Production of gene therapy vectors in engineered bacteria

Abstract

Provided herein are improved methods for engineering bacterial cells to produce circular DNA vectors, circular DNA vectors produced by such methods, and pharmaceutical compositions comprising such circular DNA vectors. The methods provided herein are suitable for large-scale production of high-purity compositions of circular DNA vectors.

Description

[Technical field]

[0001] [Cross reference] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 291,871, filed December 20, 2021, which is incorporated by reference in its entirety.

[0002] In general, the disclosure involves therapeutic vectors in engineered bacteria. [Background technology]

[0003] Gene therapy is emerging as a promising approach to treat a wide variety of diseases and disorders in human patients. Recombinant adeno-associated virus (rAAV) vectors have established a track record of highly efficient gene transfer in human patients and various model systems. The genomes of rAAV vectors have the advantage that they can persist as circular episomes in vivo for the lifespan of target cells. On the other hand, rAAV-based vectors have major disadvantages, such as maximum payload limitations, immunogenicity, and manufacturing inefficiencies.

[0004] To address some of these challenges in rAAV technology, non-viral alternatives have gained momentum in recent years. However, developing a scalable non-viral gene therapy platform with the efficiency and persistence of rAAV has proven difficult. For example, traditional bacterial plasmid DNA vectors, although versatile tools in gene delivery, are limited in that they are rich in bacterial components of plasmid DNA vectors, such as antibiotic resistance genes and transcriptional control elements, which may lead to immunogenicity and loss of gene expression due to transcriptional silencing.

[0005] Despite various efforts to improve plasmid DNA vectors by removing backbone components, there is a need for non-viral vectors with minimal bacterial elements and methods for their efficient production on a large scale. Summary of the Invention

[0006] Disclosed herein are circular DNA vectors (e.g., therapeutic circular DNA vectors) produced by bacteria, and bacterial cells (e.g., engineered bacterial cells) that can be used to produce circular DNA vectors from parent plasmids, e.g., parent plasmids in bacterial cells (e.g., engineered bacterial cells). The therapeutic circular DNA vectors provided herein contain small (e.g., less than 50 base pairs) origins of replication and lack selection markers (e.g., antibiotic resistance genes), which can reduce the risk caused by foreign sequences in the vector. Therefore, such circular DNA vectors produced by bacteria can be efficiently and large-scale produced for therapeutic applications.

[0007] Embodiments disclosed herein include an engineered bacterial cell that includes (a) a Rep gene encoding a bacterial replication protein integrated into the bacterial genome, and (b) a circular DNA vector that includes (i) a coding sequence and (ii) an origin of replication dependent on the replication protein, wherein the circular DNA vector does not include a selectable marker. In some embodiments, the origin of replication is less than 50 base pairs in length. In some embodiments, the origin of replication and the replication protein are derived from a ColE2-related plasmid. In some embodiments, the ColE2-related plasmid is ColE2-P9.

[0008] In some embodiments, the Rep gene is operably linked to a first inducible promoter. In some embodiments, the first inducible promoter is a T7 RNA polymerase-dependent promoter. In some embodiments, the engineered bacterial cell further comprises a gene encoding T7 RNA polymerase (T7RNAP) integrated into the bacterial genome. In some embodiments, the T7RNAP gene is operably linked to a second inducible promoter. In some embodiments, the second inducible promoter is a P tac It is.

[0009] In some embodiments, the engineered bacterial cell further comprises a gene encoding an exogenous restriction enzyme integrated into the bacterial genome. In some embodiments, the gene encoding the exogenous restriction enzyme is operably linked to a third inducible promoter. In some embodiments, the third inducible promoter is P bad In some embodiments, the bacterial genome does not contain a recognition sequence for an exogenous restriction enzyme.

[0010] In some embodiments, the coding sequence encoding the circular DNA vector comprises a therapeutic gene or nucleic acid, hi some embodiments, the coding sequence is a eukaryotic sequence (e.g., a sequence expressible in a mammalian cell).

[0011] In some embodiments, the origin of replication is the only bacterial sequence within the circular DNA vector.

[0012] In some embodiments, the engineered bacterial cell comprises at least 20 copies of the circular DNA vector. In some embodiments, the engineered bacterial cell is capable of maintaining the circular DNA vector through at least 20 cell divisions.

[0013] In some embodiments, the engineered bacterial cells do not contain any extragenomic circular DNA molecules other than one or more copies of the circular DNA vector.

[0014]

[0023] Embodiments disclosed herein include a culture comprising a plurality of any of the engineered bacterial cells described herein, wherein the culture has an average copy number of the circular DNA vector per engineered bacterial cell of at least 10. In some embodiments, the culture has an average copy number of the circular DNA vector per engineered bacterial cell of at least 10. 7 The engineered bacterial cell comprises

[0015] Embodiments disclosed herein include an engineered bacterial cell comprising: (a) a Rep gene encoding a bacterial replication protein integrated into the bacterial genome; and (b) a plasmid comprising: (i) a first segment comprising a coding sequence and an origin of replication dependent on the bacterial replication protein, and no selectable marker; and (ii) a second segment comprising a selectable marker, the first segment flanked on both sides by recognition sequences for at least one exogenous restriction enzyme or exogenous recombinase. In some embodiments, the recognition sequences flanking the first segment are the same. In some embodiments, the recognition sequences flanking the first segment are different. In some embodiments, the second segment further comprises an origin of replication, where the origin of replication in the second segment is orthologous to the origin of replication in the first segment.

[0016] In some embodiments, the origin of replication is less than 50 base pairs in length. In some embodiments, the origin of replication and the replication proteins are derived from a ColE2-related plasmid. In some embodiments, the ColE2-related plasmid is ColE2-P9.

[0017] In some embodiments, the Rep gene is operably linked to a first inducible promoter. In some embodiments, the first inducible promoter is a T7 RNA polymerase-dependent promoter. In some embodiments, the engineered bacterial cell further comprises a gene encoding T7 RNA polymerase (T7RNAP) integrated into the bacterial genome. In some embodiments, the T7RNAP gene is operably linked to a second inducible promoter. In some embodiments, the second inducible promoter is a P tac It is.

[0018] In some embodiments, the engineered bacterial cell further comprises a gene encoding an exogenous restriction enzyme or an exogenous recombinase integrated into the bacterial genome. In some embodiments, the gene encoding the exogenous restriction enzyme or the exogenous recombinase is operably linked to a third inducible promoter. In some embodiments, the third inducible promoter is P bad In some embodiments, the bacterial genome does not contain recognition sequences for exogenous restriction enzymes or exogenous recombinases.

[0019] In some embodiments, the coding sequence of the first segment encodes a therapeutic gene or nucleic acid, hi some embodiments, the coding sequence is a eukaryotic sequence (e.g., a sequence expressible in a mammalian cell).

[0020] In some embodiments, the engineered bacterial cell further comprises an exogenous restriction enzyme or an exogenous recombinase.

[0021] Embodiments disclosed herein include a method of making a circular DNA vector, comprising: (a) contacting a plasmid in a bacterial cell with an exogenous restriction enzyme to excise a first segment from the plasmid, where the first segment is flanked on both sides by recognition sequences for the exogenous restriction enzyme, and the first segment comprises a coding sequence and an origin of replication dependent on a bacterial replication protein, thereby generating a linear DNA fragment comprising a 5' end and a 3' end with complementary overhangs; and (b) ligating the 5' end and the 3' end of the linear DNA fragment to each other to generate a circular DNA vector. In some embodiments, prior to step (a), the plasmid comprises a second segment comprising a selectable marker. In some embodiments, the second segment further comprises an origin of replication, where the origin of replication in the second segment is orthologous to the origin of replication in the first segment. In some embodiments, the first segment does not comprise a selectable marker.

[0022] In some embodiments, contacting the plasmid in the cell with the exogenous restriction enzyme comprises inducing expression of the exogenous restriction enzyme in the cell. In some embodiments, the gene encoding the exogenous restriction enzyme is integrated into the bacterial genome operably linked to an inducible promoter. In some embodiments, the inducible promoter is P bad and inducing expression of the exogenous restriction enzyme in the cell comprises providing arabinose to the cell. In some embodiments, contacting the plasmid with the exogenous restriction enzyme in the cell comprises introducing the exogenous restriction enzyme into the cell from outside the bacterial cell.

[0023] In some embodiments, the ligation is performed by an exogenous ligase. In some embodiments, the exogenous ligase is expressed from a gene integrated into the bacterial genome. In some embodiments, the exogenous ligase is introduced into the bacterial cell from outside the bacterial cell.

[0024] In some embodiments, the bacterial cell comprises a Rep gene encoding a bacterial replication protein integrated within the genome. In some embodiments, the Rep gene is operably linked to an inducible promoter capable of expressing the bacterial replication protein at a first expression level and a second expression level, where the first expression level is lower than the second expression level. In some embodiments, the first expression level of the bacterial replication protein maintains the origin of replication at a first copy number, and the second expression level of the bacterial replication protein maintains the origin of replication at a second copy number, where the first copy number is less than 5 copies, less than 10 copies, less than 15 copies, less than 20 copies, or less than 50 copies per cell, and the second copy number is at least 20 copies, at least 50 copies, at least 100 copies, or at least 200 copies per cell. In some embodiments, the bacterial replication gene is expressed at the first expression level prior to step (b) and is not expressed at the second expression level prior to step (b). In some embodiments, the bacterial replication gene is expressed at a second expression level after step (b). In some embodiments, the inducible promoter is PT7, which is dependent on T7 RNA polymerase. In some embodiments, the bacterial cell comprises a gene encoding T7 RNA polymerase (T7RNAP) integrated into the genome. In some embodiments, the T7RNAP gene is operably linked to the inducible promoter. In some embodiments, the inducible promoter is P tac In some embodiments, the second segment of the plasmid is P tac and a LacI gene encoding a lactose inhibitor protein capable of repressing expression from the promoter, and expression of the bacterial replication genes is maintained at or below a first expression level by expression of the lactose inhibitor protein. In some embodiments, after step (b), expression of the lactose inhibitor protein is decreased, thereby inducing the bacterial replication genes to be expressed at a second expression level and maintaining the circular DNA vector at a second copy number.

[0025] In some embodiments, the method of making a circular DNA vector further comprises culturing the cells under conditions in which the selectable marker on the plasmid is not required for continued growth, thereby generating a progeny population of bacterial cells lacking the selectable marker. In some embodiments, the population maintains the circular DNA vector after at least 50 doublings. In some embodiments, the population maintains the circular DNA vector after at least 100 doublings, e.g., at least 150 doublings, at least 200 doublings, at least 250 doublings, or at least 290 doublings. In some embodiments, the population maintains the circular DNA vector at an average copy number of at least 20 copies per cell after at least 50 doublings. In some embodiments, the population maintains the circular DNA vector at an average copy number of at least 20 copies per cell after at least 100 doublings. In some embodiments, the population maintains the circular DNA vector at an average copy number of at least 20 copies per cell after at least 150 doublings, at least 200 doublings, at least 250 doublings, or at least 290 doublings. In some embodiments, the method further comprises purifying the circular DNA vector.

[0026] In some embodiments, the origin of replication is less than 50 base pairs in length (e.g., less than 45 base pairs in length, or about 40 base pairs in length). In some embodiments, the origin of replication and replication proteins are derived from a ColE2-related plasmid. In some embodiments, the ColE2-related plasmid is ColE2-P9. In some embodiments, the origin of replication comprises or consists of SEQ ID NO:2 (or a reverse complement) or a functional variant thereof (e.g., a functional variant having at least 90% sequence identity to SEQ ID NO:2 (e.g., at least 92% sequence identity to SEQ ID NO:2, at least 94% sequence identity to SEQ ID NO:2, at least 95% sequence identity to SEQ ID NO:2, at least 96% sequence identity to SEQ ID NO:2, at least 97% sequence identity to SEQ ID NO:2, at least 98% sequence identity to SEQ ID NO:2, at least 99% sequence identity to SEQ ID NO:2, or 100% sequence identity to SEQ ID NO:2)). In some embodiments, the origin of replication comprises or consists of SEQ ID NO:3 (or the reverse complement) or a functional variant thereof (e.g., a functional variant having at least 90% sequence identity to SEQ ID NO:3 (e.g., at least 92% sequence identity to SEQ ID NO:3, at least 94% sequence identity to SEQ ID NO:3, at least 95% sequence identity to SEQ ID NO:3, at least 96% sequence identity to SEQ ID NO:3, at least 97% sequence identity to SEQ ID NO:3, at least 98% sequence identity to SEQ ID NO:3, at least 99% sequence identity to SEQ ID NO:3, or 100% sequence identity to SEQ ID NO:3)). In some embodiments, the origin of replication comprises or consists of SEQ ID NO:4 (or the reverse complement) or a functional variant thereof (e.g., a functional variant having at least 90% sequence identity to SEQ ID NO:4 (e.g., at least 92% sequence identity to SEQ ID NO:4, at least 94% sequence identity to SEQ ID NO:4, at least 95% sequence identity to SEQ ID NO:4, at least 96% sequence identity to SEQ ID NO:4, at least 97% sequence identity to SEQ ID NO:4, at least 98% sequence identity to SEQ ID NO:4, at least 99% sequence identity to SEQ ID NO:4, or 100% sequence identity to SEQ ID NO:4)).

[0027] Embodiments disclosed herein include a method of making a circular DNA vector comprising: (a) obtaining any of the engineered bacterial cells described herein, comprising a parent plasmid comprising a first segment comprising a coding sequence and an origin of replication dependent on a bacterial replication protein, where the first segment does not comprise a selectable marker, and where the first segment is flanked on both sides by recognition sequences for at least one exogenous restriction enzyme; (b) contacting the plasmid with an exogenous restriction enzyme to excise the first segment of the plasmid, thereby generating a linear DNA fragment flanked on both sides by complementary overhangs; and (c) self-ligating the linear DNA fragment to generate a circular DNA vector.

[0028] Embodiments disclosed herein include a method of making a circular DNA vector comprising: (a) obtaining any of the engineered bacterial cells described herein that contain a parent plasmid comprising a first segment comprising a coding sequence and an origin of replication dependent on a bacterial replication protein, where the first segment does not comprise a selectable marker, and where the first segment is flanked on both sides by recognition sequences for at least one exogenous recombinase; and (b) contacting the plasmid with an exogenous recombinase that recognizes the recognition sequences flanking the first segment.

[0029] Embodiments disclosed herein include a pharmaceutical composition comprising: (a) a circular DNA vector produced by any of the methods described herein; and (b) a carrier suitable for use in delivering the pharmaceutical composition to a subject.

[0030] In another aspect, an engineered bacterial cell (e.g., E. coli) is provided that includes a circular DNA vector that includes a coding sequence and an origin of replication that is less than 50 base pairs in length and lacks a selectable marker. In some embodiments, the engineered bacterial cell does not include any extragenomic DNA molecules other than one or more copies of the circular DNA vector. In some embodiments, the engineered bacterial cell does not include a gene encoding a selectable marker. In some embodiments, the engineered bacterial cell does not include a selectable marker on an extragenomic DNA molecule. In some embodiments, the origin of replication is from a ColE2-related plasmid (e.g., a ColE2-P9 plasmid). In some embodiments, the engineered bacterial cell further includes a Rep gene that encodes a bacterial replication protein that recognizes the origin of replication (e.g., the Rep gene is from a ColE2-P9 plasmid (e.g., SEQ ID NO:1)). In some embodiments, the Rep gene is integrated into the bacterial genome. In some embodiments, the Rep gene is operably linked to an inducible promoter.

[0031] In some embodiments, the circular DNA vector further comprises a recombination site, such as an attL site (eg, attL-GA) or an attR site.

[0032] In some embodiments, the circular DNA vector does not contain any bacterial (or other prokaryotic or phage) sequences other than the origin of replication and, if present, the recombination sites, which together are 90 base pairs or less in length.

[0033] In some embodiments, the engineered bacterial cell further comprises a gene encoding a recombinase.

[0034] In some embodiments, the engineered bacterial cell comprises at least 10 copies of the circular DNA vector.

[0035] In another aspect, provided herein is an engineered bacterial cell comprising a plasmid comprising: (a) a first segment comprising a coding sequence and an origin of replication that is less than 50 base pairs in length and does not comprise a selectable marker; and (b) a second segment comprising a selectable marker, the first segment being flanked on either side by recognition sequences for an exogenous recombinase. In some embodiments, the engineered bacterial cell further comprises a gene encoding a Rep gene that encodes a bacterial replication protein that recognizes the origin of replication. In some embodiments, the Rep gene is integrated into the bacterial genome. In some embodiments, the Rep gene is operably linked to a first inducible promoter.

[0036] In some embodiments, the origin of replication and replication proteins are derived from a ColE2-related plasmid, such as ColE2-P9.

[0037] In some embodiments, the engineered bacterial cell further comprises a gene encoding an exogenous recombinase. In some embodiments, the gene encoding the exogenous recombinase is integrated into the bacterial genome. In some embodiments, the gene encoding the exogenous recombinase is on a plasmid or a bacterial artificial chromosome. In some embodiments, the gene encoding the exogenous recombinase is operably linked to a second inducible promoter. In some embodiments, the second inducible promoter is a cumic acid inducible promoter.

[0038] In some embodiments, the recombinase is Bxb1. In some embodiments, the recognition sequences include attP-GA and attB-GA.

[0039] In another aspect, provided herein is a method of making a circular DNA vector, comprising inducing recombination of a plasmid in an engineered bacterial cell of any one of the aspects described above, in some embodiments, inducing recombination of the plasmid comprises inducing expression of an exogenous recombinase in the engineered bacterial cell.

[0040] In another aspect, a method of producing a circular DNA vector is provided comprising inducing recombination of a plasmid in an engineered bacterial cell, where (a) the plasmid comprises (i) a first segment comprising a coding sequence and an origin of replication that is less than 50 base pairs in length and does not comprise a selectable marker, where the first segment is flanked on both sides by recognition sequences for an exogenous recombinase; (ii) a second segment comprising the selectable marker, and (b) the engineered bacterial cell comprises a gene encoding the exogenous recombinase, where induction causes recombination of the plasmids, thereby producing a circular DNA vector comprising the first segment.

[0041] In some embodiments, the engineered bacterial cell further comprises a Rep gene encoding a bacterial replication protein that recognizes an origin of replication. In some embodiments, the Rep gene is integrated into the bacterial genome. In some embodiments, the Rep gene is operably linked to a first inducible promoter.

[0042] In some embodiments, the origin of replication is a ColE2-P9 origin of replication and / or the Rep gene is a ColE2-P9 Rep gene.

[0043] In some embodiments, the exogenous recombinase is on a plasmid or a bacterial artificial chromosome. In some embodiments, the gene encoding the exogenous recombinase is operably linked to a second inducible promoter.

[0044] In some embodiments, inducing recombination of the plasmid comprises inducing expression of a gene encoding an exogenous recombinase. In some embodiments, inducing recombination of the plasmid comprises introducing the plasmid into the engineered bacterial cell, where upon introduction, the exogenous recombinase is expressed within the engineered bacterial cell. In some embodiments, the exogenous recombinase is expressed at uninduced levels upon introduction.

[0045] In some embodiments, the exogenous recombinase is Bxb1 and the recognition sequences include attP-GA and attB-GA. In some embodiments, the gene encoding Bxb1 is operably linked to a cumic acid inducible promoter, and the engineered bacterial cell is maintained in the absence of cumic acid upon introduction and Bxb1 is expressed at uninduced levels upon introduction.

[0046] In another aspect, a circular DNA vector (e.g., an engineered circular DNA vector) is provided that contains (a) a eukaryotic promoter, (b) a eukaryotic coding sequence, and (c) a bacterial origin of replication that is less than 50 bp in length, and lacks a selectable marker. In some embodiments, the 3' end of the eukaryotic coding sequence is linked to the 5' end of the promoter by a sequence that contains a bacterial origin of replication, where the sequence that contains the bacterial origin of replication is less than 100 bp in length. In some embodiments, the circular DNA vector is monomeric supercoiled.

[0047] In another aspect, a pharmaceutical composition is provided that includes a circular DNA vector lacking a selectable marker, the circular DNA vector comprising (a) a eukaryotic promoter, (b) a eukaryotic coding sequence, and (c) a bacterial origin of replication that is less than 50 bp in length. In some embodiments, the 3' end of the eukaryotic coding sequence is linked to the 5' end of the promoter by a sequence comprising a bacterial origin of replication, wherein the sequence comprising the bacterial origin of replication is less than 100 bp in length, and a carrier suitable for use in delivering the pharmaceutical composition to a subject.

[0048] In another aspect, a host cell (e.g., a mammalian cell, e.g., a human cell) is provided that comprises a circular DNA vector that includes (a) a eukaryotic promoter, (b) a eukaryotic coding sequence, and (c) a bacterial origin of replication that is less than 50 bp in length and lacks a selectable marker. In some embodiments, the 3' end of the eukaryotic coding sequence is linked to the 5' end of the promoter by a sequence that includes a bacterial origin of replication, wherein the sequence that includes the bacterial origin of replication is less than 100 bp in length. In some embodiments, the host cell expresses a protein encoded by the coding sequence. In some embodiments, the host cell is isolated in vitro. In some embodiments, the circular DNA vector is transfected into the host cell by electroporation.

[0049] The features and advantages of the present disclosure will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings, in which: [Brief description of the drawings]

[0050] [Figure 1] FIG. 1 shows a table depicting results from a stability study in which three ColE2-P9 origins of replication were tested for their ability to confer stability to plasmids upon expansion in E. coli. [Diagram 2] FIG. 1 shows an example of a method for assembling a parent plasmid that can be used in embodiments disclosed herein. [Diagram 3] FIG. 1 shows the experimental procedure for producing a test circular DNA vector according to an embodiment disclosed herein. [Figure 4]Figure 1 shows the results of agarose gel electrophoresis of extrachromosomal DNA purified from engineered bacteria grown in rich medium with ("Cm", lanes 7-12) or without ("No Cm", lanes 1-6). Lanes 2, 3, 5, 8, 9, and 11 show bands corresponding to the test circular DNA vectors produced by recombination from the test parent plasmids. Lanes 1, 4, 7, and 10 show bands corresponding to the test parent plasmids. [Diagram 5] Figure 1 shows a graph depicting the percentage of sfGFP positive cells in the indicated growth media with or without chloramphenicol ("Cm"), ZB = Zymo Broth, TB = Terrific Broth, SB = Super Broth, SOB = Super Optimal Broth, SOC = Super Optimal Broth with Catabolite Repression, LB = Luria Broth. [Figure 6] FIG. 1 shows a schematic chart illustrating an exemplary process for producing a circular DNA vector of the present invention using counter-selection. [Figure 7A-7F] 7 is a schematic diagram showing the process of expressing Bxb1 based on bacterial artificial chromosome (BAC) to produce a circular DNA vector. FIG. 7A is the Rep gene integrated into the host genome. FIG. 7B and FIG. 7C are two alternative BAC designs. Bxb1 in FIG. 7B is driven by a cumic acid inducible promoter, whereas Bxb1 in FIG. 7C is driven by an arabinose inducible promoter. FIG. 7D is the template plasmid, which upon recombination with Bxb1 results in the circular DNA vector in FIG. 7E and the by-product in FIG. 7F. The circular DNA vector contains an origin of replication and (optionally) the by-product contains a PheS counterselectable marker, so that as the host bacterium is replicated and expanded, the circular DNA vector becomes the dominant species. [Figure 8A-8B]FIG. 1 shows photographs showing the fluorescence of clones 24 and 72 hours, respectively, after transformation with BAC1696. [Figure 9A-9B] FIG. 1 shows photographs showing the fluorescence of clones 24 and 72 hours, respectively, after transformation with BAC1697. [Figure 10] FIG. 1 is a set of photographs showing the fluorescence of clones 24 hours after exposure to cumic acid inducer. [Figure 11] FIG. 1 is a set of photographs showing the fluorescence of clones 24 hours after exposure to the arabinose inducer. [Figure 12] FIG. 1 is a series of photographs showing the fluorescence of restreaked 1696 colonies incubated overnight under various conditions on LB agar plates. [Figure 13] FIG. 1 is a series of photographs showing the fluorescence of restreaked 1697 colonies incubated overnight under various conditions on LB agar plates. [Figure 14] Figure 1 shows a photograph of a gel electrophoresis experiment demonstrating the presence of circular DNA vector in counterselected cultures for both 1696 and 1697. A digestion map showing the theoretical bands is shown on the left of the photograph. [Figure 15A] FIG. 1 shows a plasmid map of an exemplary ABCA4 template plasmid. [Figure 15B] FIG. 15B shows the plasmid map of the ABCA4 circular DNA vector derived from the template plasmid of FIG. 15A. [Figure 16A] FIG. 1 shows a theoretical gel map showing the banding pattern for the circular DNA construct digest described in Example 7. [Figure 16B] FIG. 16B shows a photograph of a gel showing the actual banding pattern corresponding to FIG. 16A. [Figure 17A]FIG. 1 shows a histogram showing long-read sequencing data from purified ABCA4 circular DNA vectors produced using a 2-hour Kan resistance incubation with template plasmid. The main peaks are the BAC and dimeric circular DNA vectors. [Figure 17B] FIG. 1 shows a histogram showing long-read sequencing data from purified ABCA4 circular DNA vector produced using overnight Kan resistance incubation with template plasmid. The main peak is monomeric circular DNA vector and BAC. [Figure 18] FIG. 1 shows a plasmid map showing components of helper plasmids useful for expressing Bxb1 in bacterial hosts. [Figure 19] FIG. 19 is a photograph showing green fluorescent colonies (circled) containing circular DNA vectors without backbone by-products as a result of Bxb1 expression by the helper plasmid of FIG. 18. [Figure 20] FIG. 1 is a schematic showing the process of Bxb1 integration into the host cell genome. [Figure 21] Figure 1 shows a photograph of a gel showing two positive clones for Bxb1 integration, with the 1696 plasmid control shown in triplicate at the bottom left. [Figure 22] FIG. 1 shows a photograph of a Western blot demonstrating that HEK293T cells transfected with bacterially produced ABCA4 circular DNA vector express ABCA4 protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Provided herein are improved methods and related compositions for producing circular DNA vectors from parental plasmids in engineered bacterial cells. In the past, there have been efforts to improve plasmid DNA vectors by removing backbone components. For example, minicircles are made in bacterial cells by using recombination to remove the backbone from the plasmid, generating a minicircle vector and a circular backbone by-product. However, minicircles are difficult to produce on a large scale, as their isolation requires purification from the backbone by-product, which has a similar structure. Alternative vector types, such as nanoplasmids, have been designed to make purification by positive selection easier by including an origin of replication and a selectable marker in the vector. However, such foreign elements are relatively large, typically several hundred base pairs in length, and are foreign to patients.

[0052] Disclosed herein are circular DNA vectors (e.g., therapeutic circular DNA vectors) produced by bacteria, and bacterial cells (e.g., engineered bacterial cells) that can be used to produce circular DNA vectors from parental plasmids, e.g., parental plasmids within bacterial cells (e.g., engineered bacterial cells). The therapeutic circular DNA vectors provided herein contain small (e.g., less than 50 base pairs) origins of replication and lack selection markers (e.g., antibiotic resistance genes), which can reduce the risk caused by foreign sequences in the vector. Thus, such bacterially produced circular DNA vectors can be efficiently and large-scale produced for therapeutic applications. Furthermore, by eliminating or reducing bacterial plasmid DNA sequences, such as RNAPII arrest sites, transcriptional silencing of the circular DNA vector can be reduced or eliminated, resulting in persistence of vector sequences in individuals. In certain embodiments of the invention, immunogenic components (e.g., bacterial endotoxins, DNA or RNA, or bacterial signatures such as CpG motifs) are absent from the present circular DNA vectors or are present at very low levels suitable for pharmaceutical or laboratory use, thereby reducing the risk of stimulating a host immune response compared to conventional DNA vectors such as plasmid DNA vectors. These and other aspects of the disclosed embodiments are discussed in more detail below.

[0053] I. Definition 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. It should also be noted that the term "or" is generally used in the sense of including "and / or" unless the context clearly dictates otherwise. As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents may be used interchangeably. These terms may mean that any combination is specifically contemplated. For illustrative purposes only, the following phrase "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "A individually, B individually, C individually, A and B, B and C, A and C, and A, B, and C." The term "or" may be used either conjunctively or disjunctively unless the context specifically dictates disjunctive use.

[0054] As used in the specification and claim(s), the words "comprising" (and all forms of comprising, such as "comprise" and "comprises"), "having" (and all forms of having, such as "have" and "has"), "including" (and all forms of including, such as "includes" and "include"), or "containing" (and all forms of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Additionally, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0055] References in this specification to "some embodiments," "embodiments," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments. Certain specific details of this description are shown to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the present disclosure can be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0056] As used herein, an "inducible promoter" refers to a promoter whose expression can be turned on or increased in response to a stimulus. The stimulus can be, for example, the presence of a particular molecule or culture condition. The stimulus can also be, for example, the absence of a particular molecule or culture condition. As used herein, an "inducible promoter" includes a promoter whose expression can be turned on or increased by removing a condition, such as the presence of a particular molecule that represses expression from the promoter, or by removing another culture condition. In some embodiments, an inducible promoter is a T7 RNA polymerase-dependent promoter, P tac Promoter, P bad promoter, PT7 promoter, or a combination thereof. In some embodiments, the inducible promoter is integrated into the bacterial genome and operably linked to a gene, such as a gene encoding a Rep protein, a restriction enzyme, or a recombinase.

[0057] As used herein, "exogenous" refers to any substance introduced from or produced outside of an organism, cell, tissue, or system. For example, in embodiments described herein in which an exogenous restriction enzyme is present in an engineered bacterial cell, the exogenous restriction enzyme is one that would not be present in the engineered bacterial cell if the exogenous restriction enzyme had not been introduced into the engineered bacterial cell from outside the engineered bacterial cell. For example, an exogenous restriction enzyme can be introduced into an engineered bacterial cell by introducing a gene encoding the exogenous restriction enzyme into the bacterial cell or by introducing the restriction enzyme into the cell across the cell membrane, such as by electroporation. The embodiments described herein also include, for example, exogenous ligases and exogenous recombinases.

[0058] As used herein, a "parent plasmid" is a plasmid that contains both a vector sequence (defined below) and a "backbone sequence" (defined below). Embodiments disclosed herein include a method of making a circular DNA vector that includes removing the backbone sequence from the vector sequence.

[0059] As used herein, the "vector sequence" of a parental plasmid refers to the portion of the plasmid DNA that contains the origin of replication and the coding sequence of the gene of interest. In some descriptions of the embodiments herein, the vector sequence is referred to as the "first segment" of the plasmid.

[0060] As used herein, the "backbone sequence" of a parental plasmid refers to the portion of the plasmid DNA outside the vector sequence that contains one or more selectable markers, such as a drug resistance gene or fragments thereof. In some descriptions of the embodiments herein, the backbone sequence is referred to as the "second segment" of the plasmid.

[0061] As used herein, a "replication protein" is a protein that is required for the initiation of replication at the replication origin sequence corresponding to the replication protein. If a replication origin depends on a given replication protein for the initiation of replication at a particular replication origin sequence, the replication origin sequence corresponds to that replication protein. As an example, the replication protein encoded by the ColE2-P9 plasmid corresponds to the ColE2-P9 ori sequence. That is, the ColE2-P9 replication protein is required for the initiation of DNA replication at the ColE2-P9 ori sequence.

[0062] As used herein, a "functional variant" of a nucleic acid sequence differs from a reference nucleic acid sequence, such as a naturally occurring nucleic acid sequence, in at least one nucleic acid residue, where the relevant functional activity of the variant is at least 90% of the level of the relevant functional activity of the reference nucleic acid sequence (e.g., substantially similar to the relevant function of the reference nucleic acid sequence). In this context, the difference in at least one nucleic acid residue may be, for example, a mutation, deletion, or insertion of one nucleic acid residue to another. The variant may encode a homolog, isoform, or transcriptional variant of a therapeutic protein or a fragment thereof encoded by the reference nucleic acid sequence, where the homolog, isoform, or transcriptional variant is characterized by the degree of identity or homology, respectively, as defined herein.

[0063] In some cases, functional variants of polynucleotides or polypeptides contain at least one nucleic acid substitution (e.g., 1-100 nucleic acid or amino acid substitutions, 1-50 nucleic acid or amino acid substitutions, 1-20 nucleic acid or amino acid substitutions, 1-10 nucleic acid or amino acid substitutions, such as 1 nucleic acid or amino acid substitution, 2 nucleic acid or amino acid substitutions, 3 nucleic acid or amino acid substitutions, 4 nucleic acid or amino acid substitutions, 5 nucleic acid or amino acid substitutions, 6 nucleic acid or amino acid substitutions, 7 nucleic acid or amino acid substitutions, 8 nucleic acid or amino acid substitutions, 9 nucleic acid or amino acid substitutions, or 10 nucleic acid or amino acid substitutions). Nucleic acid substitutions that result in an expressed polypeptide with an exchange with an amino acid from the same class are referred to herein as conservative substitutions. In particular, these are amino acids with aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chain or amino acids whose side chains can form hydrogen bridges, e.g., side chains with hydroxyl functions. A conservative substitution, for example, may replace an amino acid having a polar side chain with another amino acid having a corresponding polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain may be replaced with another amino acid having a corresponding hydrophobic side chain (e.g., serine (threonine) with threonine (serine) or leucine (isoleucine) with isoleucine (leucine)).

[0064] To determine the percentage of identity of two sequences (e.g., nucleic acid sequences, e.g., DNA or amino acid sequences), the sequences can be aligned for subsequent comparison with each other. For this purpose, gaps can be inserted into the sequence of the first sequence and the elements at the corresponding positions of the second sequence can be compared. If a position in the first sequence is occupied by the same element as the corresponding position in the second sequence, the two sequences are identical at this position. The percentage of identity of two sequences is a function of the number of identical positions divided by the total number of positions. The percentage of identity of two sequences can be determined using a mathematical algorithm. A preferred but non-limiting example of a mathematical algorithm that can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877, or Altschul et al. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm can be integrated, for example, into the BLAST program. Sequences that have a certain degree of identity to the sequences of the present invention can be identified by this program.

[0065] As used herein, the terms "flank", "flank" and "flanked" refer to a pair of regions or points on a nucleic acid molecule (e.g., a plasmid) that are outside the reference region of the nucleic acid molecule. In some embodiments, the pair of regions or points on a nucleic acid that flank the reference region are adjacent to (i.e., abutting) the reference region (i.e., there are no intervening bases between the reference and flanking points). In other embodiments, the pair of regions or points on a nucleic acid molecule that flank the reference region are separated from the reference region by one or more intervening bases (e.g., up to 1000 intervening bases). For example, if a first restriction site is 200 bases upstream of a given sequence and a second restriction site is 100 bases downstream of the given sequence, the first and second restriction sites are said to flank the sequence. In some embodiments, all intervening sequences between the flanking region or points and the reference region are free of bacterial sequences. In such embodiments, the circular DNA vector produced by self-ligation of vector sequences excised from a parental plasmid at the restriction or recombination sites flanking the vector sequence is free of bacterial sequences other than the ori sequence. For example, in such embodiments, an exogenous restriction enzyme that cuts at sites flanking the vector sequence can produce a circular DNA vector that has sequences between the 5' and 3' ends of the therapeutic sequence, but that does not include bacterial sequences (e.g., drug resistance genes) in this region. Such intervening sequences can be artifacts from sticky end ligation, for example, corresponding to overhanging bases generated by the exogenous restriction enzyme.

[0066] The term "ABCA4" refers to any native ABCA4 of any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), as well as functional variants (e.g., natural variants or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof, unless otherwise indicated. Functional variants can be determined based on known ABCA4 signaling. ABCA4 encompasses not only full-length unprocessed ABCA4, but also any form of ABCA4 resulting from native processing in cells. Exemplary human ABCA4 sequences are provided as NCBI Reference Sequences: NG_009073 or NM_000350.

[0067] The term "MYO7A" refers to any native MYO7A (also known as DFNB2, MYU7A, NSRD2, USH1B, DFNA11, or MYOVIIA) of any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), as well as functional variants (e.g., natural variants or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof, unless otherwise indicated. Functional variants can be determined based on known MYO7A signaling. MYO7A encompasses not only full-length unprocessed MYO7A, but also any form of MYO7A resulting from native processing in cells. An exemplary human MYO7A sequence is provided as National Center for Biotechnology Information (NCBI) Gene ID: 4647.

[0068] As used herein, the term "self-replicating RNA molecule" refers to a self-replicating genetic element that contains RNA that replicates from a single origin of replication.

[0069] As used herein, the terms "operatively linked" or "operatively coupled" refer to an arrangement of elements where the components so described are configured to perform their normal functions. A nucleic acid is "operably linked" or "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter is operably linked to one or more heterologous genes if it affects the transcription of the one or more heterologous genes. Furthermore, a control element operably linked to a coding sequence can affect the expression of the coding sequence. Control elements need not be contiguous with the coding sequence, so long as they function to direct its expression. Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered to be "operably linked" or "operably linked" to the coding sequence.

[0070] As used herein, a "vector" refers to a nucleic acid molecule capable of carrying a sequence of interest, which can be linked to a target cell and then transcribed, replicated, processed, and / or expressed in the target cell. Once the target cell or host cell processes the sequence of interest in the vector, the sequence of interest is no longer considered a vector. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop capable of autonomous replication and comprising a bacterial backbone containing a bacterial origin of replication and a selectable marker, into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors" or "expression vectors").

[0071] As used herein, the terms "individual" and "subject" are used interchangeably and include, for example, any mammalian subject in need of treatment or prevention with the circular DNA vector or pharmaceutical composition thereof described herein. In some embodiments, the individual or subject is a human. In other embodiments, the individual or subject is a non-human mammalian subject (e.g., a non-human primate (e.g., monkey), mouse, pig, rabbit, cat, or dog). The individual or subject may be male or female.

[0072] As used herein, an "effective amount" or "effective dose" of a circular DNA vector or pharmaceutical composition thereof refers to an amount sufficient to achieve a desired biological, pharmacological, or therapeutic effect when administered to an individual, for example, according to a selected administration form, route of administration, and / or administration schedule. As will be appreciated by those of skill in the art, the absolute amount of a particular composition that is effective may vary depending on factors such as the desired biological or pharmacological endpoint, the agent being delivered, the target tissue, and the like. Those of skill in the art will further appreciate that an "effective amount" may be contacted with a cell or administered to a subject in a single dose or through the use of multiple doses. An effective amount of a composition for treating a disease may slow or stop the progression of a disease, or enhance a partial or complete response, as compared to a reference population, for example, an untreated or placebo population, or a population treated with a standard of care.

[0073] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to a clinical intervention that seeks to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, amelioration or alleviation of the disease state, and improved prognosis. In some embodiments, the circular DNA vector of the present invention is used to delay the onset of disease or to slow the progression of disease.

[0074] The terms "level of expression" or "expression level" are used interchangeably and generally refer to the amount of a polynucleotide, or an amino acid product or protein, in a biological sample (e.g., retina). "Expression" generally refers to the process by which genetically coded information is converted into structures present and operational in a cell. Thus, according to the present invention, "expression" may refer to transcription into a polynucleotide, translation into a protein, or post-translational modification of a protein. Transcribed polynucleotides, translated proteins, or post-translationally modified protein fragments shall also be considered expressed, regardless of whether they are derived from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of a protein, e.g., by proteolysis. "Expressed genes" include those that are transcribed into polynucleotides as mRNA and then translated into proteins, and also include those that are transcribed into RNA but not translated into proteins (e.g., transfer RNA and ribosomal RNA).

[0075] As used herein, the term "expression persistence" refers to the period during which a sequence of interest or a functional portion thereof (e.g., one or more coding sequences of a circular DNA vector) is expressible in a transfected cell ("intracellular persistence") or in any progeny of the transfected cell ("intergenerational persistence"). A sequence of interest, e.g., a therapeutic sequence or a functional portion thereof, may be expressible if it is not silenced, e.g., by DNA methylation and / or histone methylation and compaction. Expression persistence can be assessed by detecting or quantifying (i) mRNA transcribed from the sequence in the target cell or its progeny (e.g., via qPCR, RNA-seq, or any other suitable method), and (ii) protein translated from the sequence in the target cell or its progeny (e.g., via Western blot, ELISA, or any other suitable method). In some instances, expression persistence is assessed by detecting or quantifying the therapeutic DNA in the target cell or its progeny in conjunction with either or both of (i) the mRNA transcribed from the therapeutic sequence in the target cell or its progeny, and (ii) the protein translated from the therapeutic sequence in the target cell or its progeny (e.g., the presence of the circular DNA vector in the target cell, e.g., by episomal DNA copy number analysis). Expression persistence of the sequence of interest, or a functional portion thereof, can be quantified relative to a reference vector (e.g., a plasmid), such as a control vector having one or more bacterial signatures not present in the vector of the invention, using any method known in the art to characterize gene expression. Expression persistence can be quantified at any given time point after administration of the vector. For example, in some embodiments, expression of the circular DNA vector of the invention persists for at least two weeks after administration if it is detectable in the target cell or its progeny two weeks after administration of the circular DNA vector. In some embodiments, expression of a gene is "sustained" in a target cell if it is detectable in the target cell for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or longer following administration.In some embodiments, expression of a sequence is said to persist for a given period of time after administration if any detectable percentage of the original expression level (e.g., at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, or at least 100%) remains after the given period of time (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or longer after administration).

[0076] As used herein, "intracellular persistence" refers to the period during which a sequence or a functional portion thereof (e.g., one or more coding sequences of a circular DNA vector) is expressible in a cell into which it is transfected (e.g., a target cell, such as a postmitotic or quiescent cell). Intracellular persistence can be assessed by detecting or quantifying (i) the mRNA transcribed from the sequence in the target cell, and (ii) the protein translated from the sequence in the target cell. In some cases, intracellular persistence is assessed by detecting or quantifying either or both of (i) the mRNA transcribed from the sequence in the target cell, and (ii) the protein translated from the sequence in the target cell, in combination with DNA in the target cell (e.g., the presence of the circular DNA vector in the target cell). In some embodiments, the circular DNA vector of the present invention exhibits improved intracellular persistence compared to a reference vector (e.g., a plasmid DNA vector).

[0077] As used herein, "intergenerational persistence" refers to the period during which a sequence or a functional portion thereof (e.g., one or more coding sequences of a DNA vector) is expressible in the progeny of a cell transfected with a gene (e.g., the progeny of a target cell, such as the first, second, third, or fourth generation progeny of a cell transfected with a gene via a circular DNA vector). Intergenerational persistence can be useful for measuring vector persistence in dividing tissues over time, since it takes into account any dilution of the gene across cell divisions. In some embodiments, the circular DNA vectors of the invention show improved intergenerational persistence compared to a reference vector (e.g., a plasmid DNA vector). Intergenerational persistence can be assessed by detecting or quantifying (i) mRNA transcribed from the vector sequence in the progeny of the target cell, and (ii) protein translated from the vector sequence in the progeny of the target cell. In some cases, intracellular persistence is assessed by detecting or quantifying either or both of (i) mRNA transcribed from the sequence in the progeny of the target cell, and (ii) protein translated from the sequence in the progeny of the target cell, in combination with DNA in the progeny of the target cell (e.g., the presence of the circular DNA vector in the progeny of the target cell). In some embodiments, the circular DNA vectors of the invention exhibit improved intergenerational persistence compared to a reference vector (e.g., a plasmid DNA vector).

[0078] As used herein, the term "copy number" of a DNA molecule refers to the average copy number of the DNA molecule per cell in a given cell population.

[0079] The term "pharmaceutical acceptable" means safe for administration to a mammal, such as a human. In some embodiments, a pharmaceutical acceptable composition is approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia or other commonly recognized pharmacopoeias, for use in animals, and more particularly, in humans.

[0080] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the vector or composition of the present invention is administered. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA., 23rd edition, 2020.

[0081] As used herein, the term "about" refers to a value within a ±10% variation from a reference value, unless otherwise specified.

[0082] In the event of any conflict in definitions among various sources or references, the definitions set forth herein shall prevail.

[0083] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0084] Throughout this disclosure, numerical characteristics are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as each individual numerical value within that range, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed each individual value within that range, such as 1.1, 2, 2.3, 5, and 5.9, as well as subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention, unless the context dictates otherwise.

[0085] II. Methods for Producing Circular DNA Vectors in Bacteria The embodiments disclosed herein include methods of producing circular DNA vectors in engineered bacterial cells. The engineered bacterial cells disclosed herein can be used to produce circular DNA vectors from parental plasmids. In some embodiments, the engineered bacterial cells include a parental plasmid and a Rep gene that encodes a bacterial replication protein integrated into the bacterial genome. In some embodiments, the Rep gene is included on an extrachromosomal DNA molecule, such as a plasmid (e.g., a helper plasmid) or a bacterial artificial chromosome ("BAC"). In some embodiments, the Rep gene is included on a parental plasmid. The parental plasmid includes a vector sequence and a backbone sequence. The vector sequence includes an ori sequence corresponding to the Rep gene and does not include a selectable marker. The backbone sequence includes a selectable marker and does not include an ori sequence included in the vector sequence, although in some embodiments it may include a different ori sequence. The parental plasmid also has restriction enzyme recognition sequences or site-specific recombination sequences flanking the vector sequence, which are positioned such that the plasmid backbone sequence can be separated from the vector sequence inside the cell by restriction digestion or site-specific recombination. In the case of restriction digestion, the circular DNA vector is then formed by self-ligation of the vector sequences. In the case of site-specific recombination, the circular DNA vector is formed when the recombination is completed. If the rep protein is expressed after the separation of the vector sequences and the formation of the circular DNA vector, the circular DNA vector can be maintained at high copy number, even though it lacks a selectable marker. In contrast, the maintenance of the plasmid backbone sequence in the engineered bacterial cells after separation can be avoided by changing the culture conditions to remove the selection pressure for the selectable marker. Cultivating a bacterial cell population carrying a high copy number of the circular DNA vector under conditions in which the parent plasmid is not maintained can efficiently produce a high yield of high purity circular DNA vector.

[0086] 1. Engineered Bacterial Cells Methods for producing circular DNA vectors disclosed herein include the use of engineered bacterial cells, which may include, for example, engineered E. coli bacterial cells or other suitable bacteria. In some embodiments, the engineered bacterial cells include an exogenous Rep gene encoding a replication protein integrated into the bacterial genome and a parent plasmid having an ori sequence corresponding to the Rep gene. In some embodiments, the Rep gene is not integrated into the bacterial genome, but is present on an extrachromosomal DNA molecule, such as a plasmid or BAC.

[0087] In some embodiments, the engineered bacterial cell has an exogenous Rep gene integrated into the bacterial genome. Any suitable chromosomal integration process can be used to introduce the Rep gene into the bacterial genome, including integration cassettes and integration procedures well known in the art. In some embodiments, the Rep gene encodes a ColE2-P9 replication protein or a related protein. In some exemplary embodiments, the Rep gene encodes a ColE2-P9 replication protein having the amino acid sequence shown in SEQ ID NO:1. Other suitable replication proteins include replication proteins encoded by naturally occurring plasmids, including those related to ColE2-P9, such as, for example, ColE3-CA38. Replication proteins can be used in the embodiments described herein in combination with their corresponding replication origin sequences.

[0088] The ori sequence included in the vector sequence of the parental plasmid is selected to correspond to a Rep gene integrated into the genome of the engineered bacterial cell or otherwise present in the engineered bacterial cell, e.g., on a plasmid or BAC. In some exemplary embodiments, the ori comprises the nucleotide sequence set forth in SEQ ID NO:2. Thus, the engineered bacterial cell embodiments disclosed herein include a functional pair of replication proteins and origin of replication sequences that allow replication of the parental plasmid and / or circular DNA vector. In some embodiments, the ori sequence present in the vector sequence is a ColE2-P9 ori sequence or a functional fragment thereof. In some embodiments, the ori sequence present in the vector sequence is a functional fragment of the ColE2-P9 ori sequence having the DNA sequence set forth in SEQ ID NO:2. The 40 base pair functional fragment set forth in SEQ ID NO:2 is capable of supporting replication of the vector in cells expressing ColE2-P9. In some embodiments, shorter or longer functional fragments can be used. In some embodiments, a 31 base pair fragment of ColE2-P9 can be used. Other suitable ori sequences include, but are not limited to, ori sequences corresponding to suitable Rep proteins and functional fragments thereof, such as the ori sequence of ColE3-CA38.In some embodiments, the ori is equal to or less than 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, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or 400 nucleotides in length. In some embodiments, the ori sequence is a functionally modified version of a naturally occurring ori, such as an ori sequence that has been modified to be shorter than the corresponding naturally occurring ori sequence while still retaining the ability to support replication initiation. In some embodiments, the ori sequence is a naturally occurring ori sequence.

[0089] In some embodiments, the Rep gene is operably linked to an inducible promoter. Suitable inducible promoters include, but are not limited to, P, which is induced by T7 RNA polymerase. T7 Promoter, heat-inducible P L Promoter, P repressible by LacI (and therefore inducible by the absence or removal of LacI) tac Promoter, arabinose-inducible P badIn the embodiments disclosed herein, for example, a bacteriophage promoter (e.g., P L Other inducible promoters known in the art can also be used, including promoters such as s1con, T3, T7, SP6, and bacterial promoters (e.g., PmgrB, pLlacO, Ptrc2, pLtetO, Plac / ara, Pm). Examples of bacterial promoters that may be used according to the present disclosure include, but are not limited to, positively regulated E. coli promoters, such as positively regulated σ 70 Promoters (e.g., inducible pBad / araC promoter, Lux cassette right promoter, modified lambda Prm promoter, plac Or2-62 (positive), pBad / AraC with separate REN sites, pBad, P(Las)TetO, P(Las)CIO, P(Rhl), Pu, FecA, pRE, cadC, hns, pLas, pLux), σ S Promoters (e.g., Pdps), σ 32 Promoters (e.g., heat shock), and σ 54 promoters (e.g., glnAp2), negatively regulated E. coli promoters, e.g., negatively regulated σ 70Promoters (e.g., promoter (PRM+), modified lambda Prm promoter, TetR-TetR-4C P(Las)TetO, P(Las)CIO, P(Lac)IQ, RecA_DlexO_dLacO1, dapAp, FecA, Pspac-hy, pcI, plux-cI, plux-lac, CinR, CinL, glucose-regulated modified Pr, modified Prm+, FecA, Pcya, rec A(SOS), Rec A(SOS), EmrR_regulated (EmrR_regulated), BetI_regulated (BetI_regulated), pLac_lux, pTet_Lac, pLac / Mnt, pTet / Mnt, LsrA / cI, pLux / cI, LacI, LacIQ, pLacIQ1, pLas / cI, pLas / Lux, pLux / Las, pRecA with LexA binding site, reverse bBa_R0011, pLacI / ara-1, pLacIq, rrnB P1, cadC, hns, PfhuA, pBad / araC, nhaA, OmpF, RcnR), σ S Promoters (e.g., alternative sigma factor σ 38 Lutz-Bujard LacO with σ 32 Promoters (e.g., alternative sigma factor σ 32 Lutz-Bujard LacO with 54 promoters (e.g., glnAp2), negatively regulated B. subtilis promoters, e.g., repressible B. subtilis σ A Promoters (e.g., gram-positive IPTG-inducible, Xyl, hyper-spank), and σ B Other inducible bacterial promoters can be used in accordance with the present disclosure. For example, cumic acid inducible promoters such as pCymRC can be used in some embodiments.

[0090] In some embodiments, the expression level of the replication proteins affects the copy number of the parental plasmid or circular DNA vector containing the corresponding ori sequence. Thus, if a relatively low copy number (e.g., less than 5 copies, less than 10 copies, or less than 20 copies per cell on average) is desired, the engineered bacterial cell can be maintained in conditions where the replication proteins are expressed at relatively low levels. If a relatively high copy number (e.g., more than 20 copies, more than 50 copies, or more than 100 copies per cell on average) is desired, the engineered bacterial cell can be maintained in conditions where the replication proteins are expressed at relatively high levels from an inducible promoter. In some embodiments, the Rep gene is operably linked to an inducible promoter that provides a first expression level in non-induced conditions and can be induced to provide a higher second expression level that results in a higher copy number of the parental plasmid or circular DNA vector containing the corresponding ori sequence. In some embodiments, it is advantageous to maintain the parental plasmid in the engineered bacterial cell at a low copy number before the vector and backbone sequences of the parental plasmid are separated. In embodiments where the vector and backbone sequences are separated by restriction digestion, having a relatively low copy number can help ensure that the linearized vector sequence self-ligates rather than ligating to the backbone sequence or other copies of the vector sequence. After the vector sequence is separated from the backbone sequence and the circular DNA vector is formed, in some embodiments, it is advantageous to maintain the circular DNA vector containing the ori sequence at a relatively high copy number to produce a high yield of the circular DNA vector. Thus, in some embodiments, after the circular DNA vector is formed in the engineered bacterial cell, a higher expression level of the replication protein is induced, for example, by adding a molecule that induces higher expression from an inducible promoter operably linked to the Rep gene. In some embodiments, the inducible promoter is maintained in a non-induced state until after the vector sequence is separated from the backbone sequence. In some embodiments, the inducible promoter is induced after the vector sequence is separated from the backbone sequence.In some embodiments, the inducible promoter is induced simultaneously when the vector sequence is separated from the backbone sequence, hi some embodiments, the inducible promoter is induced before the vector sequence is separated from the backbone sequence.

[0091] In some embodiments, the copy number of the parental plasmid is at least about, at most about, or about 1 copy, about 2 copies, about 3 copies, about 4 copies, about 5 copies, about 6 copies, about 7 copies, about 8 copies, about 9 copies, about 10 copies, about 11 copies, about 12 copies, about 13 copies, about 14 copies, about 15 copies, about 16 copies, about 17 copies, about 18 copies, about 19 copies, about 20 copies, about 30 copies, about 40 copies, about 50 copies, about 60 copies, about 70 copies, about 80 copies, or about 100 copies per cell. The copy number may be about 90 copies, about 100 copies, about 110 copies, about 120 copies, about 130 copies, about 140 copies, about 150 copies, about 160 copies, about 170 copies, about 180 copies, about 190 copies, about 200 copies, about 250 copies, about 300 copies, about 350 copies, about 400 copies, about 450 copies, about 500 copies, about 550 copies, about 600 copies, about 650 copies, about 700 copies, about 750 copies, or maintained between any two of these values. In some embodiments, the copy number of the circular DNA vector is at least about, up to about, or about 10 copies, about 11 copies, about 12 copies, about 13 copies, about 14 copies, about 15 copies, about 16 copies, about 17 copies, about 18 copies, about 19 copies, about 20 copies, about 25 copies, about 30 copies, about 35 copies, about 40 copies, about 45 copies, about 50 copies, about 55 copies, about 60 copies, about 65 copies, about about 70 copies, about 75 copies, about 80 copies, about 85 copies, about 90 copies, about 95 copies, about 100 copies, about 110 copies, about 120 copies, about 130 copies, about 140 copies, about 150 copies, about 160 copies, about 170 copies, about 180 copies, about 190 copies, about 200 copies, about 250 copies, about 300 copies, about 350 copies, or about 400 copies, or between any two of these values.

[0092] In some embodiments, the Rep gene integrated into the engineered bacterial cell genome is under the control of a constitutive promoter or a non-inducible promoter. In some embodiments, expression of the replication protein is not regulated before, during, or after the vector sequence is separated from the backbone sequence. In some embodiments, the parental plasmid before separation and the circular DNA vector after separation are at least about, up to about, or about 1 copy, about 2 copies, about 3 copies, about 4 copies, about 5 copies, about 6 copies, about 7 copies, about 8 copies, about 9 copies, about 10 copies, about 11 copies, about 12 copies, about 13 copies, about 14 copies, about 15 copies, about 16 copies, about 17 copies, about 18 copies, about 19 copies, about 20 copies, about 25 copies, about 30 copies, about 35 copies, or about 36 copies per cell. In some embodiments, the copy number is about 40 copies, about 45 copies, about 50 copies, about 55 copies, about 60 copies, about 65 copies, about 70 copies, about 75 copies, about 80 copies, about 85 copies, about 90 copies, about 95 copies, about 100 copies, about 110 copies, about 120 copies, about 130 copies, about 140 copies, about 150 copies, about 160 copies, about 170 copies, about 180 copies, about 190 copies, or about 200 copies, or between any two of these values.

[0093] In addition to the parental plasmid or vector sequences separated from the parental plasmid, the engineered bacteria may also contain other extrachromosomal DNA molecules, such as helper plasmids or BACs. The extrachromosomal DNA molecules may encode, for example, exogenous recombinases, restriction enzymes, replication proteins, ligases, selectable markers, counterselectable markers, or reporter genes. In some embodiments, the circular DNA vector is purified from the culture of engineered bacterial cells after the extrachromosomal DNA molecules other than the vector sequences are removed from the engineered bacterial cells. In some embodiments, the extrachromosomal DNA molecules can be removed from the engineered bacterial cells by culturing the cells under conditions that maintain the extrachromosomal DNA molecules without applying selective pressure, or by culturing the cells under counterselective conditions that reduce or eliminate the growth of cells containing the extrachromosomal DNA molecules.

[0094] In some embodiments, the extrachromosomal DNA molecule contained in the engineered bacterial cell includes a reporter construct that can be used to track the presence of the extrachromosomal DNA molecule in the cell. For example, the backbone sequence of the parent plasmid or the helper plasmid or BAC can include a gene encoding a visually detectable protein, such as GFP or RFP. In that case, visual observation of the color of the colony under ultraviolet light can reveal whether the extrachromosomal DNA molecule is present in the cells of the colony. In this way, colonies that lack a given extrachromosomal DNA molecule can be detected. Other suitable reporter constructs that can be detected in other ways can also be used to determine whether an engineered bacterial cell or colony contains a given extrachromosomal DNA molecule.

[0095] B. Parental Plasmid Embodiments of the engineered bacteria disclosed herein include a parent plasmid (also referred to as a template plasmid or plasmid template) that includes vector and backbone sequences separated from each other by two restriction or recombination sites.

[0096] The vector sequence includes an ori sequence and a sequence of interest that, in some embodiments, is a therapeutic coding sequence, a reporter construct, or a combination thereof. The vector sequence can include any of the components of the circular DNA vector embodiments described herein. In some embodiments, the vector sequence does not include any sequences of bacterial origin other than the ori sequence. In some embodiments, the parental plasmid includes restriction or recombination sites immediately adjacent to the ori sequence and / or the therapeutic sequence or reporter construct, such that there is no foreign or non-functional DNA included in the vector sequence that results in a circular DNA vector.

[0097] In some embodiments, the backbone sequence includes a selectable marker and does not include an ori sequence corresponding to the exogenous replication protein encoded by the integrated Rep gene. In some embodiments, the backbone sequence does not correspond to the integrated Rep gene, i.e., includes an ori sequence in the vector sequence and an ori sequence that is orthologous to the integrated Rep gene. In some embodiments, the selectable marker included in the backbone sequence helps ensure that the parental plasmid is maintained in a population of engineered bacterial cells cultured under conditions in which the selectable marker is necessary for cell growth or survival. For example, in some embodiments, the selectable marker is an antibiotic resistance gene. Culturing the engineered bacterial cells in the presence of the corresponding antibiotic provides selective pressure, thereby maintaining the parental plasmid in the population of engineered bacterial cells. However, when the selective pressure is removed, such as by changing the growth medium to a medium lacking the antibiotic corresponding to the antibiotic resistance gene, DNA molecules containing the antibiotic resistance gene may be lost from the population, especially if such DNA molecules do not include an ori sequence. Thus, once the vector sequence is separated from the backbone sequence, if the culture conditions do not provide selective pressure to maintain the backbone sequence, the backbone sequence may be lost from the population or may not be maintained in large amounts.

[0098] In some embodiments, the backbone sequence comprises a counterselectable marker. The counterselectable marker may provide a method for selectively growing cells that do not contain the backbone sequence. In some embodiments, after the vector sequence is separated from the backbone sequence, the cells may be grown under counterselective conditions to increase purity and reduce the amount of backbone sequence in the culture and / or in a composition that includes the purified vector sequence. Suitable counterselectable markers are known in the art and may include, for example, pheS, sacB, thyA, lacY, gata-1, ccdB, rpsL, or tetAR.

[0099] The restriction or recombination sites flanking the vector sequences in the parent plasmid can be selected from any suitable restriction or recombination sites that are not present in the vector sequences.

[0100] C. Restriction Digestion and Ligation The embodiments disclosed herein include a step of performing a restriction digest to separate the vector sequence from the parental plasmid backbone sequence. In some embodiments, the restriction digest is performed within the engineered bacterial cell. In some embodiments, the restriction enzyme that digests the parental plasmid is an exogenous restriction enzyme expressed from an exogenous restriction enzyme gene introduced into the engineered bacterial cell. In some embodiments, the exogenous gene is integrated into the genome of the engineered bacterial cell. In some embodiments, the exogenous gene is encoded on a plasmid or BAC within the engineered bacterial cell. In some embodiments, it is necessary to suppress or delay induction of expression of the restriction enzyme until such time as it is desired to separate the vector sequence from the parental plasmid backbone sequence. Thus, in some embodiments, the exogenous gene is operably linked to an inducible promoter. When separation is desired, expression of the restriction enzyme can be induced to allow separation to proceed.

[0101] In some embodiments, the restriction enzyme used to separate the vector sequence from the backbone sequence is an exogenous restriction enzyme that is introduced into the engineered bacterial cell across the cell membrane. In some embodiments, this is accomplished by electroporation. A non-limiting example of the electroporation and digestion procedure is as follows: Engineered electrocompetent E. coli carrying the parental plasmid are cultured in SOB at 30° C. to an OD of 0.8. The bacteria are washed three times with ice-cold 10% glycerol and resuspended in 10% glycerol. 0.5 μl each of restriction enzyme and ligase are mixed with the electrocompetent cells and 1 μg of DNA is digested with 10 units of the restriction enzyme. The mixture is transferred to a cuvette (1 mm gap) and electroporated using an electroporator (BTX) with a setting of 1800 volts. The cells are rescued by growing in SOC for 1 hour at 37° C. and plated on LB agar plates without antibiotics. Grow colonies and purify DNA using a QIAGEN miniprep kit.

[0102] After the vector sequences are separated, the vector sequences can be self-ligated to form a circular DNA vector. In some embodiments, the ligation is performed within the engineered bacterial cell. In some embodiments, the ligase that joins the ends of the vector sequences is an exogenous ligase expressed from an exogenous ligase gene introduced into the engineered bacterial cell. The ligase can be, for example, T3 ligase, T4 ligase, or T7 ligase. In some embodiments, the exogenous ligase gene is integrated into the genome of the engineered bacterial cell. In some embodiments, the exogenous ligase gene is encoded on a plasmid within the engineered bacterial cell. In some embodiments, expression of the ligase is repressed or not induced until such time that separation of the vector sequences from the parental plasmid backbone sequence is achieved. Thus, in some embodiments, the exogenous ligase gene is operably linked to an inducible promoter.

[0103] In some embodiments, the ligase is an exogenous ligase that is introduced into the engineered bacterial cell across the cell membrane. In some embodiments, this is accomplished by electroporation, which can be done according to the electroporation protocol described above. In some embodiments, electroporation of the restriction enzyme and ligase is done in a single step where both the restriction enzyme and the ligase enzyme enter the cell in a single electroporation step. In some embodiments, the restriction enzyme(s) and the ligase are added to the cell separately.

[0104] In some embodiments, self-ligation of vector sequences is accomplished by an endogenous ligase produced by the engineered bacterial cell.

[0105] In some embodiments, the exogenous restriction enzyme and the exogenous ligase are present simultaneously in the engineered bacterial cell. In some embodiments, the exogenous restriction enzyme is introduced into the engineered bacterial cell (e.g., by electroporation of the exogenous restriction enzyme, transformation with a DNA molecule encoding the exogenous restriction enzyme, or induction of expression of the exogenous restriction enzyme gene under the control of an inducible promoter) before the exogenous ligase is introduced into the engineered bacterial cell (e.g., by electroporation of the exogenous ligase, transformation with a DNA molecule encoding the exogenous ligase, or induction of expression of the exogenous ligase gene under the control of an inducible promoter). In some embodiments, the exogenous restriction enzyme is introduced into the engineered bacterial cell before the exogenous ligase is introduced into the engineered bacterial cell. In some embodiments, the exogenous restriction enzyme is introduced into the engineered bacterial cell simultaneously with the exogenous ligase.

[0106] D. Site-specific recombination Site-specific recombination can be carried out using a variety of systems that result in site-specific recombination between sequences. In some embodiments, site-specific recombination requires two specific sequences that allow recombination with each other in the presence of a recombinase.

[0107] In some embodiments, the recombinase that separates the vector sequences from the plasmid sequences is an exogenous recombinase expressed from an exogenous recombinase gene introduced into the engineered bacterial cell. In some embodiments, the exogenous recombinase gene is integrated into the genome of the engineered bacterial cell. In some embodiments, the exogenous recombinase gene is encoded on a plasmid or BAC in the engineered bacterial cell. In some embodiments, it is necessary to suppress or delay induction of expression of the recombinase until such time as it is desired to separate the vector sequences from the backbone sequences of the parental plasmid. Thus, in some embodiments, the exogenous recombinase gene is operably linked to an inducible promoter, such as any of the inducible promoters disclosed herein. When separation is desired, expression of the exogenous recombinase can be induced to allow separation to proceed. In some embodiments, the exogenous recombinase is expressed when the parental plasmid is introduced into the engineered bacterial cell, thereby allowing recombination of the parental plasmid to occur without the need to induce expression of the recombinase. In some embodiments, when the parental plasmid is introduced into the engineered bacterial cell, the recombinase is expressed at a relatively low level. As an example, in some embodiments, the engineered bacterial cell may include an exogenous recombinase gene (which may be, for example, integrated into the bacterial chromosome or included on a plasmid or BAC present in the bacterial cell) operably linked to an inducible promoter that provides low levels of expression under non-induced conditions. Introduction of the parental plasmid into an engineered bacterial cell with an appropriately low expression level of the exogenous recombinase may allow colony growth on a medium selective for the selectable marker on the backbone sequence of the parental plasmid, while also inducing sufficient recombination of the parental plasmid to generate a population of cells in the colony that has the vector sequence separated from the backbone sequence.

[0108] In some embodiments, the recombinase used to separate the vector sequences from the backbone sequences is an exogenous recombinase that is introduced into the engineered bacterial cells across the cell membrane. In some embodiments, this is accomplished by electroporation. A non-limiting example of the electroporation and recombination procedure is as follows: Engineered electrocompetent E. coli carrying the parental plasmid are cultured in SOB at 30° C. to an OD of 0.8. The bacteria are washed three times with ice-cold 10% glycerol and resuspended in 10% glycerol. 1 μl of Cre (15 units, NEB, M0298M) is mixed with 50 μl of electrocompetent cells. The mixture is transferred to a cuvette (1 mm gap) and electroporated using an electroporator (BTX) with a setting of 1800 volts. The cells are rescued by growing in SOC for 1 hour at 37° C. and plated on LB agar plates without antibiotics. Grow colonies and purify DNA using a QIAGEN miniprep kit. Digest 1 μg DNA with 10 units of restriction enzyme.

[0109] The particular recombination systems used in the embodiments disclosed herein may be of different origins, in particular the particular sequences and recombinases used may belong to different structural classes, such as the integrase family of bacteriophage λ or the resolvase family of transposon Tn3.

[0110] Recombinases belonging to the bacteriophage λ integrase family include, for example, the integrases of phages lambda (Landy et al., Science 197: 1147, 1977), P22, and φ80 (Leong et al., J. Biol. Chem. 260: 4468,1985), HP1 of Haemophilus influenza (Hauser et al., J. Biol. Chem. 267 6859,1992), Cre integrase of phage P1 (which recognizes LoxP sites and causes recombination), integrase of plasmid pSAM2 (European Patent No. 350,341), or FLP recombinase of the 2μ plasmid. In embodiments in which a circular DNA vector is prepared by recombination with a site-specific system of the integrase family of bacteriophage λ, the resulting circular DNA vector generally contains sequences resulting from recombination between two att attachment sequences of the corresponding bacteriophage or plasmid.

[0111] Recombinases belonging to the transposon Tn3 family include, for example, the resolvase of transposon Tn3 or transposon Tn21 and Tn522 (Stark et al., Trends Genet, 8, 432-439, 1992), the Gin invertase of bacteriophage mu, or a plasmid resolvase such as the resolvase of the par fragment of RP4 (Albert et al., Mol. Microbiol. 12: 131, 1994). In an embodiment in which a circular DNA vector is prepared by recombination using a site-specific system of the transposon Tn3 family, the resulting circular DNA vector generally contains a sequence resulting from recombination between two recognition sequences of the resolvase of the transposon.

[0112] In some embodiments, the site-specific recombination sequences on the parental plasmid are derived from a bacteriophage. In some embodiments, these sequences are the attachment sequences of the bacteriophage integrase (attP and attB sequences) or sequences derived from such attachment sequences. These sequences are capable of specifically recombining with each other in the presence of a recombinase called integrase, with or without excisionase. The term "sequences derived from such attachment sequences" includes sequences obtained by modification(s) of the attachment sequence(s) of the bacteriophage that retain the ability to specifically recombine in the presence of the appropriate recombinase. Such sequences may therefore be shortened fragments of these sequences or fragments extended by the addition of other sequences (such as restriction sites). They may also be variants obtained by mutation, in particular point mutation, such as, for example, the attP-GA and attB-GA attachment sequences.

[0113] In some embodiments, the recognition sequences and recombinases used are derived from members of the tyrosine recombinase family, such as, for example, Flp, XerC, XerD, λ integrase, or HP1 integrase, or from members of the serine recombinase family, such as, for example, φBT1, TP901, Bxb1, MR11, A118, φK38, φC31, or Wβ.

[0114] In some embodiments, the recognition sequence and recombinase are derived from Bxb1 (eg, the exogenous recombinase is Bxb1 and the recognition sequences are attP-GA and attB-GA).

[0115] E. Amplification of circular DNA vector by cell culture After forming a circular DNA vector in an engineered bacterial cell according to the embodiments described herein, the amount of circular DNA vector produced can be increased by culturing a population of engineered bacterial cells containing the circular DNA vector. Culture conditions can be selected to maximize bacterial cell growth and production of additional copies of the circular DNA vector. In some embodiments, culture conditions are selected to induce high levels of expression of replication proteins, thereby supporting high copy numbers of the circular DNA vector with the corresponding ori sequence. In some embodiments, culture conditions are selected to remove selection pressure for the maintenance of the backbone sequence containing the selectable marker, such that the backbone sequence is not maintained through cell division. In some embodiments, culture conditions are selected to provide a counter-selection pressure for the counter-selection marker present on the backbone sequence, such that cells containing the backbone sequence have reduced growth capacity or are unable to grow.

[0116] In some embodiments, culturing a population of engineered bacterial cells containing the circular DNA vector results in maintenance of the circular DNA vector within such cultured cells through at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 cell divisions. In some embodiments, a cultured population of engineered bacterial cells contains at least 1 copy, at least 2 copies, at least 3 copies, at least 4 copies, at least 5 copies, at least 6 copies, at least 7 copies, at least 8 copies, at least 9 copies, at least 10 copies, at least 11 copies, at least 12 copies, at least 13 copies, at least 14 copies, at least 15 copies, at least 16 copies, at least 17 copies, at least 18 copies, at least 19 copies, at least 20 copies, at least 25 copies, at least 30 copies, at least 35 copies, at least 40 copies, at least 45 copies, at least 50 copies, at least 55 copies, at least 60 copies, at least 65 copies, at least 70 copies, at least 75 copies, at least 80 copies, at least 85 copies, at least 90 copies, at least 95 copies, or at least 100 copies of the engineered bacterial cells. copies, at least 6 copies, at least 7 copies, at least 8 copies, at least 9 copies, at least 10 copies, at least 11 copies, at least 12 copies, at least 13 copies, at least 14 copies, at least 15 copies, at least 16 copies, at least 17 copies, at least 18 copies, at least 19 copies, at least 20 copies (e.g., at least 1 copy per cell after at least 10 doublings (e.g., at least 5 copies per cell after at least 10 doublings, at least 10 copies per cell after at least 10 doublings, or at least 20 copies per cell after at least 10 doublings), at least 1 copy per cell after at least 20 doublings (e.g.,The circular DNA vector is maintained at at least 5 copies per cell after at least 20 doublings, at least 10 copies per cell after at least 20 doublings, or at least 20 copies per cell after at least 20 doublings), at least 1 copy per cell after at least 50 doublings (e.g., at least 5 copies per cell after at least 50 doublings, at least 10 copies per cell after at least 50 doublings, or at least 20 copies per cell after at least 50 doublings), or at least 1 copy per cell after at least 100 doublings (e.g., at least 5 copies per cell after at least 100 doublings, at least 10 copies per cell after at least 100 doublings, or at least 20 copies per cell after at least 100 doublings). In some embodiments, the average copy number of the backbone sequence after separation of the vector sequence from the backbone sequence is at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 65 times, at least 70 times, at least less than 5 copies, less than 4 copies, less than 3 copies, less than 2 copies, less than 1 copy, less than 0.5 copies, less than 0.1 copies, less than 0.01 copies, or less than 0.001 copies per cell after at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 doublings, or is undetectable (e.g., less than 0.001 copies per cell after at least 1 doubling (e.g., less than 0.001 copies per cell after at least 10 doublings, less than 0.001 copies per cell after at least 20 doublings, less than 0.001 copies per cell after at least 50 doublings, or less than 0.001 copies per cell after at least 100 doublings),less than 0.01 copy per cell after at least 1 doubling (e.g., less than 0.01 copy per cell after at least 10 doublings, less than 0.01 copy per cell after at least 20 doublings, less than 0.01 copy per cell after at least 50 doublings, or less than 0.01 copy per cell after at least 100 doublings), less than 0.1 copy per cell after at least 1 doubling (e.g., less than 0.1 copy per cell after at least 10 doublings, less than 0.1 copy per cell after at least 20 doublings, less than 0.1 copy per cell after at least 50 doublings, or less than 0.1 copy per cell after at least 100 doublings), or less than 1 copy per cell after at least 1 doubling (e.g., less than 1 copy per cell after at least 10 doublings, less than 1 copy per cell after at least 20 doublings, less than 1 copy per cell after at least 50 doublings, or less than 1 copy per cell after at least 100 doublings). In some embodiments, the average copy number of the backbone sequence after separation of the vector sequence from the backbone sequence is up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, up to 50, up to 55, up to 60, up to 65, up to 70, up to 75, up to 80, up to 85, up to 90, up to 95, or up to 100 doublings. less than 5 copies, less than 4 copies, less than 3 copies, less than 2 copies, less than 1 copy, less than 0.5 copies, less than 0.1 copies, less than 0.01 copies, or less than 0.001 copies per cell, or undetectable (e.g., less than 0.001 copies per cell after up to 1 doubling (e.g., less than 0.001 copies per cell after up to 10 doublings, less than 0.001 copies per cell after up to 20 doublings, less than 0.001 copies per cell after up to 50 doublings, or less than 0.001 copies per cell after up to 100 doublings), less than 0.01 copies per cell after up to 1 doubling (e.g.,less than 0.01 copy per cell after up to 10 doublings, less than 0.01 copy per cell after up to 20 doublings, less than 0.01 copy per cell after up to 50 doublings, or less than 0.01 copy per cell after up to 100 doublings), less than 0.1 copy per cell after up to 1 doubling (e.g., less than 0.1 copy per cell after up to 10 doublings, less than 0.1 copy per cell after up to 20 doublings, less than 0.1 copy per cell after up to 50 doublings, or less than 0.1 copy per cell after up to 100 doublings), or less than 1 copy per cell after up to 1 doubling (e.g., less than 1 copy per cell after up to 10 doublings, less than 1 copy per cell after up to 20 doublings, less than 1 copy per cell after up to 50 doublings, or less than 1 copy per cell after up to 100 doublings).

[0117] In some embodiments, culturing a population of engineered bacterial cells containing the circular DNA vector results in maintenance of the circular DNA vector in such cultured cells through at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 150, at least 200, at least 250, at least 290, at least 294, at least 300, at least 350, at least 400, at least 450, or at least 500 cell divisions (e.g., as confirmed by Sanger sequencing).In some embodiments, the cultured population of engineered bacterial cells is subjected to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 150, at least 200, at least 250, at least The circular DNA vector is maintained at an average copy number of at least 1 copy, at least 2 copies, at least 3 copies, at least 4 copies, at least 5 copies, at least 6 copies, at least 7 copies, at least 8 copies, at least 9 copies, at least 10 copies, at least 11 copies, at least 12 copies, at least 13 copies, at least 14 copies, at least 15 copies, at least 16 copies, at least 17 copies, at least 18 copies, at least 19 copies, or at least 20 copies per cell after at least 290 doublings, at least 294 copies, at least 300 copies, at least 350 copies, at least 400 copies, at least 450 copies, or at least 500 doublings.In some embodiments, the average copy number of the backbone sequence after separation of the vector sequence from the backbone sequence is at most 1 time, at most 2 times, at most 3 times, at most 4 times, at most 5 times, at most 6 times, at most 7 times, at most 8 times, at most 9 times, at most 10 times, at most 11 times, at most 12 times, at most 13 times, at most 14 times, at most 15 times, at most 16 times, at most 17 times, at most 18 times, at most 19 times, at most 20 times, at most 25 times, at most 30 times, at most 35 times, at most 40 times, at most 45 times, at most 50 times, at most 55 times, at most 60 times, at most 65 times, at most 70 times, at most 75 times, at most 80 times, at most 85 times, at most less than 5 copies, less than 4 copies, less than 3 copies, less than 2 copies, less than 1 copy, less than 0.5 copies, less than 0.1 copies, less than 0.01 copies, or less than 0.001 copies per cell after up to 90, up to 95, up to 100, 150, up to 200, up to 250, up to 290, up to 294, up to 300, up to 350, up to 400, up to 450, or up to 500 doublings, or is undetectable (e.g., less than 0.001 copies per cell after up to 1 doubling (e.g., less than 0.001 copies per cell after up to 10 doublings). less than 1 copy per cell after up to 20 doublings, less than 0.001 copy per cell after up to 50 doublings, or less than 0.001 copy per cell after up to 500 doublings), less than 0.01 copy per cell after up to 1 doubling (e.g., less than 0.01 copy per cell after up to 10 doublings, less than 0.01 copy per cell after up to 20 doublings, less than 0.01 copy per cell after up to 50 doublings, or less than 0.01 copy per cell after up to 100 doublings), less than 0.1 copy per cell after up to 10 doublings (e.g., less than 0.1 copy per cell after up to 20 doublings, less than 0.1 copy per cell after up to 50 doublings, or less than 0.1 copy per cell after up to 100 doublings), or less than 1 copy per cell after up to 1 doubling (e.g., less than 1 copy per cell after up to 10 doublings, less than 1 copy per cell after up to 20 doublings, less than 1 copy per cell after up to 50 doublings, or less than 1 copy per cell after up to 100 doublings).

[0118] Some embodiments include a culture of engineered bacterial cells in which the average copy number of the circular DNA vector or parental plasmid is at least 1 copy, at least 2 copies, at least 3 copies, at least 4 copies, at least 5 copies, at least 6 copies, at least 7 copies, at least 8 copies, at least 9 copies, at least 10 copies, at least 11 copies, at least 12 copies, at least 13 copies, at least 14 copies, at least 15 copies, at least 16 copies, at least 17 copies, at least 18 copies, at least 19 copies, at least 20 copies, at least 25 copies, at least 30 copies, at least 35 copies, at least 40 copies, at least 45 copies, at least 50 copies, at least 55 copies, at least 60 copies, at least 65 copies, at least 70 copies, at least 75 copies, at least 80 copies, at least 85 copies, at least 90 copies, at least 95 copies, or at least 100 copies per cell, or between any two of these values. In some embodiments, the culture is at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 10 or at least 10 12 In some embodiments, the culture comprises at least 10 total cells per ml, or between any two of these values. 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 10 or at least 10 10 cells, or between any two of these values.

[0119] E. Recovery of circular DNA vector The circular DNA vector produced by the embodiments disclosed herein can be recovered from the engineered bacterial culture by extraction and purification procedures known in the art. In some embodiments, at least 0.001 mg, at least 0.01 mg, at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.5 mg, at least 0.6 mg, at least 0.7 mg, at least 0.8 mg, at least 0.9 mg, at least 1.0 mg, at least 1.1 mg, at least 1.2 mg, at least 1.3 mg, at least 1.4 mg, at least 1.5 mg, at least 1.6 mg, at least 1.7 mg, at least 1.8 mg, at least 1.9 mg, at least 2.0 mg, at least 2.5 mg, at least 3.0 mg, at least 3.5 mg, at least 4.0 mg, at least 4.5 mg, or at least 5.0 mg of circular DNA vector can be recovered per liter of cultured engineered bacterial cells. In some embodiments, the circular DNA vector undergoes a purification procedure that reduces the amount of bacterial contaminants, such as endotoxins, to levels acceptable for use in pharmaceutical compositions. Suitable purification procedures include chromatographic procedures, such as anion exchange chromatography or hydrophobic interaction chromatography.

[0120] In some embodiments, the circular DNA vector is purified by gel electrophoresis to further avoid contamination with backbone sequences that may be maintained in cultures of engineered bacterial cells. In some embodiments, no purification is necessary to avoid detectable contamination of the circular DNA vector with backbone sequences.

[0121] In some embodiments, the circular DNA vector can be purified from a culture of engineered bacterial cells described herein without contamination of the purified product with backbone sequences or any other extrachromosomal DNA molecules. In some embodiments, a composition of isolated circular DNA vector purified from engineered bacterial cells disclosed herein contains less than 10 ng / ml, less than 1 ng / ml, less than 0.1 ng / ml, less than 0.01 ng / ml, less than 0.001 ng / ml, or less than 0.0001 ng / ml of DNA containing backbone sequences. In some embodiments, DNA containing backbone sequences is undetectable in the composition by quantitative PCR. In some embodiments, these purity levels are achieved without performing a gel or column purification step after isolating the circular DNA vector from the engineered bacterial cells.

[0122] In some embodiments, the methods for making circular DNA vectors disclosed herein comply with current Good Manufacturing Practices (GMP) as promulgated by the U.S. Food and Drug Administration and in accordance with the standards set forth in Title 21 Code of Federal Regulations, Sections 210 and 211, which are incorporated herein by reference in their entirety.

[0123] III. Circular DNA Vectors Provided herein is a circular DNA vector produced by any of the production methods described herein. In some cases, such circular DNA vectors remain in cells (e.g., in dividing cells or in quiescent cells such as post-mitotic cells) as episomes, for example in a manner similar to AAV vectors. In any of the embodiments described herein, the circular DNA vector may be a non-integrating vector. The circular DNA vectors provided herein may be naked DNA vectors that lack components inherent to viral vectors (e.g., viral proteins) and bacterial plasmid DNA, such as immunogenic components (e.g., immunogenic bacterial signatures (e.g., CpG islands or CpG motifs)) or additional components, or components otherwise associated with reduced persistence (e.g., CpG islands or CpG motifs). The circular DNA vectors produced as described herein may feature one or more therapeutic sequences and lack plasmid backbone elements (e.g., drug resistance genes). In some embodiments, the circular DNA vector lacks recombination sites. In some embodiments, particularly those in which recombination is used to remove vector sequences from plasmid backbone sequences, the circular DNA vector comprises recombination sites.

[0124] In some embodiments, the vector comprises DNA in which at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or essentially all) of the DNA lacks one or more elements of bacterial plasmid DNA, such as immunogenic components (e.g., immunogenic bacterial signatures (e.g., CpG motifs)) or additional components or components otherwise associated with reduced persistence (e.g., CpG islands). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or essentially all) of the DNA lacks CpG methylation. In some embodiments, the vector comprises DNA in which at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or essentially all) of the DNA lacks bacterial methylation signatures such as Dam methylation and Dcm methylation. For example, in some embodiments, the vector comprises DNA in which at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or essentially all) of the GATC sequences are unmethylated (e.g., by Dam methylase). Additionally or alternatively, the vector comprises DNA in which at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or essentially all) of the CCAGG and / or CCTGG sequences are unmethylated (e.g., by Dcm methylase).

[0125] In some embodiments, the circular DNA vector is survivable in vivo (e.g., the circular DNA vector exhibits improved expression survivability (e.g., intracellular and / or transgenerational survivability) and / or therapeutic survivability compared to a reference vector, e.g., a circular DNA vector produced in bacteria, or a vector of the invention, e.g., a circular DNA vector having one or more bacterial signatures not present in plasmid DNA). In some embodiments, the expression survivability of the circular DNA vector is 5%-50% greater, 50%-100% greater, 1-5-fold, or 5-10-fold greater (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or more) greater than the reference vector. In some embodiments, the intracellular persistence of the circular DNA vector is 5% to 50% greater, 50% to 100% greater, 1 to 5 times, or 5 to 10 times (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 1 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, or more) greater than a reference vector. In some embodiments, the intergenerational persistence of the circular DNA vector is 5% to 50% greater, 50% to 100% greater, 1 to 5 times, or 5 to 10 times (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 1 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, or more) greater than a reference vector.In some embodiments, the therapeutic survivability of the circular DNA vector is 5%-50% greater, 50%-100% greater, 1-5-fold, or 5-10-fold greater (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or more) greater than that of a reference vector. In some embodiments, a reference vector is a circular vector or plasmid that (a) has the same coding sequence as the compared circular DNA vector, and (b) has one or more bacterial signatures that are not present in the compared circular DNA vector, which signatures may include, for example, antibiotic resistance genes or other selectable markers.

[0126] In some embodiments, expression of the circular DNA vector persists for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or more after administration. In certain embodiments, the circular DNA vector exhibits intracellular and / or transgenerational persistence for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or more after administration. In some embodiments, therapeutic persistence of the circular DNA vector persists for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or more after administration.

[0127] In some embodiments, expression and / or therapeutic effect of the circular DNA vector remains for 1 to 4 weeks, 1 to 4 months, or 4 months to 1 year (e.g., at least 1 week, at least 2 weeks, at least 1 month, or more). In some embodiments, the expression level of the circular DNA vector does not decrease by more than 90%, more than 50%, or more than 10% from the level observed within the first 1, 2, or 3 days at least 1 week, e.g., 2, 3, 5, 7, 9, or more weeks, 13, or more weeks, 18, or more weeks after transfection.

[0128] The circular DNA vector may be monomeric, dimeric, trimer, tetramer, pentamer, hexamer, etc. In some preferred embodiments, the circular DNA vector is monomeric. In some embodiments, the DNA vector is supercoiled. The circular DNA vector may be supercoiled by endogenous processes in engineered bacterial cells or by treatment with a topoisomerase (e.g., gyrase). In some embodiments, the circular DNA vector is a monomeric supercoiled circular DNA molecule. In some embodiments, the circular DNA vector is nicked. In some embodiments, the circular DNA vector is open circular. In some embodiments, the circular DNA vector is double stranded circular. In some embodiments, a composition comprising a circular DNA vector comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.9% supercoiled monomers. In some embodiments, a composition comprising a circular DNA vector comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.9% supercoiled monomers without treatment with exogenous topoisomerase.

[0129] IV. Therapeutic Sequences In some embodiments, the coding sequence of the circular DNA vectors described herein comprises a therapeutic sequence, which may include one or more protein coding domains and / or one or more non-protein coding domains (e.g., a therapeutic nucleic acid).

[0130] In certain embodiments comprising a therapeutic domain encoding a therapeutic protein, the therapeutic sequence comprises a promoter and a single therapeutic protein coding domain (e.g., a single transcription unit), a promoter and two or more therapeutic protein coding domains (e.g., a multicistronic unit), or a first transcription unit and one or more additional transcription units (e.g., a multiple transcription unit) linked in a 5' to 3' direction. Any such protein-encoding therapeutic sequence may further comprise non-protein coding domains, such as polyadenylation sites, control elements, enhancers, sequences for labeling DNA (e.g., for antibody recognition), PCR amplification sites, sequences defining restriction enzyme sites, site-specific recombinase recognition sites, sequences recognized by proteins that bind and / or modify nucleic acids, linkers, splice sites, pre-mRNA binding domains, control sequences, and / or therapeutic nucleic acids (e.g., sequences encoding microRNAs). The therapeutic protein coding domain may be a full-length protein coding domain (e.g., corresponding to the original gene or a naturally occurring variant thereof) or a functional portion thereof, such as a truncated protein coding domain (e.g., a minigene).

[0131] In some embodiments, the therapeutic sequence encodes a monomeric protein (e.g., a monomeric protein that has a secondary structure under physiological conditions, e.g., a monomeric protein that has a secondary structure and a tertiary structure under physiological conditions, e.g., a monomeric protein that has a secondary structure, a tertiary structure, and a quaternary structure under physiological conditions). Additionally or alternatively, the therapeutic sequence may encode a multimeric protein (e.g., a dimeric protein (e.g., a homodimeric protein or a heterodimeric protein), a trimeric protein, etc.).

[0132] In certain cases, the therapeutic sequence includes an ocular gene. In some embodiments, the ocular gene is a gene expressed in ocular tissue, such as retinal tissue, which may include, for example, photoreceptor cells and / or retinal pigment epithelium (RPE) cells. In some embodiments, the coding sequence in the expression construct disclosed herein is a human ABCA4 gene sequence or a human MYO7A gene sequence. An exemplary human ABCA4 gene sequence is provided as National Center for Biotechnology Information (NCBI) Reference Sequence: NG_009073. An exemplary amino acid sequence of an ABCA4 protein is provided by protein accession number P78363.3. An exemplary human MYO7A gene sequence is provided as NCBI Gene ID: 4647. An exemplary amino acid sequence of an MYO7A protein is provided by protein accession number Q13402.

[0133] In some embodiments, the therapeutic sequence encodes an antibody, or a portion, fragment, or variant thereof. Antibodies include fragments capable of binding antigen, such as Fv, single chain Fv (scFv), Fab, Fab', di-scFv, sdAb (single domain antibody), (Fab')2 (including chemically linked F(ab')2), and nanobodies. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting its ability to crystallize easily. Pepsin treatment yields a F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. Antibodies also include chimeric and humanized antibodies. Additionally, for all antibody constructs provided herein, variants with sequences from other organisms are also contemplated. Thus, where a humanized version of an antibody is disclosed, one of skill in the art would know how to convert the human sequence-based antibody to mouse, rat, cat, dog, horse, etc. sequences. Antibody fragments include any orientation, such as single chain scFv, tandem di-scFv, diabodies, tandem tri-sdcFv, minibodies, nanobodies, etc. In some embodiments, for example when the antibody is an scFv, a single polynucleotide of the therapeutic gene sequence encodes a single polypeptide that includes both heavy and light chains linked together. Antibody fragments also include nanobodies (e.g., antibodies with a single monomer domain, such as sdAbs, a pair of variable domains of a heavy chain without a light chain). Multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.) are known in the art and are considered as expression products of the therapeutic gene sequences of the present invention.

[0134] In some cases, the therapeutic sequence may be at least 25 amino acids, at least 50 amino acids, at least 100 amino acids, at least 200 amino acids, at least 500 amino acids, at least 1000 amino acids, at least 1500 amino acids, at least 2000 amino acids, at least 2500 amino acids, at least 3000 amino acids, or more (e.g., 25 amino acids to 5000 amino acids, 50 amino acids to 5000 amino acids, 100 amino acids to 5000 amino acids, 200 amino acids to 5000 amino acids, 500 amino acids to 5000 amino acids, 1000 amino acids to 5000 amino acids, 1500 amino acids to 5000 amino acids, or 2000 amino acids to 5000 amino acids, such as 25 amino acids to 4000 amino acids, 50 amino acids to 50 amino acids, or more). The therapeutic sequence may encode one or more proteins (e.g., a single protein, two proteins, three proteins, four proteins, or more) having a length of 200 to 4000 amino acids, 100 to 4000 amino acids, 200 to 4000 amino acids, 500 to 4000 amino acids, 1000 to 4000 amino acids, 1500 to 4000 amino acids, or 2000 to 4000 amino acids, for example, 25 to 3000 amino acids, 50 to 3000 amino acids, 100 to 3000 amino acids, 200 to 3000 amino acids, 500 to 3000 amino acids, 1000 to 3000 amino acids, 1500 to 3000 amino acids, or 2000 to 3000 amino acids). In embodiments where such a therapeutic sequence encodes two or more proteins, the therapeutic sequence may be a multicistronic therapeutic sequence or a multiple transcription unit therapeutic sequence. Such a multicistronic therapeutic sequence may be, for example, a tricistronic cassette encoding Flt3L, IL-12, and XCL1 as described in Example 7 herein.

[0135] In embodiments that include a therapeutic sequence that does not encode a protein, the therapeutic sequence lacks a protein-coding domain (e.g., a therapeutic protein-coding domain). For example, in some embodiments, the therapeutic sequence includes a non-protein-coding therapeutic nucleic acid, such as a sequence encoding a small hairpin RNA (shRNA) or an immune stimulatory therapeutic nucleic acid (e.g., a TLR agonist).

[0136] In some embodiments, the therapeutic sequence or other sequence of interest (which may include non-therapeutic coding sequences, such as reporter genes used to measure expression or persistence) is between 0.1 Kb and 100 Kb in length (e.g., the sequence may be between 0.2 Kb and 90 Kb, 0.5 Kb and 80 Kb, 1.0 Kb and 70 Kb, 1.5 Kb and 60 Kb, 2.0 Kb and 50 Kb, 2.5 Kb and 45 Kb, 3.0 Kb and 40 Kb, 3.5 Kb and 35 Kb, 4.0 Kb and 30 Kb, 4.5 Kb and 25 Kb, 4.6 Kb and 24 Kb, 4.7 Kb and 23 Kb, 4.8 Kb and 5.9 Kb, 5.1 Kb and 5.2 Kb, 5.3 Kb and 5.4 Kb, 5.5 Kb and 5.6 Kb, 5.7 Kb and 5.8 Kb, 5.8 Kb and 5.9 Kb, 6.1 Kb and 6.2 Kb, 6.3 Kb and 6.4 Kb, 6.5 Kb and 6.6 Kb, 6.7 Kb and 6.8 Kb, 7.1 Kb and 7.2 Kb, 7.2 Kb and 7.4 Kb, 7.4 Kb and 7.6 Kb, 7.5 Kb and 7.8 Kb, 7.8 Kb and 7.9 Kb, 8.1 Kb and 8.2 Kb, 8.3 Kb and 8.4 Kb, 8.5 Kb and 8.6 Kb, 8.7 Kb and 8.8 Kb, 8.8 Kb and 8.9 Kb, 9.1 Kb and 9. 22Kb, 4.9Kb to 21Kb, 5.0Kb to 20Kb, 5.5Kb to 18Kb, 6.0Kb to 17Kb, 6.5Kb to 16Kb, 7.0Kb to 15Kb, 7.5Kb to 14Kb, 8.0Kb to 13Kb, 8.5Kb to 12.5Kb, 9.0Kb to 12.0Kb, 9.5Kb to 11.5Kb, or 10.0Kb to 11.0Kb in length, for example, 0.1Kb to 0.5Kb, 0.5Kb to 1.0Kb, 1.0Kb to 2.5Kb, 2.5Kb to 4.5Kb, 4.5Kb to 8Kb, 8Kb to 10Kb, 10Kb to 15Kb, 15Kb to 20Kb in length, More than that, for example, 0.1Kb~0.25Kb, 0.25Kb~0.5Kb, 0.5Kb~1.0Kb, 1.0Kb~1.5Kb, 1.5Kb~2.0Kb, 2.0Kb~2.5Kb, 2.5Kb~3.0Kb, 3.0Kb~3.5Kb, 3.5Kb~4.0Kb, 4.0Kb~4.5Kb, 4.5Kb~5.0Kb, 5.0Kb~5.5Kb, 5.5Kb~6.0Kb, 6.0Kb~6.5Kb, 6.5Kb~7.0Kb, 7.0Kb~7.5Kb, 7.5Kb~8.0Kb, 8.0Kb~8.5Kb, 8.5Kb~9.0Kb, 9.0Kb~9 .5Kb, 9.5Kb~10Kb, 10Kb~10.5Kb, 10.5Kb~11Kb, 11Kb~11.5Kb, 11.5Kb~12 Kb, 12Kb~12.5Kb, 12.5Kb~13Kb, 13Kb~13.5Kb, 13.5Kb~14Kb, 14Kb~14.5Kb , 14.5Kb~15Kb, 15Kb~15.5Kb, 15.5Kb~16Kb, 16Kb~16.5Kb, 16.5Kb~17Kb, 1 7Kb~17.5Kb, 17.5Kb~18Kb, 18Kb~18.5Kb, 18.5Kb~19Kb, 19Kb~19.5Kb, 19.The length is 5Kb to 20Kb, 20Kb to 21Kb, 21Kb to 22Kb, 22Kb to 23Kb, 23Kb to 24Kb, 24Kb to 25Kb or more, for example, about 4.5Kb, about 5.0Kb, about 5.5Kb, about 6.0Kb, about 6.5Kb, about 7.0Kb, about 7.5Kb, about 8.0Kb, about 8.5Kb, about 9.0Kb, about 9.5Kb, about 10Kb, about 11Kb, about 12Kb, about 13Kb, about 14Kb, about 15Kb, about 16Kb, about 17Kb, about 18Kb, about 19Kb, about 20Kb or more). In some embodiments, the therapeutic sequence is at least 10 Kb (e.g., 10 Kb to 15 Kb, 15 Kb to 20 Kb, or 20 Kb to 30 Kb, e.g., 10 Kb to 13 Kb, 10 Kb to 12 Kb, or 10 Kb to 11 Kb, e.g., 10 Kb to 11 Kb, 11 Kb to 12 Kb, 12 Kb to 13 Kb, 13 Kb to 14 Kb, or 14 Kb to 15 Kb). In some embodiments, the sequence is at least 1100 bp in length (e.g., 1100 bp to 10000 bp, 1100 bp to 8000 bp, or 1100 bp to 5000 bp in length). In some embodiments, the sequence is at least 2500 bp in length (e.g., 2500 bp to 15000 bp, 2500 bp to 10000 bp, or 2500 bp to 5000 bp in length, e.g., 2500 bp to 5000 bp, 5000 bp to 7500 bp, 7500 bp to 10000 bp, 10000 bp to 12500 bp, or 12500 bp to 15000 bp). In some embodiments, the sequence is at least 8000 bp, at least 9000 bp, at least 10000 bp, at least 11000 bp, at least 12000 bp, at least 13000 bp, at least 14000 bp, at least 15000 bp, at least 16000 bp (e.g., 11000 bp to 16000 bp, 12000 bp to 16000 bp, 13000 bp to 16000 bp, 14000 bp to 16000 bp, or 15000 bp to 16000 bp). In certain embodiments, the sequence is large enough to encode a protein and is not an oligonucleotide therapeutic (e.g., not an antisense therapeutic, siRNA therapeutic, shRNA therapeutic, etc.).

[0137] In some embodiments, the 3' end of a sequence of interest, such as a therapeutic coding sequence or a non-therapeutic coding sequence, is 50 bp or less (e.g., 3 bp to 34 bp, 4 bp to 20 bp, 5 bp to 12 bp, or 6 bp to 10 bp, e.g., 3 bp to 5 bp, 4 bp to 6 bp, 8 bp to 12 bp, 12 bp to 18 bp, 18 bp to 24 bp, 24 bp to 30 bp, 30 bp to 35 bp, or 35 bp to 40 bp, e.g., 3 bp, 4 bp, 5 bp, 6 bp, p, 7 bp, 8 bp, 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 31 bp, 32 bp, 33 bp, 34 bp, 35 bp, 36 bp, 37 bp, 38 bp, 39 bp, 40 bp, 41 bp, 42 bp, 43 bp, 44 bp, 45 bp, 46 bp, 47 bp, 48 bp, 49 bp, or 50 bp) of a non-bacterial sequence (e.g., a recombination site, such as a recombination scar).

[0138] In some embodiments, the 3' end of a sequence of interest, such as a therapeutic coding sequence or a non-therapeutic coding sequence, is connected to the 5' end of an ori sequence in the circular DNA vector by a non-bacterial sequence of 30 bp or less (e.g., 3 bp to 24 bp, 4 bp to 18 bp, 5 bp to 12 bp, or 6 bp to 10 bp, e.g., 3 bp to 5 bp, 4 bp to 6 bp, 8 bp to 12 bp, 12 bp to 18 bp, 18 bp to 24 bp, or 24 bp to 30 bp, e.g., 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 10 bp, 15 bp, 20 bp, 25 bp, or 30 bp).

[0139] In some embodiments, the sequence of interest contained in the circular DNA vector described herein comprises a reporter sequence in addition to a protein-encoding or non-protein-encoding therapeutic domain. In some embodiments, the therapeutic sequence lacks a reporter sequence. In some embodiments, the sequence of interest comprises a reporter sequence and does not comprise a therapeutic sequence. The reporter sequence can be, for example, a reporter gene. Such reporter genes can be useful, for example, to verify the expression of the therapeutic gene sequence in specific cells and tissues. Reporter sequences that can be provided in the circular DNA vector include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art. When the reporter sequence is associated with a control element that drives its expression, it produces a signal that can be detected by conventional means, including enzyme assays, radioactive assays, colorimetric assays, fluorescent assays, or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays, including enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemistry. For example, if the marker sequence is the LacZ gene, the presence of the signal-mediating vector is detected by assaying for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the signal-mediating vector can be visually measured by color or light emission in a luminometer.

[0140] As part of a therapeutic or other sequence of interest, the circular DNA vector of the present invention may include conventional control elements that regulate or improve transcription, translation, and / or expression in target cells. Suitable control elements are described in WO 2021 / 055760, which is incorporated herein by reference in its entirety.

[0141] In some cases, the self-replicating RNA molecule includes (i) a sequence encoding a replicase (e.g., an RNA sequence encoding an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule) and (ii) a heterologous regulatory gene. The polymerase can be an alphavirus replicase, e.g., an alphavirus replicase that includes one, two, three, or all four of the alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4. In some cases, the polymerase is a VEE replicase, e.g., a VEE replicase that includes one, two, three, or all four of the alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4.

[0142] In some cases of the invention, the self-replicating RNA molecule does not encode an alphavirus structural protein (e.g., capsid protein). Such a self-replicating RNA may lead to the production of its own genomic RNA copy in a cell, but not to the production of virions containing the RNA. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecule is unable to persist by itself in an infectious form. The structural proteins of the alphavirus can be replaced by a gene(s) encoding the heterologous regulatory protein(s) of interest, such that the subgenomic transcript encodes the heterologous regulatory protein(s) rather than the structural proteins of the alphavirus virion.

[0143] Thus, in some cases, a self-replicating RNA molecule of the invention may have two open reading frames: a first (5') open reading frame encoding a replicase and a second (3') open reading frame encoding one or more (e.g., two or three) therapeutic proteins. In some embodiments, the RNA may have additional (e.g., downstream) open reading frames, e.g., encoding additional genes or encoding accessory polypeptides.

[0144] Suitable self-replicating RNA molecules may have a variety of lengths. In some embodiments of the invention, the length of the self-replicating RNA molecule is between 5,000 nucleotides and 50,000 nucleotides (i.e., between 5 kb and 50 kb). In some cases, the self-replicating RNA molecule is between 5 kb and 20 kb in length (e.g., between 6 kb and 18 kb, between 7 kb and 16 kb, between 8 kb and 14 kb, or between 9 kb and 12 kb in length, e.g., between 5 kb and 6 kb, between 6 kb and 7 kb, between 7 kb and 8 kb, between 8 kb and 9 kb, between 9 kb and 10 kb, between 10 kb and 11 kb, between 11 kb and 12 kb, between 12 kb and 13 kb, between 13 kb and 14 kb, between 14 kb, etc.). about 15 kb, 15 kb to 16 kb, 16 kb to 18 kb, or 18 kb to 20 kb in length, for example, about 5 kb, about 6 kb, about 7 kb, about 8 kb, about 9 kb, about 10 kb, about 10.5 kb, about 11 kb, about 11.5 kb, about 12 kb, about 12.5 kb, about 13 kb, about 14 kb, about 15 kb, about 16 kb, about 17 kb, about 18 kb, about 19 kb, or about 20 kb in length).

[0145] The self-replicating RNA molecule may have a 3' poly A tail. Additionally, the self-replicating RNA molecule may contain a poly A polymerase recognition sequence (e.g., AAUAAA).

[0146] In certain embodiments, the RNA according to the invention does not encode a reporter molecule, such as luciferase, or a fluorescent protein, such as green fluorescent protein (GFP).

[0147] In some embodiments, the replicase encoded by the self-replicating RNA can be a variant of any of the replicases described herein, hi some embodiments, the variant is a functional fragment (e.g., a fragment of a protein that is functionally similar or functionally equivalent to the protein).

[0148] V. Pharmaceutical Compositions The increased efficiency makes the method of the present invention particularly suitable for scalable manufacturing of pharmaceutical compositions comprising circular DNA vectors. Any of the methods for producing circular DNA vectors described herein can be adapted to produce pharmaceutical compositions comprising circular DNA vectors in a pharma- ceutically acceptable carrier.

[0149] As part of any of the methods described herein, downstream purification processes can be easily applied. For example, various chromatography steps are known in the art and can be appropriately adapted to remove bacterial by-products, endotoxins, bacterial artificial chromosomes (BACs), helper plasmids, etc. In some cases, pharmaceutical compositions of bacterially produced circular DNA vectors are purified by anion exchange chromatography or hydrophobic interaction chromatography.

[0150] In some embodiments, the pharmaceutical formulation of the present invention is a pharmaceutical composition comprising at least 1.0 mg (e.g., 1.0 mg to 10 g, 1.0 mg to 5.0 g, 1.0 mg to 1.0 g, 1.0 mg to 500 mg, 1.0 mg to 200 mg, 1.0 mg to 100 mg, 1.0 mg to 50 mg, 1.0 mg to 25 mg, 1.0 mg to 20 mg, 1.0 mg to 15 mg, 1.0 mg to 10 mg, 1.0 mg to 5.0 mg, 2.0 mg to 10 g, 2.0 mg to 5.0 g, 2.0 mg to 1.0 g, 2.0 mg to 500 mg, 2.0 mg to 200 mg, 2.0 mg to 100 mg, 2.0 mg to 50 mg, 2.0 mg to 25 mg, 2.0 mg 5.0mg-20mg, 2.0mg-15mg, 2.0mg-10mg, 2.0mg-5.0mg, 5.0mg-10g, 5.0mg-5.0g, 5.0mg-1.0g, 5.0mg-500mg, 5.0mg-200mg, 5.0mg-100mg, 5.0mg-50mg, 5.0mg-25mg, 5.0mg-20mg, 5.0mg-15mg, 5.0mg-10mg, 10mg-10g, 10mg-5.0g, 10mg-1.0g, 10mg-500mg, 10mg-200mg, 10mg-100mg, 10mg-50mg, 10mg-25mg, 10mg-20mg, or 10mg-15mg) of the circular DNA vector.

[0151] In some embodiments, the pharmaceutical formulation of the present invention comprises at least 2.0 mg of the circular DNA vector in a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical formulation produced by any of the methods described herein comprises at least 5.0 mg of the circular DNA vector in a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical formulation produced by any of the methods described herein comprises at least 10.0 mg of the circular DNA vector in a pharma- ceutically acceptable carrier.

[0152] In some embodiments, the pharmaceutical formulation of the present invention is substantially free of impurities.For example, in some embodiments, the pharmaceutical formulation comprises a protein content by mass of less than 2.0% (e.g., a protein content by mass of less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.01%).In some cases, the protein content is determined by bicinchoninic acid assay.In addition or alternatively, the protein content is determined by ELISA.

[0153] In some cases, the pharmaceutical formulation comprises an RNA content by mass of less than 5.0% (e.g., an RNA content by mass of less than 4.5%, less than 4.0%, less than 3.5%, less than 3.0%, less than 2.5%, less than 2.0%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.01%). In some embodiments, the RNA content is determined by agarose gel electrophoresis. In some embodiments, the RNA content is determined by quantitative PCR. In some embodiments, the RNA content is determined by a fluorescent assay (e.g., Ribogreen).

[0154] In some embodiments, the pharmaceutical formulation comprises a gDNA content by mass of less than 5.0% (e.g., a gDNA content by mass of less than 4.5%, less than 4.0%, less than 3.5%, less than 3.0%, less than 2.5%, less than 2.0%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.01%). In some embodiments, the gDNA content is determined by agarose gel electrophoresis or capillary electrophoresis. In some embodiments, the gDNA content is determined by quantitative PCR. In some embodiments, gDNA content is determined by Southern blot.

[0155] In some embodiments, the pharmaceutical formulation contains less than 40 EU / mg endotoxin. In some embodiments, the pharmaceutical formulation contains less than 20 EU / mg endotoxin. In some embodiments, the pharmaceutical formulation contains less than 10 EU / mg endotoxin. In some embodiments, the pharmaceutical formulation contains less than 5 EU / mg endotoxin (e.g., less than 4 EU / mg endotoxin, less than 3 EU / mg endotoxin, less than 2 EU / mg endotoxin, less than 1 EU / mg endotoxin, less than 0.5 EU / mg endotoxin), for example, as measured by Limulus amebocyte extract (LAL) assay.

[0156] In some embodiments, the pharmaceutical compositions disclosed herein comply with current Good Manufacturing Practices (GMP) in accordance with the standards promulgated by the U.S. Food and Drug Administration and set forth in Title 21 Code of Federal Regulations, Sections 210 and 211, which are incorporated herein by reference in their entirety.

[0157] The pharmaceutical compositions provided herein may include one or more pharma- ceutically acceptable carriers, such as excipients and / or stabilizers, at dosages and concentrations used, that are non-toxic to the individual (e.g., human patient) being treated. In some embodiments, the pharma-ceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharma-ceutically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or non-ionic surfactants, such as tweens, polyethylene glycols (PEG), and pluronics.

[0158] When the pharmaceutical composition is provided in liquid form, the pharma- ceutically acceptable carrier can be water (e.g., pyrogen-free water), isotonic saline, or an aqueous buffer solution, such as a phosphate buffer solution or a citrate buffer solution. The pharmaceutical composition can be injected in water or a buffer solution, such as an aqueous buffer solution containing, for example, a sodium salt (e.g., at least 50 mM of a sodium salt), a calcium salt (e.g., at least 0.01 mM of a calcium salt), or a potassium salt (e.g., at least 3 mM of a potassium salt). According to certain embodiments, the sodium, calcium, or potassium salts can occur in the form of their halides, e.g., chlorides, iodides, or bromides, their hydroxides, carbonates, bicarbonates, or sulfates, and the like. Non-limiting examples of sodium salts include NaCl, NaI, NaBr, Na2CO2, NaHCO2, and Na2SO4. Examples of potassium salts include, for example, KCl, KI, KBr, K2CO2, KHCO2, and K2SO4. Examples of calcium salts include, for example, CaCl2, CaI2, CaBr2, CaCO2, CaSO4, and Ca(OH)2. In addition, organic anions of the above-mentioned cations may be contained in the buffer. According to certain embodiments, the buffer suitable for injection purposes as defined above may contain a salt selected from sodium chloride (NaCl), calcium chloride (CaCl2) or potassium chloride (KCl), and further anions may be present. CaCl2 may also be replaced by another salt, such as KCl. In some embodiments, the salts in the injection buffer are present in a concentration of at least 50 mM sodium chloride (NaCl), at least 3 mM potassium chloride (KCl), and at least 0.01 mM calcium chloride (CaCl2). The injection buffer may be hypertonic, isotonic, or hypotonic relative to a particular reference medium, i.e. the buffer may have a higher, the same, or a lower salt content relative to a particular reference medium, preferably using the above-mentioned salts in concentrations that do not cause cell damage due to osmotic or other concentration effects. The reference medium may be a liquid such as blood, lymph, interstitial fluid, other bodily fluids, or a common buffer solution, etc. Such common buffer solutions or liquids are known to those skilled in the art.Lactated Ringer's solution is an especially preferred liquid base.

[0159] One or more compatible solid or liquid fillers, diluents, or encapsulating compounds may be suitable for administration to a person. The components of the pharmaceutical composition according to the invention may be mixed with the nucleic acid vector according to the invention as defined herein in such a way that there is no interaction that would significantly reduce the pharmaceutical efficacy of the (pharmaceutical) composition according to the invention under typical conditions of use. Pharmaceutically acceptable carriers, fillers and diluents may have a sufficiently high purity and a sufficiently low toxicity to make them suitable for administration to the individual being treated. Some examples of compounds that can be used as pharma- ceutically acceptable carriers, fillers, or components thereof are sugars such as lactose, glucose, trehalose, and sucrose; starches such as corn starch or potato starch; dextrose; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and oil derived from cocoa; polyols such as polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; or alginic acid.

[0160] The choice of pharma- ceutically acceptable carrier can depend on the way the pharmaceutical composition is to be administered.

[0161] Suitable unit dosage forms for injection include sterile solutions of water, saline, and mixtures thereof. The pH of such solutions can be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid, and collagen matrices. Suitable pharma- ceutically acceptable carriers for topical application include those suitable for use in lotions, creams, gels, etc. For oral administration of pharmaceutical compositions, tablets, capsules, etc. are preferred unit dosage forms.

[0162] Further additives which may be included in the pharmaceutical composition are emulsifiers such as tweens, wetting agents such as sodium lauryl sulfate, colorants, pharmaceutical carriers, stabilizers, antioxidants, and preservatives.

[0163] The pharmaceutical composition according to the present invention can be provided in liquid or dry (e.g., lyophilized) form. In a particular embodiment, the nucleic acid vector of the pharmaceutical composition is provided in lyophilized form. The lyophilized composition comprising the nucleic acid vector of the present invention can be reconstituted before administration in a suitable buffer, preferably based on an aqueous carrier, such as lactated Ringer's solution, Ringer's solution, or phosphate buffer solution.

[0164] In certain embodiments of the invention, any of the circular DNA vectors of the invention can be complexed with one or more cationic or polycationic compounds, such as cationic or polycationic polymers, cationic or polycationic peptides or proteins, such as protamine, cationic or polycationic polysaccharides, and / or cationic or polycationic lipids.

[0165] According to certain embodiments, the circular DNA vectors of the present invention can be complexed with lipids to form one or more liposomes, lipoplexes, or lipid nanoparticles. Thus, in one embodiment, the pharmaceutical composition comprises a liposome, lipoplex, and / or lipid nanoparticle comprising the circular DNA vector.

[0166] Lipid-based formulations can be an effective delivery system for nucleic acid vectors due to their biocompatibility and ease of their large-scale production. Cationic lipids have been widely studied as synthetic materials for the delivery of nucleic acids. After mixing, the nucleic acid is condensed by the cationic lipid to form lipid / nucleic acid complexes known as lipoplexes. These lipid complexes can protect the genetic material from the action of nucleases and deliver the genetic material into cells by interacting with the negatively charged cell membrane. Lipoplexes can be created by directly mixing positively charged lipids with negatively charged nucleic acids at physiological pH.

[0167] Conventional liposomes contain a lipid bilayer that may be composed of cationic, anionic, or neutral phospholipids and cholesterol, which surrounds an aqueous core. Both the lipid bilayer and the aqueous space can incorporate hydrophobic or hydrophilic compounds, respectively. The properties and in vivo behavior of liposomes can be modified by adding hydrophilic polymer coatings, such as polyethylene glycol (PEG), to the liposome surface to provide steric stabilization. Furthermore, liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to their surface or to the ends of the attached PEG chains.

[0168] Liposomes are colloidal lipid- and surfactant-based delivery systems composed of a phospholipid bilayer surrounding an aqueous compartment. Liposomes can exist as spherical vesicles and can range in size from 20 nm to several microns. Cationic lipid-based liposomes can be complexed with negatively charged nucleic acids through electrostatic interactions, resulting in complexes that provide the biocompatibility, low toxicity, and potential for large-scale production required for in vivo clinical applications. Liposomes can fuse with the plasma membrane for uptake. Once inside the cell, the liposomes are processed by the endocytic pathway and the genetic material is subsequently released from the endosome / carrier into the cytoplasm.

[0169] Cationic liposomes can function as a delivery system for circular DNA vectors. Cationic lipids such as MAP, (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate) can form complexes or lipoplexes with negatively charged nucleic acids through electrostatic interactions to form nanoparticles, resulting in high in vitro transfection efficiency. In addition, neutral lipid-based nanoliposomes for nucleic acid vector delivery are available, such as neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based nanoliposomes.

[0170] Therefore, in one embodiment of the present invention, the circular DNA vector is complexed with cationic lipids and / or neutral lipids, thereby forming liposomes, lipid nanoparticles, lipoplexes or neutral lipid-based nanoliposomes in the pharmaceutical composition.

[0171] In a particular embodiment, the pharmaceutical composition comprises a circular DNA vector of the invention formulated with a cationic or polycationic compound and / or a polymeric carrier. Thus, in a further embodiment of the invention, the circular DNA vector as defined herein is optionally formulated with a nucleic acid vector and a cationic or polycationic compound in a ratio of about 5:1 (w / w) to about 0.25:1 (w / w), e.g., about 5:1 (w / w) to about 0.5:1 (w / w), e.g., about 4:1 (w / w) to about 1:1 (w / w) or about 3:1 (w / w) to about 1:1 (w / w), e.g., about 3:1 (w / w) to about 2:1 (w / w). The nucleic acid vector is associated or complexed with the cationic or polycationic compound or polymeric carrier at a weight ratio of about 0.1 to 10, e.g., about 0.3 to 4 or 0.3 to 1, e.g., about 0.5 to 1 or 0.7 to 1, e.g., about 0.3 to 0.9 or 0.5 to 0.9. For example, the N / P ratio of the circular DNA vector to one or more polycations is in the range of about 0.1 to 10, including the ranges of about 0.3 to 4, about 0.5 to 2, about 0.7 to 2, and about 0.7 to 1.5.

[0172] The nucleic acid vectors described herein may also be associated with vehicles, transfection agents or complexing agents to increase transfection efficiency and / or expression of the regulated genes according to the present invention.

[0173] In some embodiments, the circular DNA vector according to the present invention is complexed with one or more polycations, preferably protamine or oligofectamine. Further cationic or polycationic compounds which may be used as transfection or complexing agents include cationic polysaccharides such as chitosan, polybrene, cationic polymers such as polyethyleneimine (PEI), cationic lipids such as DOTMA: [1-(2,3-)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPE, LEAP, DOPE: dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: dioctadecylamidoglycylspermine, DIMRI: dimyristoxypropyldimethylhydroxyethylammonium bromide, MAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanol Amine chlorides, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers, such as modified polyamino acids, such as β-amino acid polymers or inverse polyamides, modified polyethylenes, such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), modified acrylates, such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), modified amidoamines, such as pAMAM (poly(amidoamine)), modified polyβ-aminoesters (PBAE), such as diamine end-modified 1,Examples of the polymer include 4-butanediol diacrylate-co-5-amino-1-pentanol polymers, dendrimers such as polypropylamine dendrimers or pAMAM-based dendrimers, polyimines such as PEI (poly(ethyleneimine), poly(propyleneimine), polyallylamine, sugar-based polymers such as cyclodextrin-based polymers, dextran-based polymers, chitosan, silane-based polymers such as PMOXA-PDMS copolymers, and block polymers consisting of a combination of one or more cationic blocks (e.g., selected from the cationic polymers described above) and one or more hydrophilic or hydrophobic blocks (e.g., polyethylene glycol).

[0174] According to a particular embodiment, the pharmaceutical composition comprises a circular DNA vector encapsulated in or attached to a polymeric carrier. The polymeric carrier used according to the present invention may be a polymeric carrier formed by disulfide bridged cationic components. The disulfide bridged cationic components may be the same as each other or different. The polymeric carrier may also contain further components. It is also particularly preferred that the polymeric carrier used according to the present invention comprises a mixture of cationic peptides, proteins or polymers crosslinked by disulfide bonds as described herein and, optionally, further components as defined herein. In this context, the disclosure of WO 2012 / 013326 is incorporated herein by reference. In this context, the cationic components forming the basis of the polymeric carrier by disulfide bridges are usually selected from any suitable cationic or polycationic peptide, protein or polymer suitable for this purpose, in particular any cationic or polycationic peptide, protein or polymer capable of complexing with the further nucleic acid contained in the nucleic acid vector or composition defined herein, thereby preferably condensing the nucleic acid vector. The cationic or polycationic peptides, proteins or polymers may be linear molecules, although branched cationic or polycationic peptides, proteins or polymers can also be used.

[0175] All disulfide-bridged cationic or polycationic proteins, peptides or polymers of the polymeric carrier that can be used to complex the circular DNA vector according to the invention, included as part of the pharmaceutical composition, can contain at least one SH moiety (e.g. at least one cysteine ​​residue, or any further chemical group exhibiting an SH moiety) capable of forming a disulfide bond upon condensation with at least one further cationic or polycationic protein, peptide or polymer as the cationic component of the polymeric carrier as mentioned herein.

[0176] Such polymeric carriers used to complex the circular DNA vector of the present invention may be formed by disulfide-bridged cationic (or polycationic) components. In particular, such cationic or polycationic peptides or proteins or polymers of the polymeric carrier containing at least one SH moiety or additionally modified to contain at least one SH moiety may be selected from proteins, peptides, and polymers as complexing agents.

[0177] In other embodiments, the circular DNA vector according to the present invention may be administered naked in a suitable buffer, without association with any additional vehicles, transfection agents, or complexing agents.

[0178] VI.How to use Provided herein (e.g., as part of a gene therapy regimen) is a method of inducing expression (e.g., residual expression) of a sequence of interest (e.g., a therapeutic sequence) in a subject in need thereof by administering to the subject any of the circular DNA vectors or pharmaceutical compositions thereof described herein. The target cells or tissues of the subject can be characterized to detect or quantify the presence (e.g., residual) of the delivered therapeutic sequence by examining the nucleic acid sequence (e.g., RNA sequence, e.g., mRNA sequence) of the host cell, such as by Southern blotting or PCR analysis. Alternatively, the expression of the therapeutic sequence in the subject can be characterized (e.g., quantitatively or qualitatively) by monitoring the progression of the disease (e.g., associated with the defect or mutation targeted by the therapeutic sequence) that is treated by delivery of the therapeutic sequence. In some embodiments, transcription or expression (e.g., residual transcription or residual expression) of the therapeutic sequence is confirmed by observing a decrease in one or more symptoms associated with the disease.

[0179] Thus, embodiments of the present invention include a method of treating a disease in a subject by administering to the subject any of the circular DNA vectors or pharmaceutical compositions thereof described herein. Any of the circular DNA vectors or pharmaceutical compositions thereof described herein may be administered in a dose of 1 μg to 10 mg of DNA (e.g., 5 μg to 5.0 mg, 10 μg to 2.0 mg, or 100 μg to 1.0 mg of DNA, e.g., 10 μg to 20 μg, 20 μg to 30 μg, 30 μg to 40 μg, 40 μg to 50 μg, 50 μg to 75 μg, 75 μg to 100 μg, 100 μg to 200 μg, 200 μg to 300 μg, 300 μg to 400 μg, 400 μg to 500 μg, 500 μg to 1.0 mg, 1.0 mg to 5.0 mg, Alternatively, the subject may be administered a dosage of 5.0 mg to 10 mg of DNA, e.g., about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 600 μg, about 700 μg, about 750 μg, about 1.0 mg, about 2.0 mg, about 2.5 mg, about 5.0 mg, about 7.5 mg, or about 10 mg of DNA.

[0180] In some embodiments, administration of a circular DNA vector or a pharmaceutical composition thereof is less likely to induce an immune response in a subject compared to administration of other gene therapy vectors (e.g., plasmid DNA vectors and viral vectors).

[0181] In some cases, the circular DNA vectors and pharmaceutical compositions thereof provided herein are suitable for repeated administration, since they can transfect target cells without eliciting an immune response or inducing a reduced immune response compared to reference vectors such as plasmid DNA vectors or AAV vectors discussed above. Thus, the present invention provides a method for repeatedly administering the circular DNA vectors and pharmaceutical compositions described herein. Any of the above-mentioned dosages can be repeated at an appropriate frequency and duration. In some embodiments, the subject is administered about twice per day, about once per day, about five times per week, about four times per week, about three times per week, about two times per week, about once per week, about two times per month, about once per month, about once every six weeks, about once every two months, about once every three months, about once every four months, twice per year, once per year, or less frequently. In some embodiments, the number of administrations and the frequency of administration correspond to the turnover rate of the target cells. It will be understood that in the long-lived postmitotic target cells transfected with the vector described herein, a single administration of the vector is sufficient to maintain the expression of heterologous genes in the target cells for a significant period of time.Therefore, in other embodiments, the circular DNA vector provided herein can be administered to a subject in a single administration.The number of times that the circular DNA vector is delivered to a subject can be the number of times required to maintain clinical (e.g., therapeutic) benefit.

[0182] The method of the present invention includes administration of the circular DNA vector or pharmaceutical composition thereof by any suitable route. The circular DNA vector or pharmaceutical composition thereof can be administered systemically or locally, for example, intravenously, intraocularly (e.g., intravitreally (e.g., by intravitreal injection), subretinal, by eye drop, intraocularly, intraorbitally), intramuscularly, intradermally, intrahepatically, intracerebrally, intramuscularly, transdermally, intraarterially, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, intratumorally, subcutaneously, subconjunctivally, intrabladder, intramucosally, intrapericardially, intraumbilically, orally, topically, transdermally, by inhalation, by aerosolization, by injection (e.g., by jet injection), by electroporation, by implantation, by injection (e.g., by continuous infusion), by localized perfusion bathing the target cells directly, by catheter, by lavage, in a cream, or in a lipid composition.

[0183] The circular DNA vectors described herein can be delivered into cells by in vivo electrotransfer (e.g., in vivo electroporation). In vivo electroporation has been demonstrated in certain tissues, such as skin, skeletal muscle, certain tumor types, and lung epithelium. Delivery of naked DNA to cells by in vivo electroporation involves administering the DNA to the target tissue followed by application of an electric field to temporarily increase cell membrane permeability in the tissue by generating pores, allowing the DNA molecules to pass through the cell membrane. As an example, delivery to skin using in vivo electroporation is described in Cha & Daud Hum. Vaccin. Immunother. 2012, 8(11):1734-1738, which is incorporated herein by reference in its entirety. In vivo electroporation of skeletal muscle is described in Sokolowska & Blachnio-Zabielska, Int. J. Molecular Sci. 2019, 20:2776, which is incorporated herein by reference in its entirety. Intratumoral delivery using in vivo electroporation is described in Aung et al. Gene Therapy 2009, 16:830-839, which is incorporated herein by reference in its entirety. In vivo electroporation of DNA into lung cells is described in Pringle et al. J. Gene Med. 2007, 9:369-380, which is incorporated herein by reference in its entirety. In vivo electrotransfer of circular DNA vectors into cells within the eye (e.g., retinal cells and / or photoreceptor cells) is described in International Patent Publication WO 2022 / 198138, which is incorporated herein by reference in its entirety.In some cases, after administration of the circular DNA vector to the eye, an electrode can be placed inside the eye (e.g., within about 1 mm of the retina) and an electric field can be delivered through the electrode into the target ocular tissue under conditions suitable for electrotransferring the circular DNA vector into the target cells (e.g., by applying 6-10 pulses of 10V-100V each). Devices and systems with electrodes suitable for delivering electric fields in mammalian tissue are commercially available and can be useful in the methods disclosed herein. In some cases, the electric field is delivered through an electrode that comprises a needle (e.g., a needle placed in the vitreous humor or subretinal space). Suitable needle electrodes include the CLINIPORATOR™ electrode sold by IGEA™ and the needle electrode sold by AMBU™. The methods of the invention include administering any of the circular DNA vectors described herein, or pharmaceutical compositions thereof, to the skin, skeletal muscle, tumors (including, e.g., melanoma), eyes, and lungs via in vivo electrotransfer.

[0184] Additionally or alternatively, the circular DNA vector or pharmaceutical composition thereof can be administered to host cells ex vivo, such as by cells explanted (or otherwise obtained, e.g., by induction of differentiation) from an individual patient, followed by reimplantation of the host cells into the patient, e.g., after selection of cells into which the vector has been introduced. Thus, in some embodiments, the present disclosure provides transfected host cells and methods of administration thereof for treating disease.

[0185] Additionally or alternatively, the invention includes a method of treating a subject suffering from a disease or disorder by administering to the subject an isolated DNA vector of the invention (or a composition thereof).

[0186] Evaluation of transfection efficiency of any of the circular DNA vectors described herein can be performed using any method known in the art or described herein. Isolation of transfected cells can also be performed according to standard techniques. For example, cells containing a therapeutic gene can express a visible marker, such as a fluorescent protein (e.g., GFP) or other reporter protein, encoded by the sequence of the heterologous gene, which aids in identifying and isolating the cell(s) containing the heterologous gene. Cells containing a therapeutic gene can also be characterized and assayed for the presence of the heterologous gene contained in the vector by examining the nucleic acid sequence (e.g., RNA sequence, e.g., mRNA sequence) of the host cell, such as by Southern blotting or PCR analysis.

[0187] Thus, the method of the present invention involves administering to a subject any of the circular DNA vectors encoding the genes described herein, followed by detecting expression of the gene in the subject. Expression can be detected 1 to 4 weeks after administration, 1 to 4 months after administration, 4 months to 1 year after administration, 1 to 5 years after administration, or 5 to 20 years after administration (e.g., at least 1 week, at least 2 weeks, at least 1 month, at least 4 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years after administration). At any of these detection times, persistence of the circular DNA vector (e.g., episomal persistence) can be observed. In some embodiments, the persistence of the circular DNA vector is 5% to 50% greater, 50% to 100% greater, 1 to 5 times, or 5 to 10 times (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 1 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, or more) greater than a reference vector (e.g., a circular vector produced in bacteria or a circular vector having one or more bacterial signatures not present in the vectors of the invention).

[0188] VII. KITS AND MANUFACTURES In another aspect of the invention, an article of manufacture or kit comprising any of the circular DNA vectors or a pharmaceutical composition thereof is described herein. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous fluid bags, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The containers can house the composition alone or in combination with another composition effective for treating, preventing, and / or diagnosing a condition, and can have a sterile access port (e.g., the container can be an intravenous fluid bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is the circular DNA vector of the present invention, or a pharmaceutical composition comprising the circular DNA vector. The label or package insert indicates that the composition is used to treat a condition treatable with its contents. Additionally, the article of manufacture includes (a) a first container in which a composition comprising the circular DNA vector or a pharmaceutical composition thereof is housed, and (b) a second container in which a composition comprising an additional therapeutic agent is housed. The article of manufacture may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further include a second (or third) container containing a pharma- ceutically acceptable carrier, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, dextrose solution, or any of the pharma- ceutically acceptable carriers disclosed above. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, or other delivery devices. EXAMPLES

[0189] Example 1. The ColE2-P9 origin of replication provides stable plasmid maintenance To test whether the ColE2-P9 origin of replication (ori) placed in the plasmid results in stable maintenance of the plasmid in E. coli, a plasmid containing one of three variants of the ori (SEQ ID NO:2 to SEQ ID NO:4), an R6K origin, and a carbenicillin resistance marker was constructed and expressed as pir +The plasmids were transformed into S1037 and selected on LB agar plates supplemented with carbenicillin. Colonies from each plate were grown in LB without antibiotics at 37°C. The overnight cultures were diluted 1000-fold in fresh LB without antibiotics and grown at 37°C. After five passages, each overnight culture was restreaked on an LB agar plate without antibiotics and grown overnight at 37°C. From each plate, 20 colonies were plated on LB agar plates with or without carbenicillin. After a total of 6 days (during which an estimated 210+ population doublings had occurred), colony sizes for all source mutants were normal and plasmids were detected in all 20 colonies for all three source mutants (Figure 1). These results indicate that the 40 bp origin of replication can keep plasmids stable over many population doublings in E. coli.

[0190] Example 2. Preparation of parent plasmids for the production of circular DNA vectors Figure 2 shows an example of a parent plasmid production process that can be used to produce a circular DNA vector according to embodiments disclosed herein. The individual components of the transcription unit are assembled into a transcription unit by Golden Gate assembly. The transcription unit is then assembled into a parent plasmid by Golden Gate assembly. In this example, the parent plasmid contains LoxP recombination site sequences that flank the vector sequences (the segment containing the ori and MYO7A genes) and separate the vector sequences from the backbone sequences (the segment containing the SpecR, KanR, and RFP genes).

[0191] Example 3. In vivo production of circular DNA vectors from parental plasmids FIG. 3 shows the experimental process for in vivo production of circular DNA vectors. The Golden Gate assembly method is used to produce a test parent plasmid containing a vector sequence flanked on both sides by LoxP recombination sites. The test vector sequence of the test parent plasmid contains the ColE2-P9 ori sequence and a reporter gene (sfGFP). For experimental purposes, the test vector sequence also contains a chloramphenicol resistance gene (CmR), although the embodiment of the circular DNA vector described herein lacks an antibiotic resistance gene. The test parent plasmid also contains a test backbone sequence containing antibiotic resistance genes SpecR and KanR and a reporter gene (RFP). The test parent plasmid was transformed into engineered E. coli cells with the ColE2-P9 Rep gene under the control of a constitutive promoter (J23119) integrated into the genome. Additional versions of the test parent plasmid were generated that had two I-PpoI, two I-SceI, two PI-SceI, two I-CeuI, or one PI-PspI and one SceI restriction site flanking the test vector sequences, instead of LoxP sites flanking the test vector sequences. The test parent plasmid with LoxP sites was called p1603, and the test parent plasmid with two PI-SceI restriction sites was called p1600.

[0192] Bacterial colonies carrying the parental test plasmid were identified by positive RFP fluorescence. Cre recombinase was electroporated into cells carrying p1603 to induce recombination at the LoxP sites to produce the test circular DNA vector. The procedure for Cre electroporation was as follows: Engineered electrocompetent E. coli carrying the parental plasmid were grown in SOB at 30°C to an OD of 0.8. E. coli were washed three times with ice-cold 10% glycerol and resuspended in 10% glycerol. 1 μl of Cre (15 units, NEB, M0298M) was mixed with 50 μl of electrocompetent cells. The mixture was transferred to a cuvette (1 mm gap) and electroporated using an electroporator (BTX) with a setting of 1800 volts. Cells were rescued by growing in SOC for 1 hour at 37°C and plated on LB agar plates without antibiotics. Colonies were grown and DNA was purified using a QIAGEN miniprep kit. Electroporated cells were spread onto LB plates without antibiotics. GFP-positive colonies on LB plates were streaked onto LB containing kanamycin (Kan) and spectinomycin (Spec) to test for loss of the test backbone sequence (Kan / Spec-sensitive colonies).

[0193] Figure 4 shows agarose gel electrophoresis of extragenomic DNA purified from cultures of individual colonies, showing that (1) the test circular DNA vector was produced by Cre electroporation of cells carrying p1603, and (2) the test circular DNA vector was maintained in the cells in the absence of selection pressure. Lanes 2, 3, and 5 show extragenomic DNA from p1603-transformed cells that were GFP-positive and Kan / Spec-sensitive after Cre electroporation and were grown in rich medium lacking chloramphenicol. The bands in these lanes run toward approximately 1500 bp, the expected size of the test circular DNA vector, indicating that Cre electroporation resulted in the production of the test circular DNA vector. Lanes 8, 9, and 11 correspond to lanes 2, 3, and 5, respectively, but were grown in rich medium containing chloramphenicol. The abundance of DNA in lanes 2, 3, and 5 was similar to lanes 8, 9, and 11, indicating that the circular DNA vector is maintained in cells without selection pressure. Lane 4 shows extragenomic DNA from p1603 transformed cells that were GFP-positive, RFP-positive, and Kan / Spec-resistant after Cre electroporation. The band in this lane runs toward approximately 5000 bp, the expected size for the test parent plasmid. Lane 10 corresponds to lane 4, but shows DNA from cells grown in medium containing chloramphenicol. Lane 1 shows extrachromosomal DNA purified from GFP-positive, RFP-positive, Kan / Spec-resistant colonies from p1600 transformed cells. The band in this lane runs toward approximately 5000 bp, the expected size for the test parent plasmid. Lane 7 corresponds to lane 1, but shows DNA from cells grown in medium containing chloramphenicol. Lane 6 shows extrachromosomal DNA purified from the same engineered E. coli cells transformed with a plasmid lacking the ColE2-P9 ori and grown in medium lacking chloramphenicol.No detectable plasmid was recovered from this culture, which may suggest that the ori is required for plasmid maintenance in the absence of selective pressure. Lane 12 shows DNA purified from a culture of the same cells used in lane 6, but grown in the presence of chloramphenicol. The recovery of the plasmid from these cells may suggest that selective pressure maintained the plasmid within the cells.

[0194] Example 4. Circular DNA vectors carrying the ColE2-P9 ori are maintained in engineered cells expressing the ColE2-P9 replication protein without selective pressure To test whether a circular DNA vector carrying an ori sequence (e.g., SEQ ID NO:2) from ColE2-P9 can be maintained in cells expressing ColE2-P9 replication proteins (e.g., SEQ ID NO:1) in the absence of selection pressure, cells carrying the test circular DNA vector produced by Cre recombination as described in Example 2 were cultured in various broths with and without chloramphenicol. The percentage of sfGFP positive cells in each culture was quantified. The results are shown in Figure 5. The test circular DNA vector was maintained at high levels in all media except SOC medium with and without chloramphenicol, where it was maintained at lower levels both with and without chloramphenicol.

[0195] The test circular DNA vector was purified and quantified from chloramphenicol-free TB and ZB cultures. TB cultures yielded 0.33 mg / L of the test circular DNA vector, and SB cultures yielded 0.45 mg / L of the test circular DNA vector. These results confirm that the circular DNA vector was maintained regardless of selection pressure.

[0196] Example 5. In vivo production process of circular DNA vectors using counterselection FIG. 6 shows an exemplary process for producing a circular DNA vector of the present invention using counter-selection. The transgene in this example is ABCA4, but it will be understood that the promoter driving ABCA4 can be replaced with other transgene cassettes. On day 0, competent engineered bacterial cells expressing the Rep gene are prepared using any of the processes described herein (e.g., by transforming the cells with a recombinase encoded on a bacterial artificial chromosome (BAC)). On day 1, the cells are plated on LB agar plates supplemented with Kan, and a template plasmid is added. In this example, the template plasmid contained the ABCA4 transgene downstream of the promoter and origin of replication (ori). This ori-ABCA4 cassette was flanked on both sides by recombination sites (attP-GA and attB-GA). On the other side of the plasmid (backbone region) were selectable markers, namely, antibiotic resistance genes SpR and KanR, counter-selectable marker PheS, and fluorescent marker RFP. On day 2, white colonies were picked from red colonies and counter-selected by growth in LB supplemented with 4CP. On day 3, the circular DNA vector was purified.

[0197] Example 6. Testing inducible Bxb1 for production of circular DNA vectors To test whether Bxb1 could be effective as an exogenous recombinase to produce circular DNA vectors, the Bxb1 recombinase was encoded on a bacterial artificial chromosome (BAC) and transformed into the host E. coli with a Rep gene (SEQ ID NO: 1) integrated into the genome driven by a constitutive promoter (Figure 7A). Two inducible Bxb1 BACs were tested: 1696 (Figure 7B, SEQ ID NO: 5) contained a cumic acid inducible promoter and a chloramphenicol (Cm) resistance (CmR) gene, and 1697 (Figure 7C, SEQ ID NO: 6) contained an arabinose inducible promoter and a CmR gene. Each BAC was transformed into S1037 cells by electroporation and plated in the presence of chloramphenicol.

[0198] Next, cells were transformed with the template plasmid carrying GFP as a reporter transgene (Figure 7D). The ColE2-P9 origin of replication (ori) was placed upstream of GFP and its promoter, and the ori-GFP cassette was flanked on both sides by recombination sites (attP-GA and attB-GA). On the other side of the plasmid (the backbone region) were selectable markers (antibiotic resistance genes SpR and KanR, counterselectable marker PheS, and fluorescent marker RFP). Thus, cells containing the template plasmid were able to express GFP. + , RFP + , Kan-resistant, Spec-resistant, and 4CP-sensitive, whereas cells containing only the circular DNA vector (Figure 7E), which does not contain the backbone by-product (Figure 7F), express only GFP. + , RFP - , Kan-sensitive, Spec-sensitive, and 4CP-resistant.

[0199] The template plasmid was electroporated into S1037 cells without inducer and plated on Cm+Kan plates. The results at 24 and 72 hours after transformation are shown in Figures 8 and 9. Figures 8A and 8B show that the majority of colonies carrying 1696BAC were green at 24 and 72 hours after transformation, respectively. A few red colonies were observed. Green colonies were selected and the presence and sequence of the circular DNA vector were confirmed by Sanger sequencing and gel electrophoresis. In contrast to 1696, Figures 9A and 9B show that most of the colonies carrying 1697BAC were yellow at 24 and 72 hours after transformation, respectively, while a few green colonies were observed at 72 hours (Figure 9B).

[0200] To evaluate the effect of Cm and arabinose inducers, colonies of various colors were picked from each plate and incubated with Cm and either cumic acid or arabinose for 24 hours. The results are shown in Figure 10 (1696) and Figure 11 (1697). Each culture was then diluted 500-fold and grown overnight in LB supplemented with 4CP with or without inducer for counter selection. The overnight cultures were restreaked onto plain LB agar plates and observed for fluorescence. The results are shown in Figure 12 (1696) and Figure 13 (1697). Single colonies from each plate were grown overnight in LB supplemented with 4CP, miniprepped, and digest mapping (BsaI) was performed. The gel electrophoresis results are shown in Figure 14. The expected bands for each expected species 1 to 4 are shown in Table 1 below:

[0201] [Table 1]

[0202] In colonies carrying either type of BAC (1696 or 1697), the addition of either inducer appeared to increase the number of GFP-expressing colonies, indicating that expression of Bxb1 occurred in the absence of inducer. Indeed, Bxb1 recombination had already occurred after transformation of the template plasmid and plating on Cm / Kan plates. In bacteria containing the 1606BAC, uninduced Bxb1 yielded more than 90% green colonies, indicating that more than 90% contained the circular DNA vector with little or no template plasmid.

[0203] Example 7. Production of circular DNA vectors containing therapeutic transgenes In this study, circular DNA vectors were generated that contained tricistronic cassettes encoding various types of therapeutic transgenes, namely (1) ABCA4, (2) IL-12, and (3) Flt3L, IL-12, and XCL1. The ABCA4 and IL-12 constructs contained a CAG promoter, and the tricistronic construct contained a CAG promoter upstream of each of the three genes. Exemplary sequences for the ABCA4 template plasmid and the resulting circular DNA vector are shown by Figure 15A (SEQ ID NO: 7) and Figure 15B (SEQ ID NO: 8), respectively.

[0204] First, S1037 cells were transformed with 1696BAC and grown overnight. One day later, cells were made competent and transformed with template plasmids encoding ABCA4, IL-12, or tricistronic cassettes. Cells were plated on LB agar plates supplemented with Kan. After 3 days of culture, small white colonies were picked (leaving red colonies) and purified after counterselection by growing overnight in LB+4CP.

[0205] The purified constructs were screened from single colonies in each group using BsaI digestion mapping. As shown in Figure 16A (theoretical gel profile) and Figure 16B (actual gel profile), all colonies obtained circular DNA vector bands of the expected size. These results support that the circular DNA vector production process described herein can be widely used to efficiently produce circular DNA vectors with transgenes of various sizes and configurations (e.g., multicistronic).

[0206] Example 8. Stability of circular DNA vectors to bacterial growth and scale-up A significant advantage of the vector system described herein is that bacterial cells harboring circular DNA vectors can be grown without a selection marker (e.g., after the selectable marker and other bacterial backbone elements are removed from the culture). To this end, Applicants tested whether circular DNA vectors containing therapeutic transgenes could be stably expressed in bacterial cultures over the many cell divisions associated with scale-up of vector production.

[0207] As in Example 7, overnight cultures (14-16 hours each) of cells identified as containing the circular DNA vector encoding the backbone-free ABCA4 were grown for seven consecutive nights. After the seventh culture, the presence of the circular DNA vector was confirmed by sequencing. Based on an average division rate of 3 divisions per hour, this culture had undergone at least 294 divisions and, surprisingly, maintained expression of the circular DNA vector despite the absence of selection.

[0208] Taking advantage of this remarkable stability, the cells were scaled up to produce larger quantities of the circular DNA vector containing the ABCA4 transgene. The cells were restreaked on LB plates and grown to produce a 2.5 L preparation. A glycerol stock was produced and the stock was restreaked to produce a 25 L preparation. The circular DNA vector was purified from this preparation, which yielded 18 mg of the circular DNA vector.

[0209] Example 9. Detection of monomeric circular DNA vectors In this study, the circular DNA vector containing the ABCA4 transgene produced using 1696BAC was sequenced to confirm that the circular DNA vector was in monomeric form (different from the dimer that can be obtained from recombination in trans with another template plasmid). In this study, S1037 bacteria were cultured in LB broth containing 25 μg / mL Kan at 37° C. for 2 hours. The cells were then transferred to a plate containing 4CP and 10 ng DNA and incubated at 37° C. overnight. The samples were analyzed by long-read sequencing (Oxford Nanopore) using conventional methods. As shown in FIG. 17A, a peak of monomers was observed, and no dimers were observed. In contrast, when the culture was incubated overnight with Kan, the circular DNA vector was mainly dimeric (FIG. 17B).

[0210] Example 10. Circular DNA vectors generated using Bxb1 helper plasmid As an alternative strategy to BAC-Bxb1, a circular DNA vector was generated using Bxb1 transformed into a host cell using a helper plasmid. An exemplary helper plasmid containing a cumic acid inducible promoter (CuO) is shown in FIG. 18. Furthermore, since the helper plasmid contains a temperature-sensitive backbone, it was possible to remove the helper plasmid after the production of the circular DNA vector. The DNA sequence of this helper plasmid is shown by SEQ ID NO: 11. The host cells used in this method were the same as in the similar examples, i.e., S1037 cells with Rep (SEQ ID NO: 1) integrated into the host genome driven by a constitutive promoter. The template plasmid was the same as in Example 6 (FIG. 7D).

[0211] In this study, the helper plasmid was transformed into S1037 cells and incubated overnight with 100 μg / mL carbenicillin (carb100). The plasmid template was then transformed and plated with carb100 and 500 μM Cuma, and the cells were grown at 30° C. Using this helper plasmid approach, several green colonies were observed (FIG. 19), indicating successful production of cells containing a circular DNA vector free of backbone by-products.

[0212] Example 11. Circular DNA vectors generated by integrating Bxb1 into the host genome Another source of recombinase provided herein is via integration into bacterial host genome. In this example, integration of Bxb1 was performed according to the process illustrated in Figure 20. First, S1037 cells carrying λred recombination helper plasmid were grown with 0.2% arabinose to make them electrocompetent. Then, 500ng of linearized 1696BAC was electroporated. After BAC1696 was integrated into the rsd-thiC locus, the λred recombination plasmid was removed.

[0213] Colony PCR was performed on the resulting colonies and positive clones were identified using gel electrophoresis (Figure 21). The resulting cells have been engineered to express both Rep and Bxb1 by genomic integration.

[0214] Example 12. Expression of ABCA4 protein using a circular DNA vector carrying ColE2-P9 ori To determine whether bacterially produced circular DNA vectors carrying a ColE2-derived origin of replication are capable of expressing proteins in human cells, in vitro studies were performed in which such circular DNA vectors carrying the human ABCA4 gene were transfected into HEK293T cells and the expression of ABCA4 protein was assessed by Western blot.

[0215] HEK293T cells were seeded at 150000 cells in 0.5 mL of standard medium in 24-well plates. Plates were incubated at 37°C for 24 hours. At the time of transfection, cells were 60%-80% confluent. Cells were transfected with circular DNA vectors using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol. The total amount of DNA added per well was 500 ng. After 24 hours of incubation at 37°C, cells were harvested and analyzed by Western blot using beta-actin as a control. The results of the Western blot are shown in Figure 22 and each lane is identified in Table 2 below.

[0216] [Table 2]

[0217] These results indicate that bacterially produced circular DNA vectors with ColE2-derived origin expressed their ABCA4 transgene in HEK293T human cells.

[0218] [Table 3] TIFF2025503476000005.tif234170TIFF2025503476000006.tif234170TIFF2025503476000007.tif234170TIFF2025503476000008.tif234170TIFF2025503476000009.tif234170TIFF2025503476000010.tif234170TIFF2025503476000011.tif234170TIFF2025503476000012.tif234170TIFF2025503476000013.tif234170TIFF2025503476000014.tif234170TIFF2025503476000015.tif234170TIFF2025503476000016.tif234170TIFF2025503476000017.tif234170TIFF2025503476000018.tif74170

Claims

1. 1. An engineered bacterial cell, comprising: (a) Rep genes encoding bacterial replication proteins integrated into the bacterial genome; (b) a circular DNA vector comprising (i) a coding sequence, and (ii) an origin of replication dependent on said replication protein, wherein the origin of replication is less than 50 base pairs in length; engineered bacterial cells, including

2. 2. The engineered bacterial cell of claim 1, wherein the origin of replication and replication proteins are derived from a ColE2-related plasmid.

3. 3. The engineered bacterial cell of claim 2, wherein the ColE2-related plasmid is ColE2-P9.

4. 1. An engineered bacterial cell, comprising: (a) Rep genes encoding bacterial replication proteins integrated into the bacterial genome; (b) a plasmid comprising (i) a first segment comprising a coding sequence and an origin of replication dependent on said bacterial replication protein, said origin of replication being less than 50 base pairs in length, and no selectable marker, and (ii) a second segment comprising a selectable marker, said first segment flanked on both sides by recognition sequences for at least one exogenous restriction enzyme or exogenous recombinase; engineered bacterial cells, including

5. 5. The engineered bacterial cell of claim 4, wherein the recognition sequences flanking the first segment are the same.

6. 5. The engineered bacterial cell of claim 4, wherein the recognition sequences flanking the first segment are different.

7. 5. The engineered bacterial cell of claim 4, wherein the second segment further comprises an origin of replication, wherein the origin of replication in the second segment is orthologous to the origin of replication in the first segment.

8. 5. The engineered bacterial cell of claim 4, wherein the origin of replication and the replication proteins are derived from a ColE2-related plasmid.

9. 5. The engineered bacterial cell of claim 4, wherein the ColE2-related plasmid is ColE2-P9.

10. 9. The engineered bacterial cell of any one of claims 4 to 8, wherein the Rep gene is operably linked to a first inducible promoter.

11. 11. The engineered bacterial cell of claim 10, wherein the first inducible promoter is a T7 RNA polymerase-dependent promoter.

12. 1. A method for producing a circular DNA vector, comprising inducing plasmid recombination in an engineered bacterial cell, wherein: (a) the plasmid (ii) a first segment comprising a coding sequence and an origin of replication that is less than 50 base pairs in length and does not comprise a selectable marker, the first segment being flanked on both sides by recognition sequences for an exogenous recombinase; (ii) a second segment comprising a selectable marker; and and (b) the engineered bacterial cell comprises a gene encoding the exogenous recombinase; wherein said induction causes recombination of said plasmids, thereby producing said circular DNA vector comprising said first segment. method.

13. the engineered bacterial cell further comprises a Rep gene encoding a bacterial replication protein that recognizes the origin of replication; Optionally, the Rep gene is integrated into the bacterial genome; Optionally, the Rep gene is operably linked to a first inducible promoter; Optionally, the Rep gene is a ColE2-P9 Rep gene; Optionally, the origin of replication is a ColE2-P9 origin of replication. The method of claim 12.

14. The method of claim 12 or 13, wherein the exogenous recombinase is Bxb1, and the recognition sequence comprises attP-GA and attB-GA.

15. The method of claim 14, wherein the gene encoding Bxb1 is operably linked to a cumic acid-inducible promoter, the engineered bacterial cell is maintained in the absence of cumic acid at the time of the introduction, and the Bxb1 is expressed at an uninduced level at the time of the introduction.

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