Synthetic partially single-stranded nucleic acid compositions and uses and methods therefor

Abstract

The present application discloses partially single-stranded deoxyribonucleic acid (ssDNA) molecules comprising at least one nucleic acid sequence of interest flanked by a at least one stem loop structure, methods of making, and methods of use for delivery and expression of a transgene in host cells.

Description

[0001]SYNTHETIC PARTIALLY SINGLE-STRANDED NUCLEIC ACID COMPOSITIONS AND USES AND METHODS THEREFOR CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No.63 / 709,155, filed on October 18, 2024, the entire contents of which is hereby incorporated by reference in its entirety. BACKGROUND Vectors derived from adeno-associated viruses AAV (e.g., recombinant AAV (rAAV) or AAV vectors) are attractive for delivering genetic material because: (i) they are able to infect (“transduce”) a wide variety of dividing as well as non-dividing cell types such as myocytes and neurons; (ii) they are devoid of the virus structural genes, thereby diminishing the host cell responses to virus infection, e.g., interferon-mediated responses; (iii) wild-type AAVs are considered non- pathologic in humans; and (iv) in contrast to wild type AAVs, which are capable of integrating into the host cell genome, replication-deficient AAV vectors lack the rep gene and generally persist as an episome, thus greatly limiting the risk of insertional mutagenesis or genotoxicity. However, there are several major drawbacks and deficiencies in using AAV particles as a gene delivery vector that stems from conventional AAV production from host cells. One major problem associated with rAAV is its limited viral packaging capacity of about 4.5 kb of heterologous DNA (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010). As a result, the use of AAV vectors has been limited to less than 150 kDa protein coding capacity due to this limitation in viral packaging. A second problem is related to the capsid immunogenicity that prevents re-administration to patients. The immune system in the patients can respond to the vector which effectively acts as a booster to stimulate the immune system generating high titer anti-AAV antibodies that preclude future treatments. Some recent reports indicate concerns with immunogenicity in high dose situations. Another notable problem is that production of AAV in host cells (e.g., insect cells) on a large scale for the manufacture of the viral genome results in a random mixture of plus (+) and minus (-) stranded vectors. This significantly decreases the strand specificity of a transgene for the much-needed therapeutic expression of the sense strand. Additionally, conventional AAV virions with capsids are produced by introducing a plasmid or plasmids containing the AAV genome, rep genes, and cap genes (Grimm et al., 1998). However, such encapsidated AAV virus vectors were found to inefficiently transduce certain cell and tissue types, and the capsids were also found to induce a severe immune response in hosts. Further, the methods of producing such AAV vectors have relied greatly upon traditional insect cell-dependent production methods (e.g., Sf9). AAV vector produced using such methods can include contaminants from the cells used to produce the vectors, which are inconvenient or costly to remove or purify away, and which may induce undesirable side effects if included in therapeutic formulations. Accordingly, there is a strong need in the gene therapy field for a therapeutic vector that is minimally immunogenic and redosable, and that allows for the large-scale production of recombinant vectors that increase transgene expression level, strand specificity, and purity while increasing the capacity of a transgene size. SUMMARY According to some aspects, the disclosure provides isolated partially single-stranded deoxyribonucleic acid (ssDNA) molecules comprising: (a) a central region, comprising at least one single-stranded region and at least one double-stranded region, wherein the central region comprises at least one nucleic acid sequence of interest; and (b) at least a first stem-loop structure at its 3’ end, wherein the first stem-loop structure comprises at least one stem and at least one loop, and / or at least a second stem-loop structure at its 5’ end, wherein the second stem-loop structure comprises at least one stem and at least one loop, wherein the first stem-loop structure and / or the second stem-loop structure flank the central region, and wherein the central region comprises a total of at least 50 base pairs (bp) of double-stranded DNA, and / or wherein the central region is at least 5% double-stranded, and wherein the partially ssDNA molecule comprises at least one hybridized oligonucleotide. In some embodiments, the ssDNA molecule comprises at least two hybridized, noncovalently-bound oligonucleotides. In some embodiments, the ssDNA molecule comprises at least three hybridized, noncovalently-bound oligonucleotides. In some embodiments, the ssDNA molecule comprises at least four hybridized, noncovalently-bound oligonucleotides. In some embodiments, the ssDNA molecule comprises at least five hybridized, noncovalently-bound oligonucleotides. In some embodiments, each oligonucleotide is less than 200 nucleotides in length. In some embodiments, each oligonucleotide is less than 100 nucleotides in length. In some embodiments, each oligonucleotide is less than 88 nucleotides in length. In some embodiments, each oligonucleotide is less than 45 nucleotides in length. In some embodiments, the at least one double-stranded region in the central region comprises one or more gaps and / or one or more nicks in at least one strand. In some embodiments the at least one double-stranded region in the central region comprises one or more gaps, and wherein the one or more gaps is one or more nucleotides long. In some embodiments, the at least one double stranded region in the central region comprises two or more gaps, and wherein the two or more gaps have the same length. In some embodiments, the at least one double stranded regions in the central region comprises two or more gaps, and wherein the two or more gaps have different length. In some embodiments, the at least one oligonucleotide is hybridized adjacent to the 3’ end of the ssDNA molecule with a gap between the oligonucleotide and the 3’end of the ssDNA molecule. In some embodiments, the at least one oligonucleotide is hybridized adjacent to the 5’ end of the ssDNA molecule with a gap between the oligonucleotide and the 5’end of the ssDNA molecule. In some embodiments, the at least one double-stranded region in the central region comprises no gaps or nicks in either strand. In some embodiments, the at least one oligonucleotide is ligated to the ssDNA. In some embodiments, the at least one oligonucleotide is ligated to the 3’end of the ssDNA. In some embodiments, the at least one oligonucleotide is ligated to the 5’end of the ssDNA. In some embodiments, the at least one oligonucleotide comprises one or more modified nucleotides. In some embodiments, the at least one oligonucleotide comprises one or more modified nucleotides at the 3’ end. In some embodiments, the at least one oligonucleotide comprises one or more modified nucleotides at the 5’ end. In some embodiments, the one or more modified nucleotides are phosphorothioate-modified (PS) nucleotides, 2’-O-methyl nucleotides, 2’-Fluoro nucleotides, or a combination thereof. In some embodiments, the at least one double-stranded region comprises a binding site for a protein. In some embodiments, the at least one oligonucleotide comprises a binding site for a protein. In some embodiments, the protein is an enzyme or a chaperone. In some embodiments, the protein is an enzyme and wherein the enzyme is a Cas enzyme. In some embodiments, the Cas enzyme is dead Cas9. In some embodiments, the protein is an enzyme and wherein the enzyme is a recombinase. In some embodiments, the at least one oligonucleotide comprises one or more functional moieties. In some embodiments, the functional moieties are selected from the group consisting of an aptamer, an antisense oligonucleotide (ASO), a ribozyme, a short- interfering RNA (siRNA), a benzylguanine (BG), a fluorophore, and a peptide. In some embodiments, the functional moiety is an aptamer, and wherein the aptamer is capable of nuclear translocation in a cell. In some embodiments, the ssDNA molecule comprises one or more hybridized oligonucleotides in the central region, and where the one or more oligonucleotides comprise at least one aptamer. In some embodiments, the ssDNA molecule comprises two or more hybridized oligonucleotides in the central region, and wherein each of the two or more oligonucleotides comprise an aptamer. In some embodiments, the ssDNA molecule comprises a gapped aptamer array. In some embodiments, the ssDNA molecule comprises a ligated aptamer array. In some embodiments, the aptamers have the same sequence. In some embodiments, the aptamers have different sequences. In some embodiments, one or more of the aptamers comprises a modified nucleotide. In some embodiments, at least one of the stem-loop structures at the 5’ and / or 3’ ends is sufficient to prime replication and / or transcription. In some embodiments, at least one of the stems at the 5’ and / or 3’ ends comprises a partial DNA duplex of about 4-1500 bp. In some embodiments, at least one of the stems at the 5’ and / or 3’ ends comprises a partial DNA duplex of about 4-10 nucleotides. In some embodiments, at least one of the loops at the 5’ and / or 3’ ends comprises about 3-500 single-stranded nucleotides. In some embodiments, each of the loops at the 5’ and / or 3’ ends comprise a minimum of 3 single-stranded nucleotides. In some embodiments, the ssDNA molecule comprises at least two stem-loop structures at each of the 5’ and / or 3’ ends. In some embodiments, the ssDNA molecule comprises at least three stem-loop structures at each of the 5’ and / or 3’ ends. In some embodiments, the ssDNA molecule comprises at least four or more stem-loop structures at each of the 5’ and / or 3’ ends. In some embodiments, at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises a hairpin DNA structure. In some embodiments, at least one of the stem- loop structures at the 5’ and / or 3’ ends comprises a DNA structure selected from the group consisting of: a cruciform DNA structure, a hammerhead DNA structure, a quadraplex DNA structure, a bulged DNA structure, and a multibranched loop structure. In some embodiments, at least one of the stem- loop structures at the 5’ and / or 3’ ends comprises a viral ITR or partial viral ITR. In some embodiments, at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises an AAV ITR or partial AAV ITR. In some embodiments, at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises an AAV2 ITR or partial AAV2 ITR. In some embodiments, at least one of the stem- loop structures at the 5’ and / or 3’ ends comprises a wild-type AAV ITR. In some embodiments, at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises an ITR modified by at least one deletion, addition, and / or substitution relative to a wild-type AAV ITR. In some embodiments, at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises at least one AAV ITR region selected from the group consisting of an A region, an A’ region, a B region, a B’ region, a C, a C’ region, a D region, and a D’ region. In some embodiments, at least one of the stem-loop structures at the 5’ and / or 3’ ends does not comprise the A or A’ regions that would be present in a wild-type AAV ITR. In some embodiments, at least one of the stem-loop structures at the 5’ and / or 3’ ends does not comprise the A, A’, D, or D’ regions that would be present in a wild-type AAV ITR. In some embodiments, at least one of the stem-loop structures at the 5’ and / or 3’ ends does not comprise the A, A’, B, B’, C, C’, D, or D’ regions that would be present in a wild-type AAV ITR. In some embodiments, at least one of the stem-loop structures at the 5’ and / or 3’ ends does not comprise a rep binding element (RBE) that would be present in a wild-type AAV ITR. In some embodiments, at least one of the stem-loop structures at the 5’ and / or 3’ ends does not comprise a terminal resolution site (trs) that would be present in a wild-type ITR. In some embodiments, the ssDNA molecule does not comprise any viral sequences. In some embodiments, at least one of the stems at the 5’ and / or 3’ ends of the ssDNA molecule comprises one or more nucleotides that are modified to be exonuclease resistant. In some embodiments, the 5’ and / or 3’ ends of the ssDNA molecule comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more nucleotides that are modified to be exonuclease resistant. In some embodiments, the nucleotides that are modified to be exonuclease resistant are phosphorothioate-modified (PS) nucleotides. In some embodiments, at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises at least one functional moiety. In some embodiments, the at least one functional moiety is selected from the group consisting of an aptamer, an antisense oligonucleotide (ASO), a ribozyme, a short-interfering RNA (siRNA), a benzylguanine (BG), a fluorophore, and a peptide. In some embodiments, the aptamer is capable of nuclear translocation in a cell. In some embodiments, the ssDNA molecule comprises at least one stem-loop structure at each of its 5’ and 3’ ends, wherein each of the at least one stem-loop structures at the 5’ and 3’ ends comprises at least one stem and at least one loop. In some embodiments, at least one of the loops at the 5’ and / or 3’ ends further comprises one or more nucleic acids that stabilize the ends. In some embodiments, at least one of the loops at the 5’ and / or 3’ ends further comprises one or more nucleic acids that are chemically modified. In some embodiments, at least one of the loops at the 5’ end and / or 3’ ends further comprises one or more triplex forming oligonucleotides. In some embodiments, at least one of the loops at the 5’ and / or 3’ ends further comprises one or more gRNAs or gDNAs. In some embodiments, at least one of the loops at the 5’ and / or 3’ end further comprises one or more molecular probes. In some embodiments, the ssDNA molecule is devoid of any viral capsid protein coding sequences. In some embodiments, the ssDNA molecule is synthetically produced in vitro. In some embodiments, the ssDNA molecule is synthetically produced in vitro in a cell-free environment. In some embodiments, the ssDNA molecule does not activate or minimally activates an immune pathway. In some embodiments, the immune pathway is an innate immune pathway. In some embodiments, the innate immune pathway is selected from the group consisting of the cGAS / STING pathway, the TLR9 pathway, an inflammasome- mediated pathway, and combinations thereof. In some embodiments, the ssDNA molecule does not induce, or minimally induces, at least one cytokine selected from the group consisting of IFNα, IFNγ, IL-6, TNFα, and IL-18, as compared to a closed-ended deoxyribonucleic acid (ceDNA) molecule. In some embodiments, the central region of the ssDNA molecule comprises at least one promoter. In some embodiments, the promoter is a cell type- or tissue-specific promoter. In some embodiments, the promoter is specific for a cell type or tissue selected from the group consisting of liver, skeletal muscle, smooth muscle, photoreceptors, retinal cells, hematopoietic stem cells (HSCs), T cells, B cells, Natural Killer (NK) cells, dendritic cells, megakaryocytes, neurons, brain, lung, heart, kidney, liver, spleen, pancreas, islet cells, prostate, testis, ovary, uterus, thyroid, thymus, adipose cells, epithelial cells, endothelial cells, bone cells, keratinocytes, fibroblasts, and salivary gland. In some embodiments, the promoter is a liver-specific promoter. In some embodiments, the promoter is a TTR promoter. In some embodiments, the promoter is a hAAT promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the central region of ssDNA molecule comprises at least one enhancer. In some embodiments, the enhancer comprises a serpin (SERP) enhancer. In some embodiments, the enhancer comprises a human SERP enhancer. In some embodiments, the enhancer comprises a 1X human SERP enhancer, a 2X human SERP enhancer, or a 3X human SERP enhancer. In some embodiments, the enhancer comprises a SERP enhancer with at least 95% sequence identity a human SERP enhancer. In some embodiments, the enhancer comprises a BB SERP enhancer comprising nucleotides 2749-2818 and / or 3068-3137 of the sequence set forth in FIG.93. In some embodiments, the enhancer comprises a 1X BB SERP enhancer, a 2X BB SERP enhancer, or a 3X BB SERP enhancer. In some embodiments, the ssDNA molecule comprises a TTR promoter and a SERP enhancer. In some embodiments, the promoter comprises a transcription start site (TSS). In some embodiments, the promoter is double-stranded. In some embodiments, the enhancer is double-stranded. In some embodiments, the TSS is double-stranded. In some embodiments, the central region comprises a total of 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, at least 1500 base pairs, at least 1600 base pairs, at least 1700 base pairs, at least 1800 base pairs, at least 1900 base pairs, at least 2000 base pairs, at least 2100 base pairs, at least 2200 base pairs, at least 2300 base pairs, at least 2400 base pairs, or at least 2500 base pairs of double-stranded DNA. In some embodiments, the central region comprises a total of fewer than 2500 base pairs, fewer than 2400 base pairs, fewer than 2300 base pairs, fewer than 2200 base pairs, fewer than 2100 base pairs, fewer than 2000 base pairs, fewer than 1900 base pairs, fewer than 1800 base pairs, fewer than 1700 base pairs, fewer than 1600 base pairs, fewer than 1500 base pairs, fewer than 1400 base pairs, fewer than 1300 base pairs, fewer than 1200 base pairs, fewer than 1100 base pairs, fewer than 1000 base pairs, fewer than 950 base pairs, fewer than 900 base pairs, fewer than 850 base pairs, fewer than 800 base pairs, fewer than 750 base pairs, fewer than 700 base pairs, fewer than 650 base pairs, fewer than 600 base pairs, fewer than 550 base pairs, fewer than 500 base pairs, fewer than 480 base pairs, fewer than 460 base pairs, fewer than 440 base pairs, fewer than 420 base pairs, fewer than 400 base pairs, fewer than 380 base pairs, fewer than 360 base pairs, fewer than 340 base pairs, fewer than 320 base pairs, fewer than 300 base pairs, fewer than 280 base pairs, fewer than 260 base pairs, fewer than 240 base pairs, fewer than 220 base pairs, fewer than 200 base pairs, fewer than 190 base pairs, fewer than 180 base pairs, fewer than 170 base pairs, fewer than 160 base pairs, fewer than 150 base pairs, fewer than 140 base pairs, fewer than 130 base pairs, fewer than 120 base pairs, fewer than 110 base pairs, fewer than 100 base pairs, fewer than 90 base pairs, fewer than 80 base pairs, fewer than 70 base pairs, or fewer than 60 base pairs of double-stranded DNA. In some embodiments, the central region comprises a total of about 50-2500 base pairs of double-stranded DNA. In some embodiments, the central region comprises a total of about 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800- 1900, 1900-2000, 2000-2100, 2100-2200, 2200-2300, 2300-2400, or 2400-2500 base pairs of double- stranded DNA. In some embodiments, the central region comprises 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, at least 1500 base pairs, at least 1600 base pairs, at least 1700 base pairs, at least 1800 base pairs, at least 1900 base pairs, at least 2000 base pairs, at least 2100 base pairs, at least 2200 base pairs, at least 2300 base pairs, at least 2400 base pairs, or at least 2500 base pairs of double-stranded DNA at the 3’ end of the ssDNA molecule. In some embodiments, the central region comprises fewer than 2500 base pairs, fewer than 2400 base pairs, fewer than 2300 base pairs, fewer than 2200 base pairs, fewer than 2100 base pairs, fewer than 2000 base pairs, fewer than 1900 base pairs, fewer than 1800 base pairs, fewer than 1700 base pairs, fewer than 1600 base pairs, fewer than 1500 base pairs, fewer than 1400 base pairs, fewer than 1300 base pairs, fewer than 1200 base pairs, fewer than 1100 base pairs, fewer than 1000 base pairs, fewer than 950 base pairs, fewer than 900 base pairs, fewer than 850 base pairs, fewer than 800 base pairs, fewer than 750 base pairs, fewer than 700 base pairs, fewer than 650 base pairs, fewer than 600 base pairs, fewer than 550 base pairs, fewer than 500 base pairs, fewer than 480 base pairs, fewer than 460 base pairs, fewer than 440 base pairs, fewer than 420 base pairs, fewer than 400 base pairs, fewer than 380 base pairs, fewer than 360 base pairs, fewer than 340 base pairs, fewer than 320 base pairs, fewer than 300 base pairs, fewer than 280 base pairs, fewer than 260 base pairs, fewer than 240 base pairs, fewer than 220 base pairs, fewer than 200 base pairs, fewer than 190 base pairs, fewer than 180 base pairs, fewer than 170 base pairs, fewer than 160 base pairs, fewer than 150 base pairs, fewer than 140 base pairs, fewer than 130 base pairs, fewer than 120 base pairs, fewer than 110 base pairs, fewer than 100 base pairs, fewer than 90 base pairs, fewer than 80 base pairs, fewer than 70 base pairs, or fewer than 60 base pairs of double-stranded DNA at the 3’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 50 base pairs of double- stranded DNA at 3’ end of the ssDNA molecule and at least 50 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region is at least about 50% single-stranded, at least about 51% single-stranded, at least about 52% single-stranded, at least about 53% single-stranded, at least about 54% single-stranded, at least about 55% single-stranded, at least about 56% single-stranded, at least about 57% single-stranded, at least about 58% single-stranded, at least about 59% single-stranded, at least about 60% single-stranded, at least about 61% single- stranded, at least about 62% single-stranded, at least about 63% single-stranded, at least about 64% single-stranded, at least about 65% single-stranded, at least about 66% single-stranded, at least about 67% single-stranded, at least about 68% single-stranded, at least about 69% single-stranded, at least about 70% single-stranded, at least about 71% single-stranded, at least about 72% single-stranded, at least about 73% single-stranded, at least about 74% single-stranded, at least about 75% single- stranded, at least about 76% single-stranded, at least about 77% single-stranded, at least about 78% single-stranded, at least about 79% single-stranded, at least about 80% single-stranded, at least about 81% single-stranded, at least about 82% single-stranded, at least about 83% single-stranded, at least about 84% single-stranded, at least about 85% single-stranded, at least about 86% single-stranded, at least about 87% single-stranded, at least about 88% single-stranded, at least about 89% single- stranded, at least about 90% single-stranded, at least about 91% single-stranded, at least about 92% single-stranded, at least about 93% single-stranded, at least about 94% single-stranded, or at least about 95% single-stranded. In some embodiments, the central region is less than about 50% single- stranded, less than about 51% single-stranded, less than about 52% single-stranded, less than about 53% single-stranded, less than about 54% single-stranded, less than about 55% single-stranded, less than about 56% single-stranded, less than about 57% single-stranded, less than about 58% single- stranded, less than about 59% single-stranded, less than about 60% single-stranded, less than about 61% single-stranded, less than about 62% single-stranded, less than about 63% single-stranded, less than about 64% single-stranded, less than about 65% single-stranded, less than about 66% single- stranded, less than about 67% single-stranded, less than about 68% single-stranded, less than about 69% single-stranded, less than about 70% single-stranded, less than about 71% single-stranded, less than about 72% single-stranded, less than about 73% single-stranded, less than about 74% single- stranded, less than about 75% single-stranded, less than about 76% single-stranded, less than about 77% single-stranded, less than about 78% single-stranded, less than about 79% single-stranded, less than about 80% single-stranded, less than about 81% single-stranded, less than about 82% single- stranded, less than about 83% single-stranded, less than about 84% single-stranded, less than about 85% single-stranded, less than about 86% single-stranded, less than about 87% single-stranded, less than about 88% single-stranded, less than about 89% single-stranded, less than about 90% single- stranded, less than about 91% single-stranded, less than about 92% single-stranded, less than about 93% single-stranded, less than about 94% single-stranded, or less than about 95% single-stranded. In some embodiments, the central region is about 50%-95% single-stranded. In some embodiments, the single-stranded region of the central region is on a (-) strand of the ssDNA molecule. In some embodiments, the single-stranded region of the central region is on a (+) strand of the ssDNA molecule. In some embodiments, the nucleic acid sequence of interest encodes at least one molecule selected from the group consisting of a peptide, a polypeptide, a ribozyme, a peptide nucleic acid (PNA), an siRNA, an RNAi, an antisense oligonucleotide, an shRNA, a micro-RNA, an mRNA, a gRNA, and an antagoMiR. In some embodiments, the nucleic acid sequence of interest encodes an antibody or antigen-binding fragment thereof. In some embodiments, the nucleic acid sequence of interest comprises at least one open-reading frame (ORF). In some embodiments, the ssDNA molecule is capable of expressing at least one therapeutic protein or a therapeutic fragment thereof. In some embodiments, 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 some embodiments, the at least one therapeutic protein is useful for treating a genetic disorder selected from the group consisting of sickle cell disease, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited disorders of hepatic metabolism, Lesch-Nyhan syndrome, thalassaemias, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharide storage diseases (e.g., Hurler syndrome (MPS Type I), Scheie syndrome (MPS Type I S), Hurler-Scheie syndrome (MPS Type I H-S), 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), Tay-Sachs disease, Metachromatic Leukodystrophy, Krabbe disease, Mucolipidosis Type I, II / III and IV, Sialidosis Types I and II, Glycogen Storage disease Types I and II (Pompe disease), Gaucher disease Types I, II and III, cystinosis, Batten disease, Aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), Lysosomal Acid Lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipidoses, 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 (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. In some embodiments, the at least one therapeutic protein is Factor VIII (FVIII). In some embodiments, the at least one nucleic acid sequence of interest comprises at least one region selected from the group consisting of a spacer, a 5’ untranslated region (UTR), a 3’ UTR, an intron, and a polyA signal. In some embodiments, rein the at least one nucleic acid sequence of interest comprises a spacer. In some embodiments, the spacer is located between the at least one stem-loop structure at the 3’ end and an enhancer, and / or between the at least one stem-loop structure at the 3’ end and a promoter. In some embodiments, the spacer is at least partially double-stranded and comprises at least about 100, 200, 300, 400, 500, or 600 base pairs. In some embodiments, the at least one nucleic acid sequence of interest comprises a 5’ UTR. In some embodiments, the at least one nucleic acid sequence of interest comprises a 3’ UTR. In some embodiments, the at least one nucleic acid sequence of interest comprises an intron. In some embodiments, the at least one nucleic acid sequence of interest comprises a polyA signal. In some embodiments, the at least one nucleic acid sequence of interest comprises a promoter, an enhancer, a TSS, an ORF, a 5’ UTR, a 3’ UTR, an intron, and a polyA signal. In one aspect, provided herein are compositions comprising the ssDNA molecule of any of the above embodiments and a lipid. In some embodiments, the ssDNA molecule is encapsulated in the lipid. Also provided herein are lipid nanoparticles comprising the ssDNA molecule of any of the above embodiments; pharmaceutical compositions comprising the lipid nanoparticles and a pharmaceutically acceptable excipient; pharmaceutical compositions comprising the ssDNA molecule of any of the above embodiments and a pharmaceutically acceptable excipient; and cells comprising the ssDNA molecule of any of the above embodiments. In some embodiments, the cells are in vitro, ex vivo, or in vivo. In one aspect, provided herein are methods of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the ssDNA molecule of any of the above embodiments, the composition any of the above embodiments, the lipid nanoparticle of any of the above embodiments, the pharmaceutical composition of any of the above embodiments, or the cell of any of the above embodiments. In some embodiments, the disease or disorder is selected from the group consisting of sickle cell disease, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited disorders of hepatic metabolism, Lesch-Nyhan syndrome, thalassaemias, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharide storage diseases (e.g., Hurler syndrome (MPS Type I), Scheie syndrome (MPS Type I S), Hurler-Scheie syndrome (MPS Type I H- S), 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), Tay-Sachs disease, Metachromatic Leukodystrophy, Krabbe disease, Mucolipidosis Type I, II / III and IV, Sialidosis Types I and II, Glycogen Storage disease Types I and II (Pompe disease), Gaucher disease Types I, II and III, cystinosis, Batten disease, Aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), Lysosomal Acid Lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipidoses, 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 (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. In one aspect, provided herein are methods of delivering a therapeutic gene and / or a therapeutic protein to a subject comprising administering to the subject a therapeutically effective amount of the ssDNA molecule of any of the above embodiments, the composition any of the above embodiments, the lipid nanoparticle of any of the above embodiments, the pharmaceutical composition of any of the above embodiments, or the cell of any of the above embodiments. In one aspect, provided herein are methods of delivering a therapeutic gene and / or a therapeutic protein to a cell comprising contacting the cell with the ssDNA molecule of any of the above embodiments, the composition of any of the above embodiments, the lipid nanoparticle of any of the above embodiments, the pharmaceutical composition of any of the above embodiments, or the cell of any of the above embodiments, thereby delivering the therapeutic gene and / or therapeutic protein to the cell. In one aspect, provided herein are method of delivering a therapeutic gene to the nucleus of a cell comprising contacting the cell with the ssDNA molecule of any of the above embodiments, the composition of any of the above embodiments, the lipid nanoparticle of any of the above embodiments, the pharmaceutical composition of any of the above embodiments, or the cell of any of the above embodiments, thereby delivering the therapeutic gene and / or therapeutic protein to the nucleus of the cell. In one aspect, provided herein are methods of minimizing an immune response in a subject, wherein the subject is being treated with a therapeutic gene or a therapeutic protein, comprising administering to the subject a therapeutically effective amount of the ssDNA molecule of any of the above embodiments, the composition of any of the above embodiments, the lipid nanoparticle of any of the above embodiments, the pharmaceutical composition of any of the above embodiments, or the cell of any of the above embodiments, wherein the nucleic acid of interest encodes the therapeutic gene or therapeutic protein. In one aspect, provided herein are methods for producing a second partially single-stranded deoxyribonucleic acid (ssDNA) molecule, the method comprising: (a) providing a first ssDNA molecule and an oligonucleotide complementary to a portion of the first ssDNA molecule, wherein the first ssDNA molecule comprises: (1) a central region, comprising at least one single-stranded region and at least one double-stranded region, wherein the central region comprises at least one nucleic acid sequence of interest; and (2) at least a first stem-loop structure at its 3’ end, wherein the first stem-loop structure comprises at least one stem and at least one loop, and / or at least a second stem-loop structure at its 5’ end, wherein the second stem-loop structure comprises at least one stem and at least one loop, wherein the first stem-loop structure and / or the second stem-loop structure flank the central region, and wherein the central region comprises a total of at least 50 base pairs (bp) of double-stranded DNA, and / or wherein the central region is at least 5% double-stranded. (b) allowing the oligonucleotide to hybridize to the first ssDNA molecule; (c) delivering the first ssDNA molecule and the hybridized oligonucleotide to a cell; and (d) allowing the hybridized oligonucleotide to extend using the first ssDNA molecule as a template, thereby generating a second ssDNA molecule. In some embodiments, the oligonucleotide is complementary to a portion of the single-stranded region within the central region of the first ssDNA molecule. In some embodiments, the oligonucleotide is complementary to a portion of the double-stranded region within the central region of the first ssDNA molecule. In some embodiments, the central region of the first ssDNA molecule comprises a promoter and wherein the oligonucleotide is complementary to a portion of the first ssDNA molecule upstream of the promoter. BRIEF DESCRIPTION OF DRAWINGS Embodiments of the present disclosure, briefly summarized above and discussed in greater detail below, can be understood by reference to the illustrative embodiments of the disclosure depicted in the appended drawings. However, the appended drawings illustrate only typical embodiments of the disclosure and are therefore not to be considered limiting of scope, for the disclosure may admit to other equally effective embodiments. FIG.1A shows that starting from double stranded ceDNA construct 10429, ssDNA vector was obtained by ssDNA endonuclease treatment. As can be seen in FIG.1A, a clear ~830 bp band was seen for ssRES ss360 control, and there was a clear degradation of ΦX174 ssDNA control. There was a loss of full sized ssRES ss429 in the well and appearance of a band that migrated at ~100 bp that is more prominent / clear in the 5U Mung Bean nuclease treatment. FIG.1B depicts schematics of the double stranded ceDNA 10429 construct (“ds429”) and the single stranded 10429 and 10360. FIG.2 shows Klenow exonuclease fill-in of ssDNA vector molecule supported the existence of 3’-OH. FIG.3 depicts schematic drawings of symmetric and asymmetric ITR oligos. The top drawing shows symmetric overhangs. The bottom drawing shows asymmetric overhangs, where the 3’ end of the left ITR has PS bonds (closer to the 3’ end of the molecule) and the right ITR has PS bonds shifted to the right by two bases. FIG.3 discloses SEQ ID NOs: 11-14, respectively, in order of appearance. FIG.4 depicts a schematic drawing of single stranded AAV synthetic vector hAAT-luciferase left and right ITR sequences with symmetric ITR oligos from constructs ss10429 and ss10483. As shown in FIG.4, a CpG free spacer was included that alters some nucleotides in the RBE of the A / A’ stem. The nick site Nb.BbvCI was engineered downstream of the terminal resolution site (trs). FIG.4 discloses SEQ ID NOs: 11-12, respectively, in order of appearance. FIG.5 depicts a schematic drawing of single stranded AAV synthetic vector hAAT-luciferase left and right ITR sequences with asymmetric ITR oligos from construct ss10485. As shown in FIG. 5, a CpG free spacer was included that alters some nucleotides in the RBE of the A / A’ stem. The nick site Nb.BbvCI was engineered downstream of the trs. FIG.5 discloses SEQ ID NOs: 13-14, respectively, in order of appearance. FIG.6 depicts a schematic drawing of single stranded AAV synthetic vector FVIII left and right ITR sequences symmetric ITR oligos from construct ss10491. The nick site Nb.BbvCI was engineered downstream of the trs. FIG.6 discloses SEQ ID NOs: 15 and 12, respectively, in order of appearance. FIG.7 depicts a schematic drawing of single stranded AAV synthetic vector FVIII left and right ITR sequences asymmetric ITR oligos from construct ss10484. The nick site Nb.BbvCI was engineered downstream of the trs. FIG.7 discloses SEQ ID NOs: 16 and 14, respectively, in order of appearance. FIG.8 is a graph that shows HepG2 cells transfected with single stranded AAV synthetic vector express luciferase. A clear dose response was seen from ceDNA and the single stranded AAV synthetic vector. FIG.9 is a graph that shows gel-extracted and Zymo column purified single stranded AAV synthetic vector induced lower innate immune responses compared to ceDNA. single stranded AAV synthetic vector induced less Lucia IFN reporter than ceDNA in WT THP1 cells at matched molecule doses. FIG.10 is a graph that shows gel-extracted and Zymo column purified single stranded AAV synthetic vector induced lower innate immune responses compared to ceDNA. In cGAS KO cells IFN response was present at highest ceDNA doses and column purified single stranded AAV synthetic vector, but otherwise was absent indicating cGAS sensing single stranded AAV synthetic vector and ceDNA. FIG.11 is a graph that shows interim longitudinal body weight in single stranded synthetic DNA (ssDNA) “SSD” (40004) dosed animals and double stranded (ds) ceDNA (10541) dosed animals. FIG.12 is a panel of graphs that show interim longitudinal body weight in single stranded synthetic DNA (ssDNA) “SSD” (40004) dosed animals and double stranded (ds) ceDNA (10541) dosed animals for each of the doses. FIG.13 shows the results of IVIS imaging performed on Day 4. FIG.14 shows the results of IVIS imaging performed on Days 4-7. FIG.15 shows the nucleic acid sequence of double-stranded (ds) ceDNA construct 10360 (SEQ ID NO: 1). As noted in FIG.15, the Factor VIII ORF is located at nucleotides 828-5219 of SEQ ID NO: 1. FIG.16 shows the nucleic acid sequence of ds ceDNA construct 10429 (SEQ ID NO: 2). As noted in FIG.16, the luciferase ORF is located at nucleotides 1449-3101 of SEQ ID NO: 2. FIG.17 shows the nucleic acid sequence of ds ceDNA construct 10483 (SEQ ID NO: 3). As noted in FIG.17, the luciferase ORF is located at nucleotides 1449-3101 of SEQ ID NO: 3. FIG.18 shows the nucleic acid sequence of ds ceDNA construct 10484 (SEQ ID NO: 4). As noted in FIG.18, the Factor VIII ORF is located at nucleotides 835-5226 of SEQ ID NO: 4. FIG.19 shows the nucleic acid sequence of ds ceDNA construct 10485 (SEQ ID NO: 5). As noted in FIG.19, the luciferase ORF is located at nucleotides 1449-3101 of SEQ ID NO: 5. FIG.20 shows the nucleic acid sequence of ds ceDNA construct 10491 (SEQ ID NO: 6). As noted in FIG.20, the Factor VIII ORF is located at nucleotides 835-5226 of SEQ ID NO: 6. FIG.21 shows the nucleic acid sequence of ds ceDNA construct 10376 (SEQ ID NO: 7). As noted in FIG.21, the luciferase ORF is located at nucleotides 1443-3095 of SEQ ID NO: 7. FIG.22 shows the nucleic acid sequence of ds ceDNA construct 10541 (SEQ ID NO: 8). As noted in FIG.22, the luciferase ORF is located at nucleotides 1449-3101 of SEQ ID NO: 8. FIG.23 shows the nucleic acid sequence of plasmid 210150 (SEQ ID NO: 9) which comprises one of the exemplary ds ceDNA sequences. As noted in FIG.23, the Factor VIII ORF is located at nucleotides 894-5285. FIG.24 shows the nucleic acid sequence of single stranded (ss) DNA construct 40004 (SEQ ID NO: 10). As noted in FIG.24, the luciferase ORF reverse complement is located at nucleotides 503-2155 of SEQ ID NO: 10. FIG.25 is a panel of graphs that show interim longitudinal body weight in single stranded synthetic DNA (ssDNA) “SSD” (ssDNA construct 40004, designated “ss004”) dosed animals and double stranded (ds) ceDNA (ds ceDNA construct 10541, designated “ds541”) dosed animals for five different doses matched by molecule number. FIG.26 is a panel of graphs that show weight loss over 5 days in single stranded synthetic DNA (ssDNA) “SSD” (40004) dosed animals and double stranded (ds) ceDNA (10541) dosed animals. As shown in FIG.26, the difference in BW reduction was most apparent at day 2, when animals that were dosed with ssDNA rapidly regained lost weight. FIG.27 is a panel of graphs showing the effect of ssDNA (construct 40004, designated “ss004”) and ds ceDNA (construct 10541, designated “ds541”) on cytokine expression after 6 hours. ds ceDNA construct 10376 (“376”) served as a positive control). The figure legend indicates whether PS bonds were present in the construct (“w / PS”) or absent (“no PS”). FIG.28 is a panel of graphs from a second experiment showing the effect of ssDNA (construct 40004, designated “ss004”) and ds ceDNA (construct 10541, designated “ds541”) on cytokine expression after 6 hours. ds ceDNA construct 10376 (“376”) served as a positive control). The figure legend indicates whether PS bonds were present in the construct (“w / PS”) or absent (“no PS”). FIG.29 is a graph that shows that ds ceDNA (construct 10541, designated “ds541”) and ss DNA (construct 40004, designated “ss004”) had similar levels of mRNA in the liver, as determined by qtPCR. FIG.30 is a panel 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 given intravenous injection of LNP-formulated mRNA, ceDNA, or ssDNA. Blood levels (in pg / mL) of IFN-α, IL-18, TNF-α, IL-6, and IFN-γ were measured 6 hours after injection. Several batches of ssDNA produced at scale for later use in non-human primates (NHP) were also tested. Groups in each graph, left to right: PBS control, mRNA (2.0 mg / kg), ceDNA (2.0 mg / kg), ssDNA Batch B1 (2.0 mg / kg), ssDNA Batch B1 (NHP scale; 0.5 mg / kg), ssDNA Batch B1 (NHP scale; 2.0 mg / kg), ssDNA Batch C (NHP scale; 0.5 mg / kg), ssDNA Batch C (NHP scale; 2.0 mg / kg). FIG.31 is a panel of graphs showing the effect of 1.0 mg / kg LNP-formulated ceDNA (“DNA”, circles), ssDNA (squares), and mRNA (triangles) cargoes on blood cytokine levels (in pg / mL) 6 hours and 24 hours after intravenous infusion into Cynomolgus monkeys. FIG.32 shows the levels of complement activation (left: C3a; right: C5b-9) measured in NHPs after administration of 1.0 mg / mL LNP-formulated ceDNA (circles), ssDNA (squares), or mRNA (triangles). FIG.33 shows the results of an in vitro PBMC assay. Human peripheral blood mononuclear cells (PBMCs) were contacted with LNP-formulated ceDNA or ssDNA, and TNF-α stimulation was measured. In both a first study (two left-hand bars) and a second study (three right-hand bars, which include an untreated control), ssDNA induced significantly lower TNF-α stimulation as compared to compared to ceDNA (ssDNA-induced TNF-α stimulation was comparable to the untreated control). FIG.34 is a panel of graphs showing durable and robust expression from LNP-formulated ssDNA. Mice were given intravenous injection of 0.25 mg / kg LNP-formulated ceDNA or ssDNA comprising a luciferase reporter gene. Luciferase expression (IVIS) was measured at one and four days post-injection (right), as well as at additional time points up to 30 days (left). FIGs.35A-35E depict schematics of ssDNA constructs having different components. All constructs comprise an hAAT-luciferase expression construct. FIG.35A: construct ss004 (left ITR derived from AAV2, with PS bonds on the bottom strand and an Nb.BbvCI nick site; right ITR derived from AAV2, with most of the A region removed, and with PS bonds at the 5’ end; expression construct minus strand). FIG.35B: construct ss020 (identical to ss004, but with no PS bonds). FIG. 35C: construct ss021 (identical to ss004, but with wild-type AAV2 ITRs, no PS bonds, and no Nb.BbvCI nick site). FIG.35D: construct ss022 (similar to ss021, except with the expression construct plus strand). FIG.35E: hAAT-luciferase expression construct with no ITR sequences. FIG.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 (normalized to cell viability) was measured at 48 hours. FIG.37 is a panel of graphs depicting the results of in vivo luciferase expression by ssDNA constructs in mice after hydrodynamic injection (HDI). Mice were injected with either ceDNA541 (5 ng or 500 ng per animal), constructs ss004, ss020, ss021, ss022, or ss011 (500 ng per animal), or a mixture of ss021+ss022 (250 ng per animal) or ss011+ss022 (250 ng / animal). IVIS luciferase expression is shown at day 1 (top left) and day 4 (bottom left), as well as a longitudinal plot (top right). FIG.38 is a graph depicting the results of in vivo luciferase expression in mice injected with LNP-formulated ssDNA constructs. Results are shown for, left to right, PBS control (upside-down triangles), ss011 (diamonds, ionizable lipid MC3), ss011 (open circles, ionizable lipid Y), ss011 (squares, ionizable lipid Z), and ss004 (ionizable lipid Z). FIGs.39A-39D depict schematics of ssDNA constructs having different PS bond configurations. All constructs comprise an hAAT-luciferase expression construct. The location of PS bonds is indicated by arrows. FIG.39A: construct ss004 (left ITR derived from AAV2, with PS bonds on the bottom strand; right ITR derived from AAV2, with most of the A region removed, and with PS bonds at the 5’ end). FIG.39B: construct 034 (identical to ss004, but PS bonds in the left ITR are on the top strand, near the 3’ end). FIG.39C: construct ss039 (identical to ss034, but right side has truncated ITR and has PS bonds on the bottom strand near the 5’ end). FIG.39D: construct ss040 (identical to ss039, but with no PS bonds). FIG.40 is a panel of graphs depicting the results of in vivo luciferase expression in mice injected with LNP-formulated ssDNA constructs. Mice were injected with ceDNA541, ceDNA654, ss004, ss034, ss039, or ss040 (0.25 mg / kg). IVIS luciferase expression is shown at day 1 (top left) and day 4 (top right), as well as a longitudinal plot (bottom). FIG.41 is a panel 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 given intravenous injection of 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, left to right: PBS control, ceDNA541, ceDNA654, ss004, ss034, ss039, and ss040. FIGs.42A-42D depict schematics of ssDNA constructs having single-stranded or double- stranded promoter regions. All constructs comprise a luciferase reporter. FIG.43A: ss004, single- stranded hAAT enhancer / promoter set. FIG.43B: ss104, double-stranded hAAT enhancer / promoter set. FIG.43C: ss102, single-stranded 1xSERP / TTR enhancer / promoter set. FIG.43D: ss104, double-stranded 1xSERP / TTR enhancer / promoter set. For all constructs in this figure, the length of the double-stranded region was counted using the alternate double-stranded counting method. FIG.43 is a panel of graphs measuring luciferase expression in mice injected via HDI with ssDNA constructs having single-stranded (ss004, ss102) or double-stranded (ss104, ss103) promoter regions. Left: luciferase expression at day 1. Middle: luciferase expression at day 7. Right: longitudinal expression from day 1 to day 7. FIG.44 is a panel of graphs showing the fold change in expression for ssDNA constructs with double-stranded promoter regions (ss104, ss103, respectively), as compared to the ssDNA constructs with single-stranded promoter regions (ss004, ss102, respectively). Left: day 1. Middle: day 7. Right: longitudinal fold change from day 1 to day 7. FIG.45 is a panel of graphs measuring luciferase expression in mice injected via HDI with ssDNA constructs having single-stranded (ss004, ss102) or double-stranded (ss104, ss103) promoter regions, as compared to expression from ceDNA541. Left: luciferase expression at day 1. Right: luciferase expression at day 7. FIG.46 shows the nucleic acid sequence of ssDNA construct ss104 (SEQ ID NO: 41). ss104 is 4980 nt in length and comprises 1381 bp of double-stranded DNA adjacent to the 3’ end. ss104 comprises a luciferase ORF (nt 503-2155) and a double-stranded liver-specific hAAT promoter. FIG.47 shows the nucleic acid sequence of ssDNA construct ss102 (SEQ ID NO: 42). ss102 is 3257 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss102 comprises a luciferase ORF (nt 992-2644) and a single-stranded liver-specific SERP / TTR promoter. FIG.48 shows the nucleic acid sequence of ssDNA construct ss103 (SEQ ID NO: 43). ss103 is 3718 nt in length and comprises 504 bp of double-stranded DNA adjacent to the 3’ end. ss103 comprises a luciferase ORF (nt 992-2644) and a double-stranded liver-specific SERP / TTR promoter. FIG.49 shows the nucleic acid sequence of ssDNA construct ss105 (SEQ ID NO: 44). ss105 is 2762 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss105 comprises a luciferase ORF (nt 503-2155) and a single-stranded liver-specific SERP / TTR promoter. FIG.50 shows the nucleic acid sequence of ssDNA construct ss106 (SEQ ID NO: 45). ss106 is 3234 nt in length and comprises 504 bp of double-stranded DNA adjacent to the 3’ end. ss106 comprises a luciferase ORF (nt 503-2155) and a double-stranded liver-specific SERP / TTR promoter. FIG.51 shows the nucleic acid sequence of ssDNA construct ss145 (SEQ ID NO: 46). ss145 is 3255 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss145 comprises a luciferase ORF (nt 990-2642) and a single-stranded liver-specific SERP / TTR promoter. FIG.52 shows the nucleic acid sequence of ssDNA construct ss146 (SEQ ID NO: 47). ss146 is 3287 nt in length and comprises 75 bp of double-stranded DNA adjacent to the 3’ end. ss146 comprises a luciferase ORF (nt 990-2642) and a partially double-stranded liver-specific SERP / TTR promoter. FIG.53 shows the nucleic acid sequence of ssDNA construct ss147 (SEQ ID NO: 48). ss147 is 3317 nt in length and comprises 106 bp of double-stranded DNA adjacent to the 3’ end. ss147 comprises a luciferase ORF (nt 990-2642) and a partially double-stranded liver-specific SERP / TTR promoter. FIG.54 shows the nucleic acid sequence of ssDNA construct ss148 (SEQ ID NO: 49). ss148 is 3390 nt in length and comprises 179 bp of double-stranded DNA adjacent to the 3’ end. ss148 comprises a luciferase ORF (nt 990-2642) and a partially double-stranded liver-specific SERP / TTR promoter. FIG.55 shows the nucleic acid sequence of ssDNA construct ss149 (SEQ ID NO: 50). ss149 is 3438 nt in length and comprises 217 bp of double-stranded DNA adjacent to the 3’ end. ss149 comprises a luciferase ORF (nt 990-2642) and a partially double-stranded liver-specific SERP / TTR promoter. FIG.56 shows the nucleic acid sequence of ssDNA construct ss150 (SEQ ID NO: 51). ss150 is 3526 nt in length and comprises 314 bp of double-stranded DNA adjacent to the 3’ end. ss150 comprises a luciferase ORF (nt 990-2642) and a partially double-stranded liver-specific SERP / TTR promoter. FIG.57 shows the nucleic acid sequence of ssDNA construct ss151 (SEQ ID NO: 52). ss151 is 3625 nt in length and comprises 413 bp of double-stranded DNA adjacent to the 3’ end. ss151 comprises a luciferase ORF (nt 990-2642) and a partially double-stranded liver-specific SERP / TTR promoter. FIG.58 shows the nucleic acid sequence of ssDNA construct ss152 (SEQ ID NO: 53). ss152 is 3644 nt in length and comprises 432 bp of double-stranded DNA adjacent to the 3’ end. ss152 comprises a luciferase ORF (nt 990-2642) and a partially double-stranded liver-specific SERP / TTR promoter. FIG.59 shows the nucleic acid sequence of ssDNA construct ss153 (SEQ ID NO: 54). ss153 is 3716 nt in length and comprises 504 bp of double-stranded DNA adjacent to the 3’ end. ss153 comprises a luciferase ORF (nt 990-2642) and a double-stranded liver-specific SERP / TTR promoter. FIG.60 shows the nucleic acid sequence of ssDNA construct ss154 (SEQ ID NO: 55). ss154 is 3255 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss154 comprises a luciferase ORF (nt 571-2223, (+) strand) and a single-stranded liver-specific SERP / TTR promoter. FIG.61 shows the nucleic acid sequence of ssDNA construct ss155 (SEQ ID NO: 56). ss155 is 3716 nt in length and comprises 504 bp of double-stranded DNA adjacent to the 3’ end. ss155 comprises a luciferase ORF (nt 571-2223, (+) strand) and a single-stranded liver-specific SERP / TTR promoter. FIG.62 shows the nucleic acid sequence of ssDNA construct ss004 (SEQ ID NO: 57). ss004 is 3635 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss004 comprises a luciferase ORF (nt 503-2155) and a single-stranded liver-specific hAAT promoter. FIG.63 shows the nucleic acid sequence of ssDNA construct ss156 (SEQ ID NO: 58). ss156 is 3667 nt in length and comprises 75 bp of double-stranded DNA adjacent to the 3’ end. ss156 comprises a luciferase ORF (nt 503-2155) and a partially double-stranded liver-specific hAAT promoter. FIG.64 shows the nucleic acid sequence of ssDNA construct ss157 (SEQ ID NO: 59). ss157 is 3698 nt in length and comprises 106 bp of double-stranded DNA adjacent to the 3’ end. ss157 comprises a luciferase ORF (nt 503-2155) and a partially double-stranded liver-specific hAAT promoter. FIG.65 shows the nucleic acid sequence of ssDNA construct ss158 (SEQ ID NO: 60). ss158 is 3771 nt in length and comprises 179 bp of double-stranded DNA adjacent to the 3’ end. ss158 comprises a luciferase ORF (nt 503-2155) and a partially double-stranded liver-specific hAAT promoter. FIG.66 shows the nucleic acid sequence of ssDNA construct ss159 (SEQ ID NO: 61). ss159 is 3809 nt in length and comprises 217 bp of double-stranded DNA adjacent to the 3’ end. ss159 comprises a luciferase ORF (nt 503-2155) and a partially double-stranded liver-specific hAAT promoter. FIG.67 shows the nucleic acid sequence of ssDNA construct ss160 (SEQ ID NO: 62). ss160 is 3906 nt in length and comprises 314 bp of double-stranded DNA adjacent to the 3’ end. ss160 comprises a luciferase ORF (nt 503-2155) and a partially double-stranded liver-specific hAAT promoter. FIG.68 shows the nucleic acid sequence of ssDNA construct ss161 (SEQ ID NO: 63). ss161 is 3998 nt in length and comprises 406 bp of double-stranded DNA adjacent to the 3’ end. ss161 comprises a luciferase ORF (nt 503-2155) and a partially double-stranded liver-specific hAAT promoter. FIG.69 shows the nucleic acid sequence of ssDNA construct ss162 (SEQ ID NO: 64). ss162 is 4025 nt in length and comprises 433 bp of double-stranded DNA adjacent to the 3’ end. ss162 comprises a luciferase ORF (nt 503-2155) and a partially double-stranded liver-specific hAAT promoter. FIG.70 shows the nucleic acid sequence of ssDNA construct ss163 (SEQ ID NO: 65). ss163 is 4096 nt in length and comprises 504 bp of double-stranded DNA adjacent to the 3’ end. ss163 comprises a luciferase ORF (nt 503-2155) and a partially double-stranded liver-specific hAAT promoter. FIG.71 shows the nucleic acid sequence of ssDNA construct ss164 (SEQ ID NO: 66). ss164 is 4479 nt in length and comprises 887 bp of double-stranded DNA adjacent to the 3’ end. ss164 comprises a luciferase ORF (nt 503-2155) and a partially double-stranded liver-specific hAAT promoter. FIG.72 shows the nucleic acid sequence of ssDNA construct ss165 (SEQ ID NO: 67). ss165 is 4838 nt in length and comprises 1246 bp of double-stranded DNA adjacent to the 3’ end. ss165 comprises a luciferase ORF (nt 503-2155) and a partially double-stranded liver-specific hAAT promoter. FIG.73 shows the nucleic acid sequence of ssDNA construct ss183 (SEQ ID NO: 68). ss183 is 5994 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss183 comprises a Factor VIII ORF (nt 990-5381) and a single-stranded liver-specific SERP / TTR promoter. FIG.74 shows the nucleic acid sequence of ssDNA construct ss184 (SEQ ID NO: 69). ss184 is 6455 nt in length and comprises 504 bp of double-stranded DNA adjacent to the 3’ end. ss184 comprises a Factor VIII ORF (nt 990-5381) and a double-stranded liver-specific SERP / TTR promoter. FIG.75 shows the nucleic acid sequence of ssDNA construct ss012 (SEQ ID NO: 70). ss012 is 6244 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss012 comprises a Factor VIII ORF (nt 987-5378) and a single-stranded liver-specific 3X SERP / TTR promoter. FIG.76 shows the nucleic acid sequence of ssDNA construct ss182 (SEQ ID NO: 71). ss182 is 6959 nt in length and comprises 758 bp of double-stranded DNA adjacent to the 3’ end. ss182 comprises a Factor VIII ORF (nt 987-5378) and a double-stranded liver-specific 3X SERP / TTR promoter. FIG.77 shows the nucleic acid sequence of ssDNA construct ss224 (SEQ ID NO: 72). ss224 is 3237 nt in length and comprises 57 bp of double-stranded DNA adjacent to the 3’ end. ss224 comprises a luciferase ORF (nt 971-2623) and a single-stranded liver-specific SERP / TTR promoter. ss224 further comprises simple hairpin ends devoid of virally-derived ITR sequences. FIG.78 shows the nucleic acid sequence of ssDNA construct ss225 (SEQ ID NO: 73). ss225 is 3711 nt in length and comprises 531 bp of double-stranded DNA adjacent to the 3’ end. ss225 comprises a luciferase ORF (nt 971-2623) and a double-stranded liver-specific SERP / TTR promoter. ss225 further comprises simple hairpin ends devoid of virally-derived ITR sequences. FIG.79 shows the nucleic acid sequence of ssDNA construct ss065 (SEQ ID NO: 74). ss065 is 3619 nt in length and comprises 57 bp of double-stranded DNA adjacent to the 3’ end. ss065 comprises a luciferase ORF (nt 495-2147) and a single-stranded liver-specific hAAT promoter. ss065 further comprises simple hairpin ends devoid of virally-derived ITR sequences. FIG.80 shows the nucleic acid sequence of ssDNA construct ss181 (SEQ ID NO: 75). ss181 is 4965 nt in length and comprises 1404 bp of double-stranded DNA adjacent to the 3’ end. ss181 comprises a luciferase ORF (nt 494-2146) and a double-stranded liver-specific hAAT promoter. ss181 further comprises simple hairpin ends devoid of virally-derived ITR sequences. FIG.81 shows the nucleic acid sequence of ssDNA construct ss205 (SEQ ID NO: 76). ss205 is 2712 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss205 comprises a luciferase ORF (nt 579-2231) and a single-stranded constitutive RSV promoter. FIG.82 shows the nucleic acid sequence of ssDNA construct ss206 (SEQ ID NO: 77). ss206 is 3041 nt in length and comprises 372 bp of double-stranded DNA adjacent to the 3’ end. ss206 comprises a luciferase ORF (nt 579-2231) and a double-stranded constitutive RSV promoter. FIG.83 shows the nucleic acid sequence of ssDNA construct ss209 (SEQ ID NO: 78). ss209 is 2579 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss209 comprises a luciferase ORF (nt 579-2231) and a single-stranded constitutive MP-135 promoter. FIG.84 shows the nucleic acid sequence of ssDNA construct ss210 (SEQ ID NO: 79). ss210 is 2775 nt in length and comprises 239 bp of double-stranded DNA adjacent to the 3’ end. ss210 comprises a luciferase ORF (nt 579-2231) and a double-stranded constitutive MP-135 promoter. FIG.85 shows the nucleic acid sequence of ssDNA construct ss201 (SEQ ID NO: 80). ss201 is 3062 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss201 comprises a luciferase ORF (nt 579-2231) and a single-stranded constitutive CMV promoter. FIG.86 shows the nucleic acid sequence of ssDNA construct ss202 (SEQ ID NO: 81). ss202 is 3741 nt in length and comprises 722 bp of double-stranded DNA adjacent to the 3’ end. ss202 comprises a luciferase ORF (nt 579-2231) and a double-stranded constitutive CMV promoter. FIG.87 shows the nucleic acid sequence of ssDNA construct ss139 (SEQ ID NO: 82). ss139 is 3511 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss139 comprises a luciferase ORF (nt 579-2231) and a single-stranded constitutive CMV / chimeric intron promoter. FIG.88 shows the nucleic acid sequence of ssDNA construct ss203 (SEQ ID NO: 83). ss203 is 4152 nt in length and comprises 684 bp of double-stranded DNA adjacent to the 3’ end. ss203 comprises a luciferase ORF (nt 579-2231) and a partially double-stranded constitutive CMV / chimeric intron promoter with a double-stranded (internal) transcription start site (TSS). FIG.89 shows the nucleic acid sequence of ssDNA construct ss204 (SEQ ID NO: 84). ss204 is 4639 nt in length and comprises 1171 bp of double-stranded DNA adjacent to the 3’ end. ss204 comprises a luciferase ORF (nt 579-2231) and a double-stranded constitutive CMV / chimeric intron promoter. FIG.90 shows the nucleic acid sequence of ssDNA construct ss207 (SEQ ID NO: 85). ss207 is 2529 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss207 comprises a luciferase ORF (nt 579-2231) and a single-stranded constitutive MP-84 promoter. FIG.91 shows the nucleic acid sequence of ssDNA construct ss208 (SEQ ID NO: 86). ss208 is 2675 nt in length and comprises 189 bp of double-stranded DNA adjacent to the 3’ end. ss208 comprises a luciferase ORF (nt 579-2231) and a double-stranded constitutive MP-84 promoter. FIG.92 shows the nucleic acid sequence of ssDNA construct ss216 (SEQ ID NO: 87). ss216 is 3007 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss216 comprises a luciferase ORF (nt 990-2642) and a single-stranded liver-specific 1xBBEnh-SCP1 promoter. FIG.93 shows the nucleic acid sequence of ssDNA construct ss217 (SEQ ID NO: 88). ss217 is 3220 nt in length and comprises 256 bp of double-stranded DNA adjacent to the 3’ end. ss217 comprises a luciferase ORF (nt 990-2642) and a double-stranded liver-specific 1xBBEnh-SCP1 promoter. FIG.94 shows the nucleic acid sequence of ssDNA construct ss218 (SEQ ID NO: 89). ss218 is 3254 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss218 comprises a luciferase ORF (nt 990-2642) and a single-stranded liver-specific hSERP / FOXA / HNF4- TTR promoter. FIG.95 shows the nucleic acid sequence of ssDNA construct ss219 (SEQ ID NO: 90). ss219 is 3714 nt in length and comprises 503 bp of double-stranded DNA adjacent to the 3’ end. ss219 comprises a luciferase ORF (nt 990-2642) and a double-stranded liver-specific hSERP / FOXA / HNF4- TTR promoter. FIG.96 shows the nucleic acid sequence of ssDNA construct ss222 (SEQ ID NO: 91). ss222 is 3345 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss222 comprises a luciferase ORF (nt 990-2642) and a single-stranded liver-specific HNF4 / 1_X1476-TTR promoter. FIG.97 shows the nucleic acid sequence of ssDNA construct ss223 (SEQ ID NO: 92). ss223 is 3896 nt in length and comprises 594 bp of double-stranded DNA adjacent to the 3’ end. ss223 comprises a luciferase ORF (nt 990-2642) and a double-stranded liver-specific HNF4 / 1_X1476-TTR promoter. FIG.98 shows the nucleic acid sequence of ssDNA construct ss214 (SEQ ID NO: 93). ss214 is 3305 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss214 comprises a luciferase ORF (nt 990-2642) and a single-stranded liver-specific SV40Enh-hAlb promoter. FIG.99 shows the nucleic acid sequence of ssDNA construct ss215 (SEQ ID NO: 94). ss215 is 3816 nt in length and comprises 554 bp of double-stranded DNA adjacent to the 3’ end. ss215 comprises a luciferase ORF (nt 990-2642) and a double-stranded liver-specific SV40Enh-hAlb promoter. FIG.100 shows the nucleic acid sequence of ssDNA construct ss220 (SEQ ID NO: 95). ss220 is 3252 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss220 comprises a luciferase ORF (nt 990-2642) and a single-stranded liver-specific 1xBBEnh-TTR promoter. FIG.101 shows the nucleic acid sequence of ssDNA construct ss221 (SEQ ID NO: 96). ss221 is 3710 nt in length and comprises 501 bp of double-stranded DNA adjacent to the 3’ end. ss221 comprises a luciferase ORF (nt 990-2642) and a double-stranded liver-specific 1xBBEnh-TTR promoter. FIG.102 shows the nucleic acid sequence of ssDNA construct ss097 (SEQ ID NO: 97). ss097 is 5815 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss097 comprises a Factor VIII ORF (nt 564-4955) and a single-stranded liver-specific 3xBBEnh / TTR promoter. ss097 is CpG-minimized, comprising 0 CpG motifs. FIG.103 shows the nucleic acid sequence of ssDNA construct ss092 (SEQ ID NO: 98). ss092 is 5815 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss092 comprises a FVIII ORF (nt 564-4955) and a single-stranded liver-specific 3xBBEnh / TTR promoter. ss092 is CpG-minimized, comprising 19 CpG motifs. ss092 further has one of the eight naturally- occurring Nb.BbvCI nick sites in the FVIII ORF ablated. FIG.104 shows the nucleic acid sequence of ssDNA construct ss244 (SEQ ID NO: 99). ss244 is 3312 nt in length and comprises 100 bp of double-stranded DNA adjacent to the 3’ end. ss244 comprises a luciferase ORF (nt 571-2223, (+) strand) and a single-stranded liver-specific SERP / TTR promoter. FIG.105 shows the nucleic acid sequence of ssDNA construct ss245 (SEQ ID NO: 100). ss245 is 3412 nt in length and comprises 200 bp of double-stranded DNA adjacent to the 3’ end. ss245 comprises a luciferase ORF (nt 571-2223, (+) strand) and a single-stranded liver-specific SERP / TTR promoter. FIG.106 shows the nucleic acid sequence of ssDNA construct ss246 (SEQ ID NO: 101). ss246 is 3512 nt in length and comprises 300 bp of double-stranded DNA adjacent to the 3’ end. ss246 comprises a luciferase ORF (nt 571-2223, (+) strand) and a single-stranded liver-specific SERP / TTR promoter. FIG.107 shows the nucleic acid sequence of ssDNA construct ss247 (SEQ ID NO: 102). ss247 is 3612 nt in length and comprises 400 bp of double-stranded DNA adjacent to the 3’ end. ss247 comprises a luciferase ORF (nt 571-2223, (+) strand) and a single-stranded liver-specific SERP / TTR promoter. FIG.108 shows the nucleic acid sequence of ssDNA construct ss248 (SEQ ID NO: 103). ss248 is 3712 nt in length and comprises 500 bp of double-stranded DNA adjacent to the 3’ end. ss248 comprises a luciferase ORF (nt 571-2223, (+) strand) and a single-stranded liver-specific SERP / TTR promoter. FIG.109 shows the nucleic acid sequence of ssDNA construct ss249 (SEQ ID NO: 104). ss249 is 3812 nt in length and comprises 600 bp of double-stranded DNA adjacent to the 3’ end. ss249 comprises a luciferase ORF (nt 571-2223, (+) strand) and a single-stranded liver-specific SERP / TTR promoter. FIG.110 shows the nucleic acid sequence of ssDNA construct ss250 (SEQ ID NO: 105). ss250 is 3255 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 5’ end. ss250 comprises a luciferase ORF (nt 1033-2685) and a single-stranded liver-specific SERT / TTR promoter. FIG.111 shows the nucleic acid sequence of ssDNA construct ss251 (SEQ ID NO: 106). ss251 is 3712 nt in length and comprises 500 bp of double-stranded DNA adjacent to the 5’ end. ss251 comprises a luciferase ORF (nt 1490-3142) and a single-stranded liver-specific SERP / TTR promoter. FIG.112 shows the nucleic acid sequence of ssDNA construct ss252 (SEQ ID NO: 107). ss252 is 3750 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss252 comprises a luciferase ORF (nt 990-2642) and a single-stranded liver-specific SERP / TTR promoter. FIG.113 shows the nucleic acid sequence of ssDNA construct ss253 (SEQ ID NO: 108). ss253 is 3807 nt in length and comprises 100 bp of double-stranded DNA adjacent to the 3’ end. ss253 comprises a luciferase ORF (nt 990-2642), a single-stranded liver-specific SERP / TTR promoter, and a 495 bp spacer sequence in front of the promoter. FIG.114 shows the nucleic acid sequence of ssDNA construct ss254 (SEQ ID NO: 109). ss254 is 3907 nt in length and comprises 200 bp of double-stranded DNA adjacent to the 3’ end. ss254 comprises a luciferase ORF (nt 990-2642), a single-stranded liver-specific SERP / TTR promoter, and a 495 bp spacer sequence in front of the promoter. FIG.115 shows the nucleic acid sequence of ssDNA construct ss255 (SEQ ID NO: 110). ss255 is 4007 nt in length and comprises 300 bp of double-stranded DNA adjacent to the 3’ end. ss255 comprises a luciferase ORF (nt 990-2642), a single-stranded liver-specific SERP / TTR promoter, and a 495 bp spacer sequence in front of the promoter. FIG.116 shows the nucleic acid sequence of ssDNA construct ss256 (SEQ ID NO: 111). ss256 is 4107 nt in length and comprises 400 bp of double-stranded DNA adjacent to the 3’ end. ss256 comprises a luciferase ORF (nt 990-2642), a single-stranded liver-specific SERP / TTR promoter, and a 495 bp spacer sequence in front of the promoter. FIG.117 shows the nucleic acid sequence of ssDNA construct ss257 (SEQ ID NO: 112). ss257 is 4207 nt in length and comprises 500 bp of double-stranded DNA adjacent to the 3’ end. ss257 comprises a luciferase ORF (nt 990-2642), a single-stranded liver-specific SERP / TTR promoter, and a 495 bp spacer sequence in front of the promoter. FIG.118 shows the nucleic acid sequence of ssDNA construct ss258 (SEQ ID NO: 113). ss258 is 4307 nt in length and comprises 600 bp of double-stranded DNA adjacent to the 3’ end. ss258 comprises a luciferase ORF (nt 990-2642), a single-stranded liver-specific SERP / TTR promoter, and a 495 bp spacer sequence in front of the promoter. FIG.119 shows the nucleic acid sequence of ssDNA construct ss259 (SEQ ID NO: 114). ss259 is 3750 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 5’ end. ss259 comprises a luciferase ORF (nt 1109-2761, (+) strand), a single-stranded liver-specific SERP / TTR promoter, and a 495 bp spacer sequence in front of the promoter. FIG.120 shows the nucleic acid sequence of ssDNA construct ss260 (SEQ ID NO: 115). ss260 is 4207 nt in length and comprises 500 bp of double-stranded DNA adjacent to the 5’ end. ss260 comprises a luciferase ORF (nt 1566-3218, (+) strand), a single-stranded liver-specific SERP / TTR promoter, and a 495 bp spacer sequence in front of the promoter. FIG.121 shows the nucleic acid sequence of ssDNA construct ss056 (SEQ ID NO: 116). ss056 is 3595 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss056 comprises a luciferase ORF (nt 483-2135), a single-stranded liver-specific hAAT promoter, and a deletion of the D region from both AAV2-derived ITRs. FIG.122 shows the nucleic acid sequence of ssDNA construct ss119 (SEQ ID NO: 117). ss119 is 3611 nt in length and comprises 47 bp of double-stranded DNA adjacent to the 3’ end. ss119 comprises a luciferase ORF (nt 491-2143) and a single-stranded liver-specific hAAT promoter. ss119 further comprises simple hairpin ends with partial AAV2 A regions and complete AAV2 D regions. FIG.123 shows the nucleic acid sequence of ssDNA construct ss263 (SEQ ID NO: 118). ss263 is 3306 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss263 comprises a luciferase ORF (nt 990-2642) and a single-stranded muscle-specific CK8 promoter. FIG.124 shows the nucleic acid sequence of ssDNA construct ss264 (SEQ ID NO: 119). ss264 is 3818 nt in length and comprises 555 bp of double-stranded DNA adjacent to the 3’ end. ss264 comprises a luciferase ORF (nt 990-2642) and a double-stranded muscle-specific CK8 promoter. FIG.125 shows the nucleic acid sequence of ssDNA construct ss265 (SEQ ID NO: 120). ss265 is 3248 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss265 comprises a luciferase ORF (nt 990-2642) and a single-stranded hematopoietic stem cell-specific MND promoter. FIG.126 shows the nucleic acid sequence of ssDNA construct ss266 (SEQ ID NO: 121). ss266 is 3702 nt in length and comprises 497 bp of double-stranded DNA adjacent to the 3’ end. ss265 comprises a luciferase ORF (nt 990-2642) and a double-stranded hematopoietic stem cell- specific MND promoter. FIG.127 shows the nucleic acid sequence of ssDNA construct ss267 (SEQ ID NO: 122). ss267 is 3328 nt in length and comprises 43 bp of double-stranded DNA adjacent to the 3’ end. ss267 comprises a luciferase ORF (nt 990-2642) and a single-stranded photoreceptor-specific RHO promoter. FIG.128 shows the nucleic acid sequence of ssDNA construct ss268 (SEQ ID NO: 123). ss268 is 3862 nt in length and comprises 577 bp of double-stranded DNA adjacent to the 3’ end. ss268 comprises a luciferase ORF (nt 990-2642) and a double-stranded photoreceptor-specific RHO promoter. FIG.129 shows schematics of ssDNA construct assembly using AAV2-derived ITRs (top) and complete nonviral hairpin ITRs (bottom). Double-stranded regions adjacent to the 3’ and 5’ ends are counted from the nearest predicted loop or helical junction (i.e., likely departure from canonical B-form DNA) and do not include double-stranded portions distal to the nearest predicted loop or helical junction (e.g., the arms of the ITR structure). Thus, the ssDNA construct with AAV2-derived ITRs (top) comprises 43 bp adjacent to the 3’ end (left side) and 7 bp adjacent to the 5’ end (right side), and the ssDNA construct with complete nonviral hairpin ITRs (bottom) comprises 57 bp adjacent to the 3’ end (left side) and 20 bp adjacent to the 5’ end (right side). FIG.130 shows a schematic of an ssDNA construct comprising AAV2-derived ITRs, 43 bp of double-stranded DNA adjacent to the 3’ end, and a single-stranded promoter. Examples of ssDNA constructs with this structure include ss205, ss209, ss201, ss139, ss207, ss216, ss218, ss222, ss214, ss220, ss102, ss105, ss145, ss183, ss004, and ss012. FIG.131 shows a schematic of an ssDNA construct comprising AAV2-derived ITRs, a variable number of bp of double-stranded DNA adjacent to the 3’ end (length depends on the length of the promoter), and a double-stranded promoter. Examples of ssDNA constructs with this structure include ss206, ss210, ss202, ss203, ss204, ss208, ss217, ss219, ss223, ss215, ss221, ss103, ss106, ss153, ss184, ss104, and ss182. FIG.132 shows a schematic of an ssDNA construct comprising complete nonviral hairpin ITRs, 57 bp of double-stranded DNA adjacent to the 3’ end, and a single-stranded promoter. Examples of ssDNA constructs with this structure include ss224 and ss065. FIG.133 shows a schematic of an ssDNA construct comprising complete nonviral hairpin ITRs, a variable number of bp of double-stranded DNA adjacent to the 3’ end (length depends on the length of the promoter), and a double-stranded promoter. Examples of ssDNA constructs with this structure include ss225 and ss181. FIG.134 shows schematics of ssDNA constructs with extended double-stranded regions adjacent to their 3’ ends, with different combinations of end structures, including virally-derived ITRs (e.g., AAV2-derived), synthetic nonviral ITRs (e.g., synthetic hammerheads), synthetic nonviral hairpin ITRs, and synthetic nonviral open-end structures. FIG.135 shows a schematic of an ssDNA construct comprising AAV2-derived ITRs, a variable number of bp of double-stranded DNA adjacent to the 3’ end comprising a spacer sequence, and a single-stranded promoter. Examples of ssDNA constructs with this structure include ss253, ss254, ss255, ss256, ss257, and ss258. FIG.136 shows a schematic of an ssDNA construct comprising AAV2-derived ITRs, a variable number of bp of double-stranded DNA adjacent to the 5’ end comprising a spacer sequence, and a single-stranded promoter ((+) strand). An example of an ssDNA construct with this structure is ss260. FIG.137 shows a schematic of an ssDNA construct comprising AAV2-derived ITRs, a variable number of bp of double-stranded DNA adjacent to the 3’ end comprising the 3’ UTR and polyA signal, and a single-stranded promoter ((+) strand). Examples of ssDNA constructs with this structure include ss244, ss245, ss246, ss247, ss248, and ss249. FIG.138 shows a schematic of an ssDNA construct comprising AAV2-derived ITRs, a variable number of bp of double-stranded DNA adjacent to the 5’ end comprising the 3’ UTR and polyA signal, and a single-stranded promoter. An example of an ssDNA construct with this structure is ss251. FIG.139A shows a schematic of an ssDNA construct comprising SERP / TTR promoter, as well as the subdomains comprising the entire promoter region. The region comprises, from left to right, the ITR base (e.g., the base of an AAV2-derived ITR comprising A and D regions), a spacer sequence, a 1X Serpin enhancer (Serp), a TTR promoter comprising HNF sites and the transcription start site (bent arrow), and a minute virus of mice (MVM) intron. Large arrows indicate endpoints of double-stranded DNA in constructs with partially-double stranded SERP / TTR promoters. FIG.139B is a table listing the lengths of double-stranded DNA adjacent to the 3’ end for constructs with partially-double-stranded promoters, and the biological reference location within the SERP / TTR promoter of the dsDNA endpoints. FIG.140 shows a schematic of an ssDNA construct comprising AAV2-derived ITRs, 43 bp of double-stranded DNA adjacent to the 3’ end, and a single-stranded SERP / TTR promoter. Examples of ssDNA constructs with this structure include ss102, ss105, ss145, and ss183. FIG.141 shows a schematic of ssDNA construct ss146 comprising AAV2-derived ITRs, 75 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded SERP / TTR promoter. FIG.142 shows a schematic of ssDNA construct ss147 comprising AAV2-derived ITRs, 106 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded SERP / TTR promoter. FIG.143 shows a schematic of ssDNA construct ss148 comprising AAV2-derived ITRs, 179 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded SERP / TTR promoter. FIG.144 shows a schematic of ssDNA construct ss149 comprising AAV2-derived ITRs, 217 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded SERP / TTR promoter. FIG.145 shows a schematic of ssDNA construct ss150 comprising AAV2-derived ITRs, 314 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded SERP / TTR promoter. FIG.146 shows a schematic of ssDNA construct ss151 comprising AAV2-derived ITRs, 413 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded SERP / TTR promoter. FIG.147 shows a schematic of ssDNA construct ss152 comprising AAV2-derived ITRs, 432 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded SERP / TTR promoter. FIG.148 shows a schematic of an ssDNA construct comprising AAV2-derived ITRs, 504 bp of double-stranded DNA adjacent to the 3’ end, and a double-stranded SERP / TTR promoter. Examples of ssDNA constructs with this structure include ss103, ss106, ss153, and ss184. FIG.149 shows a schematic of ssDNA construct ss154 comprising AAV2-derived ITRs, 43 bp of double-stranded DNA adjacent to the 3’ end, and a single-stranded SERP / TTR promoter ((+) strand). FIG.150 shows a schematic of ssDNA construct ss155 comprising AAV2-derived ITRs, 504 bp of double-stranded DNA adjacent to the 3’ end, and a single-stranded SERP / TTR promoter ((+) strand). FIG.151 shows a schematic of ssDNA construct ss004 comprising AAV2-derived ITRs, 43 bp of double-stranded DNA adjacent to the 3’ end, and a single-stranded hAAT promoter. FIG.152 shows a schematic of ssDNA construct ss156 comprising AAV2-derived ITRs, 75 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded hAAT promoter. FIG.153 shows a schematic of ssDNA construct ss157 comprising AAV2-derived ITRs, 106 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded hAAT promoter. FIG.154 shows a schematic of ssDNA construct ss158 comprising AAV2-derived ITRs, 179 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded hAAT promoter. FIG.155 shows a schematic of ssDNA construct ss159 comprising AAV2-derived ITRs, 217 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded hAAT promoter. FIG.156 shows a schematic of ssDNA construct ss160 comprising AAV2-derived ITRs, 314 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded hAAT promoter. FIG.157 shows a schematic of ssDNA construct ss161 comprising AAV2-derived ITRs, 406 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded hAAT promoter. FIG.158 shows a schematic of ssDNA construct ss162 comprising AAV2-derived ITRs, 433 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded hAAT promoter. FIG.159 shows a schematic of ssDNA construct ss163 comprising AAV2-derived ITRs, 504 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded hAAT promoter. FIG.160 shows a schematic of ssDNA construct ss164 comprising AAV2-derived ITRs, 887 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded hAAT promoter. FIG.161 shows a schematic of ssDNA construct ss165 comprising AAV2-derived ITRs, 1246 bp of double-stranded DNA adjacent to the 3’ end, and a partially double-stranded hAAT promoter. FIG.162 shows a schematic of ssDNA construct ss104 comprising AAV2-derived ITRs, 1381 bp of double-stranded DNA adjacent to the 3’ end, and a double-stranded hAAT promoter. FIG.163 shows IVIS expression in mice dosed with LNP-formulated ssDNA constructs comprising single-stranded (ssP) or double-stranded (dsP) hAAT or SERP / TTR (S / T) promoters. FIG.164 shows the % body weight change in mice one day after dosing with LNP- formulated ssDNA constructs comprising single-stranded (ssP) or double-stranded (dsP) promoters. FIG.165 shows longitudinal body weight change in mice dosed with LNP-formulated ssDNA constructs. FIGs.166A-166E show cytokine expression in mice dosed with LNP-formulated ssDNA constructs comprising single-stranded (ssP) or double-stranded (dsP) hAAT or SERP / TTR (S / T) promoters. FIG.166A: Interferon-α. FIG.166B: Interferon-γ. FIG.166C: IL-6. FIG.166D: TNF-α. FIG.166E: IL-18. FIGs.167A-167B show day 7 qPCR measurement of liver vector-derived mRNA (FIG. 167A) and liver vector DNA (FIG.167B). FIG.168 shows day 7 IVIS expression in mice dosed with constructs comprising single- stranded, partially-double stranded, and double-stranded SERP / TTR promoters via HDI. Construct ss145 was dosed at 500 ng / animal. All other constructs were dosed at amounts mole-matched to 500 ng ss145. The length of the double-stranded region adjacent to the 3’ end is plotted on the x-axis. FIG.169 shows schematics of ssDNA constructs ss119 and ss065 (left). ss119 comprises simple hairpin ends with partial AAV2 A regions, complete AAV2 D regions, 47 bp of double- stranded DNA adjacent to the 3’ end, and a single-stranded hAAT promoter. ss065 comprises complete nonviral hairpin ITRs, 57 bp of double-stranded DNA adjacent to the 3’ end, and a single- stranded hAAT promoter. Right: IVIS expression in mice dosed with LNP-formulated ssDNA constructs with simple hairpin ends and either +AD (ss119) or -AD (ss065; complete nonviral). FIG.170 shows a general schematic of an ssDNA construct comprising end structures (e.g., ITRs which may be virally-derived or completely nonviral), an ORF, and a double-stranded spacer and / or promoter (top), as well as different types of chemical and structural modifications that may be added to an ssDNA construct (bottom), including, but not limited to, structured elements (e.g., aptamers, hairpins, etc.), chemical modifications of the DNA, modification of PS bond presence and / or position within the ssDNA, and site-specific conjugation (e.g., of small or large molecule moieties, including polypeptides). FIG.171A shows a schematic of an ssDNA construct comprising a gapped oligonucleotide array designed to produce partial double-strandedness without inducing cGAS oligomerization. Oligonucleotides comprising stabilizing modifications at their 3’ ends are hybridized to the ssDNA to generate a partially double-stranded ssDNA molecule with contiguous dsDNA lengths below the functional cGAS detection limit. The ssDNA construct is shown as the (-) strand version, but may also be constructed as a (+) strand version, with a variety of different end structures (e.g., hairpins, aptamers, quadraplex, etc.). FIG.171B shows a schematic of an ssDNA construct comprising a ligated oligonucleotide array with varied modifications added the 3’ ends of some or all of the oligonucleotides, which generates an ssDNA molecule with a double-stranded region with low cGAS binding potential. The oligonucleotides may have full, intermittent, or alternating modifications, including, but not limited to, RNA, PS bonds, 2-OMe, etc.). The ssDNA construct is shown as the (-) strand version, but may also be constructed as a (+) strand version, with a variety of different end structures (e.g., hairpins, aptamers, quadraplex, etc.). FIG.171C shows a schematic of an ssDNA construct comprising an oligonucleotide with stabilizing modifications added to the 3’ end hybridized adjacent to or near the 3’ end of the double- stranded region of the ssDNA (e.g., a double-stranded promoter region). The oligonucleotide may or may not be ligated to the ssDNA. The ssDNA construct is shown as the (-) strand version, but it may also be constructed as a (+) strand version, and may comprise a variety of different end structures (e.g., hairpins, aptamers, quadraplex, etc.). FIG.171D shows a schematic of an ssDNA construct comprising oligonucleotides with stabilizing modifications added to their 3’ end and / or 5’ ends hybridized adjacent to or near the 3’ end and the 5’ end of the ssDNA. The oligonucleotide may or may not be ligated to the ssDNA and / or to each other. The ssDNA construct is shown as the (-) strand version, but it may also be constructed as a (+) strand version, and may comprise a variety of different end structures (e.g., hairpins, aptamers, quadraplex, etc.). FIG.171E shows a schematic of an ssDNA construct comprising an oligonucleotide array with stabilizing modifications added hybridized adjacent to an open 3’ end to generate a partially double stranded ssDNA molecule, with or without ligation between the oligonucleotides. The ssDNA construct is shown as the (-) strand version, but it may also be constructed as a (+) strand version, and may comprise a variety of different end structures (e.g., hairpins, aptamers, quadraplex, etc.). FIG.171F shows a schematic of an ssDNA construct comprising an oligonucleotide array with stabilizing modifications added hybridized adjacent to an open 5’ end to generate a partially double stranded ssDNA molecule, with or without ligation between the oligonucleotides. The ssDNA construct is shown as the (-) strand version, but it may also be constructed as a (+) strand version, and may comprise a variety of different end structures (e.g., hairpins, aptamers, quadraplex, etc.). FIG.171G shows schematics of additional end structures that may be included in the ssDNA constructs of FIGs.171A-171G (as well as any of the other ssDNA constructs disclosed herein), including, but not limited to, hairpins, aptamers, PS bonds, etc. FIG.172A shows a schematic of an ssDNA construct comprising double-stranded DNA islands with binding sites for host or exogenous proteins (e.g., recombinases, gene editing guide sites, etc.). The ssDNA construct is shown as the (-) strand version, but it may also be constructed as a (+) strand version, and may comprise a variety of different end structures (e.g., hammerheads, aptamers, quadraplex, etc.). FIG.172B shows schematics of an ssDNA construct comprising a functional structured region (e.g., an aptamer) hybridized to the ssDNA molecule to produce an ssDNA molecule with a partially double-stranded region and enhanced transcription and / or subcellular distribution. The structured region may be hybridized in isolated, arrayed, or gapped configurations. Top: an ssDNA construct with one hybridized structured region. Bottom: an ssDNA constructed with two hybridized structured regions. The ssDNA constructs are shown as (-) strand versions, but they may also be constructed as (+) strand versions, and may comprise a variety of different end structures (e.g., hammerheads, aptamers, quadraplex, etc.). FIG.172C shows a method for producing additional functional copies of an ssDNA construct. An oligonucleotide designed to hybridize adjacent to the 3’ end of the ssDNA and upstream of the promoter primes the synthesis of additional functional DNA copies upon administration to a cell. The oligonucleotide may be hybridized in isolated, arrayed, or gapped configurations. The starting ssDNA construct is shown as the (-) strand version, but it may also be constructed as a (+) strand version, and may comprise a variety of different end structures (e.g., hammerheads, aptamers, quadraplex, etc.). FIG.172D shows a method for producing additional functional copies of an ssDNA construct. An oligonucleotide designed to hybridize within a partially double stranded region and upstream of the promoter primes the synthesis of additional functional DNA copies upon administration to a cell via triplex or strand-displacing binding. The oligonucleotide may be hybridized in isolated, arrayed, or gapped configurations. The starting ssDNA construct is shown as the (-) strand version, but it may also be constructed as a (+) strand version, and may comprise a variety of different end structures (e.g., hammerheads, aptamers, quadraplex, etc.). FIGs.173A-173D show examples of ssDNA molecules that include partially double-stranded circular ssDNA. FIGS.173A and 173B depict representative circular ssDNA molecules that can function as templates for insertion of desired DNA sequences via homology-directed recombination (HDR) into other DNA, such as genomic DNA, by recombinase enzymes and corresponding site- specific recombination sequences (e.g., “attP” attachment site). The ssDNA molecule in FIG.173A contains phosphorothioate bonds (stars) at both the free 5’ and 3’ ends of the linear DNA strand that is hybridized to the circular DNA strand. The ssDNA molecule in FIG.173B contains phosphorothioate bonds at the free 5’ end of the linear DNA strand that is hybridized to the circular DNA strand. FIGS.173C and 174D depict circular ssDNA molecules for episomal transgene expression where the promoter region of the transgene cassette is double stranded. FIG.173C shows a circular ssDNA molecule that contains a circular (-) / non-coding DNA strand that is hybridized to a complementary linear DNA strand with phosphorothioate bonds at the 5’ end. FIG.173D depicts a circular ssDNA molecule that contains a circular (+) / coding DNA strand that is hydrogen bonded to a complementary linear DNA strand with phosphorothioate bonds at the 5’ end. FIG.174 depicts a method for synthesizing circular ssDNA containing phosphorothioate bonds at both the free 5’ and 3’ ends of the linear DNA strand to be used as a recombination template. First, a double-stranded plasmid is treated with a restriction enzyme to separate sequences needed for plasmid replication from the transgene cassette or DNA sequence of interest. Second, the resulting DNA sequence of interest is ligated with a DNA insert containing phosphorothioate bonds at both the 5’ and 3’ ends of the (+) strand to form the precursor plasmid. The precursor plasmid is treated with a nicking endonuclease (e.g., Nb.BbvCI) to nick a single DNA strand. The (+) DNA strand is degraded in both directions from the nick site by treatment with T7 exonuclease (5’ 3’ exonuclease) and Exonuclease III (3’ → 5’ exonuclease). Phosphorothioate bonds in the plus DNA strand stop both exonucleases, yielding the final partially double stranded circular ssDNA molecule. FIG.175 depicts a method for synthesizing circular ssDNA containing phosphorothioate bonds at the free 5’ ends of the linear DNA strand to be used for episomal transgene expression. A double-stranded plasmid is treated with a restriction enzyme to separate sequences needed for plasmid replication from the transgene cassette or DNA sequence of interest. The DNA sequence of interest is ligated with a DNA insert containing phosphorothioate bonds in one location on the (+) strand to form the precursor plasmid. The precursor plasmid is treated with a nicking endonuclease (e.g., Nb.BbvCI) to nick the plus DNA strand at the desired 3’ end of the double stranded region. The (+) DNA strand from the nick site is degraded by treatment with T7 exonuclease (5’ → 3’ exonuclease). Phosphorothioate bonds in the (+) DNA strand stop both exonucleases, yielding the final partially double-stranded circular ssDNA molecule. FIG.176 shows schematics showing an ssDNA molecule covalently bound to a SNAP- tag+Enzyme fusion protein. The SNAP-tag reacts through a benzylguanine (BG) modification within the ssDNA to covalently link the two together. Although the fusion protein is depicted as comprising an enzyme, the SNAP-tag may be fused to any peptide or polypeptide, depending on the desired function. FIG.177A shows schematics depicting ceDNA (ceDNA917 and ceDNA918) and ssDNA (ss261 and ss262) modified with either azide (N3) or benzylguanine (BG), as well as with Cy3 fluorophore (for imaging). FIG.177B: Incubation of azide-modified ssDNA with SNAP-tag protein results in no bioconjugation, even at large molar excess (87X). Incubation of benzylguanine (BG)- modified ssDNA with SNAP-tag protein results in efficient bioconjugation of ssDNA, yielding a covalently fused SNAP-tag and ssDNA product. FIG.178A shows schematics depicting ceDNA (ceDNA917 and ceDNA918) and ssDNA (ss261 and ss262) modified with either azide (N3) or benzylguanine (BG), as well as with Cyanine3 fluorophore (for imaging). FIG.178B: Incubation of azide-modified ceDNA with SNAP-tag protein results in no bioconjugation, even at large molar excess (174X). Incubation of benzylguanine (BG)- modified ceDNA with SNAP-tag protein results in efficient bioconjugation of ceDNA, yielding a covalently fused SNAP-tag and ceDNA product. FIG.179A shows schematics depicting ceDNA (ceDNA809 and ceDNA810) and ssDNA (ss174 and ss175) modified with either azide (N3) or benzylguanine (BG). Incubation of azide- modified ssDNA with SNAP-tag+dCas9 fusion protein results in no bioconjugation (FIG.179B), even at large molar excess (87X). Incubation of benzylguanine (BG)-modified ssDNA with SNAP- tag+dCas9 fusion protein results in efficient bioconjugation of ssDNA (FIG.179C), yielding a covalently fused SNAP-tag and ssDNA product. FIG.180 shows a schematic showing generation of ssDNA covalently fused to PCV2 HUH endonuclease + enzyme fusion protein. Covalent fusion is achieved through inclusion of a PCV2 target sequence incorporated into the ssDNA molecule. Although the fusion protein is depicted as comprising an enzyme, the SNAP-tag may be fused to any peptide or polypeptide, depending on the desired function. FIG.181A shows schematics depicting ssDNA (ss231 and ss230) with and without a PCV2 target sequence. FIG.181B: Incubation of PCV2 protein with ssDNA lacking a PCV2 target sequence results in no bioconjugation, even at large molar excess (195X). Incubation of PCV2 protein with ssDNA comprising a PCV2 target sequence results in bioconjugation, yielding a covalently fused PCV2 and ssDNA product. FIG.182 shows the nucleic acid sequence of ssDNA construct ss261 (SEQ ID NO: 124). ss261 is 3604 nt in length and comprises hairpin ends, a luciferase ORF (nt 484-2136), and a liver- specific hAAT promoter. ss261 further comprises a Cy3 fluorophore modification in the loop adjacent to the 3’ end, and a free azide (N3) modification in the loop adjacent to the 5’ end. FIG.183 shows the nucleic acid sequence of ssDNA construct ss262 (SEQ ID NO: 125). ss262 is 3604 nt in length and comprises hairpin ends, a luciferase ORF (nt 484-2136), and a liver- specific hAAT promoter. ss262 further comprises a Cy3 fluorophore modification in the loop adjacent to the 3’ end, and a benzylguanine (BG) modification in the loop adjacent to the 5’ end. FIG.184 shows the nucleic acid sequence of ssDNA construct ss174 (SEQ ID NO: 126). ss174 is 3604 nt in length and comprises hairpin ends, a luciferase ORF (nt 484-2136), and a liver- specific hAAT promoter. ss174 further comprises a free azide (N3) modification in the loop adjacent to the 5’ end. FIG.185 shows the nucleic acid sequence of ssDNA construct ss175 (SEQ ID NO: 127). ss175 is 2604 nt in length and comprises hairpin ends, a luciferase ORF (nt 484-2136), and a liver- specific hAAT promoter. ss175 further comprises a benzylguanine (BG) modification in the loop adjacent to the 5’ end. FIG.186 shows the nucleic acid sequence of ssDNA construct ss270 (SEQ ID NO: 128). ss270 is 6317 nt in length and comprises 684 bp of double-stranded DNA adjacent to the 3’ end. ss270 comprises nonviral-derived hairpin ends, a FVIII ORF (nt 532-4923) and a double-stranded liver-specific 2xBBEnh-TTR promoter. The FVIII ORF further comprises an ablated cryptic start site and eight ablated naturally-occurring Nb.BbvCI nick sites. FIG.187 is a graph depicting the results of in vivo Factor VIII expression at day 3 by ssDNA construct ss182 (which comprises 758 bp of double-stranded DNA adjacent to the 3’ end and a double-stranded liver-specific 3X SERP / TTR promoter) in mice after hydrodynamic injection (HDI) at doses of 50 ng / animal, 500 ng / animal, and 1000 ng / animal. Expression from ceDNA491 is shown as a control. FIG.188 is a graph depicting the results of in vivo luciferase expression at day 7 by a (+) ssDNA construct having double-stranded DNA adjacent to the 5’ end and a single-stranded promoter (left side), as well as different lengths of double-stranded DNA adjacent to the 3’ end (right side). Shown, left to right: (1) PBS control; (2) ss145: single-stranded SERP / TTR promoter, (-) strand, 43 bp of double-stranded DNA adjacent to the 3’ end; (3) ss155: single-stranded SERP / TTR promoter, (+) strand, 504 bp of double-stranded DNA adjacent to the 5’ end; (4) ss004: single-stranded hAAT promoter, (-) strand, 43 bp of double-stranded DNA adjacent to the 3’ end; (5) ss163: partially double-stranded hAAT promoter, (-) strand, 504 bp of double-stranded DNA adjacent to the 3’ end; (6): ss104: double-stranded hAAT promoter, (-) strand, 1381 bp of double-stranded DNA adjacent to the 3’ end. FIG.189 is a graph depicting the results of in vivo luciferase (IVIS) expression at day 7 in mice administered 500 ng / animal via hydrodynamic injection (HDI) with ssDNA constructs with different single-stranded liver-specific promoters. From left to right: PBS control, ss004 (hAAT), ss145 (1X SERP / TTR), ss214 (SV40Enh-hAlb), ss220 (1xBBEnh-TTR), ss216 (1xBBEnh-SCP1), ss218 (hSERP / FOXA / HNF4-TTR), and ss222 (HNF4 / 1_X1476-TTR). FIG.190 is a graph depicting the fold increase in Day 7 in vivo luciferase expression for ssDNA constructs having different liver-specific double-stranded promoters, as compared to ssDNA constructs having the same (single-stranded) promoters. Mice were injected via hydrodynamic injection (HDI). From left to right: ss215 (SV40Enh-hAlb), ss217 (1xBBEnh-SCP1), ss219 (hSERP / FOXA / HNF4-TTR), ss221 (1xBBEnh-TTR), ss223 (HNF4 / 1_X1476-TTR), and ss153 (1X SERP / TTR). FIG.191 is a graph depicting the results of in vivo luciferase (IVIS) expression at day 7 in mice administered 500 ng / animal via hydrodynamic injection (HDI) with ssDNA constructs with different single-stranded and double-stranded constitutive promoters. From left to right: PBS, ss205 (single-stranded RSV), ss206 (double-stranded RSV), ss139 (single-stranded CMV / chimeric intron), ss203 (partially double-stranded CMV / chimeric intron, with a double-stranded (internal) TSS), and ss204 (double-stranded CMV / chimeric intron). FIG.192 is a graph depicting the fold increase in Day 7 in vivo luciferase expression for ssDNA constructs having different constitutive double-stranded or partially double-stranded promoters, as compared to ssDNA constructs having the same (single-stranded) promoters. Mice were injected via hydrodynamic injection (HDI). From left to right: ss153 (1X SERP / TTR, liver- specific control), ss206 (RSV), ss203 (partially double-stranded CMV / chimeric intron, with a double- stranded (internal) TSS), and ss204 (CMV / chimeric intron). FIGs.193A-193C show schematic diagrams of ssDNA molecules comprising target sites for Cas9 used to enable nuclear translocation. FIG.193A: ssDNA molecule comprising a Cas target sequence (CTS) and NGG PAM site in the double-stranded region at the 3’ end of the ssDNA molecule. FIG.193B: Cas9 protein plus a gRNA is designed to bind but not nick or cleave the ssDNA. Inclusion of target sequence having a 4-8 nucleotide mismatch (*) to the 5’ end of the gRNA in the ssDNA molecule prevents cutting. Alternatively, the gRNA itself could be truncated at the 5’ end to 16 nucleotides, which would also prevent cutting, or a dead Cas9 enzyme could be used. FIG. 193C: The nuclear localization sequence of the Cas9 protein enables nuclear translocation of the bound ssDNA molecule. FIGs.194A-194C show schematic diagrams of ssDNA molecules comprising Cas target sequences (CTSs) in different configurations. FIG.194A: ssDNA molecule with a CTS in a longer double-stranded region at the 3’ end of the ssDNA molecule. FIG.194B: ssDNA molecule with two CTSs, one in each strand of the double-stranded region at the 3’ end of the ssDNA molecule. FIG. 194C: ssDNA molecule with a CTS in the double-stranded region at the 5’ end of the ssDNA molecule. FIG.195 shows schematic diagrams of ssDNA molecules oligo annealing and ligation processes that increase the proportion of a double-stranded region. FIGs.196A and 194B show an assay result validating oligo annealing and ligation process. FIG.197 shows a schematic diagram and a table depicting ssDNA constructs generated by the oligonucleotide tiling method. The schematic diagram shows the 3’ end of a double-stranded promoter containing ssDNA construct (ss153) depicting the location where a 54nt oligonucleotide (oligonucleotide Nos.1 or 2) anneals to ss153. The table describes ssDNA double-stranded constructs synthesized with the oligonucleotide tiling method (ss349, ss7183, ss350, ss7184). FIG.198 shows a panel of graphs measuring luciferase expression in mice injected via HDI with 500ng per animal of ssDNA constructs having a single-stranded promoter, a double-stranded promoter, or a double stranded promoter region that was extended by annealing of an oligonucleotide with or without ligation. Left: luciferase expression at day 1. Right: luciferase expression at day 7. For each graph from left to right: ss145 (single-stranded promoter), ss153 (double-stranded promoter), ss349 (double-stranded promoter with annealed and ligated unmodified oligonucleotide), ss7183 (double-stranded promoter with annealed and non-ligated unmodified oligonucleotide), ss350 (double-stranded promoter with annealed and ligated PS-modified oligonucleotide), ss7184 (double- stranded promoter with annealed and non-ligated PS-modified oligonucleotide). ssDNA constructs with annealed and ligated oligonucleotide (ss349, ss350) increase expression over the parent ssDNA construct (ss153). DETAILED DESCRIPTION I. Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0- 911910-19-3); Robert S. Porter et al. (eds.), Fields Virology, 6th Edition, published by Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013), Knipe, D.M. and Howley, P.M. (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1- 56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 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, 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, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties. As used herein, the term “AAV” or “adeno-associated virus” refer to single-stranded DNA parvoviruses that grow only in cells. Certain functions of AAV are provided only by co-infecting a helper virus. Thirteen serotypes of AAV have been identified. General information and review of AAV can be found, e.g., in Carter, 1989, Handbook of Parvoviruses, Vol.1, p.169-228, and Berns, 1990, Virology, pp.1743-1764, Raven Press, (New York). As used herein, the terms “ssDNA”, “ssDNA construct” “ssDNA molecule”, and “ssDNA vector” refer to a linear, substantially single-stranded DNA molecule comprising a nucleic acid sequence of interest. In some embodiments, the nucleic acid sequence of interest comprises at least one promoter or promoter set (comprising, e.g., a promoter and additional expression control elements). In some embodiments, an “ssDNA”, “ssDNA construct”, “ssDNA molecule”, or “ssDNA vector” may include at least one stem-loop structure at its 3’ end comprising at least one stem and one loop, and / or at least one stem-loop structure comprising at least one stem and one loop at its 5’ end. In some embodiments, an “ssDNA”, “ssDNA construct”, “ssDNA molecule”, or “ssDNA vector” may comprise an extended double-stranded region adjacent to its 3’ and / or 5’ end, and this double-stranded region may comprise the promoter region, including the promoter itself, one or more enhancers, the transcription start site, and / or other regulatory elements. In some embodiments, an “ssDNA”, “ssDNA construct”, “ssDNA molecule”, or “ssDNA vector” may further comprise double-stranded regions comprising hybridized, noncovalently-bound oligonucleotides. Although the terms “ssDNA”, “ssDNA construct” “ssDNA molecule”, and “ssDNA vector” generally refer to a linear DNA molecule, it should also be understood that in some embodiments, the term “circular ssDNA” molecule may be used to refer to circular, partially ssDNA molecules comprising one fully circular strand and at least one noncovalently-bound linear strand hybridized to the fully circular strand to produce one or more double-stranded regions. Although lacking the 3’ and / or 5’ stem-loop structures described elsewhere herein for ssDNA molecules, circular ssDNA molecules are nevertheless useful for the same applications as any of the instantly described ssDNA molecules. As used herein, the terms “single-stranded (ss) synthetic DNA molecules”, “single-stranded (ss) synthetic AAV vectors”, “synthetic production of ss DNA molecules” and “synthetic production of ss AAV vectors” refer to a single-stranded (ss) synthetic DNA molecule (ssDNA), a single- stranded AAV vector and synthetic production methods thereof in an entirely cell-free environment. The production may involve one or more molecules in a manner that does not involve replication or other multiplication of the molecule by or inside of a cell or using a cellular extract. Synthetic production avoids contamination of the produced molecule with cellular contaminants, e.g., cellular proteins or cellular nucleic acid, viral protein or DNA, insect protein or DNA and further avoids unwanted cellular-specific modification of the molecule during the production process, e.g., methylation or glycosylation or other post-translational modification. As used herein, the terms “gap” and “nick” are used interchangeably and refer to a discontinued portion of synthetic DNA vector of the present disclosure, creating a stretch of single stranded DNA portion in otherwise double stranded ceDNA. The gap can be 1 nucleotide to 100 nucleotides long in length. Typical gaps, designed and created by the methods described herein and synthetic vectors generated by the methods can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nucleotides (nt) in length. Exemplified gaps in the present disclosure can be 1 bp to 10 nt long, 1 to 20 nt long, 1 to 30 nt long, or any length necessary to nick double stranded DNA to allow for or to maintain efficient transcription of an expression cassette in host cells. In other embodiments, gaps may be present between oligonucleotides hybridized to an ssDNA molecule to produce a double-stranded region comprising at least one gap. According to some embodiments, gaps can be present 5’ upstream of an expression cassette. According to some embodiments, gaps can be present 3’ downstream of an expression cassette. According to some embodiments, gaps can be present 5’ upstream of an expression cassette and 3’ downstream of an expression cassette. As used herein, the term “nick” refers to a discontinuity in a double stranded DNA molecule where there is no phosphodiester bond between adjacent nucleotides of one strand typically through damage or enzyme action. It is understood that one or more nicks allow for the release of torsion in the strand during DNA replication and that nicks are also thought to play a role in facilitating binding of transcriptional machinery. As used herein, the term “ceDNA” refers to capsid-free closed-ended linear double stranded (ds) duplex DNA for non-viral gene transfer, synthetic or otherwise. Detailed description of ceDNA is described in International application of PCT / US2017 / 020828, filed March 3, 2017 (published as International patent publication No. WO2017152149A1), the entire content of which is incorporated herein by reference. Certain methods for the production of ceDNA comprising various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International applications 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 in its entirety by reference. Certain methods for the production of synthetic ceDNA vectors comprising various ITR sequences and configurations are described, e.g., in International application PCT / US2019 / 14122, filed January 18, 2019 (published as International patent publication No. WO 2019 / 143885 A1), the entire content of which is incorporated herein by reference. According to some embodiments, the ceDNA comprises one of more phosphorothioate- modified nucleotides. As used herein, the term “neDNA” or “nicked ceDNA” refers to a closed-ended DNA having a nick or a gap of 1-100 base pairs in a stem region or spacer region upstream of an open reading frame (e.g., a promoter and transgene to be expressed). As used herein, the term “stem-loop structure” refers to a nucleic acid sequence located at the 5’ and / or 3’ terminus of the ssDNA vectors disclosed herein, which comprises at least one partial duplex (referred to herein as a “stem”) and one loop (comprising 3 or more unbound, single-stranded nucleotides). According to some embodiments, the stem-loop structure may be an artificial sequence (e.g., contains no sequences derived from a virus), or it may be derived in part or entirely from a virus (e.g. an AAV such as AAV2). An ssDNA molecule may further comprise one stem-loop structure (e.g., a “hairpin”), or more than one stem-loop structure (e.g., 2, 3, 4, 5, or more stem-loop structures). For example, an ssDNA molecule may comprise two stem-loop structures (e.g., a “hammerhead”, “doggy-bone”, or “dumbbell”), three stem-loop structures (e.g., “cruciform”), or more complex structures. A stem-loop structure may further comprise an aptamer sequence or one or more chemical modifications. In some embodiments, a stem-loop structure at the 3’ and / or 5’ ends of an ssDNA molecule may be referred to as an “inverted terminal repeat” or “ITR”. For the purposes of the disclosure herein, the terms “inverted terminal repeat” and “ITR” are not intended to represent only viral-derived sequences, but are intended to refer to any sequence, fully synthetic or viral-derived, that contain inverted, palindromic sequences that can self-hybridize to form a stem-loop structure. According to some embodiments, a “stem-loop structure” or “ITR” can be artificially synthesized using a set of oligonucleotides comprising one or more desirable functional sequences (e.g., palindromic sequence). 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., ITR fragments removed from a viral genome). For example, the ITR can be derived from the family Parvoviridae, which encompasses parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin that serves as the origin of SV40 replication can be used as an ITR, which can further be modified by truncation, substitution, deletion, insertion and / or addition. Parvoviridae family viruses consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the viral family of the adeno-associated viruses (AAV) which are capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine and ovine species. Typically, ITR sequences can be derived not only from AAV, but also from Parvovirus, lentivirus, goose virus, B19, in the configurations of wildtype, “doggy bone” and “dumbbell shape”, symmetric or even asymmetric ITR orientation. Although the ITRs are typically present in both 5’ and 3’ ends of an AAV vector, in a partially single-stranded DNA (ssDNA) molecule, the ITR can be present in only one of end of the linear vector. For example, the ITR can be present on the 5’ end only. In other embodiments, the ITR can be present on the 3’ end only in a partially single-stranded DNA (ssDNA) molecule. As used herein, a “wild-type ITR” or “WT-ITR” refers to the sequence of a naturally occurring ITR sequence in an AAV genome or other dependovirus that remains, e.g., Rep binding activity and Rep nicking ability. The nucleotide sequence of a WT-ITR from any AAV serotype may slightly vary from the canonical naturally occurring sequence due to degeneracy of the genetic code or drift, and therefore WT-ITR sequences encompasses for use herein include WT-ITR sequences as result of naturally occurring changes (e.g., a replication error). As used herein, the term “substantially symmetric WT-ITRs” or a “substantially symmetric WT-ITR pair” refers to a pair of WT-ITRs within a partially single-stranded DNA (ssDNA) molecule that are both wild type ITRs that have an inverse complement sequence across their entire length. For example, an ITR can be considered to be a wild-type sequence, even if it has one or more nucleotides that deviate from the canonical naturally occurring canonical sequence, so long as the changes do not affect the physical and functional properties and overall three-dimensional structure of the sequence (secondary and tertiary structures). In some aspects, the deviating nucleotides represent conservative sequence changes. As one non-limiting example, a sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical sequence (as measured, e.g., using BLAST at default settings), and also has a symmetric three-dimensional spatial organization to the other WT-ITR such that their 3D structures are the same shape in geometrical space. The substantially symmetric WT-ITR has the same A, C-C’ and B-B’ loops in 3D space. A substantially symmetric WT-ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE’) and terminal resolution site (TRS) that pairs with the appropriate Rep protein. One can optionally test other functions, including transgene expression under permissive conditions. As used herein, the phrases of “modified ITR” or “mod-ITR” or “mutant ITR” are used interchangeably and refer to an ITR with a mutation in at least one or more nucleotides as compared to the WT-ITR from the same serotype. The mutation can result in a change in one or more of A, C, C’, B, B’ regions in the ITR, and can result in a change in the three-dimensional spatial organization (i.e., its 3D structure in geometric space) as compared to the 3D spatial organization of a WT-ITR of the same serotype. As used herein, the term “asymmetric ITRs”, also referred to as “asymmetric ITR pairs”, refers to a pair of ITRs within a single ssDNA vector that are not inverse complements across their full length. As one non-limiting example, an asymmetric ITR pair does not have a symmetric three- dimensional spatial organization to their cognate ITR such that their 3D structures are different shapes in geometrical space. Stated differently, an asymmetric ITR pair have the different overall geometric structure, i.e., they have different organization of their A, C-C’ and B-B’ loops in 3D space (e.g., one ITR may have a short C-C’ arm and / or short B-B’ arm as compared to the cognate ITR). The difference in sequence between the two ITRs may be due to one or more nucleotide addition, deletion, truncation, or point mutation. In one embodiment, one ITR of the asymmetric ITR pair may be a wild-type AAV ITR sequence and the other ITR a modified ITR as defined herein (e.g., a non-wild- type or synthetic ITR sequence). In another embodiment, neither ITRs of the asymmetric ITR pair is a wild-type AAV sequence and the two ITRs are modified ITRs that have different shapes in geometrical space (i.e., a different overall geometric structure). In some embodiments, one mod-ITRs of an 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 they have different three- dimensional spatial organization as compared to the cognate asymmetric mod-ITR. As used herein, the term “symmetric ITRs” refers to a pair of ITRs within an ssDNA vector that are mutated or modified relative to wild-type dependoviral ITR sequences and are inverse complements across their full length. Neither ITRs are wild type ITR AAV2 sequences (i.e., they are a modified ITR, also referred to as a mutant ITR), and can have a difference in sequence from the wild type ITR due to nucleotide addition, deletion, substitution, truncation, or point mutation. As used herein, the terms “substantially symmetric modified-ITRs” or a “substantially symmetric mod-ITR pair” refers to a pair of modified-ITRs within a partially single-stranded DNA (ssDNA) molecule that have an inverse complement sequence across their entire length. For example, the modified ITR can be considered substantially symmetric, even if it has some nucleotide sequences that deviate from the inverse complement sequence so long as the changes do not affect the properties and overall shape. As one non-limiting example, a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical sequence (as measured using BLAST at default settings), and also has a symmetric three-dimensional spatial organization to their cognate modified ITR such that their 3D structures are the same shape in geometrical space. Stated differently, a substantially symmetric modified-ITR pair have the same stem-loop structures organized in 3D space. In some embodiments, the ITRs from a mod-ITR pair may have different reverse complement nucleotide sequences but still have the same symmetric three-dimensional spatial organization – that is both ITRs have mutations that result in the same overall 3D shape. For example, in a viral-derived ITR, 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 mutation (e.g., if the 5’ ITR has a deletion in the C region, the cognate modified 3’ ITR from a different serotype has a deletion at the corresponding position in the C’ region), such that the modified ITR pair has the same symmetric three-dimensional spatial organization. In such embodiments, each ITR in a modified ITR pair can be from different serotypes (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) such as the combination of AAV2 and AAV6, with the modification in one ITR reflected in the corresponding position in the cognate ITR from a different serotype. In one embodiment, a substantially symmetric modified ITR pair refers to a pair of modified ITRs (mod-ITRs) so long as the difference in nucleotide sequences between the ITRs does not affect the properties or overall shape and they have substantially the same shape in 3D space. As a non-limiting example, a mod-ITR that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the canonical mod-ITR as determined by standard means well known in the art such as BLAST (Basic Local Alignment Search Tool), or BLASTN at default settings, and also has a symmetric three-dimensional spatial organization such that their 3D structure is the same shape in geometric space. A substantially symmetric mod-ITR pair has the same A, C-C’ and B-B’ loops in 3D space, e.g., if a modified ITR in a substantially symmetric mod-ITR pair has a deletion of a C-C’ arm, then the cognate mod-ITR has the corresponding deletion of the C-C’ loop and also has a similar 3D structure of the remaining A and B-B’ loops in the same shape in geometric space of its cognate mod-ITR. As used herein, the term “flanking” refers to a relative position of one nucleic acid sequence with respect to another nucleic acid sequence. Generally, in the sequence ABC, B is flanked by A and C. The same is true for the arrangement AxBxC. Thus, a flanking sequence precedes or follows a flanked sequence but need not be contiguous with, or immediately adjacent to the flanked sequence. In one embodiment, the term flanking refers to terminal repeats (e.g., a stem-loop structure) at each end of the linear single-stranded DNA (ssDNA) molecule. In some embodiments, a nucleic acid sequence may be flanked by at least one stem-loop structure at one end, but may be single-stranded at the other end. As used herein, the terms “canonical B-form DNA” or “B-form DNA” refer to the canonical right-handed DNA helix that is the most common form of double-stranded DNA. Canonical B-DNA is a double helix made of two antiparallel strands that are held together via hydrogen bonding between the A-T and G-C base pairs. As used herein, the term “closed-ended DNA” or “ceDNA” refers to a synthetic, double- stranded, linear, DNA construct with at least one covalently closed end that includes the gene of interest and other regulatory elements. As used herein, the term “closed-ended DNA vector” refers to a capsid-free DNA vector with at least one covalently closed end and where at least part of the vector has an intramolecular duplex structure. ceDNA vectors may be used, as described elsewhere herein, as starting materials for the manufacture of the partially ssDNA molecules of the instant disclosure. ceDNA vectors may also be used as controls, for example, in experiments measuring the transgene expression level and / or immune stimulation of the partially ssDNA molecules described herein. As defined herein, “reporter” or “reporters” refer to a protein or proteins that can be used to provide detectable read-outs. Reporters generally produce a measurable signal such as fluorescence, color, or luminescence. Reporter protein coding sequences encode proteins whose presence in the cell or organism is readily observed. For example, fluorescent proteins cause a cell to fluoresce when excited with light of a particular wavelength, luciferases cause a cell to catalyze a reaction that produces light, and enzymes such as β-galactosidase convert a substrate to a colored product. Exemplary reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP) and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art. As used herein, the term “effector protein” refers to a polypeptide that provides a detectable read-out, either as, for example, a reporter polypeptide, or more appropriately, as a polypeptide that kills a cell, e.g., a toxin, or an agent that renders a cell susceptible to killing with a chosen agent or lack thereof. Effector proteins include any protein or peptide that directly targets or damages the host cell’s DNA and / or RNA. For example, effector proteins can include, but are not limited to, a restriction endonuclease that targets a host cell DNA sequence (whether genomic or on an extrachromosomal element), a protease that degrades a polypeptide target necessary for cell survival, a DNA gyrase inhibitor, and a ribonuclease-type toxin. 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 range and complexity of a biological circuit system’s responsiveness. Transcriptional regulators refer to transcriptional activators and repressors that either activate or repress transcription of a gene of interest. Promoters are regions of nucleic acid that initiate transcription of a particular gene. Transcriptional activators typically bind nearby to transcriptional promoters and recruit RNA polymerase to directly initiate transcription. Repressors bind to transcriptional promoters and sterically hinder transcriptional initiation by RNA polymerase. Other transcriptional regulators may serve as either an activator or a repressor depending on where they bind and cellular and environmental conditions. Non-limiting examples of transcriptional regulator classes include, but are not limited to homeodomain proteins, zinc-finger proteins, winged-helix (forkhead) proteins, and leucine-zipper proteins. As used herein, a “repressor protein” or “inducer protein” is a protein that binds to a regulatory sequence element and represses or activates, respectively, the transcription of sequences 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. 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 can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce a toxic, an allergic, or similar untoward reaction when administered to a host. As used herein, an “input agent responsive domain” is a domain of a transcription factor that binds to or otherwise responds to a condition or input agent in a manner that renders a linked DNA binding fusion domain responsive to the presence of that condition or input. In one embodiment, the presence of the condition or input results in a conformational change in the input agent responsive domain, or in a protein to which it is fused, that modifies the transcription-modulating activity of the transcription factor. As used herein, the term “in vivo” refers to assays or processes that occur in or within an organism, such as a multicellular animal. In some of the aspects described herein, a method or use can be said to occur “in vivo” when a unicellular organism, such as a bacterium, is used. The term “ex vivo” refers to methods and uses that are performed using a living cell with an intact membrane that is outside of the body of a multicellular animal or plant, e.g., explants, cultured cells, including primary cells and cell lines, transformed cell lines, and extracted tissue or cells, including blood cells, among others. The term “in vitro” refers to assays and methods that do not require the presence of a cell with an intact membrane, such as cellular extracts, and can refer to the introducing of a programmable synthetic biological circuit in a non-cellular system, such as a medium not comprising cells or cellular systems, such as cellular extracts. As used herein, the term “promoter” refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving transcription of the nucleic acid sequence, which can be a heterologous target gene encoding a protein or an RNA. Promoters can be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter can also contain genetic elements at which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive the expression of transgenes in the single-stranded (ssDNA) molecules disclosed herein. A promoter sequence may be bounded at its 3’ terminus by the transcription initiation site and extends upstream (5’ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. As used herein, the terms “expression cassette” and “expression unit” are used interchangeably and refer to a heterologous DNA sequence that is operably linked to a promoter or other DNA regulatory sequence sufficient to direct transcription of a transgene of a DNA vector, e.g., a single-stranded (ssDNA) molecule. Suitable promoters include, for example, tissue specific promoters. Promoters can also be of AAV origin. As used herein, the term “regenerated”, when referring to a “regenerated double-stranded expression cassette” or a “regenerated double-stranded transgene” is meant to refer to the double stranded expression cassette or double-stranded transgene that is formed after a ssDNA molecule has been transported to the nucleus of a host cell and is responsive to DNA polymerase activity that creates double stranded DNA from the ssDNA by filling in the single strand portion of the ssDNA molecule. As used herein, “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. A promoter can be said to drive expression or drive transcription of the nucleic acid sequence that it regulates. The phrases “operably linked,” “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” indicate that a promoter is in a correct functional location and / or orientation in relation to a nucleic acid sequence it regulates to control transcriptional initiation and / or expression of that sequence. An “inverted promoter,” as used herein, refers to a promoter in which the nucleic acid sequence is in the reverse orientation, such that what was the coding strand is now the non-coding strand, and vice versa. Inverted promoter sequences can be used in various embodiments to regulate the state of a switch. In addition, in various embodiments, a promoter can be used in conjunction with an enhancer. The terms “DNA regulatory sequences,” “control elements,” and “regulatory elements,” 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 for and / or regulate transcription of a non-coding sequence (e.g., DNA-targeting RNA) or a coding sequence (e.g., site-directed modifying polypeptide, or Cas9 / Csn1 polypeptide) and / or regulate translation of an encoded polypeptide. The term “enhancer” as used herein refers to a cis-acting regulatory sequence (e.g., 50-1,500 base pairs) that binds one or more proteins (e.g., activator proteins, or transcription factor) to increase transcriptional activation of a nucleic acid sequence. Naturally, enhancers can be positioned up to 1,000,000 base pars upstream of the gene start site or downstream of the gene start site that they regulate. An enhancer can be positioned within an intronic region, or in the exonic region of an unrelated gene. A cis-acting enhancer sequence of 20-200 base pairs can be typically used to increase expression of a transgene. A promoter can be one naturally associated with a gene or sequence, as can be obtained by isolating the 5’ non-coding sequences located upstream of the coding segment and / or exon of a given gene or sequence. Such a promoter can be referred to as “endogenous.” Similarly, in some embodiments, an enhancer can be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. In some embodiments, a coding nucleic acid segment is positioned under the control of a “recombinant promoter” or “heterologous promoter,” both of which refer to a promoter that is not normally associated with the encoded nucleic acid sequence that it is operably linked to in its natural environment. Similarly, a “recombinant or heterologous enhancer” refers to an enhancer 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., comprise different elements of different transcriptional regulatory regions, and / or mutations that alter expression through methods of genetic engineering that are known in the art. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, promoter sequences can be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR, in connection with the synthetic biological circuits and modules disclosed herein (see, e.g., U.S. Pat. No.4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well. As described herein, an “inducible promoter” is one that is characterized by initiating or enhancing transcriptional activity when in the presence of, influenced by, or contacted by an inducer or inducing agent. An “inducer” or “inducing agent,” as defined herein, can be endogenous, or a normally exogenous compound or protein that is administered in such a way as to be active in inducing transcriptional activity from the inducible promoter. In some embodiments, the inducer or inducing agent, i.e., a chemical, a compound or a protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., an inducer can be an inducer protein expressed by another component or module), which itself can be under the control or an inducible promoter. In some embodiments, an inducible promoter is induced in the absence of certain agents, such as a repressor. Examples of inducible promoters include but are not limited to, tetracycline, metallothionine, ecdysone, mammalian viruses (e.g., the adenovirus late promoter; and the mouse mammary tumor virus long terminal repeat (MMTV-LTR)) and other steroid-responsive promoters, rapamycin responsive promoters and the like. The term “subject” as used herein refers to a human or animal, to whom treatment, including prophylactic treatment, with the single-stranded (ssDNA) molecule according to the present disclosure, is provided. Usually, the animal is a vertebrate such as, but not limited to a non-human primate, rodent, domestic animal or game animal. Non-human primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include, but are not limited to, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, but are not limited to, cows, horses, pigs, goats, sheep, llamas, camels, deer, bison, buffalo, feline species (e.g., domestic cats), canine species (e.g., dogs, foxes, and wolves), avian species (e.g., chickens, turkeys, geese, emus, ostriches, penguins, falcons, and eagles), marine mammals (e.g., dolphins, porpoises, orcas, whales, seals, sealions, walruses, and sea otters), and fish (e.g., trout, catfish, tuna, salmon, sharks, guppies, mollies, swordtails, goldfish, and carp). In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a non-human primate or a human. A subject can be male or female. Additionally, a subject can be an infant or a child. In some embodiments, the subject can be a neonate or an unborn subject, e.g., the subject is in utero. Mammals other than humans can be advantageously used as subjects that represent animal models of diseases and disorders. In addition, the methods and compositions described herein can be used for domesticated animals and / or pets, and / or for farm or game animals. A human subject can be of any age, gender, race or ethnic group, e.g., Caucasian (white), Asian, African, black, African American, African European, Hispanic, Mideastern, etc. In some embodiments, the subject can be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment. In some embodiments, the subject is an embryo, a fetus, neonate, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, human neonate, human infant, human child, human adolescent, or human adult. In some embodiments, the subject is an animal embryo, or non-human embryo or non-human primate embryo. In some embodiments, the subject is a human embryo. As used herein, the term “host cell” includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a partially single-stranded (ssDNA) molecule described by the present disclosure. As non-limiting examples, a host cell can be an isolated primary cell, pluripotent stem cells, CD34+cells, induced pluripotent stem cells, or any of a number of immortalized cell lines (e.g., HepG2 cells). Alternatively, a host cell can be an in situ or in vivo cell in a tissue, organ or organism. Furthermore, a host cell can be a target cell of, for example, a mammalian subject (e.g., human patient in need of gene therapy). As used herein, the term “exogenous” refers to a substance present in a cell other than its native source. The term “exogenous” when used herein can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term “endogenous” refers to a substance that is native to the biological system or cell. The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes single, double, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer including purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. “Oligonucleotide” generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- 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 known as “oligomers” or “oligos” and may be isolated from genes, or chemically synthesized by methods known in the art. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiments being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. According to some embodiments, the nucleic acid is a single-stranded DNA (ssDNA) molecule described by the present disclosure. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. DNA may be in the form of minicircle, plasmid, bacmid, minigene, ministring DNA (linear covalently closed DNA vector), closed-ended linear duplex DNA (CELiD or ceDNA), doggybone (dbDNA ™) DNA, dumbbell shaped DNA, minimalistic immunological-defined gene expression (MIDGE)-vector, viral vector or nonviral vectors. RNA may be in the form of small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs and / or modified residues include, without limitation, phosphorothioates, phosphorodiamidate morpholino oligomer (morpholino), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, locked nucleic acid (LNA™), and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as 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 sequence explicitly indicated. An “inhibitory polynucleotide” as used herein refers to a DNA or RNA molecule that reduces or prevents 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, e.g., RNAi that encode the actual inhibitory species, such as DNA molecules that encode ribozymes. “Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. “Bases” include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides. By “hybridizable” or “complementary” or “substantially complementary” it is meant that a nucleic acid (e.g., RNA) includes a sequence of nucleotides that enables it to non-covalently bind, i.e. form Watson-Crick base pairs and / or G / U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C). In addition, 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, G / U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. In the context of this disclosure, a guanine (G) of a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered complementary to a uracil (U), and vice versa. As such, when a G / U base-pair can be made at a given nucleotide position a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary. The term “nucleic acid construct” as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present disclosure. An “expression cassette” includes a DNA coding sequence operably linked to a promoter. As used herein, the phrases “nucleic acid therapeutic”, “therapeutic nucleic acid” and “TNA” are used interchangeably and refer to any modality of therapeutic using nucleic acids as an active component of therapeutic agent to treat a disease or disorder. 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 RNAs (RNAi), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA) and guide RNA (gRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigene, viral DNA (e.g., Lentiviral or AAV genome) or non- viral synthetic DNA vectors, closed-ended linear duplex DNA (ceDNA / CELiD), plasmids, bacmids, doggybone (dbDNA™) DNA vectors, minimalistic immunological-defined gene expression (MIDGE)-vector, nonviral ministring DNA vector (linear-covalently closed DNA vector), or dumbbell-shaped DNA minimal vector (“dumbbell DNA”). The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. As used herein, the term “sequence identity” refers to the relatedness between two nucleotide sequences. For purposes of the present disclosure, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides.times.100) / (Length of Alignment-Total Number of Gaps in Alignment). The length of the alignment is preferably at least 10 nucleotides, preferably at least 25 nucleotides more preferred at least 50 nucleotides and most preferred at least 100 nucleotides. As used herein, the term “homology” or “homologous” as used herein is defined as the percentage of nucleotide residues in the homology arm that are identical to the nucleotide residues in the corresponding sequence on the target chromosome, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleotide sequence homology can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST- 2, ALIGN, ClustalW2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In some embodiments, a nucleic acid sequence (e.g., DNA sequence), for example of 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. As used herein, a “homology arm” refers to a polynucleotide that is suitable to target a donor sequence to a genome through homologous recombination. Typically, two homology arms flank the donor sequence, wherein each homology arm comprises genomic sequences upstream and downstream of the loci of integration. As used herein, “a donor sequence” refers to a polynucleotide that is to be inserted into, or used as a repair template for, a host cell genome. The donor sequence can comprise the modification which is desired to be made 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 an alteration of the target sequence from the original target sequence to the sequence comprised by the donor sequence. Accordingly, the sequence comprised by the donor sequence can be, relative to the target sequence, an insertion, a deletion, an indel, a point mutation, a repair of a mutation, etc. The donor sequence can be, e.g., 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 arms. The editing can be RNA as well as DNA editing. The donor sequence can be endogenous to or exogenous to the host cell genome, depending upon 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 found in the native nucleic acid or protein, respectively. A heterologous nucleic acid sequence may be linked to a naturally occurring nucleic acid sequence (or a variant thereof) (e.g., by genetic engineering) to generate a chimeric nucleotide sequence encoding a chimeric polypeptide. A heterologous nucleic acid sequence may be linked to a variant polypeptide (e.g., by genetic engineering) to generate a nucleotide sequence encoding a fusion variant polypeptide. As used herein, a “vector” or “expression vector” is a replicon, such as plasmid, bacmid, phage, virus, virion, or cosmid, to which another DNA segment, i.e., an “insert” “transgene” or “expression cassette”, may be attached so as to bring about the expression or replication of the attached segment (“expression cassette”) in a cell. A vector can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral in origin in the final form. Accordingly, the term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. In some embodiments, a vector can be a recombinant vector or an expression vector. For the purposes of the present disclosure, a vector of the present invention generally refers to a partially ssDNA molecule or a circular ssDNA molecule. As used herein, the phrase “recombinant vector” means a vector that includes a heterologous nucleic acid sequence, or “transgene” that is capable of expression in vivo. It is to be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration. As used herein, the term “expression vector” refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the host cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. The expression vector may be a recombinant vector. As used herein, the term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. As used herein, the phrase “expression products” include RNA transcribed from a gene (e.g., transgene), and polypeptides obtained by translation of mRNA transcribed from a gene. As used herein, the term “gene” means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g., 5’ untranslated region (5’ UTR) or “leader” sequences and 3’ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons). As used herein, the term “gene delivery” means a process by which foreign DNA is transferred to host cells for applications of gene therapy. As used herein, the term “gene editing molecule” refers to one or more of a protein or a nucleic acid encoding for a protein, wherein the protein is selected from the group comprising a transposase, a nuclease, an integrase, a guide RNA (gRNA), a guide DNA, a ribonucleoprotein (RNP), or an activator RNA. A nuclease gene editing molecule is a protein having nuclease activity, with nonlimiting examples including: a CRISPR protein (Cas), CRISPR associated protein 9 (Cas9); a type IIS restriction enzyme; a transcription activator-like effector nuclease (TALEN); and a zinc finger nuclease (ZFN), a meganuclease, engineered site-specific nucleases or deactivated CAS for CRISPRi or CRISPRa systems. The gene editing molecule can also comprise a DNA-binding domain and a nuclease. In certain embodiments, the gene editing molecule comprises a DNA-binding domain and a nuclease. In certain embodiments, the DNA-binding domain comprises a guide RNA. In certain embodiments, the DNA-binding domain comprises a DNA-binding domain of a TALEN. In certain embodiments at least one gene editing molecule comprises one or more transposable element(s). In certain embodiments, the one or more transposable element(s) comprise a circular DNA. In certain embodiments, the one or more transposable element(s) comprise a plasmid vector or a minicircle DNA vector. In certain embodiments, the DNA-binding domain comprises a DNA-binding domain of a zinc-finger nuclease. In certain embodiments at least one gene editing molecule comprises one or more transposable element(s). In certain embodiments, the one or more transposable element(s) comprise a linear DNA. The linear recombinant and non-naturally occurring DNA sequence encoding a transposon may be produced in vitro. Linear recombinant and non-naturally occurring DNA sequences of the disclosure may be a product of restriction digest of a circular DNA. In certain embodiments, the circular DNA is a plasmid vector or a minicircle DNA vector. Linear recombinant and non-naturally occurring DNA sequences of the disclosure may be a product of a polymerase chain reaction (PCR). Linear recombinant and non-naturally occurring DNA sequences of the disclosure may be a double-stranded DOGGYBONE™ DNA sequence. DOGGYBONE™ DNA sequences of the disclosure may be produced by an enzymatic process that solely encodes an antigen expression cassette, comprising antigen, promoter, poly-A tail and telomeric ends. As used herein, the term “gene editing functionality” refers to the insertion, deletion or replacement of DNA at a specific site in the genome with a loss or gain of function. The insertion, deletion or replacement of DNA at a specific site can be accomplished e.g., by homology-directed repair (HDR) or non-homologous end joining (NHEJ), or single base change editing. In some embodiments, a donor template is used, for example for HDR, such that a desired sequence within the donor template is inserted into the genome by a homologous recombination event. In one embodiment, a “donor template” or “repair template” comprises two homology arms (e.g., a 5’ homology arm and a 3’ homology arm) flanking on either side of a donor sequence comprising a desired mutation or insertion in the nucleic acid sequence to be introduced into the host genome. The 5’ and 3’ homology arms are substantially homologous to the genomic sequence of the target gene at the site of endonuclease mediated cutting. The 3’ homology arm is generally immediately downstream of the protospacer adjacent motif (PAM) site where the endonuclease cuts (e.g., a double stranded DNA cut), or in some embodiments, nicks the DNA. As used herein, the term “gene editing system” refers to the minimum components necessary to effect genome editing in a cell. For example, a zinc finger nuclease or TALEN system may only require expression of the endonuclease fused to a nucleic acid complementary to the sequence of a target gene, whereas for a CRISPR / Cas gene editing system the minimum components may require e.g., a Cas endonuclease and a guide RNA. The gene editing system can be encoded on a single ceDNA vector or multiple vectors, as desired. Those of skill in the art will readily understand the component(s) necessary for a gene editing system. As used herein, the term “base editing moiety” refers to an enzyme or enzyme system that can alter a single nucleotide in a sequence, for example, 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 an adenine / thymine “A / T” nucleotide pair to a guanine / cytosine “G / C” nucleotide pair (see e.g., Gaudelli et al. Nature (2017), in press doi:10.1038 / nature24644, the contents of which are incorporated herein by reference in its entirety). As used herein, the term “genomic safe harbor gene” or “safe harbor gene” refers to a gene or loci that a nucleic acid sequence can be inserted such that the sequence can integrate and function in a predictable manner (e.g., express a protein of interest) without significant negative consequences to endogenous gene activity, or the promotion of cancer. In some embodiments, a safe harbor gene is also a loci or gene where an inserted nucleic acid sequence can be expressed efficiently and at higher levels than a non-safe harbor site. As used herein, the term “gene delivery” refers to a process by which foreign DNA is transferred to host cells for applications of gene therapy. As used herein, the term “CRISPR” stands for Clustered Regularly Interspaced Short Palindromic Repeats, which are the hallmark of a bacterial defense system that forms the basis for CRISPR-Cas9 genome editing technology. As used herein, the term “homologous recombination” refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. Homologous recombination also produces new combinations of DNA sequences. These new combinations of DNA represent genetic variation. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of viruses. The terms “correcting”, “genome editing” and “restoring” as used herein refers to changing a mutant gene that encodes a truncated protein or no protein at all, such that a full-length functional or partially full-length functional protein expression is obtained. Correcting or restoring a mutant gene may include replacing the region of the gene that has the mutation or replacing the entire mutant gene with a copy of the gene that does not have the mutation with a repair mechanism such as homology- directed repair (HDR). Correcting or restoring a mutant gene may also include repairing a frameshift mutation that causes a premature stop codon, an aberrant splice acceptor site or an aberrant splice donor site, by generating a double stranded break in the gene that is then repaired using non- homologous end joining (NHEJ). NHEJ may add or delete at least one base pair during repair which may restore the proper reading frame and eliminate the premature stop codon. Correcting or restoring a mutant gene may also include disrupting an aberrant splice acceptor site or splice donor sequence. Correcting or restoring a mutant gene may also include deleting a non-essential gene segment by the simultaneous action of two nucleases on the same DNA strand in order to restore the proper reading frame by removing the DNA between the two nuclease target sites and repairing the DNA break by NHEJ. The phrase “non-homologous end joining (NHEJ) pathway” as used herein refers to a pathway that repairs double-strand breaks in DNA by directly ligating the break ends without the need for a homologous template. The template-independent re-ligation of DNA ends by NHEJ is a stochastic, error-prone repair process that introduces random micro-insertions and micro-deletions (indels) at the DNA breakpoint. This method may be used to intentionally disrupt, delete, or alter the reading frame of targeted gene sequences. NHEJ typically uses short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the end of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately, yet imprecise repair leading to loss of nucleotides may also occur, but is much more common when the overhangs are not compatible “Nuclease mediated NHEJ” as used herein refers to NHEJ that is initiated after a nuclease, such as a cas9 or other nuclease, cuts double stranded DNA. In a CRISPR / CAS system NHEJ can be targeted by using a single guide RNA sequence. The terms “site-specific nuclease” or “sequence specific nuclease” as used herein refers to an enzyme capable of specifically recognizing and cleaving DNA sequences. The site-specific nuclease may be engineered. Examples of engineered site-specific nucleases include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR / Cas-based systems, that use various natural and unnatural Cas enzymes. The phrase “genetic disease” as used herein refers to a disease, partially or completely, directly or indirectly, caused by one or more abnormalities in the genome, especially a condition that is present from birth and can be treated by a single-stranded (ssDNA) molecule as described herein. The abnormality may be a mutation, an insertion or a deletion. The abnormality may affect the coding sequence of the gene or its regulatory sequence. The genetic disease may be, but not limited to phenylketonuria (PKU), sickle cell disease, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis, Huntington’s chorea, familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson’s disease, congenital hepatic porphyria, inherited disorders of hepatic metabolism, Lesch-Nyhan syndrome, thalassaemias, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, and mucopolysaccharide storage diseases (e.g., Hurler syndrome (MPS Type I), Scheie syndrome (MPS Type I S), Hurler-Scheie syndrome (MPS Type I H-S), 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), Tay-Sachs disease, Metachromatic Leukodystrophy, Krabbe disease, Mucolipidosis Type I, II / III and IV, Sialidosis Types I and II, Glycogen Storage disease Types I and II (Pompe disease), Gaucher disease Types I, II and III, cystinosis, Batten disease, Aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), Lysosomal Acid Lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipidoses, galactosialidosis. Also included in genetic disorders are 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 (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. As used herein, the term “treat” or “treating” and / or “treatment” is meant to refer to abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms of a condition, obtaining beneficial or desired clinical results. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s). In some embodiments, treating encompasses gene editing. In some embodiments, treating encompasses gene therapy. Beneficial or desired clinical results, such as pharmacologic and / or physiologic effects include, but are not limited to, preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization (i.e., not worsening) of the disease, disorder or condition, preventing spread of the disease, disorder or condition, delaying or slowing of the disease, disorder or condition progression, amelioration or palliation of the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment. As used herein, the term “increase,” “enhance,” “raise” (and like terms) generally refers to the act of increasing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition. As used herein, the term “suppress,” “decrease,” “interfere,” “inhibit” and / or “reduce” (and like terms) generally refers to the act of reducing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition. As used herein, the term “synthetic AAV vector”, “single-stranded (ss) synthetic AAV vector” and “synthetic production of AAV vector” refers to an AAV vector and synthetic production methods thereof in a cell-free environment. In some embodiments, a partially ssDNA molecule may be referred to herein as a “synthetic AAV vector”. However, it should also be understood that the term “synthetic AAV vector” may not actually comprise any AAV-derived sequences. As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, processes, and respective component(s) thereof, that are essential to the processes, methods or compositions, yet open to the inclusion of unspecified elements, whether essential or not. The use of “comprising” indicates inclusion rather than limitation. The term “consisting of” refers to compositions, methods, processes, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. 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. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g.” is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%. The present disclosure is further explained in detail by the following examples, but the scope of the disclosure should not be limited thereto. Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. In some embodiments of any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes. Other terms are defined herein within the description of the various aspects of the disclosure. All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims. Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure. The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. II. Partially Single-Stranded (ss) DNA Molecules As described herein, the present disclosure relates to isolated synthetic, partially single- stranded (ssDNA) molecules. As illustrated in the Examples and Figures herein, the partially ssDNA molecules described herein demonstrate increased transgene expression and also do not activate or minimally activate an immune response when administered to a subject (e.g., a mouse, non-human primate, or human subject). As such, the ssDNA molecules described herein are advantageous for use in vivo gene expression, e.g., transgene expression for gene therapy. In some aspects, the partially ssDNA molecules comprise one or more double-stranded regions and at least one nucleic acid sequence of interest, the partially ssDNA comprising: a central region, wherein the central region comprises at least one single stranded region and at least one double-stranded region, and wherein the central region comprises the at least one nucleic acid sequence of interest; at least a first stem-loop structure at its 3’ end, wherein the first stem-loop structure comprises at least one stem and at least one loop, and / or at least a second stem-loop structure at its 5’ end, wherein the second stem-loop structure comprises at least one stem and at least one loop, wherein the first stem-loop structure and / or the second stem-loop structure flank the central region, and wherein the central region comprises at total of at least 50 base pairs (bp) of double-stranded DNA, and / or wherein the central region is at least 5% double-stranded. In some embodiments, the partially ssDNA molecule described herein comprises a single, linear DNA molecule with some double-single stranded regions and some double-stranded regions (that is, if placed under denaturing conditions, the ssDNA molecule would comprise a single, linear DNA molecule). In some embodiments, described in more detail below, a partially ssDNA molecule may comprise one more double-stranded regions produced by hybridizing noncovalently-bound oligonucleotides to a single-stranded region of an ssDNA molecule (in which, case, if placed under denaturing conditions, the oligonucleotides would dissociate from the ssDNA molecule). A. Central Region As used herein the term “central region” of an ssDNA molecule refers to the nucleic acid sequences in between the terminal structures, e.g., between the terminal stem-loop structures. The central region is generally understood to comprise only single-stranded DNA or canonical B-form DNA (i.e., double-stranded DNA). The ends of the central region are the locations of the nearest predicted loop or helical junction (i.e., likely departure from canonical B-form DNA) in the terminal stem-loop structures. The central region does not include double-stranded portions distal to the nearest predicted loop or helical junction (e.g., the arms of a hammerhead or quadraplex structure). For example, as shown in FIG.129, top, the central region comprises the region in between the two helical junctions (each of which is at the end of a “7 bp” label), and includes, from left to right, the double-stranded regions labeled as “7 bp”, “36 bp”, and “7 bp”, as well as the single-stranded region. The central region does not include the portions of the hammerhead ITRs labeled as “9 bp”. In some embodiments, the central region may be understood to include and overlap with all or part of the stem of a terminal stem-loop structure. For example, in a construct comprising simple hairpin ends (e.g., FIG.129, bottom), the central region (which comprises, from left to right, the double-stranded regions labeled as “21 bp”, “36 bp”, and “20 bp”, as well as the single-stranded region) overlaps with the terminal stem structures, and comprises the entirety of the construct except for the terminal 3- nucleotide loops. The amount of single-strandedness of a central region of an ssDNA molecule may be expressed as the total absolute number of single-stranded (unpaired) nucleotides, or it may be expressed as a percentage of the nucleotides in the central-region that are single-stranded. Similarly, the amount of double-strandedness of a central region of an ssDNA molecule may be expressed as the total absolute number of double-stranded base pairs, or it may be expressed as a percentage of the nucleotides in the central-region that are double-stranded. It should be understood that each base pair is counted as “1” (not “2”, even though a base pair actually contains two nucleotides hybridized to each other). As used herein, the “total” amount of double-strandedness of a central region is understood to comprise all regions of double-strandedness in a central region. For example, a central region with 100 base pairs of double-stranded DNA at its 3’ end and 100 base pairs of double-stranded DNA at its 5’ end is understood to have a total of 200 base pairs of double-stranded DNA. Further, a central region with 100 base pairs of double-stranded DNA at its 3’ end and 100 base pairs of double- stranded DNA at its 5’ end is understood to have a total of 200 base pairs of double-stranded DNA. When expressed as a percentage, a central region with 100 base pairs of double-stranded DNA at its 3’ end, 200 single-stranded nucleotides, and 100 base pairs of double-stranded DNA at its 5’ end is understood to be 50% double-stranded and 50% single-stranded. The ssDNA molecules of the present disclosure may, as described elsewhere herein, comprise hybridized, non-covalently bound oligonucleotides that produce double-strandedness in portions of the central region. For example, a central region with 100 base pairs of double-stranded DNA at its 3’ end, 100 base pairs of double-stranded DNA at its 5’ end, and a hybridized 100-nucleotide oligonucleotide in between is understood to have a total of 300 base pairs of double-stranded DNA. The length of the double-stranded regions at or adjacent to the 3’ and 5’ ends is typically determined by counting the number of contiguous double-stranded base pairs from the nearest predicted loop or helical junction (i.e., likely departure from canonical B-form DNA) and does not include double-stranded portions distal to the nearest predicted loop or helical junction (e.g., the arms of a hammerhead or quadraplex ITR structure). For example, the ssDNA construct with AAV2- derived ITRs in FIG.129 (top) comprises 43 bp adjacent to the 3’ end (left side) and 7 bp adjacent to the 5’ end (right side), and the ssDNA construct with complete nonviral hairpin ITRs (FIG.129, bottom) comprises 57 bp adjacent to the 3’ end (left side) and 20 bp adjacent to the 5’ end (right side). In some embodiments, the length of the double-stranded regions adjacent to the 3’ and 5’ ends may be counted without including the double-stranded portion derived from an ITR oligonucleotide used in the manufacturing process (referred to herein as the “alternate double- stranded counting” method). In such embodiments, the ssDNA construct with AAV2-derived ITRs in FIG.129 (top) would comprise 36 bp adjacent to the 3’ end (that is, it would not include the 7 bp derived from the ITR oligonucleotide), while the ssDNA construct with complete nonviral hairpin ITRs (FIG.129, bottom) would comprise 36 bp adjacent to the 3’ end (that is, it would not include the 21 bp derived from the ITR oligonucleotide). However, unless specifically noted, the length of the double-stranded regions adjacent to the 3’ or 5’ ends is understood not to be counted using the alternate double-stranded counting method. As used herein, double-stranded regions “at the 3’ end” and “adjacent to the 3’ end” are understood to refer to the double-stranded regions that comprise, on one strand, the 3’ end of the ssDNA molecule. For example, construct ss148 (shown in FIG.148) comprises 179 base pairs of double-stranded DNA at or adjacent to its 3’ end. As used herein, double-stranded regions “at the 5’ end” and “adjacent to the 5’ end” are understood to refer to the double-stranded regions that comprise, on one strand, the 5’ end of the ssDNA molecule. For example, construct ss155 (shown in FIG.150) comprises 504 base pairs of double-stranded DNA at or adjacent to its 5’ end. Accordingly, in some embodiments, an ssDNA molecule may comprise a central region having a total of 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, at least 1500 base pairs, at least 1600 base pairs, at least 1700 base pairs, at least 1800 base pairs, at least 1900 base pairs, at least 2000 base pairs, at least 2100 base pairs, at least 2200 base pairs, at least 2300 base pairs, at least 2400 base pairs, or at least 2500 base pairs of double-stranded DNA. In some embodiments, the central region comprises a total of fewer than 2500 base pairs, fewer than 2400 base pairs, fewer than 2300 base pairs, fewer than 2200 base pairs, fewer than 2100 base pairs, fewer than 2000 base pairs, fewer than 1900 base pairs, fewer than 1800 base pairs, fewer than 1700 base pairs, fewer than 1600 base pairs, fewer than 1500 base pairs, fewer than 1400 base pairs, fewer than 1300 base pairs, fewer than 1200 base pairs, fewer than 1100 base pairs, fewer than 1000 base pairs, fewer than 950 base pairs, fewer than 900 base pairs, fewer than 850 base pairs, fewer than 800 base pairs, fewer than 750 base pairs, fewer than 700 base pairs, fewer than 650 base pairs, fewer than 600 base pairs, fewer than 550 base pairs, fewer than 500 base pairs, fewer than 480 base pairs, fewer than 460 base pairs, fewer than 440 base pairs, fewer than 420 base pairs, fewer than 400 base pairs, fewer than 380 base pairs, fewer than 360 base pairs, fewer than 340 base pairs, fewer than 320 base pairs, fewer than 300 base pairs, fewer than 280 base pairs, fewer than 260 base pairs, fewer than 240 base pairs, fewer than 220 base pairs, fewer than 200 base pairs, fewer than 190 base pairs, fewer than 180 base pairs, fewer than 170 base pairs, fewer than 160 base pairs, fewer than 150 base pairs, fewer than 140 base pairs, fewer than 130 base pairs, fewer than 120 base pairs, fewer than 110 base pairs, fewer than 100 base pairs, fewer than 90 base pairs, fewer than 80 base pairs, fewer than 70 base pairs, or fewer than 60 base pairs of double-stranded DNA. In some embodiments, the central region comprises a total of about 50-2500 base pairs of double-stranded DNA, about 100-2400 base pairs of double-stranded DNA, about 200-2300 base pairs of double-stranded DNA, about 300-2200 base pairs of double-stranded DNA, about 400-2100 base pairs of double-stranded DNA, about 500-2000 base pairs of double-stranded DNA, about 500- 1800 base pairs of double-stranded DNA, about 500-1600 base pairs of double-stranded DNA, about 500-1400 base pairs of double-stranded DNA, about 500-1200 base pairs of double-stranded DNA, about 500-1000 base pairs of double-stranded DNA, or about 500-800 base pairs of double-stranded DNA. In some embodiments, the central region comprises a total of about 100-200, 200-300, 300- 400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000, 2000-2100, 2100- 2200, 2200-2300, 2300-2400, or 2400-2500 base pairs of double-stranded DNA. In some embodiments, the central region comprises a total of about 100-300, 300-500, 500-700, 700-900, 900- 1100, 1100-1300, 1300-1500, 1500-1700, 1700-1900, 1900-2100, 2100-2300, or 2300-2500 base pairs of double-stranded DNA. In some embodiments, the central region comprises a total of about 100-500, 500-1000, 1000-1500, 1500-2000, or 2000-2500 base pairs of double-stranded DNA. In some embodiments, the central region comprises 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, at least 1500 base pairs, at least 1600 base pairs, at least 1700 base pairs, at least 1800 base pairs, at least 1900 base pairs, at least 2000 base pairs, at least 2100 base pairs, at least 2200 base pairs, at least 2300 base pairs, at least 2400 base pairs, or at least 2500 base pairs of double-stranded DNA at the 3’ end of the ssDNA molecule. In some embodiments, the central region comprises fewer than 2500 base pairs, fewer than 2400 base pairs, fewer than 2300 base pairs, fewer than 2200 base pairs, fewer than 2100 base pairs, fewer than 2000 base pairs, fewer than 1900 base pairs, fewer than 1800 base pairs, fewer than 1700 base pairs, fewer than 1600 base pairs, fewer than 1500 base pairs, fewer than 1400 base pairs, fewer than 1300 base pairs, fewer than 1200 base pairs, fewer than 1100 base pairs, fewer than 1000 base pairs, fewer than 950 base pairs, fewer than 900 base pairs, fewer than 850 base pairs, fewer than 800 base pairs, fewer than 750 base pairs, fewer than 700 base pairs, fewer than 650 base pairs, fewer than 600 base pairs, fewer than 550 base pairs, fewer than 500 base pairs, fewer than 480 base pairs, fewer than 460 base pairs, fewer than 440 base pairs, fewer than 420 base pairs, fewer than 400 base pairs, fewer than 380 base pairs, fewer than 360 base pairs, fewer than 340 base pairs, fewer than 320 base pairs, fewer than 300 base pairs, fewer than 280 base pairs, fewer than 260 base pairs, fewer than 240 base pairs, fewer than 220 base pairs, fewer than 200 base pairs, fewer than 190 base pairs, fewer than 180 base pairs, fewer than 170 base pairs, fewer than 160 base pairs, fewer than 150 base pairs, fewer than 140 base pairs, fewer than 130 base pairs, fewer than 120 base pairs, fewer than 110 base pairs, fewer than 100 base pairs, fewer than 90 base pairs, fewer than 80 base pairs, fewer than 70 base pairs, or fewer than 60 base pairs of double-stranded DNA at the 3’ end of the ssDNA molecule. In some embodiments, the central region comprises 1381 base pairs of double-stranded DNA at the 3’ end of the ssDNA molecule, or 504 base pairs of double-stranded DNA at the 3’ end of the ssDNA molecule. In some embodiments, the central region comprises a total of about 531 base pairs of double-stranded DNA at the 3’ end of the ssDNA molecule. In some embodiments, the central region comprises a total of about 1404 base pairs of double-stranded DNA at the 3’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 50 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 50 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 100 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 100 base pairs of double- stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 150 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 150 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 200 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 200 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 250 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 250 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 300 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 300 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 350 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 350 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 400 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 400 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 450 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 450 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 500 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 500 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 550 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 550 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 600 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 600 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments the central region comprises at least 650 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 650 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 700 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 700 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 750 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 750 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 800 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 800 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 900 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 900 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region comprises at least 1000 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 1000 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule. In some embodiments, the central region is at least about 50% single-stranded, at least about 51% single-stranded, at least about 52% single-stranded, at least about 53% single-stranded, at least about 54% single-stranded, at least about 55% single-stranded, at least about 56% single-stranded, at least about 57% single-stranded, at least about 58% single-stranded, at least about 59% single- stranded, at least about 60% single-stranded, at least about 61% single-stranded, at least about 62% single-stranded, at least about 63% single-stranded, at least about 64% single-stranded, at least about 65% single-stranded, at least about 66% single-stranded, at least about 67% single-stranded, at least about 68% single-stranded, at least about 69% single-stranded, at least about 70% single-stranded, at least about 71% single-stranded, at least about 72% single-stranded, at least about 73% single- stranded, at least about 74% single-stranded, at least about 75% single-stranded, at least about 76% single-stranded, at least about 77% single-stranded, at least about 78% single-stranded, at least about 79% single-stranded, at least about 80% single-stranded, at least about 81% single-stranded, at least about 82% single-stranded, at least about 83% single-stranded, at least about 84% single-stranded, at least about 85% single-stranded, at least about 86% single-stranded, at least about 87% single- stranded, at least about 88% single-stranded, at least about 89% single-stranded, at least about 90% single-stranded, at least about 91% single-stranded, at least about 92% single-stranded, at least about 93% single-stranded, at least about 94% single-stranded, or at least about 95% single-stranded. In some embodiments, the central region is less than about 50% single-stranded, less than about 51% single-stranded, less than about 52% single-stranded, less than about 53% single-stranded, less than about 54% single-stranded, less than about 55% single-stranded, less than about 56% single- stranded, less than about 57% single-stranded, less than about 58% single-stranded, less than about 59% single-stranded, less than about 60% single-stranded, less than about 61% single-stranded, less than about 62% single-stranded, less than about 63% single-stranded, less than about 64% single- stranded, less than about 65% single-stranded, less than about 66% single-stranded, less than about 67% single-stranded, less than about 68% single-stranded, less than about 69% single-stranded, less than about 70% single-stranded, less than about 71% single-stranded, less than about 72% single- stranded, less than about 73% single-stranded, less than about 74% single-stranded, less than about 75% single-stranded, less than about 76% single-stranded, less than about 77% single-stranded, less than about 78% single-stranded, less than about 79% single-stranded, less than about 80% single- stranded, less than about 81% single-stranded, less than about 82% single-stranded, less than about 83% single-stranded, less than about 84% single-stranded, less than about 85% single-stranded, less than about 86% single-stranded, less than about 87% single-stranded, less than about 88% single- stranded, less than about 89% single-stranded, less than about 90% single-stranded, less than about 91% single-stranded, less than about 92% single-stranded, less than about 93% single-stranded, less than about 94% single-stranded, or less than about 95% single-stranded. In some embodiments, the central region is about 50%-95% single-stranded, about 50%-70% single-stranded about 70%-95% single-stranded, about 50%-60% single-stranded, about 60%-70% single-stranded, about 70%-80%, about 80%-90% single-stranded, about 90%-95% single-stranded, about 50%-55% single-stranded, about 55%-60% single-stranded, about 60%-65% single-stranded, about 65%-70% single-stranded, about 70%-75% single-stranded, about 75%-80% single-stranded, about 80%-85% single-stranded, about 85%-90% single-stranded, about 90%-95% single-stranded. In some embodiments, the single-stranded region of the central region is on a (-) strand of the ssDNA molecule. As used herein, the “(-)” or “minus” strand of an ssDNA molecule refers to the strand comprising an anti-sense strand of nucleic acid sequence of interest (e.g., an open-reading frame of a transgene), while the “(+)” or “plus” strand of an ssDNA molecule refers to the strand comprising a sense strand of nucleic acid sequence of interest (e.g., an open-reading frame of a transgene). The ssDNA molecules described herein are generally constructed as (-) strand versions (e.g., versions comprising an intact (-) strand which is substantially single-stranded), unless specifically noted as being a (+) strand version (e.g., comprising an intact (+) strand which is substantially single-stranded). However, it should be understood that that the instant disclosure encompasses both (-) and (+) versions where either the (-) or the (+) is intact and substantially single- stranded. B. 3’ End As described herein, according to some aspects, the disclosure provides a partially ssDNA molecule comprising at least one nucleic acid sequence of interest flanked by at least one stem-loop structure at the 3’ end and / or 5’ end. As described herein, the stem structure at the 3’ end comprises a partial DNA duplex (e.g., with a free 3’-OH group) which may be used to prime replication or transcription. The partial DNA duplex functions, in part, to hold the stem-loop structure together. According to some embodiments, the partial DNA duplex comprises between 4-500 nucleotides, for example between 4-10 nucleotides, between 4-25 nucleotides, between 4-50 nucleotides, between 4-100 nucleotides, between 4-200 nucleotides, between 4-300 nucleotides, between 4-400 nucleotides, between 20-25 nucleotides, between 20-50 nucleotides, between 20-100 nucleotides, between 20-200 nucleotides, between 20-300 nucleotides, between 20-400 nucleotides, between 20-500 nucleotides, between 50-100 nucleotides, between 50-200 nucleotides, between 50- 300 nucleotides, between 50-400 nucleotides, between 50-500 nucleotides, 150-200 nucleotides, between 150-300 nucleotides, between 150-400 nucleotides, between 150-500 nucleotides, between 200-300 nucleotides, between 200-400 nucleotides, between 200-500 nucleotides, between 250-300 nucleotides, between 250-400 nucleotides, between 250-500 nucleotides, between 300-400 nucleotides, between 300-500 nucleotides, between 350-400 nucleotides, between 350-500 nucleotides, between 400-500 nucleotides, between 450-500 nucleotides, between 500-550 nucleotides, between 550-600 nucleotides, between 600-650 nucleotides, between 650-700 nucleotides, between 700-750 nucleotides, between 750-800 nucleotides, between 800-850 nucleotides, between 850-900 nucleotides, between 900-950 nucleotides, between 950-1000 nucleotides, between 1000-1050 nucleotides, between 1050-1100 nucleotides, between 1100-1150 nucleotides, between 1150-1200 nucleotides, between 1200-1250 nucleotides, between 1250-1300 nucleotides, between 1300-1350 nucleotides, between 1350-1400 nucleotides, between 1400-1450 nucleotides, between 1450-1500 nucleotides, between 1500-1550 nucleotides, between 1550-1600 nucleotides, between 1600-1650 nucleotides, between 1650-1700 nucleotides, between 1700-1750 nucleotides, between 1750-1800 nucleotides, between 1800-1850 nucleotides, between 1850-1900 nucleotides, between 1900-1950 nucleotides, between 1950-2000 nucleotides, between 2000-2050 nucleotides, between 2050-2100 nucleotides, between 2150-2200 nucleotides, between 2250-2300 nucleotides, between 2350-2400 nucleotides, or between 2450-2500 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, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500 or more nucleotides, and at least one loop on the 3’ end. According to some embodiments, the DNA duplex at the 3’ end may be described as being “adjacent” to the 3’ end. As used herein, the term “adjacent” to the 3’ end refers to a DNA duplex (i.e., a double-stranded region) in which one strand of the duplex (double-stranded region) comprises a free terminal nucleotide that terminates at the 3’ deoxyribose carbon atom. In some embodiments, the 3’ end comprises a free 3’-OH group. In some embodiments, the terminal nucleotide may comprise, e.g., a chemical modification or derivatization, such that it no longer comprises a free 3’- OH. Such embodiments are still referred to herein as the 3’ end, if the modification or derivatization replaces what would have been the 3’-OH. According to some embodiments, the loop structure at the 3’ end comprises a minimum of between 3-500 unbound nucleotides, for example between 3-450 nucleotides, between 3-400 nucleotides, between 3-350 nucleotides, between 3-300 nucleotides, between 3-250 nucleotides, between 3-200 nucleotides, between 3-150 nucleotides, between 3-100 nucleotides, between 3-90 nucleotides, between 3-80 nucleotides, between 3-70 nucleotides, between 3-60 nucleotides, between 3-50 nucleotides, between 3-40 nucleotides, between 3-30 nucleotides, between 3-20 nucleotides, between 3-10 nucleotides, between 3-5 nucleotides, between 10-450 nucleotides, between 10-400 nucleotides, between 10-350 nucleotides, between 10-300 nucleotides, between 10-250 nucleotides, between 10-200 nucleotides, between 10-150 nucleotides, between 10-100 nucleotides, between 10- 90 nucleotides, between 10-80 nucleotides, between 10-70 nucleotides, between 10-60 nucleotides, between 10-50 nucleotides, between 10-40 nucleotides, between 10-30 nucleotides, between 10-20 nucleotides, between 50-450 nucleotides, between 50-400 nucleotides, between 50-350 nucleotides, between 50-300 nucleotides, between 50-250 nucleotides, between 50-200 nucleotides, between 50- 150 nucleotides, between 50-100 nucleotides, between 50-90 nucleotides, between 50-80 nucleotides, between 50-70 nucleotides, between 50-60 nucleotides, between 100-450 nucleotides, between 100- 400 nucleotides, between 100-350 nucleotides, between 100-300 nucleotides, between 100-250 nucleotides, between 100-200 nucleotides, between 150-450 nucleotides, between 150-400 nucleotides, between 150-350 nucleotides, between 150-300 nucleotides, between 150-250 nucleotides, between 150-200 nucleotides, between 200-450 nucleotides, between 200-400 nucleotides, between 200-350 nucleotides, between 200-300 nucleotides, between 200-250 nucleotides, between 250-450 nucleotides, between 250-400 nucleotides, between 250-350 nucleotides, between 250-300 nucleotides, between 300-450 nucleotides, between 300-400 nucleotides, between 300-350 nucleotides, between 350-450 nucleotides, between 350-400 nucleotides, or between 400-450 nucleotides. 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. According to some embodiments, the loop further comprises one or more nucleic acids or that are used to stabilize the ends. According to other embodiments, the loop further comprises one or more nucleic acids that may be employed in therapeutic methods. According to other embodiments, the loop further comprises one or more nucleic acids that may be employed in diagnostic methods. According to other embodiments, the loop further comprises one or more nucleic acids that that may be employed for research purposes. According to some embodiments, the minimal nucleic acid structure that is necessary at the 3’ end of the ssDNA is any structure that loops back on itself, i.e., a hairpin structure. However, it is to be understood that a variety of structures are 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 may comprise at least one stem-loop structure at the 3’ end. In some embodiments, the ssDNA may comprise at least two stem-loop structures at the 3’ end. In some embodiments, the ssDNA may comprise at least three stem-loop structures at the 3’ end. In some embodiments, the ssDNA may comprise at least four stem-loop structures at the 3’ end. In some embodiments, the ssDNA may comprise at least five stem-loop structures at the 3’ end. According to some embodiments, the nucleotides at the 3’ end form a cruciform DNA structure. A DNA cruciform structure can be formed when both strands form a stem-loop structure at the same location in the molecule, and comprises a four-way junction and two closed hairpin-shaped points. According to some embodiments, the nucleotides at the 3’ end form a hairpin DNA structure. Hairpin loop structures in nucleic acids consist of a base-paired stem structure and a loop sequence with unpaired or non-Watson-Crick-paired nucleotides. According to some embodiments, the nucleotides at the 3’ end form a hammerhead DNA structure, made up of three base paired helices, separated by short linkers of conserved sequence. According to some embodiments, the nucleotides at the 3’ end form a quadraplex DNA structure. G-quadruplexes are four-stranded DNA secondary structures (G4s) that form from certain guanine-rich sequences. According to some embodiments, the nucleotides at the 3’ end form a bulged DNA structure. According to some embodiments, the nucleotides at the 3’ end form a multibranched loop.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. According to some embodiments, the 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. According to some embodiments, 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 a wild-type AAV ITR. According to some embodiments, 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 ITR. According to some embodiments, the at least one stem-loop structure at the 3’ end does not comprise a terminal resolution site (trs) that would be present in a wild-type ITR. According to some embodiments, the at least one stem loop structure at the 3’ end is devoid of any viral capsid protein coding sequences. According to some embodiments, the ssDNA molecule does not comprise any virally-derived sequences. According to some embodiments, the at least one stem-loop structure at the 3’ and / or 5’ ends is derived from an AAV ITR (e.g., an AAV2 ITR). According to some embodiments, the stem structure at the 3’ end comprises one or more nucleotides that are modified to be exonuclease resistant. 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 that are modified to be exonuclease resistant. According to some embodiments, the stem structure at the 3’ end comprises one or more phosphorothioate-modified nucleotides. According to some embodiments, the stem structure at the 3’ end comprises about 4 to about 10 phosphorothioate-modified nucleotides, e.g., about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 to about 10 or about 9 to about 10. According to some embodiments, the stem structure comprises more than 10 phosphorothioate- modified nucleotides. According to some embodiments, the phosphorothioate-modified nucleotides are located adjacent to each other. According to some embodiments, the one or more phosphorothioate-modified nucleotides of the 3’ end are resistant to exonuclease degradation. Boranophosphate modified DNA is also resistant to nuclease degradation, and may be considered as an alternative to phosphorothioate modification. According to further embodiments, the stem structure may comprise at least one functional moiety. In one embodiment, the at least one functional moiety is an aptamer sequence. In further embodiments, the aptamer sequence has a high binding affinity to a nuclear localized protein. According to some embodiments, the nucleotides in the loop are chemically modified with functional groups in order to alter their properties. According to some embodiments, the loop further comprises one or more aptamers. According to some embodiments, the aptamer is identified from the Apta-index database of aptamers available to the public (aptagen.com / apta-index). According to some embodiments, the loop further comprises one or more synthetic ribozymes. According to some embodiments, the loop further comprises one or more antisense oligonucleotides (ASOs). According to some embodiments, the loop further comprises one or more short-interfering RNAs (siRNAs). According to some embodiments, the loop further comprises one or more antiviral nucleoside analogues (ANAs). According to some embodiments, the loop further comprises one or more triplex forming oligonucleotides. According to some embodiments, the loop further comprises one or more gRNAs or gDNAs. According to some embodiments, the loop further comprises one or more molecular probes, for example nucleic acid based fluorescent probes. According to some embodiments, “click” azide-alkyne cycloaddition (Kolb et al., Angew. Chem. Int. Ed. Engl.2001, 40, 2004-2021) is used to modify the nucleotides in the loop. Click chemistry was developed to join together organic molecules under mild conditions in the presence of a diverse range of functional groups. Most click-mediated modifications are performed on the nitrogenous bases by introducing novel base analogues, attaching fluorophores or isotopic elements for molecular imaging, forming inter-strand linkages between oligonucleotides, and for the bioconjugation of molecules. The best example of click chemistry is the CuIcatalyzed version of Huisgen’s [3 + 2] azide–alkyne cycloaddition reaction (Angew. Chem., Int. Ed.1963, 2, 633–645), discovered independently by Sharpless and Meldal (the CuAAC reaction) (Angew. Chem., Int. Ed. 2002, 41, 2596–2599). According to some embodiments, the introduction of active amino or thiol groups into synthesized oligonucleotides provides acceptors for, e.g., subsequent chemical fluorescent labeling. According to some embodiments, the stem-loop structure may comprise alternative or modified nucleotides, including, but not limited to, ribonucleic acids (RNA), peptide-nucleic acids (PNA), locked nucleic acids (LNA). According to some embodiments, the loop portion of the stem- loop structure may comprise a chemical structure that does not comprise nucleic acids. According to some embodiments, the 3’ end of the partially ssDNA molecule is single- stranded, and does not comprise any double-stranded regions. As described in Example 5 and FIGs. 35-38, a fully single-stranded ssDNA molecule can induce transgene expression. In some embodiments, a fully-single-stranded ssDNA molecule with no double-stranded regions may 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, at least 3000 nucleotides in length, at least 3500 nucleotides in length, at least 4000 nucleotides in length, at least 4500 nucleotides in length, at least 5000 nucleotides in length, at least 5500 nucleotides in length, at least 6000 nucleotides in length, at least 6500 nucleotides in length, at least 7000 nucleotides in length, at least 7500 nucleotides in length, at least 8000 nucleotides in length, at least 8500 nucleotides in length, at least 9000 nucleotides in length, at least 9500 nucleotides in length, or at least 10,000 nucleotides in length. C. 5’ End As described herein, according to some aspects, the disclosure provides a partially ssDNA molecule comprising at least one nucleic acid sequence of interest flanked by at least one stem-loop structure at the 3’ end and / or 5’ end, as set forth 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 (referred to herein as “symmetric”). According to some embodiments, the DNA structure at the 5’ end is different from the DNA structure at the 3’ end (referred to herein as “asymmetric”). As described herein, the stem-loop structure at the 5’ end comprises a partial DNA duplex. The partial DNA duplex functions, in part, to hold the stem-loop structure together. According to some embodiments, the partial DNA duplex comprises between 4-500 nucleotides, for example between 4-10 nucleotides, between 4-25 nucleotides, between 4-50 nucleotides, between 4-100 nucleotides, between 4-200 nucleotides, between 4-300 nucleotides, between 4-400 nucleotides, between 20-25 nucleotides, between 20-50 nucleotides, between 20-100 nucleotides, between 20-200 nucleotides, between 20-300 nucleotides, between 20-400 nucleotides, between 20-500 nucleotides, between 50-100 nucleotides, between 50-200 nucleotides, between 50- 300 nucleotides, between 50-400 nucleotides, between 50-500 nucleotides, 150-200 nucleotides, between 150-300 nucleotides, between 150-400 nucleotides, between 150-500 nucleotides, between 200-300 nucleotides, between 200-400 nucleotides, between 200-500 nucleotides, between 250-300 nucleotides, between 250-400 nucleotides, between 250-500 nucleotides, between 300-400 nucleotides, between 300-500 nucleotides, between 350-400 nucleotides, between 350-500 nucleotides, between 400-500 nucleotides, between 450-500 nucleotides, between 500-550 nucleotides, between 550-600 nucleotides, between 600-650 nucleotides, between 650-700 nucleotides, between 700-750 nucleotides, between 750-800 nucleotides, between 800-850 nucleotides, between 850-900 nucleotides, between 900-950 nucleotides, between 950-1000 nucleotides, between 1000-1050 nucleotides, between 1050-1100 nucleotides, between 1100-1150 nucleotides, between 1150-1200 nucleotides, between 1200-1250 nucleotides, between 1250-1300 nucleotides, between 1300-1350 nucleotides, between 1350-1400 nucleotides, between 1400-1450 nucleotides, between 1450-1500 nucleotides, between 1500-1550 nucleotides, between 1550-1600 nucleotides, between 1600-1650 nucleotides, between 1650-1700 nucleotides, between 1700-1750 nucleotides, between 1750-1800 nucleotides, between 1800-1850 nucleotides, between 1850-1900 nucleotides, between 1900-1950 nucleotides, between 1950-2000 nucleotides, between 2050-2100 nucleotides, between 2150-2200 nucleotides, between 2250-2300 nucleotides, between 2350-2400 nucleotides, or between 2450-2500 nucleotides, and at least one loop on the 5’ 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, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500 or more nucleotides, and at least one loop on the 5’ end. According to some embodiments, the DNA duplex at the 5’ end may be described as being “adjacent” to the 5’ end. As used herein, the term “adjacent” to the 5’ end refers to a DNA duplex (i.e., a double-stranded region) in which one strand of the duplex (double-stranded region) comprises a free terminal nucleotide that terminates at the 5’ deoxyribose carbon atom. In some embodiments, the 5’ end further comprises a free phosphate group. In some embodiments, the terminal nucleotide may comprise, e.g., a chemical modification or derivatization, such that it no longer comprises a free phosphate group. Such embodiments are still referred to herein as the 5’ end. In some embodiments, the partially ssDNA described herein may comprise at least one stem- loop structure at the 5’ end. According to some embodiments, 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. According to some embodiments, the nucleotides at the 5’ end form a cruciform DNA structure. According to some embodiments, the nucleotides at the 5’ end form a hairpin structure. According to some embodiments, the nucleotides at the 5’ end form a hammerhead structure. According to some embodiments, the nucleotides at the 5’ end form a quadraplex structure. According to some embodiments, the nucleotides at the 5’ end form a bulged structure. According to some embodiments, the nucleotides at the 5’ end form a multibranched loop. 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. According to some embodiments, the at least one stem-loop structure at the 5’ end does not comprise the A, A’, D, and / or D’ regions that would be present in a wild-type AAV ITR. According to some embodiments, the at least one stem-loop structure at the 5’ end does not comprise the A, A’, B, B’C, C’, D, and / or D’ regions that would be present in a wild-type AAV ITR. According to some embodiments, 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. According to some embodiments, the at least one stem-loop structure at the 5’ end does not comprise a terminal resolution site (trs) that would be present in a wild-type ITR. According to some embodiments, the at least one stem loop structure at the 5’ end is devoid of any viral capsid protein coding sequences. According to some embodiments, the ssDNA molecule does not comprise any virally-derived sequences. According to some embodiments, the at least one stem-loop structure at the 5’ and / or 3’ ends is derived from an AAV ITR (e.g., an AAV2 ITR). According to some embodiments, the stem structure at the 5’ end comprises one or more nucleotides that are modified to be exonuclease resistant. 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 that are modified to be exonuclease resistant. According to some embodiments, the stem structure comprises one or more phosphorothioate-modified nucleotides. According to some embodiments, the stem structure comprises about 4 to about 10 phosphorothioate-modified nucleotides, e.g., about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 to about 10 or about 9 to about 10. According to some embodiments, the stem structure comprises more than 10 phosphorothioate- modified nucleotides. According to some embodiments, the phosphorothioate-modified nucleotides are located adjacent to each other. According to some embodiments, the one or more phosphorothioate-modified nucleotides of the are resistant to exonuclease degradation. According to some embodiments, the loop further comprises one or more nucleic acids or that are used to stabilize the ends. According to other embodiments, the loop further comprises one or more nucleic acids that may be employed in therapeutic methods. According to other embodiments, the loop further comprises one or more nucleic acids that may be employed in diagnostic methods. According to other embodiments, the loop further comprises one or more nucleic acids that that may be employed for research purposes. According to some embodiments, the nucleotides in the loop are chemically modified with functional groups in order to alter their properties. According to some embodiments, the loop further comprises one or more aptamers. According to some embodiments, the aptamer is identified from the Apta-index database of aptamers available to the public (aptagen.com / apta-index). According to some embodiments, the loop further comprises one or more synthetic ribozymes. According to some embodiments, the loop further comprises one or more antisense oligonucleotides (ASOs). According to some embodiments, the loop further comprises one or more short-interfering RNAs (siRNAs). According to some embodiments, the loop further comprises one or more antiviral nucleoside analogues (ANAs). According to some embodiments, the loop further comprises one or more triplex forming oligonucleotides. According to some embodiments, the loop further comprises one or more gRNAs or gDNAs. According to some embodiments, the loop further comprises one or more molecular probes, for example nucleic acid based fluorescent probes. According to some embodiments, “click” azide-alkyne cycloaddition (Kolb et al., Angew. Chem. Int. Ed. Engl.2001, 40, 2004–2021) is used to modify the nucleotides in the loop. Click chemistry was developed to join together organic molecules under mild conditions in the presence of a diverse range of functional groups. Most click-mediated modifications are performed on the nitrogenous bases by introducing novel base analogues, attaching fluorophores or isotopic elements for molecular imaging, forming inter-strand linkages between oligonucleotides, and for the bioconjugation of molecules. The best example of click chemistry is the CuIcatalyzed version of Huisgen’s [3 + 2] azide–alkyne cycloaddition reaction (Angew. Chem., Int. Ed.1963, 2, 633–645), discovered independently by Sharpless and Meldal (the CuAAC reaction) (Angew. Chem., Int. Ed. 2002, 41, 2596–2599). According to some embodiments, the introduction of active amino or thiol groups into synthesized oligonucleotides provides acceptors for, e.g., subsequent chemical fluorescent labeling. According to some embodiments, the stem-loop structure may comprise alternative or modified nucleotides, including, but not limited to, ribonucleic acids (RNA), peptide-nucleic acids (PNA), locked nucleic acids (LNA). According to some embodiments, the loop portion of the stem- loop structure may comprise a chemical structure that does not comprise nucleic acids. According to some embodiments, the 5’ end of the ssDNA molecule is single-stranded, and does not comprise any double-stranded regions. As described in Example 5 and FIGs.35-38, a fully single-stranded ssDNA molecule can induce transgene expression. D. Nucleic Acid Sequences of Interest The partially single-stranded DNA (ssDNA) molecules described herein have no packaging constraints imposed by the limiting space within the viral capsid. This permits the insertion of one or more genetic elements, e.g., a single-stranded enhancer, a single-stranded intron, a single-stranded posttranscriptional regulatory element, a single-stranded polyadenylation signal, and a single-stranded regulatory switch, large transgenes, multiple transgenes, etc. According to some embodiments, the “nucleic acid sequence of interest” comprises all DNA located between the 3’ and / or 5’ stem-loop structures (e.g., all DNA located in the central region). In some embodiments, the “nucleic acid sequence of interest” may be understood to comprise and / or overlap with the stem portion of the stem-loop structure (e.g., in an ssDNA molecule comprising simple hairpin ends). For example, the length of the double-stranded regions adjacent to the 3’ and 5’ ends is typically determined by counting the number of contiguous double-stranded base pairs from the nearest predicted loop or helical junction (i.e., likely departure from canonical B-form DNA) and does not include double-stranded portions distal to the nearest predicted loop or helical junction (e.g., the arms of a hammerhead or quadraplex ITR structure). According to some embodiments, the nucleic acid sequence of interest further comprises at least one single-stranded, partially double-stranded, or double-stranded promoter linked to at least one open reading frame (ORF). In other aspects of the disclosure, the single-stranded transgene cassettes find use in gene editing applications, as described in more detail herein. According to some embodiments, the nucleic acid sequence of interest (also referred to herein as a transgene) encodes a protein that is either absent, inactive, or has insufficient activity in the recipient subject or a protein having a desired biological or a therapeutic effect. The transgene can encode a gene product that can function to correct the expression of a defective gene or transcript. In principle, the expression cassette can include any gene that encodes a protein, polypeptide or RNA that is either reduced or absent due to a mutation or which conveys a therapeutic benefit when overexpressed is considered to be within the scope of the disclosure. The nucleic acid sequence of interest can comprise any sequence that is useful for treating a disease or disorder in a subject. A partially ssDNA molecule can be used to deliver and express any gene of interest in the subject, which includes but are not limited to, nucleic acids encoding polypeptides, or non-coding nucleic acids (e.g., RNAi, miRs etc.), as well as exogenous genes and nucleotide sequences, including virus sequences in a subjects’ genome, e.g., HIV virus sequences and the like. In some embodiments, ssDNA molecules disclosed herein are used for therapeutic purposes (e.g., for medical, diagnostic, or veterinary uses). In certain embodiments, ssDNA molecules are useful to express any gene of interest in the subject, which includes one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, or RNAs (coding or non-coding; e.g., siRNAs, shRNAs, micro-RNAs, mRNA or gRNA, and their antisense counterparts (e.g., antagoMiR)), antibodies, antigen binding fragments, or any combination thereof. Sequences can be codon optimized for the target host cell. As used herein, the term “codon optimized” or “codon optimization” refers to the process of modifying a nucleic acid sequence for enhanced expression in the cells of the vertebrate of interest, e.g., mouse or human, by replacing at least one, more than one, or a significant number of codons of the native sequence (e.g., a prokaryotic sequence) with codons that are more frequently or most frequently used in the genes of that vertebrate. Various species exhibit particular bias for certain codons of a particular amino acid. Typically, codon optimization does not alter the amino acid sequence of the original translated protein. Optimized codons can be determined using e.g., Aptagen’s GENEFORGE® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va.20171) or another publicly available database. In some embodiments, a transgene expressed by the partially ssDNA molecules is a therapeutic gene. In some embodiments, a therapeutic gene is an antibody, or antibody fragment, or antigen-binding fragment thereof, e.g., a neutralizing antibody or antibody fragment and the like. In particular, a therapeutic gene is one or more therapeutic agent(s), including, but not limited to, for example, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen binding fragments, as well as variants, and / or active fragments thereof, for use in the treatment, prophylaxis, and / or amelioration of one or more symptoms of a disease, dysfunction, injury, and / or disorder. Exemplary therapeutic genes are described herein in the section entitled “Method of Treatment”. According to any of the above aspects and embodiments, the partially ssDNA molecules are synthetically produced. According to any of the above aspects and embodiments, the partially ssDNA molecules are devoid of any viral capsid protein coding sequences. According to any of the above aspects the DNA is peptide nucleic acid (PNA) are synthetic mimics of DNA. III. Preparation of Partially Single-Stranded (ssDNA) Molecules As described herein, the present disclosure relates to isolated, partially single-stranded (ssDNA) molecules. In some aspects, the partially ssDNA molecules may be produced from double stranded closed-ended DNA (ceDNA) comprising phosphorothioate (PS) bonds. The PS bond substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of an oligonucleotide. Advantageously, this modification renders the internucleotide linkage resistant to nuclease degradation, and provides accuracy for targeting of the exonuclease. In some aspects, the disclosure provides a single-stranded transgene cassette comprising at least one single-stranded transgene and at least one stem-loop structure comprising one or more phosphorothioate-modified nucleotides. According to some embodiments, a partially ssDNA molecule comprises a first stem-loop structure and an optional second stem-loop structure; wherein at least one of the first stem-loop structure and the optional second stem-loop structure comprises one or more phosphorothioate-modified nucleotides. According to some aspects, the disclosure provides an isolated, linear, partially single- stranded DNA (ssDNA) molecule comprising a single-stranded transgene cassette comprising at least one single-stranded transgene; and a first stem-loop structure and a second stem-loop structure that each flank the at least one single-stranded transgene cassette; wherein at least one of the first stem- loop structure and the second stem-loop structure comprises one or more phosphorothioate-modified nucleotides. As described in more detail herein, the ssDNA molecule may be synthetically produced in vitro from dsDNA comprising phosphorothioate (PS) bonds (“starting material”) by removing one DNA strand from a specific nicking site and to a PS bonded site of the dsDNA. According to further embodiments, the partially ssDNA molecule is synthetically produced in vitro in a cell-free environment. Methods for producing the partially ssDNA molecules of the present disclosure may be found, for example, in International Patent Application No. PCT / US2023 / 082143, filed on December 1, 2023, incorporated herein by reference. Further details of methods for partially ssDNA molecules are described below. According to some embodiments, it is a feature of the present disclosure that the 3’ terminal portion of the double stranded DNA molecule (starting material) comprises a nickase recognition sequence. In one embodiment, the 3’ terminal portion of the dsDNA molecule comprises the sequence 5’-CCAA-3’. In some embodiments the 3’ terminal portion of the dsDNA molecule comprises any one or more of the sequences shown in Table 1 below. Further, since these are unique sequences, after a double stranded ceDNA with special engineered nick sites has been nicked by a nicking endonuclease as shown in the table, the resultant ssDNA molecule will also comprise any one or more of the sequences shown in Table 1 below in its 3’ terminal fragment. Table 1. According to some embodiments, the 3’ terminal fragment of the ssDNA molecule comprises a terminal residue that is hydroxylated (-OH) such that it enables polymerase activity once the ssDNA is transported to the nucleus of a host cell in which the ssDNA get convert to regenerated dsDNA that is capable of being expressed. According to some embodiments, the partially ssDNA molecule comprises a 3’ terminal fragment that comprises a terminal resolution site (trs) sequence. According to some embodiments, the partially ssDNA molecules described herein are capable of being transported across the nuclear membrane from the cytosol into the nucleus of a host cell, and reached by host cell DNA polymerase (referred to herein as “second-strand synthesis”) to generate a double stranded DNA (“regenerated dsDNA”) for expression of the transgene in the host cell. Accordingly, in some embodiments, the terminal residue that is hydroxylated (-OH) in the ssDNA molecule is necessary to be responsive towards DNA polymerase activity inside the nucleus of a host cell. According to further embodiments, the DNA polymerase generates a dsDNA molecule. In other embodiments, the terminal 3’ residue comprises a chemical modification that stabilizes the ssDNA molecule. In other embodiments, the 3’ residue does not comprise a free -OH, but is still capable of supporting transgene transcription and expression. Importantly, the ssDNA molecule does not activate or minimally activates an innate immune pathway inside a host cell. As used herein the term “the innate immune response” refers to the cellular pathways that respond to pathogen associated molecular patterns and activate a defense response through the RIG-I-like receptors, the toll-like receptors, or other pathogen associated molecular pattern receptors to activate interferon, NF-kappa-B, STAT, IRF and other response pathways that protect against pathogen infection. According to some embodiments, the innate immune pathway may be the cGAS / STING pathway, the TLR9 pathway, an inflammasome-mediated pathway, or a combination thereof. Indicators of the activation of the innate immune response include increased expression and / or phosphorylation of IRF family members, increased expression of the RIG-I like receptors, and increased expression of interferons and / or chemokines. In some embodiments, the term “minimally activates” an immune pathway refers to the lower or complete lack of induction of cytokines including, but not limited to, IFNα, IFNγ, IL-6, TNFα, and IL-18, as compared to a closed-ended deoxyribonucleic acid (ceDNA) molecule, when administered to a subject (e.g., a human, non-human primate, or mouse). According to some embodiments, the single-stranded transgene cassette further comprises at least one promoter operably linked to the at least one transgene; and the dsDNA molecule comprises a regenerated double-stranded expression cassette comprising at least one regenerated double-stranded transgene and at least one double-stranded promoter operably linked to the regenerated double- stranded transgene to control expression of the at least one regenerated double-stranded transgene. The double-stranded expression cassette is capable of being expressed in a host cell, for example a host cell in vivo. In some embodiments, the double-stranded expression cassette is capable of being expressed into at least one therapeutic protein or a fragment thereof. In further embodiments, the transgene cassette further comprises one or more genetic elements selected from the group consisting of an enhancer, an intron, a posttranscriptional regulatory element, a polyadenylation signal, and a regulatory switch. In other aspects of the disclosure, the single-stranded transgene cassettes may find use in gene editing applications. Accordingly, in some embodiments, the at least one single-stranded transgene cassette is a promoterless transgene cassette. In some embodiments, the at least one promoterless transgene is capable of being inserted at a target locus in the genome of a host cell. In further embodiments, the at least one promoterless transgene is capable of being inserted at a target locus in the genome of a host cell in vivo. In some embodiments, the at least one promoterless transgene is capable of being inserted at the target locus to replace or to supplement at least one target gene. In other embodiments, the at least one promoterless transgene is capable of being inserted at the target locus via homology-directed recombination (HDR) or microhomology-mediated end joining (MMEJ). In other further embodiments, the at least one 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 between about 10 to 2000 nt in length, for example about 100 to 2000 nt in length or about 1000 to 2000 nt in length, or about 10 to 1000 nt in length, for example 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 promoterless transgene is capable of being inserted at the target locus via non-homology end joining (NHEJ). In some embodiments, the at least one transgene is a single-stranded donor sequence; and the single-stranded transgene cassette is devoid of 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. As described in more detail herein, in some embodiments, the partially ssDNA molecule described herein is synthetically produced from a dsDNA construct by a method comprising a) contacting the dsDNA construct with one or more nicking endonucleases that nick one of the single strands of the dsDNA construct at one or more nick sites; and b) contacting the dsDNA construct with an exonuclease capable of removing nucleotides from the nicked strand of the dsDNA construct to thereby produce the partially ssDNA molecule. A. Double-Stranded (ds) Closed-Ended DNA (ceDNA) In some aspects, the present disclosure encompasses the use of double-stranded closed-ended DNA (ceDNA) comprising phosphorothioate (PS) bonds. As described herein, PS bond modifications is advantageously located in the terminal stem-loop region in a space where the exonuclease is active, and functions as a lock on the 5’ and / or 3’ ends, rendering the internucleotide linkage resistant to nuclease degradation, and ensuring the accuracy of exonuclease activity. The double-stranded ceDNA described herein is used to produce the partially ssDNA molecules described herein. Double-stranded ceDNA vectors may further be used as controls in experiments measuring the levels of transgene expression and immune response for the partially ssDNA molecules described herein. For example, in one embodiment, an ssDNA molecule may be compared to its “parent” ceDNA (as used herein, the term “parent” ceDNA refers to the double-stranded ceDNA molecule from which a specific ssDNA molecule was produced by removing part of one strand in the central region). In one aspect, the disclosure encompasses the use of an isolated double-stranded DNA (dsDNA) construct comprising a double-stranded transgene cassette comprising at least one double- stranded transgene; and a first stem-loop structure and an optional second stem-loop structure that each flank the at least one double-stranded transgene; wherein at least one of the first stem-loop structure and the optional second stem-loop structure comprises one or more phosphorothioate- modified nucleotides. According to some embodiments, the dsDNA construct comprises a nickase recognition sequence (“nick site”). In one embodiment, the dsDNA construct comprises a terminal resolution site (trs) sequence of an AAV ITR that contains a nick site. According to some embodiments, the dsDNA construct comprises one or more recognition nucleotide sequences of one or more nicking endonucleases that are each independently selected from Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BssSI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, and an isoschizomer of any of the foregoing. According to a further embodiment, the one or more recognition nucleotide sequences comprise any one or more of the following sequences shown in Table 2 below: Table 2. 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 nick sites of the one or more nicking endonucleases. According to some embodiments, the one or more nick sites are about 0 to about 20 nucleotides downstream of a terminal resolution site (trs), for example, about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1819, or 20 nucleotides downstream of the terminal resolution 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 resolution site (trs). According to some embodiments, there is just one nick site that serves as the exonuclease entry site. According to some embodiments, the dsDNA construct comprises one or more recognition nucleotide sequences of Nb.BbvCI or an isoschizomer thereof. According to some embodiments, the dsDNA construct comprises a single recognition nucleotide sequence of Nb.BbvCI or an isoschizomer thereof. According to some embodiments, the dsDNA construct comprises one or more recognition nucleotide sequences of Nb.BtsI or an isoschizomer thereof. According to some embodiments, the dsDNA construct comprises a single recognition nucleotide sequence of Nb.BtsI or an isoschizomer thereof. According to embodiments of the 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 posttranscriptional regulatory element, a double-stranded polyadenylation signal, and a double- stranded regulatory switch. According to other further embodiments, the at least one double-stranded transgene is a promoterless double-stranded transgene. As described herein the at least one double- stranded transgene is a double-stranded donor sequence; and the double-stranded transgene cassette further comprises a double-stranded 5’ 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 between about 10 to 2000 nt in length, for example about 100 to 2000 nt in length or about 1000 to 2000 nt in length, or about 10 to 1000 nt in length, for example 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. According to some embodiments, the at least one double-stranded transgene is a double- stranded donor sequence; and the double-stranded transgene cassette is devoid of a single-stranded 5’ homology arm and a single-stranded 3’ homology arm. Further, in some embodiments, the double- stranded transgene cassette is cleavable and further comprises at least a first double-stranded guide RNA (gRNA) target sequence (TS); at least a first double-stranded protospacer adjacent motif (PAM); at least a second double-stranded gRNA TS; and at least a second double-stranded PAM. As described in more detail herein, in some embodiments, the dsDNA construct is synthetically produced by a method comprising a) contacting a dsDNA template with at least one restriction endonuclease, wherein the template comprises the double-stranded transgene cassette comprising the 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 and second cleavage sites to release an insert having single-stranded overhangs at the 5’ and 3’ ends of the insert; and b) ligating the 5’ and 3’ ends of the insert to a first inverted terminal repeat (ITR) oligonucleotide and an optional second ITR oligonucleotide to form the dsDNA construct. According to some embodiments, at least one of the first ITR and the optional second ITR oligonucleotides comprises one or more phosphorothioate-modified nucleotides. According to some other embodiments, at least one of the first ITR and the optional second ITR oligonucleotides comprises the 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 to a nuclear localized protein. In another embodiment, the at least one function moiety is a nuclear localization peptide conjugated to the at least one of the ITR oligonucleotides. In another embodiment, the at least one function moiety is a fluorophore chemically conjugated to the ITR oligonucleotides. Methods for producing double-stranded ceDNA constructs may further be found, for example, in International Publication No. WO2019 / 143885 A1 and International Publication No. WO2023 / 122303 A2, the entire contents of each of which are incorporated by reference. B. Partially ssDNA molecules and vectors derived from double-stranded DNA Owing to the fact that the partially single-stranded DNA (ssDNA) molecules according to embodiments of the present disclosure may be derived from double-stranded DNA (dsDNA) constructs, and particularly double-stranded ceDNA (ds ceDNA) with phosphorothioate-modified nucleotides, physical attributes of the ds ceDNA vectors are also present in the partially single- stranded DNA (ssDNA) molecules, including, e.g., the presence of the at least one functional moiety such as an aptamer sequence, e.g., having a high binding affinity to a nuclear localized protein or a fluorophore chemically conjugated to the ITR oligonucleotides. In another embodiment, the at least one functional moiety is a fluorophore chemically conjugated to the ITR oligonucleotides. The partially single-stranded DNA (ssDNA) molecules and dsDNA constructs (e.g., ds ceDNA) produced using the synthetic process as described herein have no packaging constraints imposed by the limiting space within the viral capsid. This permits the insertion of control elements, e.g., regulatory switches as disclosed herein, large transgenes, multiple transgenes etc. According to some embodiments, the one or more phosphorothioate (PS)-modified nucleotides of the ssDNA molecule are each independently located in any region of a terminal stem- loop structure. In viral-derived terminal stem-loop structures (ITRs), the PS modified nucleotides may be located in any region selected from A, A’, B, B’, C, C’, and D’ of at least one of the first and the optional second ITRs. According to some embodiments, the one or more PS-modified nucleotides of the dsDNA construct are each independently located in any region selected from A, A’, B, B’, C, C’, D, and D’ of at least one of the first and the optional second ITRs. According to some embodiments, the one or more PS-modified nucleotides of the partially ssDNA molecule are each independently located in any part of a terminal stem-loop structure, and / or any viral-derived ITR region selected from A, A’, and D of at least one of the first and the optional second ITRs. According to some embodiments, the one or more PS-modified nucleotides of the dsDNA construct are each independently located in any part of a terminal stem-loop structure and / or any viral-derived ITR region selected from A, A’, and D of at least one of the first and the optional second ITRs. According to some embodiments, the one or more PS-modified nucleotides of the partially ssDNA molecule are each independently located in any part of a terminal stem-loop structure, and / or any viral-derived ITR region selected from A and A’ of at least one of the first and the optional second ITRs. According to some embodiments, the one or more PS-modified nucleotides of the dsDNA construct are each independently located in any part of a terminal stem-loop structure and / or any viral-derived ITR region selected from A and A’ of at least one of the first and the optional second ITRs. According to some embodiments, all of the one or more PS-modified nucleotides of the partially ssDNA molecule in a first viral-derived ITR are located in an A’ region and / or D region of the first ITR. According to some embodiments, all of the one or more PS-modified nucleotides of the dsDNA construct in a first viral-derived ITR are located in an A’ region and / or D region of the first ITR. According to some embodiments, all of the one or more PS-modified nucleotides in a first viral-derived ITR of the partially ssDNA molecule are located in an A region of the first ITR. According to some embodiments, all of the one or more PS-modified nucleotides in the first viral- derived ITR of the dsDNA construct are located in an A region of the first ITR. According to some embodiments, all of the one or more PS-modified nucleotides in a second viral-derived ITR of the partially ssDNA molecule, if present, are located in an A’ region and / or D region of the second ITR. According to some embodiments, all of the one or more PS-modified nucleotides in a second viral-derived ITR of the dsDNA construct, if present, are located in an A’ region and / or D region of the second ITR. According to some embodiments, all of the one or more PS-modified nucleotides in a second viral-derived ITR of the partially ssDNA molecule, if present, are located in an A region of the second ITR. According to some embodiments, all of the one or more PS-modified nucleotides in a second viral-derived ITR of the dsDNA construct, if present, are located in an A region of the second ITR. According to some embodiments, the one or more PS-modified nucleotides of the partially ssDNA molecule are adjacent to one another. According to some embodiments, the one or more PS- modified nucleotides of the dsDNA construct are adjacent to one another. According to some embodiments, the one or more PS-modified nucleotides of the partially ssDNA molecule are about 1 to 15 nucleotides from a B-B’ arm and C-C’ arm, if present, of the first viral-derived ITR or the optional second viral-derived ITR. According to some embodiments, the one or more phosphorothioate-modified nucleotides in the dsDNA construct are about 1 to 15 nucleotides from a B-B’ arm and C-C’ arm, if present, of the first viral-derived ITR or the optional second ITR. According to some embodiments, the one or more PS-modified nucleotides of the partially ssDNA molecule are about 1 to 10 nucleotides from aB-B’ arm and C-C’ arm, if present, of the first viral-derived ITR or the optional second viral-derived ITR. According to some embodiments, the one or more phosphorothioate-modified nucleotides dsDNA construct are about 1 to 10 nucleotides from a B-B’ arm and C-C’ arm, if present, of the first viral-derived ITR or the optional second viral-derived ITR. According to some embodiments, the one or more PS-modified nucleotides of the partially ssDNA molecule are about 1 to 5 nucleotides from a B-B’ arm and C-C’ arm, if present, of the first viral-derived ITR or the optional second viral-derived ITR. According to some embodiments, the one or more PS-modified nucleotides in the dsDNA construct are about 1 to 5 nucleotides from a B-B’ arm and C-C’ arm, if present, of the first viral-derived ITR or the optional second viral-derived ITR. According to some embodiments, the one or more PS-modified nucleotides of the partially ssDNA molecule are resistant to exonuclease degradation. According to some embodiments, the one or more PS-modified nucleotides containing dsDNA construct are resistant to exonuclease degradation at the PS bonded sequence. According to some embodiments, at least one of the first and the optional second stem-loop structures of the ssDNA molecule each comprises about 1 to about 60 PS-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 and the optional second stem-loop structures in a dsDNA construct each comprises about 1 to about 60 PS-modified nucleotides. According to some embodiments, at least one of the first and the optional second stem-loop structures of the partially ssDNA molecule each comprises about 1 to about 5 PS-modified nucleotides. According to some embodiments, at least one of the first and the optional second stem-loop structures of the partially ssDNA molecule each comprises about 1 to about 10 PS-modified nucleotides. According to some embodiments, at least one of the first and the optional second stem-loop structures of the partially ssDNA molecule each comprises about 1 to about 15 PS-modified nucleotides. According to some embodiments, at least one of the first and the optional second stem-loop structures in a dsDNA construct each comprises about 1 to about 20 PS-modified nucleotides. According to some embodiments, at least one of the first and the optional second stem-loop structures of the partially ssDNA molecule each comprises about 1 to about 25 PS-modified nucleotides. According to some embodiments, at least one of the first and the optional second stem-loop structures in a dsDNA construct each comprises about 1 to about 30 PS-modified nucleotides. According to some embodiments, the one or more PS-modified nucleotides are located at the 5’ end of the partially ssDNA molecule. According to some embodiments, the one or more PS- modified nucleotides are located at the 3’ end of the partially ssDNA molecule. According to some embodiments, the one or more PS-modified nucleotides are located at the 3’ end of the partially ssDNA molecule, the 5’ end of the partially ssDNA molecule, or both. According to some embodiments, the one or more PS-modified nucleotides are located upstream of each of the one or more nicking endonuclease recognition sequences. According to some embodiments, the one or more PS-modified nucleotides are located at the 5’ end of the first stem-loop structure and / or the optional second stem-loop structure. According to some embodiments, the partially 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 PS-modified nucleotides. According to some embodiments, wherein the partially ssDNA molecule comprises at least 1, 2, 3, 4, 5, or more PS-modified nucleotides at the 3’ end of the partially ssDNA molecule, the 5’ end of the partially ssDNA molecule, or both. According to some embodiments, the partially ssDNA molecule comprises at least 1, 2, 3, 4, 5 or more PS-modified nucleotides upstream of each of the one or more nicking endonuclease recognition sequences. According to some embodiments, the partially ssDNA molecule comprises at least 1, 2, 3, 4, 5 or more PS-modified nucleotides at the 5’ end of the first stem-loop structure and / or at least 1, 2, 3, 4, 5 or more PS-modified nucleotides at the 5’ end of the optional second stem-loop structure. According to some embodiments, at least one of the first and optional second stem-loop structures of the partially ssDNA molecule each comprises no more than about 6 PS-modified nucleotides. According to some embodiments, at least one of the first and optional second stem-loop structures of the partially ssDNA molecule each comprises no more than about 5 PS-modified nucleotides. According to some embodiments, at least one of the first and optional second stem-loop structures of the partially ssDNA molecule each comprises no more than about 4 PS-modified nucleotides. According to some embodiments, at least one of the first and optional second stem-loop structures of the partially ssDNA molecule each comprises no more than about 3 PS-modified nucleotides. According to some embodiments, at least one of the first and optional second stem-loop structures of the partially ssDNA molecule each comprises no more than about 2 PS-modified nucleotides. According to some embodiments, at least one of the first and optional second stem-loop structures of the partially ssDNA molecule each comprises no more than about 1 PS-modified nucleotides. According to some embodiments, at least one of the first and optional second stem-loop structures dsDNA construct each comprises no more than about 6 PS-modified nucleotides. According to some embodiments, at least one of the first and optional second stem-loop structures in the dsDNA construct each comprises no more than about 5 PS-modified nucleotides. According to some embodiments, at least one of the first and optional second stem-loop structures in the dsDNA construct each comprises no more than about 4 PS-modified nucleotides. According to some embodiments, at least one of the first and optional second stem-loop structures in the dsDNA construct each comprises no more than about 3 PS-modified nucleotides. According to some embodiments, at least one of the first and optional second stem-loop structures in the dsDNA construct each comprises no more than about 2 PS-modified nucleotides. According to some embodiments, at least one of the first and optional second stem-loop structures in the dsDNA construct each comprises no more than about 1 PS-modified nucleotides. According to some embodiments, at least one of the first and optional second stem-loop structures of the partially ssDNA molecule each comprises about 3, about 4, or about 5 PS-modified nucleotides. According to some embodiments, at least one of the first and optional second stem-loop structures in the dsDNA construct each comprises about 3, about 4, or about 5 PS-modified nucleotides. According to some embodiments, the first stem-loop structure and the optional second stem- loop structure of the partially ssDNA molecule are symmetric or substantially symmetric with respect to each other. According to some embodiments, the first stem-loop structure and the optional second stem-loop structure of the dsDNA construct are symmetric or substantially symmetric with respect to each other. According to some embodiments, the first stem-loop structure and the optional second stem- loop structure of the partially ssDNA molecule are asymmetric with respect to each other. According to some embodiments, the first stem-loop structure and the optional second stem-loop structure in the dsDNA construct are asymmetric with respect to each other. According to some embodiments, at least one or both of the first stem-loop structure and the optional second stem-loop structure of the partially ssDNA molecule are wild-type ITRs (e.g., wild- type AAV ITRs, including wild-type AAV2 ITRs). According to some embodiments, at least one of both of the first stem-loop structure and the optional second stem-loop structure in a dsDNA construct are wild-type ITRs. According to some embodiments, at least one of both of the first stem-loop structure and the optional second stem-loop structure of the partially ssDNA molecule are modified by a deletion, insertion, and / or base substitution in at least one of the regions selected from the group consisting of the A, A’, B, B’, C, C’, D, and D’ ITR regions. as compared to a wild-type ITR. According to some embodiments, at least one of both of the first stem-loop structure and the optional second stem-loop structure in a dsDNA construct are modified by a deletion, insertion, and / or base substitution in at least one of the regions selected from the group consisting of the A, A’, B, B’, C, C’, D, and D’ ITR regions, as compared to a wild-type ITR . According to some embodiments, the first stem-loop structure and the optional second stem- loop structure of the partially ssDNA molecule are each derived from an AAV ITR and are each independently derived from 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 stem-loop structure and the optional second stem-loop structure in a dsDNA construct are each derived from an AAV ITR and are each independently derived from 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 partially ssDNA molecule is devoid of any viral capsid protein coding sequences. According to some embodiments, the dsDNA construct is devoid of any viral capsid protein coding sequences. According to some embodiments, the partially 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. However, it should be understood that, for use in, e.g., in vivo gene therapy applications, where minimization of immune response is desirable, a partially ssDNA molecule should not comprise any viral capsid coding sequence (due to the fact that viral capsid proteins are likely to induce immune responses in vivo). C. Expression Cassettes, Transgenes and Nucleic Acid Sequences of Interest The expression cassette may comprise a transgene (a nucleic acid sequence of interest) and one or more regulatory sequences that allows and / or controls the expression of the transgene, e.g., where the expression cassette can comprise one or more of, in this order: an enhancer / promoter, an open reading frame (ORF), which may encode, e.g., a reporter gene or a therapeutic transgene, , a post-transcription regulatory element (e.g., 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. The cis-regulatory elements include, but are not limited to, a promoter, a riboswitch, an insulator, a mir-regulatable element, a post-transcriptional regulatory element, a tissue- and cell type-specific promoter and an enhancer. In some embodiments an ITR (e.g., a viral-derived ITR) may act as the promoter for the transgene. In some embodiments, the partially ssDNA molecule or a dsDNA construct described herein in part II or part III comprises additional components to regulate expression of the transgene or nucleic acid sequence of interest, for example, a regulatory switch, which are described herein in the section entitled “Regulatory Switches” for controlling and regulating the expression of the transgene, and can include if desired, a regulatory switch which is a kill switch to enable controlled cell death of a cell comprising a partially ssDNA molecule. The expression cassette or the nucleic acid sequence of interest in the partially ssDNA construct can comprise more than 1000 nucleotides, 2000 nucleotides, 3000 nucleotides, 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 more than 50,000 nucleotides. In some embodiments, the expression cassette can comprise a transgene in the range of 500 to 50,000 nucleotides in length. In some embodiments, the expression cassette can comprise a transgene in the range of 500 to 75,000 nucleotides in length. In some embodiments, the expression cassette can comprise a transgene which is in the range of 500 to 10,000 nucleotides in length. In some embodiments, the expression cassette can comprise a transgene which is in the range of 1000 to 10,000 nucleotides in length. In some embodiments, the expression cassette can comprise a transgene which is in the range of 500 to 5,000 nucleotides in length. The partially ssDNA molecules and dsDNA constructs described herein do not have the size limitations of encapsidated AAV vectors, and thus enable delivery of a large-size expression cassette to provide efficient transgene expression. In some embodiments, the partially ssDNA molecules and dsDNA constructs described herein in part II or part III are devoid of prokaryote-specific methylation. An expression cassette can include, for example, an expressible exogenous sequence (e.g., open reading frame) or transgene or nucleic acid sequence of interest that encodes a protein that is either absent, inactive, or insufficient activity in the recipient subject or a gene that encodes a protein having a desired biological or a therapeutic effect. The transgene or nucleic acid sequence of interest can encode a gene product that can function to correct the expression of a defective gene or transcript. In principle, the expression cassette can include any gene that encodes a protein, polypeptide or RNA that is either reduced or absent due to a mutation or which conveys a therapeutic benefit when overexpressed is considered to be within the scope of the disclosure. The expression cassette can comprise any transgene or nucleic acid sequence of interest useful for treating a disease or disorder in a subject. A partially ssDNA molecule or dsDNA construct described herein in part II or part III produced using the synthetic processes as described herein can be used to deliver and express any gene of interest in the subject, which includes but are not limited to, nucleic acids encoding polypeptides, or non-coding nucleic acids (e.g., RNAi, miRs etc.), as well as exogenous genes and nucleotide sequences, including virus sequences in a subjects’ genome, e.g., HIV virus sequences and the like. In some embodiments, partially 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 uses). In certain embodiments, partially ssDNA molecules and dsDNA constructs described herein in part II or part III are useful to express any gene of interest in the subject, which includes one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, or RNAs (coding or non- coding; e.g., siRNAs, shRNAs, micro-RNAs, mRNA or gRNA, and their antisense counterparts (e.g., antagoMiR)), antibodies, antigen binding fragments, or any combination thereof. The expression cassette can also encode polypeptides, sense or antisense oligonucleotides, or RNAs (coding or non-coding; e.g., siRNAs, shRNAs, micro-RNAs, and their antisense counterparts (e.g., antagoMiR)). Expression cassettes can include an exogenous sequence that encodes a reporter protein to be used for experimental or diagnostic purposes, such as β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art. Sequences provided in the expression cassette, expression construct of partially ssDNA molecules and 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 for enhanced expression in the cells of the vertebrate of interest, e.g., mouse or human, by replacing at least one, more than one, or a significant number of codons of the native sequence (e.g., a prokaryotic sequence) with codons that are more frequently or most frequently used in the genes of that vertebrate. Various species exhibit particular bias for certain codons of a particular amino acid. Typically, codon optimization does not alter the amino acid sequence of the original translated protein. Optimized codons can be determined using e.g., Aptagen’s GENEFORGE® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va.20171) or another publicly available database. In some embodiments, a transgene or nucleic acid sequence of interest expressed by the partially ssDNA molecules described herein is a therapeutic gene. In some embodiments, a therapeutic gene is an antibody, or antibody fragment, or antigen-binding fragment thereof, e.g., a neutralizing antibody or antibody fragment and the like. In particular, a therapeutic gene is one or more therapeutic agent(s), including, but not limited to, for example, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen binding fragments, as well as variants, and / or active fragments thereof, for use in the treatment, prophylaxis, and / or amelioration of one or more symptoms of a disease, dysfunction, injury, and / or disorder. Exemplary therapeutic genes are described herein in the section entitled “Method of Treatment”. There are many structural features of partially ssDNA molecules and dsDNA constructs described herein in part II or part III that differ from plasmid-based expression vectors. ssDNA molecules described herein in part II or part III and dsDNA constructs produced by the synthetic methods herein may possess one or more of the following features: the lack of original (i.e. not inserted) bacterial DNA, the lack of a prokaryotic origin of replication, being self-containing, i.e., they do not require any sequences other than the one or two terminal stem-loop structures, including the Rep binding and terminal resolution sites (RBS and TRS) present in viral-derived ITRs, and an exogenous sequence between the stem-loop structures. In general, the partially ssDNA molecule described herein do not to contain any prokaryotic DNA, but it is contemplated that some prokaryotic DNA may be inserted as an exogenous sequence, as a non-limiting example in a promoter or enhancer region. There are several advantages of using the partially ssDNA molecules described herein over plasmid-based expression vectors. Such advantages include, but are not limited to: 1) plasmids contain bacterial DNA sequences and are subjected to prokaryotic-specific methylation, e.g., 6-methyl adenosine and 5-methyl cytosine methylation, whereas the partially ssDNA molecules are of eukaryotic origin and / or are synthetic and do not undergo prokaryotic-specific methylation; as a result, partially ssDNA vectors are less likely to induce inflammatory and immune responses compared to plasmids; 2) while plasmids require the presence of a resistance gene during the production process, partially ssDNA molecules of the present disclosure do not; 3) while a circular plasmid is not delivered to the nucleus upon introduction into a cell and requires overloading to bypass degradation by cellular nucleases, partially ssDNA molecules may contain viral cis-elements, i.e., ITRs, that confer resistance to nucleases and can be designed to be targeted and delivered to the nucleus. Without intending to be limited by mechanism, it is hypothesized that the minimal defining elements indispensable for ITR function are a Rep-binding site (RBS; 5’-GCGCGCTCGCTCGCTC- 3’ (SEQ ID NO: 17) for AAV2) and a terminal resolution site (TRS; 5’-AGTTGG-3’ for AAV2) plus a variable palindromic sequence allowing for hairpin formation; and 4) partially ssDNA molecules do not have the over-representation of CpG dinucleotides often found in prokaryote-derived plasmids that reportedly binds a member of the Toll-like family of receptors, eliciting a T cell-mediated immune response. There are further advantages of using the partially ssDNA molecules described herein over double-stranded expression vectors, including ceDNA vectors. Most importantly, the partially ssDNA vectors described herein comprise enough double-stranded DNA (i.e., double-stranded DNA in the central region) to induce robust transgene expression, while minimizing immune responses, particular immune responses mediated by the double-stranded DNA-sensing cGAS-STING pathway. D. Inverted Terminal Repeats (ITRs) As set forth herein, according to some aspects, the disclosure provides a partially 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 (a “stem”) and at least one loop on the 3’ end. In some embodiments, the ssDNA molecule comprises at least one stem- loop structure comprising a partial DNA duplex (stem) and at least one loop at the 5’ end. According to some aspects, the partially ssDNA molecules (and the dsDNA constructs use to produce the ssDNA molecules) contain a transgene or heterologous nucleic acid sequence positioned between two stem-loop structures, where the stem-loop structures can be an asymmetric stem-loop structure pair or a symmetric or substantially symmetric stem-loop structure pair, as these terms are defined herein. A partially ssDNA molecule disclosed herein can comprise stem-loop structures that are selected from any of: (i) at least one WT AAV ITR and at least one modified AAV ITR (mod- ITR) (e.g., asymmetric modified ITRs); (ii) two modified ITRs where the mod-ITR pair have a different three-dimensional spatial organization with respect to each other (e.g., asymmetric modified ITRs), or (iii) symmetric or substantially symmetric WT-WT ITR pair, where each WT-ITR has the same three-dimensional spatial organization, or (iv) symmetric or substantially symmetric modified ITR pair, where each mod-ITR has the same three-dimensional spatial organization, where the methods of the present disclosure may further include a delivery system, such as but not limited to a liposome nanoparticle delivery system. A partially ssDNA molecule as described herein may further have any combination of symmetric, substantially symmetric, or asymmetric stem-loop structures, and encompasses any and all combinations or permutations of WT ITRs (e.g., WT AAV ITRs), modified ITRs (e.g., modified AAV ITRs), and fully synthetic stem-loop structures, including structures with a variety of different shapes, configurations, and / or numbers of stems and loops, as described elsewhere herein. In some embodiments, a viral-derived ITR sequence can be from viruses of the Parvoviridae family, which includes two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect insects. The subfamily Parvovirinae (referred to as the parvoviruses) includes the genus Dependovirus, the members of which, under most conditions, require coinfection with a helper virus such as adenovirus or herpes virus for productive infection. The genus Dependovirus includes adeno- associated virus (AAV), which normally infects humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4), and related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses). The parvoviruses and other members of the Parvoviridae family are generally described in Kenneth I. Berns, “Parvoviridae: The Viruses and Their Replication,” Chapter 69 in FIELDS VIROLOGY (3d Ed.1996). While some of the ITRs exemplified in the specification and Examples herein may comprise WT AAV2 ITRs and / or modified AAV2 ITRs, a partially ssDNA molecule as described herein may include ITRs from any known parvovirus, including a dependovirus such as AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genome. E.g., NCBI: NC 002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261), chimeric ITRs, or ITRs from any synthetic AAV. In some embodiments, the AAV can infect warm-blooded animals, e.g., avian (AAAV), bovine (BAAV), canine, equine, and ovine adeno-associated viruses. In some embodiments an ITR is from B19 parvovirus (GenBank Accession No: NC 000883), Minute Virus from Mouse (MVM) (GenBank Accession No. NC 001510); goose parvovirus (GenBank Accession No. NC 001701); snake parvovirus 1 (GenBank Accession No. NC 006148). In some embodiments, a 5’ ITR can be derived from one AAV serotype, and a 3’ ITR can be derived from a different serotype, as discussed herein. It should be understood that the term “derived from”, when used in the context of a viral ITR, includes both WT ITRs and viral ITRs with one or more nucleotide additions, deletions, and / or substitutions, as compared to a WT viral ITR. An ordinarily skilled artisan is aware that ITR sequences have a common structure of a double-stranded Holliday junction, which typically is a T-shaped or Y-shaped hairpin structure, where 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, (where the order of these palindromic sequences defines the flip or flop orientation of the ITR). See, for example, 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. One of ordinary skill in the art can readily determine WT-ITR sequences from any AAV serotype for use in a ssDNA molecules and dsDNA constructs based on the exemplary AAV2 ITR sequences provided herein. See, for example, the sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6, and avian AAV (AAAV) and bovine AAV (BAAV)) described in Grimm et al., J. Virology, 2006; 80(1); 426-439; that show the % identity of the left ITR of AAV2 to the left ITR 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%). According to some other embodiments, at least one of the first ITR and the optional second ITR oligonucleotides comprising one or more phosphorothioate-modified nucleotides of the present invention can further comprise one or more functional moieties. In one embodiment, the at least one function moiety is an aptamer sequence, optionally wherein the aptamer sequence has a high binding affinity to a nuclear localized protein. In another embodiment, the at least one function moiety is a nuclear localization peptide conjugated to the at least one of the ITR oligonucleotides. In another embodiment, the at least one function moiety is a fluorophore chemically conjugated to the ITR oligonucleotides. In some embodiments, ITRs as used herein may be fully synthetic, e.g., an ITR may comprise no virally-derived sequences. E. Regulatory elements The partially single-stranded DNA (ssDNA) molecules as described herein can further comprise a specific combination of cis-regulatory elements. The cis-regulatory elements include, but are not limited to, a promoter, a riboswitch, an insulator, a mir-regulatable element, a post- transcriptional regulatory element, promoter (e.g., a tissue- and / or cell type-specific promoter) and an enhancer (e.g., a tissue- and / or cell type-specific enhancer). In some embodiments, the partially ssDNA molecule described herein comprises additional components to regulate expression of the transgene or nucleic acid of interest, for example, regulatory switches as described herein, to regulate the expression of the transgene or nucleic acid of interest, or a kill switch, which can kill a cell comprising the partially ssDNA molecule described herein. Regulatory elements, including regulatory switches that can be used in the present disclosure, are more fully discussed in International application No. PCT / US18 / 49996 (published as International patent publication No. WO 2019 / 051255 A1), which is incorporated herein in its entirety by reference. According to some embodiments, the nucleotide sequence includes a regulatory sequence, and a nucleotide sequence encoding a therapeutic protein. In certain embodiments, the regulatory sequence is operably linked to the nucleotide sequence encoding the therapeutic protein. In certain embodiments, the regulatory sequence is suitable for controlling the expression of the therapeutic proteinin a host cell. In certain embodiments, the regulatory sequence includes a suitable promoter sequence, being able to direct transcription of a gene operably linked to the promoter sequence, such as a nucleotide sequence encoding the therapeutic protein of the present disclosure. In certain embodiments, the nucleotide sequence includes an intron sequence linked to the 5’ terminus of the nucleotide sequence encoding the therapeutic protein. In certain embodiments, an enhancer sequence is provided upstream of the promoter to increase the efficacy of the promoter. In certain embodiments, the regulatory sequence includes an enhancer and a promoter, wherein the nucleotide sequence includes an intron sequence upstream of the nucleotide sequence encoding a therapeutic protein, and wherein the promoter is operably linked to the nucleotide sequence encoding the therapeutic protein. The partially single-stranded DNA (ssDNA) molecules described herein can further comprise a specific combination of cis-regulatory elements such as WHP posttranscriptional regulatory element (WPRE) and BGH polyA signal. Suitable expression cassettes for use in expression constructs are not limited by the packaging constraint imposed by the viral capsid. (i) Promoters It will be appreciated by one of ordinary skill in the art that promoters used in the synthetically produced partially single-stranded DNA (ssDNA) molecules described herein should be tailored as appropriate for the specific sequences they are regulating. For example, a guide RNA may not require a promoter at all, since its function is to form a duplex with a specific target sequence on the native DNA to effect a recombination event. In contrast, a therapeutic proteinencoded by the ssDNA molecule or the dsDNA construct vector would benefit from a promoter so that it can be efficiently expressed from the vector – and, optionally, in a regulatable fashion. Expression cassettes of the present disclosure include a promoter, which can influence overall expression levels as well as cell-specificity. For transgene expression, they can include a highly active virus-derived immediate early promoter (e.g., a constitutive promoter). Expression cassettes can contain tissue-specific eukaryotic promoters to limit transgene expression to specific cell types and reduce toxic effects and immune responses resulting from unregulated, ectopic expression of exogenous proteins. In some embodiments, an expression cassette can contain a synthetic regulatory element, such as a CAG promoter. The CAG promoter comprises (i) the cytomegalovirus (CMV) early enhancer element, (ii) the promoter, the first exon and the first intron of chicken beta-actin gene, and (iii) the splice acceptor of the rabbit beta-globin gene. Alternatively, an expression cassette can contain a humam Alpha-1-antitrypsin (hAAT) promoter, a liver specific (LP1) promoter, a human elongation factor-1 alpha (EF1a) promoter, or a human transthyretin (TTR) promoter. In some embodiments, the expression cassette includes one or more constitutive promoters, for example, a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), or a cytomegalovirus (CMV) immediate early promoter (optionally with the CMV enhancer). Alternatively, an inducible promoter, a native promoter for a transgene, a tissue-specific promoter, or various promoters known in the art can be used. Suitable promoters, including those described above, can be derived from viruses and can therefore be referred to as viral promoters, or they 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 the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovir...

Claims

CLAIMS 1. An isolated partially single-stranded deoxyribonucleic acid (ssDNA) molecule comprising: (a) a central region, comprising at least one single-stranded region and at least one double-stranded region, wherein the central region comprises at least one nucleic acid sequence of interest; (b) at least a first stem-loop structure at its 3’ end, wherein the first stem-loop structure comprises at least one stem and at least one loop, and / or at least a second stem-loop structure at its 5’ end, wherein the second stem-loop structure comprises at least one stem and at least one loop, wherein the first stem-loop structure and / or the second stem-loop structure flank the central region, and wherein the central region comprises a total of at least 50 base pairs (bp) of double-stranded DNA, and / or wherein the central region is at least 5% double-stranded, and wherein the partially ssDNA molecule comprises at least one hybridized oligonucleotide.

2. The ssDNA molecule of claim 1, comprising at least two hybridized, noncovalently-bound oligonucleotides.

3. The ssDNA molecule of any one of claims 1-2, comprising at least three hybridized, noncovalently-bound oligonucleotides.

4. The ssDNA molecule of any one of claims 1-3, comprising at least four hybridized, noncovalently-bound oligonucleotides.

5. The ssDNA molecule of any one of claims 1-4, comprising at least five hybridized, noncovalently-bound oligonucleotides.

6. The ssDNA molecule of any one of claims 1-5, wherein each oligonucleotide is less than 200 nucleotides in length.

7. The ssDNA molecule of any one of claims 1-6, wherein each oligonucleotide is less than 88 nucleotides in length.

8. The ssDNA molecule of any one of claims 1-7, wherein each oligonucleotide is less than 45 nucleotides in length.

9. The ssDNA molecule of any one of claims 1-8, wherein the at least one double-stranded region in the central region comprises one or more gaps and / or one or more nicks in at least one strand.

10. The ssDNA molecule of any one of claims 1-9, wherein the at least one double-stranded region in the central region comprises one or more gaps, and wherein the one or more gaps is one or more nucleotides long.

11. The ssDNA molecule of any one of claims 1-10, wherein the at least one double stranded region in the central region comprises two or more gaps, and wherein the two or more gaps have the same length.

12. The ssDNA molecule of any one of claims 1-11, wherein the at least one double stranded region in the central region comprises two or more gaps, and wherein the two or more gaps have different length.

13. The ssDNA molecule of any one of claims 1-12, wherein the at least one oligonucleotide is hybridized adjacent to the 3’ end of the ssDNA molecule with a gap between the oligonucleotide and the 3’end of the ssDNA molecule.

14. The ssDNA molecule of any one of claims 1-13, wherein the at least one oligonucleotide is hybridized adjacent to the 5’ end of the ssDNA molecule with a gap between the oligonucleotide and the 5’end of the ssDNA molecule.

15. The ssDNA molecule of any one of claims 1-8, wherein the at least one double-stranded region in the central region comprises no gaps or nicks in either strand.

16. The ssDNA molecule of any one of claims 1-15, wherein the at least one oligonucleotide is ligated to the ssDNA.

17. The ssDNA molecule of claim 16, wherein the at least one oligonucleotide is ligated to the 3’end of the ssDNA.

18. The ssDNA molecule of claim 16, wherein the at least one oligonucleotide is ligated to the 5’end of the ssDNA.

19. The ssDNA molecule of any one of claims 1-18, wherein the at least one oligonucleotide comprises one or more modified nucleotides.

20. The ssDNA molecule of claim 19, wherein the at least one oligonucleotide comprises one or more modified nucleotides at the 3’ end.

21. The ssDNA molecule of claim 19, wherein the at least one oligonucleotide comprises one or more modified nucleotides at the 5’ end.

22. The ssDNA molecule of any one of claims 19-21, wherein the one or more modified nucleotides are phosphorothioate-modified (PS) nucleotides, 2’-O-methyl nucleotides, 2’-Fluoro nucleotides, or a combination thereof.

23. The ssDNA molecule of any one of claims 1-22, wherein the at least one double-stranded region comprises a binding site for a protein.

24. The ssDNA molecule of any one of claims 1-23, wherein the at least one oligonucleotide comprises a binding site for a protein.

25. The ssDNA molecule of claims 23 or 24, wherein the protein is an enzyme or a chaperone.

26. The ssDNA molecule of claim 25, wherein the protein is an enzyme and wherein the enzyme is a Cas enzyme.

27. The ssDNA molecule of claim 26, wherein the Cas enzyme is dead Cas9 28. The ssDNA molecule of claim 25, wherein the protein is an enzyme and wherein the enzyme is a recombinase.

29. The ssDNA molecule of any one of claims 1-28, wherein the at least one oligonucleotide comprises one or more functional moieties.

30. The ssDNA molecule of claim 29, wherein the functional moieties are selected from the group consisting of an aptamer, an antisense oligonucleotide (ASO), a ribozyme, a short-interfering RNA (siRNA), a benzylguanine (BG), a fluorophore, and a peptide.

31. The ssDNA molecule of claim 30, wherein the functional moiety is an aptamer, and wherein the aptamer is capable of nuclear translocation in a cell.

32. The ssDNA molecule of claim 29, wherein the ssDNA molecule comprises one or more hybridized oligonucleotides in the central region, and where the one or more oligonucleotides comprise at least one aptamer.

33. The ssDNA molecule of claim 32, wherein the ssDNA molecule comprises two or more hybridized oligonucleotides in the central region, and wherein each of the two or more oligonucleotides comprises an aptamer.

34. The ssDNA molecule of claim 33, wherein the ssDNA molecule comprises a gapped aptamer array.

35. The ssDNA molecule of claim 33, wherein the ssDNA molecule comprises a ligated aptamer array.

36. The ssDNA molecule of claim 33, wherein the aptamers have the same sequence.

37. The ssDNA molecule of claim 33, wherein the aptamers have different sequences.

38. The ssDNA molecule of claim 37, wherein one or more of the aptamers comprise a modified nucleotide.

39. The ssDNA molecule of any one of claims 1-38, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends is sufficient to prime replication and / or transcription.

40. The ssDNA molecule of any one of claims 1-39, wherein at least one of the stems at the 5’ and / or 3’ ends comprises a partial DNA duplex of about 4-1500 bp.

41. The ssDNA molecule of any one of claims 1-40, wherein at least one of the stems at the 5’ and / or 3’ ends comprises a partial DNA duplex of about 4-10 nucleotides.

42. The ssDNA molecule of any one of claims 1-41, wherein at least one of the loops at the 5’ and / or 3’ ends comprises about 3-500 single-stranded nucleotides.

43. The ssDNA molecule of any one of claims 1-42, wherein each of the loops at the 5’ and / or 3’ ends comprise a minimum of 3 single-stranded nucleotides.

44. The ssDNA molecule of any one of claims 1-43, wherein the ssDNA molecule comprises at least two stem-loop structures at each of the 5’ and / or 3’ ends.

45. The ssDNA molecule of any one of claims 1-44, wherein the ssDNA molecule comprises at least three stem-loop structures at each of the 5’ and / or 3’ ends.

46. The ssDNA molecule of any one of claims 1-45, wherein the ssDNA molecule comprises at least four or more stem-loop structures at each of the 5’ and / or 3’ ends.

47. The ssDNA molecule of any one of claims 1-46, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises a hairpin DNA structure.

48. The ssDNA molecule of any one of claims 1-47, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises a DNA structure selected from the group consisting of: a cruciform DNA structure, a hammerhead DNA structure, a quadraplex DNA structure, a bulged DNA structure, and a multibranched loop structure.

49. The ssDNA molecule of any one of claims 1-48, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises a viral ITR or partial viral ITR.

50. The ssDNA molecule of any one of claims 1-49, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises an AAV ITR or partial AAV ITR.

51. The ssDNA molecule of any one of claims 1-50, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises an AAV2 ITR or partial AAV2 ITR.

52. The ssDNA molecule of any one of claims 1-51, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises a wild-type AAV ITR.

53. The ssDNA any of any one of claims 1-52, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises an ITR modified by at least one deletion, addition, and / or substitution relative to a wild-type AAV ITR.

54. The ssDNA molecule of any one of claims 1-53, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises at least one AAV ITR region selected from the group consisting of an A region, an A’ region, a B region, a B’ region, a C, a C’ region, a D region, and a D’ region.

55. The ssDNA molecule of any one of claims 1-54, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends does not comprise the A or A’ regions that would be present in a wild-type AAV ITR.

56. The ssDNA molecule of any one of claims 1-55, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends does not comprise the A, A’, D, or D’ regions that would be present in a wild-type AAV ITR.

57. The ssDNA molecule of any one of claims 1-56, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends does not comprise the A, A’, B, B’, C, C’, D, or D’ regions that would be present in a wild-type AAV ITR.

58. The ssDNA molecule of any one of claims 1-57, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends does not comprise a rep binding element (RBE) that would be present in a wild-type AAV ITR.

59. The ssDNA molecule of any one of claims 1-58, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends does not comprise a terminal resolution site (trs) that would be present in a wild-type ITR.

60. The ssDNA molecule of any one of claims 1-48 or 55-59, wherein the ssDNA molecule does not comprise any viral sequences.

61. The ssDNA molecule of any one of claims 1-60, wherein at least one of the stems at the 5’ and / or 3’ ends of the ssDNA molecule comprises one or more nucleotides that are modified to be exonuclease resistant.

62. The ssDNA molecule of any one of claims 1-61, wherein the 5’ and / or 3’ ends of the ssDNA molecule comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more nucleotides that are modified to be exonuclease resistant.

63. The ssDNA molecule of claim 1-62, wherein the nucleotides that are modified to be exonuclease resistant are phosphorothioate-modified (PS) nucleotides.

64. The ssDNA molecule of any one of claims 1-63, wherein at least one of the stem-loop structures at the 5’ and / or 3’ ends comprises at least one functional moiety.

65. The ssDNA molecule of claim 64, wherein the at least one functional moiety is selected from the group consisting of an aptamer, an antisense oligonucleotide (ASO), a ribozyme, a short- interfering RNA (siRNA), a benzylguanine (BG), a fluorophore, and a peptide.

66. The ssDNA molecule of claim 65, wherein the aptamer is capable of nuclear translocation in a cell.

67. The ssDNA molecule of any one of claims 1-66, wherein the ssDNA molecule comprises at least one stem-loop structure at each of its 5’ and 3’ ends, wherein each of the at least one stem-loop structures at the 5’ and 3’ ends comprises at least one stem and at least one loop.

68. The ssDNA molecule of any one of claims 67, wherein at least one of the loops at the 5’ and / or 3’ ends further comprises one or more nucleic acids that stabilize the ends.

69. The ssDNA molecule of claims 67 or 68, wherein at least one of the loops at the 5’ and / or 3’ ends further comprises one or more nucleic acids that are chemically modified.

70. The ssDNA molecule of any one of claims 1-69, wherein at least one of the loops at the 5’ end and / or 3’ ends further comprises one or more triplex forming oligonucleotides.

71. The ssDNA molecule of any one of claims 1-70, wherein at least one of the loops at the 5’ and / or 3’ ends further comprises one or more gRNAs or gDNAs.

72. The ssDNA molecule of any one of claims 1-71, wherein at least one of the loops at the 5’ and / or 3’ end further comprises one or more molecular probes.

73. The ssDNA molecule of any one of claims 1-72, wherein the ssDNA molecule is devoid of any viral capsid protein coding sequences.

74. The ssDNA molecule of any one of claims 1-73, wherein the ssDNA molecule is synthetically produced in vitro.

75. The ssDNA molecule of any one of claims 1-74 wherein the ssDNA molecule is synthetically produced in vitro in a cell-free environment.

76. The ssDNA molecule of any one of claims 1-75, wherein the ssDNA molecule does not activate or minimally activates an immune pathway.

77. The ssDNA molecule of claim 76, wherein the immune pathway is an innate immune pathway.

78. The ssDNA molecule of claim 77, wherein the innate immune pathway is selected from the group consisting of the cGAS / STING pathway, the TLR9 pathway, an inflammasome-mediated pathway, and combinations thereof.

79. The ssDNA molecule of any one of claims 76-78, wherein the ssDNA molecule does not induce, or minimally induces, at least one cytokine selected from the group consisting of IFNα, IFNγ, IL-6, TNFα, and IL-18, as compared to a closed-ended deoxyribonucleic acid (ceDNA) molecule.

80. The ssDNA molecule of any one of claims 1-79, wherein the central region of the ssDNA molecule comprises at least one promoter.

81. The ssDNA molecule of claim 80, wherein the promoter is a cell type- or tissue-specific promoter.

82. The ssDNA molecule of claim 80 or 81, wherein the promoter is specific for a cell type or tissue selected from the group consisting of liver, skeletal muscle, smooth muscle, photoreceptors, retinal cells, hematopoietic stem cells (HSCs), T cells, B cells, Natural Killer (NK) cells, dendritic cells, megakaryocytes, neurons, brain, lung, heart, kidney, liver, spleen, pancreas, islet cells, prostate, testis, ovary, uterus, thyroid, thymus, adipose cells, epithelial cells, endothelial cells, bone cells, keratinocytes, fibroblasts, and salivary gland.

83. The ssDNA molecule of any one of claims 80-82, wherein the promoter is a liver-specific promoter.

84. The ssDNA molecule of any one of claims 80-83, wherein the promoter is a TTR promoter.

85. The ssDNA molecule of any one of claims 80-83, wherein the promoter is a hAAT promoter.

86. The ssDNA molecule of claim 80, wherein the promoter is a constitutive promoter.

87. The ssDNA molecule of any of claims 1-86, wherein the central region of ssDNA molecule comprises at least one enhancer.

88. The ssDNA molecule of claim 87, wherein the enhancer comprises a serpin (SERP) enhancer.

89. The ssDNA molecule of claim 87 or 88, wherein the enhancer comprises a human SERP enhancer.

90. The ssDNA molecule of any one of claims 87-89, wherein the enhancer comprises a 1X human SERP enhancer, a 2X human SERP enhancer, or a 3X human SERP enhancer.

91. The ssDNA molecule of claim 87 or 88, wherein the enhancer comprises a SERP enhancer with at least 95% sequence identity a human SERP enhancer.

92. The ssDNA molecule of any one of claims 87, 88, or 91, wherein the enhancer comprises a BB SERP enhancer comprising nucleotides 2749-2818 and / or 3068-3137 of the sequence set forth in FIG.

93.

93. The ssDNA molecule of claim 92, wherein the enhancer comprises a 1X BB SERP enhancer, a 2X BB SERP enhancer, or a 3X BB SERP enhancer 94. The ssDNA molecule of any one of claims 80-84 or 87-93, wherein the ssDNA molecule comprises a TTR promoter and a SERP enhancer.

95. The ssDNA molecule of any one of claims 80-94, wherein the promoter comprises a transcription start site (TSS).

96. The ssDNA molecule of any one of claims 80-95, wherein the promoter is double-stranded.

97. The ssDNA molecule of any one of claims 87-96, wherein the enhancer is double-stranded.

98. The ssDNA molecule of any one of claims 95-97, wherein the TSS is double-stranded.

99. The ssDNA molecule of any one of claims 1-98, wherein the central region comprises a total of 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, at least 1500 base pairs, at least 1600 base pairs, at least 1700 base pairs, at least 1800 base pairs, at least 1900 base pairs, at least 2000 base pairs, at least 2100 base pairs, at least 2200 base pairs, at least 2300 base pairs, at least 2400 base pairs, or at least 2500 base pairs of double- stranded DNA.

100. The ssDNA molecule of any one of claims 1-99, wherein the central region comprises a total of fewer than 2500 base pairs, fewer than 2400 base pairs, fewer than 2300 base pairs, fewer than 2200 base pairs, fewer than 2100 base pairs, fewer than 2000 base pairs, fewer than 1900 base pairs, fewer than 1800 base pairs, fewer than 1700 base pairs, fewer than 1600 base pairs, fewer than 1500 base pairs, fewer than 1400 base pairs, fewer than 1300 base pairs, fewer than 1200 base pairs, fewer than 1100 base pairs, fewer than 1000 base pairs, fewer than 950 base pairs, fewer than 900 base pairs, fewer than 850 base pairs, fewer than 800 base pairs, fewer than 750 base pairs, fewer than 700 base pairs, fewer than 650 base pairs, fewer than 600 base pairs, fewer than 550 base pairs, fewer than 500 base pairs, fewer than 480 base pairs, fewer than 460 base pairs, fewer than 440 base pairs, fewer than 420 base pairs, fewer than 400 base pairs, fewer than 380 base pairs, fewer than 360 base pairs, fewer than 340 base pairs, fewer than 320 base pairs, fewer than 300 base pairs, fewer than 280 base pairs, fewer than 260 base pairs, fewer than 240 base pairs, fewer than 220 base pairs, fewer than 200 base pairs, fewer than 190 base pairs, fewer than 180 base pairs, fewer than 170 base pairs, fewer than 160 base pairs, fewer than 150 base pairs, fewer than 140 base pairs, fewer than 130 base pairs, fewer than 120 base pairs, fewer than 110 base pairs, fewer than 100 base pairs, fewer than 90 base pairs, fewer than 80 base pairs, fewer than 70 base pairs, or fewer than 60 base pairs of double-stranded DNA.

101. The ssDNA molecule of any one of claims 1-100, wherein the central region comprises a total of about 50-2500 base pairs of double-stranded DNA.

102. The ssDNA molecule of any one of claims 1-101, wherein the central region comprises a total of about 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000- 1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000, 2000-2100, 2100-2200, 2200-2300, 2300-2400, or 2400-2500 base pairs of double- stranded DNA.

103. The ssDNA molecule of any one of claims 1-102, wherein the central region comprises 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, at least 1500 base pairs, at least 1600 base pairs, at least 1700 base pairs, at least 1800 base pairs, at least 1900 base pairs, at least 2000 base pairs, at least 2100 base pairs, at least 2200 base pairs, at least 2300 base pairs, at least 2400 base pairs, or at least 2500 base pairs of double- stranded DNA at the 3’ end of the ssDNA molecule.

104. The ssDNA molecule of any one of claims 1-103, wherein the central region comprises fewer than 2500 base pairs, fewer than 2400 base pairs, fewer than 2300 base pairs, fewer than 2200 base pairs, fewer than 2100 base pairs, fewer than 2000 base pairs, fewer than 1900 base pairs, fewer than 1800 base pairs, fewer than 1700 base pairs, fewer than 1600 base pairs, fewer than 1500 base pairs, fewer than 1400 base pairs, fewer than 1300 base pairs, fewer than 1200 base pairs, fewer than 1100 base pairs, fewer than 1000 base pairs, fewer than 950 base pairs, fewer than 900 base pairs, fewer than 850 base pairs, fewer than 800 base pairs, fewer than 750 base pairs, fewer than 700 base pairs, fewer than 650 base pairs, fewer than 600 base pairs, fewer than 550 base pairs, fewer than 500 base pairs, fewer than 480 base pairs, fewer than 460 base pairs, fewer than 440 base pairs, fewer than 420 base pairs, fewer than 400 base pairs, fewer than 380 base pairs, fewer than 360 base pairs, fewer than 340 base pairs, fewer than 320 base pairs, fewer than 300 base pairs, fewer than 280 base pairs, fewer than 260 base pairs, fewer than 240 base pairs, fewer than 220 base pairs, fewer than 200 base pairs, fewer than 190 base pairs, fewer than 180 base pairs, fewer than 170 base pairs, fewer than 160 base pairs, fewer than 150 base pairs, fewer than 140 base pairs, fewer than 130 base pairs, fewer than 120 base pairs, fewer than 110 base pairs, fewer than 100 base pairs, fewer than 90 base pairs, fewer than80 base pairs, fewer than 70 base pairs, or fewer than 60 base pairs of double-stranded DNA at the 3’ end of the ssDNA molecule.

105. The ssDNA molecule of any one of claims 1-104, wherein the central region comprises at least 50 base pairs of double-stranded DNA at 3’ end of the ssDNA molecule and at least 50 base pairs of double-stranded DNA at the 5’ end of the ssDNA molecule.

106. The ssDNA molecule of any one of claims 1-105, wherein the central region is at least about 50% single-stranded, at least about 51% single-stranded, at least about 52% single-stranded, at least about 53% single-stranded, at least about 54% single-stranded, at least about 55% single-stranded, at least about 56% single-stranded, at least about 57% single-stranded, at least about 58% single- stranded, at least about 59% single-stranded, at least about 60% single-stranded, at least about 61% single-stranded, at least about 62% single-stranded, at least about 63% single-stranded, at least about 64% single-stranded, at least about 65% single-stranded, at least about 66% single-stranded, at least about 67% single-stranded, at least about 68% single-stranded, at least about 69% single-stranded, at least about 70% single-stranded, at least about 71% single-stranded, at least about 72% single- stranded, at least about 73% single-stranded, at least about 74% single-stranded, at least about 75% single-stranded, at least about 76% single-stranded, at least about 77% single-stranded, at least about 78% single-stranded, at least about 79% single-stranded, at least about 80% single-stranded, at least about 81% single-stranded, at least about 82% single-stranded, at least about 83% single-stranded, at least about 84% single-stranded, at least about 85% single-stranded, at least about 86% single- stranded, at least about 87% single-stranded, at least about 88% single-stranded, at least about 89% single-stranded, at least about 90% single-stranded, at least about 91% single-stranded, at least about 92% single-stranded, at least about 93% single-stranded, at least about 94% single-stranded, or at least about 95% single-stranded.

107. The ssDNA molecule of any one of claims 1-106, wherein the central region is less than about 50% single-stranded, less than about 51% single-stranded, less than about 52% single-stranded, less than about 53% single-stranded, less than about 54% single-stranded, less than about 55% single- stranded, less than about 56% single-stranded, less than about 57% single-stranded, less than about 58% single-stranded, less than about 59% single-stranded, less than about 60% single-stranded, less than about 61% single-stranded, less than about 62% single-stranded, less than about 63% single- stranded, less than about 64% single-stranded, less than about 65% single-stranded, less than about 66% single-stranded, less than about 67% single-stranded, less than about 68% single-stranded, less than about 69% single-stranded, less than about 70% single-stranded, less than about 71% single- stranded, less than about 72% single-stranded, less than about 73% single-stranded, less than about 74% single-stranded, less than about 75% single-stranded, less than about 76% single-stranded, lessthan about 77% single-stranded, less than about 78% single-stranded, less than about 79% single- stranded, less than about 80% single-stranded, less than about 81% single-stranded, less than about 82% single-stranded, less than about 83% single-stranded, less than about 84% single-stranded, less than about 85% single-stranded, less than about 86% single-stranded, less than about 87% single- stranded, less than about 88% single-stranded, less than about 89% single-stranded, less than about 90% single-stranded, less than about 91% single-stranded, less than about 92% single-stranded, less than about 93% single-stranded, less than about 94% single-stranded, or less than about 95% single- stranded.

108. The ssDNA molecule of any one of claims 1-107, wherein the central region is about 50%- 95% single-stranded.

109. The ssDNA molecule of any one of claims 1-108, wherein the single-stranded region of the central region is on a (-) strand of the ssDNA molecule.

110. The ssDNA molecule of any one of claims 1-108, wherein the single-stranded region of the central region is on a (+) strand of the ssDNA molecule.

111. The ssDNA molecule of any one of claims 1-110, wherein the nucleic acid sequence of interest encodes at least one molecule selected from the group consisting of a peptide, a polypeptide, a ribozyme, a peptide nucleic acid (PNA), an siRNA, an RNAi, an antisense oligonucleotide, an shRNA, a micro-RNA, an mRNA, a gRNA, and an antagoMiR.

112. The ssDNA molecule of any one of claims 1-111, wherein the nucleic acid sequence of interest encodes an antibody or antigen-binding fragment thereof.

113. The ssDNA molecule of any one of claims 1-112, wherein the nucleic acid sequence of interest comprises at least one open-reading frame (ORF).

114. The ssDNA molecule of any one of claims 1-113, wherein the ssDNA molecule is capable of expressing at least one therapeutic protein or a therapeutic fragment thereof.

115. The ssDNA molecule of claim 114, 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.

116. The ssDNA molecule of claim 114 or 115, wherein the at least one therapeutic protein is useful for treating a genetic disorder selected from the group consisting of sickle cell disease, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited disorders of hepatic metabolism, Lesch-Nyhan syndrome, thalassaemias, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharide storage diseases (e.g., Hurler syndrome (MPS Type I), Scheie syndrome (MPS Type I S), Hurler-Scheie syndrome (MPS Type I H-S), 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), Tay-Sachs disease, Metachromatic Leukodystrophy, Krabbe disease, Mucolipidosis Type I, II / III and IV, Sialidosis Types I and II, Glycogen Storage disease Types I and II (Pompe disease), Gaucher disease Types I, II and III, cystinosis, Batten disease, Aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), Lysosomal Acid Lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipidoses, 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 (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.

117. The ssDNA molecule of any one of claims 114-116, wherein the at least one therapeutic protein is Factor VIII (FVIII).

118. The ssDNA molecule of any one of claims 1-117, wherein the at least one nucleic acid sequence of interest comprises at least one region selected from the group consisting of a spacer, a 5’ untranslated region (UTR), a 3’ UTR, an intron, and a polyA signal.

119. The ssDNA molecule of any one of claims 1-118, wherein the at least one nucleic acid sequence of interest comprises a spacer.

120. The ssDNA molecule of claim 119, wherein the spacer is located between the at least one stem-loop structure at the 3’ end and an enhancer, and / or between the at least one stem-loop structure at the 3’ end and a promoter.

121. The ssDNA molecule of claim 119 or 120, wherein the spacer is at least partially double- stranded and comprises at least about 100, 200, 300, 400, 500, or 600 base pairs.

122. The ssDNA molecule of any one of claims 1-121, wherein the at least one nucleic acid sequence of interest comprises a 5’ UTR.

123. The ssDNA molecule of any one of claims 1-122, wherein the at least one nucleic acid sequence of interest comprises a 3’ UTR.

124. The ssDNA molecule of any one of claims 1-123, wherein the at least one nucleic acid sequence of interest comprises an intron.

125. The ssDNA molecule of any one of claims 1-124, wherein the at least one nucleic acid sequence of interest comprises a polyA signal.

126. The ssDNA molecule of any one of claims 1-125, wherein the at least one nucleic acid sequence of interest comprises a promoter, an enhancer, a TSS, an ORF, a 5’ UTR, a 3’ UTR, an intron, and a polyA signal.

127. A composition comprising the ssDNA molecule of any one of claims 1-126 and a lipid.

128. The composition of claim 127, wherein the ssDNA molecule is encapsulated in the lipid.

129. A lipid nanoparticle comprising the ssDNA molecule of any one of claims 1-126.

130. A pharmaceutical composition comprising the ssDNA molecule of any one of claims 1-126 and a pharmaceutically acceptable excipient.

131. A pharmaceutical composition comprising the liquid nanoparticle of claim 129 and a pharmaceutically acceptable excipient.

132. A cell comprising the ssDNA molecule of any one of claims 1-126.

133. The cell of claim 132, wherein the cell is in vitro, ex vivo, or in vivo.

134. A method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the ssDNA molecule of any one of claims 1-126, the composition of claim 127 or 128, the lipid nanoparticle of claim 129, the pharmaceutical composition of claim 130 or 131, or the cell of claim 132 or 133.

135. The method of claim 134, wherein the disease or disorder is a genetic disease.

136. The method of claim 134 or 135, wherein the disease or disorder is selected from the group consisting of sickle cell disease, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited disorders of hepatic metabolism, Lesch-Nyhan syndrome, thalassaemias, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharide storage diseases (e.g., Hurler syndrome (MPS Type I), Scheie syndrome (MPS Type I S), Hurler-Scheie syndrome (MPS Type I H- S), 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), Tay-Sachs disease, Metachromatic Leukodystrophy, Krabbe disease, Mucolipidosis Type I, II / III and IV, Sialidosis Types I and II, Glycogen Storage disease Types I and II (Pompe disease), Gaucher disease Types I, II and III, cystinosis, Batten disease, Aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), Lysosomal Acid Lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipidoses, 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 (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.

137. A method of delivering a therapeutic gene and / or a therapeutic protein to a subject comprising administering to the subject a therapeutically effective amount of the ssDNA molecule of any one ofclaims 1-126, the composition of claim 127 or 128, the lipid nanoparticle of claim 129, the pharmaceutical composition of claim 130 or 131, or the cell of claim 132 or 133.

138. A method of delivering a therapeutic gene and / or a therapeutic protein to a cell comprising contacting the cell with the ssDNA molecule of any one of claims 1-126, the composition of claim 127 or 128, the lipid nanoparticle of claim 129, the pharmaceutical composition of claim 130 or 131, or the cell of claim 132 or 133, thereby delivering the therapeutic gene and / or therapeutic protein to the cell.

139. A method of delivering a therapeutic gene to the nucleus of a cell comprising contacting the cell with the ssDNA molecule of any one of claims 1-126, the composition of claim 127 or 128, the lipid nanoparticle of claim 129, the pharmaceutical composition of claim 130 or 131, or the cell of claim 132 or 133, thereby delivering the therapeutic gene and / or therapeutic protein to the nucleus of the cell.

140. A method of minimizing an immune response in a subject, wherein the subject is being treated with a therapeutic gene or a therapeutic protein, comprising administering to the subject a therapeutically effective amount of the ssDNA molecule of any one of claims 1-126, the composition of claim 127 or 128, the lipid nanoparticle of claim 129, the pharmaceutical composition of claim 130 or 131, or the cell of claim 132 or 133, wherein the nucleic acid of interest encodes the therapeutic gene or therapeutic protein.

141. A method for producing a second partially single-stranded deoxyribonucleic acid (ssDNA) molecule, the method comprising: (a) providing a first ssDNA molecule and an oligonucleotide complementary to a portion of the first ssDNA molecule, wherein the first ssDNA molecule comprises: (1) a central region, comprising at least one single-stranded region and at least one double-stranded region, wherein the central region comprises at least one nucleic acid sequence of interest; and (2) at least a first stem-loop structure at its 3’ end, wherein the first stem-loop structure comprises at least one stem and at least one loop, and / or at least a second stem-loop structure at its 5’ end, wherein the second stem-loop structure comprises at least one stem and at least one loop, wherein the first stem-loop structure and / or the second stem-loop structure flank the central region, and wherein the central region comprises a total of at least 50 base pairs (bp) of double-stranded DNA, and / or wherein the central region is at least 5% double- stranded.(b) allowing the oligonucleotide to hybridize to the first ssDNA molecule; (c) delivering the first ssDNA molecule and the hybridized oligonucleotide to a cell; and (d) allowing the hybridized oligonucleotide to extend using the first ssDNA molecule as a template, thereby generating a second ssDNA molecule.

142. The method of claim 141, wherein the oligonucleotide is complementary to a portion of the single-stranded region within the central region of the first ssDNA molecule.

143. The method of claim 141, wherein the oligonucleotide is complementary to a portion of the double-stranded region within the central region of the first ssDNA molecule.

144. The method of any one of claims 141-143, wherein the central region of the first ssDNA molecule comprises a promoter and wherein the oligonucleotide is complementary to a portion of the first ssDNA molecule upstream of the promoter.

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