Regulation of REP protein activity in the production of closed-end DNA (ceDNA)

Abstract

Provided herein is a method for producing a DNA vector, comprising: incubating a population of cells containing a vector polynucleotide encoding a heterogeneous nucleic acid operably positioned between first and second AAV inverted-end repeat DNA polynucleotide sequences (ITRs) in the presence of at least one Rep protein having DNA-binding and DNA-nicking functionality, under conditions and for a sufficient time to induce DNA production in cells, together with at least one ITR having a nucleotide sequence corresponding to an AAV wild-type ITR; and collecting and isolating the DNA having the resulting ITR from the cells.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 806,076, filed February 15, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which is incorporated herein by reference in its entirety. The aforementioned ASCII copy, created on February 13, 2020, is named 131698-05420_SL.txt and is 388,896 bytes in size.

[0003] The present invention relates to the field of gene therapy, which involves the delivery of exogenous DNA sequences to target cells, tissues, organs, or organisms. [Background technology]

[0004] Gene therapy aims to improve clinical outcomes for patients suffering from either genetic mutations or acquired diseases caused by abnormalities in gene expression profiles. Gene therapy involves the treatment or prevention of pathologies resulting from defective genes or dysregulation or expression, e.g., underexpression or overexpression, which can result in disorders, diseases, malignancies, etc. For example, diseases or disorders caused by defective genes can be treated, prevented, or ameliorated by delivering corrective genetic material to the patient, resulting in therapeutic expression of the genetic material in the patient's body. The basis of gene therapy is the provision of an active gene product (sometimes referred to as a transgene) in a transcription cassette, which can result in other outcomes, such as a positive gain-of-function effect, a negative loss-of-function effect, or an oncolytic effect. Human single-gene disorders can be treated by the delivery and expression of a normal gene in target cells. Delivery and expression of a corrective gene in the patient's target cells can be carried out via a number of methods, including the use of engineered viruses and viral gene delivery vectors. Among the many available virus-derived vectors (e.g., recombinant retroviruses, lentiviruses, adenoviruses, etc.), recombinant adeno-associated viruses (rAAVs) have gained popularity as versatile vectors in gene therapy.

[0005] Adeno-associated viruses (AAVs) belong to the Parvoviridae family and, more specifically, constitute the Dependoparvovirus genus. The AAV genome contains approximately 4.7 kilobases (kb) and is composed of a linear, single-stranded DNA molecule consisting of two major open reading frames (ORFs) encoding the nonstructural Rep (replication) and structural Cap (capsid) proteins. A second ORF within the cap gene has been identified that encodes the assembly-activating protein (AAP). The DNA flanking the AAV coding region is two cis-acting inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, containing an interrupted palindromic sequence that can fold into an energetically stable hairpin structure that functions as a primer for DNA replication. In addition to their role in DNA replication, ITR sequences have been shown to be involved in viral DNA integration into the cellular genome, rescue from the host genome or plasmids, and encapsidation of viral nucleic acids in mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129).

[0006] AAV-derived vectors (i.e., recombinant AAV (rAVV) or AAV vectors) are attractive for delivering genetic material because (i) they can infect (transduce) a wide variety of non-dividing and dividing cell types, including muscle cells and neurons; (ii) they lack viral structural genes, thereby reducing host cell responses to viral infection, such as interferon-mediated responses; (iii) wild-type viruses are considered non-pathogenic in humans; (iv) in contrast to wild-type AAV, which can integrate into the host cell genome, replication-deficient AAV vectors lack the rep gene and generally persist episomally, thus limiting the risk of insertional mutagenesis or genotoxicity; and (v) compared to other vector systems, AAV vectors are generally considered to be relatively poor immunogens and therefore do not elicit a significant immune response (see ii), thereby resulting in sustained vector DNA and potentially long-term expression of therapeutic transgenes. AAV vectors can also be produced and formulated at high titers and delivered via intra-arterial, intravenous, or intraperitoneal injection, enabling vector distribution and gene transfer to critical muscle regions with a single injection in rodents (Goyenvalle et al., 2004; Fougerousse et al., 2007; Koppanati et al., 2010; Wang et al., 2009) and dogs. In clinical studies to treat spinal muscular atrophy type 1, AAV vectors were delivered systemically with the intention of targeting the brain and resulting in clear clinical improvement.

[0007] However, the use of AAV particles as gene delivery vectors presents several significant drawbacks. One significant drawback associated with rAAV is the limited viral packaging capacity of approximately 4.5 kb of heterologous DNA (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010). As a result, the use of AAV vectors is limited to protein-coding capacities of less than 150 kDa. A second drawback is the need to screen rAAV gene therapy candidates for the presence of neutralizing antibodies that eliminate the vector from patients as a result of the prevalence of wild-type AAV infection in the population. A third drawback relates to capsid immunogenicity, which prevents re-administration in patients who have not been eliminated from initial treatment. In response to vectors that effectively act as a "booster" shot, the patient's immune system may stimulate the production of high-titer anti-AAV antibodies that prevent future treatment. Several recent reports have demonstrated a relationship between immunogenicity in high-dose settings. Another notable drawback is the relatively slow onset of AAV-mediated gene expression, given that single-stranded AAV DNA must be converted to double-stranded DNA before heterologous gene expression can occur. Attempts have been made to circumvent this problem by constructing double-stranded DNA vectors, but this strategy further limits the size of the transgene expression cassette that can be incorporated into the AAV vector (McCarty, 2008; Varenika et al., 2009; Foust et al., 2009).

[0008] Additionally, conventional AAV virions with capsids are produced by introducing one or more plasmids containing the AAV genome, rep gene, and cap gene (Grimm et al., 1998). Upon introduction of these helper plasmids in trans, the AAV genome is "rescued" from the host genome (i.e., released and then amplified) and further encapsidated (viral capsid), producing a biologically active AAV vector. However, such encapsidated AAV viral vectors have been found to inefficiently transduce certain cell and tissue types. The capsid also induces an immune response.

[0009] Therefore, the use of adeno-associated virus (AAV) vectors for gene therapy is limited by the single-dose requirement (due to patient immune responses), the limited range of transgene genetic material suitable for delivery in AAV vectors due to the minimal viral packaging capacity (approximately 4.5 kb) of the relevant AAV capsid, and slow AAV-mediated gene expression. The application of rAAV for clinical gene therapy is further hindered by patient-to-patient variability that is not predicted by dose-response in syngeneic mouse models or in other model species. Recombinant capsid-free AAV vectors can be obtained as isolated linear nucleic acid molecules containing an expressible transgene and a promoter region flanked by two wild-type AAV inverted terminal repeats (ITRs) containing Rep binding sites and terminal resolution sites (TRS). These recombinant AAV vectors lack the sequence encoding AAV capsid proteins and can be single-stranded, double-stranded, or duplexed, with one or both ends covalently linked through two wild-type ITR palindromic sequences (e.g., WO2012 / 123430, U.S. Patent No. 9,598,703). They have a much higher transgene capacity, a faster onset of transgene expression, and avoid many of the drawbacks of AAV-mediated gene therapy in that the patient's immune system recognizes the DNA molecule as a virus that should be cleared. However, constant expression of the transgene may not be desirable in all cases, and the AAV canonical wild-type ITRs may not be optimized for ceDNA function. Thus, there remains a significant unmet need for controllable recombinant DNA vectors and improved production and / or expression characteristics. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Muzyczka,(1992)Curr.Top.Micro.Immunol.158:97-129 Summary of the Invention [Means for solving the problem]

[0011] The invention described herein relates to improved production of non-viral, capsid-free DNA vectors with covalently closed ends (referred to herein as "closed-end DNA vectors" or "ceDNA vectors"). The ceDNA vectors produced by the methods described herein are non-capsid-containing, linear, double-stranded DNA molecules formed from continuous strands of complementary DNA (linear, continuous, non-encapsidated structures) with covalently closed ends, and contain 5' inverted terminal repeat (ITR) sequences and 3' ITR sequences that are different or asymmetric with respect to each other.

[0012] The technology described herein relates to the production of ceDNA or AAV vectors in cells (e.g., insect cells, mammalian cells) or cell-free systems with a single Rep protein species. In particular, the present disclosure is based, in part, on the unexpected discovery that either Rep78 or Rep68 alone is sufficient for the production of ceDNA or AAV vectors in cells. This is an improved, more efficient method of producing ceDNA vectors than described in the prior art, in which AAV or ceDNA vectors are produced in cells (e.g., insect cells) that require two Rep proteins, for example, at least one small Rep protein (e.g., Rep52 or Rep40) and at least one large Rep protein (e.g., Rep78 or Rep68). That is, the prior art describes the production of ceDNA or AAV vectors using two Rep proteins encoded on separate nucleic acid constructs each operably linked to a promoter, or two Rep proteins encoded on a single nucleic acid construct operably linked to a single promoter and having two initiation sites.

[0013] Thus, one embodiment of the technology described herein relates to nucleic acid constructs for producing DNA vectors, e.g., ceDNA vectors and other recombinant parvovirus (e.g., adeno-associated virus) vectors, in cells (e.g., insect cells, mammalian cells) and cell-free systems, e.g., insect cells or cell-free systems comprising a first nucleotide sequence encoding a single parvovirus Rep protein, wherein the nucleotide sequence has no open reading frame (ORF), lacks a functional start codon downstream of the first start codon, and / or lacks alternative splicing sites that prevent exon skipping, thereby allowing translation of only the single parvovirus Rep protein (e.g., Rep78 or Rep68 protein) without translating additional Rep proteins at subsequent start codons (e.g., any one or more of Rep52 or Rep40) in the insect cell or cell-free system. That is, a nucleic acid encoding Rep78 does not also produce a Rep52 protein, and similarly, a nucleic acid encoding Rep68 does not produce a Rep40 protein. Furthermore, no other Rep proteins are present or expressed in the system.

[0014] In some embodiments, the methods and compositions described herein for using a single Rep protein can be used in the production of any ceDNA vector, including, but not limited to, ceDNA vectors containing an asymmetric ITRS as disclosed in International Patent Application PCT / US18 / 49996, filed September 7, 2018 (see, e.g., Examples 1-4), ceDNA vectors for gene editing as disclosed in International Patent Application PCT / US18 / 64242, filed December 6, 2018 (see, e.g., Examples 1-7), or ceDNA vectors for the production of antibodies or fusion proteins as disclosed in International Patent Application PCT / US19 / 18016, filed February 14, 2019 (see, e.g., Examples 1-4), all of which are incorporated herein by reference in their entireties. In some embodiments, it is also contemplated that the methods and compositions described herein using a single Rep protein may be used in the synthetic production of ceDNA vectors, e.g., in cell-free or insect-free systems of ceDNA production, as disclosed in International Application PCT / US19 / 14122, filed January 18, 2019, which is incorporated herein by reference in its entirety, wherein a single Rep protein may be used for protein-assisted ligation of ITR oligonucleotides therein.

[0015] The technology described herein relates to improved methods for producing ceDNA vectors containing at least one modified AAV inverted terminal repeat (ITR) and an expressible transgene. The ceDNA vectors disclosed herein can be produced in eukaryotic cells according to the described methods and thus lack the prokaryotic DNA modifications and bacterial endotoxin contamination that insect cells suffer from.

[0016] Aspects of the invention relate to methods and compositions for producing ceDNA and AAV vectors using the single Rep proteins described herein. Other embodiments relate to the ceDNA vectors produced by the methods and compositions provided herein.

[0017] In one aspect, the non-viral capsid-free DNA vector with covalently closed ends produced by the methods described herein is preferably a linear double-stranded molecule and can be obtained from a vector polynucleotide encoding a heterologous nucleic acid operably positioned between two different inverted terminal repeats (ITRs) (e.g., AAV ITRs), at least one of which comprises a terminal resolution site and a replication protein binding site (RPS) (sometimes referred to as a replicative protein binding site), e.g., a Rep binding site, and one of the ITRs comprises a deletion, insertion, or substitution relative to the other ITR. That is, one of the ITRs is asymmetric relative to the other ITR. In one embodiment, at least one of the ITRs is an AAV ITR, e.g., a wild-type AAV ITR or a modified AAV ITR. In one embodiment, at least one of the ITRs is a modified ITR relative to the other ITR, that is, the ceDNA comprises ITRs that are asymmetric relative to each other. In one embodiment, at least one of the ITRs is a non-functional ITR.

[0018] In some embodiments, the ceDNA vector produced by the methods and compositions described herein comprises (1) an expression cassette containing cis-regulatory elements, a promoter, and at least one transgene, or (2) a promoter operably linked to at least one transgene, and (3) two self-complementary sequences, e.g., ITRs, flanking the expression cassette, wherein the ceDNA vector is not associated with a capsid protein. In some embodiments, the ceDNA vector comprises two self-complementary sequences found in the AAV genome, at least one of which comprises an operational Rep-binding element (RBE) (sometimes referred to herein as an "RBS") and an AAV terminal resolution site (trs) or a functional variant of the RBE, and one or more cis-regulatory elements operably linked to the transgene. In some embodiments, the ceDNA vector comprises additional components for regulating expression of the transgene, e.g., a regulatory switch, described herein in the section entitled "Regulatory Switch," for controlling and regulating expression of the transgene, e.g., a kill switch that enables controlled cell death of cells containing the ceDNA vector.

[0019] In some embodiments, the two self-complementary sequences can be ITR sequences from any known parvovirus, such as a dependovirus, such as AAV (e.g., AAV1-AAV12). Any AAV serotype can be used, including, but not limited to, modified AAV2 ITR sequences that retain a Rep binding site (RBS) such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) and a terminal resolution site (trs) in addition to a variable palindromic sequence that allows hairpin secondary structure formation. In some embodiments, the ITRs are synthetic ITR sequences that retain a functional Rep binding site (RBS) and a terminal resolution site (TRS) such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) in addition to a variable palindromic sequence that allows hairpin secondary structure formation. In some examples, the modified ITR sequences retain the RBS, trs sequences, and the structure and position of the Rep binding element that forms the terminal loop portion of one ITR hairpin secondary structure from the corresponding sequence of the wild-type AAV2 ITR.

[0020] Exemplary ITR sequences for use in ceDNA vectors produced by the methods and compositions described herein can be any one or more of the partial ITR sequences shown in Tables 2-10A and 10B, or SEQ ID NOs: 2, 52, 101-499, and 545-547, or Figures 26A-26B. In some embodiments, ceDNA vectors produced by the methods and compositions described herein do not have ITRs containing any sequence selected from SEQ ID NOs: 500-529.

[0021] In some embodiments, the ceDNA vectors produced by the methods and compositions described herein may comprise ITRs having ITR modifications corresponding to any of the ITR sequence or ITR subsequence modifications set forth in any one or more of Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A, and 10B herein.

[0022] As an illustrative example, a closed-end DNA vector produced by the methods and compositions described herein comprises a promoter operably linked to a transgene, the ceDNA lacks capsid protein, (a) is produced from a ceDNA-plasmid (see, e.g., Examples 1-2 and / or Figures 1A-B) encoding a mutant right AAV2 ITR having the same number of intramolecularly duplexed base pairs as SEQ ID NO:2 or a mutant left AAV2 ITR having the same number of intramolecularly duplexed base pairs as SEQ ID NO:51 in its hairpin secondary configuration (preferably excluding the deletion of any AAA or TTT terminal loops in this configuration compared to these reference sequences), and (b) is identified as a ceDNA using the assay for identification of ceDNA by agarose gel electrophoresis under native and denaturing conditions in Example 1. Examples of such modified ITR sequences are provided in Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A, and 10B herein.

[0023] The technology described herein further relates to the production of ceDNA vectors that can be used to deliver and encode one or more transgenes in target cells, for example, ceDNA vectors containing multicistronic sequences, or a transgene and its natural genomic context (e.g., a transgene, introns, and endogenous untranslated regions) are both incorporated into the ceDNA vector. A transgene can be a protein-coding transcript, a non-coding transcript, or both. The ceDNA vectors produced by the methods and compositions described herein can contain multiple coding sequences and non-canonical translation start sites, or two or more promoters for expressing protein-coding transcripts, non-coding transcripts, or both. A transgene can contain two or more protein-coding sequences or can be a non-coding transcript sequence. The expression cassette can contain, for example, more than 4,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, 20,000 nucleotides, 30,000 nucleotides, 40,000 nucleotides, or 50,000 nucleotides, or any range between about 4,000 and 10,000 nucleotides, or 10,000 and 50,000 nucleotides, or more than 50,000 nucleotides. The ceDNA vectors produced by the methods and compositions described herein do not have the size limitations of encapsidated AAV vectors, and therefore, delivery of large-sized expression cassettes can result in efficient expression of the transgene. In some embodiments, the ceDNA vectors produced by the methods and compositions described herein lack prokaryotic cell-specific methylation.

[0024] The expression cassette of the ceDNA vector produced by the methods and compositions described herein may also contain an internal ribosome entry site (IRES) and / or a 2A element. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue- and cell-type-specific promoters, and enhancers. In some embodiments, the ITRs may act as promoters for the transgene. In some embodiments, the ceDNA vector contains additional components for regulating the expression of the transgene. For example, the additional regulatory components may be the regulatory switches disclosed herein, including, but not limited to, kill switches that can kill ceDNA-infected cells, as well as other inducible and / or repressible elements, as needed.

[0025] The technology described herein further provides a novel method for efficiently producing ceDNA vectors or other AAV vectors that can selectively express one or more transgenes.The ceDNA vectors produced by the methods and compositions described herein have the ability to be incorporated into host cells and transported into the nucleus in the absence of AAV capsid.Furthermore, because the ceDNA vectors produced by the methods and compositions described herein lack capsid, they avoid the immune response that may occur in response to capsid-containing vectors.

[0026] In one embodiment, a capsid-free, non-viral DNA vector (ceDNA vector) is obtained from a plasmid (referred to herein as a "ceDNA-plasmid") containing a polynucleotide expression construct template comprising, in order, a first 5' inverted terminal repeat (e.g., AAV ITR), an expression cassette, and a 3' ITR (e.g., AAV ITR), wherein at least one of the 5' and 3' ITRs is a modified ITR, or when both the 5' and 3' ITRs are modified, they have different modifications from each other and are not the same sequence. In such an embodiment, the ceDNA vector is obtained by the process illustrated in the Examples and shown in Figures 4A-4D herein, wherein only a single Rep protein is required for production.

[0027] ceDNA vectors can be obtained by several means known to those skilled in the art after reading this disclosure. For example, the polynucleotide expression construct template used to generate the ceDNA vectors of the present invention can be a ceDNA-plasmid (e.g., see Table 12 or Figure 10B), a ceDNA-bacmid, and / or a ceDNA-baculovirus. In one embodiment, the ceDNA-plasmid contains a restriction cloning site (e.g., SEQ ID NO: 7) operably positioned between the ITRs, allowing for the insertion of, for example, an expression cassette comprising a promoter operably linked to a transgene, such as a reporter gene and / or a therapeutic gene. In some embodiments, the ceDNA vector is produced from a polynucleotide template (e.g., a ceDNA-plasmid, a ceDNA-bacmid, a ceDNA-baculovirus) containing modified ITRs compared to the corresponding adjacent AAV3 ITRs or wild-type AAV2 ITR sequences, where the modifications are any one or more of deletions, insertions, and / or substitutions.

[0028] According to some embodiments, the present disclosure provides methods for culturing insect cells (e.g., Sf9, Sf21, Trichoplusia ni cells, and High and a method for producing a ceDNA vector in insect or mammalian cells (e.g., HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1080, monocytes, and mature and immature dendritic cells), the method comprising culturing insect or mammalian cells containing a first nucleotide sequence encoding a single parvoviral Rep protein, wherein the first nucleotide sequence lacks a functional start codon downstream of the first start codon and lacks alternative splice sites that prevent exon skipping, thereby allowing translation of only the single Rep protein (e.g., Rep78) without translating additional Rep proteins at later start codons (e.g., any one or more of Rep52 or Rep40) or full-length spliced ​​variants (e.g., Rep68) in the cell.

[0029] According to some other embodiments, the present disclosure provides methods for culturing insect cells (e.g., Sf9, Sf21, Trichoplusia ni cells, and High and a method for producing a ceDNA vector in insect or mammalian cells (e.g., HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1080, monocytes, and mature and immature dendritic cells), the method comprising culturing insect or mammalian cells containing a first nucleotide sequence encoding a single parvoviral Rep protein, the first nucleotide sequence lacking a functional start codon downstream of the first start codon and containing a deletion of a carboxy-terminal splicing sequence (e.g., any portion or the full length of a c-terminal intron / skipped exon), thereby allowing translation of only the single Rep protein (e.g., Rep68) without translating additional Rep proteins (e.g., any one or more of Rep52 or Rep40) or the full length Rep72 protein at a later start codon in the cell.

[0030] According to some other aspects, the present disclosure provides methods for producing ceDNA vectors in insect cells (e.g., Sf9, Sf21, Trichoplusia ni cells, High Five cells) or mammalian cells (e.g., HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1080, monocytes, and mature and immature dendritic cells), the method comprising culturing insect or mammalian cells comprising a first nucleotide sequence encoding one or two Rep proteins (e.g., Rep78 and / or Rep68 proteins), wherein the first nucleotide sequence lacks a functional start codon and intact alternative splice sites downstream of the first start codon, thereby allowing translation of only the Rep78 and / or Rep68 protein without translating additional Rep proteins at later start codons (e.g., any one or more of Rep52 or Rep40).

[0031] The cells described in the above methods may further comprise a second nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) sequence flanking the heterologous sequence under conditions such that, when the first sequence is expressed to produce Rep78 and / or Rep68, ceDNA is produced by the Rep78 and / or Rep68 proteins in the absence of Rep52 or Rep40. The ceDNA vector can then be recovered from the cells. According to some embodiments, the nucleotide sequence comprising at least one AAV is part of an expression construct. According to some embodiments, the heterologous sequence comprises a therapeutic nucleic acid. According to some embodiments, the therapeutic nucleic acid is part of an expression construct. According to some embodiments, the cells further comprise a nucleic acid that functions as a marker. According to some embodiments, the nucleic acid that functions as a marker is part of an expression construct.

[0032] In a permissive host cell, for example, in the presence of a single Rep protein, a polynucleotide template having at least one modified ITR replicates to produce a ceDNA vector, which is produced through two steps: (i) a step in which the single Rep protein effects excision ("rescue") of the template from the template backbone (e.g., ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus genome, etc.), and (ii) a step in which the single Rep protein mediates replication of the excised ceDNA vector. The single Rep protein required for steps (i) and (ii) of excision and replication can be any Rep protein described herein. Rep proteins and Rep-binding sites of various AAV serotypes are well known to those skilled in the art.

[0033] Those skilled in the art will understand that Rep proteins are selected from serotypes that bind to and replicate nucleic acid sequences based on at least one functional ITR. For example, if the replication-competent ITRs are derived from AAV serotype 2, the corresponding Rep proteins are derived from AAV serotypes that cooperate with that serotype; for example, AAV2 ITRs cooperate with AAV2 or AAV4 Rep, but not with AAV5 Rep. During replication (i.e., after step (ii)), the covalently closed-end ceDNA vector continues to accumulate in permissive cells, and the ceDNA vector preferably accumulates in an amount of at least 1 pg / cell, preferably at least 2 pg / cell, preferably at least 3 pg / cell, more preferably at least 4 pg / cell, and even more preferably at least 5 pg / cell under standard replication conditions and in the presence of a single Rep protein, sufficiently stably for a long period of time.

[0034] Thus, one aspect of the present invention relates to a process comprising: a) incubating a population of host cells (e.g., insect cells) harboring a polynucleotide expression construct template (e.g., ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus) lacking a viral capsid-encoding sequence in the presence of a single Rep protein under conditions effective for a sufficient time to induce production of a ceDNA vector in the host cells, wherein the host cells do not contain a viral capsid-encoding sequence; and b) harvesting and isolating the ceDNA vector from the host cells. The presence of the single Rep protein induces replication of the vector polynucleotide with the modified ITR to produce the ceDNA vector in the host cells. However, viral particles (e.g., AAV virions) are not expressed. Therefore, there is no mandatory virion size limit. If the nucleic acid sequence encoding a Rep protein encodes a large Rep protein, e.g., a Rep78 or Rep68 protein, it is envisioned that the start codon of the smaller Rep protein will be modified so that only the large Rep protein is expressed in the cell.

[0035] According to some embodiments, the present disclosure provides insect cells (e.g., Sf9, Sf21, Trichoplusia ni cells, and High Five cells) or mammalian cells (e.g., HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1080, monocytes, and mature and immature dendritic cells), wherein the insect cells or mammalian cell lines comprise a first nucleotide sequence encoding a single parvoviral Rep protein, wherein the first nucleotide sequence lacks a functional start codon downstream of the first start codon and lacks alternative splice sites that prevent exon skipping, thereby allowing translation of only the single Rep protein (e.g., Rep78) without translating additional Rep proteins at later start codons (e.g., any one or more of Rep52 or Rep40) or full-length spliced ​​variants (e.g., Rep68) in the cell.

[0036] According to some other embodiments, the present disclosure provides methods for culturing insect cells (e.g., Sf9, Sf21, Trichoplusia ni cells, and High Insect or mammalian cells (e.g., HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1080, monocytes, and mature and immature dendritic cells) are provided, wherein the insect or mammalian cells comprise a first nucleotide sequence encoding a single parvoviral Rep protein, wherein the first nucleotide sequence lacks a functional start codon downstream of the first start codon and contains a deletion of a carboxy-terminal splicing sequence (e.g., any portion or the full length of a c-terminal intron / skipped exon), thereby allowing translation of only the single Rep protein (e.g., Rep68) without translating additional Rep proteins (e.g., any one or more of Rep52 or Rep40) or the full-length Rep72 protein at a later start codon in the cell.

[0037] According to some other aspects, the present disclosure provides insect cells (e.g., Sf9, Sf21, Trichoplusia ni cells, High Five cells) or mammalian cells (e.g., HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1080, monocytes, and mature and immature dendritic cells), wherein the insect cells or mammalian cell lines comprise a first nucleotide sequence encoding one or two Rep proteins (e.g., Rep78 and / or Rep68 proteins), wherein the first nucleotide sequence lacks a functional start codon and intact alternative splice sites downstream of the first start codon, thereby allowing translation of only the Rep78 and / or Rep68 proteins without translation of additional Rep proteins at later start codons (e.g., any one or more of Rep52 or Rep40).

[0038] The cells described above may further comprise a second nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) sequence flanking the heterologous sequence under conditions such that, when the first sequence is expressed to produce Rep78 and / or Rep68, ceDNA is produced by the Rep78 and / or Rep68 proteins without the presence of Rep52 or Rep40. The ceDNA vector may then be recovered from the cells. According to some embodiments, the nucleotide sequence comprising at least one AAV is part of an expression construct. According to some embodiments, the heterologous sequence comprises a therapeutic nucleic acid. According to some embodiments, the therapeutic nucleic acid is part of an expression construct. According to some embodiments, the cells further comprise a nucleic acid that functions as a marker. According to some embodiments, the nucleic acid that functions as a marker is part of an expression construct.

[0039] According to some aspects, the present disclosure provides a cell-free system comprising a first nucleotide sequence encoding a single parvoviral Rep protein, wherein the nucleotide sequence lacks a functional start codon downstream of the first start codon and / or lacks alternative splicing sites that prevent exon skipping, thereby allowing translation of only the single parvoviral Rep protein (e.g., a Rep78 or Rep68 protein) in the cell-free system without translating additional Rep proteins at later start codons (e.g., one or more of Rep52 or Rep40). According to some embodiments, a nucleic acid encoding Rep78 also does not produce a Rep52 or Rep40 protein. According to some embodiments, a nucleic acid encoding Rep68 does not produce a Rep52 or Rep40 protein. According to some embodiments, the insect cell, mammalian cell, or cell-free system does not express any other Rep proteins.

[0040] A ceDNA vector produced according to the methods described herein using a single Rep protein may be isolated from a host cell and its presence confirmed by digesting the DNA isolated from the host cell with a restriction enzyme that has a single recognition site on the ceDNA vector, and analyzing the digested DNA material on denaturing and native gels to confirm the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA. The present invention provides, for example, the following items. (Item 1) A DNA vector obtained from a vector polynucleotide, wherein the vector polynucleotide encodes a heterologous nucleic acid operably disposed between a first inverted terminal repeat DNA polynucleotide sequence (ITR) and a second ITR, and at least one of the first ITR and the second ITR comprises a nucleotide sequence corresponding to an AAV Rep binding sequence for inducing replication of the DNA vector in a cell in the presence of a single species of Rep protein, and the DNA vector comprises: a. incubating a population of cells harboring said vector polynucleotide lacking a viral capsid coding sequence in the presence of a single species of Rep protein having at least DNA binding and DNA nicking functionality under conditions effective and for a time sufficient to induce production of said DNA vector in said cells, wherein said cells do not contain viral capsid coding sequences and are free of other species of Rep protein; b. Harvesting and isolating the resulting DNA vector from said cell. (Item 2) 2. The DNA vector of item 1, wherein the cell is not contacted with a nucleotide sequence encoding a second Rep protein. (Item 3) 2. The DNA vector of item 1, wherein the single Rep protein further has helicase, ligase, and ATPase functionalities. (Item 4) 2. The DNA vector of item 1, wherein the Rep protein is an AAV Rep protein. (Item 5) 5. The DNA vector of item 4, wherein the Rep protein is selected from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 Rep proteins. (Item 6) 5. The DNA vector of item 4, wherein the Rep protein is the AAV2 Rep68 protein. (Item 7) 5. The DNA vector of item 4, wherein the Rep protein is AAV2 Rep78 protein. (Item 8) 8. The DNA vector of item 7, wherein the Rep78 protein is encoded by a mutant Rep78 nucleotide sequence that does not have a functional translation initiation codon for Rep52. (Item 9) 9. The DNA vector of item 8, wherein the mutant Rep78 nucleotide sequence encodes a mutant Rep78 protein containing a mutation at amino acid position 225 of SEQ ID NO: 530. (Item 10) 10. The DNA vector of item 9, wherein amino acid position 225 of SEQ ID NO: 530 is mutated to glycine (Gly) or threonine (Thr). (Item 11) 9. The DNA vector of item 8, wherein the mutated Rep78 nucleotide sequence comprises the sequence of SEQ ID NO: 530 or a sequence having at least 95% sequence identity to SEQ ID NO: 530, has at least DNA binding and DNA nicking functionality, and does not express a second Rep protein. (Item 12) 2. The DNA vector according to item 1, wherein the ITRs are parvovirus ITRs. (Item 13) Item 13. The DNA vector of item 12, wherein the parvovirus is a dependovirus. (Item 14) 2. The DNA vector according to item 1, wherein the DNA vector is a non-viral, capsid-free, double-stranded DNA vector (ceDNA vector) with covalently closed ends. (Item 15) 15. The DNA vector of item 14, wherein the presence of the ceDNA vector isolated from the cell can be confirmed by digesting the DNA isolated from the cell with a restriction enzyme that has a single recognition site on the DNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA. (Item 16) A DNA vector derived from a vector polynucleotide, wherein the vector polynucleotide encodes a heterologous nucleic acid operably disposed between two different inverted terminal repeats (ITRs), at least one of the ITRs is a functional ITR comprising a functional terminal resolution site and a Rep binding site, and the presence of a single species of Rep protein induces replication of the vector polynucleotide in a cell and production of the DNA vector, and the DNA vector is a. incubating a population of cells harboring said vector polynucleotide lacking a viral capsid coding sequence in the presence of a single species of Rep protein having at least DNA binding and DNA nicking functionality under conditions effective and for a time sufficient to induce production of said DNA vector in said cells, wherein said cells do not contain any nucleic acid encoding Rep52 or Rep40 within said cells and other species of Rep are not present in said cells; b. Harvesting and isolating said DNA vector from said cell. (Item 17) A polynucleotide for generating a DNA vector comprising a nucleotide sequence encoding a single species of Rep protein amino acid sequence having at least DNA-binding and DNA-nicking functionalities operably linked to at least one expression control sequence. (Item 18) 18. The polynucleotide of item 17, wherein the Rep protein has helicase, ligase, and ATPase functionality. (Item 19) 18. The polynucleotide of claim 17, wherein the Rep protein is an AAV Rep protein. (Item 20) 20. The polynucleotide of claim 19, wherein the AAV Rep protein is selected from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 Rep proteins. (Item 21) 20. The polynucleotide of claim 19, wherein the AAV Rep protein is an AAV2 Rep protein. (Item 22) 20. The polynucleotide of item 19, wherein the AAV Rep protein is an AAV2 Rep78 protein. (Item 23) 23. The polynucleotide of item 22, wherein the Rep78 protein is encoded by a mutant Rep78 nucleotide sequence that does not have a functional start codon for Rep52. (Item 24) 24. The polynucleotide of item 23, wherein the mutant Rep78 nucleotide sequence encodes a mutant Rep78 protein comprising a mutation at amino acid position 225 of SEQ ID NO: 530. (Item 25) 25. The polynucleotide of item 24, wherein amino acid 225 of SEQ ID NO: 530 is mutated to glycine (Gly) or threonine (Thr). (Item 26) 24. The polynucleotide of claim 23, wherein the mutant Rep78 nucleotide sequence comprises the sequence of SEQ ID NO: 530 or a sequence having at least 95% sequence identity to SEQ ID NO: 530, has at least DNA binding and DNA nicking functionality, and does not express a second Rep protein. (Item 27) 18. The polynucleotide of claim 17, wherein the at least one expression control sequence encodes an IE promoter, a ΔIE promoter, or a CMV promoter. (Item 28) 18. The polynucleotide of item 17, wherein the DNA vector is a non-viral capsid-free double-stranded DNA vector (ceDNA vector) with covalently closed ends. (Item 29) 29. The polynucleotide of claim 28, wherein the presence of the ceDNA vector isolated from the cell can be confirmed by digesting the DNA isolated from the cell with a restriction enzyme that has a single recognition site on the DNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA. (Item 30) 1. A method for producing a DNA vector, comprising: Cells, (1) a nucleotide sequence encoding a single species of AAV Rep protein (Rep78 and / or Rep68) having at least DNA binding and DNA nicking functionality linked to at least one expression control sequence, wherein the cell does not express or is not contacted with any other species of Rep protein; (2) A double-stranded DNA construct, an expression cassette; a first ITR upstream (5' end) of the expression cassette; a second ITR downstream (3' end) of the expression cassette; and (3) harvesting the DNA vector. (Item 31) 31. The method of claim 30, wherein the Rep protein is an AAV Rep protein. (Item 32) 32. The method of claim 31, wherein the AAV Rep protein is selected from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 Rep proteins. (Item 33) 32. The method of claim 31, wherein the AAV Rep protein is an AAV2 Rep68 protein. (Item 34) 32. The method of claim 31, wherein the AAV Rep protein is an AAV2 Rep78 protein. (Item 35) 35. The method of item 34, wherein the Rep78 protein is encoded by a mutant Rep78 nucleotide sequence that does not have a functional start codon for Rep52. (Item 36) 36. The method of claim 35, wherein the mutant Rep78 nucleotide sequence encodes a mutant Rep78 protein comprising a mutation at amino acid position 225 of SEQ ID NO: 530. (Item 37) 37. The method of claim 36, wherein amino acid 225 of SEQ ID NO: 530 is mutated to glycine (Gly) or threonine (Thr). (Item 38) 36. The method of claim 35, wherein the mutant Rep78 nucleotide sequence comprises the sequence of SEQ ID NO: 530 or a sequence having at least 95% sequence identity to SEQ ID NO: 530, and has at least DNA binding and DNA nicking functionality. (Item 39) 31. The method of claim 30, wherein the at least one expression control sequence encodes an IE promoter, a ΔIE promoter, or a CMV promoter. (Item 40) 40. The method of any one of items 30 to 39, wherein the double-stranded DNA construct is a bacmid, a plasmid, a minicircle, or a linear double-stranded DNA molecule. (Item 41) 41. The method according to any one of items 30 to 40, wherein the first ITR upstream of the expression cassette is a wild-type ITR. (Item 42) 42. The method of any one of items 30 to 41, wherein the first ITR upstream of the expression cassette and the second ITR downstream of the expression cassette are symmetric or substantially symmetric, or asymmetric with respect to each other. (Item 43) 43. The method of any one of items 30 to 42, wherein the ITR sequences are selected from any of those listed in Tables 2, 4A, 4B and 5 of International Patent Application PCT / US18 / 65242. (Item 44) 42. The method of claim 41, wherein the wild-type ITR comprises the polynucleotide of SEQ ID NO: 51. (Item 45) 45. The method according to any one of items 30 to 44, wherein the second ITR downstream of the expression cassette is a modified ITR. (Item 46) 46. ​​The method of claim 45, wherein the modified ITR comprises the polynucleotide of SEQ ID NO: 2. (Item 47) 41. The method according to any one of items 30 to 40, wherein the first ITR upstream of the expression cassette is a modified ITR. (Item 48) 48. The method of claim 47, wherein the modified ITR comprises the polynucleotide of SEQ ID NO: 52. (Item 49) 49. The method according to any one of items 47 to 48, wherein the second ITR downstream of the expression cassette is a wild-type ITR. (Item 50) 50. The method of claim 49, wherein the wild-type ITR comprises the polynucleotide of SEQ ID NO: 1. (Item 51) 51. The method of any one of items 30 to 50, wherein the ITR is replicable. (Item 52) 52. The method of any one of items 30 to 51, wherein the ITRs are AAV ITRs. (Item 53) 53. The method of any one of items 30 to 52, wherein the expression cassette comprises a cis-regulatory element. (Item 54) 54. The method of item 53, wherein the cis-regulatory element is selected from the group consisting of a post-transcriptional regulatory element and a BGH polyA signal. (Item 55) 55. The method of item 54, wherein the post-transcriptional regulatory element comprises a WHP post-transcriptional regulatory element (WPRE). (Item 56) 40. The method according to any one of items 30 to 39, wherein the expression cassette further comprises a promoter selected from the group consisting of a CAG promoter, an AAT promoter, an LP1 promoter, and an EF1a promoter. (Item 57) 57. The method according to any one of items 30 to 56, wherein the expression cassette comprises the polynucleotides of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. (Item 58) 58. The method of any one of items 30 to 57, wherein the expression cassette further comprises an exogenous sequence. (Item 59) 59. The method of claim 58, wherein the exogenous sequence comprises at least 2000 nucleotides. (Item 60) 60. The method of claim 58 or claim 59, wherein the exogenous sequence encodes a protein. (Item 61) 59. The method of claim 58, wherein the exogenous sequence encodes a reporter protein, a therapeutic protein, an antigen, a gene-editing protein, or a cytotoxic protein. (Item 62) 62. The method according to any one of items 30 to 61, wherein the DNA vector has a linear and continuous structure. (Item 63) A DNA vector produced by the method according to any one of items 30 to 62. (Item 64) 64. A pharmaceutical composition comprising the DNA vector of item 63 and, optionally, an excipient. (Item 65) A kit for producing a DNA vector, comprising: an expression construct comprising at least one restriction site for inserting at least one heterologous nucleotide sequence, or a regulatory switch, or both, wherein said at least one restriction site is operably positioned between asymmetric inverted terminal repeats (asymmetric ITRs), at least one of said asymmetric ITRs comprising a functional terminal resolution site and a Rep binding site; a vector comprising a polynucleotide sequence encoding a single species of Rep protein, the vector being suitable for expressing said single species of Rep protein in an insect cell. (Item 66) Item 66. The kit according to Item 65, which is suitable for producing the DNA vector according to Item 63. (Item 67) 67. The kit of claim 65 or 66, further comprising a population of insect cells lacking a viral capsid coding sequence and capable of inducing production of the ceDNA vector in the presence of a single species of Rep protein. (Item 68) A cell comprising a nucleotide sequence encoding a single species of AAV Rep protein (Rep78 and / or Rep68) having at least DNA-binding and DNA-nicking functionality, operably linked to at least one expression control sequence, wherein the cell does not express any other parvovirus Rep protein (Rep52 or Rep40) and is not in contact with any other species of Rep protein, and optionally a double-stranded DNA construct comprising an expression cassette, a first ITR upstream (5' end) of the expression cassette, and a second ITR downstream (3' end) of the expression cassette. (Item 69) 69. The cell of item 68, wherein the cell is an insect cell. (Item 70) 70. The cell of item 69, wherein the insect cell is selected from the group consisting of Sf9, Sf21, Trichoplusia ni cells, and High Five cells. (Item 71) 71. The cell of item 70, wherein the insect cell is an Sf9 cell. (Item 72) 71. The cell of item 70, wherein the insect cell is a High Five cell. (Item 73) 69. The cell of item 68, wherein the cell is a mammalian cell. (Item 74) 74. The cell of item 73, wherein the mammalian cell is selected from the group consisting of HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1080, monocytes, and mature and immature dendritic cells. (Item 75) 75. The cell of item 74, wherein the mammalian cell is HEK293. (Item 76) 69. The cell of item 68, wherein the nucleotide sequence encoding a single species of AAV Rep protein encodes Rep78 and / or Rep68. (Item 77) 77. The cell of item 76, wherein the nucleotide sequence does not have a functional start codon for Rep52 or Rep40. (Item 78) 78. The cell of item 77, wherein the nucleotide sequence encodes a Rep78 protein. (Item 79) 78. The cell of item 77, wherein the nucleotide sequence encodes a Rep68 protein. (Item 80) 78. The cell of item 77, wherein the nucleotide sequence encodes a mutant Rep78 or Rep68 protein comprising a mutation at amino acid position 225 of SEQ ID NO: 530. (Item 81) 81. The method of item 80, wherein amino acid 225 (methionine) of SEQ ID NO: 530 is mutated to glycine (Gly) or threonine (Thr). (Item 82) 81. The method of claim 80, wherein the nucleotide sequence further comprises one or more modifications at the carboxy-terminal alternative splice site to prevent splicing events that lead to the production of Rep68, thereby allowing the production of only Rep78. (Item 83) 78. The cell of item 77, wherein the nucleotide sequence is full length and contains an intact alternative splice site at the carboxy terminus, resulting in the production of both Rep78 and Rep68. (Item 84) 78. The cell of item 77, wherein the nucleotide sequence containing a deletion of a carboxy-terminal intron sequence results in the production of only Rep68. (Item 85) 78. The cell of item 77, wherein the nucleotide sequence comprises the sequence of SEQ ID NO: 530 or a sequence having at least 95% sequence identity to SEQ ID NO: 530, and has at least DNA binding and DNA nicking functionality. [Brief explanation of the drawings]

[0041] [Figure 1A]1 shows an exemplary structure of a ceDNA vector produced using a single Rep protein according to the methods and compositions described herein. In this embodiment, the exemplary ceDNA vector includes an expression cassette containing a CAG promoter, a WPRE, and BGHpA. An open reading frame (ORF) encoding a transgene is inserted into the cloning site (R3 / R4) between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs)—a wild-type AAV2 ITR upstream (5' end) of the expression cassette and a modified ITR downstream (3' end). Thus, the two ITRs flanking the expression cassette are asymmetric with respect to each other. Those skilled in the art will understand that any ITRs can be used. For illustrative purposes, the ITRs of the ceDNA constructs in this figure and in the examples herein are modified ITRs and WT ITRs. However, encompassed herein is a ceDNA vector containing a heterologous nucleic acid sequence (e.g., a transgene) positioned between two inverted terminal repeat (ITR) sequences, and the ITR sequences may be an asymmetric ITR pair or a symmetric or substantially symmetric ITR pair, as these terms are defined herein. The ceDNA vectors disclosed herein may include ITR sequences selected from: (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (mod-ITR) (e.g., an asymmetric modified ITR), (ii) two modified ITRs (e.g., an asymmetric modified ITR) in which the mod-ITR pair has a different three-dimensional spatial configuration relative to each other, or (iii) a symmetric or substantially symmetric WT-WT ITR pair in which each WT-ITR has the same three-dimensional spatial configuration, or (iv) a symmetric or substantially symmetric modified ITR pair in which each mod-ITR has the same three-dimensional spatial configuration. In some embodiments, the methods of the present disclosure involve using a single rep protein to produce a ceDNA vector that is formulated into a composition comprising a delivery system, such as, but not limited to, a liposomal nanoparticle delivery system. [Figure 1B]1A shows an exemplary structure of a ceDNA vector produced using a single Rep protein according to the methods and compositions described herein, which includes an expression cassette containing a CAG promoter, a WPRE, and BGHpA. An open reading frame (ORF) encoding a luciferase transgene is inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs)—a modified ITR upstream (5' end) and a wild-type ITR downstream (3' end) of the expression cassette. As discussed in FIG. 1A, one skilled in the art can easily select ITR sequences to form an asymmetric ITR pair or a symmetric or substantially symmetric ITR pair, as these terms are defined herein. [Figure 1C] 1A shows an exemplary structure of a ceDNA vector produced using a single Rep protein according to the methods and compositions described herein. The ceDNA vector comprises an expression cassette containing an enhancer / promoter and an open reading frame (ORF) for inserting a transgene, a post-transcriptional element (WPRE), and a poly(A) signal. The open reading frame (ORF) allows for insertion of a transgene into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs) that are asymmetric with respect to each other: a modified ITR upstream (5' end) and a modified ITR downstream (3' end) of the expression cassette, where both the 5' and 3' ITRs are modified ITRs but have different modifications (i.e., do not have the same modifications). As discussed in FIG. 1A, one skilled in the art can easily select ITR sequences to result in an asymmetric ITR pair or a symmetric or substantially symmetric ITR pair, as these terms are defined herein. [Figure 2A]The T-shaped stem-loop structure of the wild-type left ITR of AAV2 (SEQ ID NO: 538) is provided, with identification of the A-A' arm, the B-B' arm, the C-C' arm, and two Rep binding sites (RBE and RBE'), and the terminal resolution site (trs) is also shown. The RBE contains a series of four double tetramers that are thought to interact with either Rep78 or Rep68. Furthermore, the RBE' is also thought to interact with the Rep complex assembled on the wild-type or variant ITR in the construct. The D and D' regions contain transcription factor binding sites and other conserved structures. [Figure 2B] 1 shows the proposed Rep catalytic nicking and ligation activity in the wild-type left ITR (SEQ ID NO: 539), including the T-shaped stem-loop structure of the wild-type left ITR of AAV2, with identification of the A-A' arm, the B-B' arm, the C-C' arm, two Rep binding sites (RBE and RBE'), and the terminal resolution site (TRS), and also shows the D and D' regions, which contain several transcription factor binding sites, and other conserved structures. [Figure 3A] 1 provides the primary structure (polynucleotide sequence) (left) and secondary structure (right) of the RBE-containing portions of the A-A' arm and the C-C' and B-B' arms of the wild-type left AAV2 ITR (SEQ ID NO: 540). [Figure 3B] 1 shows an exemplary mutant ITR (also referred to as a modified ITR) sequence for the left ITR. The primary structure (left) and predicted secondary structure (right) of the RBE portions of the A-A', C, and B-B' arms of an exemplary mutant left ITR (ITR-1, left) (SEQ ID NO: 113) are shown. [Figure 3C] The primary structure (left) and secondary structure (right) of the A-A' loop and the RBE-containing portions of the B-B' and C-C' arms of the wild-type right AAV2 ITR (SEQ ID NO: 541) are shown. [Figure 3D]An exemplary right-modified ITR is shown. The primary structure (left) and predicted secondary structure (right) of the A-A' arm and the RBE-containing portion of the B-B' and C arms of an exemplary mutant right ITR (ITR-1, right) (SEQ ID NO: 114) are shown. Any combination of left and right ITRs (e.g., AAV2 ITRs or other viral serotype or synthetic ITRs) can be used, as long as the left ITR is asymmetric or different from the right ITR. Each of the polynucleotide sequences in Figures 3A-3D refers to the sequence used in the plasmid or bacmid / baculovirus genome used to produce ceDNA as described herein. The corresponding ceDNA secondary structure, deduced from the ceDNA vector configuration in the plasmid or bacmid / baculovirus genome and predicted Gibbs free energy values, is also included in each of Figures 3A-3D. [Figure 4A]

[0023] Figure 4B is a schematic showing the upstream process for generating baculovirus-infected insect cells (BIIC), which are useful for producing ceDNA in the process described in the schematic of Figure 4B. In this embodiment, two bacmids are generated by transposing a ceDNA plasmid or a Rep plasmid (encoding a single Rep protein) into a baculovirus expression vector to generate a ceDNA vector bacmid (i.e., bacmid-1) and a single Rep bacmid (Rep-bacmid), which are used to transfect insect cells to produce baculovirus-injected insect cells, BIIC-1 and BICC-2 (single Rep), respectively. [Figure 4B] FIG. 1 is a schematic diagram of an exemplary ceDNA production method using insect cells (e.g., BICC-2) containing a Rep-bacmid containing the nucleic acid sequence of a single Rep protein. [Figure 4C] Using the single Rep protein method described herein, we present a biochemical method and process for confirming ceDNA vector production. [Figure 4D]Figure 4B is a schematic diagram illustrating a process for identifying the presence of ceDNA in DNA collected from cell pellets obtained during the ceDNA production process. Figure 4E shows DNA with a discontinuous structure. ceDNA can be cleaved by a restriction endonuclease with a single recognition site on the ceDNA vector, generating two DNA fragments of different sizes (1 kb and 2 kb) under both neutral and denaturing conditions. Figure 4E also shows ceDNA with a linear and continuous structure. The ceDNA vector can be cleaved by a restriction endonuclease to generate two DNA fragments migrating as 1 kb and 2 kb under neutral but not denaturing conditions, while the strands remain connected, producing single strands migrating as 2 kb and 4 kb. Figure 4D shows the schematic expected bands of an exemplary ceDNA, either uncut or digested with a restriction endonuclease, after electrophoresis on either a native or denaturing gel. The leftmost schematic is a native gel, showing multiple bands, suggesting that in its duplex and uncut form, ceDNA exists in at least two states: a faster-migrating, smaller monomer and a slower-migrating dimer that is twice the size of the monomer. The second schematic from the left shows that when ceDNA is cut with a restriction endonuclease, the original band disappears and a faster-migrating (e.g., smaller) band appears, corresponding to the expected fragment size remaining after cleavage. Under denaturing conditions, the original duplex DNA is single-stranded and migrates as a species twice as large as that observed on the native gel because the complementary strand is covalently linked. Thus, in the second schematic from the right, the digested ceDNA shows a banding distribution similar to that observed on the native gel, but the bands migrate as fragments twice the size of their native gel counterparts. The schematic on the far right shows that uncut ceDNA under denaturing conditions migrates as a single-stranded open circle, and therefore the observed band is twice the size of that observed under native conditions where the circle is not open.In this figure, "kb" is used to indicate the relative size of a nucleotide molecule based on the length of the nucleotide chain (e.g., for single-stranded molecules observed in a denatured state) or the number of base pairs (e.g., for double-stranded molecules observed in a native state), depending on the context. [Figure 4E]Figure 4B is a schematic diagram illustrating a process for identifying the presence of ceDNA in DNA collected from cell pellets obtained during the ceDNA production process. Figure 4E shows DNA with a discontinuous structure. ceDNA can be cleaved by a restriction endonuclease with a single recognition site on the ceDNA vector, generating two DNA fragments of different sizes (1 kb and 2 kb) under both neutral and denaturing conditions. Figure 4E also shows ceDNA with a linear and continuous structure. The ceDNA vector can be cleaved by a restriction endonuclease to generate two DNA fragments migrating as 1 kb and 2 kb under neutral but not denaturing conditions, while the strands remain connected, producing single strands migrating as 2 kb and 4 kb. Figure 4D shows the schematic expected bands of an exemplary ceDNA, either uncut or digested with a restriction endonuclease, after electrophoresis on either a native or denaturing gel. The leftmost schematic is a native gel, showing multiple bands, suggesting that in its duplex and uncut form, ceDNA exists in at least two states: a faster-migrating, smaller monomer and a slower-migrating dimer that is twice the size of the monomer. The second schematic from the left shows that when ceDNA is cut with a restriction endonuclease, the original band disappears and a faster-migrating (e.g., smaller) band appears, corresponding to the expected fragment size remaining after cleavage. Under denaturing conditions, the original duplex DNA is single-stranded and migrates as a species twice as large as that observed on the native gel because the complementary strand is covalently linked. Thus, in the second schematic from the right, the digested ceDNA shows a banding distribution similar to that observed on the native gel, but the bands migrate as fragments twice the size of their native gel counterparts. The schematic on the far right shows that uncut ceDNA under denaturing conditions migrates as a single-stranded open circle, and therefore the observed band is twice the size of that observed under native conditions where the circle is not open.In this figure, "kb" is used to indicate the relative size of a nucleotide molecule based on the length of the nucleotide chain (e.g., for single-stranded molecules observed in a denatured state) or the number of base pairs (e.g., for double-stranded molecules observed in a native state), depending on the context. [Figure 5] Illustrative diagram of an example denaturing gel run of ceDNA vectors with (+) or without (-) digestion with endonucleases (EcoRI for ceDNA constructs 1 and 2, BamH1 for ceDNA constructs 3 and 4, SpeI for ceDNA constructs 5 and 6, and XhoI for ceDNA vectors 7 and 8). The sizes of the bands highlighted with asterisks were determined and are indicated below the figure. [Figure 6A] Shown are results from an in vitro protein expression assay measuring luciferase activity (y-axis, RQ(Luc)) in HEK293 cells 48 hours after transfection with 400 ng (black), 200 ng (gray), or 100 ng (white) of the constructs identified on the x-axis (Construct-1, Construct-3, Construct-5, Construct-7) (Table 12). [Figure 6B] Shown is the luciferase activity (y-axis, RQ(Luc)) measured in HEK293 cells 48 hours after transfection with 400 ng (black), 200 ng (gray), or 100 ng (white) of the constructs (Construct-2, Construct-4, Construct-6, Construct-8) identified on the x-axis (Table 12). Also provided is the luciferase activity measured in HEK293 cells treated with Fugene ("Fugene") or untreated ("Untreated") without any plasmid. [Figure 7A] Shown is the viability of HEK293 cells (y-axis) 48 hours after transfection with 400 ng (black), 200 ng (gray), or 100 ng (white) of constructs identified on the x-axis (construct-1, construct-3, construct-5, construct-7). [Figure 7B]Shown is the viability of HEK293 cells (y-axis) 48 hours after transfection with 400 ng (black), 200 ng (gray), or 100 ng (white) of constructs identified on the x-axis (construct-2, construct-4, construct-6, construct-8). [Figure 8A]

[0039] An exemplary Rep-bacmid of the pFBDLSR plasmid contains the nucleic acid sequence of a modified Rep78 protein, where the modified Rep78 protein is a modification of amino acid residue 225 (Met) of SEQ ID NO: 530, where amino acid residue 225 is changed to glycine (Gly) (e.g., M225G or Met225Gly) or threonine (Thr) (e.g., M225T or Met225Thr). This exemplary Rep-bacmid contains an IE1 promoter fragment (SEQ ID NO: 66), a Rep78 nucleotide sequence encoding a modified Rep78 protein lacking a functional start codon downstream of the first start codon, thereby allowing translation of a single Rep78 protein. As one of skill in the art will appreciate, a nucleic acid encoding any single Rep protein (e.g., Rep68, Rep52, Rep40) that has been modified to have a single start codon and thus encodes a single Rep protein can be used to modify this modified Rep78 bacmid or modified Rep78 plasmid. [Figure 8B] FIG. 1 is a schematic diagram of an exemplary ceDNA-plasmid-1, carrying wt-L ITRs, a CAG promoter, a luciferase transgene, a WPRE and polyadenylation sequence, and a mod-R ITR. [Figure 9A] 1 shows the predicted lowest energy structure of the RBE-containing portions of the A-A' and C-C' arms of an exemplary modified left ITR ("ITR-2 (left)," SEQ ID NO: 101). [Figure 9B] The predicted lowest energy structures of the RBE-containing portions of the A-A' and C-C' arms of an exemplary modified right ITR ("ITR-2 (right)" SEQ ID NO: 102) are shown. They are predicted to form a structure with a single arm (C-C') and a single unpaired loop. Their Gibbs unfolding free energy is predicted to be -72.6 kcal / mol. [Figure 10A] 1 shows the predicted lowest energy structures of the RBE-containing portions of the A-A' and B-B' arms of an exemplary modified left ITR ("ITR-3 (left)," SEQ ID NO: 103). [Figure 10B] The predicted lowest energy structures of the RBE-containing portions of the A-A' and B-B' arms of an exemplary modified right ITR ("ITR-3 (right)," SEQ ID NO: 104) are shown. They are predicted to form a structure with a single arm (B-B') and a single unpaired loop. Their Gibbs unfolding free energy is predicted to be -74.8 kcal / mol. [Figure 11A] 1 shows the predicted lowest energy structure of the RBE-containing portions of the A-A' and C-C' arms of an exemplary modified left ITR ("ITR-4 (left)," SEQ ID NO: 105). [Figure 11B] The predicted lowest energy structures of the RBE-containing portions of the A-A' and C-C' arms of an exemplary modified right ITR ("ITR-4 (right)" SEQ ID NO: 106) are shown. They are predicted to form a structure with a single arm (C-C') and a single unpaired loop. Their Gibbs unfolding free energy is predicted to be -76.9 kcal / mol. [Figure 12A] The predicted lowest energy structure of the A-A' arm of an exemplary modified left ITR, as well as the RBE-containing portions of the C-C' and B-B' portions, is shown, demonstrating complementary base pairing of the C-B' and C'-B portions ("ITR-10 (left)" SEQ ID NO: 107). [Figure 12B] The predicted lowest energy structures of the RBE-containing portions of the A-A' arm and the B-B' and C-C' portions of an exemplary modified right ITR are shown, showing complementary base pairing of the B-C' and B'-C portions ("ITR-10 (right)" SEQ ID NO: 108). They are predicted to form a structure with a single arm (C'-B and C-B' portions or B'-C and B-C' portions) and a single unpaired loop. Their Gibbs unfolding free energy is predicted to be -83.7 kcal / mol. [Figure 13A]1 shows the predicted lowest energy structure of the A-A' arm and the RBE-containing portions of the C-C' and B-B' portions of an exemplary modified left ITR ("ITR-17(left)" SEQ ID NO: 109). [Figure 13B] The predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C-C' and B-B' portions of an exemplary modified right ITR ("ITR-17(Right)" SEQ ID NO: 110) are shown. Both ITR-17(Left) and ITR-17(Right) are predicted to form structures with a single arm (B-B') and a single unpaired loop. Their Gibbs unfolding free energies are predicted to be -73.3 kcal / mol. [Figure 14A] 1 shows the predicted lowest energy structure of the RBE-containing portion of the A-A' arm of an exemplary modified ITR ("ITR-6 (left)," SEQ ID NO: 111). [Figure 14B] 1 shows the predicted lowest energy structures of the RBE-containing portion of the A-A' arm of an exemplary modified ITR ("ITR-6 (right)," SEQ ID NO: 112). Both ITR-6 (left) and ITR-6 (right) are predicted to form structures with a single arm. Their Gibbs unfolding free energy is predicted to be -54.4 kcal / mol. [Figure 15A] 1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C and B-B' arms of an exemplary modified left ITR ("ITR-1 (left)," SEQ ID NO: 113). [Figure 15B] The predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C and B-B' arms of an exemplary modified right ITR ("ITR-1 (right)," SEQ ID NO: 114) are shown. Both ITR-1 (left) and ITR-1 (right) are predicted to form structures with two arms, one of which is truncated. Their Gibbs unfolding free energies are predicted to be -74.7 kcal / mol. [Figure 16A]1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C' and B-B' arms of an exemplary modified left ITR ("ITR-5 (left)," SEQ ID NO: 545). [Figure 16B] The predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the B-B' and C' arms of an exemplary modified right ITR ("ITR-5(right)" SEQ ID NO: 116) are shown. Both ITR-5(left) and ITR-5(right) are predicted to form structures with two arms, one of which (e.g., the C' arm) is truncated. Their Gibbs unfolding free energies are predicted to be -73.4 kcal / mol. [Figure 17A] 1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-7 (left)" SEQ ID NO: 117). [Figure 17B] The predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-7(Right)" SEQ ID NO: 118) are shown. Both ITR-17(Left) and ITR-17(Right) are predicted to form structures with two arms, one of which (e.g., the B-B' arm) is truncated. Their Gibbs unfolding free energies are predicted to be -89.6 kcal / mol. [Figure 18A] 1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-8 (left)" SEQ ID NO: 119). [Figure 18B] The predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-8(Right)" SEQ ID NO: 120) are shown. Both ITR-8(Left) and ITR-8(Right) are predicted to form structures with two arms, one of which is truncated. Their Gibbs unfolding free energies are predicted to be -86.9 kcal / mol. [Figure 19A] 1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-9 (left)" SEQ ID NO: 121). [Figure 19B] The predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-9(Right)" SEQ ID NO: 122) are shown. Both ITR-9(Left) and ITR-9(Right) are predicted to form structures with two arms, one of which is truncated. Their Gibbs unfolding free energies are predicted to be -85.0 kcal / mol. [Figure 20A] 1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-11 (left)," SEQ ID NO: 123). [Figure 20B] The predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-11 (right)," SEQ ID NO: 124) are shown. Both ITR-11 (left) and ITR-11 (right) are predicted to form structures with two arms, one of which is truncated. Their Gibbs unfolding free energies are predicted to be -89.5 kcal / mol. [Figure 21A] 1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-12 (left)," SEQ ID NO: 125). [Figure 21B]The predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-12(Right)" SEQ ID NO: 126) are shown. Both ITR-12(Left) and ITR-12(Right) are predicted to form structures with two arms, one of which is truncated. Their Gibbs unfolding free energies are predicted to be -86.2 kcal / mol. [Figure 22A] 1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-13 (left)," SEQ ID NO: 127). [Figure 22B] The predicted lowest energy structures of the RBE-containing portions of the A-A' arm, and the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-13 (right)," SEQ ID NO: 128) are shown. Both ITR-13 (left) and ITR-13 (right) are predicted to form structures with two arms, one of which (e.g., the C-C' arm) is truncated. Their Gibbs unfolding free energy is predicted to be -82.9 kcal / mol. [Figure 23A] 1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-14 (left)," SEQ ID NO: 129). [Figure 23B] The predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-14 (right)," SEQ ID NO: 130) are shown. Both ITR-14 (left) and ITR-14 (right) are predicted to form structures with two arms, one of which (e.g., the C-C' arm) is truncated. Their Gibbs unfolding free energies are predicted to be -80.5 kcal / mol. [Figure 24A]1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C-C' and B-C' arms of an exemplary modified left ITR ("ITR-15 (left)" SEQ ID NO: 131). [Figure 24B] The predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-15(right)" SEQ ID NO: 132) are shown. Both ITR-15(left) and ITR-15(right) are predicted to form structures with two arms, one of which (e.g., the C-C' arm) is truncated. Their Gibbs unfolding free energies are predicted to be -77.2 kcal / mol. [Figure 25A] 1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C-C' and B-C' arms of an exemplary modified left ITR ("ITR-16 (left)," SEQ ID NO: 133). [Figure 25B] The predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-16 (right)," SEQ ID NO: 134) are shown. Both ITR-16 (left) and ITR-16 (right) are predicted to form structures with two arms, one of which (e.g., the C-C' arm) is truncated. Their Gibbs unfolding free energies are predicted to be -73.9 kcal / mol. [Figure 26A-1] The predicted structures of the A-A' arms, and the RBE-containing portions of the modified B-B' arms and / or modified C-C' arms of exemplary modified right ITRs listed in Table 10A are shown. [Figure 26A-2] The predicted structures of the A-A' arms, and the RBE-containing portions of the modified B-B' arms and / or modified C-C' arms of exemplary modified right ITRs listed in Table 10A are shown. [Figure 26B]

[0049] Figure 10B shows predicted structures of the A-A' arm and the RBE-containing portion of the modified C-C' arm and / or modified B-B' arm of exemplary modified left ITRs. The structures shown are the predicted lowest free energy structures. Color code: red = >99% probability, orange = 99%-95% probability, beige = 95%-90% probability, dark green = 90%-80%, light green = 80%-70%, light blue = 70%-60%, dark blue = 60%-50%, and pink = less than 50%. [Figure 27] Figure 1 shows the luciferase activity of Sf9 GlycoBac insect cells transfected with asymmetric ITR mutant variants selected from Tables 10A and 10B. The ceDNA vectors had a luciferase gene flanked by wild-type ITRs and modified asymmetric ITRs selected from Tables 10A or 10B. "ITR-50 right no rep" is a known rescuable mutant without coinfection of a Rep-containing baculovirus. The "mock" condition is transfection reagent only, without donor DNA. [Figure 28] A native agarose gel (1% agarose, 1x TAE buffer) of representative crude ceDNA extracts from Sf9 insect cell cultures transfected with a ceDNA-plasmid containing the left wt-ITR along with other ITRs selected from the various mutant right ITRs disclosed in Table 10A is shown. 2 μg of total extract was loaded per lane. From left to right: Lane 1) 1 kb + ladder; Lane 2) ITR-18 right; Lane 3) ITR-49 right; Lane 4) ITR-19 right; Lane 5) ITR-20 right; Lane 6) ITR-21 right; Lane 7) ITR-22 right; Lane 8) ITR-23 right; Lane 9) ITR-24 right; Lane 10) ITR-25 right; Lane 11) ITR-26 right; Lane 12) ITR-27 right; Lane 13) ITR-28 right; Lane 14) ITR-50 right; Lane 15) 1 kb + ladder. [Figure 29]A denaturing gel (0.8% alkaline agarose) of a representative construct from the ITR mutant library is shown. The ceDNA vectors were generated from a plasmid construct containing the left wt-ITR along with other ITRs selected from the various mutant right ITRs disclosed in Table 10A. From left to right, lane 1) 1 kb+ DNA ladder; lane 2) ITR-18 right uncut; lane 3) ITR-18 right restriction digest; lane 4) ITR-19 right uncut; lane 5) ITR-19 right restriction digest; lane 6) ITR-21 right uncut; lane 7) ITR-21 right restriction digest; lane 8) ITR-25 right uncut; lane 9) ITR-25 right restriction digest. The extract was treated with EcoRI restriction endonuclease. Each mutant ceDNA is expected to have a single EcoRI recognition site that produces two characteristic fragments, approximately 2,000 bp and 3,000 bp (reaching approximately 4,000 and 6,000 bp under denaturing conditions, respectively). Untreated ceDNA extracts are expected to be approximately 5,000 bp and migrate at approximately 11,000 bp under denaturing conditions. [Figure 30] In vitro luciferase activity in HEK293 cells of ITR mutants ITR-18 right, ITR-19 right, ITR-21 right, and ITR-25 right, and ITR-49, where the left ITR in the ceDNA vector is the WT ITR, is shown. The "mock" condition is transfection reagent only, without donor DNA, and the negative control is untreated. [Figure 31] 1 is a table showing various properties and activities (e.g., DNA binding, DNA nicking, helicase activity, ATPase activity, and Zn finger activity) of different Rep protein species (e.g., wild-type Rep78, wild-type Rep68, wild-type Rep52, and wild-type Rep40) and modified Rep68 species, where, for example, the amino acids of the Rep78 protein are modified to either Y156, K340H, or Met→Gly(M225G). The Met→Gly(M225G) modification of Rep78 maintained all of the properties and activities of the wild-type Rep78 protein. [Figure 32A]Figure 32A shows a non-denaturing gel showing the presence of highly stable DNA vectors and characteristic bands, confirming the presence of highly stable closed-end DNA (ceDNA) vectors generated with a single Rep protein using the methods described herein. In Figure 32A, compared to production using nucleic acid encoding wild-type Rep78 (lane 5), a higher amount of ceDNA vector was produced using modified Rep78 nucleic acids with the Rep78 modification Met→Gly(M225G) (lane 1) or Rep Met→Thr(M225T) (lane 2), which express both Rep78 / 68 and Rep52 / 40 proteins. Rep78 binding / nicking mutants containing the Gly(Y156F) (lane 3) or Thr(Y156F) (lane 4) modifications did not produce ceDNA vectors. In Figure 32B, the Rep68 Met→Gly (M225G) and Rep68 Met→Thr (M225T) mutations also produced ceDNA vectors at levels equal to or greater than the amounts of ceDNA vectors produced using modified Rep78 nucleic acids with the Met→Gly (M225G) or Rep Met→Thr (M225T) Rep78 modifications. DLSR: Plasmid construct expressing long (Rep78) and short (Rep52) Rep proteins in tandem. pIE78: Wild-type full-length Rep78 sequence. Rep78M→G: Full-length Rep78 containing a single M225G mutation. Rep78M→T: Full-length Rep78 containing a single M225T mutation. Rep78Y156F: Full-length Rep78 with a single mutation in the nickase domain. [Figure 32B]Figure 32A shows a non-denaturing gel showing the presence of highly stable DNA vectors and characteristic bands, confirming the presence of highly stable closed-end DNA (ceDNA) vectors generated with a single Rep protein using the methods described herein. In Figure 32A, compared to production using nucleic acid encoding wild-type Rep78 (lane 5), a higher amount of ceDNA vector was produced using modified Rep78 nucleic acids with the Rep78 modification Met→Gly(M225G) (lane 1) or Rep Met→Thr(M225T) (lane 2), which express both Rep78 / 68 and Rep52 / 40 proteins. Rep78 binding / nicking mutants containing the Gly(Y156F) (lane 3) or Thr(Y156F) (lane 4) modifications did not produce ceDNA vectors. In Figure 32B, the Rep68 Met→Gly (M225G) and Rep68 Met→Thr (M225T) mutations also produced ceDNA vectors at levels equal to or greater than the amounts of ceDNA vectors produced using modified Rep78 nucleic acids with the Met→Gly (M225G) or Rep Met→Thr (M225T) Rep78 modifications. DLSR: Plasmid construct expressing long (Rep78) and short (Rep52) Rep proteins in tandem. pIE78: Wild-type full-length Rep78 sequence. Rep78M→G: Full-length Rep78 containing a single M225G mutation. Rep78M→T: Full-length Rep78 containing a single M225T mutation. Rep78Y156F: Full-length Rep78 with a single mutation in the nickase domain. DETAILED DESCRIPTION OF THE INVENTION

[0042] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is understood that the present invention is not limited to the specific methodology, protocols, and reagents, etc., described herein, and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, 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, 6 thEdition,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)、および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,4 thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414), Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X), Laboratory Methods in Enzymology: DNA, Jon Lorsch(ed.)Elsevier,2013(ISBN 0124199542), Current Protocols in Molecular Biology(CPMB),Frederick M.Ausubel(ed.),John Wiley and Sons,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, A.D.A. M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of all of which are incorporated herein by reference in their entireties.

[0043] As used herein, the terms "heterologous nucleotide sequence" and "transgene" are used interchangeably and refer to a nucleic acid of interest (other than a nucleic acid encoding a capsid polypeptide) that can be incorporated into, delivered, and expressed by, the ceDNA vectors disclosed herein. Transgenes of interest include, but are not limited to, nucleic acids encoding polypeptides, preferably therapeutic (e.g., for medical, diagnostic, or veterinary applications) or immunogenic polypeptides (e.g., for vaccines). In some embodiments, the nucleic acid of interest comprises a nucleic acid that is transcribed into a therapeutic RNA. Transgenes included for use in the ceDNA vectors of the present invention include, but are not limited to, those that express or encode one or more polypeptides, peptides, ribozymes, aptamers, peptide nucleic acids, siRNAs, RNAi, miRNAs, IncRNAs, antisense oligonucleotides or polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0044] As used herein, the terms "expression cassette" and "transcription cassette" are used interchangeably and refer to a linear stretch of nucleic acid that includes a transgene operably linked to one or more promoters or other regulatory sequences sufficient to direct transcription of the transgene, but does not include capsid-encoding sequences, other vector sequences, or inverted terminal repeat regions. An expression cassette may additionally include one or more cis-acting sequences (e.g., promoters, enhancers, or repressors), one or more introns, and one or more post-transcriptional regulatory elements.

[0045] As used herein, the term "terminal repeat" or "TR" includes any viral or synthetic terminal sequence that contains at least one minimally necessary origin of replication and a region containing a palindromic hairpin structure. The Rep binding sequence ("RBS") (also referred to as an RBE (Rep binding element)) and terminal resolution site ("TRS") together constitute the "minimally necessary origin of replication," and thus a TR contains at least one RBS and at least one TRS. TRs that are reverse complements of each other within a given stretch of a polynucleotide sequence are typically referred to as "inverted terminal repeats" or "ITRs," respectively. In the context of viruses, ITRs mediate replication, viral packaging, integration, and proviral rescue. As unexpectedly found in the present invention, TRs that are not reverse complements over their entire length can still perform the traditional functions of ITRs, and therefore the term ITR is used herein to refer to TRs in a ceDNA genome or ceDNA vector that are capable of mediating replication of the ceDNA vector. It will be understood by those skilled in the art that there may be more than two ITRs or asymmetric ITR pairs in a composite ceDNA vector construct. The ITRs may be AAV or non-AAV ITRs, or AAV ITRs or non-AAV ITRs. ITRs can be derived from ITRs. For example, ITRs can be derived from the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin, which serves as the origin of SV40 replication, can be used as an ITR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. The Parvoviridae family of viruses is composed of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses, which are capable of replicating in vertebrate hosts, including, but not limited to, humans, primates, bovine, canine, equine, and ovine species.For convenience herein, an ITR located 5' to (upstream of) an expression cassette in a ceDNA vector is referred to as the "5' ITR" or "left ITR," and an ITR located 3' to (downstream of) an expression cassette in a ceDNA vector is referred to as the "3' ITR" or "right ITR."

[0046] "Wild-type ITR" or "WT-ITR" refers to the sequence of a naturally occurring ITR sequence in an AAV or other dependant virus, e.g., that retains Rep binding activity and Rep nicking ability. The nucleotide sequence of a WT-ITR from any AAV serotype may differ slightly from the naturally occurring canonical sequence due to degeneracy of the genetic code or drift; therefore, WT-ITR sequences encompassed for use herein include WT-ITR sequences that result from naturally occurring variations (e.g., replication errors) that occur during the production process.

[0047] As used herein, the term "substantially symmetric WT-ITR" or "substantially symmetric WT-ITR pair" refers to a pair of WT-ITRs within a single ceDNA genome or ceDNA vector, both of which are wild-type ITRs with reverse-complementary sequences throughout their entire length. For example, an ITR can be considered to have a wild-type sequence even if it has one or more nucleotides that deviate from the naturally occurring canonical sequence, as long as the changes do not affect the sequence's characteristics and overall three-dimensional structure. In some embodiments, the deviating nucleotides represent conservative sequence changes. As a non-limiting example, a sequence has at least 95%, 96%, 97%, 98%, or 99% sequence identity (e.g., as measured using BLAST with default settings) to the canonical sequence and has a symmetric three-dimensional spatial organization relative to the other WT-ITR, such that their three-dimensional structures have the same shape in geometric space. A substantially symmetric WT-ITR has the same A, C-C', and B-B' loops in three-dimensional space. Substantially symmetric WT ITRs can be functionally confirmed as WT by determining that they have an operable Rep binding site (RBE or RBE') and terminal resolution site (trs) that pairs with the appropriate Rep protein. Optionally, other functions can be tested, including transgene expression under permissive conditions.

[0048] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutant ITR" are used interchangeably herein and refer to an ITR that has a mutation in at least one or more nucleotides compared to the WT-ITR from the same serotype. The mutation may result in a change in one or more of the A, C, C', B, and B' regions of the ITR, resulting in a change in the three-dimensional spatial configuration (i.e., its three-dimensional structure in geometric space) compared to the three-dimensional spatial configuration of the WT-ITR of the same serotype.

[0049] As used herein, the term "asymmetric ITR," also referred to as an "asymmetric ITR pair," refers to a pair of ITRs within a single ceDNA genome or ceDNA vector that are not reverse-complementary across their entire length. As a non-limiting example, an asymmetric ITR pair does not have a symmetrical three-dimensional spatial configuration relative to its cognate ITR, such that their three-dimensional structures are different shapes in geometric space. In other words, an asymmetric ITR pair differs in overall geometric structure, i.e., the configuration of their A, C-C', and B-B' loops in three-dimensional space (e.g., compared to the cognate ITR), differs. The sequence difference between the two ITRs may be due to one or more nucleotide additions, deletions, truncations, or point mutations. In one embodiment, one ITR of the asymmetric ITR pair may be a wild-type AAV ITR sequence, and the other ITR may be a modified ITR (e.g., a non-wild-type or synthetic ITR sequence) as defined herein. In another embodiment, neither ITR of the asymmetric ITR pair is a wild-type AAV sequence, but rather the two ITRs are modified ITRs with different shapes in geometric space (i.e., different overall geometric structures). In some embodiments, one mod-ITR of the asymmetric ITR pair can have a short C-C' arm, and the other ITR can have a different modification (e.g., a single arm, or a short B-B' arm, etc.) such that they have a different three-dimensional spatial organization compared to their cognate asymmetric mod-ITR.

[0050] As used herein, the term "symmetric ITRs" refers to a pair of ITRs within a single ceDNA genome or ceDNA vector that are mutated or modified relative to the wild-type Dependvirus ITR sequence and are reverse-complementary across their entire length. Neither ITR is the wild-type ITR AAV2 sequence (i.e., they are modified ITRs, also referred to as mutant ITRs), and their sequence may differ from the wild-type ITR due to nucleotide additions, deletions, substitutions, truncations, or point mutations. For convenience, the ITR located 5' (upstream) of the expression cassette in the ceDNA vector is referred to as the "5' ITR" or "left ITR," and the ITR located 3' (downstream) of the expression cassette in the ceDNA vector is referred to as the "3' ITR" or "right ITR."

[0051] As used herein, the term "substantially symmetric modified ITR" or "substantially symmetric mod-ITR pair" refers to a pair of modified ITRs within a single ceDNA genome or ceDNA vector, both of which have reverse-complementary sequences throughout their entire length. For example, modified ITRs can be considered substantially symmetric even if there are some nucleotide sequence deviations from the reverse-complementary sequence, as long as the changes do not affect the properties and overall shape. As a non-limiting example, the sequences have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity (measured using BLAST with default settings) to the canonical sequence and have a symmetric three-dimensional spatial organization relative to their cognate modified ITR, such that their three-dimensional structures have the same shape in geometric space. In other words, a substantially symmetric modified ITR pair has the same A, C-C', and B-B' loops organized in three-dimensional space. In some embodiments, the ITRs from a mod-ITR pair may have different reverse-complementary nucleotide sequences but still have the same symmetrical three-dimensional spatial organization. That is, both ITRs have mutations that result in the same overall three-dimensional shape. For example, one ITR (e.g., the 5' ITR) of a mod-ITR pair may be derived from one serotype, and the other ITR (e.g., the 3' ITR) may be derived from a different serotype, but both may have the same corresponding mutation (e.g., if the 5' ITR has a deletion in the C region, the cognate modified 3' ITR of the different serotype will have a deletion in the corresponding position in the C' region), thereby resulting in the modified ITR pair having the same symmetrical three-dimensional spatial organization. In such embodiments, each ITR of a modified ITR pair can be derived from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAV2 and AAV6, and modifications in one ITR are reflected in the corresponding position of the cognate ITR of 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 nucleotide sequence differences between the ITRs do not affect their properties or overall shape and they have substantially the same shape in three-dimensional space.As a non-limiting example, a mod-ITR has at least 95%, 96%, 97%, 98%, or 99% sequence identity to a canonical mod-ITR, as determined by standard means well known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN with default settings, and has a symmetrical three-dimensional spatial organization such that their three-dimensional structures have the same shape in geometric space. A substantially symmetric mod-ITR pair will have the same A, C-C', and B-B' loops in three-dimensional space. For example, if the modified ITR of a substantially symmetric mod-ITR pair has a deletion of the C-C' arm, the cognate mod-ITR will have a corresponding deletion of the C-C' loop and a similar three-dimensional structure of the remaining A and B-B' loops that are the same shape in geometric space as its cognate mod-ITR.

[0052] The term "flanking" refers to the relative position of one nucleic acid sequence with respect to another. Generally, in the sequence ABC, B is flanked on either side by A and C. The same is true for the arrangement AxBxC. Thus, a flanking sequence may precede or follow the flanked sequence, but need not be contiguous with or immediately adjacent to the flanked sequence. In one embodiment, the term flanking refers to the terminal repeats at each end of a linear, double-stranded ceDNA vector.

[0053] As used herein, the term "ceDNA genome" refers to an expression cassette that further incorporates at least one inverted terminal repeat region. The ceDNA genome may further comprise one or more spacer regions. In some embodiments, the ceDNA genome is incorporated into a plasmid or viral genome as an intermolecular double-stranded polynucleotide of DNA.

[0054] As used herein, the term "ceDNA spacer region" refers to an intervening sequence that separates functional elements in a ceDNA vector or ceDNA genome. In some embodiments, a ceDNA spacer region maintains two functional elements at a desired distance for optimal functionality. In some embodiments, a ceDNA spacer region provides or increases the genetic stability of a ceDNA genome, for example, within a plasmid or baculovirus. In some embodiments, a ceDNA spacer region facilitates easy genetic manipulation of a ceDNA genome by providing a convenient location for a cloning site or the like. For example, in certain aspects, an oligonucleotide "polylinker" containing several restriction endonuclease sites, or a non-open reading frame sequence designed to lack known protein (e.g., transcription factor) binding sites, can be positioned in a ceDNA genome to separate cis-acting elements, e.g., inserting a 6-mer, 12-mer, 18-mer, 24-mer, 48-mer, 86-mer, 176-mer, etc., between the terminal resolution site and the upstream transcriptional regulatory element. Similarly, a spacer can be incorporated between a polyadenylation signal sequence and a 3' terminal resolution site.

[0055] As used herein, "Rep binding site," "Rep binding element," "RBE," and "RBS" are used interchangeably and refer to the binding site of a Rep protein (e.g., AAV Rep78 or AAV Rep68), which, upon binding by the Rep protein, allows the Rep protein to perform its site-specific endonuclease activity on the sequence incorporating the RBS. An RBS sequence and its reverse complement together form a single RBS. RBS sequences are known in the art and include, for example, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531), the RBS sequence identified in AAV2. Any known RBS sequence may be used in embodiments of the present invention, including other known AAV RBS sequences and other naturally occurring or synthetic RBS sequences. Without being bound by theory, it is believed that the nuclease domain of the Rep protein binds to the double-stranded nucleotide sequence GCTC, and thus two known AAV Rep proteins directly bind and stably assemble to the double-stranded oligonucleotide 5'-(GCGC)(GCTC)(GCTC)(GCTC)-3' (SEQ ID NO: 531). In addition, soluble aggregated conformers (i.e., an indefinite number of interrelated Rep proteins) dissociate and bind to oligonucleotides containing the Rep binding site. Each Rep protein interacts with both the nitrogenous bases and the phosphodiester backbone on each strand. The interactions with the nitrogenous bases provide sequence specificity, while the interactions with the phosphodiester backbone are non- or low-sequence specific, stabilizing the protein-DNA complex.

[0056] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably herein and refer to the region where Rep forms a tyrosine-phosphodiester bond with the 5'-thymidine, generating a 3'-OH that serves as a substrate for DNA elongation via a cellular DNA polymerase, such as DNA pol δ or DNA pol ε. Alternatively, the Rep-thymidine complex can participate in a coordinate ligation reaction. In some embodiments, the TRS minimally includes a non-base-paired thymidine. In some embodiments, the nicking efficiency of the TRS can be controlled, at least in part, by its distance from the RBS within the same molecule. When the acceptor substrate is a complementary ITR, the resulting product is an intermolecular duplex. TRS sequences are known in the art and include, for example, the hexanucleotide sequence 5'-GGTTGA-3' (SEQ ID NO: 45) identified in AAV2. Any known TRS sequence can be used in embodiments of the invention, including other known AAV TRS sequences, other naturally known or synthetic TRS sequences such as AGTT (SEQ ID NO:46), GGTTGG (SEQ ID NO:47), AGTTGG (SEQ ID NO:48), AGTTGA (SEQ ID NO:49), and other motifs such as RRTTRR (SEQ ID NO:50).

[0057] As used herein, the term "ceDNA-plasmid" refers to a plasmid that contains a ceDNA genome as an intermolecular duplex.

[0058] As used herein, the term "ceDNA-bacmid" refers to an infectious baculovirus genome that contains a ceDNA genome as an intermolecular duplex that can be propagated as a plasmid in E. coli, thereby manipulating it as a baculovirus shuttle vector.

[0059] As used herein, the term "ceDNA-baculovirus" refers to a baculovirus that contains a ceDNA genome as an intermolecular duplex within the baculovirus genome.

[0060] As used herein, the terms "ceDNA-baculovirus-infected insect cells" and "ceDNA-BIIC" are used interchangeably and refer to invertebrate host cells (including but not limited to insect cells (e.g., Sf9 cells)) infected with a ceDNA-baculovirus.

[0061] As used herein, the term "ceDNA" refers to capsid-free, closed-ended, linear, double-stranded (ds) duplex DNA for synthetic or other non-viral gene transfer. A detailed description of ceDNA is provided in International Application No. PCT / US2017 / 020828, filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Certain methods for producing ceDNA containing various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Application Nos. PCT / US18 / 49996, filed September 7, 2018, and PCT / US2018 / 064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Certain methods for the production of synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Application No. PCT / US2019 / 14122, filed January 18, 2019, the entire contents of which are incorporated herein by reference.

[0062] As used herein, the term "closed-end DNA vector" refers to a capsid-free DNA vector that has at least one covalently closed end and at least a portion of the vector has an intramolecular double-stranded structure.

[0063] As used herein, the terms "ceDNA vector" and "ceDNA" are used interchangeably and refer to a closed-end DNA vector containing at least one terminal palindrome. In some embodiments, the ceDNA contains two covalently closed ends.

[0064] As used herein, the term "neDNA" or "nicked ceDNA" refers to closed-end DNA that has a nick or gap of 1 to 100 base pairs in the stem or spacer region 5' upstream of the open reading frame (e.g., the promoter and transgene to be expressed).

[0065] As used herein, the terms "gap" and "nick" are used interchangeably and refer to an interrupted portion of the synthetic DNA vector of the invention, creating a stretch of single-stranded DNA in the otherwise double-stranded cDNA. The gap can be from 1 base pair to 100 base pairs in length for one strand of the double-stranded DNA. Exemplary gaps designed and created by the methods described herein, and synthetic vectors generated thereby, 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 bp in length. Exemplary gaps in the present disclosure can be 1 bp to 10 bp in length, 1 to 20 bp in length, or 1 to 30 bp in length.

[0066] As defined herein, a "reporter" refers to a protein that can be used to provide a detectable readout. Reporters generally produce a measurable signal, such as fluorescence, color, or luminescence. A reporter protein coding sequence encodes a protein whose presence in a cell or organism is easily observed. For example, fluorescent proteins cause cells to fluoresce when excited with a particular wavelength of light, luciferase causes cells to catalyze a light-producing reaction, and enzymes such as β-galactosidase convert a substrate into a colored product. Exemplary reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP) and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0067] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable readout, for example, as a reporter polypeptide, or more appropriately, as a cell-killing polypeptide, such as a toxin or a drug that renders cells susceptible to killing by a selected agent or its deletion. Effector proteins include any protein or peptide that directly targets or damages host cell DNA and / or RNA. For example, effector proteins include, but are not limited to, restriction endonucleases (whether genomic or extrachromosomal) that target host cell DNA sequences, proteases that target polypeptides required for cell survival, DNA gyrase inhibitors, and ribonuclease-type toxins. In some embodiments, the expression of an effector protein controlled by a synthetic biological circuit described herein can participate as a factor in another synthetic biological circuit, thereby expanding the scope and complexity of the reactivity of the biological circuit system.

[0068] Transcriptional regulators refer to transcriptional activators and repressors that activate or suppress the transcription of a gene of interest. A promoter is a region of nucleic acid that initiates the transcription of a specific gene. A transcriptional activator typically binds near the transcriptional promoter and recruits RNA polymerase to directly initiate transcription. A repressor binds to the transcriptional promoter and sterically hinders the transcription initiation by RNA polymerase. Other transcriptional regulators can serve as either activators or repressors 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.

[0069] As used herein, a "repressor protein" or an "inducer protein" is a protein that binds to a regulatory sequence element and represses or activates, respectively, the transcription of a sequence operably linked to the regulatory sequence element. Preferred repressor and inducer proteins described herein are sensitive to the presence or absence of at least one input agent or environmental input. Preferred proteins described herein are modular in form, for example, containing separable DNA binding and input agent binding or reactive elements or domains.

[0070] 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, etc. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar adverse reactions when administered to a host.

[0071] As used herein, an "input agent-responsive domain" is a domain of a transcription factor that responds to a condition or input agent such that it binds to or otherwise renders the 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 the protein to which it is fused, which modifies the transcriptional regulatory activity of the transcription factor.

[0072] The term "in vivo" refers to an assay or process that occurs in or within an organism, such as a multicellular animal. In some of the embodiments described herein, a method or use may 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 carried out using living cells with intact membranes outside the body of a multicellular animal or plant, such as explants, cultured cells (including primary cells and cell lines), transformed cell lines, and extracted tissues or cells (including blood cells), among others. The term "in vitro" refers to assays and methods that do not require the presence of cells with intact membranes, such as cell extracts, and can refer to introducing a programmable synthetic biological circuit into a non-cellular system, e.g., a medium that does not contain cells or cell systems, such as a cell extract.

[0073] As used herein, the term "promoter" refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of that nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. A promoter can be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence where the initiation and rate of transcription of the remainder of the nucleic acid sequence are controlled. A promoter can also contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. In some embodiments of the aspects described herein, a promoter can drive the expression of a transcription factor that regulates its own expression or of another promoter used in another modular component of the synthetic biological circuit described herein. Within the promoter sequence, one will find a transcription initiation site, as well as protein binding domains involved in the binding of RNA polymerase. Eukaryotic promoters often, but not necessarily, contain "TATA" and "CAT" boxes. A variety of promoters, including inducible promoters, can be used to drive the expression of transgenes in the ceDNA vectors disclosed herein.

[0074] As used herein, the term "enhancer" refers to a cis-acting regulatory sequence (e.g., 50 to 1,500 base pairs) that binds to one or more proteins (e.g., activator proteins or transcription factors) to increase transcriptional activation of a nucleic acid sequence. Enhancers can be located up to 1,000,000 base pairs upstream of the start site of the gene they regulate or downstream of the start site of the gene. Enhancers can be located within intronic or exon regions of unrelated genes.

[0075] A promoter can be said to drive the expression or transcription of the nucleic acid sequence it regulates. The phrases "operably linked," "operably positioned," "operably linked," "under control," and "under transcriptional control" indicate that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence and regulates to control transcription initiation and / or expression of that sequence. As used herein, an "inverted promoter" refers to a promoter in which a nucleic acid sequence is in the reverse orientation, whereby what was the coding strand is now the non-coding strand, and vice versa. Inverted promoter sequences are used in various embodiments to regulate the state of a switch. Additionally, in various embodiments, a promoter can be used in conjunction with an enhancer.

[0076] A promoter can be one that is naturally associated with a gene or sequence, which can be obtained by isolating the 5' non-coding sequence 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 that is naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.

[0077] In some embodiments, the encoding nucleic acid segment is placed under the control of a "recombinant promoter" or "heterologous promoter," both of which refer to a promoter that is not normally associated with an operably linked encoded nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a given nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and synthetic promoters or enhancers that are not "naturally occurring," i.e., may contain different elements of different transcriptional regulatory regions and / or mutations that alter expression through methods of genetic engineering known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, promoter sequences can be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, for the synthetic biological circuits and modules disclosed herein (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference). Furthermore, it is contemplated that regulatory sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, etc., can be similarly employed.

[0078] As described herein, an "inducible promoter" is characterized by initiating or enhancing transcriptional activity when in the presence of, affected by, or contacted by an inducer or inducer agent. As defined herein, an "inducer" or "inducer agent" can be endogenous or can be a compound or protein, usually exogenous, administered in such a manner that it is active in inducing transcriptional activity from an inducible promoter. In some embodiments, the inducer or inducer agent, i.e., a chemical, compound, or protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., the inducer can be an inducer protein expressed by another component or module) and can itself be under the control of an inducible promoter. In some embodiments, an inducible promoter is induced in the absence of certain agents, such as repressors. Examples of inducible promoters include, but are not limited to, tetracycline, metallothionine, ecdysone, mammalian viruses (e.g., adenovirus late promoter and mouse mammary tumor virus long terminal repeat (MMTV-LTR)), as well as other steroid-responsive promoters, rapamycin-responsive promoters, and the like.

[0079] As used herein, the term "subject" refers to a human or animal to which treatment, including prophylactic treatment, with a ceDNA vector according to the present invention is provided. Typically, the animal is a vertebrate, such as, but not limited to, a primate, a rodent, a domestic animal, or a game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, felines, such as domestic cats, canines, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, such as a primate or human. The subject may be male or female. Additionally, the subject may be an infant or child. In some embodiments, the subject may be a neonatal or fetal subject, e.g., the subject is present in utero. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Non-human mammals may be advantageously used as subjects representing animal models of diseases and disorders. Additionally, the methods and compositions described herein may be used with domestic animals and / or pets. Human subjects may be of any age, sex, race, or ethnic group, e.g., Caucasian (white), Asian, African, Black, African-American, African-European, Latino, Middle Eastern, etc. In some embodiments, the subject may be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment.

[0080] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the same antigen as the intact antibody. In one embodiment, an antibody or antibody fragment comprises an immunoglobulin chain or antibody fragment and at least one immunoglobulin variable domain sequence. Examples of antibodies or fragments thereof include, but are not limited to, Fv, scFv, Fab fragment, Fab', F(ab')2, Fab'-SH, single-domain antibodies (dAbs), heavy chain, light chain, heavy and light chain, complete antibodies (e.g., including each of Fc, Fab, heavy chain, light chain, variable region, etc.), bispecific antibodies, diabodies, linear antibodies, single-chain antibodies, intrabodies, monoclonal antibodies, chimeric antibodies, multispecific antibodies, or multimeric antibodies. The antibody or fragment thereof can be of any class, including but not limited to IgA, IgD, IgE, IgG, and IgM, and any subclass thereof, including but not limited to IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Furthermore, the antibody can be derived from any mammal, such as a primate, human, rat, mouse, horse, goat, etc. In one embodiment, the antibody is human or humanized. In some embodiments, the antibody is a modified antibody. In some embodiments, components of the antibody can be expressed separately such that the antibody self-assembles after expression of the protein components. In some embodiments, the antibody is "humanized" to reduce immunogenic responses in humans. In some embodiments, the antibody has a desired function, e.g., desired protein interaction and inhibition, for purposes of treating a disease or disease symptom. In one embodiment, the antibody or antibody fragment comprises a framework region or F domain. c Includes the area.

[0081] As used herein, the term "antigen-binding domain" of an antibody molecule refers to the portion of an antibody molecule, e.g., an immunoglobulin (Ig) molecule, that is involved in antigen binding. In embodiments, the antigen-binding site is formed by amino acid residues in the variable (V) regions of the heavy (H) and light (L) chains. Three highly divergent stretches within the variable regions of the heavy and light chains are called hypervariable regions and are located between more conserved adjacent stretches called "framework regions" (FRs). FRs are amino acid sequences naturally found between and adjacent to the hypervariable regions of immunoglobulins. In embodiments, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface that is complementary to the three-dimensional surface of a bound antigen. The three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions" or "CDRs." Framework regions and CDRs are defined and described, for example, in Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variable heavy chain and variable light chain) typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0082] As used herein, the term "full length antibody" refers to an immunoglobulin (Ig) molecule (e.g., an IgG antibody), e.g., that occurs in nature and is formed by recombinant processes of conventional immunoglobulin gene fragments.

[0083] As used herein, the term "functional antibody fragment" refers to a fragment that binds to the same antigen recognized by an intact (e.g., full-length) antibody. The term "antibody fragment" or "functional fragment" also includes isolated fragments consisting of the variable regions, such as an "Fv" fragment consisting of the variable regions of the heavy and light chains, or a recombinant single-chain polypeptide molecule in which the light and heavy chain variable regions are connected by a peptide linker ("scFv protein"). In some embodiments, an antibody fragment does not include a portion of an antibody without antigen-binding activity, such as an Fc fragment or a single amino acid residue.

[0084] As used herein, "immunoglobulin variable domain sequence" refers to an amino acid sequence capable of forming the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two, or more N- or C-terminal amino acids, or may include other alterations compatible with forming a protein structure.

[0085] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. An "oligonucleotide" generally refers to a polynucleotide of about 5 to about 100 nucleotides, either single- or double-stranded DNA. However, for purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides, also known as "oligomers" or "oligos," can be isolated from genes or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides, as applicable to the described embodiments. DNA can be in the form of, for example, antisense molecules, plasmid DNA, DNA-DNA duplexes, precondensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. DNA can be in the form of minicircles, plasmids, bacmids, minigenes, ministring DNA (linear covalently closed DNA vectors), closed-end linear double-stranded DNA (CELiD or ceDNA), doggybone (dbDNA™) DNA, dumbbell-shaped DNA, minimally immunologically defined gene expression (MIDGE) vectors, viral vectors, or non-viral vectors. RNA can be in the form of small interfering RNA (siRNA), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof.Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties to the reference nucleic acid. Examples of such analogs and / or modified residues include phosphorothioates, phosphorodiamidate morpholino oligomers (morpholinos), phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNA™), and peptide nucleic acids (PNAs). Unless otherwise specified, this term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequence.

[0086] A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group.

[0087] "Base" includes purines and pyrimidines, which further include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogues, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0088] "Hybridizable" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA) contains a sequence of nucleotides that allows it to non-covalently bind, i.e., form Watson-Crick base pairs and / or G / U base pairs, to "anneal" or "hybridize" to another nucleic acid (i.e., the nucleic acid specifically binds to a complementary nucleic acid) in a sequence-specific, antiparallel manner under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base pairings include adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C). Additionally, it is known in the art that guanine (G) bases pair with uracil (U) for hybridization between two RNA molecules (e.g., dsRNA). For example, G / U base pairing, in the context of tRNA anticodon base pairing with codons in mRNA, is partially responsible for the degeneracy (i.e., redundancy) of the genetic code. In the context of this disclosure, guanine (G) in the protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered complementary to uracil (U), and vice versa. Thus, if a G / U base pair can be made at a given nucleotide position in the protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule, that position is not considered non-complementary, but instead is considered complementary.

[0089] As used herein, the term "nucleic acid construct" refers to a single- or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or that is modified to contain a segment of nucleic acid in a manner that does not otherwise occur in nature, or that is synthetic. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences necessary for expression of the coding sequence of the present disclosure. An "expression cassette" comprises a DNA coding sequence operably linked to a promoter.

[0090] As used herein, the terms "nucleic acid therapy," "therapeutic nucleic acid," and "TNA" are used interchangeably and refer to any modality of treatment that uses nucleic acids as the active ingredient of a therapeutic agent to treat a disease or disorder. As used herein, these terms refer to RNA-based therapeutic agents and DNA-based therapeutic agents. Non-limiting examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigene, viral DNA (e.g., lentivirus or AAV genome) or nonviral synthetic DNA vector, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmid, bacmid, doggybone (dbDNA™) DNA vector, minimally immunologically defined gene expression (MIDGE) vector, nonviral ministring DNA vector (linear covalently closed DNA vector), or dumbbell-shaped DNA minimal vector ("dumbbell DNA").

[0091] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include coded and non-coded amino acids, amino acids that are chemically or biochemically modified or derivatized, and polypeptides with modified peptide backbones.

[0092] As used herein, the terms "synthetic AAV vector" and "synthetic production of AAV vector" refer to AAV vectors and methods for their synthetic production in an entirely cell-free environment.

[0093] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective components that are essential to the method or composition, but are open to the inclusion of non-specified elements, whether essential or not.

[0094] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristics of that embodiment.

[0095] The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any elements not recited in the description of the embodiments.

[0096] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those of ordinary skill in the art upon reading this disclosure, etc. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with "for example."

[0097] Except in working examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." The term "about," when used in connection with percentages, may mean ±1%. The present invention is further illustrated by the following examples, but the scope of the present invention should not be limited thereto.

[0098] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0099] II. Replication Initiator (Rep) Proteins As described herein, the technology described herein relates to compositions and improved methods for producing DNA vectors, e.g., ceDNA vectors described herein, or AAV vectors having a single Rep protein species. According to some aspects, the present disclosure provides methods for producing DNA vectors, e.g., ceDNA vectors described herein, or AAV vectors using a single Rep protein, where the Rep protein is not Rep52 or Rep40. According to some embodiments, the single Rep protein is Rep78. According to some embodiments, the single Rep protein is Rep68. This is an improved, more efficient method for producing ceDNA vectors, producing superior ceDNA vector yields than methods described in the prior art that use two Rep proteins, including Rep78 or 68 and Rep52 or 40 (e.g., Rep78 and Rep52, see FIG. 32). Indeed, prior to the present invention, it was believed that two Rep proteins, one long (e.g., Rep78 or Rep68) and one short (e.g., Rep52 or Rep40), must be present to produce AAV particles. In particular, it was thought that production of AAV particles required the presence of Rep78 and Rep52 as a single unit, or using a single coding sequence for the Rep78 and Rep52 proteins.

[0100] Therefore, one aspect of the technology described herein relates to a method for producing a DNA vector, such as a ceDNA vector described herein, or an AAV vector that uses a single Rep protein instead of two Rep proteins. According to some embodiments, the single Rep protein is Rep78. According to some embodiments, the single Rep protein is Rep68. According to some embodiments, the Rep protein can be Rep78 and Rep68, but not Rep52 or Rep40.

[0101] Another aspect of the technology described herein relates to a composition comprising a nucleic acid construct comprising a first nucleotide sequence encoding a single parvoviral Rep protein, wherein the nucleotide sequence does not have an open reading frame (ORF), lacks a functional initiation codon downstream of the first initiation codon, and / or lacks alternative splicing sites that prevent exon skipping, thereby allowing translation of the single parvoviral Rep protein (e.g., Rep78 or Rep68 protein) without translating additional Rep proteins (e.g., any one or more of Rep52 or Rep40) in insect cells or cell-free systems. That is, a nucleic acid encoding Rep78 will not also produce Rep52 protein, and similarly, a nucleic acid encoding Rep68 will not produce Rep40 protein. Furthermore, no other Rep proteins are present or expressed in the system. For nucleic acid constructs for producing DNA vectors, cells (e.g., insect cells, mammalian cells) and cell-free systems, e.g., ceDNA vectors and other recombinant parvovirus (e.g., adeno-associated virus) vectors in insect cells or cell-free systems.

[0102] Common Rep proteins The Rep gene functions to replicate the viral genome. In wild-type nucleic acids encoding Rep78 or Rep68, splicing events in either Rep78 or Rep68 Rep open reading frames result in two Rep proteins, Rep52 and Rep40, respectively, during translation. That is, Rep78 protein and Rep68 protein are encoded by a single nucleic acid that undergoes differential splicing to produce both Rep78 and Rep68. Similarly, Rep52 protein and Rep40 protein are encoded by a single nucleic acid that undergoes differential splicing to produce both Rep52 and Rep40 proteins. Rep78 is a full-length protein produced from the original first translation initiation site, while Rep52 is a translation product from the downstream internal "second (AUG)" translation initiation site. Thus, when a full-length wild-type AAV genome is expressed, all four Rep proteins are typically present (e.g., Rep78, Rep68, Rep52, and Rep40), primarily due to two distinct translation initiation sites and alternative splicing sites near the carboxy terminus. Rep proteins each contain various functionalities, such as DNA nicking, DNA binding, helicase, ligase, and ATPase activity. The functionality of a given Rep protein is further described in Figure 31. It has previously been reported that both Rep78 and Rep52 proteins are required for the production of AAV vectors or ceDNA vectors in various systems, such as insect and mammalian cell lines. However, as discussed herein, the inventors demonstrate that a combination of at least the long Rep proteins (Rep78 and Rep68), rather than a single Rep protein alone or the short Rep proteins (Rep52 and Rep40), can be used for the production of AAV vectors or ceDNA vectors. A single Rep protein useful in the compositions and methods described herein comprises all three functions: DNA nicking, DNA binding, and DNA ligation. In certain embodiments, the single Rep protein further comprises helicase and ATPase functionality.

[0103] In some embodiments, a single Rep protein useful in the compositions and methods described herein is an AAV2 Rep protein when the ITRs are from serotype 2 (e.g., AAV2). In alternative embodiments, the single Rep protein can be from any of the 42 AAV serotypes, or more preferably, an AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 Rep protein. In some embodiments, a single Rep protein encompassed for use in the methods and compositions disclosed herein corresponds to an animal parvovirus Rep protein when the ITRs are from serotype 2 (e.g., AAV2). The Rep protein functions as part of a system that includes the ITRs, binding to the ITRs to initiate terminal-resolving replication and catalyzing the formation of a closed-end ceDNA vector molecule.

[0104] In some embodiments, a single Rep protein useful in the compositions and methods described herein is Rep78. In alternative embodiments, a single Rep protein useful in the compositions and methods described herein is Rep68. In alternative embodiments, a single species of Rep protein is Rep52 or Rep40 modified to contain the functionality of Rep78 or Rep68, e.g., to have DNA binding, DNA nicking, helicase, and ATPase activity. Alternatively, in some embodiments, a Rep protein useful in the compositions and methods described herein can be a combination of long Rep proteins (e.g., Rep78 and Rep68) that does not include the short Rep proteins Rep52 or Rep40.

[0105] Another aspect of the technology described herein relates to a nucleic acid construct encoding a single Rep protein, where the nucleic acid does not induce or allow expression of a second Rep protein. Thus, in one aspect, a nucleic acid construct encoding a single Rep protein is modified to lack a functional start codon for another Rep protein.

[0106] In one embodiment, the presence of a single Rep species (e.g., the absence of other species) is determined by a specific mutation that prevents translation of p19 Rep and by the absence of other Rep species on a Western blot using anti-Rep antibodies known in the art.

[0107]

[0108] Nucleic acid constructs encoding modified Rep proteins In one embodiment, a single Rep protein is encoded by a nucleotide sequence encoding a modified Rep protein, e.g., a modified Rep78 protein, but the nucleotide sequence does not have a functional start codon for encoding a Rep52 protein or a splicing site for exon skipping to produce Rep68 or Rep40. For example, a modified Rep78 nucleotide sequence includes a modification or mutation in the start codon of Rep52 (e.g., AUG) so that it no longer encodes methionine but rather encodes a different amino acid. In some embodiments, the start codon of Rep52 (Met) in the Rep78 nucleic acid sequence is mutated to encode the amino acid glycine (e.g., AUG is mutated to one of GGU, GGC, GGA, GGG, which encodes GIy) or threonine (e.g., AUG is mutated to one of ACT, ACC, ACA, and ACG, which encode Thr).

[0109] Modified Rep proteins In some embodiments, the modified Rep78 nucleotide sequence encodes a modified Rep78 protein comprising a modification of amino acid residue 225 (Met) of SEQ ID NO: 530, where amino acid residue 225 is changed to glycine (Gly) (e.g., M225G or Met225Gly) or threonine (Thr) (e.g., M225T or Met225Thr). In one embodiment, the mutant Rep78 protein comprises the sequence of SEQ ID NO: 530 or a sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 530, wherein the amino acid at position 225 is not Met, the modified Rep protein has at least DNA binding and DNA nicking functionality, and the gene encoding it does not promote the production of a second Rep protein. One skilled in the art would be able to generate point mutations using, for example, site-directed mutagenesis, and sequence alignments of modified Rep proteins (i.e., Rep proteins containing point mutations) compared to wild-type Rep proteins can be performed to assess whether the nucleotide sequence mutations were generated correctly.

[0110] In one embodiment, a nucleotide sequence encoding a single Rep protein useful in the compositions and methods disclosed herein comprises an expression control sequence, such as a promoter, cis-regulatory element, or regulatory switch described herein, located upstream of the start codon of a nucleotide sequence encoding a parvoviral Rep78 protein, and the nucleic acid sequence lacks a functional start codon for Rep52. In one embodiment, a nucleotide sequence encoding a single Rep protein useful in the compositions and methods disclosed herein comprises an expression control sequence upstream of the start codon of a nucleotide sequence encoding a parvoviral Rep78 protein, and the nucleic acid sequence lacks a functional splice site for encoding Rep68.

[0111] That is, in some embodiments, the nucleic acid encoding Rep78 has only one start codon, thereby allowing translation of only the Rep78 protein or the Rep68 protein. In such embodiments, the Rep78 nucleic acid has a first functional start codon that allows translation of the Rep78 protein, but start codons downstream of the first start codon are modified (or non-functional), such that Rep52 is not expressed.

[0112] In all cases, no other vectors encoding a different Rep are used, and Rep proteins are not already present in the insect or mammalian cells used in the methods for producing DNA vectors, e.g., ceDNA vectors or AAV vectors, according to the methods described herein.

[0113] In one embodiment, the single Rep protein useful in the compositions and methods disclosed herein is derived from the Parvoviridae family. In another embodiment, the single Rep protein useful in the compositions and methods disclosed herein is preferably from a Dependovirinae virus Rep. In another embodiment, the single Rep protein useful in the compositions and methods disclosed herein is more preferably an AAV Rep.

[0114] In one embodiment, a nucleotide sequence of the invention comprises expression control sequences encoding an AAV Rep68 protein, wherein the nucleic acid sequence lacks a functional start codon for Rep40 but has a deletion in its carboxy-terminal intron sequence, resulting in Rep68. In another embodiment, the nucleic acid sequence lacks a deletion in the intron sequence of full-length Rep78, resulting in no other functional splice site, resulting in a transcript that can be translated only into Rep68. That is, in some embodiments, the nucleic acid encoding Rep68 has only one start codon, thereby allowing translation of only the Rep68 protein with the C-terminal intron sequence deleted. In such embodiments, the Rep68 nucleic acid has a first functional start codon that allows for translation of the Rep68 protein, but the start codon downstream of the first start codon is modified by mutation or is non-functional (e.g., M225G or M225T, resulting in Rep40 not being expressed. Alternatively, the nucleic acid encoding Rep68 has a second start codon that is modified by mutation (e.g., M225G or M225T) or is non-functional, but the downstream c-terminal splice site is engineered to allow for expression of the Rep78 and Rep68 proteins.

[0115] A sequence that is substantially identical to the nucleotide sequence of SEQ ID NO:530 is a sequence that has at least 60%, 70%, 80% or 90% identity to SEQ ID NO:530.

[0116] III. Detailed Method for Producing ceDNA Vectors Using a Single Rep Protein A. General production As described herein, ceDNA vectors can be obtained by a process using only one Rep protein, as opposed to two or more, for example, two Rep proteins.Accordingly, one aspect of the present invention relates to a method comprising:a) incubating a population of host cells (e.g., insect cells) harboring a polynucleotide expression construct template (e.g., ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus) lacking a viral capsid-encoding sequence in the presence of a single Rep protein for a sufficient period of time and under conditions effective to induce the production of a ceDNA vector in the host cells, wherein the host cells do not contain a viral capsid-encoding sequence; andb) harvesting and isolating the ceDNA vector from the host cells.The presence of a single Rep protein induces replication of the vector polynucleotide with modified ITRs to produce a ceDNA vector in the host cells.However, viral particles (e.g., AAV virions) are not expressed.Therefore, there are no size limitations, such as those naturally imposed on AAV or other viral vectors.

[0117] The presence of a ceDNA vector isolated from a host cell can be confirmed by digesting the DNA isolated from the host cell with a restriction enzyme that has a single recognition site on the ceDNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of a characteristic linear and continuous DNA band compared to linear and discontinuous DNA.

[0118] In yet another aspect, the present invention provides the use of host cell lines that stably integrate a DNA vector polynucleotide expression template (ceDNA template) into their own genome in the production of non-viral DNA vectors, as described, for example, in Lee, L. et al. (2013) Plos One 8(8):e69879. Preferably, Rep is added to the host cells at an MOI of about 3. When the host cell line is a mammalian cell line, such as HEK293 cells, the cell line may have a stably integrated polynucleotide vector template, and a second vector, such as a herpesvirus, may be used to introduce Rep proteins into the cells, allowing for excision and amplification of ceDNA in the presence of Rep and a helper virus.

[0119] In one embodiment, the host cells used to generate the ceDNA vectors described herein are insect cells, and a baculovirus is used to deliver both a polynucleotide encoding a single Rep protein and a non-viral DNA vector polynucleotide expression construct template for ceDNA, e.g., as described in Figures 4A-4C and Example 1. In some embodiments, the host cells are engineered to express a single Rep protein.

[0120] The ceDNA vector is then harvested and isolated from the host cells. The time for harvesting the ceDNA vector described herein from cells can be selected and optimized to achieve high-yield production of the ceDNA vector. For example, the harvest time can be selected taking into account cell viability, cell morphology, cell growth, etc. In one embodiment, the cells are grown under sufficient conditions and harvested after a sufficient amount of time has passed since baculovirus infection to produce the ceDNA vector, but before the majority of the cells begin to die due to the toxicity of the baculovirus. The DNA vector can be isolated using a plasmid purification kit such as the Qiagen Endo-Free Plasmid Kit. Other methods developed for plasmid isolation can also be adapted to DNA vectors. Generally, any nucleic acid purification method can be employed.

[0121] DNA vector can be purified by any means known to those skilled in the art for DNA purification.In one embodiment, ceDNA vector is purified as DNA molecule.In another embodiment, ceDNA vector is purified as exosome or microparticle.

[0122] The presence of ceDNA vectors can be confirmed by digesting vector DNA isolated from cells with a restriction enzyme that has a single recognition site on the DNA vector and analyzing both the digested and undigested DNA material using gel electrophoresis to confirm the presence of characteristic linear and continuous DNA compared to linear and discontinuous DNA. Figures 4C and 4E show one embodiment for identifying the presence of closed-end ceDNA vectors produced by the process herein. For example, Figure 5 is a gel confirming the production of ceDNA from multiple plasmid constructs using one embodiment for producing these vectors described in the Examples.

[0123] B.ceDNA Plasmid The ceDNA-plasmid is a plasmid used for the later production of the ceDNA vector. In some embodiments, the ceDNA-plasmid is constructed using known techniques to provide at least (1) a 5'ITR sequence, (2) an expression cassette containing cis-regulatory elements, such as a promoter, an inducible promoter, a regulatory switch, an enhancer, and the like, and (3) a 3'ITR sequence as operably linked components in the transcription direction, and the 3'ITR sequence is asymmetric with respect to the 5'ITR sequence. In some embodiments, the expression cassette flanked by ITRs contains a cloning site for introducing an exogenous sequence. The expression cassette replaces the rep and cap coding regions of the AAV genome.

[0124] In one aspect, the ceDNA vector is derived from a plasmid, referred to herein as a "ceDNA-plasmid," that encodes, in that order, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), an expression cassette containing a transgene, and a mutant or modified AAV ITR, wherein the ceDNA-plasmid lacks the AAV capsid protein coding sequence. In an alternative embodiment, the ceDNA-plasmid encodes, in that order, a first (or 5') modified or mutant AAV ITR, an expression cassette containing a transgene, and a second (or 3') wild-type AAV ITR, wherein the ceDNA-plasmid lacks the AAV capsid protein coding sequence, and the 5' and 3' ITRs are asymmetric with respect to each other. In an alternative embodiment, the ceDNA-plasmid encodes, in that order, a first (or 5') modified or mutated AAV ITR, an expression cassette containing a transgene, and a second (or 3') mutated or modified AAV ITR, wherein the ceDNA-plasmid lacks the AAV capsid protein coding sequence, and the 5' and 3' modified ITRs are different and do not have the same modifications.

[0125] In further embodiments, the ceDNA-plasmid system lacks viral capsid protein coding sequences (i.e., it lacks not only the AAV capsid gene but also the capsid gene of other viruses). Additionally, in certain embodiments, the ceDNA-plasmid also lacks the AAV Rep protein coding sequence. Thus, in preferred embodiments, the ceDNA-plasmid lacks functional AAV cap and AAV rep genes (GG-3' in the case of AAV2) as well as a variable palindrome sequence that allows hairpin formation.

[0126] The ceDNA-plasmids of the present invention can be generated using the native nucleotide sequence of the genome of any AAV serotype known in the art. In one embodiment, the ceDNA-plasmid backbone is derived from the AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes. See, e.g., NCBI: NC002077, NC001401, NC001729, NC001829, NC006152, NC006260, NC006261; Kotin and Smith, The Springer Index of Viruses, available at the URL maintained by Springer (www web address: oesys.springer.de / viruses / database / mkchapter.asp?virID=42.04) (Note - references to a URL or database refer to the URL or database contents as of the effective filing date of this application). In certain embodiments, the ceDNA-plasmid backbone is derived from the AAV2 genome. In another specific embodiment, the ceDNA-plasmid backbone is a synthetic backbone engineered to include 5' and 3' ITRs from one of these AAV genomes.

[0127] The ceDNA-plasmid may optionally contain a selectable or selectable marker for use in establishing a ceDNA vector-producing cell line. In one embodiment, the selectable marker may be inserted downstream (i.e., 3') of the 3' ITR sequence. In another embodiment, the selectable marker may be inserted upstream (i.e., 5') of the 5' ITR sequence. Suitable selectable markers include, for example, those that confer drug resistance. The selectable marker may be, for example, the blasticidin S resistance gene, kanamycin, geneticin, etc. In a preferred embodiment, the drug selectable marker is the blasticidin S resistance gene.

[0128] Exemplary ceDNA (e.g., rAAV0) is produced from an rAAV plasmid. A method for producing an rAAV vector can include (a) providing a host cell with an rAAV plasmid as described above, where both the host cell and the plasmid lack a capsid protein-encoding gene, (b) culturing the host cell under conditions that allow for the production of a ceDNA genome, and (c) harvesting the cells and isolating the AAV genome produced from the cells.

[0129] C. Exemplary Methods for Producing ceDNA Vectors from ceDNA Plasmids Also provided herein are methods for producing capsid-free ceDNA vectors, particularly methods with yields high enough to provide sufficient vector for in vivo experiments.

[0130] In some embodiments, a method for producing a ceDNA vector includes the steps of: (1) introducing a nucleic acid construct containing an expression cassette and two asymmetric ITR sequences into host cells (e.g., Sf9 cells); (2) optionally establishing a clonal cell line, for example, by using a selectable marker present on the plasmid; (3) introducing a Rep-encoding gene into the insect cells (either by transfection or infection with a baculovirus carrying the gene); and (4) harvesting the cells and purifying the ceDNA vector. For the production of capsid-free AAV vectors, the nucleic acid construct containing the expression cassette and two ITR sequences described above can be in the form of a cfAAV-plasmid, or a bacmid or baculovirus generated with the cfAAV-plasmid as described below. The nucleic acid construct can be introduced into host cells by transfection, viral transduction, stable integration, or other methods known in the art.

[0131] D. Cell lines: Host cell lines used in the production of ceDNA vectors can include insect cell lines derived from Spodoptera frugiperda (Sf9, Sf21, etc.) or Trichoplusia ni cells, or other eukaryotic cell lines, including other invertebrate, vertebrate, or mammalian cells. Other cell lines known to those skilled in the art can also be used, such as HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1080, monocytes, and mature and immature dendritic cells. Host cell lines can be transfected for stable expression of ceDBA-plasmids for high-yield ceDNA vector production.

[0132] The ceDNA-plasmid can be introduced into Sf9 cells by transient transfection using reagents known in the art (e.g., liposomes, calcium phosphate) or physical means (e.g., electroporation). Alternatively, stable Sf9 cell lines that have stably integrated the ceDNA-plasmid into their genome can be established. Such stable cell lines can be established by incorporating a selection marker into the above-mentioned ceDNA-plasmid. If the ceDNA-plasmid used to transfect the cell line contains a selection marker such as an antibiotic, cells that have been transfected with the ceDNA-plasmid and have integrated the ceDNA-plasmid DNA into their genome can be selected by adding the antibiotic to the cell growth medium. Resistant clones of cells can then be isolated and propagated by single-cell dilution or colony transfer techniques.

[0133] Isolate and purify E. ceDNA vector Examples of processes for obtaining and isolating ceDNA vectors are described in Figures 4A-4E and in the specific examples below. The ceDNA vectors disclosed herein can be obtained from producer cells expressing a single AAV Rep protein and further transformed with a ceDNA plasmid, ceDNA bacmid, or ceDNA baculovirus. Plasmids useful for producing ceDNA vectors include those shown in Figure 8A (useful for Rep BIIC production) and Figure 8B (plasmids used to obtain ceDNA vectors).

[0134] In one embodiment, the polynucleotide encodes a single AAV Rep protein (Rep78 or 68) delivered to a producer cell in a plasmid (Rep-plasmid), bacmid (Rep-bacmid), or baculovirus (Rep-baculovirus). The Rep-plasmid, Rep-bacmid, and Rep-baculovirus can be generated by the methods described above.

[0135] A method for producing an exemplary ceDNA vector, a ceDNA vector, is described herein. The expression construct used to generate the ceDNA vector of the present invention can be a plasmid (e.g., a ceDNA-plasmid), a bacmid (e.g., a ceDNA-bacmid), and / or a baculovirus (e.g., a ceDNA-baculovirus). By way of example only, a ceDNA vector can be generated from cells co-infected with a ceDNA-baculovirus and a Rep-baculovirus. The Rep protein produced from the Rep-baculovirus replicates the ceDNA-baculovirus to generate a ceDNA vector. Alternatively, a ceDNA vector can be generated from cells stably transfected with a construct containing a sequence encoding a single AAV Rep protein (e.g., Rep78, Rep68, or Rep52) delivered in a Rep-plasmid, Rep-bacmid, or Rep-baculovirus. The ceDNA-baculovirus can be transiently transfected into cells and replicated by the Rep protein to produce a ceDNA vector.

[0136] The bacmid (e.g., ceDNA-bacmid) can be transfected into permissive insect cells, such as Sf9, Sf21, Tni (Trichoplusia ni) cells, or High Five cells, to generate a ceDNA-baculovirus, a recombinant baculovirus containing sequences comprising the asymmetric ITRs and an expression cassette. The ceDNA-baculovirus can be reinfected into insect cells to obtain the next generation of recombinant baculovirus. Optionally, this step can be repeated one or more times to produce larger quantities of recombinant baculovirus.

[0137] The time for harvesting the ceDNA vector described herein from cells can be selected and optimized to achieve high-yield production of the ceDNA vector. For example, the harvest time can be selected taking into account cell viability, cell morphology, cell growth, etc. Typically, cells can be harvested after sufficient time has passed since baculovirus infection to produce the ceDNA vector (e.g., the ceDNA vector), but before a portion of the cells begin to die due to viral toxicity. The ceDNA vector can be isolated from Sf9 cells using a plasmid purification kit, such as the Qiagen ENDO-FREE PLASMID® kit. Other methods developed for plasmid isolation can also be adapted for ceDNA vectors. Generally, any nucleic acid purification method known in the art, as well as commercially available DNA extraction kits, can be employed.

[0138] Alternatively, purification can be achieved by subjecting the cell pellet to an alkaline lysis process, centrifuging the resulting lysate, and performing chromatographic separation. As a non-limiting example, this process can be carried out by loading the supernatant onto an ion exchange column (e.g., SARTOBIND Q®) that retains nucleic acids, then eluting (e.g., with a 1.2 M NaCl solution), and performing further chromatographic purification on a gel filtration column (e.g., 6 Fast Flow GE). The capsid-free AAV vector is then recovered, for example, by precipitation.

[0139] In some embodiments, ceDNA vectors can also be purified in the form of exosomes or microparticles. It is known in the art that many cell types release not only soluble proteins but also complex protein / nucleic acid cargoes through the shedding of membrane microvesicles (Cocucci et al., 2009, EP10306226.1). Such vesicles include microvesicles (also called microparticles) and exosomes (also called nanovesicles), both of which contain proteins and RNA as cargo. Microvesicles are generated by direct budding of the plasma membrane, and exosomes are released into the extracellular environment upon fusion of multivesicular endosomes with the plasma membrane. Therefore, microvesicles and / or exosomes containing ceDNA vectors can be isolated from cells transduced with ceDNA plasmids or bacmids or baculoviruses produced with ceDNA plasmids.

[0140] Microvesicles can be isolated by filtration or ultracentrifugation of the culture medium at 20,000 x g and exosomes at 100,000 x g. The optimal duration of ultracentrifugation can be determined experimentally and will depend on the specific cell type from which vesicles are isolated. Preferably, the culture medium is first cleared by low-speed centrifugation (e.g., 2000 x g for 5-20 minutes) and then subjected to spin concentration, for example, using an AMICON® spin column (Millipore, Watford, UK). Microvesicles and exosomes can be further purified via FACS or MACS by using specific antibodies that recognize specific surface antigens present on microvesicles and exosomes. Other microvesicle and exosome purification methods include, but are not limited to, immunoprecipitation, affinity chromatography, filtration, and magnetic beads coated with specific antibodies or aptamers. During purification, vesicles are washed, for example, with phosphate-buffered saline. One advantage of using microvesicles or exosomes to deliver ceDNA-containing vesicles is that these vesicles can be targeted to various cell types by containing on them membrane proteins that are recognized by specific receptors on each cell type (see also EP10306226).

[0141] Another aspect of the present invention relates to a method for purifying ceDNA vectors from host cell lines that have stably integrated ceDNA constructs into their genomes. In one embodiment, the ceDNA vectors are purified as DNA molecules. In another embodiment, the ceDNA vectors are purified as exosomes or microparticles.

[0142] Figure 5 shows a gel confirming the production of ceDNA from multiple ceDNA plasmid constructs using the methods described in the Examples. The ceDNA is confirmed by the characteristic band pattern in the gel, as discussed with respect to Figure 4D in the Examples. Other features of the ceDNA production process and intermediates are summarized in Figures 6A and 6B and Figures 7A and 7B, as described in the Examples.

[0143] IV. ceDNA Vector As described herein, methods and compositions using a single Rep protein are useful in producing capsid-free ceDNA molecules having covalently closed ends (ceDNA) vectors. In some embodiments, these ceDNA vectors are produced in permissive host cells containing a single Rep protein and are produced from expression constructs (e.g., ceDNA-plasmids, ceDNA-bacmids, ceDNA-baculoviruses, or integrated cell lines) that contain a heterologous gene (transgene) located between two inverted terminal repeat (ITR) sequences, which can be an asymmetric ITR pair or a symmetric or substantially symmetric ITR pair, as these terms are defined herein. A ceDNA vector comprising an NLS as disclosed herein may comprise an ITR sequence selected from any of: (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (mod-ITR) (e.g., an asymmetric modified ITR); (ii) two modified ITRs (e.g., an asymmetric modified ITR) in which the mod-ITR pair has a different three-dimensional spatial configuration relative to each other; or (iii) a symmetric or substantially symmetric WT-WT ITR pair in which each WT-ITR has the same three-dimensional spatial configuration; or (iv) a symmetric or substantially symmetric modified ITR pair in which each mod-ITR has the same three-dimensional spatial configuration; and the methods of the present disclosure may further comprise a delivery system, such as, but not limited to, a liposomal nanoparticle delivery system.

[0144] The ceDNA vector is preferably double-stranded, e.g., self-complementary, over at least a portion of the molecule, such as an expression cassette (e.g., the ceDNA is not a double-stranded circular molecule). The ceDNA vector has covalently closed ends and is therefore resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III), e.g., for greater than 1 hour at 37°C.

[0145] The ceDNA vectors produced according to the methods and compositions using the single Rep protein disclosed herein do not have the packaging constraints imposed by the limited space within the viral capsid. In contrast to the enclosed AAV genome, ceDNA vectors represent a versatile eukaryotic alternative to prokaryotically produced plasmid DNA vectors. This allows the insertion of control elements, such as the regulatory switches disclosed herein, large transgenes, multiple transgenes, etc.

[0146] In one aspect, a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR, wherein the first and second ITRs are asymmetric with respect to each other, i.e., they are different from each other. In an exemplary embodiment, the first ITR can be a wild-type ITR and the second ITR can be a mutant or modified ITR. In some embodiments, the first ITR can be a mutant or modified ITR and the second ITR is a wild-type ITR. In another embodiment, the first and second ITRs are both modified but have different sequences, different modifications, or are not identical modified ITRs. In other words, the ITRs are asymmetric, meaning that any change in one ITR is not reflected in the other ITR, or the ITRs are different from each other. Exemplary ITRs in ceDNA vectors for use in generating ceDNA-plasmids are described below in the section entitled "ITRs."

[0147] The wild-type or mutant or otherwise modified ITR sequences provided herein represent DNA sequences included in an expression construct (e.g., ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus) for the production of a ceDNA vector. Thus, the ITR sequences actually contained in a ceDNA vector produced from a ceDNA-plasmid or other expression construct may or may not be identical to the ITR sequences provided herein as a result of naturally occurring variations (e.g., replication errors) that occur during the production process.

[0148] In some embodiments, the ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein comprises an expression cassette having a transgene, which may be, for example, a regulatory sequence, a nucleic acid-encoding sequence (e.g., miR or antisense sequence), or a polypeptide-encoding sequence (e.g., transgene). In one embodiment, the transgene may be operably linked to one or more regulatory sequences that enable or control expression of the transgene. In one embodiment, the polynucleotide comprises a first ITR sequence and a second ITR sequence, and the nucleotide sequence of interest is flanked by the first and second ITR sequences, and the first and second ITR sequences are asymmetric with respect to each other.

[0149] In one embodiment of each of these aspects, the expression cassette comprises, in order, a promoter located between two ITRs and operably linked to the transgene, a post-transcriptional regulatory element, and one or more polyadenylation and termination signals. In one embodiment, the promoter is regulatable—inducible or repressible. The promoter can be any sequence that promotes transcription of the transgene. In one embodiment, the promoter is a CAG promoter (e.g., SEQ ID NO: 03) or a variant thereof. The post-transcriptional regulatory element is a sequence that regulates expression of the transgene, including, but not limited to, any sequence that creates a tertiary structure that enhances expression of the transgene.

[0150] In one embodiment, the post-transcriptional regulatory element comprises WPRE (e.g., SEQ ID NO: 08). In one embodiment, the polyadenylation and termination signal comprises BGH polyA (e.g., SEQ ID NO: 09). Any cis-regulatory element known in the art, or combinations thereof, can additionally be used, such as the SV40 late polyA signal upstream enhancer sequence (USE) or other post-transcriptional processing elements (including, but not limited to, the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV)). In one embodiment, the length of the expression cassette in the 5' to 3' orientation exceeds the maximum length known to be encapsidated in AAV virions. In one embodiment, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb. Various expression cassettes are exemplified herein.

[0151] The expression cassette of a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein can contain more than 4,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, 20,000 nucleotides, 30,000 nucleotides, 40,000 nucleotides, or 50,000 nucleotides, or any range of about 4,000 to 10,000 nucleotides, or 10,000 to 50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, the expression cassette can contain a transgene or nucleic acid in the range of 500 to 50,000 nucleotides in length. In some embodiments, the expression cassette can contain a transgene or nucleic acid in the range of 500 to 75,000 nucleotides in length. In some embodiments, the expression cassette can contain a transgene or nucleic acid in the range of 500 to 10,000 nucleotides in length. In some embodiments, the expression cassette can contain a transgene or nucleic acid in the range of 1,000 to 10,000 nucleotides in length. In some embodiments, the expression cassette can contain a transgene or nucleic acid ranging in length from 500 to 5,000 nucleotides. ceDNA vectors do not have the size limitations of encapsidated AAV vectors, allowing delivery of large expression cassettes resulting in efficient expression of the transgene. In some embodiments, the ceDNA vector lacks prokaryotic cell-specific methylation.

[0152] In some embodiments, the expression cassette in a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein may also contain an internal ribosome entry site (IRES) and / or a 2A element. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue- and cell-type-specific promoters, and enhancers. In some embodiments, the ITRs may act as promoters for the transgene. In some embodiments, the ceDNA vector may contain additional components for regulating the expression of the transgene, such as one or more regulatory switches for controlling and regulating the expression of the transgene, as described herein in the section entitled "Regulatory Switches," and, if desired, a regulatory switch that is a kill switch that enables controlled cell death of cells containing the ceDNA vector.

[0153] 1A-1C show schematic diagrams of non-limiting exemplary ceDNA vectors, or the corresponding sequences of ceDNA plasmids. The ceDNA vector is derived from a capsid-free plasmid encoding a first ITR, an expressible transgene cassette, and a second ITR, in that order, where at least one of the first and / or second ITR sequences is mutated relative to the corresponding wild-type AAV2 ITR sequence. The expressible transgene cassette preferably includes, in that order, one or more of an enhancer / promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA).

[0154] The expression cassette of a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein can include any transgene of interest. Transgenes of interest include, but are not limited to, nucleic acids encoding polypeptides or non-coding nucleic acids (e.g., RNAi, miR, etc.), preferably therapeutic (e.g., for medical, diagnostic, or veterinary applications) or immunogenic polypeptides (e.g., for vaccines). In certain embodiments, the transgene in the expression cassette encodes one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof. In some embodiments, the transgene is a therapeutic gene or a marker protein. In some embodiments, the transgene is an agonist or antagonist. In some embodiments, the antagonist targets a mimic or antibody or antibody fragment, or an antigen-binding fragment thereof, such as a neutralizing antibody or antibody fragment. In some embodiments, the transgene encodes an antibody, including a full-length antibody or antibody fragment, as defined herein. In some embodiments, an antibody is an antigen binding domain or an immunoglobulin variable domain sequence, as defined herein.

[0155] In particular, the transgene is one or more therapeutic agents, including, but not limited to, proteins, polypeptides, peptides, enzymes, antibodies, antigen-binding fragments, and variants and / or active fragments thereof, for use in the treatment, prevention, and / or amelioration of, for example, one or more symptoms of a disease, dysfunction, injury, and / or disorder. Exemplary therapeutic genes are described herein in the section entitled "Methods of Treatment."

[0156] There are many structural features of ceDNA vectors produced according to the present methods and compositions using the single Rep protein disclosed herein that distinguish them from plasmid-based expression vectors. ceDNA vectors may have one or more of the following characteristics: lack of original (i.e., uninserted) bacterial DNA, lack of a prokaryotic replication origin, be self-contained (i.e., do not require any sequences other than the two ITRs containing the Rep binding and terminal resolution sites (RBS and TRS), and do not require any exogenous sequences between the ITRs), have the presence of hairpin-forming ITR sequences of eukaryotic origin (i.e., are produced in eukaryotic cells), and lack bacterial-type DNA methylation, or indeed any other methylation that would be considered abnormal by a mammalian host. Generally, it is preferred that the present vectors do not contain any prokaryotic DNA, although it is contemplated that some prokaryotic DNA may be inserted as exogenous sequences, such as, but not limited to, promoter or enhancer regions. Another important feature that distinguishes ceDNA vectors from plasmid expression vectors is that ceDNA vectors are single-stranded linear DNA with closed ends, whereas plasmids are always double-stranded DNA.

[0157] The ceDNA vectors produced according to the methods and compositions using the single Rep protein disclosed herein preferably have a linear, continuous, rather than discontinuous, structure as determined by restriction enzyme digestion assays ( FIG. 4D ). A linear, continuous structure is believed to be more stable against attack by cellular endonucleases and less likely to recombine and cause mutagenesis. Therefore, a ceDNA vector with a linear, continuous structure is a preferred embodiment. Continuous, linear, single-stranded intramolecular duplex ceDNA vectors may have covalently linked termini without sequences encoding AAV capsid proteins. These ceDNA vectors are structurally distinct from plasmids (including the ceDNA plasmids described herein), which are circular, double-stranded nucleic acid molecules of bacterial origin. While the complementary strands of a plasmid can be separated following denaturation to produce two nucleic acid molecules, a ceDNA vector, although having complementary strands, is a single DNA molecule and therefore remains a single molecule even when denatured. In some embodiments, the ceDNA vectors described herein, unlike plasmids, can be produced without DNA base methylation in prokaryotic cell types. Thus, ceDNA vectors and ceDNA-plasmids differ both in terms of structure (particularly linear vs. circular) and the methods used to produce and purify these different entities (see below), and in terms of their DNA methylation, which is that of prokaryotic cell types in the case of ceDNA-plasmids and eukaryotic cell types in the case of ceDNA vectors.

[0158] Some advantages of the ceDNA vectors described herein over plasmid-based expression vectors include, but are not limited to, the following: 1) Plasmids contain bacterial DNA sequences and are subject to prokaryotic cell-specific methylation, such as 6-methyladenosine and 5-methylcytosine methylation, whereas capsid-free AAV vector sequences are of eukaryotic origin and are not subject to prokaryotic cell-specific methylation. As a result, capsid-free AAV vectors are less likely to induce inflammation and immune responses compared to plasmids. 2) Plasmids require the presence of resistance genes during the production process, whereas ceDNA vectors do not. 3) Circular plasmids are not delivered to the nucleus upon introduction into cells and require overloading to bypass degradation by cellular nucleases, whereas ceDNA vectors contain viral cis elements, i.e., ITRs, that confer resistance to nucleases and can be designed to be targeted and delivered to the nucleus. We hypothesize that the minimal defining elements essential for ITR function are a Rep binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) for AAV2) and a terminal resolution site (TRS, 5'-AGTTGG-3' (SEQ ID NO: 48) for AAV2), plus a variable palindromic sequence that allows hairpin formation. 4) ceDNA vectors do not have the excessive representation of CpG dinucleotides often found in prokaryotic-derived plasmids that reportedly bind members of the Toll-like family of receptors and elicit T cell-mediated immune responses. In contrast, transduction with the capsid-free AAV vectors disclosed herein can use a variety of delivery reagents and efficiently target cells and tissue types that are difficult to transduce with conventional AAV virions.

[0159] V.ITR The ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein contain a heterologous gene located between two inverted terminal repeat (ITR) sequences that differ from each other (i.e., are asymmetric ITRs). In some embodiments, at least one of the ITRs is modified by deletion, insertion, and / or substitution compared to the wild-type ITR sequence (e.g., AAV ITR), and at least one of the ITRs contains a functional Rep binding site (RBS, e.g., 5'-GCGCGCTCGCTCGCTC-3', SEQ ID NO: 531 for AAV2) and a functional terminal resolution site (TRS, e.g., 5'-AGTT-3', SEQ ID NO: 46). In one embodiment, at least one of the ITRs is a non-functional ITR. In one embodiment, the different ITRs are not wild-type ITRs from different serotypes.

[0160] ITRs are exemplified in the specification, and examples herein are AAV2 ITRs, but one of skill in the art will recognize that, as noted above, ITRs from any known parvovirus, e.g., dependovirus, such as AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes can be used; see, e.g., NCBI:NC002077;NC001401;NC001729;NC001829;NC006152;NC006260;NC006261), chimeric ITRs, or ITRs from any synthetic AAV. In some embodiments, the AAV can infect warm-blooded animals, such as avian (AAAV), bovine (BAAV), canine, equine, and ovine adeno-associated viruses. In some embodiments, the ITRs are derived from B19 parvovirus (GenBank accession number NC000883), minute virus of mice (MVM) (GenBank accession number NC001510), goose parvovirus (GenBank accession number NC001701), or snake parvovirus 1 (GenBank accession number NC006148).

[0161] In some embodiments, the ITR sequences of ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein can be derived from viruses of the Parvoviridae family, which includes two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect insects. Parvovirinae (also called parvoviruses) comprises the Dependovirus genus, whose members, under most conditions, require co-infection with a helper virus, such as an adenovirus or herpesvirus, for productive infection. The Dependovirus family includes adeno-associated viruses (AAVs), which typically infect humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4), as well as related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses). Parvoviruses and other members of the family Parvoviridae are generally described in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996).

[0162] A skilled practitioner will appreciate that ITR sequences have the general structure of a double-stranded Holliday junction, typically a T- or Y-shaped hairpin structure (e.g., Figures 2A and 3A). Each 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 (the order of these palindromic sequences defines the flip or flop orientation of the ITR). Based on the exemplary AAV2 ITR sequences provided herein, skilled practitioners can easily determine corresponding modified ITR sequences from any AAV serotype for use in ceDNA vectors or ceDNA-plasmids. See, for example, structural analysis and sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6). Grimm et al., J. Virology, 2006;80(1);426-439, Yan et al., J. Virology, 2005;364-379, Duan et al., Virology 1999;261;8-14.

[0163] Although specific modifications and mutations in ITRs are described in detail herein, in the context of ITRs, "modified" or "mutated" indicates that nucleotides have been inserted, deleted, and / or substituted relative to the wild-type, reference, or original ITR sequence, and may be modified relative to other adjacent ITRs in a ceDNA vector having two adjacent ITRs. A modified or mutant ITR may be an engineered ITR. As used herein, "engineered" refers to an aspect that has been manipulated by human hands. For example, a polypeptide is considered "engineered" when at least one aspect of the polypeptide, such as its sequence, has been manipulated by human hands and is different from its naturally occurring aspect.

[0164] In some embodiments, the ITRs of a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein can be synthetic. In one embodiment, the synthetic ITRs are based on ITR sequences from two or more AAV serotypes. In another embodiment, the synthetic ITRs do not contain AAV-based sequences. In yet another embodiment, the synthetic ITRs preserve the ITR structure described above but have little or no AAV-origin sequences. In some aspects, the synthetic ITRs may preferentially interact with wild-type Rep or Rep of a specific serotype, or in some cases, are not recognized by wild-type Rep but are recognized only by mutant Rep.

[0165] ITR sequences have the general structure of a double-stranded Holliday junction, typically a T-shaped or Y-shaped hairpin structure (see, e.g., Figures 2A and 3A). Each 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 (the order of these palindromic sequences defines the flip or flop orientation of the ITR). Based on the exemplary AAV2 ITR sequences provided herein, those skilled in the art can easily determine ITR sequences or modified ITR sequences from any AAV serotype for use in ceDNA vectors or ceDNA-plasmids. See, for example, the sequence comparison of ITRs from different AAV serotypes (AAV1 to AAV6, as well as avian AAV (AAAV) and bovine AAV (BAAV)) described in Grimm et al., J. Virology, 2006;80(1);426-439. This shows the % identity of the left ITR of AAV2 to left ITRs from other serotypes: AAV-1 (84%), AAV-3 (86%), AAV-4 (79%), AAV-5 (58%), AAV-6 (left ITR) (100%), and AAV-6 (right ITR) (82%).

[0166] Thus, although AAV2 ITRs are used as exemplary ITRs for ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein, ceDNA vectors can be prepared with or based on ITRs from any known AAV serotype, including, for example, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12). Those skilled in the art can determine corresponding sequences in other serotypes by known means. For example, determine whether there are changes in the A, A', B, B', C, C', or D regions to determine the corresponding region in another serotype. Corresponding sequences can be determined using BLAST® (Basic Local Alignment Search Tool) or other homology alignment programs in their default settings. The present invention further provides a collection and a plurality of ceDNA vectors comprising ITRs from a combination of different AAV serotypes. That is, one ITR can be derived from one AAV serotype and the other ITR can be derived from a different serotype. Without being bound by theory, in one embodiment, one ITR may be derived from or based on an AAV2 ITR sequence, and the other ITR of the ceDNA vector may be derived from or based on any one or more ITR sequences of AAV serotype 1 (AAV1), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12).

[0167] Any parvovirus ITR can be used as an ITR or as a basic ITR for modification. Preferably, the parvovirus is a dependovirus. More preferably, it is an AAV. The serotype selected can be based on the tissue tropism of the serotype. AAV2 has broad tissue tropism, AAV1 preferentially targets neurons and skeletal muscle, and AAV5 targets neurons, retinal pigment epithelium, and photoreceptors. AAV6 preferentially targets skeletal muscle and lung. AAV8 preferentially targets liver, skeletal muscle, heart, and pancreatic tissue. AAV9 preferentially targets liver, skeletal, and lung tissue. In one embodiment, the modified ITR is based on the AAV2 ITR, e.g., selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:52. In one embodiment of each of these aspects, the vector polynucleotide comprises a pair of ITRs selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:52 and SEQ ID NO:2 and SEQ ID NO:51. In one embodiment of each of these aspects, the vector polynucleotide or non-viral capsid-free DNA vector with covalently closed ends comprises a different pair of ITRs selected from the group consisting of SEQ ID NO: 101 and SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106, SEQ ID NO: 107 and SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110, SEQ ID NO: 111 and SEQ ID NO: 112, SEQ ID NO: 113 and SEQ ID NO: 114, and SEQ ID NO: 115 and SEQ ID NO: 116. In some embodiments, the modified ITRs are selected from any of the ITR or partial ITR sequences of SEQ ID NOs: 2, 52, 63, 64, 101-499, or 545-547.

[0168] In some embodiments, ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein may comprise an ITR having an ITR sequence or ITR subsequence set forth in any one or more of Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A, and 10B herein, or an ITR modification corresponding to any of the modifications of the sequences set forth in Figures 26A or 26B.

[0169] In some embodiments, ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein can form intramolecular double-stranded secondary structures. The secondary structures of the first ITR and asymmetric second ITR are illustrated in the context of wild-type ITRs (see, e.g., Figures 2A, 3A, and 3C) and modified ITR structures (see, e.g., Figures 2B and 3B and 3D). The secondary structures are inferred or predicted based on the ITR sequence of the plasmid used to produce the ceDNA vector. Exemplary secondary structures of modified ITRs in which part of the stem-loop structure is deleted are shown in Figures 9A-25B and 26A-26B, and also in Tables 10A and 10B. Exemplary secondary structures of modified ITRs containing a single stem and two loops are shown in Figures 9A-13B. Exemplary secondary structures of modified ITRs with a single stem and a single loop are shown in Figure 14. In some embodiments, secondary structure can be predicted using a thermodynamic method based on nearest neighbor rules, which predicts structural stability as quantified by folding free energy change, as shown herein. For example, structures can be predicted by finding the lowest free energy structure. In some embodiments, ITR structures can be predicted using the algorithm disclosed in Reuter, JS, & Mathews, DH (2010) RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics. 11, 129, and implemented in the RNAstructure software (available at the World Wide Web address "rna.urmc.rochester.edu / RNAstructureWeb / index.html"). This algorithm can also include both free energy change parameters at 37°C and enthalpy change parameters derived from experimental literature, allowing for the prediction of conformational stability at any temperature.Using RNAstructure software, several modified ITR structures can be predicted as modified T-shaped stem-loop structures with estimated Gibbs unfolding free energies (ΔG) under physiological conditions as shown in Figures 3A-3D. Using RNAstructure software, three types of modified ITRs are predicted to have higher Gibbs unfolding free energies than the wild-type AAV2 ITR (-92.9 kcal / mol), as follows: (a) the single-arm / single-unpaired loop structure provided herein is predicted to have a Gibbs unfolding free energy in the range of -85 to -70 kcal / mol; (b) the modified ITR with a single hairpin structure provided herein is predicted to have a Gibbs unfolding free energy in the range of -70 to -40 kcal / mol; and (c) the modified ITR with a two-arm structure provided herein is predicted to have a Gibbs unfolding free energy in the range of -90 to -70 kcal / mol. Without wishing to be bound by theory, structures with high Gibbs free energy are easier to unfold for replication by Rep68 or Rep78 replication proteins. Therefore, modified ITRs with high Gibbs unfolding free energy (e.g., single-arm / single-unpaired loop structure, single hairpin structure, truncated structure) tend to be replicated more efficiently than wild-type ITRs.

[0170] In one embodiment, the left ITR of a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein is modified or mutated with respect to the wild-type (wt) AAV ITR structure, and the right ITR is the wild-type AAV ITR. In one embodiment, the right ITR of a ceDNA vector is modified with respect to the wild-type AAV ITR structure, and the left ITR is the wild-type AAV ITR. In such embodiments, the modification of an ITR (e.g., the left or right ITR) can be generated by deletion, insertion, or substitution of one or more nucleotides from the wild-type ITR derived from the AAV genome.

[0171] The ITRs used herein can be degradable or non-degradable, and those selected for use in ceDNA vectors are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5, 6, 7, 8, and 9 being preferred. Degradable AAV ITRs do not require wild-type ITR sequences (e.g., endogenous or wild-type AAV ITR sequences may be altered by insertion, deletion, truncation, and / or missense mutation), as long as the terminal repeats mediate the desired function (e.g., replication, viral packaging, integration, and / or proviral rescue, etc.). Typically, but not necessarily, the ITRs are derived from the same AAV serotype. For example, both ITR sequences in a ceDNA vector are derived from AAV2. The ITRs may also be synthetic sequences that function as AAV inverted terminal repeats, such as the "double D sequence" described in U.S. Patent No. 5,478,745 to Samulski et al. Although not required, the ITRs can be derived from the same parvovirus, e.g., both ITR sequences are derived from AAV2.

[0172] In one embodiment, a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein may contain an ITR structure that is mutated with respect to one of the wild-type ITRs disclosed herein, but where the mutated or modified ITR still retains an operable Rep binding site (RBE or RBE') and terminal resolution site (trs). In one embodiment, the mutant ceDNA ITR contains a functional replication protein site (RPS-1), and a replicative protein that binds to the RPS-1 site is used for production.

[0173] In one embodiment, at least one of the ITRs of a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein is defective with respect to Rep binding and / or Rep nicking. In one embodiment, the defect is at least 30% relative to the wild-type reduced ITR, and in other embodiments, at least 35%, 50%, 65%, 75%, 85%, 90%, 95%, 98%, or a complete lack of function or any point in between. The host cell does not express viral capsid proteins, and the polynucleotide vector template lacks viral capsid coding sequences. In one embodiment, the polynucleotide vector template and host cell lacking AAV capsid genes, and the resulting proteins, also do not encode or express capsid genes of other viruses. Additionally, in certain embodiments, the nucleic acid molecule also lacks AAV Rep protein coding sequences.

[0174] In some embodiments, the structural element of the ITR can be any structural element involved in the functional interaction of the ITR with a single large Rep protein (e.g., Rep78 or Rep68). In certain embodiments, the structural element provides selectivity for the interaction of the ITR with a single large Rep protein, i.e., determines, at least in part, which Rep protein functionally interacts with the ITR. In other embodiments, the structural element physically interacts with a single large Rep protein when the Rep protein is bound to the ITR. Each structural element can be, for example, the secondary structure of the ITR, the nucleotide sequence of the ITR, the space between two or more elements, or any combination of the above. In one embodiment, the structural element is selected from the group consisting of A and A' arms, B and B' arms, C and C' arms, D arm, Rep binding site (RBE) and RBE' (i.e., complementary RBE sequences), and terminal resolution site (trs).

[0175] More specifically, the ability of structural elements of the ITRs of ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein to functionally interact with a particular single Rep protein, e.g., a large Rep protein or a small Rep protein, can be altered by modifying the structural elements. For example, the nucleotide sequence of the structural elements can be modified compared to the wild-type sequence of the ITR. In one embodiment, structural elements of an ITR (e.g., the A arm, A' arm, B arm, B' arm, C arm, C' arm, D arm, RBE, RBE', and trs) can be removed and replaced with wild-type structural elements from a different parvovirus. For example, the replacement structure can be from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (e.g., royal python parvovirus), bovine parvovirus, caprine parvovirus, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus, or insect AAV. For example, the ITRs can be AAV2 ITRs, and the A or A' arms or RBEs can be replaced with structural elements from AAV5. In another example, the ITRs can be AAV5 ITRs, and the C or C' arms, RBE, and trs can be replaced with structural elements from AAV2. In another example, the AAV ITRs can be AAV5 ITRs, and the B and B' arms are replaced with AAV2 ITR B and B' arms.

[0176] By way of example only, Table 1 shows exemplary modifications (e.g., deletions, insertions, and / or substitutions) of at least one nucleotide in a region of a modified ITR, where X indicates the modification (e.g., deletion, insertion, and / or substitution) of at least one nucleic acid in that section relative to the corresponding wild-type ITR. In some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in any of the C and / or C' and / or B and / or B' regions retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. For example, if the modification results in either a single-arm ITR (e.g., a single C-C' arm or a single B-B' arm), or a modified C-B' arm or C'-B arm, or a two-arm ITR with at least one truncated arm (e.g., a truncated C-C' arm and / or a truncated B-B' arm), at least the single arm, or at least one of the arms of the two-arm ITR (one arm may be truncated), retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. In some embodiments, the truncated C-C' arm and / or the truncated B-B' arm has three consecutive T nucleotides (i.e., TTT) in the terminal loop. [Table 1]

[0177] In some embodiments, modified ITRs for use in ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein may include any one of the combinations of modifications shown in Table 1, as well as a modification of at least one nucleotide in any one or more of the regions selected from between A' and C, between C and C', between C' and B, between B and B', and between B' and A. In some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the C or C' or B or B' region still preserves the terminal loop of the stem-loop. In some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide between C and C' and / or B and B' retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. In alternative embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide between C and C' and / or between B and B' preserves three consecutive "A" nucleotides (i.e., AAA) in at least one terminal loop. In some embodiments, modified ITRs for use in ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein may include any one of the combinations of modifications shown in Table 1, as well as a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in any one or more of the regions selected from A', A, and / or D. For example, in some embodiments, modified ITRs for use herein may include any one of the combinations of modifications shown in Table 1, as well as at least one modification (e.g., deletion, insertion, and / or substitution) in the A region. In some embodiments, modified ITRs for use in ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein may include any one of the combinations of modifications shown in Table 1, as well as a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the A' region.In some embodiments, modified ITRs for use herein may include any one of the combinations of modifications shown in Table 1, as well as a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the A and / or A' region. In some embodiments, modified ITRs for use herein may include any one of the combinations of modifications shown in Table 1, as well as a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the D region.

[0178] In one embodiment, the nucleotide sequence of a structural element of an ITR can be modified (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides, or any range therein) to produce a modified structural element. In one embodiment, specific modifications to the ITRs of ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein are exemplified herein (e.g., SEQ ID NOs: 2, 52, 63, 64, 101-499, or 545-547). In some embodiments, the ITRs of ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein can be modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides, or any range therein). In other embodiments, the ITRs of a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the modified ITRs of SEQ ID NOs: 469-499 or 545-547, or to one of the RBE-containing sections of the A-A' arms and the C-C' and B-B' arms of SEQ ID NOs: 101-134 or 545-547.

[0179] In some embodiments, modified ITRs in ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein can include, for example, removal or deletion of all of a particular arm, e.g., all or part of the A-A' arm, or all or part of the B-B' arm, or all or part of the C-C' arm, or removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs that form the stem of a loop (e.g., see ITR-6), so long as a final loop capping the stem (e.g., a single arm) is still present. In some embodiments, modified ITRs can include removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. In some embodiments, modified ITRs can include removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm. In some embodiments, the modified ITR can include the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm and the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. Any combination of base pair removal is contemplated; for example, six base pairs in the C-C' arm and two base pairs in the B-B' arm can be removed. As an illustrative example, Figures 13A-13B show an exemplary modified ITR having at least seven base pairs deleted from each of the C and C' portions, a substitution of a nucleotide in the loop between the C and C' regions, and the deletion of at least one base pair from each of the B and B' regions, such that the modified ITR comprises two arms in which at least one arm (e.g., C-C') is truncated. Note that in this example, the modified ITR contains a deletion of at least one base pair from each of the B and B' regions, so that the B-B' arm is also truncated relative to the WT ITR.

[0180] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more complementary base pairs are removed from each of the C and C' portions of the C-C' arm, thereby cleaving the C-C' arm. That is, when a base in the C portion of the C-C' arm is removed, a complementary base pair in the C' portion is removed, thereby cleaving the C-C' arm. In such embodiments, 2, 4, 6, 8, or more base pairs are removed from the C-C' arm, thereby cleaving the C-C' arm. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs are removed from the C portion of the C-C' arm, thereby leaving only the C' portion of the arm. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs are removed from the C' portion of the C-C' arm, thereby leaving only the C portion of the arm.

[0181] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more complementary base pairs are removed from each of the B and B' portions of the B-B' arm, thereby cleaving the B-B' arm. That is, when a base in the B portion of the B-B' arm is removed, a complementary base pair in the B' portion is removed, thereby cleaving the B-B' arm. In such embodiments, 2, 4, 6, 8, or more base pairs are removed from the B-B' arm, thereby cleaving the B-B' arm. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs are removed from the B portion of the B-B' arm, thereby leaving only the B' portion of the arm. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs are removed from the B' portion of the B-B' arm, thereby leaving only the B portion of the arm.

[0182] In some embodiments, the modified ITRs of ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein can have a 1-50 (e.g., 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, or 50) nucleotide deletion relative to the full-length wild-type ITR sequence. In some embodiments, the modified ITRs can have a 1-30 nucleotide deletion relative to the full-length wild-type ITR sequence. In some embodiments, the modified ITRs have a 2-20 nucleotide deletion relative to the full-length wild-type ITR sequence.

[0183] In some embodiments, the modified ITR forms two opposing longitudinally asymmetric stem-loops, e.g., the C-C' loop is a different length from the B-B' loop. In some embodiments, one of the opposing longitudinally asymmetric stem-loops of the modified ITR has a C-C' and / or B-B' stem portion ranging from 8 to 10 base pairs in length and a loop portion having 2 to 5 unpaired deoxyribonucleotides (e.g., between C and C' or between B and B'). In some embodiments, one longitudinally asymmetric stem-loop of the modified ITR has a C-C' and / or B-B' stem portion less than 8, or less than 7, 6, 5, 4, 3, 2, or 1 base pairs in length, and a loop portion having 0 to 5 nucleotides (e.g., between C and C' or between B and B'). In some embodiments, the modified ITR with the longitudinally asymmetric stem-loop has a C-C' and / or B-B' stem portion less than 3 base pairs in length.

[0184] In some embodiments, the modified ITRs of ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein do not contain any nucleotide deletions in the RBE-containing portions of the A or A' regions so as not to interfere with DNA replication (e.g., binding to the RBE by the Rep protein or nicking at the terminal resolution site). In some embodiments, modified ITRs encompassed for use in ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein have one or more deletions in the B, B', C, and / or C regions described herein. Some non-limiting examples of modified ITRs are shown in Figures 9A-26B.

[0185] In some embodiments, modified ITRs of ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein can include a deletion of the B-B' arm, leaving only a C-C' arm. See, for example, exemplary ITR-2 (left) and ITR-2 (right), and ITR-4 (left) and ITR-4 (right) shown in Figures 9A-9B (Figures 11A-11B). In some embodiments, modified ITRs can include a deletion of the C-C' arm, leaving only a B-B' arm. See, for example, exemplary ITR-3 (left) and ITR-3 (right), shown in Figures 10A-10B. In some embodiments, modified ITRs can include a deletion of the B-B' and C-C' arms, leaving only a single stem-loop. See, for example, exemplary ITR-6 (left) and ITR-6 (right), and ITR-21 and ITR-37 shown in Figures 14A-14B. In some embodiments, modified ITRs of ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein can include a deletion of the C' region, leaving a truncated C' loop and B-B' arm. See, for example, exemplary ITR-1 (left) and ITR-1 (right) shown in Figures 15A-15B. Similarly, in some embodiments, modified ITRs can include a deletion of the C region, leaving a truncated C' loop and B-B' arm. See, for example, exemplary ITR-5 (left) and ITR-5 (right) shown in Figures 16A-16B.

[0186] In some embodiments, the modified ITRs of ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein can include deletion of base pairs in any one or more of the C, C', B, or B' portions, thereby allowing complementary base pairing between the C-B' and C'-B portions to produce a single arm. See, e.g., ITR-10(right) and ITR-10(left) (Figures 12A-12B).

[0187] In some embodiments, in addition to modification of one or more nucleotides in the C, C', B, and / or B' regions, modified ITRs for use in ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein may include modification (e.g., deletion, substitution, or addition) of at least 1, 2, 3, 4, 5, or 6 nucleotides in one or more regions selected from between A' and C, between C and C', between C' and B, between B and B', and between B' and A. For example, the nucleotides between B' and C in the modified right ITR can be substituted from A to G, C, or A, deleted, or one or more nucleotides can be added. The nucleotides between C' and B in the modified left ITR can be changed from T to G, C, or A, deleted, or one or more nucleotides can be added.

[0188] In certain embodiments, ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein do not have a modified ITR consisting of a nucleotide sequence selected from any of SEQ ID NOs: 550-557. In certain embodiments, ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein do not have a modified ITR comprising a nucleotide sequence selected from any of SEQ ID NOs: 550-557.

[0189] In some embodiments, the ceDNA vector comprises a regulatory switch as disclosed herein and a selected modified ITR having a nucleotide sequence selected from the group consisting of any of SEQ ID NOs: 550-557.

[0190] In another embodiment, the structure of the structural elements of the ITRs of ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein can be modified. For example, the structural elements can be altered by varying the height of the stem and / or the number of nucleotides in the loop. For example, the height of the stem can be about 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides or more, or any range therein. In one embodiment, the height of the stem can be about 5 nucleotides to about 9 nucleotides and can functionally interact with Rep. In another embodiment, the height of the stem can be about 7 nucleotides and can functionally interact with Rep. In another example, the loop can have a length of 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more, or any range therein.

[0191] In another embodiment, the number of GAGY-binding sites or GAGY-related binding sites in an RBE or extended RBE can be increased or decreased. In one example, an RBE or extended RBE can contain 1, 2, 3, 4, 5, 6, or more GAGY-binding sites, or any range therein. Each GAGY-binding site can independently be the exact GAGY sequence or a sequence similar to GAGY, so long as the sequence is sufficient to bind to the Rep protein.

[0192] In another embodiment, the spacing between two elements (such as, but not limited to, an RBE and a hairpin) can be altered (e.g., increased or decreased) to alter their functional interactions with a single large Rep protein. For example, the spacing can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides or more, or any range therein.

[0193] ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein and described herein may contain ITR structures that are modified with respect to the wild-type AAV2 ITR structure disclosed herein, while still retaining operable RBE, trs, and RBE' portions. Figures 2A and 2B illustrate one possible mechanism for engineering the trs portion within the wild-type ITR structure of a ceDNA vector. In some embodiments, the ceDNA vector contains one or more functional ITR polynucleotide sequences, including a Rep-binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) for AAV2) and a terminal resolution site (TRS, 5'-AGTT (SEQ ID NO: 46)). In some embodiments, at least one ITR (wild-type or modified ITR) is functional. In alternative embodiments, where the ceDNA vector contains two modified ITRs that are different or asymmetric from each other, at least one modified ITR is functional and at least one modified ITR is non-functional.

[0194] In some embodiments, ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein do not have modified ITRs selected from any sequence consisting of or consisting essentially of SEQ ID NOs: 500-529, as provided herein. In some embodiments, ceDNA vectors do not have ITRs selected from any sequence selected from SEQ ID NOs: 500-529.

[0195] In some embodiments, the modified ITR (e.g., left or right ITR) of a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein has a modification in the loop arm, cleavage arm, or spacer. Exemplary sequences of ITRs with modifications in the loop arm, cleavage arm, or spacer are listed in Table 2.

[0196] In some embodiments, the modified ITR (e.g., left or right ITR) of a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein has modifications in the loop arm and cleavage arm. Exemplary sequences of ITRs with modifications in the loop arm and cleavage arm are listed in Table 3.

[0197] In some embodiments, the modified ITR (e.g., left or right ITR) of a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein has modifications in the loop arm and spacer. Exemplary sequences of ITRs with modifications in the loop arm and spacer are listed in Table 4.

[0198] In some embodiments, the modified ITR (e.g., left or right ITR) of a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein has modifications within the cleavage arm and spacer. Exemplary sequences of ITRs with modifications within the cleavage arm and spacer are listed in Table 5.

[0199] In some embodiments, the modified ITRs (e.g., left or right ITRs) of ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein have modifications within the loop arm, cleavage arm, and spacer. Exemplary sequences of ITRs with modifications within the loop arm, cleavage arm, and spacer are listed in Table 6.

[0200] In some embodiments, an ITR (e.g., left or right ITR) of a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein is modified so that it contains the lowest unfolding energy (a "low energy structure"). A low energy would have a reduced Gibbs free energy compared to the wild-type ITR. Exemplary sequences of ITRs modified to have a lower (i.e., reduced) unfolding energy are presented herein in Tables 7-9.

[0201] In some embodiments, the modified ITRs of the ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein are selected from any of those shown in Tables 2-9, 10A, or 10B, or a combination thereof. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7]

[0202] As disclosed herein, modified ITRs of ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein can be generated to contain one or more nucleotide deletions, insertions, or substitutions from the wild-type ITRs derived from the AAV genome. The modified ITRs can be generated by genetic modification during propagation in a plasmid in Escherichia coli, or as a baculovirus genome in Spodoptera frugiperda cells, or by other biological methods, for example, using the polymerase chain reaction or chemical synthesis in vitro.

[0203] In some embodiments, the modified ITRs of ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein may contain a deletion, insertion, or substitution of one or more nucleotides from the AAV2 (left) wild-type ITR (SEQ ID NO: 51) or the AAV2 (right) wild-type ITR (SEQ ID NO: 1). Specifically, one or more nucleotides are deleted, inserted, or substituted from B-C' or C-C' of the T-shaped stem-loop structure. Furthermore, the modified ITRs do not contain modifications to the Rep binding element (RBE) and terminal resolution site (trs) of the AAV2 wild-type ITR, although the RBE' (TTT) may or may not be present depending on whether the template has undergone one round of replication, thereby converting the AAA triplet to the complementary RBE'-TTT.

[0204] Three types of modified ITRs are exemplified: (1) modified ITRs with the lowest energy structure containing a single arm and a single unpaired loop ("single arm / single unpaired loop structure"), (2) modified ITRs with the lowest energy structure containing a single hairpin ("single hairpin structure"), and (3) modified ITRs with the lowest energy structure containing two arms (one of which is truncated) ("truncated structure").

[0205] Modified ITR with a single arm / single unpaired loop structure Wild-type ITRs can be modified to form secondary structures containing a single arm and a single unpaired loop (i.e., "single arm / single unpaired loop structures"). The Gibbs unfolding free energies (ΔG) of the structures can range from -85 kcal / mol to -70 kcal / mol. Exemplary structures of modified ITRs are provided.

[0206] Modified ITRs predicted to form a single-arm / single unpaired loop structure may contain deletions, insertions, or substitutions of one or more nucleotides from the wild-type ITR in the sequences forming the B and B' arms and / or the C and C' arms. Modified ITRs may be produced by genetic modification or by biosynthesis and / or chemical synthesis.

[0207] For example, ITR-2 left and right (SEQ ID NOs: 101 and 102) provided in Figures 9A-9B were generated to have two nucleotides deleted from the C-C' arm and 16 nucleotides deleted from the B-B' arm in the wild-type ITR of AAV2. The remaining three nucleotides in the B-B' arm of the modified ITR do not form complementary pairs. Therefore, ITR-2 left and right have the lowest energy structure with a single C-C' arm and a single unpaired loop. The Gibbs free energy of unfolding the structure is predicted to be approximately -72.6 kcal / mol.

[0208] The ITR-3 left and right (SEQ ID NOs: 103 and 104) provided in Figures 10A and 10B were generated to contain a 19-nucleotide deletion in the C-C' arm from the wild-type ITR of AAV2. The remaining three nucleotides in the B-B' arm of the modified ITR do not form a complementary pair. Therefore, the ITR-3 left and right have the lowest energy structure with a single B-B' arm and a single unpaired loop. The Gibbs free energy of unfolding the structure is predicted to be approximately -74.8 kcal / mol.

[0209] The ITR-4 left and right (SEQ ID NOs: 105 and 106) provided in Figures 11A and 11B were generated to contain a 19-nucleotide deletion in the B-B' arm from the wild-type ITR of AAV2. The remaining three nucleotides in the B-B' arm of the modified ITR do not form a complementary pair. Therefore, the ITR-4 left and right have the lowest energy structure with a single C-C' arm and a single unpaired loop. The Gibbs free energy of unfolding the structure is predicted to be approximately -76.9 kcal / mol.

[0210] The ITR-10 left and right (SEQ ID NOs: 107 and 108) provided in Figures 12A and 12B were generated to contain an 8-nucleotide deletion in the B-B' arm from the wild-type ITR of AAV2. The remaining nucleotides in the B-B' and C-C' arms create new complementary bonds between the B and C' motifs (ITR-10 left) or between the C and B' motifs (ITR-10 right). Thus, the ITR-10 left and right have the lowest energy structures with a single B-C' or C-B' arm and a single unpaired loop. The Gibbs free energy of unfolding the structure is predicted to be approximately -83.7 kcal / mol.

[0211] The ITR-17 left and right (SEQ ID NOs: 109 and 110) provided in Figures 13A and 13B were generated to contain a 14-nucleotide deletion in the C-C' arm from the wild-type ITR of AAV2. The remaining 8 nucleotides in the C-C' arm do not form a complementary bond. As a result, the ITR-17 left and right have the lowest energy structure with a single B-B' arm and a single unpaired loop. The Gibbs free energy of unfolding the structure is predicted to be approximately -73.3 kcal / mol.

[0212] The sequences of the wild-type ITR left or right (top) and various modified ITR left or right (bottom) forms predicted to form a single arm / single unpaired loop structure are aligned and provided in Table 7 below. [Table 7-1] [Table 7-2] [Table 7-3]

[0213] Modified ITR with a single hairpin structure The wild-type ITR can be modified to have a lowest energy structure, including a single hairpin structure. The Gibbs unfolding free energy (ΔG) of the structure can range from -70 kcal / mol to -40 kcal / mol. Exemplary structures of modified ITRs are provided in Figures 14A and 14B.

[0214] Modified ITRs predicted to form a single hairpin structure may contain deletions, insertions, or substitutions of one or more nucleotides from the wild-type ITR in the sequences forming the B and B' arms and / or the C and C' arms. Modified ITRs may be produced by genetic modification or by biosynthesis and / or chemical synthesis.

[0215] For example, ITR-6 left and right (SEQ ID NOs: 111 and 112) provided in Figures 14A and 14B contain 40 nucleotide deletions in the B-B' and C-C' arms from the wild-type ITR of AAV2. The remaining nucleotides in the modified ITR are predicted to form a single hairpin structure. The Gibbs free energy of unfolding the structure is approximately -54.4 kcal / mol.

[0216] The sequences of the wild-type ITR and ITR-6 (both left and right) were aligned and are provided in Table 8 below. [Table 8]

[0217] Modified ITR with a truncated structure The wild-type ITR can be modified to have a lowest-energy structure containing two cleavable arms. Their Gibbs unfolding free energies (ΔG) range from -90 to -70 kcal / mol. Therefore, their Gibbs unfolding free energies are lower than those of the wild-type ITR of AAV2.

[0218] Modified ITRs may include deletions, insertions, or substitutions of one or more nucleotides from the wild-type ITR in the sequences forming the B and B' arms and / or the C and C' arms. In some embodiments, modified ITRs may include, for example, the complete removal of a particular loop, e.g., the A-A' loop, the B-B' loop, or the C-C' loop, or alternatively, the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs forming the stem of the loop, so long as the final loop at the end of the stem is still present. Modified ITRs may be produced by genetic modification or biosynthesis and / or chemical synthesis.

[0219] Exemplary structures of modified ITRs with truncated structures are provided in Figures 15A-15B.

[0220] The sequences of various modified ITRs predicted to form cleavage structures are aligned with the sequence of the wild-type ITR and are provided in Table 9 below. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5]

[0221] Additional exemplary modified ITRs of each of the above classes for use in ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein are provided in Tables 10A and 10B. The predicted secondary structures of the right-modified ITRs in Table 10A are shown in Figure 26A, and the predicted secondary structures of the left-modified ITRs in Table 10B are shown in Figure 26B.

[0222] Tables 10A and 10B show exemplary right and left modified ITRs in ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein. [Table 10A-1] [Table 10A-2] [Table 10B-1] [Table 10B-2]

[0223] In an embodiment of the present invention, a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein does not have a modified ITR having a nucleotide sequence selected from any of the group of SEQ ID NOs: 550, 551, 552, 553, 553, 554, 555, 556, 557.

[0224] To the extent that a ceDNA vector produced in accordance with the methods and compositions using a single Rep protein disclosed herein has modified ITRs, one of the modifications in the B, B', C, or C' regions set forth in SEQ ID NOS:550-557, as defined within any invention defined in any one or more of the claims of this application, or in any amended claims that may be hereafter filed in this application or in any patent derived therefrom, and to the extent the law of any relevant country(ies) to that claim(s) applies, we hereby reserve the right to disclaim such disclosure from the claims of this application or any patent derived therefrom to the extent necessary to prevent the invalidity of this application or any patent derived therefrom.

[0225] For example, without limitation, we reserve the right to disclaim any one of the following subject matter from any claim of this application (as currently or hereafter amended) or any patent proceeding therefrom:

[0226] A. Modified ITRs selected from the group consisting of SEQ ID NOs: 2, 52, 63 64, 113, 114, 550, 551; 552, 553, 553, 554, 555, 556, 557 for use in ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein without a regulatory switch.

[0227] B. A modified ITR as specified in A. above of a ceDNA vector produced according to the methods and compositions using a single Rep protein disclosed herein without regulatory sequences, wherein the heterologous nucleic acid encodes ABCA4, USA2A var1, VEGFR, CEP290, BDD Factor VIII (FVIII), Factor VIII, vWF_His, vWF, lecithin cholesterol acetyltransferase, PAH, G6PC, or CFTR.

[0228] VI. Regulatory Elements. A composition useful in the method for producing a DNA vector, for example, a ceDNA vector or AAV vector described herein, comprises a nucleic acid sequence encoding a single modified Rep protein, and can further comprise a regulatory element, for example, a cis-regulatory element described herein, upstream of or operably linked to the nucleic acid encoding the single modified Rep protein. For example, a nucleotide sequence encoding a modified Rep protein, for example, a modified Rep78 protein, but not including a functional initiation codon for encoding a Rep52 protein, or a splicing site for exon skipping to produce Rep68 or Rep40, is operably linked to a regulatory element, for example, a cis-regulatory element.

[0229] In one embodiment, a nucleotide sequence encoding a single Rep protein useful in the compositions and methods disclosed herein comprises an expression control sequence, such as a promoter, cis-regulatory element, or regulatory switch described herein, located upstream of the start codon of a nucleotide sequence encoding a parvoviral Rep78 protein, and the nucleic acid sequence does not have a functional start codon for Rep52 and / or a splice site for exon skipping to produce Rep68 or Rep40. In one embodiment, a nucleotide sequence encoding a single Rep protein useful in the compositions and methods disclosed herein comprises an expression control sequence upstream of the start codon of a nucleotide sequence encoding a parvoviral Rep78 protein, and the nucleic acid sequence does not have a functional splice site for encoding Rep68.

[0230] Similarly, ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein can be produced from expression constructs that further contain a specific combination of cis-regulatory elements. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue- and cell-type-specific promoters, and enhancers. In some embodiments, the ITRs can act as promoters for transgenes. In some embodiments, the ceDNA vectors contain additional components for regulating the expression of transgenes, such as regulatory switches described herein, to regulate the expression of the transgenes or kill switches that can kill cells containing the ceDNA vectors.

[0231] Similarly, ceDNA vectors produced according to the methods and compositions using a single Rep protein disclosed herein can be produced from expression constructs that further include specific combinations of cis-regulatory elements, such as the WHP posttranscriptional regulatory element (WPRE) (e.g., SEQ ID NO: 8) and BGH polyA (SEQ ID NO: 9). Suitable expression cassettes for use in expression constructs are not limited by packaging constraints imposed by viral capsids. Expression cassettes of the present invention contain promoters that can affect cell specificity as well as overall expression levels. In the case of transgene expression, they can include highly active immediate-early promoters from viruses. Expression cassettes can contain tissue-specific eukaryotic promoters to restrict transgene expression to specific cell types and reduce toxic effects and immune responses resulting from unregulated ectopic expression.

[0232] In a preferred embodiment, a promoter or regulatory element for use in expressing a modified single Rep protein or in an expression cassette of a ceDNA vector produced by the methods disclosed herein may contain a synthetic regulatory element such as the CAG promoter (SEQ ID NO:3). The CAG promoter includes (i) a cytomegalovirus (CMV) early enhancer element, (ii) the promoter, the first exon and first intron of the chicken β-actin gene, and (iii) the splice acceptor of the rabbit β-globin gene. Alternatively, a promoter or regulatory element for use in expressing a modified single Rep protein or in an expression cassette of a ceDNA vector produced by the methods disclosed herein may contain the alpha-1-antitrypsin (AAT) promoter (SEQ ID NO:4 or SEQ ID NO:74), the liver-specific (LP1) promoter (SEQ ID NO:5 or SEQ ID NO:16), or the human elongation factor-1 alpha (EF1a) promoter (e.g., SEQ ID NO:6 or SEQ ID NO:15). In some embodiments, promoters or regulatory elements for use in expressing a modified single Rep protein or in the expression cassette of a ceDNA vector produced by the methods disclosed herein are selected from one or more constitutive promoters, such as the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), or the cytomegalovirus (CMV) immediate early promoter (optionally with a CMV enhancer, e.g., SEQ ID NO: 22). Alternatively, an inducible promoter, a transgene's native promoter, a tissue-specific promoter, or various promoters known in the art can be operably linked to a nucleic acid encoding a modified single Rep protein or can be present in the expression cassette of a ceDNA vector produced by the methods disclosed herein.

[0233] Suitable promoters, including those described above, can be derived from viruses and therefore can be referred to as viral promoters, or they can be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters that may be operably linked to a nucleic acid encoding a modified single Rep protein or that may be present in the expression cassette of a ceDNA vector produced by the methods disclosed herein include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal sequence (LTR) promoter, the adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, e.g., the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the human U6 micronucleus promoter (U6, e.g., SEQ ID NO: 18) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31 (17)), the human H1 promoter (H1) (e.g., SEQ ID NO: 19), the CAG promoter, the human alpha 1-antitrypsin (HAAT) promoter (e.g., SEQ ID NO: 21), and the like. In embodiments, these promoters are modified at their downstream intron-containing ends to contain one or more nuclease cleavage sites, hi embodiments, the DNA containing the nuclease cleavage sites is exogenous to the promoter DNA.

[0234] A promoter can further enhance expression and / or alter its spatial and / or temporal expression by including one or more specific transcriptional regulatory sequences. A promoter can also include distal enhancer or repressor elements, which can be located thousands of base pairs away from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can constitutively or differentially regulate the expression of gene components in relation to the cell, tissue, or organ in which expression occurs, or in relation to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters that can be operably linked to the nucleic acid encoding the modified single Rep protein or that can be present in the expression cassette of the ceDNA vector produced by the method disclosed herein include, but are not limited to, bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and CMV IE promoter, and the promoters listed below. Such promoters and / or enhancers can be used to express any gene of interest, such as a gene editing molecule, a donor sequence, a therapeutic protein, etc. For example, the vector can include a promoter operably linked to a nucleic acid sequence encoding a therapeutic protein.The promoter operably linked to the therapeutic protein coding sequence can be a promoter derived from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as the long terminal repeat (LTR) promoter of bovine immunodeficiency virus (BIV), a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter from a human gene, such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter can also be a natural or synthetic tissue-specific promoter, such as a liver-specific promoter, such as human α1-antitrypsin (HAAT). In one embodiment, delivery to the liver can be achieved using endogenous ApoE-specific targeting of a composition comprising a ceDNA vector to hepatocytes via the low-density lipoprotein (LDL) receptor present on the surface of hepatocytes.

[0235] In one embodiment, the promoter used is the native promoter of the gene encoding the therapeutic protein.The promoter and other regulatory sequences of each gene encoding the therapeutic protein are known and characterized.The promoter region used may further comprise one or more additional regulatory sequences (e.g., native), such as enhancers (e.g., SEQ ID NO: 22 and SEQ ID NO: 23).

[0236] Non-limiting examples of suitable promoters for use in the expression of modified single Rep proteins or in ceDNA vectors produced by the methods disclosed herein include, for example, the CAG promoter (SEQ ID NO: 3), the hAAT promoter (SEQ ID NO: 21), the human EF1-α promoter (SEQ ID NO: 6) or a fragment of the EF1a promoter (SEQ ID NO: 15), the IE2 promoter (e.g., SEQ ID NO: 20), and the rat EF1-α promoter (SEQ ID NO: 24).

[0237] Polyadenylation Sequence: In some embodiments, a sequence encoding a polyadenylation sequence may be operably linked to a nucleic acid encoding a modified single Rep protein or may be present in a ceDNA vector produced by the methods disclosed herein to stabilize the expressed mRNA and / or assist in nuclear export and translation. In one embodiment, a construct comprising a nucleic acid encoding a modified single Rep protein, or a ceDNA vector produced by the methods disclosed herein, does not comprise a polyadenylation sequence. In alternative embodiments, a construct comprising a nucleic acid encoding a modified single Rep protein, or a ceDNA vector produced by the methods disclosed herein, comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, or more adenine dinucleotides. In some embodiments, the polyadenylation sequence comprises about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range therebetween.

[0238] Constructs containing nucleic acids encoding modified single Rep proteins, or ceDNA vectors produced by the methods disclosed herein, can include polyadenylation sequences known in the art or variations thereof, such as naturally occurring sequences isolated from bovine BGHpA (e.g., SEQ ID NO: 74) or viral SV40pA (e.g., SEQ ID NO: 10), or synthetic sequences (e.g., SEQ ID NO: 27). Some expression cassettes can also include an SV40 late polyA signal upstream enhancer (USE) sequence. In some embodiments, the USE can be used in combination with SV40pA or a heterologous polyA signal.

[0239] The expression cassette may also contain post-transcriptional elements to increase transgene expression. In some embodiments, the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) (e.g., SEQ ID NO: 8) is used to increase transgene expression. Other post-transcriptional processing elements, such as those from the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV), can be used. A secretory sequence can be linked to the transgene, for example, the VH-02 and VK-A26 sequences, e.g., SEQ ID NO: 25 and SEQ ID NO: 26.

[0240] VI. Adjustment switch A molecular regulatory switch generates a measurable change in state in response to a signal. Such a regulatory switch can be usefully combined with a construct comprising a nucleic acid encoding a modified single Rep protein or a ceDNA vector produced by the methods disclosed herein to control the output of the ceDNA vector. In some embodiments, a construct comprising a nucleic acid encoding a modified single Rep protein or a ceDNA vector produced by the methods disclosed herein includes a regulatory switch that serves to fine-tune the expression of the single Rep protein or transgene in the ceDNA vector. For example, it can serve as a biological containment function for the ceDNA vector. In some embodiments, the switch is an "on / off" switch designed to start or stop (i.e., shut down) the controllable and regulatable expression of a gene of interest in the ceDNA vector. In some embodiments, the switch can include a "kill switch" that, once activated, can instruct cells containing the ceDNA vector to undergo programmed cell death.

[0241] A. Binary adjustment switch In some embodiments, the ceDNA vector comprises a regulatory switch that can controllably adjust the expression of the transgene.In such embodiments, the expression cassette located between the ITRs of the ceDNA vector can additionally comprise a regulatory region, such as a promoter, a cis-element, a repressor, an enhancer, etc., operably linked to the gene of interest, and this regulatory region is regulated by one or more cofactors or exogenous agents.Therefore, in one embodiment, the transcription and expression of the gene of interest from the ceDNA vector only occurs when one or more cofactors or exogenous agents are present in the cell.In another embodiment, the transcription and expression of the gene of interest can be derepressed using one or more cofactors or exogenous agents.

[0242] Any nucleic acid regulatory region known by those skilled in the art can be used in a ceDNA vector designed to contain a regulatory switch. By way of example only, the regulatory region can be regulated by a small molecule switch or an inducible or repressible promoter. Non-limiting examples of inducible promoters include hormone-inducible or metal-inducible promoters. Other exemplary inducible promoter / enhancer elements include, but are not limited to, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, and metallothionein promoters. Classical tetracycline-based or other antibiotic-based switches, including those disclosed in (Fussenegger et al., Nature Biotechnol. 18:1203-1208 (2000)), are also contemplated for use.

[0243] B. Small Molecule Regulatory Switches Various small molecule-based regulatory switches known in the art can be combined with the ceDNA vectors known in the art and disclosed herein to form a ceDNA vector controlled by the regulatory switch. In some embodiments, the regulatory switch is an orthogonal ligand / nuclear receptor pair, such as retinoid receptor variants / LG335 and GRQCIMFI (with an artificial promoter controlling the expression of an operably linked transgene, such as that disclosed in Taylor, et al., BMC Biotechnology 10 (2010): 15); an engineered steroid receptor, such as a modified progesterone receptor with a C-terminal truncation that cannot bind progesterone but binds RU486 (mifepristone) (U.S. Patent No. 5,364,791); the ecdysone receptor from Drosophila and its ecdysteroid ligands (Saez, et al., PNAS, 97 (26) (2000), 14512-14517), or Sando R 3 rd; switches controlled by the antibiotic trimethoprim (TMP) as disclosed in Nat Methods. 2013, 10(11):1085-8.

[0244] Other small molecule-based regulatory switches known by those skilled in the art are also contemplated for use in controlling transgene expression of ceDNA, including, but not limited to, those disclosed in Buskirk et al., Cell; Chem and Biol., 2005; 12(2); 151-161; abscisic acid-sensitive ON switches, such as those disclosed in Liang, F.-S., et al., (2011) Science Signaling, 4(164); exogenous L-arginine-sensitive ON switches, such as those disclosed in Hartenbach, et al. Nucleic Acids Research, 35(20), 2007; synthetic bile acid-sensitive ON switches, such as those disclosed in Rossger et al., Metab Eng. 2014, 21:81-90; and biotin-sensitive ON switches, such as those disclosed in Weber et al., Metab. Eng. 2009 Mar; 11(2): 117-124. Dual-input food additive benzoate / vanillin-sensitive regulatory switches, such as those disclosed in Xie et al., Nucleic Acids Research, 2014; 42(14); e116; 4-hydroxytamoxifen-sensitive switches, such as those disclosed in Giuseppe et al., Molecular Therapy, 6(5), 653-663; and flavonoid (phloretin)-sensitive regulatory switches, such as those disclosed in Gitzinger et al., Proc. Natl. Acad. Sci. US A. 2009 Jun 30; 106(26): 10638-10643.

[0245] In some embodiments, the regulatory switch for controlling the transgene expressed by the ceDNA vector is a prodrug activation switch, such as those disclosed in U.S. Pat. Nos. 8,771,679 and 6,339,070.

[0246] Exemplary regulatory switches for use in ceDNA vectors include, but are not limited to, those in Table 11.

[0247] C. "Passcode" adjustment switch In some embodiments, the regulatory switch may be a "passcode switch" or "passcode circuit." Passcode switches allow for fine-tuning of the control of transgene expression from a ceDNA vector when specific conditions occur. That is, a combination of conditions must be present for transgene expression and / or repression to occur. For example, at least conditions A and B must occur for transgene expression to occur. A passcode regulatory switch may be any number of conditions, e.g., at least two, or at least three, or at least four, or at least five, or at least six, or at least seven or more conditions must be present for transgene expression to occur. In some embodiments, at least two conditions (e.g., conditions A and B) must occur, and in some embodiments, at least three conditions (e.g., conditions A, B, and C or A, B, and D) must occur. By way of example only, conditions A, B, and C must be present for gene expression from a ceDNA having a passcode "ABC" regulatory switch to occur. Conditions A, B, and C may be as follows: Condition A is the presence of a pathology or disease, Condition B is a hormonal response, and Condition C is a response to transgene expression. As merely illustrative examples, if the transgene is insulin, condition A occurs when the subject has diabetes, condition B occurs when blood sugar levels are high, and condition C is when endogenous insulin levels are not expressed in the required amount. When sugar levels decrease or a desired level of insulin is achieved, the transgene (e.g., insulin) is turned off again and turned back on until one of the three conditions occurs. In another illustrative example, if the transgene is EPO, condition A is the presence of chronic kidney disease (CKD), condition B occurs when the subject has hypoxia in the kidney, and condition C is when erythropoietin-producing cell (EPC) mobilization in the kidney is insufficient, or alternatively, when HIF-2 activation is insufficient. When oxygen levels increase or a desired level of EPO is achieved, the transgene (e.g., EPO) is turned off again and turned back on until one of the three conditions occurs.

[0248] Passcode regulatory switches are useful for fine-tuning transgene expression from ceDNA vectors. For example, passcode regulatory switches can be modular in that they include multiple switches, such as tissue-specific and inducible promoters that are turned on only in the presence of certain levels of a metabolite. In such embodiments, for transgene expression from the ceDNA vector to occur, an inducer must be present in the desired cell type (condition B) (condition A), and the metabolite must be above or below a certain threshold (condition C). In alternative embodiments, passcode regulatory switches can be designed such that transgene expression is on when conditions A and B are present, but off when condition C is present. Such embodiments are useful when condition C occurs as a direct result of an expressed transgene. That is, condition C acts as a positive feedback loop to turn off transgene expression from the ceDNA vector when the transgene has a sufficient amount of the desired therapeutic effect.

[0249] In some embodiments, the passcode regulatory switch included for use in ceDNA vectors is disclosed in WO2017 / 059245 (incorporated herein by reference in its entirety), which describes a switch referred to as a "passcode switch," or "passcode circuit," or "passcode kill switch," which is a synthetic biological circuit that uses hybrid transcription factors (TFs) to engineer complex environmental requirements for cell survival. The passcode regulatory switch described in WO2017 / 059245 is particularly useful for use in ceDNA vectors because it is modular and customizable, both in terms of the environmental conditions that control circuit activation and the output module that controls cell fate. Furthermore, passcode circuits are particularly useful for use in ceDNA vectors because, without the appropriate "passcode" molecule, transgene expression is only possible when the required predetermined conditions are present. If the cell encounters a problem or if further transgene expression is no longer desired for some reason, an associated kill switch (i.e., a deadman switch) can be triggered.

[0250] In some embodiments, the passcode regulatory switches or "passcode circuits" included for use in ceDNA vectors comprise hybrid transcription factors (TFs) to expand the range and complexity of environmental signals used to define biological containment conditions. Unlike deadman switches, which trigger cell death in the presence of predetermined conditions, "passcode circuits" allow cell survival or transgene expression when a particular "passcode" is present, and can be easily reprogrammed to allow transgene expression and / or cell survival only when a given environmental condition or passcode is present.

[0251] In one embodiment, a "passcode" system for restricting cell growth to the presence of a predetermined set of at least two selected drugs comprises one or more nucleic acid constructs encoding expression modules including: i) a toxin expression module encoding a toxin that is toxic to host cells, wherein the toxin-encoding sequence is operably linked to a promoter P1 that is repressed by the binding of a first hybrid repressor protein, hRP1; and ii) a first hybrid repressor protein expression module encoding a first hybrid repressor protein, hRP1, wherein expression of hRP1 is controlled by an AND gate formed by two hybrid transcription factors, hTF1 and hTF2, whose binding or activity is responsive to drugs A1 and A2, such that both drugs A1 and A2 are required for expression of hRP1; and wherein the absence of either A1 or A2 results in insufficient expression of hRP1 to repress the toxin promoter module P1 and toxin production, resulting in death of the host cell. In this system, the hybrid factors hTF1, hTF2, and hRP1 each contain an environmental sensing module from one transcription factor and a DNA recognition module from a different transcription factor, which makes the binding of their respective pathway regulatory switches sensitive to the presence of environmental substances A1 or A2, different from those to which each subunit normally binds naturally.

[0252] Thus, a ceDNA vector can contain a "passcode regulatory circuit" that requires the presence and / or absence of a specific molecule to activate the output module. In some embodiments, in which a gene encoding a cytotoxin is placed in the output module, this passcode regulatory circuit can be used not only to regulate transgene expression, but also to create a kill switch mechanism in which the circuit kills a cell if the cell behaves in an undesirable manner (e.g., leaves a specific environment defined by the sensor domain or differentiates into another cell type). In one non-limiting example, the modularity of the hybrid transcription factor, circuit architecture, and output module allows the circuit to be reconfigured to sense and respond to other environmental signals and control other functions in the cell in addition to induced cell death, as understood in the art.

[0253] Any and all combinations of the regulatory switches disclosed herein, such as small molecule switches, nucleic acid-based switches, small molecule-nucleic acid hybrid switches, post-transcriptional transgene-regulated switches, post-translational regulated radiation-controlled switches, hypoxia-mediated switches, and other regulatory switches disclosed herein known to those of skill in the art, can be used in the passcode regulatory switches disclosed herein. Regulatory switches encompassed for use are also discussed in the review article Kis et al., JR Soc Interface. 12:20141000 (2015) and summarized in Table 1 of Kis. In some embodiments, regulatory switches for use in the passcode system can be selected from any of the switches in Table 11, or combinations thereof.

[0254] D. Nucleic Acid-Based Regulatory Switches for Controlling Transgene Expression In some embodiments, the regulatory switch for controlling the transgene expressed by ceDNA is based on nucleic acid-based control mechanism.Exemplary nucleic acid control mechanism is known in the art and can be used.For example, such mechanism includes riboswitch, for example, those disclosed in US2009 / 0305253, US2008 / 0269258, US2017 / 0204477, WO2018 / 026762A1, U.S. Patent No. 9,222,093 and European Patent Application No. EP288071 (all of which are incorporated herein by reference in their entirety), and those disclosed in the review by Villa JK et al., Microbiol Spectr.2018 May;6(3) (which are incorporated herein by reference in their entirety). Also included are metabolite-responsive transcriptional biosensors, such as those disclosed in WO2018 / 075486 and WO2017 / 147585 (incorporated herein by reference in their entirety). Other mechanisms known in the art that may be used include silencing a transgene by siRNA or RNAi molecules (e.g., miR, shRNA). For example, a ceDNA vector may contain a regulatory switch encoding an RNAi molecule that is complementary to the transgene expressed by the ceDNA vector. If such an RNAi is expressed even when expressed by the ceDNA vector, the transgene will be silenced by the complementary RNAi molecule; if the RNAi is not expressed when the transgene is expressed by the ceDNA vector, the transgene will not be silenced by the RNAi. Examples of such RNAi molecules that control gene expression or serve as regulatory switches are disclosed in US2017 / 0183664. In some embodiments, the regulatory switch comprises a repressor that blocks expression of the transgene from the ceDNA vector.In some embodiments, the on / off switch is a small transcription activating RNA (STAR)-based switch, such as those disclosed in Chappell J. et al., Nat Chem Biol. 2015 Mar;11(3):214-20; and Chappell et al., Microbiol Spectr. 2018 May;6(3). In some embodiments, the regulatory switch is a toehold switch, such as those disclosed in US2009 / 0191546, US2016 / 0076083, WO2017 / 087530, US2017 / 0204477, WO2017 / 075486, and Green et al, Cell, 2014;159(4);925-939, all of which are incorporated herein by reference in their entireties.

[0255] In some embodiments, the regulatory switch is a tissue-specific self-inactivating regulatory switch, e.g., as disclosed in US2002 / 0022018, whereby the regulatory switch purposefully switches off transgene expression in sites where transgene expression would otherwise be detrimental. In some embodiments, the regulatory switch is a recombinase reversible gene expression system, e.g., as disclosed in US2014 / 0127162 and U.S. Patent No. 8,324,436.

[0256] In some embodiments, the regulatory switch for controlling the transgene or gene of interest that is expressed by ceDNA vector is the hybrid of nucleic acid-based control mechanism and small molecule regulatory system.Such system is well known to those skilled in the art and is intended for use herein.Examples of such regulatory switch include but are not limited to LTRi system or " Lac-Tet-RNAi " system, as disclosed in, for example, US2010 / 0175141 and DEANS T.ET AL., CELL.,2007,130(2);363-372, WO2008 / 051854 and US Patent No. 9,388,425.

[0257] In some embodiments, the regulatory switch for controlling the transgene or gene of interest expressed by the ceDNA vector involves circular permutation, as disclosed in U.S. Patent No. 8,338,138. In such embodiments, the molecular switch is multistable, i.e., can switch between at least two states, or bistable, i.e., can be either "on" or "off," e.g., can emit light or not, can bind or not, can catalyze or not, can transfer electrons or not, etc. In another aspect, the molecular switch uses fusion molecules, so the switch can switch between three or more states. For example, in response to a specific threshold state exhibited by the insert sequence or acceptor sequence, each other sequence in the fusion can exhibit a range of states (e.g., a range of binding activity, a range of enzyme catalysis, etc.). Thus, rather than switching from "on" or "off," the fusion molecule can exhibit a graded response to a stimulus.

[0258] In some embodiments, the nucleic acid-based regulatory switch can be selected from any of the switches in Table 11 or a combination thereof.

[0259] E. Post-transcriptional and post-translational regulatory switches In some embodiments, the regulatory switch for controlling the transgene or gene of interest expressed by ceDNA vector is a post-transcriptional modification system.For example, such regulatory switch can be an aptazyme riboswitch that is sensitive to tetracycline or theophylline, as disclosed in US2018 / 0119156, GB2011 / 07768, WO2001 / 064956A3, EP2707487, and Beilstein et al., ACS Synth.Biol.,2015,4(5),pp 526-534, Zhong et al., Elife.2016 Nov 2;5.pii:e18858.In some embodiments, it is assumed that a person skilled in the art can code both the transgene and the inhibitory siRNA that contains a ligand-sensitive (off-switch) aptamer, and the final result is a ligand-sensitive on-switch.

[0260] In some embodiments, the regulatory switch for controlling the transgene or gene of interest expressed by ceDNA vector is a post-translational modification system.In alternative embodiments, the gene or protein of interest is expressed as a proprotein or preproprotein, or has a signal response element (SRE) or destabilization domain (DD) attached to the expressed protein, thereby preventing correct protein folding and / or activity until post-translational modification occurs.In the case of a destabilization domain (DD) or SRE, the destabilization domain is cleaved after translation in the presence of an exogenous drug or small molecule.Those skilled in the art can utilize the control method disclosed in U.S. Patent No. 8,173,792 and PCT Application No. WO2017 / 180587. Other post-transcriptional regulatory switches envisioned for use in ceDNA vectors to control functional transgene activity are disclosed in Rakhit et al., Chem Biol. 2014;21(9):1238-52 and Navarro et al., ACS Chem Biol. 2016;19;11(8):2101-2104A.

[0261] In some embodiments, the regulatory switch for controlling the transgene or gene of interest expressed by ceDNA vector is a post-translational modification system that incorporates a ligand-sensitive intein into the transgene coding sequence, thereby inhibiting the transgene or expressed protein before splicing.For example, this has been demonstrated using both 4-hydroxytamoxifen and thyroid hormone (see, for example, U.S. Patent Nos. 7,541,450, 9,200,045, 7,192,739, Buskirk, et al., Proc Natl Acad Sci USA.2004 Jul 20;101(29):10505-10510, ACS Synth Biol.2016 Dec 16;5(12):1475-1484, and 2005 Feb;14(2):523-532).In some embodiments, the nucleic acid-based regulatory switch can be selected from any one of the switches in Table 11 or their combinations.

[0262] F. Other Exemplary Adjustment Switches Any known regulatory switch can be used in ceDNA vector to control the gene expression of the transgene expressed by ceDNA vector, including those triggered by environmental changes. Additional examples include, but are not limited to, the BOC method of Suzuki et al., Scientific Reports 8;10051(2018), genetic code expansion and non-physiological amino acid, radiation-controlled or ultrasound-controlled on / off switch (see, for example, Scott S et al., Gene Ther.2000 July;7(13):1121-5, U.S. Patent Nos. 5,612,318, 5,571,797, 5,770,581, 5,817,636, and WO1999 / 025385A1). In some embodiments, the regulatory switch is controlled by an implantable system, e.g., as disclosed in U.S. Pat. No. 7,840,263, US2007 / 0190028A1, and gene expression is controlled by one or more forms of energy, including electromagnetic energy, that activate a promoter operably linked to a transgene in a ceDNA vector.

[0263] In some embodiments, the regulatory switch envisioned for use in the ceDNA vector is a hypoxia-mediated or stress-activated switch, such as those disclosed in WO1999 / 060142A2, U.S. Patent Nos. 5,834,306, 6,218,179, 6,709,858, US2015 / 0322410, Greco et al., (2004) Targeted Cancer Therapies 9, S368, as well as FROG, TOAD, and NRSE elements, and conditionally inducible silence elements (e.g., hypoxia response element (HRE), inflammatory response element (IRE), and shear stress activation element (SSAE) disclosed in U.S. Patent No. 9,394,526). Such embodiments are useful for turning on the expression of transgenes from ceDNA vectors after ischemia or in ischemic tissues and / or tumors.

[0264] In some embodiments, the regulatory switch contemplated for use in the ceDNA vector is an optogenetic (e.g., light-controlled) regulatory switch, such as one of the switches reviewed in Polesskaya et al., BMC Neurosci. 2018;19(Suppl 1):12, which are also contemplated for use herein. In such embodiments, the ceDNA vector may contain a genetic element that is light-sensitive and can regulate transgene expression in response to visible wavelengths (e.g., blue, near-infrared). ceDNA vectors containing optogenetic regulatory switches are useful for expressing transgenes in locations of the body that can receive a light source, such as the skin, eyes, and muscles, and can also be used when the ceDNA vector expresses a transgene in internal organs and tissues where a light signal can be provided by suitable means (e.g., an implantable device as disclosed herein). Such optogenetic regulatory switches include light-responsive elements, or light-induced transcriptional effectors (LITEs) (disclosed, for example, in 2014 / 0287938), the Light-On system (disclosed, for example, in Wang et al., Nat Methods. 2012 Feb 12;9(3):266-9, which has been reported to allow in vivo control of expression of the insulin transgene, the Cry2 / CIB1 system (disclosed, for example, in Kennedy et al., Nature Methods;7,973-975 (2010)), and the FKF1 / GIGANTEA system (disclosed, for example, in Yazawa et al., Nat Biotechnol. 2009 Oct;27(10):941-5).

[0265] G. Kill Switch Another embodiment of the present invention relates to a ceDNA vector comprising a kill switch. The kill switch disclosed herein can cause cells containing the ceDNA vector to be killed or undergo programmed cell death as a means of permanently removing the introduced ceDNA vector from a subject's system. Those skilled in the art will understand that the use of a kill switch in the ceDNA vector of the present invention is typically linked to targeting the ceDNA vector to a limited number of cells that a subject can tolerately lose, or to a cell type (e.g., cancer cells) for which apoptosis is desired. In all aspects, the "kill switch" disclosed herein is designed to result in rapid and robust cell killing of cells containing the ceDNA vector in the absence of an input survival signal or other specified condition. In other words, the kill switch encoded by the ceDNA vector herein can restrict cell survival of cells containing the ceDNA vector to an environment defined by a specific input signal. Such a kill switch serves as a biological containment function when it is desirable to remove the ceDNA vector from a subject or ensure that the encoded transgene is not expressed. Thus, the kill switch is a synthetic biological circuit within the ceDNA vector that links an environmental signal to the conditional survival of cells containing the ceDNA vector. In some embodiments, different ceDNA vectors can be designed with different kill switches, allowing control over which transgene-expressing cells are killed when a cocktail of ceDNA vectors is used.

[0266] In some embodiments, the ceDNA vector may include a kill switch, a modular biological containment circuit. In some embodiments, a kill switch incorporated for use in a ceDNA vector is disclosed in WO 2017 / 059245, which describes a switch called a "deadman kill switch" that includes a mutually inhibitory arrangement of at least two repressible sequences such that an environmental signal represses the activity of a second molecule in the construct (e.g., using a small molecule-binding transcription factor to create a "survival" state due to the suppression of toxin production). In cells containing a ceDNA vector containing a deadman kill switch, loss of the environmental signal permanently switches the circuit to a "dead" state, derepressing the toxin, resulting in the production of the toxin and killing the cell. In another embodiment, a synthetic biological circuit called a "passcode circuit" or "passcode kill switch" is provided that uses hybrid transcription factors (TFs) to construct complex environmental requirements for cell survival. The deadman and passcode kill switches described in WO2017 / 059245 are particularly useful for use with ceDNA vectors because they are modular and customizable, both in terms of the environmental conditions that control circuit activation and the output modules that control cell fate. With the appropriate selection of toxins, including but not limited to endonucleases, such as EcoRI, the passcode circuits present in ceDNA vectors can be used to not only kill host cells containing the ceDNA vector, but also degrade their genomes and accompanying plasmids.

[0267] Other kill switches known to those of skill in the art are encompassed for use in the ceDNA vectors as disclosed herein, e.g., those disclosed in US2010 / 0175141, US2013 / 0009799, US2011 / 0172826, US2013 / 0109568, as well as the kill switches disclosed in Jusiak et al, Reviews in Cell Biology and molecular Medicine;2014;1-56, Kobayashi et al., PNAS,2004;101;8419-9, Marchisio et al., Int. Journal of Biochem and Cell Biol.,2011;43;310-319, and Reinshagen et al., Science Translational Medicine,2018,11.

[0268] Thus, in some embodiments, the ceDNA vector can include a kill-switch nucleic acid construct comprising a nucleic acid encoding an effector toxin or reporter protein, where expression of the effector toxin (e.g., death protein) or reporter protein is controlled by a predetermined condition. For example, the predetermined condition can be the presence of an environmental substance, e.g., an exogenous substance, without which the cell will by default express the effector toxin (e.g., death protein) and be killed. In an alternative embodiment, the predetermined condition is the presence of two or more environmental substances, e.g., the cell will survive only if two or more necessary exogenous substances are provided, and in the absence of either, the cell containing the ceDNA vector will be killed.

[0269] In some embodiments, the ceDNA vector is modified to incorporate a kill switch that destroys cells containing the ceDNA vector, effectively terminating the in vivo expression of the transgene expressed by the ceDNA vector (e.g., a therapeutic gene, protein, or peptide). Specifically, the ceDNA vector is further engineered to express a switch protein that does not function in mammalian cells under normal physiological conditions. Only upon administration of a drug or environmental condition that specifically targets this switch protein are cells expressing the switch protein destroyed, thereby terminating the expression of the therapeutic protein or peptide. For example, it has been reported that cells expressing HSV-thymidine kinase can be killed by administering drugs such as ganciclovir and cytosine deaminase. See, for example, Dey and Evans, "Suicide Gene Therapy by Herpes Simplex Virus-1 Thymidine Kinase (HSV-TK)," in "Targets in Gene Therapy," edited by You (2011), and Beltinger et al., "Proc. Natl. Acad. Sci. USA 96(15):8699-8704 (1999)." In some embodiments, the ceDNA vector can include an siRNA kill switch called DISE (death by excision of survival genes) (Murmann et al., Oncotarget. 2017;8:84643-84658. Induction of DISE in ovarian cancer cells in vivo).

[0270] In some embodiments, a deadman kill switch is a biological circuit or system that sensitizes a cellular response to a predetermined condition, such as the absence of an exogenous agent, in the cell's growth environment. Such a circuit or system can include a nucleic acid construct that includes an expression module that forms a deadman regulatory circuit that is sensitive to the predetermined condition. The construct can include an expression module that forms a regulatory circuit, and the construct can include: i) a first repressor protein expression module, wherein the first repressor protein binds to a first repressor protein nucleic acid binding element and represses transcription from a coding sequence comprising the first repressor protein binding element, and wherein the repression activity of the first repressor protein is sensitive to inhibition by a first exogenous agent, the presence or absence of the first exogenous agent establishing a predetermined condition; ii) a second repressor protein expression module, wherein the second repressor protein binds to a second repressor protein nucleic acid binding element and represses transcription from a coding sequence that includes the second repressor protein binding element, and the second repressor protein is different from the first repressor protein; and iii) an effector expression module comprising a nucleic acid sequence encoding an effector protein operably linked to a genetic element comprising a binding element for a second repressor protein, such that expression of the second repressor protein causes repression of effector expression from the effector, wherein the second expression module comprises a first repressor protein nucleic acid binding element that enables repression of transcription of the second repressor protein when the element is bound by the first repressor protein, and wherein in the absence of the first exogenous agent, the first repressor protein is produced from the first repressor protein expression module and the second repressor an effector expression module, each of which forms a regulatory circuit to repress transcription from a protein expression module, thereby relieving repression of effector expression by a second repressor protein and resulting in expression of the effector protein; however, in the presence of a first exogenous agent, activity of the first repressor protein is inhibited, allowing expression of the second repressor protein and maintaining expression of the effector protein expression in an "off" state; thereby, the first exogenous agent is required in the circuit to maintain expression of the effector protein in an "off" state; and removal or absence of the first exogenous agent does not result in expression of the effector protein.

[0271] In some embodiments, the effector is a toxin or protein that induces a cell death program. Any protein that is toxic to host cells can be used. In some embodiments, the toxin kills only those cells in which it is expressed. In other embodiments, the toxin kills other cells of the same host organism. Any of a number of products that lead to cell death can be used in a dead-man kill switch. Agents that inhibit DNA replication, protein translation, or other processes, or that degrade host cell nucleic acids, for example, are particularly useful. To identify an efficient mechanism for killing host cells upon activation of the circuit, several toxin genes that directly damage host cell DNA or RNA were tested. The endonuclease ecoRI21 , DNA gyrase ccdB 22 , and the ribonuclease-type toxin MazF 23 These were tested because they are well characterized, native to E. coli, and offer a range of killing mechanisms. To increase circuit robustness and provide independent methods of circuit-dependent cell death, the system can be further adapted to express, for example, targeted proteases or nucleases that further interfere with the repressor that maintains the death gene in an "off" state. Loss or withdrawal of the survival signal would more efficiently remove repression of the death gene, for example, by active degradation of the repressor protein or its message. As a non-limiting example, mf-Lon protease was used to target essential proteins for degradation in addition to degrading LacI. The mf-Lon degradation tag pdt#1 was used to identify proteins whose protein products are affected by mf-Lon degradation. 20 We were able to add a nucleotide sequence to the 3' end of five essential genes that are particularly sensitive to ATc, and measured cell viability after removal of ATc. Among the essential gene targets tested, the peptidoglycan biosynthesis gene murC provided the strongest and fastest cell death phenotype (viability <1 x 10 within 6 hours). -4 ).

[0272] As used herein, the term "predetermined input" refers to an agent or condition that affects the activity of a transcription factor polypeptide in a known manner. Generally, such agents can bind to and / or alter the conformation of a transcription factor polypeptide, thereby altering the activity of the transcription factor polypeptide. Examples of predetermined inputs include, but are not limited to, environmental input agents that are not required for the survival of a given host organism (i.e., in the absence of the synthetic biology circuit described herein). Conditions under which predetermined inputs can be provided include temperature (e.g., when the activity of one or more factors is temperature-sensitive), the presence or absence of light (including light of a given wavelength spectrum), and concentrations of gases, salts, metals, or minerals. Environmental input agents include small molecules, biological agents (such as pheromones, hormones, growth factors, metabolites, nutrients, etc.) and their analogs, chemicals, environmental by-products, concentrations of metal ions and other such molecules or agents, light levels, temperature, mechanical stress or pressure, or electrical signals (such as current and voltage).

[0273] In some embodiments, a reporter is used to quantify the strength or activity of a signal received by a module or programmable synthetic biology circuit of the invention. In some embodiments, the reporter may be fused in-frame to other protein-coding sequences to identify where the protein is located in a cell or organism. Luciferase can be used as an effector protein in various embodiments described herein, for example, to measure low levels of gene expression, since cells tend to have little or no background luminescence in the absence of luciferase. In other embodiments, enzymes that produce colored substrates can be quantified using a spectrophotometer or other instrument capable of measuring absorbance, including a plate reader. Similar to luciferase, enzymes such as β-galactosidase tend to amplify low signals and can therefore be used to measure low levels of gene expression. In some embodiments, the effector protein can be an enzyme that can degrade or otherwise destroy a given toxin. In some embodiments, the effector protein can be an odorant enzyme that converts a substrate into an odorant product. In some embodiments, the effector protein can be an enzyme that phosphorylates or dephosphorylates either a small molecule or other proteins, or an enzyme that methylates or demethylates other proteins or DNA.

[0274] In some embodiments, the effector protein can be a receptor, a ligand, or a soluble protein. Receptors tend to have three domains: an extracellular domain for binding a ligand such as a protein, peptide, or small molecule, a transmembrane domain, and an intracellular or cytoplasmic domain that can frequently be involved in certain signaling events such as phosphorylation. In some embodiments, transporter, channel, or pump gene sequences are used as effector proteins. Non-limiting examples and sequences of effector proteins for use with the kill switches described herein can be found on the World Wide Web at the Registry of Standard Biological Parts at parts.igem.org.

[0275] As used herein, a "modulator protein" is a protein that regulates expression from a target nucleic acid sequence. Modulator proteins include, for example, transcription factors, including transcription activators and repressors, among others, as well as proteins that bind to or modify transcription factors to affect their activity. In some embodiments, modulator proteins include, for example, proteases that degrade protein factors involved in regulating expression from a target nucleic acid sequence. Preferred modulator proteins include, for example, modulator proteins in which the DNA-binding and input agent-binding or response elements or domains are separable and transferable, such that, for example, fusing the DNA-binding domain of a first modulator protein to the input agent-responsive domain of a second modulator protein results in a new protein that binds the DNA sequence recognized by the first protein but is sensitive to the input agent to which the second protein normally responds. Thus, as used herein, the terms "modulator polypeptide," and more specifically, "repressor polypeptide," include, in addition to specific polypeptides, for example, "Lacl (repressor) polypeptides," variants, or derivatives of such polypeptides that respond to different or variant input agents. Thus, in the case of LacI polypeptides, LacI mutants or variants that bind agents other than lactose or IPTG are included. A wide variety of such agents are known in the art. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6]

[0276] VII. Pharmaceutical Compositions In another aspect, a pharmaceutical composition is provided. Typically, the pharmaceutical composition comprises a ceDNA vector disclosed herein and a pharmaceutically acceptable carrier or diluent.

[0277] The DNA vectors disclosed herein can be incorporated into pharmaceutical compositions suitable for administration to a subject for in vivo delivery to the subject's cells, tissues, or organs. Typically, the pharmaceutical composition comprises the ceDNA vectors disclosed herein and a pharmaceutically acceptable carrier. For example, the ceDNA vectors described herein can be incorporated into pharmaceutical compositions suitable for the desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transduction via high-pressure intravenous or intraarterial injection, as well as intracellular injection, such as intranuclear microinjection or intracytoplasmic injection, are also contemplated. Pharmaceutical compositions for therapeutic purposes can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA vector concentrations. A sterile injectable solution can be prepared by combining the required amount of ceDNA vector compounds in an appropriate buffer with one or a combination of the above-listed components and sterilizing by filtration.

[0278] The ceDNA vector-containing pharmaceutically active composition can be formulated to deliver the transgene in the nucleic acid to the recipient's cells, resulting in therapeutic expression of the transgene therein. The composition can also include a pharmaceutically acceptable carrier.

[0279] The ceDNA vectors disclosed herein can be incorporated into pharmaceutical compositions suitable for local, systemic, intra-amniotic, intrathecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intratissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (e.g., extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, intrainterstitial, intracameral, and intravitreal), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via high-pressure intravenous or intra-arterial injection, as well as intracellular injection, e.g., intranuclear microinjection or intracytoplasmic injection, are also contemplated.

[0280] Pharmaceutical compositions for therapeutic purposes must typically be sterile and stable under the conditions of manufacture and storage.Compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA vector concentrations.Sterile injectable solutions can be prepared by incorporating the required amount of ceDNA vector compounds in a suitable buffer with one or a combination of the above-listed components, and then filtering and sterilizing.

[0281] Various techniques and methods for delivering nucleic acid to cells are known in the art.For example, nucleic acid such as ceDNA can be formulated in lipid nanoparticle (LNP), lipidoid, liposome, lipid nanoparticle, lipoplex or core-shell nanoparticle.Typically, LNP is composed of nucleic acid (for example, ceDNA) molecule, one or more ionized or cationic lipid (or their salt), one or more non-ionic or neutral lipid (for example, phospholipid), aggregation prevention molecule (for example, PEG or PEG-lipid complex), and optionally sterol (for example, cholesterol).

[0282] Another method for delivering nucleic acid such as ceDNA into cells is by complexing nucleic acid with a ligand that can be internalized by cells.For example, the ligand can bind to a receptor on the cell surface and be internalized through endocytosis.The ligand can be covalently bound to the nucleotide in nucleic acid.Exemplary complexes for delivering nucleic acid into cells are described in, for example, WO2015 / 006740, WO2014 / 025805, WO2012 / 037254, WO2009 / 082606, WO2009 / 073809, WO2009 / 018332, WO2006 / 112872, WO2004 / 090108, WO2004 / 091515 and WO2017 / 177326.

[0283] Nucleic acids such as ceDNA can also be delivered to cells by transfection. Useful transfection methods include, but are not limited to, lipid-mediated transfection, cationic polymer-mediated transfection, or calcium phosphate precipitation. Transfection reagents are well known in the art and include TurboFect transfection reagent (Thermo Fisher Scientific), Pro-Ject reagent (Thermo Fisher Scientific), TRANSPASS™ P protein transfection reagent (New England Biolabs), CHARIOT™ protein delivery reagent (Active Motif), PROTEOJUICE™ protein transfection reagent (EMD Millipore), 293fectin, LIPOFECTAMINE™ 2000, LIPOFECTAMINE™ 3000 (Thermo Fisher Scientific), LIPOFECTAMINE™ (Thermo Fisher Scientific), LIPOFECTIN™ (Thermo Fisher Scientific), DMRIE-C, CELLFECTIN™ (Thermo Fisher Scientific), OLIGOFECTAMINE™ (Thermo Fisher Scientific), and LIPOFECTAMINE™ (Thermo Fisher Scientific). Scientific), LIPOFECTACE(TM), FUGENE(TM)(Roche, Basel, Switzerland), FUGENE(TM) HD(Roche), TRANSFECTAM(TM)(Transfectam, Promega, Madison, Wis.), TFX-10(TM)(Pr omega), TFX-20(TM)(Promega), TFX-50(TM)(Promega), TRANSFECTIN(TM)(BioRad, Hercules, Calif.), SILENTFECT(TM)(Bio-Rad), Effectene(TM)(Qiagen, Valencia, Calif.).), DC-chol (Avanti Polar Lipids), GENEPORTER™ (Gene Therapy Systems, San Diego, Calif.), DHARMAFECT 1™ (Dharmacon, Lafayette, Colo.), DHARMAFECT 2™ (Dharmacon), DHARMAFECT 3™ (Dharmacon), DHARMAFECT 4™ (Dharmacon), ESCORT™ III (Sigma, St. Louis, Mo.), and ESCORT™ IV (Sigma Chemical Co.). Nucleic acids, such as cDNA, can also be delivered to cells via microfluidics methods known to those skilled in the art.

[0284] Non-viral methods for in vivo or ex vivo delivery of nucleic acids include electroporation, lipofection (U.S. Pat. Nos. 5,049,386, 4,946,787, and commercially available reagents such as Transfectam™ and Lipofectin™), microinjection, biolistics, virosomes, liposomes (e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820(1992), U.S. Patent No. 4,186,183, U.S. Patent No. 4,217,344, U.S. Patent No. 4,235,871, U.S. Patent No. 4,261,975, U.S. Patent No. 4,485,054, U.S. Patent No. 4,501,728, U.S. Patent No. 4,774,085, U.S. Patent No. 4,837,028 and U.S. Patent No. 4,946,787), immunoliposome, polycation or lipid: nucleic acid complex, naked DNA and the drug-enhanced uptake of DNA.For example, sonoporation using Sonitron 2000 system (Rich-Mar) can also be used for the delivery of nucleic acid.

[0285] The ceDNA vector described herein can also be directly administered to organism for in vivo cell transduction.Administered by any of the routes that are usually used to finally bring molecules into contact with blood or tissue cells, including but not limited to infusion, injection, topical application and electroporation.Suitable methods for administering such nucleic acid are available and well known to those skilled in the art, and two or more routes can be used to administer certain compositions, although certain routes can often bring about more immediate and effective responses than other routes.

[0286] The nucleic acid vectors disclosed herein can be delivered to, for example, hematopoietic stem cells by the ceDNA vector introduction method, for example, by the method described in US Pat. No. 5,928,638.

[0287] The ceDNA vector of the present invention can be added to liposomes for delivery to cells or target organs in a subject. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic agent delivery in the context of pharmaceutical development. They act by fusing with cell membranes and rearranging their lipid structure to deliver drugs or active pharmaceutical ingredients (APIs). Liposome compositions for such delivery are composed of compounds with phospholipids, particularly phosphatidylcholine, although these compositions may also contain other lipids.

[0288] In some embodiments, the present disclosure provides liposomal formulations containing one or more compounds with polyethylene glycol (PEG) functional groups (so-called "PEGylated compounds"), which can reduce immunogenicity / antigenicity, provide hydrophilic and hydrophobic properties to the compounds, and reduce dosing frequency. Alternatively, the liposomal formulations simply contain polyethylene glycol (PEG) polymers as additional components. In such embodiments, the molecular weight of the PEG or PEG functional group can be between 62 Da and about 5,000 Da.

[0289] In some embodiments, the present disclosure provides liposomal formulations that will deliver APIs with extended- or controlled-release profiles over a period of hours to weeks. In some related embodiments, the liposomal formulations may include aqueous chambers bounded by a lipid bilayer. In other related embodiments, the liposomal formulations encapsulate APIs with components that undergo a physical transition at elevated temperatures, releasing the API over a period of hours to weeks.

[0290] In some embodiments, the liposomal formulation comprises sphingomyelin and one or more lipids disclosed herein. In some embodiments, the liposomal formulation comprises an Optisome.

[0291] In some aspects, the present disclosure provides a method for preparing a lipid-rich lipid complex comprising administering to a subject a lipid-rich lipid complex comprising: N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, (distearoyl-sn-glycero-phosphoethanolamine), MPEG (methoxypolyethylene glycol) conjugated lipid, HSPC (hydrogenated soy phosphatidylcholine); PEG (polyethylene glycol); DSPE (distearoyl-sn-glycero-phosphoethanolamine); DSPC (distearoylphosphatidylcholine); DOPC (dioleoylphosphatidylcholine); DPPG (dipalmitoylphosphatidylglycerol); EPC (egg phosphatidylcholine); DOPS (dioleoylphosphatidylcholine); dioleoyl-sn-glycero-phosphoethanolamine), cholesteryl sulfate (CS), dipalmitoylphosphatidylglycerol (DPPG), DOPC (dioleoyl-sn-glycero-phosphoethanolamine), or any combination thereof.

[0292] In some embodiments, the present disclosure provides a liposome formulation comprising a phospholipid, cholesterol, and a PEGylated lipid in a molar ratio of 56:38:5. In some embodiments, the total lipid content of the liposome formulation is 2-16 mg / mL. In some embodiments, the present disclosure provides a liposome formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group, and a PEGylated lipid. In some embodiments, the present disclosure provides a liposome formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group, and a PEGylated lipid in a molar ratio of 3:0.015:2, respectively. In some embodiments, the present disclosure provides a liposome formulation comprising a lipid containing a phosphatidylcholine functional group, cholesterol, and a PEGylated lipid. In some embodiments, the present disclosure provides a liposome formulation comprising a lipid containing a phosphatidylcholine functional group and cholesterol. In some embodiments, the PEGylated lipid is PEG-2000-DSPE. In some embodiments, the present disclosure provides a liposome formulation comprising DPPG, soy PC, an MPEG-DSPE lipid conjugate, and cholesterol.

[0293] In some embodiments, the present disclosure provides a liposome formulation comprising one or more lipids containing a phosphatidylcholine functional group and one or more lipids containing an ethanolamine functional group. In some embodiments, the present disclosure provides a liposome formulation comprising one or more lipids containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group, and a sterol, such as cholesterol. In some embodiments, the liposome formulation comprises DOPC / DEPC and DOPE.

[0294] In some embodiments, the present disclosure provides liposome formulations that further comprise one or more pharmaceutical excipients, for example, sucrose and / or glycine.

[0295] In some embodiments, the present disclosure provides liposome formulations that have either a unilamellar or multilamellar structure. In some embodiments, the present disclosure provides liposome formulations that include multivesicular particles and / or effervescent particles. In some embodiments, the present disclosure provides liposome formulations that are larger in size relative to typical nanoparticles, approximately 150-250 nm in size. In some embodiments, the liposome formulation is a lyophilized powder.

[0296] In some embodiments, the present disclosure provides liposomal formulations prepared and loaded with the ceDNA vectors disclosed or described herein by adding a weak base to a mixture containing isolated ceDNA outside the liposome. This addition increases the pH outside the liposome to approximately 7.3, allowing the API to enter the liposome. In some embodiments, the present disclosure provides liposomal formulations having an acidic pH inside the liposome. In such cases, the inside of the liposome may be pH 4 to 6.9, more preferably pH 6.5. In other embodiments, the present disclosure provides liposomal formulations prepared using intraliposomal drug stabilization techniques. In such cases, a polymeric or non-polymeric highly charged anion and an intraliposomal entrapment agent, such as polyphosphate or sucrose octasulfate, are utilized.

[0297] In another aspect, the present disclosure provides a liposome formulation comprising a phospholipid, a lecithin, a phosphatidylcholine, and a phosphatidylethanolamine.

[0298] For example, delivery reagents such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., can be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, nucleic acids can be formulated for delivery or encapsulated in lipid particles, liposomes, vesicles, nanoparticles, gold particles, etc. Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids disclosed herein.

[0299] ceDNA vector can be delivered in vitro or in vivo using various delivery methods known in the art or their modifications.For example, in some embodiments, ceDNA vector is delivered by creating temporary punctures in cell membrane by mechanical, electrical, ultrasonic, hydrodynamic or laser-based energy, thereby facilitating DNA entry into target cells.For example, ceDNA vector can be delivered by squeezing cells through size-restricted channels or by other means known in the art to temporarily disrupt cell membrane.In some cases, ceDNA vector is directly injected into skin, thyroid, cardiac muscle, skeletal muscle or liver cells as naked DNA alone.

[0300] In some cases, cDNA vectors are delivered by gene gun: gold or tungsten spherical particles (1–3 μm diameter) coated with capsid-free AAV vectors are accelerated to high velocities by pressurized gas and can penetrate into target tissue cells.

[0301] In some embodiments, electroporation is used to deliver ceDNA vector.Electroporation causes the temporary destabilization of cell membrane target cell tissue by inserting electrode pair into tissue, so that the DNA molecules in the surrounding medium of destabilized membrane can penetrate into the cytoplasm and nucleoplasm of cell.Electroporation has been used in many tissue types in vivo, such as skin, lung and muscle.

[0302] In some cases, ceDNA vectors are delivered by hydrodynamic injection, a simple and highly efficient method for the direct intracellular delivery of any water-soluble compound and particle to skeletal muscles throughout the viscera and limbs.

[0303] In some cases, ceDNA vectors are delivered by ultrasound, creating nanoscopic pores in the membrane to facilitate intracellular delivery of DNA particles to organ or tumor cells, so the size and concentration of the plasmid DNA play a major role in the efficiency of this system. In some cases, ceDNA vectors are delivered by magnetofection, using a magnetic field to concentrate nucleic acid-containing particles in target cells.

[0304] In some cases, chemical delivery systems can be used, for example, by using nanomer complexes, including the compression of negatively charged nucleic acids with cationic liposomes / micelles or polycationic nanomer particles belonging to cationic polymers.Cationic lipids used in delivery methods include, but are not limited to, monovalent cationic lipids, polyvalent cationic lipids, guanidine-containing compounds, cholesterol-derivative compounds, cationic polymers (e.g., poly(ethyleneimine), poly-L-lysine, protamine, other cationic polymers), and lipid-polymer hybrids.

[0305] A. Exosomes: In some embodiments, the ceDNA vectors disclosed herein are delivered by packaging them into exosomes. Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane. Their surface consists of a lipid bilayer from the plasma membrane of the donor cell; they contain cytosol from the cell that produced the exosome and exhibit membrane proteins from the parent cell on their surface. Exosomes are produced by various cell types, including epithelial cells, B and T lymphocytes, mast cells (MCs), and dendritic cells (DCs). In some embodiments, exosomes with diameters of 10 nm to 1 μm, 20 nm to 500 nm, 30 nm to 250 nm, or 50 nm to 100 nm are contemplated for use. Exosomes can be isolated for delivery to target cells either by using their donor cells or by introducing specific nucleic acids into them. Various approaches known in the art can be used to produce exosomes containing the capsid-free AAV vectors of the invention.

[0306] B. Microparticles / Nanoparticles: In some embodiments, the ceDNA vectors disclosed herein are delivered by lipid nanoparticles. Generally, lipid nanoparticles comprise an ionizable amino lipid (e.g., heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA, phosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol, and a coating lipid (polyethylene glycol-dimyristolglycerol, PEG-DMG), as disclosed, for example, by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3):498-507.

[0307] In some embodiments, the lipid nanoparticles have an average diameter of about 10 to about 1000 nm. In some embodiments, the lipid nanoparticles have a diameter of less than 300 nm. In some embodiments, the lipid nanoparticles have a diameter of about 10 to about 300 nm. In some embodiments, the lipid nanoparticles have a diameter of less than 200 nm. In some embodiments, the lipid nanoparticles have a diameter of about 25 to about 200 nm. In some embodiments, the lipid nanoparticle preparation (e.g., a composition comprising a plurality of lipid nanoparticles) has a size distribution, with an a...

Claims

1. A DNA vector obtained from a vector polynucleotide, said vector polynucleotide encoding a heterologous nucleic acid sequence spaced between a first inverted terminal repeat (ITR) and a second ITR, at least one of said first ITR and said second ITR comprising a nucleic acid sequence corresponding to a replication (Rep) protein binding site for inducing replication of said DNA vector in a cell in the presence of a single species of Rep protein having at least DNA binding and DNA nicking functionality, said single species of Rep protein being an AAV Rep78 protein or an AAV Rep68 protein, said DNA vector comprising: incubating a population of cells harboring the vector polynucleotide lacking a viral capsid coding sequence in the presence of the single species of replication (Rep) protein under conditions effective and for a time sufficient to induce production of the DNA vector in the cells, wherein the cells do not contain a viral capsid coding sequence, other species of Rep protein are absent, and the cells have not been contacted with a nucleic acid sequence encoding a second Rep protein; b. harvesting and isolating the resulting DNA vector from said cell; A DNA vector, wherein the cell does not express Rep52 or Rep40.

2. said single Rep protein further possesses helicase, ligase and ATPase functionalities; and / or the single species of AAV Rep protein is selected from the group consisting of an AAV1 Rep protein, an AAV2 Rep protein, an AAV3 Rep protein, an AAV4 Rep protein, an AAV5 Rep protein, an AAV6 Rep protein, an AAV7 Rep protein, an AAV8 Rep protein, an AAV9 Rep protein, an AAV10 Rep protein, an AAV11 Rep protein, and an AAV12 Rep protein; The DNA vector of claim 1.

3. The DNA vector of claim 1 , wherein the AAV Rep68 protein is an AAV2 Rep68 protein.

4. The DNA vector of claim 1 , wherein the AAV Rep78 protein is an AAV2 Rep78 protein.

5. the AAV Rep78 protein is encoded by a mutated Rep78 nucleic acid sequence, the mutated Rep78 nucleic acid sequence lacking a functional translation initiation codon for Rep52; said mutated Rep78 nucleic acid sequence encodes a mutated Rep78 protein comprising a mutation at amino acid position 225 of SEQ ID NO: 588; and / or The mutant Rep78 nucleic acid sequence comprises a sequence having at least 95% sequence identity to SEQ ID NO: 588 and encodes a mutant Rep78 protein having at least DNA binding and DNA nicking functionality; The DNA vector of claim 1.

6. The DNA vector of claim 5, wherein amino acid position 225 of SEQ ID NO:588 is mutated to glycine (Gly) or threonine (Thr).

7. 2. The DNA vector of claim 1, wherein the first ITR and the second ITR are each derived from a virus selected from the group consisting of a parvovirus, a dependovirus, and an adeno-associated virus (AAV).

8. 8. The DNA vector of claim 7, wherein the AAV is an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

9. The DNA vector of claim 1, wherein the DNA vector is a non-viral capsid-free double-stranded DNA vector with covalently closed ends (ceDNA vector).

10. A DNA vector derived from a vector polynucleotide, said vector polynucleotide encoding a heterologous nucleic acid sequence disposed between two different inverted terminal repeats (ITRs), at least one of said two different ITRs being a functional ITR comprising a functional terminal resolution site and a replication (Rep) protein binding site that induces replication of said vector polynucleotide in a cell and production of said DNA vector in the presence of a single species of Rep protein having at least DNA binding and DNA nicking functionalities, said single species of Rep protein being an AAV Rep78 protein or an AAV Rep68 protein, and said DNA vector comprising: a. incubating a population of cells harboring the vector polynucleotide lacking a viral capsid coding sequence in the presence of the single species of Rep protein under conditions effective and for a time sufficient to induce production of the DNA vector in the cells, wherein the cells do not contain any nucleic acid encoding Rep52 or Rep40 within the cells, no other species of Rep protein is present in the cells, and the cells have not been contacted with a nucleic acid sequence encoding a second Rep protein; b. harvesting and isolating said DNA vector from said cell; A DNA vector, wherein the cell does not express Rep52 or Rep40.

11. 1. A polynucleotide for generating a DNA vector comprising a nucleic acid sequence encoding a single species of replication (Rep) protein amino acid sequence having at least DNA binding and DNA nicking functionalities linked to at least one expression control sequence, wherein the single species of Rep protein is an AAV Rep78 protein or an AAV Rep68 protein, and the DNA vector is generated in a cell in which no other species of Rep protein is present, and the cell is not contacted with a nucleic acid sequence encoding a second Rep protein; The polynucleotide, wherein the cell does not express Rep52 or Rep40.

12. said single Rep protein has helicase, ligase and ATPase functionalities; and / or the single species of AAV Rep protein is selected from the group consisting of an AAV1 Rep protein, an AAV2 Rep protein, an AAV3 Rep protein, an AAV4 Rep protein, an AAV5 Rep protein, an AAV6 Rep protein, an AAV7 Rep protein, an AAV8 Rep protein, an AAV9 Rep protein, an AAV10 Rep protein, an AAV11 Rep protein, and an AAV12 Rep protein; The polynucleotide of claim 11.

13. The polynucleotide of claim 11 , wherein the AAV Rep78 protein is an AAV2 Rep78 protein.

14. The polynucleotide of claim 11 , wherein the AAV Rep68 protein is an AAV2 Rep68 protein.

15. the AAV Rep78 protein is encoded by a mutated Rep78 nucleic acid sequence, the mutated Rep78 nucleic acid sequence lacking a functional start codon for Rep52; said mutated Rep78 nucleic acid sequence encodes a mutated Rep78 protein comprising a mutation at amino acid position 225 of SEQ ID NO: 588; and / or The mutant Rep78 nucleic acid sequence comprises a sequence having at least 95% sequence identity to SEQ ID NO: 588 and encodes a mutant Rep78 protein having at least DNA binding and DNA nicking functionality; The polynucleotide of claim 11.

16. The polynucleotide of claim 11, wherein the DNA vector is a non-viral capsid-free double-stranded DNA vector with covalently closed ends (ceDNA vector).

17. 1. A method for producing a DNA vector, comprising: (a) a cell; (1) A nucleic acid sequence encoding a single AAV replication (Rep) protein having at least DNA binding and DNA nicking functionality, said single Rep protein being an AAV Rep78 protein or an AAV Rep68 protein, said nucleic acid sequence being linked to at least one expression control sequence, said cell not expressing or being in contact with any other Rep protein, and said cell not expressing Rep52 or Rep40; and (2) A double-stranded DNA construct comprising: An expression cassette; a first ITR upstream (5' end) of the expression cassette; a second ITR downstream (3' end) of the expression cassette; (b) harvesting the DNA vector.

18. the single species of AAV Rep protein is selected from the group consisting of an AAV1 Rep protein, an AAV2 Rep protein, an AAV3 Rep protein, an AAV4 Rep protein, an AAV5 Rep protein, an AAV6 Rep protein, an AAV7 Rep protein, an AAV8 Rep protein, an AAV9 Rep protein, an AAV10 Rep protein, an AAV11 Rep protein, and an AAV12 Rep protein; 20. The method of claim 17.

19. 18. The method of claim 17, wherein the AAV Rep68 protein is an AAV2 Rep68 protein.

20. 18. The method of claim 17, wherein the AAV Rep78 protein is an AAV2 Rep78 protein.

21. 18. The method of claim 17, wherein the at least one expression control sequence encodes a promoter selected from the group consisting of an IE promoter, a ΔIE promoter, and a CMV promoter.

22. 18. The method of claim 17, wherein the double-stranded DNA construct is selected from the group consisting of a bacmid, a plasmid, a minicircle, and a linear double-stranded DNA molecule.

23. The first ITR is a wild-type ITR or a modified ITR; The second ITR is a wild-type ITR or a modified ITR. The first ITR and the second ITR are (i) are symmetrical with respect to each other; (ii) have reverse complements of each other over their entire length; or (iii) are asymmetric with respect to each other; the first ITR and the second ITR are replicable; At least one of the first and second ITRs comprises a nucleic acid sequence corresponding to a Rep protein binding site that directs replication of the DNA vector in the cell; and / or the first ITR and the second ITR are AAV ITRs; 20. The method of claim 17.

24. The wild-type ITR comprises SEQ ID NO:

51. The modified ITR comprises SEQ ID NO:

2. the modified ITR comprises SEQ ID NO:52, and / or The wild-type ITR comprises SEQ ID NO:

1.

24. The method of claim 23.

25. 18. The method of claim 17, wherein the expression cassette comprises a cis-regulatory element and / or a promoter selected from the group consisting of a CAG promoter, an AAT promoter, an LP1 promoter, and an EF1a promoter.

26. 26. The method of claim 25, wherein the cis-regulatory element is selected from the group consisting of a post-transcriptional regulatory element and a BGH polyA signal.

27. 27. The method of claim 26, wherein the post-transcriptional regulatory element comprises a WHP post-transcriptional regulatory element (WPRE).

28. 18. The method of claim 17, wherein the expression cassette comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:

9.

29. 20. The method of claim 17, wherein the expression cassette further comprises an exogenous sequence.

30. The exogenous sequence is Contains at least 2000 nucleotides; Encodes a protein; and / or encoding a reporter protein, a therapeutic protein, an antigen, a gene editing protein, or a cytotoxic protein; 30. The method of claim 29.

31. The method of claim 17 , wherein the DNA vector has a linear and contiguous structure.

32. 20. A DNA vector produced by the method of claim 17.

33. A pharmaceutical composition comprising the DNA vector of claim 32.

34. A kit for producing a DNA vector, comprising: an expression construct comprising at least one restriction site for inserting at least one heterologous nucleic acid sequence, or a regulatory switch, or both, said at least one restriction site being located between asymmetric inverted terminal repeats (asymmetric ITRs), at least one of said asymmetric ITRs comprising a functional terminal resolution site and a replication (Rep) protein binding site; a vector comprising a nucleic acid sequence encoding a single Rep protein, said single Rep protein being an AAV Rep78 protein or an AAV Rep68 protein, said vector being suitable for expressing said single Rep protein in an insect cell, said insect cell not expressing Rep52 or Rep40; wherein the DNA vector is produced in a cell that does not have a Rep protein of another species present, and the cell has not been contacted with a nucleic acid sequence encoding a second Rep protein.

35. 35. The kit of claim 34, further comprising a population of insect cells lacking a viral capsid coding sequence and capable of inducing production of the DNA vector in the presence of a single species of Rep protein.

36. A cell comprising a nucleic acid sequence encoding a single AAV replication (Rep) protein having at least DNA binding and DNA nicking functionality linked to at least one expression control sequence, wherein said single Rep protein is an AAV Rep78 protein or an AAV Rep68 protein, said cell does not express any other parvoviral Rep protein and is not in contact with any other Rep protein, and said cell does not express Rep52 or Rep40.

37. 37. The cell of claim 36, wherein the cell is an insect cell or a mammalian cell.

38. 38. The cell of claim 37, wherein the insect cell is selected from the group consisting of an Sf9 cell, an Sf21 cell, a Trichoplusia ni cell, and a High Five cell.

39. 38. The cell of claim 37, wherein the mammalian cell is selected from the group consisting of HEK293 cells, Huh-7 cells, HeLa cells, HepG2 cells, Hep1A cells, 911 cells, CHO cells, COS cells, MeWo cells, NIH3T3 cells, A549 cells, HT1080 cells, monocytes, and mature and immature dendritic cells.

40. 37. The cell of claim 36, wherein the nucleic acid sequence does not have a functional start codon for Rep52 or Rep40.

41. the nucleic acid sequence encodes an AAV2 Rep78 protein or an AAV2 Rep68 protein; The nucleic acid sequence encodes a mutated Rep78 protein comprising a mutation at amino acid position 225 of SEQ ID NO:

588. the nucleic acid sequence is full length and contains an intact alternative splice site at its carboxy terminus, resulting in the production of both Rep78 and Rep68; the nucleic acid sequence comprises a deletion of a carboxy-terminal intron sequence, resulting in the production of Rep68 alone; and / or The nucleic acid sequence comprises a sequence having at least 95% sequence identity to SEQ ID NO: 588 and encodes a Rep78 protein having at least DNA binding and DNA nicking functionality. The cell of claim 40.

42. 34. The pharmaceutical composition of claim 33, further comprising an excipient.

43. Expression cassette, a first ITR upstream (5' end) of the expression cassette; and A second ITR downstream (3' end) of the expression cassette 37. The cell of claim 36, further comprising a double-stranded DNA construct comprising:

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