 Figure 2 is a graph showing that gel-extracted and Zymo column-purified single-stranded AAV synthetic vectors induce lower innate immune responses compared to ceDNA. In cGAS KO cells, an IFN response was present at the highest dose of ceDNA and column-purified single-stranded AAV synthetic vectors, but absent otherwise, indicating that cGAS senses both single-stranded AAV synthetic vectors and ceDNA.

[0050] Figure 11 Graph showing median longitudinal body weight of animals dosed with single-stranded synthetic DNA (ssDNA) "SSD" (40004) and animals dosed with double-stranded (ds)ceDNA (10541).

[0051] Figure 12 is a set of graphs showing median longitudinal body weights for each of the doses in animals dosed with single-stranded synthetic DNA (ssDNA) "SSD" (40004) and animals dosed with double-stranded (ds)ceDNA (10541).

[0052] Figure 13 Results of IVIS imaging performed on day 4 are shown.

[0053] Figure 14 Shown are the results of IVIS imaging performed on days 4-7.

[0054] Figure 15 The nucleic acid sequence of double-stranded (ds) ceDNA construct 10360 (SEQ ID NO: 1) is shown. Figure 15 As indicated in SEQ ID NO: 1, Factor VIII ORF is located at nucleotides 828-5219 of SEQ ID NO: 1.

[0055] Figure 16 The nucleic acid sequence of ds ceDNA construct 10429 (SEQ ID NO: 2) is shown. Figure 16 As indicated in FIG, the luciferase ORF is located at nucleotides 1449-3101 of SEQ ID NO: 2.

[0056] Figure 17 The nucleic acid sequence of ds ceDNA construct 10483 (SEQ ID NO: 3) is shown. Figure 17 As indicated in FIG, the luciferase ORF is located at nucleotides 1449-3101 of SEQ ID NO: 3.

[0057] Figure 18 The nucleic acid sequence of ds ceDNA construct 10484 (SEQ ID NO: 4) is shown. Figure 18 As indicated in SEQ ID NO: 4, Factor VIII ORF is located at nucleotides 835-5226 of SEQ ID NO: 4.

[0058] Figure 19 The nucleic acid sequence of ds ceDNA construct 10485 (SEQ ID NO: 5) is shown. Figure 19 As indicated in FIG, the luciferase ORF is located at nucleotides 1449-3101 of SEQ ID NO:5.

[0059] Figure 20 The nucleic acid sequence of ds ceDNA construct 10491 (SEQ ID NO: 6) is shown. Figure 20 As indicated in SEQ ID NO: 6, Factor VIII ORF is located at nucleotides 835-5226 of SEQ ID NO: 6.

[0060] Figure 21 The nucleic acid sequence of ds ceDNA construct 10376 (SEQ ID NO: 7) is shown. Figure 21 As indicated in FIG, the luciferase ORF is located at nucleotides 1443-3095 of SEQ ID NO:7.

[0061] Figure 22 The nucleic acid sequence of ds ceDNA construct 10541 (SEQ ID NO: 8) is shown. Figure 22 As indicated in FIG, the luciferase ORF is located at nucleotides 1449-3101 of SEQ ID NO:8.

[0062] Figure 23 The nucleic acid sequence of plasmid 210150 (SEQ ID NO: 9) is shown, which contains one of the exemplary ds ceDNA sequences. Figure 23 As indicated in , Factor VIII ORF is located at nucleotides 894-5285.

[0063] Figure 24 The nucleic acid sequence of the single-stranded (ss) DNA construct 40004 (SEQ ID NO: 10) is shown. Figure 24 As indicated in , the reverse complement of the luciferase ORF is located at nucleotides 503-2155 of SEQ ID NO: 10.

[0064] Figure 25 is a set of graphs showing median longitudinal body weight in animals dosed with single-stranded synthetic DNA (ssDNA) "SSD" (ssDNA construct 40004, designated "ss004") and double-stranded (ds)ceDNA (dsceDNA construct 10541, designated "ds541") for five different doses matched by molecular number.

[0065] Figure 26 is a set of graphs showing weight loss over 5 days in animals administered with single-stranded synthetic DNA (ssDNA) "SSD" (40004) and double-stranded (ds)ceDNA (10541). Figure 26 As shown, the difference in BW loss was most pronounced on day 2, when animals dosed with ssDNA rapidly regained their lost weight.

[0066] Figure 27 Figure 1 is a set of graphs showing the effects of ssDNA (construct 40004, designated "ss004") and ds ceDNA (construct 10541, designated "ds541") on cytokine expression after 6 hours. ds ceDNA construct 10376 ("376") was used as a positive control. The legend indicates whether the PS linkage was present ("w / PS") or absent ("no PS") in the construct.

[0067] Figure 28 Figure 1 is a set of graphs from a second experiment showing the effects of ssDNA (construct 40004, designated "ss004") and ds ceDNA (construct 10541, designated "ds541") on cytokine expression after 6 hours. ds ceDNA construct 10376 ("376") was used as a positive control. The legend indicates whether the PS linkage was present ("w / PS") or absent ("no PS") in the construct.

[0068] Figure 29 is a graph showing that ds ceDNA (construct 10541, designated "ds541") and ssDNA (construct 40004, designated "ss004") have similar levels of mRNA in the liver as determined by qtPCR.

[0069] Figure 30: is a set of figures showing the effects of LNP-formulated mRNA, ceDNA, and ssDNA cargo on blood cytokine levels in mice. Mice (n = 5 per group) were intravenously injected with LNP-formulated mRNA, ceDNA, or ssDNA. Blood levels of IFN-α, IL-18, TNF-α, IL-6, and IFN-γ were measured 6 hours after injection (in pg / mL). Several batches of ssDNA (NHP) produced later for non-human primates were also tested. Groups in each figure, from 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).

[0070] Figure 31 is a set of graphs showing the effects of 1.0 mg / kg of LNP-formulated ceDNA ("DNA", circles), ssDNA (squares), and mRNA (triangles) cargo on blood cytokine levels (in pg / mL) 6 and 24 hours after intravenous infusion into cynomolgus monkeys.

[0071] Figure 32 Shown are the levels of complement activation (left: C3a; right: C5b-9) measured in NHPs following administration of 1.0 mg / mL LNP-formulated ceDNA (circles), ssDNA (squares), or mRNA (triangles).

[0072] Figure 33 Results of an in vitro PBMC assay are shown. Human peripheral blood mononuclear cells (PBMCs) were contacted with LNP-formulated ceDNA or ssDNA, and TNF-α stimulation was measured. In both the first study (two left-hand bars) and the second study (three right-hand bars, which included untreated controls), ssDNA induced significantly lower TNF-α stimulation compared to ceDNA (TNF-α stimulation induced by ssDNA was comparable to that of the untreated control).

[0073] Figure 34 It is a group of figures showing the persistent and robust expression of ssDNA prepared through LNP. 0.25 mg / kg of ceDNA prepared through LNP or ssDNA comprising a luciferase reporter gene was injected intravenously into mice. Luciferase expression (IVIS) was measured one day and four days after injection (right) and at other time points (left) up to 30 days.

[0074] Figures 35A-35E Schematic diagram of ssDNA constructs with different components is depicted. All constructs contain the hAAT-luciferase expression construct. Figure 35A : Construct ss004 (left ITR derived from AAV2 with the PS bond on the bottom strand and with a Nb.BbvCI nicking site; right ITR derived from AAV2 with most of the A region removed and the PS bond at the 5' end; expression construct minus strand). Figure 35B : Construct ss020 (same as ss004, but without the PS linkage). Figure 35C : Construct ss021 (same as ss004, but with wild-type AAV2 ITRs, no PS linkage, and no Nb.BbvCI nicking site). Figure 35D : Construct ss022 (similar to ss021 except the construct plus strand is expressed). Figure 35E : hAAT-luciferase expression construct without ITR sequences.

[0075] Figure 36 is a graph of in vitro luciferase expression by ssDNA constructs. HepG2 cells were transfected with 100 ng or 200 ng each of ss004 or ss011, and luciferase expression was measured at 48 hours (normalized to cell viability).

[0076] Figure 37 It is a group of figures depicting the result of the in vivo luciferase expression by ssDNA constructs in mice after hydrodynamic injection (HDI). Mice were injected with a mixture of ceDNA541 (5 ng or 500 ng per animal), constructs ss004, ss020, ss021, ss022 or ss011 (500 ng per animal), or ss021+ss022 (250 ng per animal), or ss011+ss022 (250 ng per animal). IVIS luciferase expression is shown at day 1 (upper left) and day 4 (lower left), and in longitudinal view (upper right).

[0077] Figure 38 Graph depicting the results of in vivo luciferase expression in mice injected with LNP-formulated ssDNA constructs. Shown from left to right are the results for PBS control (inverted triangles), ss011 (diamonds, ionizable lipid MC3), ss011 (open circles, ionizable lipid Y), ss011 (squares, ionizable lipid Z), and ss004 (ionizable lipid Z).

[0078] Figures 39A-39DSchematic diagrams of ssDNA constructs with different PS bond configurations are depicted. All constructs contain a hAAT-luciferase expression construct. The position of the PS bond is indicated by an arrow. Figure 39A : Construct ss004 (left ITR derived from AAV2 with the PS bond on the bottom strand; right ITR derived from AAV2 with most of the A region removed and the PS bond at the 5' end). Figure 39B : Construct 034 (same as ss004, but the PS bond in the left ITR is on the top strand, near the 3' end). Figure 39C : Construct ss039 (same as ss034, but with a truncated ITR on the right side and the PS linkage on the bottom strand, near the 5' end). Figure 39D : Construct ss040 (same as ss039, but without the PS linkage).

[0079] Figure 40 Is a set of graphs depicting the results of in vivo luciferase expression in mice injected with ssDNA constructs formulated with LNPs. Mice were injected with ceDNA541, ceDNA654, ss004, ss034, ss039, or ss040 (0.25 mg / kg). IVIS luciferase expression is shown at day 1 (top left) and day 4 (top right), as well as in the longitudinal graph (bottom).

[0080] Figure 41 Figure 1 is a set of graphs showing the effects of LNP-formulated mRNA, ceDNA, and ssDNA cargoes on blood cytokine levels in mice. Mice (n = 5 per group) were intravenously injected with LNP-formulated ceDNA or ssDNA constructs. Blood levels (in pg / mL) of IFN-α (top left), IFN-γ (top right), IL-6 (middle left), TNF-α (middle right), and IL-18 (bottom left) were measured 6 hours after injection. Groups in each graph, from left to right: PBS control, ceDNA541, ceDNA654, ss004, ss034, ss039, and ss040.

[0081] Figures 42A-42D Schematic diagrams of ssDNA constructs with single-stranded or double-stranded promoter regions are depicted. All constructs contain a luciferase reporter. Figure 43 A: ss004, single-chain hAAT enhancer / promoter group. Figure 43 B: ss104, double-stranded hAAT enhancer / promoter group. Figure 43 C: ss102, single-stranded 1xSERP / TTR enhancer / promoter group. Figure 43 D: ss104, double-stranded 1xSERP / TTR enhancer / promoter group.

[0082] Figure 43 Figure 1 shows a set of graphs measuring luciferase expression in mice injected via HDI with ssDNA constructs containing single-stranded (ss004, ss102) or double-stranded (ss104, ss103) promoter regions. Left: Luciferase expression on day 1. Middle: Luciferase expression on day 7. Right: Longitudinal expression from day 1 to day 7.

[0083] Figure 44 Figure 1 shows the fold change in expression of ssDNA constructs with double-stranded promoter regions (ss104 and ss103, respectively) compared to ssDNA constructs with single-stranded promoter regions (ss004 and ss102, respectively). Left: Day 1. Middle: Day 7. Right: Vertical fold change from Day 1 to Day 7.

[0084] Figure 45 Figure 1 shows a set of graphs measuring luciferase expression in mice injected via HDI with ssDNA constructs containing single-stranded (ss004, ss102) or double-stranded (ss104, ss103) promoter regions, compared to expression from ceDNA541. Left: Luciferase expression on day 1. Right: Luciferase expression on day 7. DETAILED DESCRIPTION

[0085] I. Definition

[0086] Unless otherwise defined herein, scientific and technical terms used in conjunction with this application shall have the meanings commonly understood by those of ordinary skill in the art of the present disclosure. It should be understood that the present disclosure is not limited to the specific methods, protocols, and reagents described herein and may vary accordingly. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present disclosure, which is limited only by the claims. Definitions of commonly used terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th ed., Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), Fields Virology, 6th ed., Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013); Knipe, DM and Howley, PM (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, Blackwell Science Ltd. Ltd., 1999–2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc.), 1995 (ISBN 1-56081-569-8); Werner Luttmann, Immunology, 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 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, 2013 (ISBN 0124199542); Sons), 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E.Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.), John Wiley and Sons, 2003) (ISBN 0471142735, 9780471142737), the contents of which are all incorporated herein by reference in their entirety.

[0087] As used herein, the term "AAV" or "adeno-associated virus" refers to a single-stranded DNA parvovirus that grows only in cells. Some of the functions of AAV are provided only by co-infection with a helper virus. Thirteen serotypes of AAV have been identified. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228 and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York).

[0088] As used herein, the terms "single-stranded (ss) synthetic DNA molecules," "single-stranded (ss) synthetic AAV vectors," "synthetic production of ssDNA molecules," and "synthetic production of ssAAV 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. Production may involve one or more molecules in a manner that does not involve replication or other proliferation of the molecules by or within cells or using cell extracts. Synthetic production avoids contamination of the produced molecules with cellular contaminants (e.g., cellular proteins or cellular nucleic acids, viral proteins or DNA, insect proteins or DNA), and further avoids undesirable cell-specific modifications of the molecules during the production process, such as methylation or glycosylation or other post-translational modifications.

[0089] As used herein, the terms "gap" and "nick" are used interchangeably and refer to an interruption in a synthetic DNA vector of the present disclosure that creates an extension of a single-stranded DNA portion in an otherwise double-stranded ceDNA. The length of the gap can be 1 base pair to 100 base pairs. 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 long, 1 to 20 bp long, 1 to 30 bp long, or any length required to nick the double-stranded DNA to allow or maintain efficient transcription of the expression cassette in the host cell. According to some embodiments, the gap can be present 5' upstream of the expression cassette. According to some embodiments, the gap can be present 3' downstream of the expression cassette. According to some embodiments, the gap can be present 5' upstream of the expression cassette and 3' downstream of the expression cassette.

[0090] As used herein, the term "nick" refers to a discontinuity in a double-stranded DNA molecule in which the phosphodiester bond between adjacent nucleotides of one strand is absent, typically by damage or enzymatic action. It will be understood that one or more nicks allow for the release of torsion in the strand during DNA replication, and nicks are also believed to play a role in facilitating the binding of the transcription machinery.

[0091] As used herein, the term "ceDNA" refers to non-capsid-terminated linear double-stranded (ds) duplex DNA for non-viral gene transfer, synthetic or otherwise. A detailed description of ceDNA is described in International Application No. PCT / US2017 / 020828, filed on March 3, 2017 (published as International Patent Publication No. WO2017152149A1), the entire contents of which are incorporated herein by reference. Certain methods for generating 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, filed September 7, 2018 (published as International Patent Publication No. WO 2019 / 051255 A1) and PCT / US2018 / 064242, filed December 6, 2018 (published as International Patent Publication No. WO 2019 / 113310 A1), each of which is incorporated herein by reference in its entirety. Certain methods for generating synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Application PCT / US2019 / 14122, filed January 18, 2019 (published as International Patent Publication No. WO 2019 / 143885 A1), the entire contents of which are incorporated herein by reference. According to some embodiments, the ceDNA comprises one or more phosphorothioate-modified nucleotides.

[0092] As used herein, the term "neDNA" or "nicked ceDNA" refers to capped DNA having a nick or gap of 1-100 base pairs in the stem or spacer region upstream of the open reading frame (e.g., the promoter and transgene to be expressed).

[0093] 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 the ssDNA vector disclosed herein, which comprises at least one stem-loop structure comprising a partial duplex and at least one loop. According to some embodiments, the ITR can be an artificial sequence (e.g., does not contain sequences derived from a virus). The ITR can further comprise a stem-loop structure (e.g., a "hairpin") or more than one stem-loop structure. For example, the ITR can comprise two stem-loop structures (e.g., a "hammerhead," "doggy-bone," or "dumbbell"), three stem-loop structures (e.g., a "cross"), or a more complex structure. The ITR can comprise an aptamer sequence or one or more chemical modifications.

[0094] According to some embodiments, "ITR" can be artificially synthesized using a set of oligonucleotides comprising one or more desired functional sequences (e.g., palindromic sequences). The ITR sequence can be an artificial AAV ITR, an artificial non-AAV ITR, or an ITR physically derived from a viral AAV ITR (e.g., an ITR fragment taken out of a viral genome). For example, ITR can be derived from the Parvoviridae family, which covers parvoviruses and dependent viruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or an SV40 hairpin acting as an SV40 origin of replication can be used as an ITR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. The Parvoviridae family of viruses consists of two subfamilies: the Parvovirinae family that infects vertebrates and the Densovirinae family that infects invertebrates. The genus Dependaviridae is a family of viruses that includes adeno-associated viruses (AAVs), which are capable of replicating in vertebrate hosts, including but not limited to humans, primates, cattle, dogs, horses, and sheep species. Generally speaking, ITR sequences can be derived not only from AAV, but also from parvoviruses, lentiviruses, goose viruses, B19, in configurations of wild-type, "doggy bone" and "dumbbell-shaped", symmetrical or even asymmetrical ITR orientations. Although ITRs are typically present at the 5' and 3' ends of AAV vectors, in single-stranded DNA (ssDNA) molecules, ITRs can only be present at one end of a linear vector. For example, ITRs can only be present at the 5' end. In some other cases, ITRs can only be present at the 3' end in single-stranded DNA (ssDNA) molecules. For convenience herein, the ITR located 5' ("upstream") of the expression cassette in the single-stranded DNA (ssDNA) molecule is referred to as the "5' ITR" or "left ITR", and the ITR located 3' ("downstream") of the expression cassette in the single-stranded DNA (ssDNA) molecule is referred to as the "3' ITR" or "right ITR".

[0095] As used herein, "wild-type ITR" or "WT-ITR" refers to a sequence of an ITR sequence naturally occurring in the AAV genome or other dependent virus that retains, for example, Rep binding activity and Rep cleavage ability. Due to the degeneracy or drift of the genetic code, the nucleotide sequence of the WT-ITR from any AAV serotype may differ slightly from the typical naturally occurring sequence, and therefore, the WT-ITR sequences contemplated for use herein include those resulting from naturally occurring variations (e.g., replication errors).

[0096] As used herein, the term "substantially symmetrical WT-ITR" or "substantially symmetrical WT-ITR pair" refers to a pair of WT-ITRs within a single-stranded DNA (ssDNA) molecule (e.g., a synthetic AAV vector, e.g., a single-stranded (ss) synthetic AAV vector) that are two wild-type ITRs with inverted complement sequences throughout their entire length. For example, even if an ITR has one or more nucleotides that deviate from a typical, naturally occurring canonical sequence, the ITR can be considered a wild-type sequence as long as these changes do not affect the physical and functional properties and overall three-dimensional structure (secondary and tertiary structure) of the sequence. In some aspects, the deviated nucleotides represent conservative sequence changes. As a non-limiting example, a sequence has at least 95%, 96%, 97%, 98% or 99% sequence identity to the canonical sequence (as measured, for example, using BLAST at default settings) and also has a symmetrical three-dimensional spatial organization with another WT-ITR such that its 3D structure has the same shape in geometric space. Substantially symmetrical WT-ITRs have identical A, C-C' and B-B' loops in 3D space. Substantially symmetrical WT-ITRs can be functionally confirmed to be WT by confirming that they have an operable Rep binding site (RBE or RBE') and a terminal melting site (TRS) that pairs with the appropriate Rep protein.Other functions can optionally be tested, including transgene expression under permissive conditions.

[0097] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutant ITR" are used interchangeably and refer to an ITR that has a mutation in at least one or more nucleotides compared to a WT-ITR from the same serotype. The mutation can result in a change in one or more of the A, C, C', B, or B' regions of the ITR and can result in a change in the three-dimensional organization (i.e., its 3D structure in geometric space) compared to the 3D organization of the WT-ITR of the same serotype.

[0098] As used herein, the term "asymmetric ITR", also referred to as an "asymmetric ITR pair", refers to a pair of ITRs within a single ssDNA vector that are not reverse complementary over their entire length. As a non-limiting example, an asymmetric ITR does not have a symmetrical three-dimensional spatial organization with its homologous ITR, such that its 3D structure has a different shape in geometric space. In other words, the asymmetric ITR pair has a different overall geometric structure, i.e., it has a different A, C-C' and B-B' loop organization in 3D space (e.g., one ITR may have a short C-C' arm and / or a short B-B' arm compared to the homologous ITR). The sequence difference between the two ITRs may be due to one or more nucleotide additions, deletions, truncations, or point mutations. In one embodiment, one ITR in the asymmetric ITR pair may be a wild-type AAV ITR sequence, and the other ITR is a modified ITR as defined herein (e.g., a non-wild-type or synthetic ITR sequence). In another embodiment, neither ITR in the asymmetric ITR pair is a wild-type AAV sequence, and the two ITRs are modified ITRs having different shapes in geometric space (i.e., different overall geometric structures). In some embodiments, one mod-ITR in 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, etc.) such that it has a different three-dimensional spatial organization than the homologous asymmetric mod-ITR.

[0099] As used herein, the term "symmetric ITR" refers to a pair of ITRs in an ssDNA vector that are mutated or modified relative to a wild-type dependent viral ITR sequence and are reversely complementary over their full length. These two ITRs are not wild-type ITR AAV2 sequences (i.e., they are modified ITRs, also referred to as mutant ITRs), and are different from wild-type ITRs in sequence due to nucleotide additions, deletions, substitutions, truncations, or point mutations. For convenience herein, the ITR at the 5' (upstream) of the expression cassette in a single-stranded DNA (ssDNA) molecule is referred to as "5' ITR" or "left ITR," and the ITR at the 3' (downstream) of the expression cassette in a single-stranded DNA (ssDNA) molecule is referred to as "3' ITR" or "right ITR."

[0100] As used herein, the term "substantially symmetrical modified ITR" or "substantially symmetrical mod-ITR pair" refers to a pair of modified ITRs within a single-stranded DNA (ssDNA) molecule (e.g., a synthetic AAV vector, e.g., a single-stranded (ss) synthetic AAV vector) that have reverse complement sequences throughout their entire length. For example, even if the modified ITR has some nucleotide sequence that deviates from the reverse complement sequence, it can be considered to be substantially symmetrical as long as these changes do not affect the properties and overall shape. As a non-limiting example, the sequence has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the typical sequence (as measured using BLAST at default settings) and also has a symmetrical three-dimensional spatial organization with its cognate modified ITR, such that their 3D structures have the same shape in geometric space. In other words, the substantially symmetrical modified ITR pair has the same stem-loop structure organized in 3D 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 produce the same overall 3D shape. For example, in viral ITRs, one ITR (e.g., 5' ITR) in a mod-ITR pair can be from one serotype, and the other ITR (e.g., 3' ITR) can be from a different serotype, however, both can have the same corresponding mutations (e.g., if the 5' ITR has a deletion in the C region, the homologously modified 3' ITR from a different serotype also has a deletion at the corresponding position in the C' region), such that the modified ITR pair has the same symmetrical three-dimensional spatial organization. In such embodiments, each ITR in the modified ITR pair can be from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAV2 and AAV6, wherein the modification in one ITR is reflected in the corresponding position in the homologous ITR from the different serotype. In one embodiment, a substantially symmetrical modified ITR pair refers to a pair of modified ITRs (mod-ITRs) as long as the difference in nucleotide sequence between the ITRs does not affect the properties or overall shape and they have substantially the same shape in 3D space. As non-limiting examples, the mod-ITRs have at least 95%, 96%, 97%, 98% or 99% sequence identity to a typical mod-ITR as determined by standard methods well known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN under default settings, and also have a symmetrical three-dimensional spatial organization such that their 3D structures have the same shape in geometric space.A substantially symmetric mod-ITR pair has identical A, C-C', and BB' loops in 3D space. For example, if a modified ITR in a substantially symmetric mod-ITR pair lacks the C-C' arm, then the corresponding homologous mod-ITR lacks the C-C' loop and also has a similar 3D structure with the remaining A and BB' loops being identically shaped in the geometric space of its cognate mod-ITR.

[0101] As used herein, the term "flanking" refers to the relative position of one nucleic acid sequence relative to another nucleic acid sequence. Typically, in the sequence ABC, B is flanked by A and C. This is also true for the arrangement AxBxC. Thus, a flanking sequence precedes or follows the flanking sequence, but need not be adjacent to or immediately adjacent to the flanking sequence. In one embodiment, the term flanking refers to the terminal repeats at each end of a linear single-stranded DNA (ssDNA) molecule.

[0102] As used herein, the term "capped DNA" or "ceDNA" refers to a synthetic double-stranded linear DNA construct with at least one covalently blocked end, including a gene of interest and other regulatory elements.

[0103] As used herein, the term "capped DNA vector" refers to a capsid-free DNA vector having at least one covalently blocked end, wherein at least a portion of the vector has an intramolecular duplex structure.

[0104] As defined herein, "reporter" or "reporters" refer to one or more proteins that can be used to provide detectable readings. Reporter factors typically produce measurable signals, such as fluorescence, color, or luminescence. The presence of a reporter protein coding sequence in a cell or organism is easily observable. For example, fluorescent proteins can cause cells to fluoresce when excited by light of a specific wavelength, luciferases cause cells to catalyze the production of light, and enzymes such as beta-galactosidase convert substrates into colored products. Exemplary reporter polypeptides that can be used for experiments or diagnostic purposes include, but are not limited to, beta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP) and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and other well-known in the art.

[0105] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable readout, such as a reporter polypeptide, or more appropriately, a cell-killing polypeptide, such as a toxin, or an agent that renders a cell susceptible to a selected agent or killed in the absence of a selected agent. Effector proteins include any protein or peptide that directly targets or damages the DNA and / or RNA of a host cell. For example, effector proteins may 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. In some embodiments, the expression of an effector protein controlled by a synthetic biological circuit as described herein can participate as a factor in another synthetic biological circuit to thereby expand the scope and complexity of the response of the biological circuit system.

[0106] Transcriptional regulatory factors refer to transcriptional activators and repressors that activate or repress the transcription of a gene of interest. A promoter is a nucleic acid region that initiates transcription of a specific gene. Transcriptional activators typically bind near a transcriptional promoter and recruit RNA polymerase to directly initiate transcription. Repressors bind to transcriptional promoters and spatially hinder RNA polymerase from initiating transcription. Other transcriptional regulatory factors can act as activators or repressors depending on their binding location and cell and environmental conditions. Non-limiting examples of transcriptional regulatory factor categories include, but are not limited to, homeodomain proteins, zinc finger proteins, winged helix (forkhead) proteins, and leucine zipper proteins.

[0107] As used herein, a "repressor protein" or "inducer protein" is a protein that binds to a regulatory sequence element and represses or activates, respectively, transcription of a sequence operatively linked to the regulatory sequence element. Preferred repressor and inducer proteins as described herein are sensitive to the presence or absence of at least one input agent or environmental input. Preferred proteins as described herein are modular in form, comprising, for example, separable DNA binding and input agent binding or responsive elements or domains.

[0108] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, 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 may 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.

[0109] As used herein, an "input 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 causes an attached DNA binding fusion domain to respond to the presence of the condition or input agent. In one embodiment, the presence of the condition or input agent causes a conformational change in the input response domain or a protein to which it is fused, which alters the transcriptional regulatory activity of the transcription factor.

[0110] As used herein, the term "in vivo" refers to an assay or process performed in or within an organism, such as a multicellular animal. In some of the aspects described herein, when a single-cell organism, such as a bacterium, is used, the method or use can be said to occur "in vivo." The term "ex vivo" refers to methods and uses performed using living cells with intact membranes that are outside of a multicellular animal or plant, for example, explants, cultured cells, including primary cells and cell lines, transformed cell lines, and extracted tissues or cells, including blood cells, etc. 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 into non-cellular systems, such as media that do not contain cells or cell systems, such as cell extracts.

[0111] As used herein, the term "promoter" refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of a nucleic acid sequence, which can be a heterologous target gene encoding a protein or RNA. A promoter can be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence, where the initiation and transcription rate of the remainder of the nucleic acid sequence are controlled. A promoter can also contain genetic elements that can bind to regulatory proteins and molecules such as RNA polymerase and other transcription factors. A transcription start site will be found within the promoter sequence, as well as a protein binding domain responsible for RNA polymerase binding. Eukaryotic promoters will often, but not always, contain a "TATA" box and a "CAT" box. Various promoters, including inducible promoters, can be used to drive the expression of transgenics in the single-stranded (ssDNA) molecules disclosed herein. The promoter sequence can be defined by a transcription start site at its 3' end and extend upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background.

[0112] 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 transgene transcription of a DNA vector, e.g., a single-stranded (ssDNA) molecule. Suitable promoters include, for example, tissue-specific promoters. The promoter may also be of AAV origin.

[0113] As used herein, when referring to a "regenerated double-stranded expression cassette" or a "regenerated double-stranded transgene," the term "regeneration" means a double-stranded expression cassette or double-stranded transgene that is formed after the ssDNA molecule has been transported to the nucleus of a host cell and responds to DNA polymerase activity that generates double-stranded DNA from the ssDNA by filling in the single-stranded portions of the ssDNA molecule.

[0114] As used herein, "operably connected" refers to a juxtaposition in which the components described are in a relationship that allows them to function in an expected manner. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably connected to the coding sequence. A promoter can be said to drive the expression of the nucleic acid sequence it regulates or to drive its transcription. The phrases "operably connected," "operably positioned," "operably connected," "under control," and "under transcriptional control" indicate that a promoter is in correct functional position and / or orientation relative to the nucleic acid sequence it regulates, to control the start of transcription and / or expression of the sequence. As used herein, a "reverse promoter" refers to a nucleic acid sequence that is in an opposite orientation so that the coding strand now becomes the promoter of the non-coding strand, and vice versa. Reverse promoter sequences can be used in various embodiments to regulate the state of a switch. Additionally, in various embodiments, a promoter can be used in combination with an enhancer.

[0115] The terms "DNA regulatory sequence," "control element," and "regulatory element," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide 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.

[0116] As used herein, the term "enhancer" refers to a cis-acting regulatory sequence (e.g., 50-1,500 base pairs) that binds to one or more proteins (e.g., activator proteins or transcription factors) to enhance transcriptional activation of a nucleic acid sequence. Naturally, an enhancer can be positioned up to 1,000,000 base pairs upstream of the gene start site or downstream of the gene start site that it regulates. An enhancer can be positioned within an intron region, or in an exon region of an unrelated gene. Cis-acting enhancer sequences of 20-200 base pairs are typically used to increase transgenic expression.

[0117] A promoter can be one naturally associated with a gene or sequence, such as one that can be obtained by isolating 5' non-coding sequences upstream of the coding segment and / or exons of a given gene or sequence. Such promoters can be referred to as "endogenous." Similarly, in some embodiments, an enhancer can be one naturally associated with a nucleic acid sequence, located downstream or upstream of the sequence. In some embodiments, the coding nucleic acid segment is positioned under the control of a "recombinant promoter" or a "heterologous promoter," both of which refer to promoters that are not normally associated with the coding nucleic acid sequence to which they are operably linked in their 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., those that have been engineered to contain different elements of different transcriptional regulatory regions that alter expression and / or have been mutated by methods known in the art. In addition to synthetically generating promoter and enhancer nucleic acid sequences, promoter sequences can also be generated using recombinant cloning and / or nucleic acid amplification techniques, including PCR, in conjunction with the synthetic biology circuits and modules disclosed herein (see, e.g., U.S. Patent No. 4,683,202, U.S. Patent No. 5,928,906, each of which is incorporated herein by reference). Furthermore, it is contemplated that control sequences that direct transcription and / or expression within non-nuclear organelles, such as mitochondria and chloroplasts, can also be employed.

[0118] As described herein, an "inducible promoter" is a promoter characterized in that when an inducer or inducer is present, affected by it, or contacted by it, transcriptional activity is initiated or enhanced. As defined herein, an "inducer" or "inducer" can be endogenous, or a generally exogenous compound or protein that is administered in a manner capable of inducing transcriptional activity from an inducible promoter. In some embodiments, the inducer or inducer, i.e., a chemical substance, compound, or protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., the inducer can be an inducible protein expressed by another component or module), and transcription or expression itself can be under the control of an inducible promoter. In some embodiments, an inducible promoter is induced in the absence of certain agents, such as repressors. Examples of inducible promoters include, but are not limited to, tetracycline, metallothionein, ecdysone, mammalian viruses (e.g., adenovirus late promoter; and mouse mammary tumor virus long terminal repeat (MMTV-LTR)) and other steroid-responsive promoters, rapamycin-responsive promoters, and the like.

[0119] As used herein, the term "subject" refers to a human or animal to whom treatment with a single-stranded (ssDNA) molecule according to the present disclosure is provided, including prophylactic treatment. Generally speaking, an animal is a vertebrate, such as, but not limited to, a primate, a rodent, a domestic animal, or a wild animal. Primates include, but are not limited to, chimpanzees, crab-eating macaques, spider monkeys, and macaques, for example, rhesus macaques. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Livestock and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species (e.g., house cats), canine species (e.g., dogs, foxes, wolves), avian species (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate or a human. The subject can be male (male) or female (female). In addition, the subject can be an infant or a child. In some embodiments, the subject can be a newborn or unborn subject, for example, while the subject is still in the womb. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. Mammals other than humans can advantageously be used as subjects representing animal models of diseases and illnesses. In addition, the methods and compositions described herein can be used for domestic animals and / or pets. The human subject can be any age, sex, race or ethnic group, for example, Caucasian (white race), Asian, African, black race, African American, African European, Hispanic, Middle Eastern etc. In certain embodiments, the subject can be other subjects in a patient or clinical setting. In certain embodiments, the subject has been treated. In certain embodiments, the subject is an embryo, fetus, newborn, infant, child, teenager or adult. In certain embodiments, the subject is a human fetus, human newborn, human infant, human child, human teenager or human adult. In certain embodiments, the subject is an animal embryo, or a non-human embryo or a non-human primate embryo. In certain embodiments, the subject is a human embryo.

[0120] 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 herein. As non-limiting examples, the host cell can be an isolated primary cell, a pluripotent stem cell, a CD34 + Cells, induced pluripotent stem cells or any of many immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell can be an in situ or in vivo cell in a tissue, organ or organism. In addition, the host cell can be a target cell of, for example, a mammalian subject (e.g., a human patient in need of gene therapy).

[0121] As used herein, the term "exogenous" refers to a substance that is present in a cell other than its natural source. When used herein, the term "exogenous" can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced into a biological system, such as a cell or organism, by a process involving human hands, in which the nucleic acid or polypeptide is not normally found, and in which it is desired to introduce the nucleic acid or polypeptide into such cell or organism. Alternatively, "exogenous" can refer to a nucleic acid or polypeptide that has been introduced into a biological system, such as a cell or organism, by a process involving human hands, in which the nucleic acid or polypeptide is found in relatively low amounts, and in which it is desired to increase the amount of the nucleic acid or polypeptide in the cell or organism, for example to produce ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is natural to a biological system or cell.

[0122] The terms "polynucleotide" and "nucleic acid" used interchangeably herein refer to nucleotides of any length, either ribonucleotides or deoxyribonucleotides in polymeric form. Thus, this term encompasses single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. "Oligonucleotide" generally refers to a polynucleotide of about 5 to about 100 nucleotides of single-stranded or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also referred to as "oligomers" or "oligos" and can be isolated from genes or chemically synthesized by methods known in the art. It should be understood that the terms "polynucleotide" and "nucleic acid" include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides, if the described embodiments are applicable. According to some embodiments, nucleic acid is a single-stranded DNA (ssDNA) molecule described in this disclosure. The DNA can be in the form of, for example, an antisense molecule, plasmid DNA, DNA-DNA duplex, precondensed DNA, PCR product, vector (P1, PAC, BAC, YAC, artificial chromosome), expression cassette, chimeric sequence, chromosomal DNA, or derivatives and combinations of these groups. The DNA can be in the form of a minicircle, plasmid, bacmid, minigene, ministring DNA (linear covalently enclosed DNA vector), capped linear duplex DNA (CELiD or ceDNA), doggybone (dbDNA), or a combination of these groups. TM) DNA, dumbbell-shaped DNA, simple 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, small 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 bonds, which are synthetic, naturally occurring and non-naturally occurring, and have binding properties similar to reference nucleic acids. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, diamidophosphorothioate morpholino oligomers (morpholinos), phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNAs) TM ) and peptide nucleic acids (PNA). Unless otherwise 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 conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly indicated.

[0123] As used herein, an "inhibitory polynucleotide" refers to a DNA 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" further includes DNA and RNA molecules, for example, RNAi molecules that encode the actual inhibitory species, such as DNA molecules that encode ribozymes.

[0124] A "nucleotide" contains the sugar deoxynucleoside (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are linked together by the phosphate groups.

[0125] "Bases" include purines and pyrimidines, and further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including but not limited to modifications that place new reactive groups such as but not limited to amines, alcohols, thiols, carboxylates, and alkyl halides.

[0126] "Hybridizable" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA) comprises a nucleotide sequence that enables it to non-covalently bind to another nucleic acid sequence under conditions of appropriate temperature and solution ionic strength in vitro and / or in vivo, i.e., to form Watson-Crick base pairs and / or G / U base pairs, "anneal" or "hybridize" in a sequence-specific antiparallel manner (i.e., a nucleic acid that specifically binds to a complementary nucleic acid). As is known in the art, standard Watson-Crick base pairs comprise: adenine (A) pairs with thymidine (T), adenine (A) pairs with uracil (U), and guanine (G) pairs with cytosine (C). Additionally, it is also known in the art that for hybridization between two RNA molecules (e.g., dsRNA), guanine (G) base pairs with uracil (U). For example, in the case of tRNA anticodon base pairing with codons in mRNA, G / U base pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code. In the context of the present disclosure, a guanine (G) of the protein-binding segment (dsRNA duplex) of an RNA molecule targeting a subject DNA is considered to be complementary to a uracil (U), and vice versa. Thus, when a G / U base pair can be formed at a given nucleotide position of a protein-binding segment (dsRNA duplex) of an RNA molecule targeting a subject DNA, that position is not considered to be non-complementary, but rather is considered to be complementary.

[0127] As used herein, the term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a manner not otherwise found in nature or synthesized to contain a segment of nucleic acid. When the nucleic acid construct contains the control sequences required for expression of the coding sequence of the present disclosure, the term nucleic acid construct is synonymous with the term "expression cassette." An "expression cassette" comprises a DNA coding sequence operably linked to a promoter.

[0128] As used herein, the phrases "nucleic acid therapeutic," "therapeutic nucleic acid," and "TNA" are used interchangeably and refer to any modality of therapy that uses nucleic acids as the active component of a therapeutic agent for treating a disease or condition. As used herein, these phrases refer to RNA-based therapeutics and DNA-based therapeutics. Non-limiting examples of RNA-based therapeutics include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), and guide RNA (gRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentiviral or AAV genomes) or non-viral synthetic DNA vectors, capped linear duplex DNA (ceDNA / CELiD), plasmids, bacmids, doggybone (dbDNA TM ) DNA vectors, minimalist immunologically defined gene expression (MIDGE) vectors, non-viral ministring DNA vectors (linear covalently enclosed DNA vectors), or dumbbell-shaped minimal DNA vectors ("dumbbell DNA").

[0129] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to a polymeric form 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 with modified peptide backbones.

[0130] As used herein, term " sequence identity " refers to the dependency between two nucleotide sequences.For the purpose of the present disclosure, the sequence identity degree between two deoxyribonucleotide sequences is determined using Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, the same), described algorithm is as in EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, the same), preferably implemented in the Needle program of version 3.0.0 or newer. The optional parameters used are gap open penalty 10, gap extension penalty 0.5 and EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output (obtained using -nobrief option) of Needle labeled "longest identity" is used as identity percentage, and is calculated as follows: (identical deoxyribonucleotides multiplied by 100) / (total number of gaps in the length-alignment of comparison). 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.

[0131] As used herein, term "homology" or "homology" as used herein is defined as the percentage of the nucleotide residues identical with the nucleotide residues in the corresponding sequence on the target chromosome in the alignment sequence and, when necessary, introducing a gap for realizing maximum sequence identity percentage. The comparison carried out for the purpose of determining nucleotide sequence homology percentage can be realized in the various ways within 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 alignment sequences, including any algorithm for realizing maximum alignment on the full length of the compared sequence. In some embodiments, a nucleic acid sequence (e.g., a DNA sequence), such as a homology arm of a repair template, is considered "homologous" when 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.

[0132] As used herein, "homology arms" refer to polynucleotides suitable for targeting a donor sequence to a genome by homologous recombination. Typically, two homology arms flank the donor sequence, wherein each homology arm comprises genomic sequences upstream and downstream of the integration locus.

[0133] As used herein, "donor sequence" refers to a polynucleotide to be inserted into the host cell genome or used as a repair template for the host cell genome. The donor sequence may include modifications that are desired to be performed during gene editing. The sequence to be incorporated can be introduced into the target nucleic acid molecule via homology-directed repair at the target sequence, thereby causing the target sequence to change from the original target sequence to the sequence contained in the donor sequence. Therefore, the sequence contained in the donor sequence can be an insertion, deletion, indel, point mutation, mutation repair, etc. relative to the target sequence. The donor sequence can be, for example, a single-stranded DNA molecule; a double-stranded DNA molecule; a DNA / RNA hybrid molecule; and a DNA / modRNA (modified RNA) hybrid molecule. In one embodiment, the donor sequence is foreign to the homology arm. Editing can be RNA as well as DNA editing. The donor sequence can be endogenous or exogenous to the host cell genome, depending on the nature of the desired gene editing. As used herein, the term "heterologous" as used herein means a nucleotide or polypeptide sequence that is not present in a natural nucleic acid or protein, respectively. Heterologous nucleic acid sequences can be connected (e.g., by genetic engineering) to a naturally occurring nucleic acid sequence (or variant thereof) to produce a chimeric nucleotide sequence encoding a chimeric polypeptide. Heterologous nucleic acid sequences can be connected (e.g., by genetic engineering) to a variant polypeptide to produce a nucleotide sequence encoding a fusion variant polypeptide.

[0134] As used herein, a "vector" or "expression vector" is a replicon, such as a plasmid, bacmid, phage, virus, virion or cosmid, which can be connected to another DNA segment, i.e., an "insert," "transgene," or "expression cassette," to cause expression or replication of the attached A 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 viral or non-viral in its final form. However, for the purposes of this disclosure, a "vector" generally refers to a synthetic AAV, such as a single-stranded (ss) synthetic AAV vector or a nicked ceDNA vector. Thus, the term "vector" encompasses any genetic element that is capable of replication and can transfer a gene sequence to a cell when associated with appropriate control elements. In some embodiments, a vector can be a recombinant vector or an expression vector.

[0135] As used herein, the phrase "recombinant vector" means a vector comprising a heterologous nucleic acid sequence or "transgene" that can be expressed in vivo. It should be understood that in some embodiments, the vectors described herein can be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of suitable episomal vectors provides a method for maintaining the nucleotides of interest to the subject in a high copy number of extrachromosomal DNA, thereby eliminating the potential effects of chromosome fusions.

[0136] As used herein, the term "expression vector" refers to a vector that directs the expression of an RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The expressed sequence will generally, but not necessarily, be heterologous to the host cell. An expression vector may include additional elements, for example, an expression vector may have two replication systems so that it can be maintained in two organisms, for example, expression in human cells and cloning and amplification in a prokaryotic host. The expression vector may be a recombinant vector.

[0137] As used herein, the term "expression" refers to the cellular processes involved in the production of RNA and protein, and secretion of protein when appropriate, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, as applicable.

[0138] As used herein, the phrase "expression product" includes RNA transcribed from a gene (eg, a transgene), as well as polypeptides obtained by translation of mRNA transcribed from a gene.

[0139] As used herein, the term "gene" means a nucleic acid sequence that is transcribed (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, for example, a 5' untranslated region (5'UTR) or "leader" sequence and a 3'UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0140] As used herein, the term "gene delivery" means the process of transferring foreign DNA into host cells to administer gene therapy.

[0141] 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 consisting of a transposase, a nuclease, an integrase, a guide RNA (gRNA), a guide DNA, a ribonucleoprotein (RNP), or an activator RNA. Nuclease gene editing molecules are proteins with nuclease activity, non-limiting examples of which include: CRISPR protein (Cas), CRISPR-associated protein 9 (Cas9); type IIS restriction enzymes; transcription activator-like effector nucleases (TALEN); and zinc finger nucleases (ZFNs), large-range nucleases, engineered site-specific nucleases, or inactivated CAS for CRISPRi or CRISPRa systems. The 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 TALEN. In certain embodiments, at least one gene editing molecule includes one or more transposable elements. In certain embodiments, one or more transposable elements include circular DNA. In certain embodiments, one or more transposable elements comprise a plasmid vector or a minicircle DNA vector. In certain embodiments, the DNA binding domain comprises the 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, one or more transposable elements comprise linear DNA. Linear recombinant and non-naturally occurring DNA sequences encoding transposons can be produced in vitro. The linear recombinant and non-naturally occurring DNA sequences of the present disclosure can 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 and non-naturally occurring DNA sequences of the present disclosure can be the product of polymerase chain reaction (PCR). The linear recombinant and non-naturally occurring DNA sequences of the present disclosure can be double-stranded DOGGYBONE TM DNA sequence. DOGGYBONE of the present disclosure TM A DNA sequence can be generated by an enzymatic process that encodes only the antigen expression cassette, which comprises the antigen, promoter, poly-A tail and telomeric ends.

[0142] As used herein, the term "gene editing function" refers to the insertion, deletion or substitution of DNA at a specific site in the genome, and loss of function or acquisition. The insertion, deletion or substitution of DNA at a specific site can be realized, for example, by homology directed repair (HDR) or non-homologous end joining (NHEJ) or single base change editing. In certain embodiments, a donor template is used, for example, for HDR, so that the desired sequence in the donor template is inserted into the genome by a homologous recombination event. In one embodiment, "donor template" or "repair template" includes two homology arms (for example, 5' homology arms and 3' homology arms) flanking either side of the donor sequence, and the two homology arms include the desired mutation or insertion in the nucleic acid sequence to be introduced into the host genome. 5' and 3' homology arms are substantially homologous to the genomic sequence of the target gene at the endonuclease-mediated cleavage site. 3' homology arms are generally adjacent to endonuclease cutting (for example, double-stranded DNA cutting) or in some embodiments cut the downstream of the original spacer adjacent motif (PAM) site of DNA.

[0143] As used herein, the term "gene editing system" refers to the minimum components necessary to achieve genome editing in a cell. For example, a zinc finger nuclease or TALEN system may only require expression of an endonuclease fused to a nucleic acid complementary to the sequence of the target gene, while for a CRISPR / Cas gene editing system, the minimum components may require, for example, a Cas endonuclease and a guide RNA. The gene editing system can be encoded on a single ceDNA vector or multiple vectors as needed. Those skilled in the art will readily understand the (multiple) components required for the gene editing system.

[0144] As used herein, the term "base editing moiety" refers to an enzyme or enzyme system that can change a single nucleotide in a sequence, for example, changing a cytosine / guanine nucleotide pair "G / C" to an adenine and thymine "T" / uridine "U" nucleotide pair (A / T, U) (see, e.g., 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 entirety) or changing an adenine / thymine "A / T" nucleotide pair to a guanine / cytosine "G / C" nucleotide pair (see, e.g., Gaudelli et al. Nature (2017), at publication doi: 10.1038 / nature24644, the contents of which are incorporated herein by reference in their entirety).

[0145] 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 (e.g., express a protein of interest) in a predictable manner without significantly negatively affecting endogenous gene activity or promoting cancer. In some embodiments, a safe harbor gene is also a locus or gene into which an inserted nucleic acid sequence can be expressed efficiently and at a higher level than a non-safe harbor site.

[0146] As used herein, the term "gene delivery" refers to the process of transferring foreign DNA into host cells to administer gene therapy.

[0147] As used herein, the term "CRISPR" means Clustered Regularly Interspaced Short Palindromic Repeats, a hallmark of the bacterial defense system that forms the basis of the CRISPR-Cas9 genome editing technology.

[0148] 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 DNA molecules. Homologous recombination also produces new combinations of DNA sequences. These new combinations of DNA represent genetic variations. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and virus species.

[0149] As used herein, the terms "correction", "genome editing" and "restoration" refer to changes in mutant genes encoding truncated proteins or proteins that are not encoded at all, so that full-length functional or partially full-length functional protein expression is obtained. Correction or restoration of mutant genes can include replacing the region of the gene with a copy of a gene that does not have a mutation with a repair mechanism such as homology-directed repair (HDR) or replacing the entire mutant gene. Correction or restoration of mutant genes can also include repairing the frameshift mutation that causes premature stop codons, abnormal splicing acceptor sites or abnormal splicing donor sites by producing a double-strand break in the gene, and then repairing it using non-homologous end joining (NHEJ). NHEJ can add or delete at least one base pair during repair, which can restore the correct reading frame and eliminate premature stop codons. Correction or restoration of mutant genes can also include destroying abnormal splicing acceptor sites or splicing donor sequences. Correction or restoration of mutant genes can also include deleting non-essential gene segments by the simultaneous action of two nucleases on the same DNA chain so as to restore the appropriate reading frame by removing the DNA between the two nuclease target sites and repairing the DNA break by NHEJ.

[0150] As used herein, phrase " non-homologous end joining (NHEJ) pathway " refers to a pathway for repairing double-strand breaks in DNA by directly connecting the broken ends without the need for a homologous template. The template-independent reconnection of the DNA ends performed by NHEJ is a random, error-prone repair process that introduces random micro-insertions and micro-deletions (indels) at the DNA breakpoints. This method can be used to intentionally destroy, delete, or change the reading frame of the targeted gene sequence. NHEJ typically uses a short homologous DNA sequence called microhomology to guide repair. These microhomologies typically exist as single-stranded overhangs on the ends of double-strand breaks. When the overhangs are fully compatible, NHEJ typically accurately repairs the break, but imprecise repairs that cause nucleotide loss may also occur, but when the overhangs are incompatible, as used herein, " nuclease-mediated NHEJ " refers to NHEJ initiated after nucleases such as cas9 or other nucleases cut double-stranded DNA, which is much more common. In CRISPR / CAS systems, NHEJ can be targeted by using a single guide RNA sequence.

[0151] As used herein, the term "site-specific nuclease" or "sequence-specific nuclease" refers to an enzyme that can specifically recognize and cut a DNA sequence. 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 using various natural and non-natural Cas enzymes.

[0152] As used herein, the phrase "genetic disease" refers to a disease that is caused, directly or indirectly, in part or in whole, by one or more abnormalities in the genome, particularly conditions that are present from birth and that can be treated by single-stranded (ssDNA) molecules as described herein. The abnormality can be a mutation, insertion, or deletion. The abnormality can affect the coding sequence of a gene or its regulatory sequences. The genetic disease may be, but is not limited to, phenylketonuria (PKU), sickle cell anemia, melanoma, hemophilia A (coagulation factor VIII (FVIII) deficiency) and hemophilia B (coagulation factor IX (FIX) deficiency), cystic fibrosis, Huntington's disease, familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited liver metabolic disorders, Leschnihan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, and mucopolysaccharidoses (e.g., Hurler syndrome (MPS type I), Shay syndrome (MPS type I S), Hurler-Shay syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo A, B, C, and D (MPS MPS III A, B, C, and D), Morquer A and B (MPS IVA and MPS IVB), Mallory-Lami syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPS Type IX)), Niemann-Pick disease types A / B, C1, and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff 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, aspartate glucosamineuria, sialidosis, Dannon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipid storage diseases, and galactosialidosis. Genetic diseases 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 congenital amaurosis (LCA, e.g., LCA10 [CEP290]), Stargardt macular dystrophy (ABCA4), or cathepsin A deficiency.

[0153] As used herein, the terms "treat" or "treating" and / or "treatment" mean eliminating, substantially inhibiting, slowing 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, obtaining a beneficial or desired clinical outcome. Treatment further refers to achieving one or more of the following: (a) reducing the severity of the condition; (b) limiting the development of symptoms characteristic of the condition being treated; (c) limiting the worsening of symptoms characteristic of the condition being treated; (d) limiting the recurrence of the condition in patients who previously had the condition; and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic for the condition. In some embodiments, treatment encompasses gene editing. In some embodiments, treatment encompasses gene therapy.

[0154] Beneficial or desired clinical results, such as pharmacological and / or physiological effects, include, but are not limited to: preventing the development of a disease, disorder or condition in a subject who may be susceptible to the disease, disorder or condition but who does not yet experience or exhibit symptoms of the disease (prophylactic or preventive treatment); alleviating the symptoms of the disease, disorder or condition; reducing the extent of the disease, disorder or condition; stabilizing the disease, disorder or condition (i.e., not worsening); preventing the spread of the disease, disorder or condition; delaying or slowing the progression of the disease, disorder or condition; ameliorating or palliating the disease, disorder or condition; and combinations thereof, and prolonging survival as compared to expected survival if not receiving treatment.

[0155] As used herein, the terms "increase," "enhance," "elevate" (and similar terms) generally refer to the act of directly or indirectly increasing the concentration, level, function, activity or behavior relative to native conditions, expected conditions or average conditions, or relative to control conditions.

[0156] As used herein, the terms "suppress," "reduce," "interfere with," "inhibit," and / or "reduce" (and similar terms) generally refer to the act of directly or indirectly decreasing the concentration, level, function, activity, or behavior relative to natural, expected, or average conditions, or relative to control conditions.

[0157] As used herein, the terms "synthetic AAV vector" and "single-stranded (ss) synthetic AAV vector" and "synthetic production of AAV vectors" refer to AAV vectors and methods for their synthetic production in a cell-free environment.

[0158] As used herein, the term "comprising" or "comprises" is used when referring to a composition, method, process, and its corresponding components that are essential to the process, method, or composition, but remains open to the inclusion of unspecified elements, whether essential or not. The use of "comprising" indicates inclusion rather than limitation.

[0159] The term "consisting of refers to compositions, methods, processes, and corresponding components thereof as described herein, excluding any elements not recited in the description of the embodiment.

[0160] As used herein, the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the disclosure.

[0161] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents 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 one skilled in the art after reading this disclosure and so forth. Similarly, the word "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.

[0162] The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example".

[0163] Except in the operating examples, or where otherwise indicated, all numbers used herein expressing quantities of ingredients or reaction conditions should be understood as modified by the term "about" in all cases. The term "about" when used with a percentage can mean ±1%. The following examples further explain the present disclosure in detail, but the scope of the present disclosure should not be limited thereto.

[0164] The grouping of the alternative elements of the present disclosure disclosed herein or embodiments should not be construed as limiting.Each group member can be mentioned and claimed individually or with any combination of other elements found by other members of the group or this paper.For convenience and / or patentability reasons, one or more members in a group may be included in a group or therefrom deleted.When any such inclusion or deletion occurs, description described in this article is considered to contain the group of modification, thereby meeting the written description of all Markush groups (Markush group) used in the appended claims.

[0165] In some embodiments of any aspect, 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 may cause suffering to them without any substantial medical benefit to humans or animals, and animals resulting from such processes.

[0166] Additional terms are defined herein within the description of various aspects of the disclosure.

[0167] All patents and other publications cited in this application throughout, including references, granted patents, published patent applications, and co-pending patent applications, are expressly incorporated herein by reference to describe and disclose, for example, methods described in these publications that can be used in combination 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 inventor has no right to advance the disclosure by virtue of prior disclosure or for any other reason. All statements about the dates or contents of these documents are based on information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0168] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications can be made within the scope of the present disclosure as will be appreciated by those skilled in the relevant art. For example, although method steps or functions are presented in a given order, alternative embodiments can perform functions in a different order or can perform functions substantially simultaneously. The teachings of the present disclosure provided herein can be appropriately applied to other processes or methods. The various embodiments described herein can be combined to provide additional embodiments. If necessary, aspects of the present disclosure can be modified to provide yet further embodiments of the present disclosure using the compositions, functions, and concepts in the above-mentioned references and applications. In addition, due to considerations of biological functional equivalence, some changes can be made in the protein structure without affecting the type or quantity of the biological effect. These and other changes can be made to the present disclosure based on the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0169] The specific elements of any of the foregoing embodiments may be combined with or replace elements in other embodiments. In addition, although the advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages and not all embodiments need to exhibit such advantages to fall within the scope of the present disclosure.

[0170] The technology described herein is further illustrated by the following examples, but these examples should not be construed as further limiting. It should be understood that the present disclosure is not limited to the specific methods, protocols, reagents, etc. described herein and can vary accordingly. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.

[0171] II. Single-stranded (ss) DNA molecules

[0172] As described herein, the present disclosure relates to synthetic single-stranded (ssDNA) molecules. According to some aspects, the present disclosure provides a single-stranded deoxyribonucleic acid (ssDNA) molecule comprising at least one nucleic acid sequence of interest flanked at its 3' end by at least one stem-loop structure. According to some embodiments, the ssDNA molecule further comprises a 5' end comprising at least one stem-loop structure.

[0173] In some embodiments, the ssDNA molecules described herein are linear single-stranded DNA molecules that are completely single-stranded along their entire length (ie, they contain no double-stranded regions).

[0174] A.3' end

[0175] As described herein, according to some aspects, the present disclosure provides an ssDNA molecule comprising at least one nucleic acid sequence of interest flanked at its 3' end by at least one stem-loop structure. As described herein, the stem structure comprises a partial DNA duplex (e.g., having a free 3'-OH group) to initiate replication or transcription. Part of the function of the partial DNA duplex is to hold the stem-loop structure together.

[0176] According to some embodiments, the portion of the DNA duplex comprises 4-500 nucleotides, 4-10 nucleotides, 4-25 nucleotides, 4-50 nucleotides, 4-100 nucleotides, 4-200 nucleotides, 4-300 nucleotides, 4-400 nucleotides, 20-25 nucleotides, 20-50 nucleotides, 20-100 nucleotides, 20-200 nucleotides, 20-300 nucleotides, 20-400 nucleotides, 20-500 nucleotides, 50-100 nucleotides, 50-200 nucleotides, 50-300 nucleotides, 50-400 nucleotides, 5 0-500 nucleotides, 150-200 nucleotides, 150-300 nucleotides, 150-400 nucleotides, 150-500 nucleotides, 200-300 nucleotides, 200-400 nucleotides, 200-500 nucleotides, 250-300 nucleotides, 250-400 nucleotides, 250-500 nucleotides, 300-400 nucleotides, 300-500 nucleotides, 350-400 nucleotides, 350-500 nucleotides, 400-500 nucleotides or 450-500 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 at least one loop at the 3' end.

[0177] According to some embodiments, the loop structure at the 3' end comprises a minimum of 3-500 unbound nucleotides, such as 3-450 nucleotides, 3-400 nucleotides, 3-350 nucleotides, 3-300 nucleotides, 3-250 nucleotides, 3-200 nucleotides, 3-150 nucleotides, 3-100 nucleotides, 3-90 nucleotides, 3-80 nucleotides, 3-70 nucleotides, 3-60 nucleotides, 3-50 nucleotides, 3-40 nucleotides, 3-30 nucleotides, 3-20 nucleotides, 3-10 nucleotides, 3-5 nucleotides, 10-450 nucleotides, 10-400 nucleotides, 10-350 nucleotides, 10-300 nucleotides, 10-250 nucleotides, 10-200 nucleotides, 10-150 nucleotides, 10-100 nucleotides, 10-90 nucleotides, 10-80 nucleotides, 10-70 nucleotides, 10-60 nucleotides, 10-50 nucleotides, 10-40 nucleotides, 10-30 nucleotides, 10-20 nucleotides, 50-450 nucleotides, 50-400 nucleotides, 50-350 nucleotides, 50-300 nucleotides, 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 nucleotides, 150-250 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.

[0178] According to some embodiments, the stem portion of the stem-loop is 4-500 nucleotides in length, and the loop portion of the stem-loop is 3-500 nucleotides in length. According to some embodiments, the stem portion of the stem-loop is 4-50 nucleotides in length, and the loop portion of the stem-loop is 3-50 nucleotides in length. According to some embodiments, the stem portion of the stem-loop is 4-20 nucleotides in length, and the loop portion of the stem-loop is 3-20 nucleotides in length. According to some embodiments, the stem portion of the stem-loop is 4-10 nucleotides in length, and the loop portion of the stem-loop is 3-10 nucleotides in length.

[0179] According to some embodiments, the loop further comprises one or more nucleic acids or nucleic acids used to stabilize the ends. According to other embodiments, the loop further comprises one or more nucleic acids that can be used in therapeutic methods. According to other embodiments, the loop further comprises one or more nucleic acids that can be used in diagnostic methods. According to other embodiments, the loop further comprises one or more nucleic acids that can be used for research purposes.

[0180] According to some embodiments, the minimum nucleic acid structure required at the 3' end of the ssDNA is a structure that can loop back on itself, i.e., a hairpin structure. However, it should be understood that various structures can be envisioned 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 can include at least one stem-loop structure at the 3' end. In some embodiments, the ssDNA can include at least two stem-loop structures at the 3' end. In some embodiments, the ssDNA can include at least three stem-loop structures at the 3' end. In some embodiments, the ssDNA can include at least four stem-loop structures at the 3' end. In some embodiments, the ssDNA can include at least five stem-loop structures at the 3' end.

[0181] According to some embodiments, the nucleotides at the 3' end form a cruciform DNA structure. When the two strands form a stem-loop structure at the same position in the molecule, a DNA cruciform structure can be formed, which contains a four-way junction and two closed hairpin-shaped points.

[0182] According to some embodiments, the nucleotides at the 3' end form a hairpin DNA structure.A hairpin loop structure in a nucleic acid consists of a base-paired stem structure and a loop sequence with unpaired or non-Watson-Crick paired nucleotides.

[0183] According to some embodiments, the nucleotides at the 3' end form a hammerhead DNA structure consisting of three base-paired helices separated by short linkers of conserved sequence.

[0184] According to some embodiments, the nucleotides at the 3' end form a quadruplex DNA structure. A G-quadruplex is a four-stranded DNA secondary structure (G4s) formed by certain guanine-rich sequences.

[0185] According to some embodiments, the nucleotides at the 3' end form a bulged DNA structure.

[0186] According to some embodiments, the nucleotides at the 3' end form a multi-branched loop.

[0187] According to some embodiments, the nucleotides at the 3' end do not form a 2-stem-loop structure. In one embodiment, the nucleotides at the 3' end do not form an AAV ITR structure.

[0188] According to some embodiments, at least one stem-loop structure at the 3' end does not comprise the A, A', D, and D' regions that would be present in a wild-type AAV ITR.

[0189] According to some embodiments, at least one stem-loop structure at the 3' end does not comprise the A, A', B, B', C, C', D, and D' regions that would be present in a wild-type AAV ITR.

[0190] According to some embodiments, at least one of the stem-loop structures at the 3' end does not comprise a rep binding element (RBE) that would be present in a wild-type ITR. According to some embodiments, at least one of the stem-loop structures at the 3' end does not comprise a terminal melting site (trs) that would be present in a wild-type ITR. According to some embodiments, at least one of the stem-loop structures at the 3' end lacks any viral capsid protein coding sequence. According to some embodiments, the ssDNA molecule does not comprise any viral-derived sequence.

[0191] According to some embodiments, the stem structure at the 3' end comprises one or more nucleotides modified to be resistant to nucleases. According to some embodiments, the stem structure at the 3' end comprises two or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more or 20 or more nucleotides modified to be resistant to nucleases.

[0192] According to some embodiments, the stem structure at 3 ' end comprises the nucleotides modified by one or more thiophosphates.According to some embodiments, the stem structure at 3 ' end comprises the nucleotides modified by one or more thiophosphates.According to some embodiments, the stem structure at 3 ' end comprises the nucleotides modified by approximately 4 to approximately 10 thiophosphates, for example, approximately 4 to approximately 5, approximately 4 to approximately 6, approximately 4 to approximately 7, approximately 4 to approximately 8, approximately 4 to approximately 9, approximately 4 to approximately 10, approximately 5 to approximately 6, approximately 5 to approximately 7, approximately 5 to approximately 8, approximately 5 to approximately 9, approximately 5 to approximately 10, approximately 6 to approximately 7, approximately 6 to approximately 8, approximately 6 to approximately 9, approximately 6 to approximately 10, approximately 7 to approximately 8, approximately 7 to approximately 9, approximately 7 to approximately 10, approximately 8 to approximately 9, approximately 8 to approximately 10 or approximately 9 to approximately 10.According to some embodiments, the stem structure comprises the nucleotides modified by more than 10 thiophosphates.

[0193] According to some embodiments, the phosphorothioate modified nucleotides are positioned adjacent to each other.

[0194] According to some embodiments, the 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 modifications.

[0195] According to another embodiment, the stem structure can include at least one functional portion. In one embodiment, the at least one functional portion is an aptamer sequence. In another embodiment, the aptamer sequence has a high binding affinity for nuclear localization proteins.

[0196] According to some embodiments, the nucleotides in the loop are chemically modified with functional groups to alter their properties.

[0197] According to some embodiments, the loop further comprises one or more aptamers. According to some embodiments, the aptamers are identified from the publicly available Apta-index database of aptamers (aptagen.com / apta-index).

[0198] According to some embodiments, the loop further comprises one or more synthetic ribozymes.

[0199] According to some embodiments, the loop further comprises one or more antisense oligonucleotides (ASOs).

[0200] According to some embodiments, the loop further comprises one or more short interfering RNAs (siRNAs).

[0201] According to some embodiments, the ring further comprises one or more antiviral nucleoside analogs (ANA).

[0202] According to some embodiments, the loop further comprises one or more triplex-forming oligonucleotides.

[0203] According to some embodiments, the loop further comprises one or more gRNAs or gDNAs.

[0204] According to some embodiments, the ring further comprises one or more molecular probes, such as nucleic acid-based fluorescent probes.

[0205] According to some embodiments, a "click" azide-alkyne cycloaddition (Kolb et al., Angew. Chem. Int. Ed. Engl. 2001, 40, 2004-2021) is used to modify nucleotides in the ring. 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 interchain bonds between oligonucleotides, and for bioconjugation of molecules. The best example of click chemistry is Huisgen's [3+2] azide-alkyne cycloaddition reaction of Cu I Catalytic version (International Journal of Chemical Applications 1963, 2, 633–645), independently discovered by Sharpless and Meldal (CuAAC reaction) (International Journal of Chemical Applications 2002, 41, 2596–2599).

[0206] According to some embodiments, reactive amino or thiol groups are introduced into synthetic oligonucleotides to provide receptors for, for example, subsequent chemifluorescent labeling.

[0207] 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 comprise a nucleic acid.

[0208] According to some embodiments, the 3' 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 fully single-stranded ssDNA molecule can induce transgene expression. In some embodiments, the fully single-stranded ssDNA molecule without double-stranded regions can be at least 200 nucleotides in length, 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, or at least 3000 nucleotides in length. A length of at least 3500 nucleotides, a length of at least 4000 nucleotides, a length of at least 4500 nucleotides, a length of at least 5000 nucleotides, a length of at least 5500 nucleotides, a length of at least 6000 nucleotides, a length of at least 6500 nucleotides, a length of at least 7000 nucleotides, a length of at least 7500 nucleotides, a length of at least 8000 nucleotides, a length of at least 8500 nucleotides, a length of at least 9000 nucleotides, a length of at least 9500 nucleotides or a length of at least 10,000 nucleotides.

[0209] B.5' end

[0210] As described herein, according to some aspects, the present disclosure provides an ssDNA molecule comprising at least one nucleic acid sequence of interest flanked at its 3' end by at least one stem-loop structure, 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.

[0211] For example, in some embodiments, the ssDNA described herein may comprise at least one stem-loop structure at the 5' end. According to some embodiments, the ssDNA may comprise at least two stem-loop structures at the 5' end. According to some embodiments, the ssDNA may comprise at least three stem-loop structures at the 5' end. According to some embodiments, the ssDNA may comprise at least four stem-loop structures at the 5' end. According to some embodiments, the ssDNA may comprise at least five stem-loop structures at the 5' end.

[0212] According to some embodiments, the nucleotides at the 5' end form a cruciform DNA structure.

[0213] According to some embodiments, the nucleotides at the 5' end form a hairpin structure.

[0214] According to some embodiments, the nucleotides at the 5' end form a hammerhead structure.

[0215] According to some embodiments, the nucleotides at the 5' end form a quadruplex structure.

[0216] According to some embodiments, the nucleotides at the 5' end form a bulge structure.

[0217] According to some embodiments, the nucleotides at the 5' end form a multi-branched loop.

[0218] According to some embodiments, the nucleotides at the 5' end do not form a 2-stem-loop structure. In one embodiment, the nucleotides at the 5' end do not form an AAV ITR structure.

[0219] According to some embodiments, at least one stem-loop structure at the 5' end does not comprise the A, A', D, and D' regions that would be present in a wild-type AAV ITR.

[0220] According to some embodiments, at least one stem-loop structure at the 5' end does not comprise the A, A', B, B', C, C', D, and D' regions that would be present in a wild-type AAV ITR.

[0221] According to some embodiments, at least one of the stem-loop structures at the 5' end does not comprise a rep binding element (RBE) that would be present in a wild-type ITR. According to some embodiments, at least one of the stem-loop structures at the 5' end does not comprise a terminal melting site (trs) that would be present in a wild-type ITR. According to some embodiments, at least one of the stem-loop structures at the 5' end lacks any viral capsid protein coding sequence. According to some embodiments, the ssDNA molecule does not comprise any viral-derived sequence.

[0222] According to some embodiments, the stem structure at the 5' end comprises one or more nucleotides modified to be resistant to nucleases. According to some embodiments, the stem structure at the 5' end comprises two or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more or 20 or more nucleotides modified to be resistant to nucleases.

[0223] According to some embodiments, stem structure comprises the nucleotides modified by one or more thiophosphates.According to some embodiments, stem structure comprises the nucleotides modified by one or more thiophosphates.According to some embodiments, stem structure comprises approximately 4 to approximately 10 thiophosphates modified nucleotides, for example, approximately 4 to approximately 5, approximately 4 to approximately 6, approximately 4 to approximately 7, approximately 4 to approximately 8, approximately 4 to approximately 9, approximately 4 to approximately 10, approximately 5 to approximately 6, approximately 5 to approximately 7, approximately 5 to approximately 8, approximately 5 to approximately 9, approximately 5 to approximately 10, approximately 6 to approximately 7, approximately 6 to approximately 8, approximately 6 to approximately 9, approximately 6 to approximately 10, approximately 7 to approximately 8, approximately 7 to approximately 9, approximately 7 to approximately 10, approximately 8 to approximately 9, approximately 8 to approximately 10 or approximately 9 to approximately 10.According to some embodiments, stem structure comprises the nucleotides modified by more than 10 thiophosphates.

[0224] According to some embodiments, the phosphorothioate modified nucleotides are positioned adjacent to each other. According to some embodiments, the one or more phosphorothioate modified nucleotides are resistant to exonuclease degradation.

[0225] According to some embodiments, the loop further comprises one or more nucleic acids or nucleic acids used to stabilize the ends. According to other embodiments, the loop further comprises one or more nucleic acids that can be used in therapeutic methods. According to other embodiments, the loop further comprises one or more nucleic acids that can be used in diagnostic methods. According to other embodiments, the loop further comprises one or more nucleic acids that can be used for research purposes.

[0226] According to some embodiments, the nucleotides in the loop are chemically modified with functional groups to alter their properties.

[0227] According to some embodiments, the loop further comprises one or more aptamers. According to some embodiments, the aptamers are identified from the publicly available Apta-index database of aptamers (aptagen.com / apta-index).

[0228] According to some embodiments, the loop further comprises one or more synthetic ribozymes.

[0229] According to some embodiments, the loop further comprises one or more antisense oligonucleotides (ASOs).

[0230] According to some embodiments, the loop further comprises one or more short interfering RNAs (siRNAs).

[0231] According to some embodiments, the ring further comprises one or more antiviral nucleoside analogs (ANA).

[0232] According to some embodiments, the loop further comprises one or more triplex-forming oligonucleotides.

[0233] According to some embodiments, the loop further comprises one or more gRNAs or gDNAs.

[0234] According to some embodiments, the ring further comprises one or more molecular probes, such as nucleic acid-based fluorescent probes.

[0235] According to some embodiments, a "click" azide-alkyne cycloaddition (Kolb et al., International Edition of Chemical Applications 2001, 40, 2004-2021) is used to modify nucleotides in the ring. 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 interchain bonds between oligonucleotides, and for bioconjugation of molecules. The best example of click chemistry is Huisgen's [3+2] azide-alkyne cycloaddition reaction of Cu I Catalytic version (International Journal of Chemical Applications 1963, 2, 633–645), independently discovered by Sharpless and Meldal (CuAAC reaction) (International Journal of Chemical Applications 2002, 41, 2596–2599).

[0236] According to some embodiments, reactive amino or thiol groups are introduced into synthetic oligonucleotides to provide receptors for, for example, subsequent chemifluorescent labeling.

[0237] 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 comprise a nucleic acid.

[0238] 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, all single-stranded ssDNA molecules can induce transgene expression.

[0239] C. Nucleic acid sequence of interest

[0240] The single-stranded DNA (ssDNA) molecules described herein are not restricted by the limited packaging space within the viral capsid. This allows for the insertion of one or more genetic elements, such as single-stranded enhancers, single-stranded introns, single-stranded post-transcriptional regulatory elements, single-stranded polyadenylation signals and single-stranded regulatory switches, large transgenes, multiple transgenes, and the like.

[0241] 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.

[0242] In other aspects of the disclosure, the single-stranded transgene cassettes can be used in gene editing applications, as described in more detail herein.

[0243] According to some embodiments, the nucleic acid sequence of interest (also referred to herein as a transgene) encodes a protein that is absent, inactive, or insufficiently active in the recipient subject, or a gene encoding a protein that has a desired biological or therapeutic effect. A transgene can encode a gene product that can act to correct the expression of a defective gene or transcript. In principle, an expression cassette can include any gene encoding a protein, polypeptide, or RNA that is reduced or absent due to a mutation, or that delivers a therapeutic benefit when overexpressed, and is considered within the scope of the present disclosure.

[0244] The nucleic acid sequence of interest may comprise any sequence that can be used to treat a disease or condition in a subject. ssDNA molecules can be used to deliver and express any gene of interest in a subject, including but not limited to nucleic acids encoding polypeptides or non-coding nucleic acids (e.g., RNAi, miR, etc.), as well as exogenous genes and nucleotide sequences, including viral sequences in the genome of the subject, for example, HIV viral sequences, etc. In some embodiments, the ssDNA molecules disclosed herein are used for therapeutic purposes (e.g., for medical, diagnostic, or veterinary use). In certain embodiments, ssDNA molecules can be used to express any gene of interest in a subject, including one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNA, RNAi, antisense oligonucleotides, antisense polynucleotides, or RNA (coding or non-coding; for example, siRNA, shRNA, microRNA, mRNA, or gRNA and their antisense counterparts (e.g., antagoMiR)), antibodies, antigen-binding fragments, or any combination thereof.

[0245] Sequences can be codon optimized for target host cells. As used herein, the term "codon optimized" or "codon optimization" refers to the process of modifying a nucleic acid sequence to enhance expression in the cells of a vertebrate, such as a mouse or human, by replacing at least one, more than one, or a large number of codons of a native sequence (e.g., a prokaryotic sequence) with codons that are more frequently used or most frequently used in the genes of the vertebrate being studied. Various species show specific preferences for certain codons for specific amino acids. Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be modified using, for example, Aptagen's (Aptagen) Codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd., Suite 300, Herndon, Va. 20171) or another publicly available database were used to determine the codon.

[0246] In some embodiments, the transgene expressed by the ssDNA molecule 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.

[0247] Specifically, therapeutic genes are one or more therapeutic agents, including but not limited to, for example, proteins, polypeptides, peptides, enzymes, antibodies, antigen binding fragments and variants and / or active fragments thereof for treating, preventing and / or improving one or more symptoms of a disease, dysfunction, injury and / or illness. Exemplary therapeutic genes are described herein in the section entitled "Therapeutic Methods."

[0248] According to any of the above aspects and embodiments, the ssDNA molecule is produced synthetically.

[0249] According to any of the above aspects and embodiments, the ssDNA molecule lacks any viral capsid protein coding sequence.

[0250] According to any of the above aspects, the DNA is peptide nucleic acid (PNA), a synthetic mimetic of DNA.

[0251] III. Single-stranded synthetic AAV vectors

[0252] As described herein, the present disclosure relates to single-stranded (ssDNA) molecules. In some aspects, the ssDNA molecule is, for example, a synthetic AAV vector, e.g., a single-stranded (ss) synthetic AAV vector, produced by double-stranded capped DNA comprising a phosphorothioate (PS) bond. The PS bond replaces the non-bridging oxygen in the phosphate backbone of the oligonucleotide with a sulfur atom. Advantageously, this modification renders the internucleotide bond resistant to nuclease degradation and provides accuracy for targeting of exonucleases.

[0253] 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' terminal segment comprising a terminal melting site (trs) sequence.

[0254] According to some aspects, the present disclosure provides an isolated linear and 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 of the first ITR and the second ITR 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.

[0255] As described in more detail herein, ssDNA molecules are synthetically produced in vitro from dsDNA containing phosphorothioate (PS) bonds ("starting material") by removing one DNA strand from a specific nicking site of the dsDNA and to the PS bonding site. According to further embodiments, the ssDNA molecules are synthetically produced in vitro in a cell-free environment.

[0256] According to some embodiments, the present disclosure is characterized in that the 3' terminal portion of the double-stranded DNA molecule (starting material) contains a nicking enzyme recognition sequence. In one embodiment, the 3' terminal portion of the dsDNA molecule contains the sequence 5'-CCAA-3'. In some embodiments, the 3' terminal portion of the dsDNA molecule contains any one or more of the sequences shown in Table 1 below. In addition, since these are unique sequences after the double-stranded ceDNA with a special engineered nicking site is nicked by a nicking endonuclease as shown in the table, the resulting ssDNA molecule also contains any one or more of the sequences shown in the table below in its 3' terminal fragment.

[0257] Table 1.

[0258]

[0259]

[0260] According to some embodiments, the 3' terminal segment of the ssDNA molecule comprises a hydroxylated (-OH) terminal residue, enabling polymerase activity once the ssDNA is transported into the nucleus of a host cell where the ssDNA is converted into regenerated dsDNA capable of being expressed.

[0261] According to some embodiments, the ssDNA molecule comprises a 3' terminal segment comprising a terminal melting site (trs) sequence.

[0262] A key discovery of the present disclosure is that the ssDNA molecules described herein can be transported from the cytosol across the nuclear membrane 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, the hydroxylated (-OH) terminal residue in the ssDNA molecule is crucial for responsiveness to DNA polymerase activity within the nucleus of the host cell. According to further embodiments, the DNA polymerase generates the dsDNA molecule.

[0263] Importantly, the ssDNA molecules do not activate or minimally activate innate immune pathways within 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 defense response through RIG-I-like receptors, toll-like receptors, or other pathogen-associated molecular pattern receptors to activate interferon, NF-κ-B, STAT, IRF, and other response pathways that prevent pathogen infection. According to some embodiments, the innate immune pathway can be a cGAS / STING pathway, a TLR9 pathway, an inflammasome-mediated pathway, or a combination thereof. Indicators of activation of the innate immune response 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.

[0264] 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 regeneration double-stranded expression cassette comprising at least one regeneration double-stranded transgene and at least one double-stranded promoter operably linked to the regeneration double-stranded transgene to control expression of the at least one regeneration double-stranded transgene. The double-stranded expression cassette is capable of expression in a host cell (e.g., an in vivo host cell). In some embodiments, the double-stranded expression cassette is capable of being expressed as at least one therapeutic protein or fragment thereof.

[0265] In further embodiments, 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.

[0266] In other aspects of the disclosure, single-stranded transgene cassettes can be used in gene editing applications.

[0267] 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 regenerative promoterless double-stranded transgene. In some embodiments, the at least one regenerative promoterless double-stranded transgene is capable of inserting into a target locus in the genome of a host cell. In further embodiments, the at least one regenerative promoterless double-stranded transgene is capable of inserting into a target locus in the genome of a host cell in vivo. In some embodiments, the at least one regenerative promoterless double-stranded transgene is capable of inserting into a target locus to replace or supplement at least one target gene. In other embodiments, the at least one regenerative promoterless double-stranded transgene is capable of inserting into a target locus via homology-directed 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' homology arm and a single-stranded 3' homology arm flanking the single-stranded donor sequence. The single-stranded 5' homology arm and the single-stranded 3' homology arm are each about 10 nt to 2000 nt in length, such as about 100 to 2000 nt in length or about 1000 to 2000 nt in length, or about 10 to 1000 nt in length, such as 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. In some embodiments, the at least one regenerated promoterless double-stranded transgene is capable of insertion into the target locus via non-homologous end joining (NHEJ). In some embodiments, the 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.

[0268] 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 nicking sites; and b) contacting the dsDNA construct with an exonuclease that is capable of removing nucleotides from the nicked strand of the dsDNA construct, thereby producing the ssDNA molecule.

[0269] A. Double-stranded (ds) capped DNA (ceDNA)

[0270] In some aspects, the present disclosure provides double-stranded capped DNA (ceDNA) comprising phosphorothioate (PS) bonds. As described herein, this modification is advantageously located in the ITR region in a space where exonucleases are active and acts 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 generate the ssDNA molecules described herein.

[0271] 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 of the first ITR and the optional second ITR flanking the at least one double-stranded transgene cassette; wherein at least one of the first ITR and the second ITR comprises one or more phosphorothioate-modified nucleotides. According to some embodiments, the dsDNA construct comprises a nicking enzyme recognition sequence ("nicking site").

[0272] In one embodiment, the dsDNA construct comprises a terminal melting site (trs) sequence of an AAV ITR containing a nicking site. According to some embodiments, the dsDNA construct comprises one or more recognition nucleotide sequences of one or more nicking endonucleases each independently selected from the group consisting of 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 one 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:

[0273] Table 2.

[0274] sequence Nicking endonuclease 5'-GCTGAGG-3' (Nb.BbvCI) 5'NGCATTC-3' (Nb.BsmI) N can be G, C, A or T 5'-NNCATTGC-3' (Nb.BsrDI) 5'-CTCGTG-3' (Nb.BssSI) 5'-NNCACTGC-3' (Nb.BtsI) 5'-GGATCNNNNN-3' (Nt.AlwI) 5'-CCTCAGC-3' (Nt.BbvCI) 5'-GTCTCNN-3' (Nt.BsmI) 5'-GTCTCNN-3' (Nt.BsmI) 5'-GCTCTTCN-3' (Nt.BspQI) 5'-GAGTCNNNNN-3' (Nt.BstNBI) 5'-CCD-3' (Nt.CviPII)D can be A or G or T

[0275] According to some embodiments, the one or more recognition nucleotide sequences are each an engineered sequence.According to further embodiments, the one or more recognition nucleotide sequences each comprise one or more nicking sites for one or more nicking endonucleases.

[0276] According to some embodiments, the one or more nicking sites are about 0 to about 20 nucleotides downstream of the terminal melting site (trs), for example, 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 terminal melting site (trs), or for example about 0 to about 15, about 0 to 10, about 0 to 5, about 5 to 15, about 10 to 20, about 15 to 20, about 10 to 20, about 5 to 20 nucleotides downstream of the terminal melting site (trs). According to some embodiments, there is only one nicking site that serves as an exonuclease entry site.

[0277] According to some embodiments, the dsDNA construct comprises one or more recognition nucleotide sequences of Nb.BbvCI or an isoschizomer thereof.

[0278] According to some embodiments, the dsDNA construct comprises a single recognition nucleotide sequence for Nb.BbvCI or an isoschizomer thereof.

[0279] According to some embodiments, the dsDNA construct comprises one or more recognition nucleotide sequences for Nb.BtsI or its isoschizomer.

[0280] According to some embodiments, the dsDNA construct comprises a single recognition nucleotide sequence for Nb.BtsI or an isoschizomer thereof.

[0281] 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 to control 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 yet 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' homology arm and a double-stranded 3' homology arm flanking the double-stranded donor sequence. According to some embodiments, the double-stranded 5' homology arm and the double-stranded 3' homology arm are each about 10 to 2000 nt in length, such as about 100 to 2000 nt in length or about 1000 to 2000 nt in length, or about 10 to 1000 nt in length, such as 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.

[0282] 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. In addition, 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);

[0283] at least a second double-stranded gRNA TS; and at least a second double-stranded PAM.

[0284] As described in more detail herein, in some embodiments, a dsDNA construct is synthetically produced by a 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 one double-stranded transgene; a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the double-stranded transgene cassette; and 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 cleavage site and the second cleavage site 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. 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, the at least one functional moiety is an aptamer sequence, optionally wherein the aptamer sequence has a high binding affinity for a nuclear localization protein. In another embodiment, the at least one functional moiety is a nuclear localization peptide conjugated to at least one of the ITR oligonucleotides. In another embodiment, the at least one functional moiety is a fluorophore chemically conjugated to the ITR oligonucleotide.

[0285] B. Single-stranded DNA molecules or vectors derived from double-stranded DNA

[0286] Because the 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, and specifically double-stranded ceDNA (dsceDNA) having phosphorothioate-modified nucleotides, the physical properties of the dsceDNA vectors are also present in the single-stranded DNA (ssDNA) molecules, including, for example, the presence of at least one functional moiety, such as an aptamer sequence, e.g., having high binding affinity for a nuclear localization 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.

[0287] 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 packaging constraints imposed by the confined space within the viral capsid. This allows for the insertion of control elements, e.g., regulatory switches, large transgenes, multiple transgenes, etc., as disclosed herein.

[0288] 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', B, B', C, C', 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', B, B', C, C', D, and D' of at least one of the first ITR and the optional second ITR.

[0289] 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.

[0290] 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.

[0291] 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' region and / or the D region 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' region and / or the D region of the first ITR.

[0292] According to some embodiments, all of the one or more phosphorothioate modified nucleotides in the first ITR of the ssDNA molecule are located in region A 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 region A of the first ITR.

[0293] 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 and / or the D region 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' region and / or the D region of the second ITR.

[0294] 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 region A 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 region A of the second ITR.

[0295] According to some embodiments, the one or more phosphorothioate modified nucleotides of the ssDNA molecule are adjacent to each other. According to some embodiments, the one or more phosphorothioate modified nucleotides of the dsDNA construct are adjacent to each other.

[0296] According to some embodiments, the one or more phosphorothioate modified nucleotides of the ssDNA molecule are about 1 to 15 nucleotides away from the B-B' arm and the C-C' arm of the first ITR or the optional second ITR, if present. According to some embodiments, the one or more phosphorothioate modified nucleotides of the dsDNA construct are about 1 to 15 nucleotides away from the B-B' arm and the C-C' arm of the first ITR or the optional second ITR, if present.

[0297] According to some embodiments, the one or more phosphorothioate modified nucleotides of the ssDNA molecule are about 1 to 10 nucleotides away from the B-B' arm and the C-C' arm of the first ITR or the optional second ITR, if present. According to some embodiments, the one or more phosphorothioate modified nucleotides of the dsDNA construct are about 1 to 10 nucleotides away from the B-B' arm and the C-C' arm of the first ITR or the optional second ITR, if present.

[0298] According to some embodiments, the one or more phosphorothioate modified nucleotides of the ssDNA molecule are about 1 to 5 nucleotides away from the B-B' arm and the C-C' arm of the first ITR or the optional second ITR, if present. According to some embodiments, the one or more phosphorothioate modified nucleotides of the dsDNA construct are about 1 to 5 nucleotides away from the B-B' arm and the C-C' arm of the first ITR or the optional second ITR, if present.

[0299] According to some embodiments, the one or more phosphorothioate-modified nucleotides of the ssDNA molecule are resistant to exonuclease degradation. According to some embodiments, the one or more phosphorothioate-modified nucleotides containing dsDNA constructs are resistant to exonuclease degradation at the PS bonding sequence.

[0300] According to some embodiments, at least one of the first ITR and the optional second ITR of the ssDNA molecule each comprises about 1 to about 60 phosphorothioate modified nucleotides, e.g., about 1 to about 3, about 1 to about 5, about 1 to about 7, about 1 to about 10, about 1 to about 20, about 1 to about 30, about 1 to about 40, about 1 to about 50, about 10 to about 20, about 10 to about 30, about 10 to about 40, about 10 to about 50, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 30 to about 40, about 30 to about 50, about 40 to about 50, about 25 to about 50, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25. According to some embodiments, at least one of the first ITR and the optional second ITR dsDNA construct 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 ITR and optional second ITR of the ssDNA molecule each comprises about 1 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 30 phosphorothioate-modified nucleotides.

[0301] According to some embodiments, the one or more phosphorothioate modified nucleotides are located at the 5' end of the ssDNA molecule. According to some embodiments, the one or more phosphorothioate modified nucleotides are located at the 3' end of the ssDNA molecule. According to some embodiments, the 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.

[0302] According to some embodiments, one or more phosphorothioate modified nucleotides are located upstream of each of the one or more nicking endonuclease recognition sequences.

[0303] According to some embodiments, one or more phosphorothioate modified nucleotides are located at the 5' end of the first ITR and / or the optional second ITR.

[0304] 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, wherein 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.

[0305] According to some embodiments, at least one of the first ITR and optional second ITR of the ssDNA molecule each comprises no more than about 6 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and optional second ITR of the ssDNA molecule each comprises no more than 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 no more than about 4 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and optional second ITR of the ssDNA molecule each comprises no more than about 3 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and optional second ITR of the ssDNA molecule each comprises no more than about 2 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and optional second ITR of the ssDNA molecule each comprises no more than about 1 phosphorothioate-modified nucleotide. According to some embodiments, at least one of the first ITR and optional second ITR dsDNA constructs each comprises no more than about 6 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR dsDNA construct each comprises no more than 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 no more than about 4 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR dsDNA construct each comprises no more than about 3 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR dsDNA construct each comprises no more than about 2 phosphorothioate-modified nucleotides. According to some embodiments, at least one of the first ITR and the optional second ITR dsDNA construct each comprises no more than about 1 phosphorothioate-modified nucleotides.

[0306] 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.

[0307] According to some embodiments, the first ITR and the second ITR of the ssDNA molecule are symmetrical or substantially symmetrical with each other. According to some embodiments, the first ITR and the second ITR of the dsDNA construct are symmetrical or substantially symmetrical with each other.

[0308] According to some embodiments, the first ITR and the second ITR of the ssDNA molecule are asymmetric relative to each other. According to some embodiments, the first ITR and the second ITR of the dsDNA construct are asymmetric relative to each other.

[0309] According to some embodiments, at least one of the first and second ITRs of the ssDNA molecule is a wild-type ITR. According to some embodiments, at least one of the first and second ITRs of the dsDNA construct is a wild-type ITR.

[0310] According to some embodiments, at least one of the first ITR and the second ITR of the ssDNA molecule is modified by a deletion, insertion, and / or base substitution in at least one of the regions selected from A, A', B, B', C, C', D, and D'. According to some embodiments, at least one of the first ITR and the second ITR of the dsDNA construct is modified by a deletion, insertion, and / or base substitution in at least one of the regions selected from A, A', B, B', C, C', D, and D'.

[0311] According to some embodiments, the first ITR and the second ITR of the ssDNA molecule are each an AAV ITR and are each independently an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. According to some embodiments, the first ITR and the second ITR dsDNA construct are each an AAV ITR and are each independently an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0312] 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.

[0313] 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.

[0314] C. Expression Cassettes, Transgenes, and Nucleic Acid Sequences of Interest

[0315] The expression cassette can comprise a transgene (a nucleic acid sequence of interest) and one or more regulatory sequences that allow and / or control expression of the transgene, for example, wherein the expression cassette can comprise, in this order, one or more of the following: an enhancer / promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (e.g., a WPRE), and a polyadenylation and termination signal (e.g., BGH polyA). The expression cassette can also comprise 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, the ITR can serve as a promoter for the transgene. In some embodiments, the ssDNA molecules or dsDNA constructs described herein in Section II or Section III comprise additional components to regulate expression of the transgene or nucleic acid sequence of interest, for example, a regulatory switch, such as described herein in the section entitled "Regulatory Switches" for controlling and regulating expression of the transgene, and, if desired, a regulatory switch that is a kill switch to effect controlled cell death of cells containing the ssDNA molecule.

[0316] The expression cassette or nucleic acid sequence of interest in the ssDNA construct may comprise greater than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any range between about 4000-10,000 nucleotides or 10,000-50,000 nucleotides, or greater than 50,000 nucleotides. In some embodiments, the expression cassette may comprise a transgene ranging in length from 500 to 50,000 nucleotides. In some embodiments, the expression cassette may comprise a transgene ranging in length from 500 to 75,000 nucleotides. In some embodiments, the expression cassette may comprise a transgene ranging in length from 500 to 10,000 nucleotides. In some embodiments, the expression cassette may comprise a transgene ranging in length from 1000 to 10,000 nucleotides. In some embodiments, the expression cassette may comprise a transgene ranging in length from 500 to 5,000 nucleotides. The ssDNA molecules and dsDNA construct vectors described herein do not have the size limitations of encapsulated AAV vectors and are therefore capable of delivering large expression cassettes to provide efficient transgenesis. In some embodiments, the ssDNA molecules and dsDNA constructs described herein in Section II or Section III lack prokaryotic-specific methylation.

[0317] The expression cassette can include, for example, an expressible exogenous sequence (for example, an open reading frame) or a transgenic or coding nucleic acid sequence of a protein that does not exist in a recipient subject, is inactive or is not active enough, or a gene encoding a protein with a desired biological or therapeutic effect. Transgenic or the nucleic acid sequence of a protein that is paid attention to can encode a gene product that can work to correct the expression of a defective gene or transcript. In principle, an expression cassette can include any gene encoding a protein, polypeptide, or RNA that reduces or does not exist due to a sudden change, or that transmits a therapeutic benefit when overexpression and is considered within the scope of the present disclosure.

[0318] The expression cassette can comprise any transgene or nucleic acid sequence of interest that can be used to treat a disease or condition in a subject. The ssDNA molecules or dsDNA constructs described herein 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 polypeptide-encoding nucleic acids or non-coding nucleic acids (e.g., RNAi, miR, etc.), as well as exogenous genes and nucleotide sequences, including viral sequences in the genome of the subject, e.g., HIV viral sequences, etc. In some embodiments, the ssDNA molecules and dsDNA constructs described herein in Part II or Part III are used for therapeutic purposes (e.g., for medical, diagnostic, or veterinary use). In certain embodiments, the ssDNA molecules and dsDNA constructs described herein in Section II or Section III can be used to express any gene of interest in a subject, including one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNA, RNAi, antisense oligonucleotides, antisense polynucleotides, or RNA (coding or non-coding; e.g., siRNA, shRNA, microRNA, mRNA, or gRNA and their antisense counterparts (e.g., antagoMiR)), antibodies, antigen-binding fragments, or any combination thereof.

[0319] The expression cassette can also encode a polypeptide, a sense or antisense oligonucleotide, or an RNA (coding or non-coding; for example, siRNA, shRNA, microRNA, and their antisense counterparts (e.g., antagoMiR). The expression cassette can include exogenous sequences encoding reporter proteins 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 well known in the art.

[0320] The sequences provided in the expression cassettes and expression constructs of the ssDNA molecules described herein and the dsDNA constructs described herein in Part II or Part III 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 to enhance expression in the cells of a vertebrate of interest, such as a mouse or a human, by replacing at least one, more than one, or a large number of codons of a native sequence (e.g., a prokaryotic sequence) with codons that are more frequently used or most frequently used in the genes of the vertebrate of interest. Various species show specific preferences for certain codons for specific amino acids. Typically, codon optimization does not change the amino acid sequence of the originally translated protein. The optimized codons can be modified using, for example, Aptagen's Codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Road, Suite 300, Herdon, VA 20171) or another publicly available database were used to determine the codons.

[0321] 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 antigen-binding fragment thereof, e.g., a neutralizing antibody or antibody fragment, etc.

[0322] Specifically, therapeutic genes are one or more therapeutic agents, including but not limited to, for example, proteins, polypeptides, peptides, enzymes, antibodies, antigen binding fragments and variants and / or active fragments thereof for treating, preventing and / or improving one or more symptoms of a disease, dysfunction, injury and / or illness. Exemplary therapeutic genes are described herein in the section entitled "Therapeutic Methods."

[0323] The ssDNA molecules and dsDNA constructs described herein in Section II or Section III have a number of structural features that differ from plasmid-based expression vectors. The ssDNA molecules and dsDNA constructs described herein in Section II or Section III produced by the synthetic methods herein can have one or more of the following features: lack of native (i.e., no inserted) bacterial DNA; lack of a prokaryotic origin of replication; are self-sufficient, i.e., they do not require any sequences other than the two ITRs, including the Rep binding site and the terminal melting sites (RBS and TRS) and exogenous sequences between the ITRs; the presence of ITR sequences that form hairpins; and the absence of bacterial-type DNA methylation or any other methylation that is actually associated with the production of a given cell type and is considered abnormal by a mammalian host. Generally, it is preferred that the vectors of the present invention do not contain any prokaryotic DNA, but it is contemplated that some prokaryotic DNA can be inserted as an exogenous sequence, as a non-limiting example in the promoter or enhancer region.

[0324] Compared to plasmid-based expression vectors, the use of the ssDNA molecules and dsDNA constructs described herein in Section II or Section III has several advantages. Such advantages include, but are not limited to: 1) plasmids contain bacterial DNA sequences and undergo prokaryotic-specific methylation, for example, 6-methyladenosine and 5-methylcytosine methylation, while the capsid-less AAV vector sequences are of eukaryotic origin and do not undergo prokaryotic-specific methylation; therefore, capsid-less AAV vectors are less likely to induce inflammatory and immune responses than plasmids; 2) while plasmids require the presence of resistance genes during the production process, the ssDNA molecules or dsDNA constructs of the present disclosure do not; 3) while circular plasmids are not delivered to the nucleus after introduction into cells and require overloading to circumvent degradation by cellular nucleases, ssDNA molecules and dsDNA constructs contain viral cis elements, i.e., ITRs, which confer nuclease resistance and can be designed to be targeted and delivered to the nucleus. The minimal defined elements hypothesized to be essential for ITR function are the Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' for AAV2) and the terminal melting site (TRS; 5'-AGTTGG-3' for AAV2) plus a variable palindromic sequence that allows hairpin formation; and 4) the ssDNA molecules and dsDNA construct vectors do not have an overrepresentation of CpG dinucleotides frequently found in prokaryotic plasmids that have been reported to bind to members of the Toll-like receptor family, thereby eliciting an immune response mediated by T cells.

[0325] D. Inverted terminal repeats (ITR)

[0326] As shown herein, according to some aspects, the present disclosure provides a single-stranded deoxyribonucleic acid (ssDNA) molecule comprising at least one nucleic acid sequence of interest flanked by at least one stem-loop structure comprising a partial DNA duplex and at least one loop at 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 at the 5' end.

[0327] According to some aspects, the ssDNA molecules and dsDNA constructs contain a transgene or heterologous nucleic acid sequence positioned between two inverted terminal repeat (ITR) sequences, wherein the ITR sequence 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 an ITR sequence selected from any one of the following: (i) at least one WT ITR and at least one modified AAV inverted terminal repeat sequence (mod-ITR) (e.g., an asymmetric modified ITR); (ii) two modified ITRs, wherein the mod-ITR pair has a different three-dimensional spatial organization relative to each other (e.g., an asymmetric modified ITR); or (iii) a symmetric or substantially symmetric WT-WT ITR pair, wherein each WT-ITR has the same three-dimensional spatial organization; or (iv) a symmetric or substantially symmetric modified ITR pair, wherein each mod-ITR has the same three-dimensional spatial organization, wherein the methods of the present disclosure can further include a delivery system, such as, but not limited to, a liposome nanoparticle delivery system.

[0328] In certain embodiments, the ITR sequence can be from a virus of the Parvoviridae family, which includes two subfamilies: the Parvovirinae of infecting vertebrates and the Densovirinae of infecting insects. The Parvovirinae (referred to as Parvovirus) includes the Dependaviridae genus, whose members need to be co-infected with helper viruses such as adenovirus or herpesvirus in most cases to carry out productive infection. The Dependaviridae genus includes adeno-associated viruses (AAVs) that usually infect humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4), and related viruses (e.g., adeno-associated viruses of cattle, dogs, horses, and sheep) that infect other warm-blooded animals. Other members of Parvoviridae and Parvoviridae are generally described in Kenneth I. Berns, " Parvoviridae: Viruses and Their Replication ", Chapter 69 in " Field of Virology " (3rd edition 1996).

[0329] Although the ITRs exemplified in the specification and examples herein are AAV2 WT-ITRs, one of ordinary skill in the art will appreciate that, as described above, ITRs from any known parvovirus, such as AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes. For example, NCBI: NC 002077; NC 001401; NC 001729; NC 001829; NC 006152; NC 006260; NC 006261), chimeric ITRs, or ITRs from any synthetic AAV can be used. In some embodiments, AAV can infect warm-blooded animals, such as avian (AAAV), bovine (BAAV), canine, equine, and ovine adeno-associated viruses. In some embodiments, the ITR is from B19 parvovirus (GenBank Accession No. NC 000883), minute virus of mice (MVM) (GenBank Accession No. NC 001510); goose parvovirus (GenBank Accession No. NC 001701); snake parvovirus 1 (GenBank Accession No. NC 006148). In some embodiments, the 5' WT-ITR can be from one serotype and the 3' WT-ITR from a different serotype, as discussed herein.

[0330] Those of ordinary skill are aware that ITR sequences have a common structure of a double-stranded Holliday junction, which is typically a T-shaped or Y-shaped hairpin structure, wherein each WT-ITR is formed by two palindromic arms or loops (B-B' and C-C') embedded in a larger palindromic arm (A-A') and a single-stranded D sequence (wherein the order of these palindromic sequences defines the flip or flip orientation of the ITR). See, for example, the structural analysis and sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6) and described in 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. Based on the exemplary AAV2 ITR sequences provided herein, one skilled in the art can readily determine the WT-ITR sequence from any AAV serotype for use in ssDNA molecules and dsDNA constructs. See, for example, a sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6 and avian AAV (AAAV) and bovine AAV (BAAV)) is described in Grimm et al., J. Virol., 2006; 80(1); 426-439; which shows the % 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%).

[0331] According to some other embodiments, at least one of the first ITR and optional second ITR oligonucleotides comprising one or more phosphorothioate-modified nucleotides of the present invention may further comprise one or more functional moieties. In one embodiment, at least one functional moiety is an aptamer sequence, optionally wherein the aptamer sequence has a high binding affinity to a nuclear localization 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.

[0332] In some embodiments, the ITRs as used herein may be completely synthetic, eg, the ITRs may not contain viral-derived sequences.

[0333] E. Regulatory elements

[0334] Single-stranded DNA (ssDNA) molecule as described herein in Part II or Part III can further include a specific combination of cis-regulatory elements.Cis-regulatory elements include but are not limited to promoters, riboswitches, insulators, mir controllable elements, post-transcriptional regulatory elements, tissue and cell type specific promoters and enhancers. In certain embodiments, single-stranded DNA (ssDNA) molecule as described herein in Part II or Part III includes other components to regulate the expression of transgenic or nucleic acid of interest, for example, a regulatory switch as described herein, to regulate the expression of transgenic or nucleic acid of interest, or a kill switch, the kill switch can kill the cell of single-stranded DNA (ssDNA) molecule as described herein comprising Part II or Part III. The regulatory elements including the regulatory switch that can be used for the present disclosure are discussed more fully in International Application No. PCT / US18 / 49996 (disclosed as International Patent Publication No. WO 2019 / 051255A1), which is incorporated herein by reference in its entirety.

[0335] According to some embodiments, the second nucleotide sequence comprises a regulatory sequence and a nucleotide sequence encoding a nuclease. In certain embodiments, a gene regulatory sequence is operably connected to a nucleotide sequence encoding a nuclease. In certain embodiments, a regulatory sequence is suitable for controlling the expression of a nuclease in a host cell. In certain embodiments, a regulatory sequence comprises a suitable promoter sequence, and the suitable promoter sequence can guide the transcription of a gene operably connected to a promoter sequence, such as a nucleotide sequence encoding a nuclease of the present disclosure. In certain embodiments, the second nucleotide sequence comprises an intron sequence connected to the 5' end of the nucleotide sequence encoding the nuclease. In certain embodiments, an enhancer sequence is arranged on the upstream of the promoter to increase the effect of the promoter. In certain embodiments, the regulatory sequence comprises an enhancer and a promoter, wherein the second nucleotide sequence is included in an intron sequence upstream of the nucleotide sequence encoding the nuclease, wherein the intron comprises one or more nuclease cleavage sites, and wherein the promoter is operably connected to the nucleotide sequence encoding the nuclease.

[0336] The single-stranded DNA (ssDNA) molecules and dsDNA molecules described herein in Part II or Part III produced using the synthetic processes described herein may further comprise specific combinations of cis-regulatory elements such as the WHP post-transcriptional regulatory element (WPRE) and the BGH poly A. Suitable expression cassettes for use in expression constructs are not limited by packaging constraints imposed by viral capsids.

[0337] (i) Promoter

[0338] Those skilled in the art will appreciate that the promoters used in the synthetically produced single-stranded DNA (ssDNA) molecules described herein and the dsDNA molecules disclosed herein in Section II or Section III should be appropriately tailored to the specific sequence they are promoting. For example, a guide RNA may not require a promoter at all, as its function is to form a duplex with a specific target sequence on native DNA to effect a recombination event. In contrast, a nuclease encoded by an ssDNA molecule or dsDNA construct vector will benefit from a promoter so that the nuclease can be efficiently expressed from the vector and, optionally, expressed in a regulatable manner.

[0339] The expression cassette of the present disclosure includes a promoter that can affect overall expression levels and cell specificity. For transgenic expression, the promoter can include a highly active viral-derived immediate early promoter. The expression cassette can contain a tissue-specific eukaryotic promoter to limit transgenic expression to specific cell types and reduce the toxic effects and immune responses caused by unregulated ectopic expression. In a preferred embodiment, the expression cassette can contain a synthetic regulatory element, such as the CAG promoter. The CAG promoter comprises (i) a cytomegalovirus (CMV) early enhancer element, (ii) the promoter, first exon and first intron of the chicken β-actin gene, and (iii) a splice acceptor of the rabbit β-globin gene. Alternatively, the expression cassette can contain an α-1-antitrypsin (AAT) promoter, a liver-specific (LP1) promoter, a human elongation factor-1α (EF1a) promoter, or a human transthyretin (TTR) promoter. In some embodiments, the expression cassette includes 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, a natural promoter of the transgene, a tissue-specific promoter, or various promoters known in the art can be used.

[0340] Suitable promoters, including those described above, can be derived from viruses and therefore can be referred to as viral promoters, or the promoter can be derived from any organism, including prokaryotic or eukaryotic organisms. 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, SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1; 31 (17)), human H1 promoter (H1), CAG promoter, human α1-antitrypsin (HAAT) promoter, etc. In certain embodiments, these promoters are altered 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 foreign to the promoter DNA.

[0341] In one embodiment, the promoter used is the natural promoter of the gene encoding the therapeutic protein. The promoter and other regulatory sequences of the corresponding gene encoding the therapeutic protein are known and have been characterized. The promoter region used may further include one or more additional regulatory sequences (e.g., natural enhancers). Preferably, the gap is located 5' upstream of the promoter.

[0342] 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 can be a serpin enhancer (e.g., 1xSERP, 2xSERP, or 3xSERP). As shown in Example 7 and Figures 42-45, including a double-stranded promoter, enhancer, and / or TSS can significantly increase expression from the ssDNA molecules described herein.

[0343] Thus, in some embodiments, the length of the double-stranded region comprising the promoter, the enhancer and / or the 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, 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.

[0344] In some embodiments, the length of the double-stranded region comprising the promoter, the enhancer and / or the 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 38 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.

[0345] In some embodiments, the double-stranded region comprising the promoter, the enhancer and / or the TSS is about 30-1500 base pairs in length, about 40-1400 base pairs in length, about 50-1300 base pairs in length, about 60-1200 base pairs in length, about 70-1100 base pairs in length, about 80-1000 base pairs in length, about 90-900 base pairs in length, or about 100-1500 base pairs in length. is about 90-900 base pairs in length, about 100-800 base pairs in length, about 110-700 base pairs in length, about 120-600 base pairs in length, about 130-500 base pairs in length, about 140-400 base pairs in length, about 150-300 base pairs in length, about 160-200 base pairs in length, about 1381 base pairs in length or about 499 base pairs in length.

[0346] (ii) Polyadenylation sequence

[0347] A sequence encoding a polyadenylation sequence can be included in a synthetically produced AAV vector to stabilize and facilitate nuclear export and translation of mRNA expressed from a single-stranded DNA (ssDNA) molecule (e.g., a synthetic AAV vector, e.g., a single-stranded (ss) synthetic AAV vector). In one embodiment, the synthetically produced AAV vector does not include a polyadenylation sequence. In other embodiments, the vector includes 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 comprises about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range therebetween.

[0348] The expression cassette may include a polyadenylation sequence known in the art or a variant thereof, such as a naturally occurring sequence or a synthetic sequence isolated from bovine BGHpA or viral SV40pA. Some expression cassettes may also include an SV40 late polyA signal upstream enhancer (USE) sequence. In some embodiments, the USE sequence may be used in combination with SV40pA or a heterologous poly-A signal.

[0349] The expression cassette can also include post-transcriptional elements to increase transgenic expression. In some embodiments, the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) is used to increase transgenic expression. Other post-transcriptional processing elements, such as the thymidine kinase gene from herpes simplex virus or the post-transcriptional element of hepatitis B virus (HBV), can be used. The secretory sequence can be linked to the transgenic, for example, VH-02 and VK-A26 sequences.

[0350] (iii) Nuclear localization sequence

[0351] In certain embodiments, the vector encoding the RNA-guided endonuclease comprises one or more nuclear localization sequences (NLS), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS. In certain embodiments, the one or more NLS are located at or near the amino terminal, at or near the carboxyl terminal, or a combination of these (for example, one or more NLS at the amino terminal and / or one or more NLS at the carboxyl terminal). When there is more than one NLS, each can be selected independently of one another so that a single NLS is present in more than one copy and / or in combination with one or more other NLS present in one or more copies. Non-limiting examples of NLS are shown in Table 3 below.

[0352] Table 3: Exemplary Nuclear Localization Sequences (NLS)

[0353]

[0354]

[0355] F. Additional Components

[0356] The single-stranded DNA (ssDNA) molecules and dsDNA molecules described herein in Part II or Part III produced using a synthetic process as described herein can contain nucleotides encoding other components for gene expression. For example, in order to select a specific gene targeting event, protective shRNA can be embedded in microRNA and inserted into the recombinant single-stranded DNA (ssDNA) molecules described herein in Part II or Part III, which are designed to be site-specifically integrated into highly active loci such as albumin loci. Such embodiments can provide systems for in vivo selection and amplification 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 herein in Part II or Part III of the present disclosure can contain one or more selectable markers that allow selection of transformed, transfected, transduced, or similar cells. Selectable markers are genes whose products provide biocide or viral resistance, resistance to heavy metals, prototrophy for auxotrophs, NeoR, etc. In certain embodiments, positive selectable markers are incorporated into donor sequences such as NeoR. Negative selectable markers can be incorporated downstream of the donor sequence, for example, the nucleic acid sequence HSV-tk encoding the negative selectable marker can be incorporated into the nucleic acid construct downstream of the donor sequence.

[0357] In embodiments, the single-stranded DNA (ssDNA) molecules and dsDNA molecules described herein in Part II or Part III produced using the synthetic processes described herein can be used for gene editing, e.g., as disclosed in International Application PCT / US2018 / 064242, filed December 6, 2018 (published as International Patent Publication No. WO 2019 / 113310 A1), which is incorporated herein by reference in its entirety, and can include one or more of the following: a 5' homology arm, a 3' homology arm, and a polyadenylation site upstream and proximal to the 5' homology arm. Exemplary homology arms are 5' and 3' albumin homology arms or CCR5 5' and 3' homology arms.

[0358] G.Switch

[0359] A molecular regulatory switch is a switch that generates a measurable change in state in response to a signal. Such regulatory switches can be effectively combined with single-stranded DNA (ssDNA) molecules and dsDNA molecules as described herein in Part II or Part III produced using a synthetic process as described herein to control the output of the expression of transgenics from single-stranded DNA (ssDNA) molecules as described herein in Part II or Part III. In certain embodiments, the single-stranded DNA (ssDNA) molecules as described herein in Part II or Part III include a regulatory switch that is used to fine-tune the expression of transgenics. For example, the regulatory switch can be used as a bio-sequestration function of single-stranded DNA (ssDNA) molecules as described herein in Part II or Part III. In certain embodiments, the switch is an "on / off" switch that is designed to start or stop (i.e., close) the expression of the gene of interest in synthesizing AAV in a controllable and regulatable manner. In certain embodiments, the switch can include a "kill switch," which can indicate that cells containing single-stranded DNA (ssDNA) molecules as described herein in Part II or Part III experience programmed cell death after the switch is activated. Exemplary regulatory switches for use in the single-stranded DNA (ssDNA) molecules described herein, encompassed in Section II or Section III, can be used to regulate expression of transgenes and are discussed more fully in International Application PCT / US18 / 49996 (published as International Patent Publication No. WO 2019 / 051255A1), which is incorporated herein by reference in its entirety.

[0360] (i) Binary control switch

[0361] In some embodiments, the single-stranded DNA (ssDNA) molecules described herein in Part II or Part III produced using a synthetic process as described herein comprise a regulatory switch that can be used to controllably regulate the expression of a transgenic gene. For example, the expression cassette between the ITRs of the single-stranded DNA (ssDNA) molecules described herein in Part II or Part III can additionally comprise a regulatory region operably connected to the gene of interest, such as a promoter, a cis-element, a repressor, an enhancer, etc., wherein the regulatory region is regulated by one or more cofactors or exogenous agents. As an 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 or metal-inducible promoters. Other exemplary inducible promoter / enhancer elements include, but are not limited to, RU486 inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, and metallothionein promoter.

[0362] (ii) Small molecule control switches

[0363] A variety of art-known small molecule-based regulatory switches are known in the art and can be combined with the synthetically produced single-stranded DNA (ssDNA) molecules described herein in Section II or Section III disclosed herein to form the single-stranded DNA (ssDNA) molecules controlled by the regulatory switches described herein in Section II or Section III. In some embodiments, the regulatory switch can be selected from any one or a combination of the following: an orthogonal ligand / nuclear receptor pair, such as retinoic acid receptor variant / LG335 and GRQCIMFI, and an artificial promoter that controls expression of an operably linked transgene, as disclosed in Taylor et al., BMC Biotechnology, 10 (2010): 15; an engineered steroid receptor, such as a modified progesterone receptor with a C-terminal truncation that cannot bind progesterone but does bind RU486 (mifepristone) (U.S. Patent No. 5,364,791); an ecdysone receptor from Drosophila and its ecdysteroid ligand (Saez et al., PNAS, 97(26) (2000), 14512–14517); or a switch controlled by the antibiotic trimethoprim (TMP), such as disclosed in Sando R, 3rd ed., Nat Methods. Methods). 2013, 10(11): 1085-8. In some embodiments, the regulatory switch that controls expression of a transgene or 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 the activation switches disclosed in U.S. Patents 8,771,679 and 6,339,070.

[0364] (iii) “Password” control switch

[0365] In some embodiments, the regulatory switch can be a "code switch" or "code loop." When specific conditions occur, the code switch allows fine-tuning of the control of expression of the transgene from the synthetically produced single-stranded DNA (ssDNA) molecules described herein in Section II or Section III, i.e., a combination of conditions must occur for transgene expression and / or repression to occur. For example, in order for transgene expression to occur, at least conditions A and B must occur. The code regulatory switch can be any number of conditions that must occur, for example, at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7 or more conditions for transgene expression to occur. In some embodiments, at least 2 conditions (e.g., conditions A and B) must occur, and in some embodiments, at least 3 conditions (e.g., A, B, and C, or A, B, and D) must occur. By way of example only, in order for gene expression from a synthetic AAV with a code "ABC" regulatory switch to occur, conditions A, B, and C must be present. 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 hypoxic conditions in the kidneys, and condition C is impaired recruitment of erythropoietin-producing cells (EPCs) in the kidneys; or alternatively, impaired HIF-2 activation. Once oxygen levels increase or the desired EPO level is reached, the transgene is turned off again until all three conditions occur, at which point it is turned back on.

[0366] In some embodiments, the code-regulated switches or "code circuits" used in the synthetically produced single-stranded DNA (ssDNA) molecules described herein, encompassed in Section II or Section III, comprise hybrid transcription factors (TFs) to expand the range and complexity of environmental signals used to define biocontainment conditions. In contrast to disable switches that trigger cell death in the presence of predetermined conditions, "code circuits" allow cell survival or transgene expression in the presence of a specific "code" and can be easily reprogrammed to allow transgene expression and / or cell survival only in the presence of predetermined environmental conditions or codes.

[0367] Any and all combinations of regulatory switches disclosed herein, for example, small molecule switches, nucleic acid-based switches, small molecule-nucleic acid hybrid switches, post-transcriptional transgenic regulatory switches, post-translational regulation, radiation-controlled switches, hypoxia-mediated switches, and other regulatory switches known to those of ordinary skill in the art as disclosed herein, can be used in the cryptographic regulatory switches disclosed herein. Regulatory switches contemplated for use are also discussed in the review article Kis et al., J. R. Soc. Interface. 12:20141000 (2015) and are summarized in Table 1 of Kis et al. In some embodiments, the regulatory switches used in the cryptographic system can be selected from any one or combination of the switches listed in Table 4 below.

[0368] (iv) Nucleic acid-based regulatory switches to control transgene expression

[0369] In some embodiments, the regulatory switch for controlling the expression of a transgene from a synthetically produced single-stranded DNA (ssDNA) molecule described herein in Section II or Section III is based on a nucleic acid-based control mechanism. Exemplary nucleic acid control mechanisms are known in the art and are contemplated for use. For example, such mechanisms include riboswitches, such as those disclosed in US2009 / 0305253, US2008 / 0269258, US2017 / 0204477, WO2018026762A1, U.S. Patent No. 9,222,093, and EP Application No. EP288071, and are also disclosed in Billa JK et al., Microbiol Spectr. May 2018; 6(3). Metabolite-responsive transcriptional biosensors, such as those disclosed in WO2018 / 075486 and WO2017 / 147585, are also included. Other mechanisms known in the art that are contemplated for use include silencing the transgene with siRNA or RNAi molecules (e.g., miR, shRNA). For example, the single-stranded DNA (ssDNA) molecules described herein in Section II or Section III can contain a regulatory switch encoding an RNAi molecule that is complementary to the transgene expressed by the single-stranded DNA (ssDNA) molecules described herein in Section II or Section III. Even if the transgene is expressed by the single-stranded DNA (ssDNA) molecules described herein in Section II or Section III, when such RNAi is expressed, the transgene will be silenced by the complementary RNAi molecule, and when the RNAi is not expressed when the transgene is expressed by the single-stranded DNA (ssDNA) molecules described herein in Section II or Section III, the transgene will not be silenced by the RNAi.

[0370] In some embodiments, the regulatory switch is a tissue-specific self-inactivating regulatory switch, for example, as disclosed in US 2002 / 0022018, whereby the regulatory switch intentionally turns off transgene expression at a site where transgene expression may otherwise be detrimental. In some embodiments, the regulatory switch is a recombinase reversible gene expression system, for example, as disclosed in US 2014 / 0127162 and U.S. Patent 8,324,436.

[0371] (v) Post-transcriptional and post-translational regulatory switches.

[0372] In some embodiments, the regulatory switch that controls the expression of a transgene or gene of interest from a synthetically produced single-stranded DNA (ssDNA) molecule described herein in Section II or Section III is a post-transcriptional modification system. For example, such a regulatory switch can be an aptamer enzyme riboswitch that is sensitive to tetracycline or theophylline, such as disclosed in US2018 / 0119156, GB201107768, WO2001 / 064956A3, EP Patent 2707487, and Beilstein et al., ACS Synthetic Biology (ACS Synth. Biol)., 2015, 4(5), pp. 526–534; Zhong et al., Elife., November 2, 2016; 5.pii:e18858. In some embodiments, it is contemplated that one of ordinary skill in the art can encode both a transgene and an inhibitory siRNA containing a ligand-sensitive (off-switch) aptamer, with the net result being a ligand-sensitive on-switch.

[0373] (vi) Other Exemplary Control Switches

[0374] Any known regulatory switch can be used in synthetically produced ssDNA molecules to control gene expression of transgenes expressed from the single-stranded DNA (ssDNA) molecules described herein in Part II or Part III, including gene expression triggered by environmental changes. Additional examples include, but are not limited to, the BOC approach of Suzuki et al., Scientific Reports 8;10051 (2018); genetic code expansion and non-physiological amino acids; radiation-controlled or ultrasound-controlled on / off switches (see, e.g., Scott S et al., Gene Therapy 2000 Jul;7(13):1121-5; U.S. Patents 5,612,318; 5,571,797; 5,770,581; 5,817,636; and WO1999 / 025385A1. In some embodiments, the regulatory switch is controlled by an implantable system, e.g., as disclosed in U.S. Patents 7,840,263; US2007 / 0190028A1, wherein gene expression is controlled by one or more forms of energy, including electromagnetic energy, that activates a promoter of a transgene operably linked to a single-stranded DNA (ssDNA) molecule described herein in Section II or Section III.

[0375] In some embodiments, the regulatory switches contemplated for use in the synthetically produced single-stranded DNA (ssDNA) molecules described herein in Part II or Part III are hypoxia-mediated or stress-activated switches, for example, as disclosed in WO1999060142A2, U.S. Patents 5,834,306; 6,218,179; 6,709,858; US2015 / 0322410; Greco et al. (2004) Targeted Cancer Therapies 9, S368, as well as FROG, TOAD, and NRSE elements, and conditionally inducible silencing elements, including hypoxia-responsive elements (HREs), inflammatory-responsive elements (IREs), and shear-stress-activated elements (SSAEs), for example, as disclosed in U.S. Patent 9,394,526. Such embodiments can be used to turn on expression of transgenes from the single-stranded DNA (ssDNA) molecules described herein in Part II or Part III after ischemia or in ischemic tissue and / or tumors.

[0376] H. Kill switch

[0377] Other embodiments of the present disclosure relate to synthetically produced single-stranded DNA (ssDNA) molecules described herein in Section II or Section III and dsDNA molecules comprising a kill switch. The kill switch as disclosed herein enables cells comprising the single-stranded DNA (ssDNA) molecules described herein in Section II or Section III to be killed or undergo programmed cell death as a means of permanently removing the introduced single-stranded DNA (ssDNA) molecules described herein in Section II or Section III from the subject's system. One of ordinary skill in the art will appreciate that the use of a kill switch in the synthetically produced single-stranded DNA (ssDNA) molecules described herein in Section II or Section III of the present disclosure is typically combined with targeting the single-stranded DNA (ssDNA) molecules described herein in Section II or Section 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 switch" as disclosed herein is designed to provide rapid and robust cell killing of cells comprising the single-stranded DNA (ssDNA) molecules described herein in Section II or Section III in the absence of an input survival signal or other specified conditions. In other words, the kill switch encoded by the single-stranded DNA (ssDNA) molecule described herein in Section II or Section III herein can restrict the cell survival of cells comprising the single-stranded DNA (ssDNA) molecule described herein in Section II or Section III to an environment defined by a specific input signal. Such a kill switch serves as a biological biosequestration function if it is desired to remove the synthetically produced single-stranded DNA (ssDNA) molecule described herein in Section II or Section III from a subject or to ensure that the molecule does not express an encoded transgene.

[0378] Thus, a kill switch is a synthetic biological circuit in an ssDNA molecule or dsDNA construct that couples an environmental signal to conditional survival of a cell comprising the ssDNA molecule or dsDNA construct. In some embodiments, different ssDNA molecules or dsDNA constructs can be designed with different kill switches.

[0379] In some embodiments, the single-stranded DNA (ssDNA) molecule described herein in Part II or Part III can include a kill switch, which is a modular biological sealing circuit. In some embodiments, the kill switch for ssDNA molecules or dsDNA constructs is disclosed in WO2017 / 059245, which describes a switch referred to as a "disability kill switch", wherein the switch includes a mutual inhibition arrangement of at least two repressor sequences so that the environmental signal represses the activity of the second molecule in the construct (for example, a small molecule binding transcription factor is used to produce a "survival" state due to the repression of toxins). In cells comprising single-stranded DNA (ssDNA) molecules described herein in Part II or Part III comprising a disabling kill switch, when the environmental signal is lost, the circuit in which the toxin is now suppressed is permanently switched to a "death" state, thereby producing a toxin that kills cells. In another embodiment, a synthetic biological circuit referred to as a "password circuit" or "password kill switch" is provided, which uses a hybrid transcription factor (TF) to construct a complex environmental requirement for cell survival. The incapacitating and codon-killing switches described in WO2017 / 059245 are particularly suitable for use in the single-stranded DNA (ssDNA) molecules described herein in Section II or Section III because the kill switches are modular and customizable, both in terms of the environmental conditions that control circuit activation and in the output modules that control cell fate. By appropriate selection of toxins, including but not limited to nucleases such as EcoRI, the codon-killing circuits present in ssDNA molecules or dsDNA constructs can be used to not only kill host cells containing ssDNA molecules or dsDNA constructs, but also degrade their genome and accompanying plasmids.

[0380] Other kill switches known to those of ordinary skill in the art are contemplated for use in the single-stranded DNA (ssDNA) molecules described herein as in Section II or Section III as disclosed herein, for example, as disclosed in US2010 / 0175141; US2013 / 0009799; US2011 / 0172826; US2013 / 0109568, and the kill switches disclosed in Jusiak et al., Reviews in Cell Biology and Molecular Medicine; 2014; 1-56; Kobayashi et al., Proceedings of the National Academy of Sciences of the United States of America, 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. Medicine), 2018, 11.

[0381] Thus, in some embodiments, the single-stranded DNA (ssDNA) molecules described herein in Part II or Part III can comprise a kill switch nucleic acid construct comprising a nucleic acid encoding an effector toxin or a reporter protein, wherein the expression of the effector toxin (e.g., a death protein) or the reporter protein is controlled by a predetermined condition. For example, the predetermined condition can be the presence of an environmental agent, such as an exogenous agent, in the absence of which the cell will express the effector toxin (e.g., a death protein) by default and be killed. In alternative embodiments, the predetermined condition is the presence of two or more environmental agents, for example, the cell will only survive when two or more necessary exogenous agents are supplied, and in the absence of any one of which, the cell comprising the single-stranded DNA (ssDNA) molecules described herein in Part II or Part III is killed.

[0382] In some embodiments, the single-stranded DNA (ssDNA) molecules described herein in Section II or Section III are modified to incorporate a kill switch to destroy cells containing the single-stranded DNA (ssDNA) molecules described herein 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 herein in Section II or Section III are further genetically 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 such a switch protein will cells expressing the switch protein be 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, e.g., Dey and Evans, Suicide Gene Therapy by Herpes SimplexVirus-1 Thymidine Kinase (HSV-TK), Targets in Gene Therapy, You ed. (2011); and Beltinger et al., Proc. Natl. Acad. Sci. USA 96(15):8699-8704 (1999). In some embodiments, the ssDNA molecule or dsDNA construct may comprise an siRNA kill switch known as DISE (death induced by survival gene elimination) (Murmann et al., Oncotarget. 2017;8:84643-84658. Induction of DISE in ovarian cancer cells in vivo).

[0383] In some aspects, a disabling kill switch is a biological circuit or system that sensitizes a cellular response to a predetermined condition, such as the absence of an agent, e.g., an exogenous agent, in the cell's growth environment. Such a circuit or system can comprise a nucleic acid construct comprising an expression module that forms a disabling regulatory circuit that is sensitive to the predetermined condition, the construct comprising an expression module that forms a regulatory circuit, the construct comprising: a first repressor protein expression module, wherein the first repressor protein binds to a first repressor protein nucleic acid binding element and represses transcription from a coding sequence comprising the first repressor protein binding element, and wherein the repressive activity of the first repressor protein is sensitive to inhibition by a first exogenous agent, the presence or absence of the first exogenous agent establishing the predetermined condition;

[0384] 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 comprising the second repressor protein binding element, wherein the second repressor protein is different from the first repressor protein; and

[0385] iii) an effector expression module comprising a nucleic acid sequence encoding an effector protein, said effector expression module being operably linked to a genetic element comprising a binding element for a second repressor protein, such that expression of the second repressor protein results in repression of effector expression from the effector expression module, wherein the second expression module comprises a first repressor protein nucleic acid binding element which, when bound by the first repressor protein, allows repression of transcription of the second repressor protein, such that the respective modules form a regulatory loop such that, in the absence of the first exogenous agent, The first repressor protein is produced by the first repressor protein expression module and represses transcription from the second repressor protein expression module, so that the repression of effector expression by the second repressor protein is relieved, thereby allowing effector protein expression, but in the presence of the first exogenous agent, the activity of the first repressor protein is inhibited, thereby allowing the second repressor protein to be expressed, which maintains the expression of the effector protein expression in an "off" state, such that the circuit requires the first exogenous agent to maintain the effector protein expression in the "off" state, and the removal or absence of the first exogenous agent defaults to effector protein expression.

[0386] 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 those cells in which it is expressed. In other embodiments, the toxin kills other cells of the same host organism. Any of a large number of products that will cause cell death can be used in a disabling kill switch. Agents that inhibit DNA replication, protein translation, or other processes, or that, for example, degrade the nucleic acids of the host cell, are particularly useful. To identify highly effective mechanisms for killing host cells after loop activation, several toxin genes that directly damage the DNA or RNA of the host cell were tested. The endonuclease ecoRI, the DNA gyrase inhibitor ccdB, and the ribonuclease-type toxin mazF were tested because the toxin genes are well characterized, native to E. coli, and provide a range of killing mechanisms. To increase the stability of the loop and provide an independent method for loop-dependent cell death, the system can be further adapted to express, for example, a targeted protease or nuclease that further interferes with the repressor that maintains the death gene in an "off" state. After the survival signal is lost or withdrawn, the death gene repression is even more efficiently removed by, for example, active degradation of the repressor protein or its message. As a non-limiting example, the mf-Lon protease is used not only to degrade LacI, but also to target essential proteins for degradation. The mf-Lon degradation tag pdt#1 can be attached to the 3' end of five essential genes whose protein products are particularly sensitive to mf-Lon degradation, and cell viability is measured after removal of aTc. Among the essential gene targets tested, the peptidoglycan biosynthesis gene murC provided the strongest and fastest cell death phenotype (survival rate <1x 10 in 6 hours). -4 ).

[0387] 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. Typically, such agents can bind to and / or alter the conformation of a transcription factor polypeptide to thereby alter the activity of the transcription factor polypeptide. Examples of predetermined inputs include, but are not limited to, environmental input agents that are not required for the survival of a given host organism (i.e., in the absence of a synthetic biological circuit as described herein). Conditions that can provide predetermined inputs include, for example, temperature, such as where the activity of one or more factors is temperature sensitive, the presence or absence of light, including light of a given wavelength spectrum, and the concentration of gases, salts, metals, or minerals. Environmental input agents include, for example, small molecules, biological agents, such as pheromones, hormones, growth factors, metabolites, nutrients, and the like, and the like; concentrations of chemicals, environmental byproducts, metal ions, and other such molecules or agents; light levels; temperature; mechanical stress or pressure; or electrical signals, such as current and voltage.

[0388] In some embodiments, a reporter is used to quantify the intensity or activity of a signal received by a module or programmable synthetic biological circuit of the present disclosure. In some embodiments, a reporter can be fused in-frame to other protein-coding sequences to identify the location of a protein in a cell or organism. For various embodiments described herein, luciferase can be used as an effector protein, for example, to measure low levels of gene expression, since cells often have little or no background luminescence in the absence of luciferase. In other embodiments, enzymes that produce colored substrates can be quantified using a spectrophotometer or other instrumentation that can obtain absorbance measurements, including plate readers. Like luciferase, enzymes such as β-galactosidase can be used to measure low levels of gene expression, since such enzymes often amplify low signals. 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 small molecules or other proteins, or an enzyme that methylates or demethylates other proteins or DNA.

[0389] In some embodiments, the effector protein can be a receptor, a ligand, or a lytic protein. Receptors often have three domains: an extracellular domain for binding to a ligand such as a protein, peptide, or small molecule; a transmembrane domain, and an intracellular or cytoplasmic domain that can often participate in certain signal transduction events such as phosphorylation. In some embodiments, a transporter, channel, or pump gene sequence is used as an effector protein. Non-limiting examples and sequences of effector proteins for use with a kill switch as described herein can be found in the Registry of Standard Biological Parts on the World Wide Web at parts.igem.org.

[0390] As used herein, "regulatory protein" is a protein that regulates the expression of a target nucleic acid sequence. Regulatory proteins include, for example, transcription factors, including transcriptional activators and repressors, and proteins that bind to or modify transcription factors and affect their activity. In certain embodiments, regulator proteins include, for example, proteases that degrade protein factors involved in regulating the expression of target nucleic acid sequences. Preferred regulator proteins include modular proteins, wherein, for example, DNA binding elements and input agent binding elements or responsive elements or domains are separable and transferable, such that, for example, the fusion of the DNA binding domain of a first regulator protein with the input agent responsiveness domain of a second regulator protein produces a new protein that is sensitive to the input agent that is normally responded to the second protein and is combined with the DNA sequence recognized by the first protein. Therefore, as used herein, in addition to the specified polypeptide, the term "regulatory polypeptide" and more specifically "repressor polypeptide" include, for example, "LacI (repressor) polypeptide", variants, or derivatives of such polypeptides that respond to different or variant input agents. Therefore, for LacI polypeptides, LacI mutants or variants that are combined with agents other than lactose or IPTG are included. A wide range of such agents are known in the art.

[0391] Table 4. Exemplary regulatory switches. b On-switchability by effector; except for removal of the effector that confers the off-state. c Off-switchability by effector; except for removal of the effector that confers the on-state. d Ligand or other physical stimulus (e.g., temperature, electromagnetic radiation, electricity) that stabilizes the switch in its on or off state. e Refers to reference numbers cited in Kis et al., J. Royal Society Interface 12:20141000 (2015), both of which are hereby incorporated by reference in their entirety.

[0392] Table 4.

[0393]

[0394]

[0395]

[0396]

[0397] IV. Synthetic Production Methods

[0398] The methods and compositions provided herein are based, in part, on the discovery of synthetic and cell-free production processes and methods for producing single-stranded (ssDNA) molecules described herein in Section II or Section III. According to some embodiments, in the methods for producing ssDNA molecules, PS bonds replace the non-bridging oxygen in the phosphate backbone of the oligonucleotide with a sulfur atom. Advantageously, this modification stabilizes the nucleic acid and renders the internucleotide bond resistant to nuclease degradation.

[0399] The present disclosure provides methods for the synthetic production of single-stranded DNA (ssDNA) molecules described herein and double-stranded capped DNA (ceDNA) described herein for use in Part II or Part III.

[0400] According to some embodiments, the methods and / or production steps of the present disclosure are performed entirely in a cell-free environment. According to some embodiments, the methods and / or production steps of the present disclosure are performed in part in a cell-free environment. According to some embodiments, the dsDNA construct (e.g., linear double-stranded ceDNA) is synthetically produced in vitro. According to some embodiments, the dsDNA construct (e.g., double-stranded ceDNA) is synthetically produced in vitro in a cell-free environment.

[0401] According to some embodiments, the ssDNA molecule is synthetically produced in vitro. According to other embodiments, the ssDNA molecule is synthetically produced 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 other embodiments, the dsDNA construct comprises a double-stranded transgene cassette comprising at least one double-stranded transgene; a first ITR; and optionally, a second ITR. According to yet other embodiments, the dsDNA construct comprises a double-stranded transgene cassette comprising at least one double-stranded transgene; a first ITR; and a second ITR.

[0402] According to one aspect, the present disclosure provides a method for synthetically producing an ssDNA molecule described herein (e.g., a synthetic AAV vector, e.g., a single-stranded (ss) synthetic AAV vector) from a double-stranded DNA (dsDNA) construct, the 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 nicking 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. In some embodiments, the method does not include a purification step between steps a) and b). In other embodiments, the method does include a purification step between steps a) and b). An advantage of the present disclosure is that the method for synthetically producing ssDNA molecules does not include a purification step after step a), which reduces or minimizes the loss of DNA.

[0403] According to some embodiments, step a) comprises contacting the dsDNA construct with a single nicking endonuclease. According to further embodiments, the single nicking endonuclease produces a single nick on one of the single strands of the dsDNA construct. In other further embodiments, the single nicking endonuclease is Nb.BbvCI or an isoschizomer thereof, or the single nicking endonuclease is Nb.BtsI or an isoschizomer thereof.

[0404] According to some embodiments, the exonuclease is capable of removing the nicked strand of the dsDNA construct starting from one or more nicking sites and ending at one or more phosphorothioate-modified nucleotides. The exonuclease can be selected from, but is not limited to, T7 exonuclease, lambda exonuclease, T5 exonuclease, and exonuclease V. According to some embodiments, the exonuclease is T7 exonuclease.

[0405] According to some embodiments, step a) and step b) are performed simultaneously or sequentially in a single reaction vessel. According to some embodiments, when T7 exonuclease is used, step a) and step b) are performed sequentially due to the short degradation reaction of T7 exonuclease.

[0406] According to some embodiments, the one or more nicking 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 an isoschizomer of any of the foregoing. In some embodiments, the one or more nicking endonucleases comprise Nb.BbvCI or an isoschizomer thereof. In some embodiments, the one or more nicking endonucleases comprise Nb.BtsI or an isoschizomer thereof.

[0407] According to some embodiments, the one or more nicking sites are about 0 to about 20 nucleotides downstream of the terminal melting site (trs), for example, 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 terminal melting site (trs), or for example, about 0 to about 15, about 0 to 10, about 0 to 5, about 5 to 15, about 10 to 20, about 15 to 20, about 10 to 20, about 5 to 20 nucleotides downstream of the terminal melting site (trs). According to some embodiments, there is only one nicking site.

[0408] According to other aspects, the present disclosure provides a method for synthetically producing a dsDNA construct (e.g., ds-capped DNA) as described herein, 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 one double-stranded transgene; a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the double-stranded transgene cassette; and 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 cleavage site and the second cleavage site 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, optionally, a second ITR oligonucleotide to form a dsDNA construct.

[0409] The single-stranded DNA (ssDNA) molecules as described herein in Part II or Part III are superior to other vectors because 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 HEK 293). That is, since the resulting vectors do not contain bacterial or insect cell contaminants, the production of linear vectors by such cell-free methods can potentially minimize undesirable side effects. Synthetic production methods can also produce higher purity of the desired vectors. Synthetic production methods can also be more efficient and / or more cost-effective than traditional cell-based production methods for such vectors. Vectors synthesized as described herein can express any desired transgene, such as a transgene that treats or cures a given disease. One of ordinary skill in the art will readily recognize that any transgene used in conventional gene therapy methods with conventional recombinant vectors can be adapted for expression by single-stranded DNA (ssDNA) molecules and dsDNA constructs (e.g., ds ceDNA), such as those prepared by the methods described herein, particularly when not limited by the size capacity of the transgenic insert.

[0410] In the present disclosure, it should be understood that the production process of the present disclosure can be potentially carried out in a completely cell-free environment if necessary. However, depending on the starting material, some DNA components can be derived from nucleotide fragments (e.g., plasmid-ceDNA, AAV vectors produced by insect cells) initially prepared in cells. In certain embodiments, non-viral ssDNA can be prepared by introducing an incision at a desired position and with a desired length in an existing double-stranded ceDNA vector produced by cell replication (e.g., in an insect or mammalian cell line), wherein the existing double-stranded ceDNA vector has a design sequence of an incision endonuclease binding site at the stem of the ITR and comprises one or more phosphorothioate-modified nucleotides that form a PS bond in one or two ITRs. In other embodiments, single-stranded DNA (ssDNA) molecules (e.g., synthetic AAV vectors, e.g., single-stranded (ss) synthetic AAV vectors) are synthesized in a cell-free method by double-stranded ceDNA with a PS bond. As described herein, one result after adding a phosphorothioate bond is the stability of the ITR to ensure the accuracy of the position where the exonuclease works in the production of ssDNA.

[0411] Those skilled in the art will appreciate that one or more of the one or more enzymes or oligonucleotide components used in the synthetic production method can be produced by cells and used in the methods of the present disclosure in purified form. Thus, in some embodiments, the synthetic production method is a cell-free method, however, the restriction enzyme and / or ligase can be produced by cells.

[0412] In one embodiment, restriction endonucleases and / or proteins with ligation ability can be expressed or provided from expression vectors in cells, such as bacterial cells. In one embodiment, cells such as bacterial cells containing expression vectors expressing one or more restriction endonucleases or ligases may be present. Therefore, although the methods disclosed herein primarily relate to cell-free synthesis methods for producing ssDNA molecules disclosed herein, in some embodiments, it is also encompassed that cells, such as bacterial cells, are present, but insect cells are not present, and that one or more of the enzymes required for the expression method are synthesized and produced. In such embodiments, cells expressing restriction endonucleases and / or ligation ability proteins are not insect cells. In all embodiments in which cells are present and express one or more restriction endonucleases or ligation ability proteins, cells do not replicate single-stranded DNA (ssDNA) molecules (e.g., synthetic AAV vectors, e.g., single-stranded (ss) synthetic AAV vectors). In other words, the intracellular machinery of the cell does not replicate or participate in the replication of single-stranded DNA (ssDNA) molecules (e.g., synthetic AAV vectors, e.g., single-stranded (ss) synthetic AAV vectors).

[0413] A. Isolation and Purification

[0414] Described herein is a method for generating and separating single-stranded DNA (ssDNA) molecules (e.g., synthetic AAV vectors, e.g., single-stranded (ss) synthetic AAV vectors) and dsDNA constructs (e.g., dsceDNA). For example, single-stranded DNA (ssDNA) molecules and dsDNA constructs (e.g., dsceDNA) as described herein in Part II or Part III produced by synthetic methods as described herein can be harvested or collected at the appropriate time after the last ligation reaction, and can be optimized to achieve high yield production of vectors. ssDNA molecules and dsDNA constructs (e.g., dsceDNA) can be purified by any means for purifying DNA known to those skilled in the art. In one embodiment, ssDNA molecules or dsDNA constructs (e.g., dsceDNA) are purified into DNA molecules. Typically, any nucleic acid purification method as known in the art, and commercially available DNA extraction kits can be used.

[0415] Purification can be performed by subjecting the reaction mixture to chromatography. As a non-limiting example, the process can be performed by loading the reaction mixture onto an ion exchange column (e.g., SARTOBIND ) and then eluted (e.g., using 1.2 M NaCl solution) and further chromatographically purified on a gel filtration column (e.g., 6 fast flow GE). The DNA vector is then recovered by, for example, precipitation.

[0416] The presence of an ssDNA molecule or a dsDNA construct (e.g., dsceDNA) can be readily confirmed by digesting the vector DNA with a restriction enzyme that has a single recognition site for the DNA vector and analyzing the digested and undigested DNA material using gel electrophoresis to confirm the presence of characteristic bands of linear and continuous DNA compared to linear and non-continuous single-stranded DNA known in the art.

[0417] In some embodiments, the ssDNA molecules or dsDNA constructs can be delivered to target cells in vitro or in vivo by various suitable methods as discussed herein. The vector can be applied or injected alone. The vector can be delivered to the cell without the aid of a transfection agent or other physical means. Alternatively, the vector can be delivered using a transfection agent or other physical means that promotes DNA entry into the cell, such as liposomes, alcohols, polylysine-rich compounds, arginine-rich compounds, calcium phosphate, microvesicles, microinjections, and the like.

[0418] V. Gene Editing Applications

[0419] In some aspects, the present disclosure provides a gene editing system comprising an ssDNA molecule as described herein, at least one guide RNA (gRNA); and at least one site-specific nuclease or a messenger RNA (mRNA) encoding the at least one site-specific nuclease.

[0420] A. Nucleic acid-guided endonucleases

[0421] 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.

[0422] 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 complementary to a selected target DNA sequence, such that the RNA molecule binds to the selected sequence to guide the endonuclease activity to the selected target DNA sequence.

[0423] In one embodiment, the RNA-guided endonuclease is a CRISPR enzyme. In some embodiments, the RNA-guided endonuclease comprises a nicking enzyme activity. In some embodiments, the RNA-guided endonuclease guides the cutting of one or two chains at the position of the target sequence, such as within the target sequence and / or within the complementary sequence of the target sequence. In some embodiments, the RNA-guided endonuclease guides the cutting of one or two chains 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 nicking enzyme activity relates to one or more sequences on the ceDNA vector itself, for example, to loosen sequence constraints so that the HDR template is exposed to perform HDR interactions with the genomic sequence of the target gene.

[0424] In certain embodiments, it is envisioned that the nicking enzyme cuts at least 1 site, at least 2 sites, at least 3 sites, at least 4 sites, at least 5 sites, at least 6 sites, at least 7 sites, at least 8 sites, at least 9 sites, at least 10 sites or more on the desired nucleic acid sequence (e.g., one or more regions of an ssDNA molecule or a dsDNA construct). In another embodiment, it is envisioned that the nicking enzyme cuts at 1 and / or 2 sites via trans-nicking. Trans-nicking can enhance genome editing by high-fidelity HDR, introduce fewer errors, and therefore reduce unwanted off-target effects.

[0425] 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 mutated endonuclease lacks the ability to cleave one strand of a target polynucleotide containing a target sequence.

[0426] In certain embodiments, the nucleic acid sequence encoding the RNA-guided endonuclease is codon-optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells can be derived from specific organisms, such as mammals. Non-limiting examples of mammals can include people, mice, rats, rabbits, dogs, or non-human primates. Generally, codon optimization refers to replacing at least one codon (e.g., about or greater than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of a native sequence with a codon more frequently used or most frequently used in the gene of a host cell of interest to modify the nucleic acid sequence to enhance expression in the host cell while maintaining the process of the native amino acid sequence.

[0427] In some embodiments, the RNA-guided endonuclease is a part of a fusion protein comprising one or more heterologous protein domains (e.g., in addition to the endonuclease, about or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains). The RNA-guided endonuclease fusion protein can include any additional protein sequence, 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 having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity,...

Claims

1. 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) Lipids.

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

3. The LNP according to any one of claims 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. The LNP of any one of claims 1 to 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, or at least 5000 nucleotides in length. The nucleic acid sequence of the present invention may be a nucleic acid sequence of 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.

6. The LNP according to any one of claims 1 to 5, wherein the at least one nucleic acid sequence of interest is flanked at its 3' end by at least one stem-loop structure, wherein 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 initiate replication and / or transcription.

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

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

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

11. The LNP according to any one of claims 6 to 10, wherein the at least one loop at the 3' end comprises a minimum of 3 unbound nucleotides. 12 . The LNP according to claim 6 , wherein the ssDNA molecule comprises at least two stem-loop structures at the 3′ end.

13. The LNP according to 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 according to 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 according to 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 according to 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 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 comprise an A or A' region as 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 comprise an 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 comprise an A, A', B, B', C, C', D, or D' region 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 comprise 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 comprise a terminal melting site (trs) that would be present in a wild-type ITR.

21. The LNP according to any one of claims 6 to 20, wherein the ssDNA molecule does not comprise any viral-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 confer exonuclease resistance.

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 confer exonuclease resistance.

24. The LNP of claim 23, wherein the nucleotide modified to have exonuclease resistance 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. The LNP of any one of claims 25 or 26, wherein the at least one functional moiety is an aptamer.

28. The LNP of claim 27, wherein the aptamer is capable of nuclear translocation 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, wherein the at least one stem-loop structure at the 5' end comprises at least one stem and at least one loop.

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 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 comprise an A or A' region as 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 comprise an 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 comprise an A, A', B, B', C, C', D, or D' region 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 comprise 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 comprise a terminal melting site (trs) that would be 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 confer exonuclease resistance.

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 confer exonuclease resistance.

42. The LNP of claim 29, wherein the nucleotide modified to have exonuclease resistance 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. ​​The LNP of claim 45, wherein the at least one functional moiety is an aptamer.

47. The LNP of claim 46, wherein the aptamer is capable of nuclear translocation in a cell.

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

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

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

51. 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 the 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. The LNP of any one of claims 1 to 57, wherein the ssDNA molecule does not activate or minimally activates an immune pathway.

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

60. The LNP of claim 59, wherein the innate immune pathway is selected from the group consisting of a cGAS / STING pathway, a TLR9 pathway, an 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 the 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 to 62 or 64 to 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 ssDNA molecule is capable of expressing at least one therapeutic protein or therapeutic fragment thereof.

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 coagulation factor, a transcription factor, a replication factor, a growth factor, a hormone, and a fusion protein.

72. The LNP of any one of claims 70 or 71, wherein the at least one therapeutic protein is useful for treating a genetic disease 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 disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi's anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidoses (e.g.,Hurler syndrome (MPS type I), Scheie syndrome (MPS type I S), Hurler-Scheie syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo types A, B, C, and D (MPS types 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 (MPS type IX), Niemann-Pick Disease types A / B, C1, and C2, Fabry disease disease), Schindle disease, GM2-gangliosidosis type II (Sandhoff 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 (Pompedisease), Gaucher disease types I, II, and III, Fabry disease, cystinosis, Battendisease, aspartate glucosamineuria, sialic acid storage disease disease), Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipid storage diseases, galactosialidosis, 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 dystrophies (DMD),BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber congenital amaurosis, 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. A pharmaceutical composition comprising the LNP according to 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. The LNP of any one of claims 1 to 72 or 74, further comprising a sterol.

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

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

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

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

80. The LNP of claim 79, wherein the non-cationic lipid is selected from the group consisting of: distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (DPPC), OPC), 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), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1 -trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dieucoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), ditransoleoyl 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysolecithin, lysophatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine and mixtures thereof.

81. The LNP of any one of claims 79 or 80, wherein the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (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 LNP of claim 82, wherein the at least one PEGylated lipid is selected from the group consisting of: PEG-dilauryloxypropyl; PEG-dimyristoyloxypropyl; PEG-dipalmitoyloxypropyl, PEG-distearoyloxypropyl; 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (DMG-PEG); PEG-dilaurylglycerol; PEG-dipalmitoylglycerol; PEG-distearoylglycerol; PEG-dilaurylglycamide; PEG-dimyristyl Glycylamide; PEG-dipalmitoyl glycylamide; PEG-distearoyl glycylamide; (1-[8'-(cholest-5-en-3[β]-oxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol)) (PEG-cholesterol); 3,4-ditetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether (PEG-DMB); and 1,2-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 of any one of claims 82 to 84, wherein the at least one PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, or both.

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

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

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

90. The lipid nanoparticle of any one of claims 88 or 89, wherein the antibody, the antibody fragment or the antibody derivative is a scFv.

91. The LNP of any one of claims 86 to 90, wherein the tissue and / or cell type specific targeting moiety is covalently linked 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 tetraantennary GalNAc covalently linked to DSPE-PEG2000.

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

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

95. The LNP of any one of claims 93 or 94, wherein the ionizable lipid is selected from the group consisting of: 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Di-γ-linoleyloxy-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-Dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP); 1,2-Dioleoyl-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-[bis(3-amino-propyl)amino]butylcarboxamidoethyl]-3,4-di[oleyloxy]-benzamide (MVL5); dioctadecylamido-glycyl spermine (DOGS); 3b-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol); dioctadecyldimethylammonium bromide (DDAB); Saint lipids (e.g., SAINT-2,N-methyl-4-(dioleyl)methylpyridinium ion); 1,2-dimyristoyl Oxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORIE); 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI); dialkylated amino acids (DILA2) (e.g., C18:1-norArg-C16); dioleyldimethylammonium chloride (DODAC); 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC); and 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC).In some variations, the condensing agent, e.g., cationic lipid, is a lipid such as dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,31]-dioxolane (DLin-KC2-DMA), heptathriacontane-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), 1,2-dioleo ...2,2-dioleoyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,31]-dioxolane (DLin- propane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), morpholino cholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G) and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentane-1-ammonium 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 at a molar percentage of about 30% to about 80%.

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

98. The LNP of any one of claims 79 to 97, wherein the non-cationic lipid is present at a molar percentage 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 at a molar percentage 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 at a molar percentage 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 a 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 a 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 a 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 according to any one of claims 1 to 72 or 74 to 109, and a pharmaceutically acceptable excipient.

111. A method of treating a genetic disease in a subject, the method 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. The method of claim 111, wherein the subject is a human.

113. The method of any one of claims 111 or 112, wherein the genetic disease 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 disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Leschnehan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharidoses (e.g., Hurler syndrome (MPS type I), Shay syndrome (MPS type I S), Hurler-Shay syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo A, B, C, and D (MPS MPS III A, B, C, and D), Morquer A and B (MPS IVA and MPS IVB), Mallory-Lami syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPS Type IX), Niemann-Pick disease A / B, C1, and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff 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, aspartate glucosamineuria, sialidosis, Dannon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, 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 congenital amaurosis, 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. The method of any one of claims 111 to 113, wherein the genetic disease is hemophilia A.

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

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

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

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

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

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

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

122. The method of any one of claims 111 to 113, wherein the genetic disease 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 of delivering a therapeutic gene and / or therapeutic protein to a subject, the method comprising administering to the subject a therapeutically effective amount of the LNP according to any one of claims 1 to 72 or 74 to 109 or the pharmaceutical composition according to claim 73 or claim 110.

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

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

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

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

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

132. A method of 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 nucleic acid of interest encodes the therapeutic gene or the therapeutic protein.

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

134. The method of any one of claims 111 to 122, 126 to 129, or 131 to 133, wherein the dose of the ssDNA molecule administered to the subject is 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 dose of the ssDNA molecule administered to the subject is selected from the group consisting of 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, about 0.75 mg / kg, about 0.8 mg / kg, about 0.85 mg / kg, about 0.9 mg / kg, about 0.95 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, 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 mg / 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 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.25 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg kg, about 3.6 mg / kg, 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. The method of any one of claims 111 to 122, 126 to 129, or 131 to 135, wherein the dose of the ssDNA molecule administered to the subject is less than about 4.0 mg / kg.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

164. The method of any one of claims 111 to 122, 126 to 129, or 131 to 163, further comprising administering four or more doses of the LNP or the 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 flanked at its 3' end by at least one stem-loop structure, wherein the at least one stem-loop structure comprises at least one stem and at least one loop.

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

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 4-500 nucleotides.

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 4-5 nucleotides.

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

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 3 unbound nucleotides.

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. 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. 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. 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. 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 DNA structure, and a multi-branched loop structure.

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 comprise an A or A' region as would be present in a wild-type AAV ITR.

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 comprise an A, A', D, or D' region that would be present in a wild-type AAV ITR.

178. The ssDNA molecule of any one of claims 165 to 177, wherein the at least one stem-loop structure at the 3' end does not comprise the A, A', B, B', C, C', D, and D' regions that would be present in wild-type AAV ITRs.

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

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

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

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 confer exonuclease resistance.

183. The ssDNA molecule of any one of claims 165 to 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 confer exonuclease resistance.

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

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

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

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

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, wherein the at least one stem-loop structure at the 5' end comprises at least one stem and at least one loop.

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

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. 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. 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. 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 DNA structure, and a multi-branched loop structure.

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 comprise an A or A' region as would be present in a wild-type AAV ITR.

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 comprise an A, A', D, or D' region as would be present in a wild-type AAV ITR.

197. The ssDNA molecule of any one of claims 189 to 196, wherein the at least one stem-loop structure at the 5' end does not comprise the A, A', B, B', C, C', D, and D' regions that would be present in wild-type AAV ITRs.

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 comprise a rep binding element (RBE) that would be present in a wild-type ITR.

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

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 confer exonuclease resistance.

201. The ssDNA molecule of any one of claims 189 to 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 confer exonuclease resistance.

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

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

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

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

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

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

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

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

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

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

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. The ssDNA molecule of any one of claims 165 to 217, wherein the ssDNA molecule does not activate or minimally activates an immune pathway.

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

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

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

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

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

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

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

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

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

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

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

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

231. The ssDNA molecule of any one of claims 228 to 230, wherein the length of the double-stranded region comprising the promoter, the enhancer and / or the 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, 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, 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.

232. The ssDNA molecule of any one of claims 228 to 231, wherein the length of the double-stranded region comprising the promoter, the enhancer and / or the 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 5 less than 400 base pairs, less than 380 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 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 therapeutic fragment thereof.

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 coagulation factor, a transcription factor, a replication factor, a growth factor, a hormone, and a fusion protein.

253. The ssDNA molecule of any one of claims 251 or 252, wherein the at least one therapeutic protein is useful for treating a genetic disease 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 disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Leschnehan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidoses (e.g., Hurler syndrome (MPS type I), Shay syndrome (MPS type I S), Hurler-Shay syndrome (MPS type I HS), Hunter syndrome (MPS II), Sanfilippo A, B, C, and D (MPS III A, B, C, and D), Morque A and B (MPS IVA and MPS IVB), Mallory-Lami 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 (Sandhoff 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, aspartate glucosamineuria, sialidosis, Dannon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), LS), 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 congenital amaurosis, 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. The ssDNA molecule of any one of claims 165 to 253, further comprising a lipid.

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

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

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

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

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

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

263. A method of 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 ssDNA molecule of any one of claims 165 to 256 or the pharmaceutical composition of claim 257, wherein the nucleic acid of interest encodes the therapeutic gene or the therapeutic protein.

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

265. The LNP of any one of claims 68 to 109 or 264, wherein the length of the double-stranded region comprising the promoter, the enhancer and / or the 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, 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, 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.

266. The LNP of any one of claims 68 to 72, 74 to 109, or 263 to 265, wherein the length of the double-stranded region comprising the promoter, the enhancer, and / or the 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, base pairs, less than 400 base pairs, less than 380 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 17 0 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 to 72, 74 to 109, or 263 to 266, wherein the double-stranded region comprising the promoter, the enhancer, and / or the TSS is about 30-1500 base pairs in length.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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