Production of gene therapy vectors in engineered bacteria

Abstract

Provided herein are improved methods for producing engineered bacterial cells of circular DNA vectors, circular DNA vectors produced by such methods, and pharmaceutical compositions containing such circular DNA vectors. The methods provided herein are suitable for large scale production of high purity compositions of circular DNA vectors.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 291,871, filed on December 20, 2021, the entire contents of which are incorporated herein by reference. Technical Field

[0003] In general, the present disclosure relates to therapeutic vectors in engineered bacteria. Background Art

[0004] Gene therapy is becoming a promising method for treating a variety of diseases and conditions in human patients. Recombinant adeno-associated virus (rAAV) vectors have a good record of efficient gene transfer in human patients and a variety of model systems. The advantage of the genome of rAAV vectors is their ability to persist in vivo as a circular episome during the life cycle of target cells. On the other hand, there are many disadvantages based on rAAV vectors, such as limited maximum payload, immunogenicity and production efficiency.

[0005] To address some of these challenges in rAAV technology, non-viral alternatives have received extensive attention in recent years. However, the development of scalable non-viral gene therapy platforms with the efficiency and durability of rAAV has proven elusive. For example, traditional bacterial plasmid DNA vectors represent a versatile tool in gene delivery but are limited by the abundant bacterial components in plasmid DNA vectors, such as antibiotic resistance genes and transcriptional regulatory elements, which can lead to immunogenicity and loss of gene expression through transcriptional silencing.

[0006] Despite various efforts to improve plasmid DNA vectors by removing backbone components, there remains a need for non-viral vectors with minimal bacterial elements and methods for their efficient production on a large scale. Summary of the invention

[0007] Disclosed herein are circular DNA vectors (e.g., therapeutic circular DNA vectors) and bacterial cells (e.g., engineered bacterial cells) produced by bacteria, which can be used to produce them from parent plasmids, such as parent plasmids in bacterial cells (e.g., engineered bacterial cells). The therapeutic circular DNA vectors provided herein include small (e.g., less than 50 base pairs) replication origins and lack selection markers (e.g., antibiotic resistance genes), which can reduce the risk of exogenous sequence introduction in the vector. Therefore, the circular DNA vectors produced by such bacteria can be effectively and mass-produced for therapeutic applications.

[0008] Embodiments disclosed herein include an engineered bacterial cell comprising: (a) a Rep gene encoding a bacterial replication protein integrated into the bacterial genome; (b) a circular DNA vector comprising: (i) a coding sequence; and (ii) a replication origin that depends on the replication protein; wherein the circular DNA vector does not contain a selective marker. In some embodiments, the replication origin is less than 50 base pairs in length. In some embodiments, the replication origin and the replication protein are from a ColE2-related plasmid. In some embodiments, the ColE2-related plasmid is ColE2-P9.

[0009] In some embodiments, the Rep gene is operably coupled 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 a T7 RNA polymerase (T7 RNAP) integrated into the bacterial genome. In some embodiments, the T7 RNAP gene is operably coupled to a second inducible promoter. In some embodiments, the second inducible promoter is Ptac.

[0010] 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 coupled to a third inducible promoter. In some embodiments, the third inducible promoter is Pbad. In some embodiments, the bacterial genome does not comprise a recognition sequence for an exogenous restriction enzyme.

[0011] In some embodiments, the coding sequence encoding the circular DNA vector comprises a therapeutic gene or nucleic acid. In some embodiments, the coding sequence is a eukaryotic sequence (eg, a sequence that can be expressed in mammalian cells).

[0012] In some embodiments, the origin of replication is the only bacterial sequence in the circular DNA vector.

[0013] 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 for at least 20 rounds of cell division.

[0014] In some embodiments, the engineered bacterial cell does not contain any extragenomic circular DNA molecules other than the one or more copies of the circular DNA vector.

[0015] Embodiments disclosed herein include a culture comprising a plurality of any engineered bacterial cells described herein, wherein the average copy number of the circular DNA vector per engineered bacterial cell is at least 10. In some embodiments, the culture contains at least 10 7 Engineered bacterial cells.

[0016] Embodiments disclosed herein include an engineered bacterial cell comprising: (a) a Rep gene encoding a bacterial replication protein integrated into a bacterial genome; (b) a plasmid comprising: (i) a first segment comprising a coding sequence and a replication origin that depends on a bacterial replication protein, wherein the first segment does not comprise a selective marker; and (ii) a second segment comprising a selective marker; wherein the first segment is flanked by at least one recognition sequence of an exogenous restriction enzyme or an exogenous recombinase. In some embodiments, the recognition sequences flanking the first segment are identical. In some embodiments, the recognition sequences flanking the first segment are different. In some embodiments, the second segment further comprises a replication origin, wherein the replication origin in the second segment is directly homologous to the replication origin in the first segment.

[0017] In some embodiments, the replication origin is less than 50 base pairs in length. In some embodiments, the replication origin and replication protein are from a ColE2-related plasmid. In some embodiments, the ColE2-related plasmid is ColE2-P9.

[0018] In some embodiments, the Rep gene is operably coupled 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 a T7 RNA polymerase (T7 RNAP) integrated into the bacterial genome. In some embodiments, the T7 RNAP gene is operably coupled to a second inducible promoter. In some embodiments, the second inducible promoter is Ptac.

[0019] 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 an exogenous restriction enzyme or an exogenous recombinase is operatively coupled to a third inducible promoter. In some embodiments, the third inducible promoter is Pbad. In some embodiments, the bacterial genome does not comprise a recognition sequence for an exogenous restriction enzyme or an exogenous recombinase.

[0020] In some embodiments, the coding sequence of the first segment encodes a therapeutic gene or nucleic acid. In some embodiments, the coding sequence is a eukaryotic sequence (eg, a sequence that can be expressed in mammalian cells).

[0021] In some embodiments, the engineered bacterial cell further comprises an exogenous restriction enzyme or an exogenous recombinase.

[0022] Embodiments disclosed herein include a method for preparing a circular DNA vector, the method comprising: (a) contacting a plasmid in a bacterial cell with an exogenous restriction enzyme to excise a first segment from the plasmid, wherein the first segment is flanked by a recognition sequence of an exogenous restriction enzyme, and wherein the first segment comprises a coding sequence and a replication origin that depends on a bacterial replication protein, thereby producing a linear DNA fragment comprising a 5' end and a 3' end and a complementary overhang; and (b) connecting the 5' end and the 3' end of the linear DNA fragment together to produce a circular DNA vector. In some embodiments, prior to step (a), the plasmid comprises a second segment containing a selective marker. In some embodiments, the second segment also comprises a replication origin, wherein the replication origin in the second segment is directly homologous to the replication origin in the first segment. In some embodiments, the first segment does not include a selective marker.

[0023] In some embodiments, contacting the plasmid in the cell with an 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 coupled to an inducible promoter. In some embodiments, the inducible promoter is a 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 in the cell with the exogenous restriction enzyme comprises introducing the exogenous restriction enzyme into the cell from outside the bacterial cell.

[0024] In some embodiments, the ligation is performed by an exogenous ligase. In some embodiments, the exogenous ligase is expressed by a gene integrated into the bacterial genome. In some embodiments, the exogenous ligase is introduced into the bacterial cell from outside the bacterial cell.

[0025] In some embodiments, the bacterial cell comprises a Rep gene encoding a bacterial replication protein integrated into the genome. In some embodiments, the Rep gene is operably coupled to an inducible promoter capable of expressing a bacterial replication protein at a first expression level and a second expression level, wherein 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 replication origin at a first copy number, and the second expression level of the bacterial replication protein maintains the replication origin at a second copy number, wherein the first copy number is lower than 5, 10, 15, 20 or 50 copies / cell, and the second copy number is at least 20, 50, 100 or 200 copies / cell. In some embodiments, the bacterial replication gene is expressed at a first expression level before step (b), and is not expressed at a second expression level before 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 dependent on T7 RNA polymerase. In some embodiments, the bacterial cell comprises a gene (T7RNAP) encoding T7 RNA polymerase integrated into the genome. In some embodiments, the T7 RNAP gene is operably coupled to an inducible promoter. In some embodiments, the inducible promoter is P tac In some embodiments, the second segment of the plasmid further comprises a gene encoding a gene capable of inhibiting the expression of tac The invention relates to a LacI gene of a lactose repressor protein expressed by a promoter, and wherein the expression of the bacterial replication gene is maintained at a first expression level or below the first expression level by the expression of the lactose repressor protein. In some embodiments, after step (b), the expression of the lactose repressor protein is reduced, thereby inducing the bacterial replication gene to be expressed at a second expression level and maintaining the circular DNA vector at a second copy number.

[0026] In some embodiments, the method for preparing a circular DNA vector also includes culturing cells under conditions where continuous growth does not require a selective marker on a plasmid, thereby producing a bacterial cell progeny population lacking a selective marker. In some embodiments, after at least 50 doublings, the colony maintains a circular DNA vector. In some embodiments, after at least 100 doublings, for example, at least 150 doublings, at least 200 doublings, at least 250 doublings, or at least 290 doublings, the colony maintains a circular DNA vector. In some embodiments, after at least 50 doublings, the colony maintains the circular DNA vector at an average copy number of at least 20 copies / cell. In some embodiments, after at least 100 doublings, the colony maintains the circular DNA vector at an average copy number of at least 20 copies / cell. In some embodiments, after at least 150 doublings, at least 200 doublings, at least 250 doublings, or at least 290 doublings, the colony maintains the circular DNA vector at an average copy number of at least 20 copies / cell. In some embodiments, the method further comprises purifying the circular DNA vector.

[0027] In some embodiments, the replication origin 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 replication origin and replication protein are from a ColE2-related plasmid. In some embodiments, the ColE2-related plasmid is ColE2-P9. In some embodiments, the replication origin comprises or consists of SEQ ID NO: 2 (or the reverse complementary sequence) 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 replication origin comprises or consists of SEQ ID NO: 3 (or the reverse complementary sequence) 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 replication origin comprises or consists of SEQ ID NO:4 (or the reverse complementary sequence) 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)).

[0028] Embodiments disclosed herein include a method for preparing a circular DNA vector, the method comprising: (a) obtaining any engineered bacterial cell described herein comprising a parent plasmid, the parent plasmid comprising a first segment, the first segment comprising a coding sequence and a replication origin that depends on a bacterial replication protein, wherein the first segment does not comprise a selectable marker, and wherein the first segment is flanked by a recognition sequence for at least one exogenous restriction enzyme; and (b) contacting the plasmid with an exogenous restriction endonuclease to excise the first segment of the plasmid, thereby generating a linear DNA fragment flanked by complementary overhangs; and (c) self-ligating the linear DNA fragment to generate a circular DNA vector.

[0029] Embodiments disclosed herein include a method for preparing a circular DNA vector, the method comprising: (a) obtaining any engineered bacterial cell described herein comprising a parent plasmid, the parent plasmid comprising a first segment, the first segment comprising a coding sequence and a replication origin that depends on a bacterial replication protein, wherein the first segment does not comprise a selectable marker, and wherein the first segment is flanked by a recognition sequence for at least one exogenous recombinase; (b) contacting the plasmid with an exogenous recombinase that recognizes the recognition sequence flanking the first segment.

[0030] Embodiments disclosed herein include a pharmaceutical composition comprising (a) a circular DNA vector produced by any of the methods described herein; and (b) a suitable carrier for delivering the pharmaceutical composition to a subject.

[0031] On the other hand, a kind of engineered bacterial cell (for example, Escherichia coli (E.coli)) is provided, it comprises the circular DNA vector containing the replication origin of coding sequence and length less than 50 base pairs, wherein said circular DNA vector lacks selective marker.In some embodiments, said engineered bacterial cell does not comprise any extragenomic DNA molecule except one or more copies of circular DNA vector.In some embodiments, said engineered bacterial cell does not comprise the gene encoding the selective marker.In some embodiments, said engineered bacterial cell does not comprise the selective marker on the extragenomic DNA molecule.In some embodiments, said replication origin is from ColE2 related plasmid (for example, ColE2-P9 plasmid).In some embodiments, said engineered bacterial cell also comprises the Rep gene (for example, Rep gene is from ColE2-P9 plasmid (for example, SEQ ID NO:1)) of the bacterial replication protein encoding recognition replication origin.In some embodiments, said Rep gene is integrated into the bacterial genome.In some embodiments, said Rep gene is operably coupled to an inducible promoter.

[0032] In some embodiments, the circular DNA vector further comprises a recombination site (eg, an attL site (eg, attL-GA) or an attR site).

[0033] In some embodiments, the circular DNA vector does not comprise any bacterial (or other prokaryotic or bacteriophage) sequences except an origin of replication and a recombination site (when present). In some embodiments, the length of the origin of replication and the recombination site together does not exceed 90 base pairs.

[0034] In some embodiments, the engineered bacterial cell further comprises a gene encoding a recombinase.

[0035] In some embodiments, the engineered bacterial cell comprises at least 10 copies of the circular DNA vector.

[0036] On the other hand, an engineered bacterial cell is provided herein, comprising a plasmid comprising: (a) a first segment comprising a coding sequence and a replication origin of less than 50 base pairs in length, wherein the first segment does not comprise a selective marker; and (b) a second segment comprising a selective marker; wherein the first segment is flanked by a recognition sequence of an exogenous recombinase. In some embodiments, the engineered bacterial cell further comprises a gene encoding a Rep gene, which encodes a bacterial replication protein that recognizes the replication origin. In some embodiments, the Rep gene is integrated into the bacterial genome. In some embodiments, the Rep gene is operably coupled to a first inducible promoter.

[0037] In some embodiments, the replication origin and replication proteins are from a ColE2-related plasmid, such as ColE2-P9.

[0038] 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 located on a plasmid or a bacterial artificial chromosome. In some embodiments, the gene encoding the exogenous recombinase is operatively coupled to a second inducible promoter. In some embodiments, the second inducible promoter is a cuminic acid-inducible promoter.

[0039] In some embodiments, the recombinase is Bxb1. In some embodiments, the recognition sequence comprises attP-GA and attB-GA.

[0040] On the other hand, the present invention provides a method for preparing a circular DNA vector, wherein the method comprises inducing recombination of a plasmid in an engineered bacterial cell of any one of the foregoing aspects. In some embodiments, inducing recombination of a plasmid comprises inducing expression of an exogenous recombinase in an engineered bacterial cell.

[0041] In another aspect, a method for producing a circular DNA vector is provided, the method comprising inducing recombination of a plasmid in an engineered bacterial cell, wherein: (a) the plasmid comprises: (i) a first segment comprising a coding sequence and a replication origin of less than 50 base pairs in length, wherein the first segment does not comprise a selectable marker, wherein the first segment is flanked by a recognition sequence for an exogenous recombinase; and (ii) a second segment comprising a selectable marker; and (b) the engineered bacterial cell comprises a gene encoding an exogenous recombinase; wherein the induction causes recombination of the plasmid, thereby producing a circular DNA vector comprising the first segment.

[0042] In some embodiments, the engineered bacterial cell further comprises a Rep gene encoding a bacterial replication protein that recognizes a replication origin. In some embodiments, the Rep gene is integrated into the bacterial genome. In some embodiments, the Rep gene is operably coupled to a first inducible promoter.

[0043] In some embodiments, the origin of replication is the ColE2-P9 origin of replication and / or the Rep gene is the ColE2-P9 Rep gene.

[0044] In some embodiments, the exogenous recombinase is located on a plasmid or a bacterial artificial chromosome. In some embodiments, the gene encoding the exogenous recombinase is operably coupled to a second inducible promoter.

[0045] In some embodiments, the induction of the reorganization of the plasmid comprises inducing the expression of a gene encoding an exogenous recombinase. In some embodiments, the induction of the reorganization of the plasmid comprises introducing the plasmid into an engineered bacterial cell, wherein the exogenous recombinase is expressed in the engineered bacterial cell when introduced. In some embodiments, the exogenous recombinase is expressed at a non-induced level when introduced.

[0046] In some embodiments, the exogenous recombinase is Bxb1, and the recognition sequence comprises attP-GA and attB-GA. In some embodiments, the gene encoding Bxb1 is operably coupled to a cuminate inducible promoter, the engineered bacterial cell is maintained in the absence of cuminate upon introduction, and the Bxb1 is expressed at a non-induced level upon introduction.

[0047] On the other hand, a circular DNA vector (e.g., an engineered circular DNA vector) is provided, comprising (a) a eukaryotic promoter; (b) a eukaryotic coding sequence; and (c) a bacterial replication origin of less than 50 bp in length, wherein the circular DNA vector lacks a selective marker. In some embodiments, the 3' end of the eukaryotic coding sequence is connected to the 5' end of the promoter via a sequence comprising a bacterial replication origin, wherein the sequence comprising the bacterial replication origin is less than 100 bp in length. In some embodiments, the circular DNA vector is monomeric supercoiled.

[0048] On the other hand, a pharmaceutical composition is provided, comprising a circular DNA vector, the circular DNA vector comprising (a) a eukaryotic promoter; (b) a eukaryotic coding sequence; and (c) a bacterial replication origin of less than 50 bp in length, wherein the circular DNA vector lacks a selective marker. In some embodiments, the 3' end of the eukaryotic coding sequence is connected to the 5' end of the promoter via a sequence comprising a bacterial replication origin, wherein the sequence length comprising the bacterial replication origin is less than 100 bp.

[0049] On the other hand, a host cell (e.g., a mammalian cell, e.g., a human cell) is provided, comprising a circular DNA vector comprising (a) a eukaryotic promoter; (b) a eukaryotic coding sequence; and (c) a bacterial replication origin of less than 50 bp in length, wherein the circular DNA vector lacks a selective marker. In some embodiments, the 3' end of the eukaryotic coding sequence is connected to the 5' end of the promoter by a sequence comprising a bacterial replication origin, wherein the sequence length comprising the bacterial replication origin is less than 100 bp. 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description setting forth illustrative embodiments, in which the principles of the present disclosure are utilized, and in the accompanying drawings:

[0051] Figure 1 is a table showing the results from a stability study in which the three ColE2-P9 origins of replication were tested for their ability to confer plasmid stability in E. coli amplification.

[0052] Figure 2 Examples of methods for assembling parental plasmids that can be used in embodiments disclosed herein are described.

[0053] Figure 3An experimental procedure for producing and testing circular DNA vectors according to embodiments disclosed herein is described.

[0054] Figure 4 The results of agarose gel electrophoresis of extrachromosomal DNA purified from engineered bacteria grown in rich medium containing chloramphenicol ("Cm"; lanes 7-12) or without chloramphenicol ("no Cm"; lanes 1-6) are shown. Lanes 2, 3, 5, 8, 9, and 11 show bands corresponding to the test circular DNA vectors produced by recombination of the test parent plasmids. Lanes 1, 4, 7, and 10 show bands corresponding to the test parent plasmids.

[0055] Figure 5 is a graph showing the percentage of sfGFP-positive cells in the indicated growth media with or without chloramphenicol ("Cm"). ZB = fermentation broth; TB = best broth; SB = super broth; SOB = super optimal broth; SOC = super optimal broth with catabolite repression; LB = Luria broth.

[0056] Figure 6 is a schematic diagram showing an exemplary method for producing a circular DNA vector of the present invention using counter selection.

[0057] 7A to 7F is a schematic diagram showing the contents of a bacterial artificial chromosome (BAC) based method for expressing Bxb1 to generate a circular DNA vector. Fig. 7A It is the Rep gene integrated into the host genome. Figure 7B and Figure 7C There are two alternative BAC designs; Figure 7B Bxb1 is driven by a cuminate-inducible promoter, while Figure 7C Bxb1 is driven by an arabinose-inducible promoter. Fig.7D is a template plasmid, which, after recombination by Bxb1, becomes Fig. 7E Circular DNA vectors and Figure 7F Because the circular DNA vector contains a replication origin and (optionally) the byproduct contains a PheS counter-selectable marker, the circular DNA vector becomes the dominant species when the host bacteria replicate and amplify.

[0058] Fig. 8A and Figure 8B are photographs showing the fluorescence of clones 24 hours and 72 hours after transformation with BAC 1696, respectively.

[0059] Fig.9A and Fig. 9B are photographs showing the fluorescence of clones 24 hours and 72 hours after transformation with BAC 1697, respectively.

[0060] Fig.10 is a set of photographs showing the fluorescence of clones after exposure to cuminate inducer for 24 hours.

[0061] Fig.11 is a set of photographs showing the fluorescence of clones after exposure to the arabinose inducer for 24 hours.

[0062] Fig.12 is a series of photographs showing the fluorescence of restreaked 1696 colonies incubated overnight on LB agar plates under various conditions.

[0063] Fig.13 is a series of photographs showing the fluorescence of restreaked 1697 colonies incubated overnight on LB agar plates under various conditions.

[0064] Fig.14 is a photograph of a gel electrophoresis experiment showing the presence of circular DNA vectors in the counter-selected cultures of 1696 and 1697. The digestion profile showing theoretical bands is shown on the left side of the photograph.

[0065] Fig.15A is a plasmid map of an exemplary ABCA4 template plasmid.

[0066] Fig. 15B Is Fig.15A Plasmid map of the ABCA4 circular DNA vector generated from the template plasmid.

[0067] Fig.16A is a theoretical gel image showing the banding pattern of the circular DNA construct digest described in Example 7. Fig. 16B Is displayed corresponding to Fig.16A Photograph of the gel with the actual banding pattern.

[0068] Fig.17A is a histogram showing long read sequencing data from purified ABCA4 circular DNA vector generated using a 2-hour Kan resistance incubation with template plasmid. The major peaks are BAC and dimeric circular DNA vectors.

[0069] Fig. 17B is a histogram showing long read sequencing data from purified ABCA4 circular DNA vector generated using overnight Kan resistance incubation with template plasmid. The major peaks are monomeric circular DNA vector and BAC.

[0070] Fig.18 is a plasmid map showing the components of a helper plasmid used to express Bxb1 in a bacterial host.

[0071] Fig.19 is a photograph showing green fluorescent colonies (circles) containing circular DNA vectors that have not been Fig.18The helper plasmid for Bxb1 expression produces the backbone byproduct.

[0072] Fig. 20 is a set of diagrams depicting the process of integration of Bxb1 into the host cell genome.

[0073] Fig.21 is a photograph of a gel showing two positive clones for Bxb1 integration. A 1696 plasmid control is shown in triplicate at the bottom left.

[0074] Fig. 22 is a photograph of a western blot showing that HEK293T cells transfected with a bacterially produced ABCA4 circular DNA vector expressed ABCA4 protein. DETAILED DESCRIPTION

[0075] Provided herein is an improved method for producing circular DNA vectors from parent plasmids in engineered bacterial cells, and related compositions. In the past, people have made efforts to improve plasmid DNA vectors by removing main chain components. For example, reorganization is used in bacterial cells to remove the main chain from plasmids to make minicircles, thereby producing minicircle vectors and circular main chain byproducts. However, minicircles are difficult to mass production because their separation requires purification from main chain byproducts with similar structures. By including replication origins and selective markers in vectors, alternative vector types have been designed, such as nanoplasmids, so as to be more easily purified by positive selection. But such foreign elements are relatively large (usually hundreds of base pairs long) and are exogenous to patients.

[0076] Disclosed herein are circular DNA vectors (e.g., therapeutic circular DNA vectors) and bacterial cells (e.g., engineered bacterial cells) produced by bacteria, which can be used to produce them from parent plasmids, such as parent plasmids in bacterial cells (e.g., engineered bacterial cells). The therapeutic circular DNA vectors provided herein include small (e.g., less than 50 base pairs) replication origins, and lack selection markers (e.g., antibiotic resistance genes), which can reduce the risk of exogenous sequence introduction in the vector. Therefore, the circular DNA vectors produced by such bacteria can be effectively and mass-produced for therapeutic applications. In addition, by eliminating or reducing bacterial plasmid DNA sequences (such as RNAPII arrest sites), the transcriptional silencing of the circular DNA vector can be reduced or eliminated, thereby allowing the persistent presence of the vector sequence in an individual. In a specific embodiment of the present invention, immunogenic components (e.g., bacterial endotoxins, DNA or RNA, or bacterial markers, such as CpG motifs) are not present in the circular DNA vector of the present invention, or are present at extremely low levels suitable for pharmaceutical or laboratory applications; therefore, the risk of stimulating host immune response is reduced relative to conventional DNA vectors (such as plasmid DNA vectors). These and other aspects of the disclosed embodiments are discussed in greater detail below.

[0077] I. Definitions

[0078] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in its sense including "and / or" unless the content 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 indicate that any combination is specifically contemplated. For purposes of illustration only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C". The term "or" may be used conjunctively or disjunctively unless the context specifically dictates disjunctive use.

[0079] As used in this specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form 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 in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0080] References in the specification to "some embodiments", "an embodiment" or "other embodiments" mean that a particular feature, structure or characteristic described in conjunction with the embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments. Certain specific details of this specification are set forth in order to provide a thorough understanding of the various embodiments. However, those skilled in the art will understand that the present disclosure can be practiced without these details. In other cases, well-known structures are not shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0081] As used herein, "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 specific molecule or culture condition. The stimulus can also be, for example, the absence of a specific molecule or culture condition. As used herein, "inducible promoter" includes promoters whose expression can be turned on or increased by removing conditions that inhibit expression from the promoter, such as the presence of a specific molecule or other culture condition. In some embodiments, the inducible promoter is a T7 RNA polymerase-dependent promoter, a P tac Promoter, P bad In some embodiments, the inducible promoter is integrated into the bacterial genome and operably linked to a gene (such as, for example, a gene encoding a Rep protein, a restriction endonuclease, or a recombinase).

[0082] As used herein, "exogenous" refers to any substance introduced from the outside of an organism, cell, tissue or system or produced outside an organism, cell, tissue or system. For example, in embodiments described herein, wherein an exogenous restriction enzyme is present in an engineered bacterial cell, the exogenous restriction enzyme is not an enzyme that will be present in the engineered bacterial cell when the exogenous restriction enzyme is not introduced into the engineered bacterial cell from the outside of the engineered bacterial cell. Exogenous restriction enzymes can be introduced into engineered bacterial cells by, for example, introducing genes encoding exogenous restriction enzymes into bacterial cells or introducing restriction enzymes into cells through cell membranes (such as by electroporation). Embodiments described herein also include, for example, exogenous ligases and recombinases.

[0083] As used herein, a "parent plasmid" is a plasmid containing both a vector sequence (as defined below) and a "backbone sequence" (as defined below). Embodiments disclosed herein include methods of preparing a circular DNA vector comprising removing the backbone sequence from the vector sequence.

[0084] As used herein, the "vector sequence" of a parent plasmid refers to a portion of the plasmid DNA that includes an origin of replication and the coding sequence of a gene of interest. In some descriptions of the embodiments herein, the vector sequence is referred to as the "first segment" of the plasmid.

[0085] As used herein, the "backbone sequence" of a parent plasmid refers to a portion of the plasmid DNA outside the vector sequence that includes one or more selectable markers (such as a drug resistance gene or a fragment thereof). In some descriptions of the embodiments herein, the backbone sequence is referred to as the "second segment" of the plasmid.

[0086] As used herein, a "replication protein" is a protein that is necessary to initiate replication at a replication sequence origin that corresponds to the replication protein. A particular replication sequence origin corresponds to a given replication protein if the replication origin depends on the replication protein to initiate replication at the replication sequence origin. For 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 necessary for initiating DNA replication at the ColE2-P9 ori sequence.

[0087] 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, at least one nucleic acid residue, wherein 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 can include, for example, a mutation, deletion or insertion of one nucleic acid residue into another. The variant can encode a homologue, isoform or transcript variant of a therapeutic protein or fragment thereof encoded by a reference nucleic acid sequence, wherein the homologue, isoform or transcript variant is characterized by the degree of identity or homology, respectively, as defined herein.

[0088] In some cases, the functional variant of a polynucleotide or polypeptide includes 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, e.g., 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 cause the expressed polypeptide to have exchange amino acids 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 chains, or amino acids whose side chains can form hydrogen bridges, such as side chains with hydroxyl functional groups. By conservative substitution, for example, an amino acid having a polar side chain can be substituted by another amino acid having a corresponding polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain can be substituted by another amino acid having a corresponding hydrophobic side chain (e.g., serine (threonine) is replaced by threonine (serine), or leucine (isoleucine) is replaced by isoleucine (leucine)).

[0089] In order to determine the percentage of two sequences (e.g., nucleic acid sequences, such as DNA or amino acid sequences) being identical, the sequences can be aligned so that they can be subsequently compared to each other. To this end, a vacancy can be inserted into the sequence of the first sequence, and the components at the corresponding positions of the second sequence can be compared. If a position in the first sequence is occupied by the same component as the corresponding position in the second sequence, the two sequences are identical at this position. The percentage of two sequences being identical is a function of the number of identical positions divided by the total number of positions. The percentage of two sequences being identical can be determined using a mathematical algorithm. Preferred but non-limiting examples of mathematical algorithms that can be used are algorithms such as Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res., 25:3389-3402. Such algorithms can be integrated into, for example, the BLAST program. Sequences identical to the sequences of the present invention to a certain extent can be identified by this program.

[0090] As used herein, the term "flank, flanking and flanked" refers to a pair of regions or points on a nucleic acid molecule (e.g., a plasmid) outside a reference region of a nucleic acid molecule. In some embodiments, a pair of regions or points of a reference region on a flanking nucleic acid are adjacent to (i.e., adjacent to) the reference region (i.e., there is no intervening base between the reference point and the flanking point). In other embodiments, a pair of regions or points on a nucleic acid molecule flanking a reference region are separated from the reference region by one or more intervening bases (e.g., up to 1,000 intervening bases). For example, if the first restriction site is 200 bases upstream of a given sequence, and the second restriction site is 100 bases downstream of a given sequence, the first restriction site and the second restriction site are said to flank a given sequence. In some embodiments, all intervening sequences between a flanking region or point and a reference region do not contain bacterial sequences. In such embodiments, there are no bacterial sequences other than ori sequences in the circular DNA vector produced by cutting a self-ligated vector sequence from a parental plasmid at a restriction site or recombination site of a flanking vector sequence. For example, in such embodiments, an exogenous restriction enzyme that cuts a site flanking a vector sequence can produce a circular DNA vector having a sequence between the 5' and 3' ends of the therapeutic sequence; however, this region does not contain bacterial sequences (e.g., drug resistance genes). Such intervening sequences can be artifacts resulting from sticky end ligation, e.g., corresponding to overhanging bases generated by an exogenous restriction enzyme.

[0091] Unless otherwise indicated, the term "ABCA4" refers to any native ABCA4 from any vertebrate source, 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 or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isoforms, splice variants, and fragments thereof. Functional variants can be determined based on known ABCA4 signaling. ABCA4 encompasses full-length, unprocessed ABCA4, as well as any form of ABCA4 produced by natural processing in cells. Exemplary human ABCA4 sequences are provided as NCBI reference sequences: NG_009073 or NM_000350.

[0092] Unless otherwise indicated, the term "MYO7A" refers to any native MYO7A (also known as DFNB2, MYU7A, NSRD2, USH1B, DFNA11 or MYOVIIA) from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), and functional variants (e.g., natural or synthetic variants), such as mutants, mutant proteins, analogs, subunits, receptor complexes, isoforms, splice variants and fragments thereof. Functional variants can be determined based on known MYO7A signaling. MYO7A encompasses full-length, unprocessed MYO7A, as well as any form of MYO7A produced by natural processing in cells. An exemplary human MYO7A sequence is provided as National Center for Biotechnology Information (NCBI) gene ID: 4647.

[0093] As used herein, the term "self-replicating RNA molecule" refers to a self-replicating genetic element comprising RNA that replicates from one origin of replication.

[0094] As used herein, the term "operably linked" or "operably coupled" refers to an arrangement of elements in which the described components are constructed to perform their usual functions. A nucleic acid is "operably linked" or "operably coupled" when it is in a functional relationship with another nucleic acid sequence. For example, if a promoter affects the transcription of one or more heterologous genes, the promoter is operably linked to one or more heterologous genes. In addition, regulatory elements that are operably linked to a coding sequence can affect the expression of the coding sequence. Regulatory elements do not have to be adjacent to the coding sequence, as long as they have the function of directing their expression. Thus, for example, intervening untranslated but transcribed sequences can be present between a promoter sequence and a coding sequence, and the promoter sequence can still be considered to be "operably linked" or "operably coupled" to the coding sequence.

[0095] As used herein, "vector" refers to such a nucleic acid molecule, which can carry the sequence of interest connected thereto into the target cell, and then the sequence of interest can be transcribed, replicated, processed and / or expressed in the target cell. After the target cell or host cell processes the sequence of interest of the vector, the sequence of interest is not considered to be a vector. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop that can replicate autonomously and contain a bacterial backbone, wherein the bacterial backbone includes a bacterial replication origin and a selective marker, wherein an additional DNA segment can be connected. Another type of vector is a bacteriophage vector. Another type of vector is a viral vector, wherein an additional DNA segment can be connected to the viral genome. Some vectors can replicate autonomously in the host cell it is introduced into (for example, a bacterial vector with a bacterial replication origin and an additional mammalian vector). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated with the host genome. In addition, some vectors can guide the expression of the gene operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors" or "expression vectors").

[0096] As used herein, the terms "individual" and "subject" are used interchangeably and include any mammal in need of treatment or prevention, such as treatment or prevention by a circular DNA vector or a pharmaceutical composition thereof, as described herein. In some embodiments, the individual or subject is a human. In other embodiments, the individual or subject is a non-human mammal (e.g., a non-human primate (e.g., monkey), mouse, pig, rabbit, cat, or dog). The individual or subject can be male or female.

[0097] As used herein, an "effective amount" or "effective dose" of a circular DNA vector or a pharmaceutical composition thereof refers to an amount sufficient to achieve a desired biological, pharmacological or therapeutic effect, for example, when administered to an individual according to a selected form of administration, route and / or schedule. As will be appreciated by those of ordinary skill in the art, the absolute amount of an effective particular composition may vary according to factors such as the desired biological or pharmacological endpoint, the agent to be delivered, the target tissue, and the like. Those of ordinary skill in the art will further appreciate that an "effective amount" may be contacted with cells or administered to a subject in a single dose or by using multiple doses. Relative to a reference population (e.g., an untreated or placebo population, or a population receiving standard care treatment), an effective amount of a composition for treating a disease may slow or prevent disease progression, or increase a partial or complete response.

[0098] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and the clinical intervention can be directed to prevention or during clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, ameliorating or alleviating the disease state, and improving prognosis. In some embodiments, the circular DNA vectors of the present invention are used to delay the development of the disease or slow the progression of the disease.

[0099] The terms "level of expression" or "expression level" are used interchangeably and generally refer to the amount of polynucleotides or amino acid products or proteins in a biological sample (e.g., retina). "Expression" generally refers to the method by which the information encoded by a gene is converted into a structure that exists and operates in a cell. Therefore, according to the present invention, "expression" can refer to transcription into polynucleotides, translation into proteins, or post-translational modification of proteins. Fragments of transcribed polynucleotides, translated proteins, or post-translationally modified proteins should also be considered as expressed, whether they are derived from alternative splicing to produce transcripts or degraded transcripts, or from post-translational processing of proteins (e.g., by proteolysis). "Expressed genes" include those genes that are transcribed into polynucleotides in the form of mRNA and then translated into proteins, as well as those genes that are transcribed into RNA but not translated into proteins (e.g., transfer RNA and ribosomal RNA).

[0100] As used herein, the term "expression persistence" refers to the duration that a sequence of interest or a functional portion thereof (e.g., one or more coding sequences of a circular DNA vector) can be expressed in a cell into which it is transfected ("intracellular persistence") or can be expressed in any progeny of a transfected cell ("transgenerational persistence"). A sequence of interest (such as a therapeutic sequence) or a functional portion thereof can be expressible if it is not silenced (e.g., silenced by DNA methylation and / or histone methylation and compression). Expression persistence can be assessed by detecting or quantifying (i) mRNA transcribed from a sequence in a target cell or its progeny (e.g., by qPCR, RNA-seq, or any other suitable method) and (ii) protein translated from a sequence in a target cell or its progeny (e.g., by Western blot, ELISA, or any other suitable method). In some cases, expression persistence is assessed by detecting or quantifying therapeutic DNA in target cells or their progeny (e.g., whether there is a circular DNA vector in the target cell, e.g., by additional DNA copy number analysis) and any one or both of the following: (i) mRNA transcribed from the therapeutic sequence in the target cell or its progeny and (ii) protein translated from the therapeutic sequence in the target cell or its progeny. Expression persistence of the sequence of interest or its functional part can be quantified relative to a reference vector using any gene expression characterization method known in the art, such as a control vector with one or more bacterial markers not present in the vector of the present invention (e.g., plasmid). Expression persistence can be quantified at any given time point after the administration of the vector. For example, in some embodiments, if the circular DNA vector can be detected in the target cell or its progeny two weeks after the administration of the circular DNA vector of the present invention, the circular DNA vector continues for at least two weeks after administration. In some embodiments, if the expression of a gene can be detected in the target cell one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year or more after administration, the expression of the gene is "persistent" in the target cell. In some embodiments, if there is any detectable portion 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% of the original expression level) after a given period of time (e.g., one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year or more after administration), the expression of the sequence is considered to continue for the given period of time after administration.

[0101] As used herein, "intracellular persistence" refers to the duration that a sequence or a functional portion thereof (e.g., one or more coding sequences of a circular DNA vector) can be expressed in a cell (e.g., a target cell, such as a post-mitotic cell or a resting cell) to which it is transfected. Intracellular persistence can be assessed by detecting or quantifying the following: (i) mRNA transcribed from a sequence in a target cell and (ii) protein translated from a sequence in a target cell. In some cases, intracellular persistence is assessed by detecting or quantifying DNA in a target cell (e.g., whether a circular DNA vector is present in a target cell) and any one or both of the following: (i) mRNA transcribed from a sequence in a target cell and (ii) protein translated from a sequence in a target cell. In some embodiments, the circular DNA vector of the present invention exhibits improved intracellular persistence relative to a reference vector (e.g., a plasmid DNA vector).

[0102] As used herein, "transgenerational persistence" refers to the duration that a sequence or a functional portion thereof (e.g., one or more coding sequences of a DNA vector) can be expressed in the progeny of a gene-transfected cell (e.g., the progeny of a target cell, such as the first, second, third, or fourth generation of a cell transfected (e.g., by a circular DNA vector). Transgenerational persistence accounts for any dilution of a gene during cell division and can therefore be used to measure the persistence of a vector in a dividing tissue over time. In some embodiments, the circular DNA vectors of the present invention exhibit improved transgenerational persistence relative to a reference vector (e.g., a plasmid DNA vector). Transgenerational persistence can be assessed by detecting or quantifying (i) mRNA transcribed from a vector sequence in the progeny of a target cell and (ii) protein translated from a vector sequence in the progeny of a target cell. In some cases, intracellular persistence is assessed by detecting or quantifying DNA in the progeny of the target cell (e.g., whether the circular DNA vector is present in the progeny of the target cell) and 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 some embodiments, the circular DNA vectors of the invention exhibit improved transgenerational persistence relative to a reference vector (e.g., a plasmid DNA vector).

[0103] As used herein, the term "copy number" of a DNA molecule refers to the average number of copies of the DNA molecule per cell in a given population of cells.

[0104] The term "pharmaceutically acceptable" means safe for administration to mammals, such as humans. In some embodiments, a pharmaceutically acceptable composition is approved by a regulatory agency of the federal or state government, or is listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, more specifically in humans.

[0105] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the vector or composition of the invention is administered. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA., 23rd edition, 2020.

[0106] As used herein, unless otherwise specified, the term "about" refers to a value that varies within ±10% from a reference value.

[0107] For any conflicts in definitions between different sources or references, the definitions provided herein shall prevail.

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below.

[0109] Throughout the disclosure, numerical features are presented in range format. It should be understood that the description of the range format is only for convenience and simplicity, and should not be interpreted as an unchangeable limitation to the scope of any embodiment. Therefore, unless the context clearly stipulates otherwise, the description of the range should be considered to have specifically disclosed all possible sub-ranges and a single numerical value within the range to one-tenth of the lower limit unit. For example, the description of a range such as from 1 to 6 should be considered to have a specifically disclosed sub-range, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and a single value within the range, such as 1.1, 2, 2.3, 5 and 5.9. This applies to the width of any range. The upper and lower limits of these intervening ranges can be independently included in a smaller range, and are also encompassed within the present invention, subject to the constraints of any specifically excluded limits within the stated range. Unless clearly stipulated otherwise in the context, otherwise in the case where the range includes one or two limits, the scope that does not include one or two of these included limits is also included in the present invention.

[0110] II. Methods for producing circular DNA vectors in bacteria

[0111] Embodiments disclosed herein include methods for producing circular DNA vectors in engineered bacterial cells. Engineered bacterial cells disclosed herein can be used to produce circular DNA vectors from parent plasmids. In some embodiments, engineered bacterial cells include Rep genes encoding bacterial replication proteins integrated into bacterial genomes and parent plasmids. In some embodiments, the Rep gene is contained on an extrachromosomal DNA molecule, such as, for example, a plasmid (e.g., a helper plasmid) or a bacterial artificial chromosome ("BAC"). In some embodiments, the Rep gene is contained on a parent plasmid. The parent plasmid includes a vector sequence and a backbone sequence. The vector sequence includes an ori sequence corresponding to the Rep gene, but does not include a selective marker. The backbone sequence includes a selective marker, but does not include an ori sequence included in the vector sequence, but in some embodiments, different ori sequences may be included. The parent plasmid also has a restriction enzyme recognition sequence or a site-specific recombination sequence flanking the vector sequence that is arranged so that the plasmid backbone sequence can be separated from the vector sequence in the cell by restriction digestion or site-specific recombination. In the case of restriction digestion, a circular DNA vector is then formed by self-ligation of the vector sequence. In the case of site-specific recombination, when the recombination is completed, a circular DNA vector is formed. After vector sequence isolation and circular DNA vector formation, the expression of rep protein can maintain the high copy number of circular DNA vector, despite the lack of selectable markers. Conversely, by changing the culture conditions to remove the selective pressure of the selectable marker, the retention of plasmid backbone sequences in engineered bacterial cells after isolation can be avoided. Cultivating bacterial cell populations with high copy number circular DNA vectors under conditions where the parental plasmid is not maintained can effectively produce high yields of high purity circular DNA vectors.

[0112] 1. Engineering bacterial cells

[0113] The method for producing circular DNA vector disclosed herein includes 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.

[0114] In some embodiments, the engineered bacterial cell has an exogenous Rep gene integrated into the bacterial genome. Any suitable chromosomal integration method can be used to incorporate the Rep gene into the bacterial genome, including integration boxes and procedures well known in the art. In some embodiments, the Rep gene encodes the ColE2-P9 replication protein or related proteins. In some exemplary embodiments, the Rep gene encodes the ColE2-P9 replication protein with the amino acid sequence shown in SEQ ID NO:1. Other suitable replication proteins include replication proteins encoded by naturally occurring plasmids, including, for example, those replication proteins associated with ColE2-P9, such as ColE3-CA38. Replication proteins can be used in the embodiments described herein together with their corresponding replication origin sequences.

[0115] The ori sequence contained in the vector sequence of the parent plasmid is selected so that it corresponds to the Rep gene integrated into the genome of the engineered bacterial cell, or is otherwise present in the engineered bacterial cell, such as on a plasmid or BAC. In some exemplary embodiments, the ori comprises the nucleotide sequence shown in SEQ ID NO:2. Therefore, the embodiments of the engineered bacterial cells disclosed herein include a functional pair of replication proteins and replication origin sequences that allow replication of the parent 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, which has the DNA sequence shown in SEQ ID NO:2. The 40 base pair functional fragment given in SEQ ID NO:2 is capable of supporting vector replication in cells expressing ColE2-P9. In some embodiments, shorter or longer functional fragments may be used. In some embodiments, a 31 base pair fragment of ColE2-P9 may 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, for example, the ori sequence of ColE3-CA38. In some embodiments, the ori is no more than 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, for example, an ori sequence 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.

[0116] In some embodiments, the Rep gene is operably linked to an inducible promoter. Suitable inducible promoters include, but are not limited to, P promoters induced by T7 RNA polymerase. T7 Promoter, heat-inducible P L promoter, P repressible by LacI tac promoter (and thus can be induced by the deletion or removal of LacI), the P badOther inducible promoters known in the art may also be used in the embodiments disclosed herein, including, for example, bacterial phage promoters (e.g., P L s1con, T3, T7, SP6) and bacterial promoters (e.g., PmgrB, pLlacO, Ptrc2, pLtetO, Plac / ara, Pm). Examples of bacterial promoters used in accordance with the present disclosure include, but are not limited to, positively regulated E. coli promoters, such as the positively regulated s1con, T3, T7, SP6, and PmgrB. 70 Promoters (e.g., inducible pBad / araC promoter, Lux cassette right promoter, modified λ Prm promoter, placOr2-62 (positive), pBad / AraC with an additional REN site, pBad, P(Las)TetO, P(Las)CIO, P(Rhl), Pu, FecA, pRE, cadC, hns, pLas, pLux), σ S Promoter (e.g., Pdps), σ 32 Promoter (e.g., heat shock) and σ 54 promoter (e.g., glnAp2); negatively regulated E. coli promoters, such as the negatively regulated σ 70 Promoters (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 control, modified Pr, modified Prm+, FecA, Pcya, rec A(SOS), Rec A(SOS), EmrR_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 Promoter (e.g., with alternative σ factor σ 38 Lutz-Bujard LacO), σ 32 Promoter (e.g., with alternative σ factor σ 32 Lutz-Bujard LacO) and σ 54promoter (eg, glnAp2); a negatively regulated B. subtilis promoter, such as the repressible B. subtilis σ A Promoters (e.g., Gram-positive IPTG-inducible, Xyl, hyper-spank) and σ B Promoters. Other inducible bacterial promoters can be used in accordance with the present disclosure. For example, in some embodiments, a cuminate inducible promoter such as pCymRC can be used.

[0117] In some embodiments, the expression level of replication protein affects the copy number of the parent plasmid or circular DNA vector containing the corresponding ori sequence. Therefore, when a relatively low copy number is required (for example, less than 5, 10 or 20 copies / cell on average), the engineered bacterial cell can be maintained under the condition that the replication protein is expressed at a relatively low level. When a relatively high copy number is required (for example, more than 20, 50 or 100 copies / cell on average), the engineered bacterial cell can be maintained under the condition that the replication protein is expressed at a relatively high level from an inducible promoter. In some embodiments, the Rep gene is operably connected to an inducible promoter, and the promoter provides a first level of expression under non-inducing conditions, and can be induced to provide a second, higher level of expression, which results in a higher copy number of the parent plasmid or circular DNA vector containing the corresponding ori sequence. In some embodiments, before separating the vector sequence and the main chain sequence of the parent plasmid, it is advantageous to maintain the parent plasmid in the engineered bacterial cell with a low copy number. In embodiments where the vector sequence and the backbone sequence are separated by restriction digestion, having a relatively low copy number helps to ensure that the linearized vector sequence is self-connected, rather than connected to other copies of the backbone sequence or the vector sequence. After separating the vector sequence from the backbone sequence and forming a circular DNA vector, in some embodiments, it is advantageous for the circular DNA vector containing the ori sequence to maintain a relatively high copy number to produce a high-yield circular DNA vector. Therefore, 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 by, for example, adding a molecule that induces higher expression from an inducible promoter operably connected to the Rep gene. In some embodiments, the inducible promoter maintains an uninduced state until 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 while the vector sequence is separated from the backbone sequence. In some embodiments, the inducible promoter is induced before the vector sequence is separated from the backbone sequence.

[0118] In some embodiments, the copy number of the parental plasmid is maintained at at least about, at most about, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 copies / cell, or a copy number between any two of these values. In some embodiments, the copy number of the circular DNA vector is maintained at at least about, at most about, or at about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 copies per cell, or between any two of these values.

[0119] In some embodiments, the Rep gene integrated into the genome of the engineered bacterial cell is under the control of a constitutive or non-inducible promoter. In some embodiments, the expression of the replication protein is not regulated before, during or after separation of the vector sequence from the backbone sequence. In some embodiments, the parental plasmid before isolation and the circular DNA vector after isolation are maintained at at least about, at most about, or at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 copies / cell, or a copy number between any two of these values.

[0120] In addition to the parent plasmid or the carrier sequence separated from the parent plasmid, the engineered bacteria can also include other extrachromosomal DNA molecules, such as helper plasmid or BAC.Extrachromosomal DNA molecules can encode for example exogenous recombinase, restriction endonuclease, replication protein, ligase, selective marker, reverse selective marker or reporter gene.In some embodiments, before the circular DNA vector is purified from the culture of the engineered bacterial cell, the extrachromosomal DNA molecules except the carrier sequence are removed from the engineered bacterial cell.In some embodiments, can be by not applying selective pressure to maintain the extrachromosomal DNA molecule under the condition of culturing cells, or by reducing or eliminating the cell growth comprising the extrachromosomal DNA molecule under the reverse selection condition of culturing cells, remove the extrachromosomal DNA molecule from the engineered bacterial cell.

[0121] In some embodiments, the extrachromosomal DNA molecule included 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 auxiliary plasmid or BAC can include a gene encoding a visually detectable protein (such as GFP or RFP). In this case, visual observation of the bacterium colony color under UV light can reveal whether the extrachromosomal DNA molecule is present in the cell of the bacterium colony. In this way, the bacterium colony lacking 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 the engineered bacterial cell or bacterium colony contain a given extrachromosomal DNA molecule.

[0122] B. Parental plasmid

[0123] Embodiments of the engineered bacteria disclosed herein include a parent plasmid (also referred to as a template plasmid or plasmid template) comprising a vector sequence and a backbone sequence separated from each other by two restriction sites or recombination sites.

[0124] The vector sequence includes an ori sequence and a sequence of interest, and in some embodiments, the sequence of interest is a therapeutic coding sequence, a reporter gene construct, or a combination thereof. The vector sequence may include any component of the circular DNA vector embodiment described herein. In some embodiments, the vector sequence does not include the sequence of any bacterial origin except the ori sequence. In some embodiments, the parent plasmid includes a restriction site or a recombination site next to the ori sequence and / or the therapeutic sequence or the reporter gene construct, so that there is no foreign or non-functional DNA in the vector sequence that becomes the circular DNA vector.

[0125] In some embodiments, the main chain sequence includes a selective marker, but does not include an ori sequence corresponding to an exogenous replication protein encoded by an integrated Rep gene. In some embodiments, the main chain sequence includes an ori sequence that does not correspond to an integrated Rep gene, i.e., is directly homologous to the ori sequence and the integrated Rep gene in the vector sequence. In some embodiments, the selective marker contained in the main chain sequence helps to ensure that the parent plasmid is maintained in an engineered bacterial cell population cultured under conditions where the selective marker is necessary for cell growth or survival. For example, in some embodiments, the selective marker is an antibiotic resistance gene. Cultivating engineered bacterial cells in the presence of corresponding antibiotics applies selective pressure so that the parent plasmid is maintained in the engineered bacterial cell population. However, once the selective pressure is removed, such as by changing the growth medium to a medium lacking an antibiotic corresponding to an antibiotic resistance gene, the DNA molecule containing the antibiotic resistance gene may be lost from the population, especially if such DNA molecule does not include an ori sequence. Therefore, after the vector sequence is separated from the main chain sequence, if the culture conditions do not apply selective pressure to maintain the main chain sequence, the main chain sequence may be lost from the population or cannot be maintained in large quantities.

[0126] In some embodiments, the backbone sequence comprises a counter selection marker. The counter selection marker can provide a way to selectively grow cells that do not contain backbone sequences. In some embodiments, after separating the vector sequence from the backbone sequence, culturing cells under counter selection conditions can improve purity and reduce the amount of the backbone sequence in the culture and / or the composition comprising the purified vector sequence. Suitable counter selection markers are known in the art and can include, for example, pheS, sacB, thyA, lacY, gata-1, ccdB, rpsL or tetAR.

[0127] The restriction sites or recombination sites in the parental plasmid flanking the vector sequences may be selected from any suitable restriction sites or recombination sites not present in the vector sequences.

[0128] C. Restriction Digestion and Ligation

[0129] Embodiments disclosed herein include a restriction digestion step to separate the vector sequence from the main chain sequence of the parent plasmid. In some embodiments, restriction digestion occurs in engineered bacterial cells. In some embodiments, the restriction enzyme that digests the parent plasmid is an exogenous restriction enzyme expressed by 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 in the engineered bacterial cell. In some embodiments, it is necessary to suppress or delay the induction of restriction enzyme expression until the vector sequence is separated from the main chain sequence of the parent plasmid. Therefore, in some embodiments, the exogenous gene is operably connected to an inducible promoter. When separation is required, the expression of the restriction enzyme can be induced and then separated.

[0130] In some embodiments, the restriction enzyme for separating the vector sequence from the backbone sequence is an exogenous restriction enzyme introduced into the engineered bacterial cell through the cell membrane. In some embodiments, this is achieved by electroporation. The non-limiting examples of electroporation and digestion procedures are as follows: the electrocompetent engineered Escherichia coli containing the parent plasmid is cultivated to OD of 0.8 at 30°C in SOB. The bacteria are washed three times with ice-cold 10% glycerol and resuspended in 10% glycerol. Each restriction enzyme of 0.5 μl and ligase are mixed with electrocompetent cells to digest 1 μg of DNA with 10 units of restriction enzymes. The mixture is transferred to a cuvette (1mm gap), and electroporation is performed using an electroporator (BTX) using 1800 volts. Cells are saved by growing in SOC for 1 hour at 37°C, and are plated on LB agar plates without antibiotics. QIAGEN miniprep kit is used to grow colonies and purify DNA.

[0131] After the separation of the vector sequence, a circular DNA vector can be formed by the self-ligation of the vector sequence. In some embodiments, the connection occurs in an engineered bacterial cell. In some embodiments, the ligase at the end of the connection vector sequence is an exogenous ligase expressed by an exogenous ligase gene introduced into the engineered bacterial cell. The ligase can be, for example, a T3 ligase, a T4 ligase, or a 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 in the engineered bacterial cell. In some embodiments, the expression of the ligase is suppressed or not induced until the separation of the vector sequence from the main chain sequence of the parent plasmid is completed. Therefore, in some embodiments, the exogenous ligase gene is operably connected to an inducible promoter.

[0132] In some embodiments, the ligase is an exogenous ligase introduced into the engineered bacterial cell through the cell membrane. In some embodiments, this is accomplished by electroporation, which can be accomplished according to the electroporation protocol described above. In some embodiments, the electroporation of the restriction enzyme and the ligase is accomplished in a single step, and the restriction enzyme and the ligase enter the cell in a single electroporation step. In some embodiments, the restriction enzyme and the ligase are added to the cell separately.

[0133] In some embodiments, self-ligation of vector sequences is accomplished by endogenous ligases produced by the engineered bacterial cells.

[0134] In some embodiments, exogenous restriction enzyme and exogenous ligase are present in engineered bacterial cells simultaneously. In some embodiments, before exogenous ligase is introduced into engineered bacterial cells (for example, by electroporation of exogenous restriction enzyme, by transformation with DNA molecules encoding exogenous restriction enzyme or by inducing expression of exogenous restriction enzyme gene under the control of inducible promoter), exogenous restriction enzyme is introduced into engineered bacterial cells (for example, by electroporation of exogenous ligase, by transformation with DNA molecules encoding exogenous ligase or by inducing expression of exogenous ligase gene under the control of inducible promoter). In some embodiments, before exogenous ligase is introduced into engineered bacterial cells, exogenous restriction enzyme is introduced into engineered bacterial cells. In some embodiments, exogenous restriction enzyme and exogenous ligase are introduced into engineered bacterial cells simultaneously.

[0135] D. Site-specific recombination

[0136] Site-specific recombination can be performed using various systems that result in site-specific recombination between sequences. In some embodiments, site-specific recombination involves two specific sequences that are capable of recombining with each other in the presence of a recombinase.

[0137] In some embodiments, the recombinase separating the carrier sequence from the plasmid sequence is an exogenous recombinase expressed by an exogenous recombinase gene introduced into an 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 the induction of recombinase expression until the carrier sequence is separated from the main chain sequence of the parent plasmid. Therefore, in some embodiments, the exogenous recombinase gene is operably connected to an inducible promoter, such as any inducible promoter disclosed herein. When separation is required, the expression of the exogenous recombinase can be induced, and then separated. In some embodiments, the exogenous recombinase is expressed when the parent plasmid is introduced into the engineered bacterial cell, which can cause the parent plasmid to experience restructuring without inducing the expression of the recombinase. In some embodiments, when the parent plasmid is introduced into the engineered bacterial cell, the recombinase is expressed at a relatively low level. For example, in some embodiments, the engineered bacterial cells may include an exogenous recombinase gene operatively coupled to an inducible promoter that provides low-level expression under non-inducing conditions (e.g., it may be integrated into the bacterial chromosome or contained on a plasmid or BAC present in the bacterial cell before the parent plasmid is introduced into the engineered bacterial cell). Introducing the parent plasmid into the engineered bacterial cells with an appropriately low level of exogenous recombinase expression can allow the colonies to grow on a medium that is selective for the selectable marker on the parent plasmid backbone sequence, while also inducing sufficient recombination of the parent plasmid to produce a cell population in the colonies with the vector sequence separated from the backbone sequence.

[0138] In some embodiments, the recombinase for separating the vector sequence from the main chain sequence is an exogenous recombinase introduced into the engineered bacterial cell through the cell membrane. In some embodiments, this is achieved by electroporation. The non-limiting examples of electroporation and recombinant procedures are as follows: the electrocompetent engineered Escherichia coli containing the parent plasmid is cultivated to OD of 0.8 at 30°C in SOB. 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 (1mm gap) and electroporated using an electroporator (BTX) using 1800 volts. Cells are saved by growing in SOC for 1 hour at 37°C and are plated on LB agar plates without antibiotics. QIAGEN miniprep kit is used to grow colonies and purify DNA. 1 μg of DNA is digested with 10 units of restriction enzymes.

[0139] The specific recombination systems used in the embodiments disclosed herein can have different origins. In particular, the specific sequences and recombinases used can belong to different structural classes, such as the integrase family of bacteriophage lambda or the resolvase family of transposon Tn3.

[0140] Recombinases belonging to the integrase family of bacteriophage lambda include, for example, bacteriophage lambda (Landy et al., Science 197:1147, 1977), P22 and (Leong et al., J. Biol. Chem. 260:4468, 1985), HP1 of Haemophilus influenzae (Hauser et al., J. Biol. Chem. 267:6859, 1992), Cre integrase of phage P1 (which recognizes and causes recombination of LoxP sites), integrase of plasmid pSAM2 (EP 350,341), or alternatively FLP recombinase of 2μ plasmid. In embodiments where circular DNA vectors are prepared by site-specific systematic recombination of the integrase family of bacteriophage λ, the resulting circular DNA vector typically comprises sequences resulting from recombination between two att attachment sequences of the corresponding bacteriophage or plasmid.

[0141] 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 μ, or the resolvase of alternative geoparticles, such as the resolvase of the par fragment of RP4 (Albert et al., Mol. Microbiol. 12: 131, 1994). In embodiments in which circular DNA vectors are prepared by site-specific system recombination of the transposon Tn3 family, the resulting circular DNA vectors typically contain sequences generated by recombination between two recognition sequences of the resolvase of the transposon in question.

[0142] In some embodiments, the site-specific recombination sequence on the parent plasmid is derived from a bacteriophage. In some embodiments, the sequence is the attachment sequence (attP and attB sequences) of the bacteriophage integrase or a sequence derived from such an attachment sequence. These sequences are able to specifically recombinate with each other in the presence of a recombinase known as an integrase, with or without an excision enzyme. The term "sequence derived from such an attachment sequence" includes sequences obtained by modifying the attachment sequence of a bacteriophage, which retains the ability to specifically recombine in the presence of a suitable recombinase. Therefore, such sequences can be reduced fragments of these sequences, or alternatively, fragments extended by adding other sequences (restriction sites, etc.). They can also be variants obtained by mutation, particularly by point mutation, such as attP-GA and attB-GA attachment sequences.

[0143] In some embodiments, the recognition sequences and recombinases used are from members of the tyrosine recombinase family, such as, for example, Flp, XerC, XerD, λ integrase, or HP1 integrase, or members of the serine recombinase family, such as, for example, φBT1, TP901, Bxb1, MR11, A118, φK38, φC31, or Wβ.

[0144] In some embodiments, the recognition sequence and the recombinase are from Bxb1 (eg, the exogenous recombinase is Bxb1 and the recognition sequences are attP-GA and attB-GA).

[0145] E. Amplification of circular DNA vectors by cultured cells

[0146] According to the embodiments described herein, after the circular DNA vector is formed in the engineered bacterial cell, the amount of the circular DNA vector produced can be increased by culturing the engineered bacterial cell colony containing the circular DNA vector. Culture conditions can be selected to maximize the production of bacterial cell growth and additional copies of the circular DNA vector. In some embodiments, culture conditions are selected to induce high-level expression of replication proteins, thereby supporting high copy numbers of circular DNA vectors with corresponding ori sequences. In some embodiments, culture conditions are selected to remove the selective pressure of the main chain sequence containing a selective marker so that the main chain sequence is not maintained in several rounds of cell division. In some embodiments, culture conditions are selected to provide reverse selective pressure for the reverse selection marker present on the main chain sequence so that the cell including the main chain sequence has a reduced growth potential or cannot grow.

[0147] In some embodiments, culturing a population of engineered bacterial cells comprising a circular DNA vector results in maintaining the circular DNA vector in such cultured cells for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 rounds of cell division. In some embodiments, the cultured population of engineered bacterial cells maintains the circular DNA vector at an average copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 copies / cell after at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 copies / cell (e.g., at least 1 copy / cell after at least 10 doublings, at least 5 copies / cell after at least 10 doublings, at least 10 copies / cell after at least 10 doublings, at least 10 copies / cell after at least 10 doublings, at least 10 copies / cell after at least 10 doublings, at least 10 copies / cell after at least 10 doublings, at least 10 copies / cell after at least 10 doublings, at least 10 copies / cell after at least 10 doublings, at least 10 copies / cell after at least 10 doublings, at least 10 copies / cell after at least 10 doublings, at least 10 copies / cell after at least 10 doublings, at least 10 copies / cell after at least 10 doublings, at least 10 copies / cell after at least 10 doublings, at least 10 copies / cell after at least 10 doublings, at least 10 copies / cell after at least 10 doublings The invention further comprises at least 1 copy / cell after at least 20 doublings (e.g., at least 5 copies / cell after at least 20 doublings, at least 10 copies / cell after at least 20 doublings, or at least 20 copies / cell after at least 20 doublings), at least 1 copy / cell after at least 50 doublings (e.g., at least 5 copies / cell after at least 50 doublings, at least 10 copies / cell after at least 50 doublings, or at least 20 copies / cell after at least 50 doublings), or at least 1 copy / cell after at least 100 doublings (e.g., at least 5 copies / cell after at least 100 doublings, at least 10 copies / cell after at least 100 doublings, or at least 20 copies / cell after at least 100 doublings)).In some embodiments, after separation of the vector sequence from the backbone sequence, the average copy number of the backbone sequence is less than 5, 4, 3, 2, 1, 0.5, 0.1, 0.01, or 0.001 copies / cell, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 001 copies / cell after at least 10 doublings, less than 0.001 copies / cell after at least 20 doublings, less than 0.001 copies / cell after at least 50 doublings, or less than 0.001 copies / cell after at least 10 doublings. less than .001 copies / cell after at least 10 doublings, less than .01 copies / cell after at least 20 doublings, less than .01 copies / cell after at least 50 doublings, or less than .01 copies / cell after at least 100 doublings), less than .1 copies / cell after at least 1 doubling (e.g., less than .1 copies / cell after at least 10 doublings, less than .01 copies / cell after at least 100 doublings), less than .001 copies / cell after at least 10 doublings (e.g., less than .0 ... The invention relates to a method for expressing a gene in a cell, wherein the gene has a plurality of copies / cells, a plurality of copies / cells, a plurality of copies / cells, a plurality of copies / cells, and a plurality of copies / cells. The method further relates to a method for expressing a gene in a cell, wherein the gene has a plurality of copies / cells, a plurality of copies / cells, a plurality of copies / cells, and a plurality of copies / cells. The method further relates to a method for expressing a gene in a cell, wherein the gene has a plurality of copies / cells, a plurality of copies / cells, a plurality of copies / cells, and a plurality of copies / cells.In some embodiments, after separation of the vector sequence from the backbone sequence, the average copy number of the backbone sequence is less than 5, 4, 3, 2, 1, 0.5, 0.1, 0.01, or 0.001 copies / cell, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 001 copies / cell after up to 10 doublings, less than .001 copies / cell after up to 20 doublings, less than .001 copies / cell after up to 50 doublings, or less than .001 copies / cell after up to 100 doublings. less than .001 copies / cell after up to 10 doublings), less than .01 copies / cell after up to 10 doublings (e.g., less than .01 copies / cell after up to 10 doublings, less than .01 copies / cell after up to 20 doublings, less than .01 copies / cell after up to 50 doublings, or less than .01 copies / cell after up to 100 doublings), less than .1 copies / cell after up to 1 doubling (e.g., less than .1 copies / cell after up to 10 doublings), less than .01 copies / cell after up to 10 doublings (e.g., less than .01 copies / cell after up to 10 doublings). The invention further provides that the number of copies / cells present in the genome of the gene is less than 1 copy / cell after up to 10 doublings, less than 1 copy / cell after up to 20 doublings, less than 1 copy / cell after up to 50 doublings, or less than 1 copy / cell after up to 100 doublings) or less than 1 copy / cell after up to 1 doubling (e.g., less than 1 copy / cell after up to 10 doublings, less than 1 copy / cell after up to 20 doublings, less than 1 copy / cell after up to 50 doublings, or less than 1 copy / cell after up to 100 doublings)).

[0148] In some embodiments, culturing a population of engineered bacterial cells comprising a circular DNA vector results in maintenance of the circular DNA vector in such cultured cells for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 290, 294, 300, 350, 400, 450, or 500 rounds of cell division (e.g., as confirmed by Sanger sequencing). In some embodiments, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 290 times. After 1, 294, 300, 350, 400, 450 or 500 doublings, the cultured population of engineered bacterial cells maintains the circular DNA vector at an average copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 copies / cell.In some embodiments, after separation of the vector sequence from the backbone sequence, the average copy number of the backbone sequence is less than 5, 4, 3, 2, 1, 0.5, 0.1, 0.01, or 0.001 copies / cell, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 001 copies / cell after up to 1 doubling (e.g., less than .001 copies / cell after up to 10 doublings, less than .001 copies / cell after up to 20 doublings, less than .001 copies / cell after up to 30 doublings, less than .001 copies / cell after up to 40 doublings, less than .001 copies / cell after up to 50 doublings, less than .001 copies / cell after up to 60 doublings, less than .001 copies / cell after up to 70 doublings, less than .001 copies / cell after up to 80 doublings, less than .001 copies / cell after up to 10 doublings, less than .001 copies / cell after up to 20 doublings, , less than .001 copies / cell after up to 50 doublings or less than .001 copies / cell after up to 500 doublings), less than .01 copies / cell after up to 1 doubling (e.g., less than .01 copies / cell after up to 10 doublings, less than .01 copies / cell after up to 20 doublings, less than .01 copies / cell after up to 50 doublings or less than .01 copies / cell after up to 100 doublings), less than .1 copies / cell after up to 1 doubling (e.g., less than .01 copies / cell after up to 10 doublings, less than .01 copies / cell after up to 20 doublings, less than .01 copies / cell after up to 50 doublings or less than .01 copies / cell after up to 100 doublings). less than 1 copy / cell after up to 10 doublings, less than 1 copy / cell after up to 20 doublings, less than 1 copy / cell after up to 50 doublings, or less than 1 copy / cell after up to 100 doublings) or less than 1 copy / cell after up to 1 doubling (e.g., less than 1 copy / cell after up to 10 doublings, less than 1 copy / cell after up to 20 doublings, less than 1 copy / cell after up to 50 doublings, or less than 1 copy / cell after up to 100 doublings)).

[0149] Some embodiments include cultures of engineered bacterial cells wherein the average copy number of the circular DNA vector or parent plasmid is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 copies / cell, or between any two of these values. In some embodiments, the culture comprises at least 10 5 10 6 10 7 10 8 109 10 10 10 11 or 10 12 total cells, or between any two of these values. In some embodiments, the culture comprises at least 10 4 10 5 10 6 10 7 10 8 10 9 or 10 10 cells / ml, or between any two of these values.

[0150] E. Recovery of circular DNA vector

[0151] By extraction and purification procedures known in the art, the circular DNA vector produced by the embodiments disclosed herein can be recovered from the culture of engineered bacteria. In some embodiments, the engineered bacterial cells of every liter of culture can recover at least 0.001mg, 0.01mg, 0.1mg, 0.2mg, 0.3mg, 0.4mg, 0.5mg, 0.6mg, 0.7mg, 0.8mg, 0.9mg, 1.0mg, 1.1mg, 1.2mg, 1.3mg, 1.4mg, 1.5mg, 1.6mg, 1.7mg, 1.8mg, 1.9mg, 2.0mg, 2.5mg, 3.0mg, 3.5mg, 4.0mg, 4.5mg or 5.0mg of circular DNA vector. In some embodiments, the circular DNA vector is subjected to purification procedures to reduce the amount of bacterial contaminants (such as endotoxins) to an acceptable level that can be used in a pharmaceutical composition. Suitable purification methods include chromatographic procedures, such as anion exchange chromatography or hydrophobic interaction chromatography.

[0152] In some embodiments, the circular DNA vector is purified by gel electrophoresis to further avoid contamination by backbone sequences that may be maintained in engineered bacterial cell cultures. In some embodiments, purification is not required to avoid detectable contamination of the circular DNA vector by backbone sequences.

[0153] In some embodiments, the circular DNA vector can be purified from the culture of the engineered bacterial cells described herein, and the purified product will not be contaminated by the backbone sequence or any other extrachromosomal DNA molecules. In some embodiments, the composition of the isolated circular DNA vector purified from the engineered bacterial cells disclosed herein comprises less than 10ng / ml, 1ng / ml, 0.1ng / ml, 0.01ng / ml, 0.001ng / ml or 0.0001ng / ml of DNA comprising the backbone sequence. In some embodiments, the DNA comprising the backbone sequence is not detected in the composition by quantitative PCR. In some embodiments, after the circular DNA vector is isolated from the engineered bacterial cells, these purity levels can be achieved without gel purification or column purification steps.

[0154] In some embodiments, the methods of preparing circular DNA vectors disclosed herein comply with current good manufacturing practices (GMP) according to the standards promulgated by the U.S. Food and Drug Administration and set forth in 21 CFR Parts 210 and 211, the entire contents of which are incorporated herein by reference.

[0155] III. Circular DNA Vectors

[0156] Provided herein is a circular DNA vector produced by any of the production methods described herein. In some cases, such circular DNA vectors persist in cells (e.g., in dividing cells or resting cells, such as post-mitotic cells) in episomal form, for example, persist in a manner similar to that of an AAV vector. In any embodiment described herein, a circular DNA vector can be a non-integrating vector. Provided herein is a circular DNA vector that can be a naked DNA vector, free of components inherent to viral vectors (e.g., viral proteins) and bacterial plasmid DNA, such as immunogenic components (e.g., immunogenic bacterial markers (e.g., CpG islands or CpG motifs)) or additionally or otherwise with components associated with reduced persistence (e.g., CpG islands or CpG motifs). The circular DNA vector produced as described herein is characterized by one or more therapeutic sequences, and may lack plasmid backbone elements (e.g., drug resistance genes). In some embodiments, a circular DNA vector lacks a recombination site. In some embodiments, particularly in embodiments where recombination is used to remove a vector sequence from a plasmid backbone sequence, a circular DNA vector includes a recombination site.

[0157] In some embodiments, the vector contains DNA wherein 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 substantially all) of the DNA lacks one or more elements of bacterial plasmid DNA, such as immunogenic components (e.g., immunogenic bacterial markers (e.g., CpG motifs)) or components that are otherwise or 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 substantially all) of the DNA lacks CpG methylation. In some embodiments, the vector contains DNA wherein 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 substantially all) of the DNA lacks bacterial methylation markers, such as Dam methylation and Dcm methylation. For example, in some embodiments, the vector contains 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 substantially all) of the GATC sequence is unmethylated (e.g., by a Dam methylase). Additionally or alternatively, the vector contains 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 substantially all) of the CCAGG sequence and / or the CCTGG sequence is unmethylated (e.g., by a Dcm methylase).

[0158] In some embodiments, the circular DNA vector persists in vivo (e.g., the circular DNA vector exhibits improved expression persistence (e.g., intracellular persistence and / or transgenerational persistence) and / or therapeutic persistence relative to a reference vector (e.g., a circular DNA vector produced in bacteria or having one or more bacterial markers not present in the vectors of the invention, e.g., plasmid DNA). In some embodiments, the expression persistence of the circular DNA vector is 5% to 50%, 50% to 100%, one to five times, or five to ten times (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, one, two, three, four, five, six, seven, eight, nine, ten times, or more) greater than that of the reference vector. In some embodiments, the intracellular persistence of the circular DNA vector is 5% to 50%, 50% to 100%, one to five times, or five to ten times higher than that of a reference vector (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, one, two, three, four, five, six, seven, eight, nine, ten times, or more). In some embodiments, the transgenerational persistence of the circular DNA vector is 5% to 50%, 50% to 100%, one to five times, or five to ten times higher than that of a reference vector (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, one, two, three, four, five, six, seven, eight, nine, ten times, or more). In some embodiments, the therapeutic persistence of the circular DNA vector is 5% to 50%, 50% to 100%, one to five times, or five to ten times greater (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, one, two, three, four, five, six, seven, eight, nine, ten times, or more) than that of a reference vector. In some embodiments, the reference vector is a circular vector or plasmid that (a) has the same coding sequence as the circular DNA vector to which it is compared, and (b) has one or more bacterial markers that are not present in the circular DNA vector to which it is compared, which markers can include, for example, antibiotic resistance genes or other selectable markers.

[0159] In some embodiments, expression of the circular DNA vector continues for one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year or more after administration. In specific embodiments, the intracellular persistence and / or transgenerational persistence exhibited by the circular DNA vector after administration is one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year or more. In some embodiments, the therapeutic persistence of the circular DNA vector continues for one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year or more after administration.

[0160] In some embodiments, the expression and / or therapeutic effect of the circular DNA vector lasts from one week to four weeks, one month to four months, or four months to one year (e.g., at least one week, at least two weeks, at least one month or longer). In some embodiments, the expression level of the circular DNA vector is reduced by no more than 90%, no more than 50%, or no more than 10% within 1 week or more (e.g., 2 weeks, 3 weeks, 5 weeks, 7 weeks, 9 weeks or more, 13 weeks or more, 18 weeks or more) after transfection compared to the level observed within the first 1, 2, or 3 days.

[0161] Circular DNA vectors can be monomeric, dimeric, trimeric, tetrameric, pentamer, hexameric, etc. In some preferred embodiments, the circular DNA vector is monomeric. In some embodiments, the DNA vector is supercoiled. Due to endogenous processes in engineered bacterial cells or due to treatment with topoisomerases (e.g., gyrase), circular DNA vectors can be supercoiled. In some embodiments, circular DNA vectors are monomeric, supercoiled circular DNA molecules. In some embodiments, circular DNA vectors are nicked. In some embodiments, circular DNA vectors are open circular. In some embodiments, circular DNA vectors are double-stranded circular. In some embodiments, the composition comprising the circular DNA vector comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% supercoiled monomers. In some embodiments, the composition comprising a circular DNA vector comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% supercoiled monomers that have not been treated with an exogenous topoisomerase.

[0162] IV. Therapeutic Sequence

[0163] In some embodiments, the coding sequence of the circular DNA vectors described herein contains a therapeutic sequence, which may include one or more protein coding domains and / or one or more non-protein coding domains (eg, a therapeutic nucleic acid).

[0164] In specific embodiments involving therapeutic protein encoding therapeutic domains, the therapeutic sequence comprises: a promoter and a single therapeutic protein encoding domain (e.g., a single transcription unit); a promoter and two or more therapeutic protein encoding domains (e.g., a polycistronic unit); or a first transcription unit and one or more additional transcription units (e.g., multiple transcription units) connected in a 5' to 3' direction. Any such protein encoding therapeutic sequence may additionally comprise non-protein coding domains, such as polyadenylation sites, regulatory elements, enhancers, sequences for marking 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 to and / or modify nucleic acids, linkers, splice sites, pre-mRNA binding domains, regulatory sequences, and / or therapeutic nucleic acids (e.g., microRNA coding sequences). The therapeutic protein encoding domains may be full-length protein encoding domains (e.g., corresponding to a native gene or a natural variant thereof) or functional portions thereof, such as truncated protein encoding domains (e.g., minigenes).

[0165] In some embodiments, the therapeutic sequence encodes a monomeric protein (e.g., a monomeric protein having a secondary structure under physiological conditions, e.g., a monomeric protein having a secondary structure and a tertiary structure under physiological conditions, e.g., a monomeric protein having 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.).

[0166] In certain instances, the therapeutic sequence comprises an ocular gene. In some embodiments, the ocular gene is a gene expressed in ocular tissue, such as, for example, retinal tissue, which may include, for example, photoreceptor cells and / or retinal pigment epithelial (RPE) cells. In some embodiments, the coding sequence in the expression construct disclosed herein is a human ABCA4 or MYO7A gene sequence. An exemplary human ABCA4 gene sequence is provided as the National Center for Biotechnology Information (NCBI) Reference Sequence: NG_009073. The amino acid sequence of an exemplary ABCA4 protein is given by protein accession number P78363.3. An exemplary human MYO7A gene sequence is provided as NCBI Gene ID: 4647. The amino acid sequence of an exemplary MYO7A protein is given by protein accession number Q13402.

[0167] In some embodiments, the therapeutic sequence encodes an antibody or a portion, fragment or variant thereof. Antibodies include fragments capable of binding to antigens, such as Fv, single-chain Fv (scFv), Fab, Fab', double scFv, sdAb (single domain antibody), (Fab')2 (including chemically linked F(ab')2) and nanobodies. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with one antigen-binding site, and the remaining "Fc" fragments, whose names reflect their ability to be easily crystallized. Pepsin treatment produces F(ab')2 fragments with two antigen-binding sites and still capable of cross-linking antigens. Antibodies also include chimeric antibodies and humanized antibodies. In addition, for all antibody constructs provided herein, variants with sequences from other organisms are also envisioned. Therefore, if the human form of an antibody is disclosed, those skilled in the art will understand how to convert antibodies based on human sequences into sequences of mice, rats, cats, dogs, horses, etc. Antibody fragments also include single-chain scFvs, tandem double scFvs, bivalent antibodies, tandem three sdcFvs, miniantibodies, nanobodies, etc. in any orientation. In some embodiments, such as when the antibody is an scFv, a single polynucleotide of the therapeutic gene sequence encodes a single polypeptide comprising both heavy and light chains linked together. Antibody fragments also include nanobodies (e.g., sdAbs, antibodies with a single monomer domain (such as a pair of variable domains with a heavy chain but no light chain)). Multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.) are known in the art and are considered to be expression products of the therapeutic gene sequences of the present invention.

[0168] In some cases, the therapeutic sequence encodes one or more proteins (e.g., one protein, two proteins, three proteins, four proteins, or more proteins), each protein having a length of 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 1,000 amino acids, at least 1,500 amino acids, at least 2,000 amino acids, at least 2,500 amino acids, at least 3,000 amino acids, or more (e.g., 25 to 5,000 amino acids, 50 to 5,000 amino acids, 100 to 5,000 amino acids, 200 to 5,000 amino acids, 500 to 5,000 amino acids, 1,000 to 5,000 amino acids, 4,000 amino acids, 1,500 to 5,000 amino acids, or 2,000 to 5,000 amino acids; e.g., 25 to 4,000 amino acids, 50 to 4,000 amino acids, 100 to 4,000 amino acids, 200 to 4,000 amino acids, 500 to 4,000 amino acids, 1,000 to 4,000 amino acids, 1,500 to 4,000 amino acids, or 2,000 to 4,000 amino acids; e.g., 25 to 3,000 amino acids, 50 to 3,000 amino acids, 100 to 3,000 amino acids, 200 to 3,000 amino acids, 500 to 3,000 amino acids, 1,000 to 3,000 amino acids, 1,500 to 3,000 amino acids, or 2,000 to 3,000 amino acids). In embodiments where the therapeutic sequence encodes two or more proteins, the therapeutic sequence can be a polycistronic therapeutic sequence or a multi-transcription unit therapeutic sequence. Such a polycistronic therapeutic sequence can be, for example, a tricistronic cassette encoding Flt3L, IL-12, and XCL1, as described in Example 7 herein.

[0169] In embodiments involving non-protein encoding therapeutic sequences, the therapeutic sequence lacks a protein encoding domain (e.g., a therapeutic protein encoding domain). For example, in some embodiments, the therapeutic sequence comprises a non-protein encoding therapeutic nucleic acid, such as a short hairpin RNA (shRNA) encoding sequence or an immune activating therapeutic nucleic acid (e.g., a TLR agonist).

[0170] In some embodiments, the therapeutic sequence or other sequence of interest (which may include non-therapeutic coding sequences, such as reporter genes for measuring expression or persistence) is 0.1 Kb to 100 Kb in length (e.g., 0.2 Kb to 90 Kb, 0.5 Kb to 80 Kb, 1.0 Kb to 70 Kb, 1.5 Kb to 60 Kb, 2.0 Kb to 50 Kb, 2.5 Kb to 45 Kb, 3.0 Kb to 40 Kb, 3.5 Kb to 35 Kb, 4.0 Kb to 30 Kb, 4.5 Kb to 25 Kb, 4.6 Kb to 24 Kb, 4.7 Kb to 23 Kb, 4.8 Kb to 22 Kb, 4.9 Kb to 21 Kb, 5.0 Kb to 50 Kb, 5.5 Kb to 55 Kb, 5.6 Kb to 56 Kb, 5.7 Kb to 58 Kb, 5.8 Kb to 59 Kb, 6.9 Kb to 60 Kb, 6.9 Kb to 61 Kb, 6.1 Kb to 62 Kb, 6.1 Kb to 63 Kb, 6.1 Kb to 64 Kb, 6.1 Kb to 65 Kb, 6.1 Kb to 66 Kb, 6.1 Kb to 67 Kb, 6.1 Kb to 68 Kb, 6.1 Kb to 69 Kb, 7.1 Kb to 70 Kb, 7.1 Kb to 71 Kb, 7.1 Kb to 72 Kb, 7.1 Kb to 73 Kb, 7.1 Kb to 74 Kb 20 Kb, 5.5 Kb to 18 Kb, 6.0 Kb to 17 Kb, 6.5 Kb to 16 Kb, 7.0 Kb to 15 Kb, 7.5 Kb to 14 Kb, 8.0 Kb to 13 Kb, 8.5 Kb to 12.5 Kb, 9.0 Kb to 12.0 Kb, 9.5 Kb to 11.5 Kb or 10.0 Kb to 11.0 Kb, for example, 0.1 Kb to 0.5 Kb, 0.5 Kb to 1.0 Kb, 1.0 Kb to 2.5 Kb, 2.5 Kb to 4.5 Kb, 4.5 Kb to 8 Kb, 8 Kb to 10 Kb, 10 Kb to 15 Kb, 15 Kb to 20 Kb or longer, for example, 0.1 Kb to 0.25 Kb in length , 0.25Kb to 0.5Kb, 0.5Kb to 1.0Kb, 1.0Kb to 1.5Kb, 1.5Kb to 2.0Kb, 2.0Kb to 2.5Kb, 2.5Kb to 3.0Kb, 3.0Kb to 3.5Kb, 3.5Kb to 4.0Kb, 4.0Kb to 4.5Kb, 4.5Kb to 5.0Kb, 5.0Kb to 5.5Kb, 5.5Kb to 6.0Kb, 6.0Kb to 6.5Kb, 6.5Kb to 7.0Kb, 7.0Kb to 7.5Kb, 7.5Kb to 8.0Kb, 8.0Kb to 8.5Kb, 8.5Kb to 9.0Kb, 9.0Kb to 9.5Kb, 9.5Kb to 10Kb, 10Kb to 10.5Kb, 10.5Kb to 11Kb, 11Kb to 11.5Kb, 11.5Kb to 12Kb, 12Kb to 12.5Kb, 12.5Kb to 13Kb, 13Kb to 13.5Kb, 13.5Kb to 14Kb, 14Kb to 14.5Kb, 14.5Kb to 15Kb, 15Kb to 15.5Kb, 15.5Kb to 16Kb, 16Kb to 16.5Kb, 16.5Kb to 17Kb, 17Kb to 17.5Kb, 17.5Kb to 18Kb, 18Kb to 18.5Kb, 18.5Kb to 19Kb, 19Kb to 19.5Kb, 19.Kb, about 11 Kb, about 12 Kb, about 13 Kb, about 14 Kb, about 15 Kb, about 16 Kb, about 17 Kb, about 18 Kb, about 19 Kb, about 20 Kb or more in length). In some embodiments, the therapeutic sequence is at least 10Kb (e.g., 10Kb to 15Kb, 15Kb to 20Kb, or 20Kb to 30Kb; e.g., 10Kb to 13Kb, 10Kb to 12Kb, or 10Kb to 11Kb; e.g., 10 to 11Kb, 11 to 12Kb, 12 to 13Kb, 13 to 14Kb, or 14 to 15Kb). In some embodiments, the sequence is at least 1,100bp in length (e.g., 1,100bp to 10,000bp, 1,100bp to 8,000bp, or 1,100bp to 5,000bp in length). In some embodiments, the sequence is at least 2,500 bp in length (e.g., 2,500 bp to 15,000 bp, 2,500 bp to 10,000 bp, or 2,500 bp to 5,000 bp in length; e.g., 2,500 bp to 5,000 bp, 5,000 bp to 7,500 bp, 7,500 bp to 10,000 bp, 10,000 bp to 12,500 bp, or 12,500 bp to 15,000 bp). In some embodiments, the sequence is at least 8,000 bp, at least 9,000 bp, at least 10,000 bp, at least 11,000 bp, at least 12,000 bp, at least 13,000 bp, at least 14,000 bp, at least 15,000 bp, at least 16,000 bp (e.g., 11,000 bp to 16,000 bp, 12,000 bp to 16,000 bp, 13,000 bp to 16,000 bp, 14,000 bp to 16,000 bp, or 15,000 bp to 16,000 bp). In specific embodiments, the length of the sequence is sufficient to encode a protein and is not an oligonucleotide therapeutic (e.g., not an antisense, siRNA, shRNA therapeutic, etc.). .

[0171] In some embodiments, the 3' end of the sequence of interest (such as a therapeutic or non-therapeutic coding sequence) is separated by no more than 50 bp (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 A non-bacterial sequence (e.g., a recombination site, such as a recombination scar) of 5', 7bp, 8bp, 10bp, 15bp, 20bp, 25bp, 30bp, 31bp, 32bp, 33bp, 34bp, 35bp, 36bp, 37bp, 38bp, 39bp, 40bp, 41bp, 42bp, 43bp, 44bp, 45bp, 46bp, 47bp, 48bp, 49bp or 50bp is linked to the 5' end of the ori sequence in the circular DNA vector.

[0172] In some embodiments, the 3' end of the sequence of interest (such as a therapeutic or non-therapeutic coding sequence) is linked to the 5' end of the ori sequence in the circular DNA vector through a non-bacterial sequence of no more than 30 bp (e.g., 3 bp to 24 bp, 4 bp to 18 bp, 5 bp to 12 bp, or 6 bp to 10 bp; for example, 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; for example, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 10 bp, 15 bp, 20 bp, 25 bp, or 30 bp).

[0173] In some embodiments, in addition to the therapeutic protein coding domain or the therapeutic non-protein coding domain, the sequence of interest contained in the circular DNA vector described herein also includes a reporter sequence. In some embodiments, the therapeutic sequence lacks a reporter sequence. In some embodiments, the sequence of interest includes a reporter sequence, but does not include a therapeutic sequence. The reporter sequence can be, for example, a reporter gene. Such reporter genes can be used to verify the expression of therapeutic gene sequences, for example, in specific cells and tissues. The reporter sequence that can be provided by reference to the entire incorporation into the circular DNA vector includes, but is not limited to, DNA sequences encoding beta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and other sequences well known in the art. When associated with a regulatory element that drives expression, the reporter sequence provides a signal that can be detected by conventional means, including enzymatic, radioimaging, colorimetric, fluorescence or other spectrometric assays, fluorescence activated cell sorting assays, and immunoassays (including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemical assays). For example, when the marker sequence is the LacZ gene, the presence of the signal-carrying vector is detected by a β-galactosidase activity assay. When the transgene is green fluorescent protein or luciferase, the signal-carrying vector can be visually measured by color or light production with a luminometer.

[0174] As part of the therapeutic sequence or other sequence of interest, the circular DNA vector of the present invention may contain conventional regulatory elements that regulate or improve transcription, translation and / or expression in the target cell. Suitable regulatory elements are described in International Publication WO 2021 / 055760, which is incorporated herein by reference in its entirety.

[0175] In some cases, the self-replicating RNA molecule comprises (i) a replicase encoding sequence (e.g., an RNA sequence encoding an RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA molecule) and (ii) a heterologous regulatory gene. The polymerase can be an alphavirus replicase, for example, an alphavirus replicase comprising 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, for example, a VEE replicase comprising one, two, three, or all four of the alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4.

[0176] In some cases of the present invention, the self-replicating RNA molecule does not encode alphavirus structural proteins (e.g., capsid proteins). Such self-replicating RNA can produce copies of its own genomic RNA in cells, but does not produce RNA-containing viral particles. The inability to produce these viral particles means that, unlike wild-type alphaviruses, the self-replicating RNA molecule cannot be continued in an infectious form. Alphavirus structural proteins can be replaced with genes encoding heterologous regulatory proteins of interest so that the subgenomic transcripts encode heterologous regulatory proteins rather than structural alphavirus particle proteins.

[0177] Thus, in some cases, the self-replicating RNA molecules of the invention can have two open reading frames. The first (5') open reading frame encodes a replicase; the second (3') open reading frame encodes one or more (e.g., two or three) therapeutic proteins. In some embodiments, the RNA can have additional (e.g., downstream) open reading frames, for example, to encode other genes or to encode accessory polypeptides.

[0178] Suitable self-replicating RNA molecules can have different lengths. In some embodiments of the invention, the length of the self-replicating RNA molecule is 5,000 to 50,000 nucleotides (i.e., 5 kb to 50 kb). In some cases, the length of the self-replicating RNA molecule is 5 kb to 20 kb (e.g., 6 kb to 18 Kb, 7 kb to 16 Kb, 8 kb to 14 kb, or 9 kb to 12 kb, for example, 5 kb to 6 Kb, 6 kb to 7 Kb, 7 kb to 8 Kb, 8 kb to 9 Kb, 9 kb to 10 Kb, 10 kb to 11 Kb, 11 kb to 12 Kb, 12 kb to 13 Kb, 13 kb to 14 Kb, 16 kb to 15 Kb, 17 kb to 18 Kb, 18 kb to 19 Kb, 19 kb to 20 Kb, 20 kb to 21 Kb, 21 kb to 22 Kb, 22 kb to 23 Kb, 24 kb to 25 Kb, 26 kb to 27 Kb, 27 kb to 28 Kb, 28 kb to 29 Kb, 29 kb to 30 Kb, 31 kb to 32 Kb, 33 kb to 34 Kb, 35 kb to 36 Kb, 37 kb to 38 Kb, 39 kb to 40 Kb, 41 kb to 42 Kb, 43 kb to 44 Kb, 45 kb to 46 Kb, 47 kb to 48 Kb, 49 kb to 50 Kb 14 Kb, about 15 Kb, about 16 Kb, about 17 Kb, about 18 Kb, about 19 Kb or about 20 kb in length).

[0179] The self-replicating RNA molecule may have a 3' poly-A tail. In addition, the self-replicating RNA molecule may comprise a poly-A polymerase recognition sequence (eg, AAUAAA).

[0180] In a particular embodiment, 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).

[0181] In some embodiments, the replicase encoded by the self-replicating RNA can be a variant of any one of the replicase described herein. In some embodiments, the variant is a functional fragment (e.g., a fragment of a protein that is similar to or functionally identical to a protein).

[0182] V. Pharmaceutical Compositions

[0183] The increased efficiency makes the methods of the present invention particularly suitable for the scalable preparation of pharmaceutical compositions containing circular DNA vectors.Any of the methods for producing circular DNA vectors described herein can be applied to produce pharmaceutical compositions containing circular DNA vectors in a pharmaceutically acceptable carrier.

[0184] As part of any method described herein, downstream purification processes can be readily applied. For example, various chromatography steps are known in the art and can be suitable for removing bacterial byproducts, 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.

[0185] In some embodiments, the pharmaceutical formulations of the invention contain at least 1.0 mg of the circular DNA vector (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) in a pharmaceutically acceptable carrier. g, 2.0 mg to 20 mg, 2.0 mg to 15 mg, 2.0 mg to 10 mg, 2.0 mg to 5.0 mg, 5.0 mg to 10 g, 5.0 mg to 5.0 g, 5.0 mg to 1.0 g, 5.0 mg to 500 mg, 5.0 mg to 200 mg, 5.0 mg to 100 mg, 5.0 mg to 50 mg, 5.0 mg to 25 mg, 5.0 mg to 20 mg, 5.0 mg to 15 mg, 5.0 mg to 10 mg, 10 mg to 10 g, 10 mg to 5.0 g, 10 mg to 1.0 g, 10 mg to 500 mg, 10 mg to 200 mg, 10 mg to 100 mg, 10 mg to 50 mg, 10 mg to 25 mg, 10 mg to 20 mg, or 10 mg to 15 mg).

[0186] In some embodiments, the pharmaceutical preparations of the present invention contain at least 2.0 mg of circular DNA vector in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical preparations produced by any of the methods described herein contain at least 5.0 mg of circular DNA vector in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical preparations produced by any of the methods described herein contain at least 10.0 mg of circular DNA vector in a pharmaceutically acceptable carrier.

[0187] In some embodiments, the pharmaceutical preparation of the present invention is substantially free of impurities. For example, in some embodiments, the pharmaceutical preparation comprises a protein content of <2.0 mass % (for example, <1.9 mass %, <1.8 mass %, <1.7 mass %, <1.6 mass %, <1.5 mass %, <1.4 mass %, <1.3 mass %, <1.2 mass %, <1.1 mass %, <1.0 mass %, <0.9 mass %, <0.8 mass %, <0.7 mass %, <0.6 mass %, <0.5 mass %, <0.4 mass %, <0.3 mass %, <0.2 mass %, <0.1 mass %, <0.05 mass % or <0.01 mass % protein content). In some cases, protein content is determined by bicinchoninic acid determination. Additionally or alternatively, protein content is determined by ELISA.

[0188] In some cases, the pharmaceutical formulation comprises an RNA content of <5.0% by mass (e.g., <4.5% by mass, <4.0% by mass, <3.5% by mass, <3.0% by mass, <2.5% by mass, <2.0% by mass, <1.9% by mass, <1.8% by mass, <1.7% by mass, <1.6% by mass, <1.5% by mass, <1.4% by mass, <1.3% by mass, <1.2% by mass, <1.1% by mass, <1.0% by mass, <0.9% by mass, <0.8% by mass, <0.7% by mass, <0.6% by mass, <0.5% by mass, <0.4% by mass, <0.3% by mass, <0.2% by mass, <0.1% by mass, <0.05% by mass, or <0.01% by mass of RNA content). 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, RNA content is determined by fluorescence assay (eg, Ribogreen).

[0189] In some embodiments, the pharmaceutical formulation contains <5.0% gDNA content by mass (e.g., 4.5%, <4.0%, <3.5%, <3.0%, <2.5%, <2.0%, <1.9%, <1.8%, <1.7%, <1.6%, <1.5%, <1.4%, <1.3%, <1.2%, <1.1%, <1.0%, <0.9%, <0.8%, <0.7%, <0.6%, <0.5%, <0.4%, <0.3%, <0.2%, <0.1%, <0.05%, or <0.01% gDNA content by mass). In some embodiments, the gDNA content is determined by agarose gel electrophoresis or capillary electrophoresis. In some embodiments, gDNA content is determined by quantitative PCR.In some embodiments, gDNA content is determined by Southern blot.

[0190] In some embodiments, the pharmaceutical preparation contains <40EU / mg endotoxin. In some embodiments, the pharmaceutical preparation contains <20EU / mg endotoxin. In some embodiments, the pharmaceutical preparation contains <10EU / mg endotoxin. In some embodiments, the pharmaceutical preparation contains <5EU / mg endotoxin (e.g., <4EU / mg endotoxin, <3EU / mg endotoxin, <2EU / mg endotoxin, <1EU / mg endotoxin, <0.5EU / mg endotoxin), e.g., as measured by a Limulus amoeba lysate (LAL) assay.

[0191] In some embodiments, the pharmaceutical compositions disclosed herein comply with current good manufacturing practices (GMP) according to the standards promulgated by the U.S. Food and Drug Administration and set forth in 21 CFR Parts 210 and 211, the entire contents of which are incorporated herein by reference.

[0192] The pharmaceutical compositions provided herein may include one or more pharmaceutically acceptable carriers, such as excipients and / or stabilizers, which are nontoxic to the individual (e.g., human patient) being treated at the dose and concentration employed. In some embodiments, the pharmaceutically acceptable carrier is a pH buffered aqueous solution. Examples of pharmaceutically acceptable carriers include buffers such as phosphates, citrates, 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 nonionic surfactants such as tweens, polyethylene glycols (PEGs), and pluronics.

[0193] If the pharmaceutical composition is provided in liquid form, the pharmaceutically acceptable carrier can be water (e.g., pyrogen-free water), isotonic saline or a buffered aqueous solution (e.g., a phosphate buffer solution or a citrate buffer solution). The injection of the pharmaceutical composition can be carried out in water or a buffer, such as an aqueous buffer, for example, containing a sodium salt (e.g., at least 50 mM of sodium salt), a calcium salt (e.g., at least 0.01 mM of calcium salt) or a potassium salt (e.g., at least 3 mM of potassium salt). According to a specific embodiment, the sodium salt, calcium salt or potassium salt can exist in the form of their halides, such as chlorides, iodides or bromides, or in the form of their hydroxides, carbonates, bicarbonates or sulfates. Examples of sodium salts include, but are not limited to, 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, the organic anions of the aforementioned cations may be contained in the buffer. According to a specific embodiment, the buffer suitable for injection purposes as defined above may contain a salt selected from the following: sodium chloride (NaCl), calcium chloride (CaCl2) or potassium chloride (KCl), in which other anions may be present. CaCl2 may also be replaced by another salt (such as KCl). In some embodiments, the salt in the injection buffer is provided at a concentration of at least 50mM sodium chloride (NaCl), at least 3mM potassium chloride (KCl) and at least 0.01mM calcium chloride (CaCl2). Relative to a specific reference culture medium, the injection buffer may be hypertonic, isotonic or hypotonic, that is, relative to a specific reference culture medium, the buffer may have a higher, equal or lower salt content, wherein preferably these concentrations of the aforementioned salts are used, and damage to the cell will not occur due to osmotic or other concentration effects. The reference culture medium may be a liquid, such as blood, lymph, cytoplasmic fluid, other body fluids or a common buffer. Such common buffers or liquids are known to those skilled in the art. Ringer's lactate solution is a particularly preferred base liquid.

[0194] One or more compatible solid or liquid fillers, diluents or encapsulated compounds may be suitable for administration to the human body. The ingredients of the pharmaceutical composition according to the present invention can be mixed with the nucleic acid carrier according to the present invention as defined herein, and this mixing method will not occur under typical conditions of use, significantly reducing the interaction of the pharmaceutical effectiveness of the (drug) composition according to the present invention. Pharmaceutically acceptable carriers, fillers and diluents may have sufficiently high purity and sufficiently low toxicity so that they are suitable for administration to the individual receiving treatment. Some examples of compounds that can be used as pharmaceutically acceptable carriers, fillers, or ingredients thereof are sugars such as lactose, glucose, trehalose, and sucrose; starches such as corn starch or potato starch; glucose; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; beef 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 cocoa butter; polyols such as polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; or alginic acid.

[0195] The choice of a pharmaceutically acceptable carrier may be determined according to the manner in which the pharmaceutical composition is to be administered.

[0196] Unit dosage forms suitable for injection include sterile solutions of water, saline and mixtures thereof. The pH of such solutions can be adjusted to about 7.4. Supporting agents suitable for injection include hydrogels, controlled release or delayed release devices, polylactic acid and collagen matrix. Pharmaceutically acceptable carriers suitable for topical application include those suitable for lotions, creams, gels, etc. If the pharmaceutical composition is administered orally, tablets, capsules, etc. are preferred unit dosage forms.

[0197] Other additives that 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.

[0198] Pharmaceutical composition according to the present invention can be provided in liquid or dry (e.g., lyophilized) form. In a specific 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 redissolved in a suitable buffer before administration, advantageously based on an aqueous carrier, such as Ringer-lactate solution (Ringer-Lactate solution), Ringer solution (Ringer solution) or phosphate buffer.

[0199] In certain embodiments of the invention, any of the circular DNA vectors of the invention may be complexed with one or more cationic or polycationic compounds (e.g., cationic or polycationic polymers, cationic or polycationic peptides or proteins (e.g., protamine), cationic or polycationic polysaccharides and / or cationic or polycationic lipids).

[0200] According to a specific embodiment, the circular DNA vector of the present invention can be complexed with lipids to form one or more liposomes, lipid complexes or lipid nanoparticles. Therefore, in one embodiment, the pharmaceutical composition comprises liposomes, lipid complexes and / or lipid nanoparticles containing the circular DNA vector.

[0201] Lipid-based formulations can be an effective delivery system for nucleic acid vectors due to their biocompatibility and ease of large-scale production. Cationic lipids have been widely studied as synthetic materials for nucleic acid delivery. After mixing, nucleic acids are condensed by cationic lipids to form lipid / nucleic acid complexes called lipoplexes. These lipoplexes are able to protect genetic material from the action of nucleases and deliver genetic material to cells by interacting with negatively charged cell membranes. Lipoplexes can be prepared by directly mixing positively charged lipids at physiological pH with negatively charged nucleic acids.

[0202] Conventional liposomes include a lipid bilayer that can be composed of cations, anions or neutral phospholipids and cholesterol, and the lipid bilayer encapsulates an aqueous core. The lipid bilayer and the aqueous space can contain hydrophobic or hydrophilic compounds respectively. The characteristics and in vivo behavior of liposomes can be modified by adding a hydrophilic polymer coating (e.g., polyethylene glycol (PEG)) to the liposome surface (to give steric stability). In addition, liposomes can be used to achieve specific targeting by attaching a part (e.g., antibody, peptide and carbohydrate) to its surface or attaching to the end of the attached PEG chain.

[0203] Liposomes are colloidal lipid-based delivery systems and surfactant-based delivery systems, which consist of a phospholipid bilayer surrounding an aqueous compartment. They can be provided as spherical vesicles and range in size from 20 nm to several microns. Liposomes based on cationic lipids can be complexed with negatively charged nucleic acids through electrostatic interactions, making the complexes biocompatible, low toxicity, and having the possibility of large-scale production required for in vivo clinical applications. Liposomes can fuse with the plasma membrane and be absorbed; once inside the cell, the liposomes are processed through the endocytic pathway, and then the genetic material is released from the endosome / carrier into the cytoplasm.

[0204] Cationic liposomes can be used as delivery systems 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-methylammonium sulfate) can form complexes or lipid complexes with negatively charged nucleic acids to form nanoparticles through electrostatic interactions, thereby providing high in vitro transfection efficiency. In addition, neutral lipid-based nanoliposomes (e.g., nanoliposomes based on neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)) for nucleic acid vector delivery are also available.

[0205] Therefore, in one embodiment of the present invention, the circular DNA vector is complexed with a cationic lipid and / or a neutral lipid to form a liposome, a lipid nanoparticle, a lipid complex or a neutral lipid-based nanoliposome in the pharmaceutical composition of the present invention.

[0206] In a specific embodiment, the pharmaceutical composition comprises a circular DNA vector of the invention formulated with a cationic or polycationic compound and / or a polymer carrier. Therefore, in another embodiment of the invention, the circular DNA vector as defined herein is optionally associated or complexed with a weight ratio of nucleic acid vector to cationic or polycationic compound and / or polymer carrier selected from the following ranges: about 5:1 (w / w) to about 0.25:1 (w / w), for example, about 5:1 (w / w) to about 0.5:1 (w / w), for example, about 4:1 (w / w) to about 1:1 (w / w) or about 3:1 (w / w). 1 (w / w) to about 1:1 (w / w), for example, about 3:1 (w / w) to about 2:1 (w / w); or optionally in a range of about 0.1 to 10, for example, in a range of about 0.3 to 4 or 0.3 to 1, for example, in a range of about 0.5 to 1 or 0.7 to 1, for example, in a range of about 0.3 to 0.9 or 0.5 to 0.9. The nitrogen / phosphate (N / P) ratio of the nucleic acid vector to the cationic or polycationic compound and / or the polymer carrier is associated or complexed. 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 in the range of about 0.3 to 4, about 0.5 to 2, about 0.7 to 2, and about 0.7 to 1.5.

[0207] The nucleic acid vectors described herein may also be combined with a vehicle, a transfection agent, or a complexing agent to increase transfection efficiency and / or regulate gene expression according to the present invention.

[0208] In some cases, the circular DNA vector according to the present invention is complexed with one or more polycations, preferably with protamine or oligotransfectamine. Other cationic or polycationic compounds that can be used as transfection agents or complexing agents may include cationic polysaccharides (e.g., chitosan, polybrene), cationic polymers (e.g., polyethyleneimine (PEI)), cationic lipids (e.g., DOTMA: [1-(2,3-sialyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPE, LEAP, DOPE: dioleoylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, D OGS: dioctadecylamidoglycerylamine spermine, DIMRI: dimyristoyl-oxypropyl dimethylhydroxyethyl ammonium bromide, MAP: dioleoyloxy-3-(trimethylammonium)propane, DC-6-14: O,O-ditetradecanoyl-N-(α-trimethylammonium hexadecyl)diethanolamine chloride, CLIP1: racemic-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: racemic-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium, CLIP9: racemic-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium

[0063] trimethylammonium, oligomeric transfectamine) or a cationic or polycationic polymer (e.g., a modified polyamino acid such as a β-amino acid polymer or a reverse polyamide, a modified polyethylene such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), a modified acrylate such as pDMAEMA (poly(dimethylaminoethyl methacrylate)), a modified amidoamine such as pAMAM (poly(amidoamine)), a modified polyβ-amino ester (PBAE) such as diamine-terminated 1,4-butylene glycol diacrylate-co-5-amino-1- amyl alcohol polymers, etc., dendrimers such as polypropylamine dendrimers or pAMAM-based dendrimers, etc., polyimines such as PEI: poly(ethyleneimine), poly(propyleneimine), etc., polyallylamine, sugar-based polymers such as cyclodextrin-based polymers, dextran-based polymers, chitosan, etc., silane-based polymers such as PMOXA-PDMS copolymers, etc., block polymers composed of a combination of one or more cationic blocks (e.g., selected from the cationic polymers mentioned above) and one or more hydrophilic or hydrophobic blocks (e.g., polyethylene glycol); and the like.

[0209] According to a specific embodiment, the pharmaceutical composition includes a circular DNA vector encapsulated in or attached to a polymer carrier. The polymer carrier used according to the present invention can be a polymer carrier formed by a disulfide cross-linked cationic component. The disulfide cross-linked cationic components can be the same or different from each other. The polymer carrier can also contain other components. It is also particularly preferred that the polymer carrier used according to the present invention comprises a mixture of cationic peptides, proteins or polymers and optionally other components as defined herein, which are cross-linked by disulfide bonds as described herein. In the context of the present invention, the disclosure of WO 2012 / 013326 is incorporated herein by reference. In the context of the present invention, the cationic component forming the polymer carrier base by disulfide cross-linking is generally selected from any suitable cationic or polycationic peptide, protein or polymer suitable for this purpose, especially any cationic or polycationic peptide, protein or polymer that can make other nucleic acids contained in the nucleic acid carrier or composition as defined herein be compounded, thereby preferably making the nucleic acid carrier condensed. Cationic or polycationic peptides, proteins or polymers can be linear molecules; however, branched cationic or polycationic peptides, proteins or polymers can also be used.

[0210] Each disulfide-crosslinked cationic or polycationic protein, peptide or polymer of the polymer carrier (which can be used to complex the circular DNA vector according to the present invention included as part of the pharmaceutical composition) can contain at least one SH moiety (e.g., at least one cysteine ​​residue or any other chemical group exhibiting an SH moiety) that is capable of forming a disulfide bond upon condensation with at least one other cationic or polycationic protein, peptide or polymer (as the cationic component of the polymer carrier as referred to herein).

[0211] Such polymer carriers for complexing the circular DNA vector of the present invention can be formed by disulfide cross-linked cationic (or polycationic) components. In particular, such cationic or polycationic peptides or proteins or polymers (comprising or otherwise modified to comprise at least one SH moiety) of the polymer carrier can be selected from proteins, peptides and polymers as complexing agents.

[0212] In other embodiments, the circular DNA vector according to the present invention can be administered naked in a suitable buffer without being combined with any other vehicle, transfection agent or complexing agent.

[0213] VI. How to use

[0214] Provided herein is a method for inducing expression (e.g., sustained expression) of a sequence of interest (e.g., a therapeutic sequence) in a subject in need thereof by administering any of the circular DNA vectors described herein or their pharmaceutical compositions to a subject (e.g., as part of a gene therapy regimen). The target cells or target tissues of the subject can be characterized by examining the nucleic acid sequence (e.g., RNA sequence, e.g., mRNA sequence) of the host cell, such as by Southern blot or PCR analysis of the presence (e.g., persistent presence) of the delivered therapeutic sequence. Alternatively, the expression of the therapeutic sequence in the subject can be characterized (e.g., quantitatively or qualitatively) by delivery of the therapeutic sequence to monitor the progression of the disease being treated (e.g., associated with a defect or mutation targeted by the therapeutic sequence). In some embodiments, transcription or expression (e.g., sustained transcription or sustained expression) of the therapeutic sequence is confirmed by observing a reduction in one or more symptoms associated with the disease.

[0215] Therefore, embodiments of the present invention include methods of treating a disease in a subject by administering any of the circular DNA vectors described herein or their pharmaceutical compositions to the subject. Any of the circular DNA vectors described herein or their pharmaceutical compositions can be administered in an amount of 1 μg to 10 mg DNA (e.g., 5 μg to 5.0 mg, 10 μg to 2.0 mg, or 100 μg to 1.0 mg 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, or 5.0 mg to 10 mg In some embodiments, the present invention relates to a method for treating a subject with a DNA (e.g., a dose of 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 DNA) is administered to a subject.

[0216] 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 than administration of other gene therapy vectors (eg, plasmid DNA vectors and viral vectors).

[0217] In some cases, the circular DNA vectors provided herein and their pharmaceutical compositions are suitable for repeated administration, because relative to reference vectors (such as plasmid DNA vectors or AAV vectors), they can transfect target cells without triggering an immune response or inducing a reduction in immune response, as discussed above. Therefore, the present invention provides a method for repeatedly administering circular DNA vectors and pharmaceutical compositions as described herein. Any of the aforementioned administration doses can be repeated with a suitable frequency and duration. In some embodiments, the subject receives about twice a day, about once a day, about five times a week, about four times a week, about three times a week, about twice a week, about once a week, about twice a month, about once a month, about once every six weeks, about once every two months, about once every three months, about once every four months, twice a year, once a year or a dose of lower frequency. In some embodiments, the number and frequency of dosage correspond to the conversion rate of target cells. It should be understood that in long-lived postmitotic target cells transfected with vectors as described herein, a single dose of vector can be sufficient to maintain the expression of heterologous genes in target cells for a considerable period of time. Therefore, in other embodiments, the circular DNA vectors provided herein can be administered to subjects in a single dose. The circular DNA vector can be delivered to the subject as many times as necessary to maintain a clinical (eg, therapeutic) beneficial effect.

[0218] The method of the present invention includes applying circular DNA vector or its pharmaceutical composition by any suitable approach. Circular DNA vector or its pharmaceutical composition can be systemically or locally applied, such as intravenously, ocularly (e.g., intravitreal (e.g., by intravitreal injection), subretinal, by eye drops, intraocular, intraorbital), intramuscularly, intradermally, intrahepatically, intracerebrally, intramuscularly, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, intratumorally, subcutaneously, subconjunctivally, intracapsularly, intramucosally, intrapericardially, subumbilically, orally, topically, transdermally, by inhalation, by aerosol, by injection (e.g., by rapid injection), by electroporation, by transplantation, by infusion (e.g., by continuous infusion), by local perfusion directly soaking target cells, by catheter, by lavage, with cream or with lipid composition application.

[0219] Circular DNA vectors described herein can be delivered to cells by in vivo electrotransfer (e.g., in vivo electroporation). In vivo electroporation has been demonstrated in specific tissues (such as skin, skeletal muscle, certain tumor types, and lung epithelium). Delivery of naked DNA to cells by in vivo electroporation involves applying DNA to target tissues, and then applying an electric field to temporarily increase the cell membrane permeability in the tissue by creating pores, thereby allowing DNA molecules to pass through the cell membrane. As an example, in vivo electroporation is used to deliver to the skin in Cha & Daud Hum. Vaccin. Immunother. 2012, 8 (11): 1734-1738, which is incorporated by reference as a whole. In vivo electroporation of skeletal muscle is described in Sokolowska & Blachnio-Zabielska, Int. J. Molecular Sci. 2019, 20: 2776, which is incorporated by reference as a whole. Intratumoral delivery using in vivo electroporation is described in Aung et al., Gene Therapy 2009, 16: 830-839, which is incorporated by reference as a whole. In vivo electroporation of DNA to lung cells is described in Pringle et al., J. Gene Med. 2007, 9: 369-380, which is incorporated by reference as a whole. In vivo electrotransfer of circular DNA vectors to intraocular cells (e.g., retinal cells and / or photoreceptor cells) is described in International Patent Publication WO 2022 / 198138, which is incorporated by reference as a whole. In some cases, after the circular DNA vector is applied to the eye, an electrode can be placed inside the eye (e.g., within about 1 mm from the retina), and the electric field can be transmitted to the target eye tissue by an electrode under conditions suitable for electrotransferring the circular DNA vector to the target cell (e.g., by applying six to ten pulses, 10V to 100V per pulse). Devices and systems having electrodes suitable for transmitting electric fields in mammalian tissue are commercially available and can be used in the methods disclosed herein. In some cases, the electric field is transmitted by an electrode comprising a needle (e.g., a needle placed in the vitreous body or in the subretinal space). Suitable needle electrodes include those composed of Sales Electrode and The methods of the invention include administering any of the circular DNA vectors described herein or their pharmaceutical compositions to the skin, skeletal muscle, tumors (including, for example, melanoma), eyes, and lungs by in vivo electrotransfer.

[0220] In addition, or alternatively, the circular DNA vector or its pharmaceutical composition can be administered to the host cell ex vivo, such as by transplanting (or otherwise deriving, e.g., inducing differentiation) cells from an individual patient, and then reimplanting the host cell into the patient, e.g., after selecting cells that have incorporated the vector. Thus, in some aspects, the present disclosure provides transfected host cells and methods of administering the same for treating a disease.

[0221] Additionally or alternatively, the invention includes methods of treating a subject suffering from a disease or disorder by administering an isolated DNA vector of the invention (or composition thereof) to the subject.

[0222] The evaluation of the transfection efficiency of any of the circular DNA vectors described herein can be performed using any method known in the art or described herein. The separation of transfected cells can also be performed according to standard techniques. For example, cells containing therapeutic genes can express visible markers, such as fluorescent proteins (e.g., GFP) or other reporter proteins, which are encoded by the sequence of the heterologous gene, which helps to identify and separate one or more cells containing the heterologous gene. Cells containing therapeutic genes can also be characterized by examining the nucleic acid sequence (e.g., RNA sequence, e.g., mRNA sequence) of the host cell, such as by Southern blots or PCR analysis of the presence of heterologous genes contained in the assay vector.

[0223] Therefore, the method of the present invention includes, after any circular DNA vector encoding a gene described herein is administered to a subject, then detecting the expression of the gene in the subject. Expression can be detected one to four weeks after administration, one month to four months after administration, four months to one year after administration, one year to five years after administration, or five to twenty years after administration (e.g., at least one week, at least two weeks, at least one month, at least four months, at least one year, at least two years, at least five years, at least ten years) after administration. At any of these detection time points, the persistence (e.g., additional persistence) of the circular DNA vector can be observed. In some embodiments, the persistence of the circular DNA vector is 5% to 50% higher, 50% to 100% higher, one to five times higher, or five to ten times higher (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, one time, two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more) than a reference vector (e.g., a circular vector produced in bacteria or having one or more bacterial markers not present in the vectors of the invention).

[0224] VII. Kits and Articles

[0225] In another aspect of the present invention, an article or kit containing any circular DNA vector or a pharmaceutical composition thereof is described herein. The article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials (such as glass or plastic). The container holds a composition that is effective for treating, preventing and / or diagnosing a condition by itself or in combination with another composition, and the container can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a circular DNA vector of the present invention or a pharmaceutical composition comprising a circular DNA vector. The label or package insert indicates that the composition can be used to treat a condition that the contents of the package can treat. In addition, the article can include (a) a first container containing a composition, wherein the composition contains a circular DNA vector or a pharmaceutical composition thereof; and (b) a second container containing a composition, wherein the composition includes an additional therapeutic agent. The article can also include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further include a second (or third) container comprising a pharmaceutically acceptable carrier, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, dextrose solution, or any of the pharmaceutically acceptable carriers disclosed above. It may also include other materials required by business and users, including other buffers, diluents, filters, needles, syringes or other delivery devices.

[0226] Example

[0227] Example 1. ColE2-P9 replication origin confers stable plasmid maintenance

[0228] In order to test whether the ColE2-P9 replication origin (ori) located in the plasmid can confer stable maintenance of the plasmid in Escherichia coli, a plasmid containing one of the three variants of ori (SEQ ID NO: 2-4), the R6K origin and the carbenicillin resistance marker was constructed and prepared in pir+ cells. The plasmid was transformed into S1037 and selected on an LB agar plate supplemented with carbenicillin. The colonies from each plate were cultured at 37°C in LB without antibiotics. Overnight cultures were diluted 1000 times in fresh LB without antibiotics and cultured at 37°C. After five passages, each overnight culture was re-streaked on an LB agar plate without antibiotics and grown overnight at 37°C. 20 colonies on each plate were spread on LB agar plates with or without carbenicillin. After a total of six days (during which an estimated ≥210 population doublings occurred), colony size was normal for all origin variants, and plasmid was detected in all 20 colonies for all three origin variants ( Figure 1 ). These results indicate that the 40-bp origin of replication allows the plasmid to remain stable in E. coli after multiple population doublings.

[0229] Example 2. Production of parent plasmids for producing circular DNA vectors

[0230] Figure 2 Shown is an example of a production process for a parent plasmid that can be used to produce a circular DNA vector according to an embodiment disclosed herein. The individual components of the transcription unit are assembled into the transcription unit by Golden Gate assembly. The transcription unit is then assembled into the parent plasmid by Golden Gate assembly. In this embodiment, the parent plasmid includes a LoxP recombination site sequence flanking the vector sequence (a segment comprising ori and MYO7A genes), and separates the vector sequence from the backbone sequence (a segment comprising SpecR, KanR, and RFP genes).

[0231] Example 3. In vivo production of circular DNA vectors from parent plasmids

[0232] Figure 3 The experimental process of producing circular DNA vector in vivo is shown.The test parent plasmid of the vector sequence containing the flanking LoxP recombination site is produced using the Golden Gate assembly method.The test vector sequence of the test parent plasmid includes ColE2-P9 ori sequence and reporter gene (sfGFP).For the purpose of experiment, the test vector sequence also includes chloramphenicol resistance gene (CmR), although the embodiment of circular DNA vector described herein lacks antibiotic resistance gene.The test parent plasmid also includes test backbone sequence, which includes antibiotic resistance gene SpecR and KanR and reporter gene (RFP).The test parent plasmid is transformed into the through engineering approaches escherichia coli cell with ColE2-P9 Rep gene under the control of the constitutive promoter (J23119) that is integrated into the genome. Other versions of the test parent plasmid were prepared that had two I-PpoI restriction sites, two I-SceI restriction sites, two PI-SceI restriction sites, two I-CeuI restriction sites, or one PI-PspI restriction site and one SceI restriction site instead of the LoxP sites flanking the test vector sequence. The test parent plasmid with LoxP sites was named p1603, and the test parent plasmid with two PI-SceI restriction sites was named p1600.

[0233] Bacterial colonies containing parental test plasmids are identified by being positive for RFP fluorescence.Cre recombinase is electroporated into cells containing p1603 to induce recombination and produce test circular DNA vectors at the LoxP site. The procedure for Cre electroporation is as follows: electrocompetent engineered Escherichia coli containing parental plasmids are cultured in SOB at 30°C to an OD of 0.8. Escherichia coli is 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 (1mm gap) and electroporated using an electroporator (BTX) with 1800 volts. Cells are rescued by growing in SOC for 1 hour at 37°C and are plated on LB agar plates without antibiotics. Colonies are grown and DNA is purified using QIAGEN miniprep kits. Electroporated cells are plated on 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).

[0234] Figure 4Agarose gel electrophoresis of extragenomic DNA purified from the culture of a single bacterium colony is shown, and (1) cells containing p1603 by Cre electroporation produce test circular DNA vectors, and (2) test circular DNA vectors are maintained in cells without selective pressure. Lanes 2, 3 and 5 show extragenomic DNA from p1603 transformed cells, which are GFP positive and sensitive to Kan / Spec after Cre electroporation, and grow in a rich medium lacking chloramphenicol. The bands in these lanes run to about 1500bp, which is the expected size of the test circular DNA vector, thereby showing that Cre electroporation causes the generation of the test circular DNA vector. Lanes 8, 9 and 11 correspond to lanes 2, 3 and 5, respectively, but grow in a rich medium containing chloramphenicol. The DNA abundance in lanes 2, 3 and 5 is similar to lanes 8, 9 and 11, thereby showing that the circular DNA vector is maintained in cells without selective pressure. Swimming lane 4 shows the extragenomic DNA of the p1603 transformed cells of GFP positive, RFP positive and Kan / Spec resistance after Cre electroporation. The band in this swimming lane runs to about 5000bp, which is the expected size of the test parent plasmid. Swimming lane 10 corresponds to swimming lane 4, but shows the DNA from the cells grown in the chloramphenicol-containing medium. Swimming lane 1 shows the extrachromosomal DNA purified from the GFP positive, RFP positive, Kan / Spec resistance bacterium colonies of the p1600 transformed cells. The band in this swimming lane runs to about 5000bp, which is the expected size of the test parent plasmid. Swimming lane 7 corresponds to swimming lane 1, but shows the DNA from the cells grown in the chloramphenicol-containing medium. Swimming lane 6 shows the extrachromosomal DNA purified from the same through engineering approaches Escherichia coli cells transformed with the plasmid lacking ColE2-P9 ori and grown in the medium lacking chloramphenicol. From this culture, no detectable plasmid is recovered, which may indicate that ori is necessary to maintain plasmid when there is no selective pressure. Lane 12 shows DNA purified from a culture of the same cells used for lane 6 but grown in the presence of chloramphenicol. Plasmid was recovered from these cells, possibly indicating selective pressure to maintain the plasmid in the cells.

[0235] Example 4. Circular DNA vector with ColE2-P9 ori is maintained in engineered cells expressing ColE2-P9 replication protein without selective pressure

[0236] To test the ability of a circular DNA vector with an ori sequence from ColE2-P9 (e.g., SEQ ID 2) to be maintained in cells expressing the ColE2-P9 replication protein (e.g., SEQ ID 1) in the absence of selective pressure, cells containing a test circular DNA vector generated 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 Table 1. Figure 5 The tested circular DNA vector was maintained at high levels in all media with and without chloramphenicol, except for SOC medium, which had lower maintenance levels with and without chloramphenicol.

[0237] The test circular DNA vector was purified and quantified from TB and ZB cultures without chloramphenicol. TB culture produced 0.33 mg / L of the test circular DNA vector, and SB culture produced 0.45 mg / L of the test circular DNA vector. These results indicate that the circular DNA vector is maintained regardless of the selective pressure.

[0238] Example 5. In vivo production of circular DNA vectors with counter-selection

[0239] Figure 6 An exemplary method for producing a circular DNA vector of the present invention using counter selection is shown. The transgene in this example is ABCA4, but it should be understood that the promoter driving ABCA4 can be replaced with other transgenic cassettes. On day 0, competent engineered bacterial cells expressing the Rep gene are prepared using any of the methods described herein (e.g., by transforming 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 includes the ABCA4 transgene and the replication origin (ori) downstream of the promoter. This ori-ABCA4 cassette is flanked by recombination sites (attP-GA and attB-GA). On the other side of the plasmid (the backbone region) are selectable markers: antibiotic resistance genes SpR and KanR, counter selection marker PheS, and fluorescent marker RFP. On day 2, white colonies are picked out from the red colonies and grown in LB supplemented with 4CP for counter selection. On day 3, the circular DNA vector is purified.

[0240] Example 6. Testing inducible Bxb1 to generate circular DNA vectors

[0241] To test whether Bxb1 as an exogenous recombinase can effectively generate circular DNA vectors, the Bxb1 recombinase was encoded on a bacterial artificial chromosome (BAC) and transformed into a host Escherichia coli that has the Rep gene (SEQ ID NO: 1) integrated into its genome and driven by a constitutive promoter ( Fig. 7A Two inducible Bxb1 BACs were tested: 1696 ( Figure 7B ; SEQ ID NO: 5) includes a cuminic acid inducible promoter and a chloramphenicol (Cm) resistance (CmR) gene, and 1697 ( Figure 7C ; SEQ ID NO: 6) including an arabinose inducible promoter and a CmR gene. Each BAC was transformed into S1037 cells by electroporation and plated in the presence of chloramphenicol.

[0242] Next, cells were transformed with a template plasmid carrying GFP as a reporter transgene ( Fig.7D ). The ColE2-P9 replication origin (ori) is located upstream of GFP and its promoter, and the recombination sites (attP-GA and attB-GA) are flanked by the ori-GFP cassette. On the other side of the plasmid (the backbone region) are selectable markers (antibiotic resistance genes SpR and KanR, counter-selection marker PheS, and fluorescent marker RFP). Therefore, cells containing the template plasmid have GFP+, RFP+, Kan resistance, Spec resistance, and 4CP sensitivity, while cells containing only the circular DNA vector ( Fig. 7E )(No main chain byproducts( Figure 7F ))'s cells are GFP+, RFP-, Kan-sensitive, Spec-sensitive and 4CP-resistant.

[0243] The template plasmid was electroporated into S1037 cells without inducer and plated on Cm+Kan plates. Figures 8 and 9 show the results 24 hours and 72 hours after transformation. Fig. 8A and Figure 8B It was shown that most of the colonies containing 1696 BAC were green at 24 hours and 72 hours after transformation, respectively. Some 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, Fig.9A and Fig. 9B It showed that most of the colonies containing 1697BAC were yellow at 24 and 72 hours after transformation, respectively, while some green colonies were observed at 72 hours ( Fig. 9B ).

[0244] To evaluate the effects of Cm and arabinose inducers, colonies of different colors were picked from each plate and incubated with Cm and cuminate or arabinose for 24 h. Fig.10 (1696) and Fig.11 (1697). Next, each culture was diluted 500-fold and grown overnight in LB supplemented with 4CP for counterselection with or without inducer. Overnight cultures were restreaked on regular LB agar plates and observed for fluorescence. The results are shown in Fig.12 (1696) and 13 (1697). A single colony from each plate was grown overnight in LB supplemented with 4CP, miniprepped, and digested for mapping (BsaI). The gel electrophoresis results are shown in Fig.14 The following Table 1 shows the predicted bands for each expected species 1-4:

[0245] Table 1. Predicted bands for each digested species

[0246] Lane number Species Stripe size 1 1696 6427bp; 4593bp; 1673bp 2 697 6225bp; 5288bp; 1673bp 3 Template plasmid 5166bp; 855bp 4 Circular DNA vector 855bp; 90bp

[0247] For colonies containing either type of BAC (1696 or 1697), the addition of either inducer appeared to increase GFP expressing colonies, indicating that expression of Bxb1 occurred in the absence of inducer. In fact, Bxb1 recombination had occurred after transformation of the template plasmid and plating on Cm / Kan plates. In bacteria containing the 1606 BAC, non-induced Bxb1 resulted in >90% green colonies, indicating that more than 90% contained the circular DNA vector with little or no template plasmid.

[0248] Example 7. Production of circular DNA vectors containing therapeutic transgenes

[0249] In this study, circular DNA vectors were made to include various types of therapeutic transgenes: (1) ABCA4, (2) IL-12, and (3) tricistronic cassettes encoding Flt3L, IL-12, and XCL1. The ABCA4 and IL-12 constructs included a CAG promoter, and the tricistronic construct included a CAG promoter upstream of each of the three genes. Exemplary sequences of the ABCA4 template plasmid and the resulting circular DNA vector are provided by Fig.15A (SEQ ID NO:7) and Fig. 15B (SEQ ID NO:8) is given.

[0250] First, S1037 cells were transformed with 1696BAC and grown overnight. After one day, the cells were made competent and transformed with template plasmids encoding ABCA4, IL-12 or tricistronic cassettes. The cells were plated on LB agar plates supplemented with Kan. After three days of cultivation, white microcolonies were selected (leaving red colonies) and grown overnight in LB+4CP for reverse selection before purification.

[0251] Individual colonies from each group were screened for purified constructs using BsaI digestion mapping. Fig.16A As shown in 16A (theoretical gel distribution diagram) and 16B (actual gel distribution diagram), all colonies produced circular DNA vector bands of the expected size. These results indicate that the circular DNA vector production method described herein can be widely used to effectively produce circular DNA vectors with transgenes (e.g., polycistronic) of various sizes and configurations.

[0252] Example 8. Stability of circular DNA vectors during bacterial growth and amplification

[0253] One significant advantage of the vector systems described herein is the ability to culture bacterial cells carrying circular DNA vectors in the absence of a selectable marker (e.g., after removing the selectable marker and other bacterial backbone elements from the culture). To this end, Applicants tested whether circular DNA vectors containing therapeutic transgenes could be stably expressed in bacterial culture over many cell divisions as vector production was scaled up.

[0254] As in Example 7, cells identified as containing a circular DNA vector encoding ABCA4 without a backbone were cultured overnight (14-16 hours each) for seven consecutive nights. After the seventh culture, the presence of the circular DNA vector was confirmed by sequencing. Based on an average rate of three 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.

[0255] Taking advantage of this remarkable stability, the cells were scaled up to produce larger quantities of circular DNA vector containing the ABCA4 transgene. The cells were restreaked and grown on LB plates to produce a 2.5 L preparation. A glycerol stock was produced, and this stock was restreaked to produce a 25 L preparation. The circular DNA vector was purified from this preparation to yield 18 mg of circular DNA vector.

[0256] Example 9. Detection of monomeric circular DNA vectors.

[0257] In this study, the circular DNA vector containing the ABCA4 transgene generated using the 1696BAC was sequenced to confirm that the circular DNA vector was in monomeric form (as opposed to dimers, which can be generated by trans-recombination with another template plasmid). In this study, S1037 bacteria were cultured in LB broth containing 25ug / mL Kan at 37°C for two hours. Next, the cells were transferred to plates containing 4CP and 10ng DNA and incubated overnight at 37°C. The samples were analyzed by long-read sequencing using conventional methods (Oxford Nanopore). As Fig.17AAs shown in , a monomer peak was observed, and no dimer was observed. In contrast, when the culture was incubated overnight with Kan, the circular DNA vector was mainly a dimer ( Fig. 17B ).

[0258] Example 10. Circular DNA vector prepared using Bxb1 helper plasmid

[0259] As an alternative strategy to BAC-Bxb1, a circular DNA vector was prepared by transforming Bxb1 into host cells using a helper plasmid. Fig.18 An exemplary helper plasmid is shown, which includes a cumate inducible promoter (CuO). In addition, the helper plasmid includes a temperature-sensitive backbone to allow removal of the helper plasmid after the circular DNA vector is generated. The DNA sequence of this helper plasmid is given by SEQ ID NO: 11. The host cells used in this method are the same as in the similar examples - S1037 cells with Rep (SEQ ID NO: 1) integrated into the host genome and driven by a constitutive promoter. The template plasmid is the same as in Example 6 ( Fig.7D ).

[0260] In this study, the helper plasmid was transformed into S1037 cells and incubated overnight with 100ug / mL carbenicillin (carb100). The plasmid template was then transformed and plated with carb100 and 500uM Cuma, and the cells were grown at 30°C. Using this helper plasmid method, several green colonies were observed ( Fig.19 ), indicating successful production of cells containing the circular DNA vector without backbone byproducts.

[0261] Example 11. Circular DNA vector produced by integrating Bxb1 into the host genome

[0262] Another source of recombinases provided herein is by integration into the bacterial host genome. In this embodiment, according to Fig. 20 The process shown was performed for integration of Bxb1. First, S1037 cells containing the lambda red recombination helper plasmid were grown with 0.2% arabinose and made electrocompetent. Then 500 ng of linearized 1696 BAC was electroporated. After integration of BAC 1696 into the rsd-thiC locus, the lambda red recombination plasmid was removed.

[0263] The resulting colonies were subjected to colony PCR and positive clones were identified using gel electrophoresis ( Fig.21 ). The resulting cells were engineered to express both Rep and Bxb1 by genomic integration.

[0264] Example 12. Expression of ABCA4 protein by a circular DNA vector with ColE2-P9 ori.

[0265] To determine whether bacterially produced circular DNA vectors with a ColE2-derived replication origin 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 ABCA4 protein expression was assessed by Western blotting.

[0266] HEK293T cells were seeded at 150,000 cells in 0.5 mL of standard culture medium in a 24-well plate. The plates were incubated at 37°C for 24 hours. At the time of transfection, the cells were 60%-80% confluent. Following the manufacturer's protocol, cells were transfected with circular DNA vectors using Lipofectamine 3000 (Invitrogen). The total amount of DNA added per well was 500 ng. After incubation at 37°C for 24 hours, cells were collected for Western blot analysis using β-actin as a control. Western blot results are shown in Fig. 22 and each lane is identified in Table 2 below.

[0267] Table 2: Sample identification for protein expression assay

[0268] Lanes sample 1 and 2 Lipofectamine only (negative control) 3 and 4 Synthetic circular DNA (positive control) 5-14 Bacterial-derived circular DNA

[0269] These results show that bacterially produced circular DNA vectors with ColE2-derived origin expressed their ABCA4 transgene in HEK293T human cells.

[0270] Sequence Listing

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Claims

1. An engineered bacterial cell comprising: (a) The Rep gene encoding a bacterial replication protein integrated into the bacterial genome; (b) a circular DNA vector comprising: (i) a coding sequence; and (ii) an origin of replication that is dependent on said replication protein.

2. The engineered bacterial cell of claim 1, wherein the origin of replication is less than 50 base pairs in length.

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

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

5. The engineered bacterial cell of any one of claims 1 to 4, wherein the Rep gene is operably coupled to a first inducible promoter.

6. The engineered bacterial cell of claim 5, wherein the first inducible promoter is a T7 RNA polymerase-dependent promoter.

7. The engineered bacterial cell of any one of claims 1 to 6, further comprising a gene encoding T7 RNA polymerase (T7 RNAP) integrated into the bacterial genome.

8. The engineered bacterial cell of claim 7, wherein the T7 RNAP gene is operably coupled to a second inducible promoter.

9. The engineered bacterial cell of claim 8, wherein the second inducible promoter is Ptac.

10. The engineered bacterial cell of any one of claims 1 to 9, further comprising a gene encoding an exogenous restriction enzyme integrated into the bacterial genome.

11. The engineered bacterial cell of claim 10, wherein the gene encoding the exogenous restriction enzyme is operably coupled to a third inducible promoter.

12. The engineered bacterial cell of claim 11, wherein the third inducible promoter is Pbad.

13. The engineered bacterial cell of claim 11 or 12, wherein the bacterial genome does not comprise a recognition sequence for the exogenous restriction enzyme.

14. The engineered bacterial cell of any one of claims 1 to 13, wherein the coding sequence encodes a therapeutic gene or nucleic acid.

15. The engineered bacterial cell of any one of claims 1 to 14, wherein the coding sequence is a eukaryotic sequence.

16. The engineered bacterial cell of any one of claims 1 to 15, wherein the origin of replication is the only bacterial sequence in the circular DNA vector.

17. The engineered bacterial cell of any one of claims 1 to 16, wherein the engineered bacterial cell comprises at least 20 copies of the circular DNA vector.

18. The engineered bacterial cell of any one of claims 1 to 17, wherein the engineered bacterial cell is capable of maintaining the circular DNA vector for at least 20 rounds of cell division.

19. The engineered bacterial cell of any one of claims 1 to 18, wherein the engineered bacterial cell does not contain any extragenomic circular DNA molecules other than one or more copies of the circular DNA vector.

20. A culture comprising a plurality of the engineered bacterial cells of any one of claims 1 to 19, wherein the average copy number of the circular DNA vector per engineered bacterial cell is at least 10.

21. The culture of claim 20, comprising at least 10 7 Engineered bacterial cells.

22. An engineered bacterial cell comprising: (a) The Rep gene encoding a bacterial replication protein 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, wherein said first segment does not comprise a selectable marker; and (ii) a second segment comprising a selectable marker; The first segment is flanked by at least one recognition sequence for an exogenous restriction enzyme or an exogenous recombinase.

23. The engineered bacterial cell of claim 22, wherein the recognition sequences flanking the first segment are identical.

24. The engineered bacterial cell of claim 22, wherein the recognition sequences flanking the first segment are different.

25. The engineered bacterial cell of claim 22, 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.

26. The engineered bacterial cell of claim 22, wherein the origin of replication is less than 50 base pairs in length.

27. The engineered bacterial cell of claim 22, wherein the replication origin and the replication protein are from a ColE2-related plasmid.

28. The engineered bacterial cell of claim 22, wherein the ColE2-related plasmid is ColE2-P9.

29. The engineered bacterial cell of any one of claims 22 to 27, wherein the Rep gene is operably coupled to a first inducible promoter.

30. The engineered bacterial cell of claim 29, wherein the first inducible promoter is a T7 RNA polymerase-dependent promoter.

31. The engineered bacterial cell of any one of claims 22 to 30, further comprising a gene encoding T7 RNA polymerase (T7 RNAP) integrated into the bacterial genome.

32. The engineered bacterial cell of claim 31 , wherein the T7 RNAP gene is operably coupled to a second inducible promoter.

33. The engineered bacterial cell of claim 32, wherein the second inducible promoter is Ptac.

34. The engineered bacterial cell of any one of claims 22 to 33, further comprising a gene encoding the exogenous restriction enzyme or exogenous recombinase integrated into the bacterial genome.

35. The engineered bacterial cell of claim 34, wherein the gene encoding the exogenous restriction enzyme or exogenous recombinase is operably coupled to a third inducible promoter.

36. The engineered bacterial cell of claim 35, wherein the third inducible promoter is Pbad.

37. The engineered bacterial cell of any one of claims 22 to 36, wherein the bacterial genome does not comprise a recognition sequence for the exogenous restriction enzyme or the exogenous recombinase.

38. The engineered bacterial cell of any one of claims 22 to 37, wherein the coding sequence encodes a therapeutic gene or nucleic acid.

39. The engineered bacterial cell of any one of claims 1 to 19, wherein the coding sequence is a eukaryotic sequence.

40. The engineered bacterial cell of any one of claims 22 to 39, further comprising an exogenous restriction enzyme or an exogenous recombinase.

41. A method for preparing a circular DNA vector, the method comprising: (a) contacting a plasmid in a bacterial cell with an exogenous restriction enzyme to excise a first segment from the plasmid, wherein the first segment is flanked by a recognition sequence for the exogenous restriction enzyme and wherein 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 and complementary overhangs; as well as (b) ligating the 5' end and the 3' end of the linear DNA fragment together to produce the circular DNA vector.

42. The method of claim 41, wherein prior to step (a), the plasmid comprises a second segment comprising a selectable marker.

43. The method of claim 42, 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.

44. The method of any one of claims 41 to 43, wherein the first segment does not comprise a selectable marker.

45. The method of any one of claims 41 to 44, wherein contacting the plasmid within the cell with the exogenous restriction enzyme comprises inducing expression of the exogenous restriction enzyme within the cell.

46. ​​The method of claim 45, wherein the gene encoding the exogenous restriction enzyme is integrated into the bacterial genome operably coupled to an inducible promoter.

47. The method of claim 46, wherein the inducible promoter is P bad , and wherein inducing expression of the exogenous restriction enzyme in the cell comprises providing arabinose to the cell.

48. The method of any one of claims 41 to 44, wherein contacting the plasmid within the cell with the exogenous restriction enzyme comprises introducing the exogenous restriction enzyme into the cell from outside the bacterial cell.

49. The method of any one of claims 41 to 48, wherein the ligation is performed by an exogenous ligase.

50. The method of claim 49, wherein the exogenous ligase is expressed from a gene integrated into the bacterial genome.

51. The method of claim 50, wherein the exogenous ligase is introduced into the bacterial cell from outside the bacterial cell.

52. The method of any one of claims 41 to 51, wherein the bacterial cell comprises a Rep gene encoding the bacterial replication protein integrated into the genome.

53. The method of claim 52, wherein the Rep gene is operably coupled to an inducible promoter capable of expressing the bacterial replication protein at a first expression level and a second expression level, wherein the first expression level is lower than the second expression level.

54. The method of claim 53, wherein the first expression level of the bacterial replication protein maintains the replication origin at a first copy number, and wherein the second expression level of the bacterial replication protein maintains the replication origin at a second copy number, wherein the first copy number is less than 5, 10, 15, 20, or 50 copies / cell, and the second copy number is at least 20, 50, 100, or 200 copies / cell.

55. The method of claim 53, wherein 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).

56. The method of claim 54 or 55, wherein the bacterial replication gene is expressed at the second expression level after step (b).

57. The method of any one of claims 53 to 56, wherein the inducible promoter is PT7 which is dependent on T7 RNA polymerase.

58. The method of any one of claims 41 to 57, wherein the bacterial cell comprises a gene encoding T7 RNA polymerase (T7 RNAP) integrated into the genome.

59. The method of claim 58, wherein the T7 RNAP gene is operably coupled to an inducible promoter.

60. The method of claim 59, wherein the inducible promoter is P tac .

61. The method of claim 60, wherein the second segment of the plasmid further comprises a coding region capable of inhibiting the expression of tac The invention relates to a LacI gene of a lactose repressor protein expressed by a promoter, and wherein the expression of the bacterial replication gene is maintained at or below the first expression level by the expression of the lactose repressor protein.

62. The method of claim 61, wherein after step (b), expression of the lactose repressor protein is reduced, thereby inducing expression of the bacterial replication gene at the second expression level and maintaining the circular DNA vector at the second copy number.

63. The method of any one of claims 41 to 62, further comprising culturing the cells under conditions where continued growth does not require the selective marker on the plasmid, thereby producing a progeny population of the bacterial cells lacking the selective marker.

64. The method of claim 63, wherein after at least 50 doublings, the population maintains the circular DNA vector at an average copy number of at least 20 copies / cell.

65. The method of claim 63 or 64, further comprising purifying the circular DNA vector.

66. The method of any one of claims 41 to 65, wherein the length of the replication origin is less than 50 base pairs.

67. The method of any one of claims 41 to 66, wherein the replication origin and the replication protein are from a ColE2-related plasmid.

68. The method of claim 67, wherein the ColE2-related plasmid is ColE2-P9.

69. A method for preparing a circular DNA vector, the method comprising: (a) obtaining the engineered bacterial cell according to any one of claims 22 to 40; (b) contacting the plasmid with an exogenous restriction enzyme to excise the first segment of the plasmid, thereby generating a linear DNA fragment flanked by complementary overhangs; and (c) self-ligating the linear DNA fragment to generate the circular DNA vector.

70. A method for preparing a circular DNA vector, the method comprising: (a) obtaining the engineered bacterial cell according to any one of claims 22 to 40; (b) contacting the plasmid with an exogenous recombinase that recognizes the recognition sequences flanking the first segment.

71. A pharmaceutical composition comprising: (a) a circular DNA vector produced by the method of any one of claims 41 to 70; and (b) a suitable carrier for delivering the pharmaceutical composition to a subject.

72. An engineered bacterial cell comprising a circular DNA vector comprising a coding sequence and an origin of replication less than 50 base pairs in length, wherein the circular DNA vector lacks a selectable marker.

73. The engineered bacterial cell of claim 72, wherein the engineered bacterial cell does not contain any extragenomic DNA molecules other than one or more copies of the circular DNA vector.

74. The engineered bacterial cell of claim 72 or 73, wherein the engineered bacterial cell does not comprise a gene encoding a selectable marker.

75. The engineered bacterial cell of any one of claims 72 to 74, wherein the engineered bacterial cell does not comprise a selectable marker on an extragenomic DNA molecule.

76. The engineered bacterial cell of any one of claims 72 to 75, wherein the origin of replication is from a ColE2-P9 plasmid.

77. The engineered bacterial cell of any one of claims 72 to 76, further comprising a Rep gene encoding a bacterial replication protein that recognizes an origin of replication.

78. The engineered bacterial cell of claim 77, wherein the Rep gene is from a ColE2-P9 plasmid.

79. The engineered bacterial cell of claim 77 or 78, wherein the Rep gene is integrated into the bacterial genome.

80. The engineered bacterial cell of any one of claims 77 to 79, wherein the Rep gene is operably coupled to an inducible promoter.

81. The engineered bacterial cell of any one of claims 72 to 80, wherein the circular DNA vector further comprises a recombination site.

82. The engineered bacterial cell of any one of claims 72 to 81, wherein the circular DNA vector does not comprise any bacterial [or other prokaryotic or bacteriophage] sequences other than the replication origin and the recombination site (when present).

83. The engineered bacterial cell of claim 81, wherein the origin of replication and the recombination site together are no more than 90 base pairs in length.

84. The engineered bacterial cell of any one of claims 72 to 83, further comprising a gene encoding a recombinase.

85. The engineered bacterial cell of any one of claims 72 to 83, wherein the bacterial cell comprises at least 10 copies of the circular DNA vector.

86. An engineered bacterial cell comprising a plasmid comprising: (a) a first segment comprising a coding sequence and an origin of replication of less than 50 base pairs in length, wherein the first segment does not comprise a selectable marker; and (b) a second segment comprising a selectable marker; The first segment is flanked by a recognition sequence for an exogenous recombinase.

87. The engineered bacterial cell of claim 86, further comprising a gene encoding a Rep gene, wherein the Rep gene encodes a bacterial replication protein that recognizes the replication origin.

88. The engineered bacterial cell of claim 87, wherein the Rep gene is integrated into the bacterial genome.

89. The engineered bacterial cell of claim 87 or 88, wherein the Rep gene is operably coupled to a first inducible promoter.

90. The engineered bacterial cell of any one of claims 87 to 89, wherein the replication origin and the replication protein are from a ColE2-related plasmid.

91. The engineered bacterial cell of any one of claims 87 to 90, wherein the ColE2-related plasmid is ColE2-P9.

92. The engineered bacterial cell of any one of claims 86 to 91, further comprising a gene encoding the exogenous recombinase.

93. The engineered bacterial cell of claim 92, wherein the gene encoding the exogenous recombinase is integrated into the bacterial genome.

94. The engineered bacterial cell of claim 92, wherein the gene encoding the exogenous recombinase is on a plasmid or a bacterial artificial chromosome.

95. The engineered bacterial cell of any one of claims 92 to 94, wherein the gene encoding the exogenous recombinase is operatively coupled to a second inducible promoter.

96. The engineered bacterial cell of claim 95, wherein the second inducible promoter is a cuminate inducible promoter.

97. The engineered bacterial cell of any one of claims 92 to 96, wherein the recombinase is Bxb1.

98. The engineered bacterial cell of claim 97, wherein the recognition sequence comprises attP-GA and attB-GA.

99. A method for preparing a circular DNA vector, comprising inducing recombination of the plasmid in the engineered bacterial cell of any one of claims 86 to 98.

100. The method of claim 99, wherein inducing recombination of the plasmid comprises inducing expression of the exogenous recombinase in the engineered bacterial cell.

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

102. The method of claim 101, wherein the engineered bacterial cell further comprises a Rep gene encoding a bacterial replication protein that recognizes an origin of replication.

103. The method of claim 101 or 102, wherein the Rep gene is integrated into the bacterial genome.

104. The method of claim 102 or 103, wherein the Rep gene is operably coupled to a first inducible promoter.

105. The method of any one of claims 101 to 104, wherein the replication origin is the ColE2-P9 replication origin.

106. The method of any one of claims 102 to 105, wherein the Rep gene is the ColE2-P9 Rep gene.

107. The method of any one of claims 101 to 106, wherein the exogenous recombinase is located on a plasmid or a bacterial artificial chromosome.

108. The method of any one of claims 101 to 107, wherein the gene encoding the exogenous recombinase is operably coupled to a second inducible promoter.

109. The method of claim 108, wherein inducing recombination of the plasmid comprises inducing expression of the gene encoding the exogenous recombinase.

110. The method of any one of claims 101 to 108, wherein the inducing recombination of the plasmid comprises introducing the plasmid into the engineered bacterial cell, wherein the exogenous recombinase is expressed in the engineered bacterial cell upon the introduction.

111. The method of claim 110, wherein the exogenous recombinase is expressed at a non-induced level upon introduction.

112. The method of any one of claims 101 to 111, wherein the exogenous recombinase is Bxb1 and the recognition sequence comprises attP-GA and attB-GA.

113. The method of claim 112, wherein the gene encoding Bxb1 is operably coupled to a cuminate inducible promoter, the engineered bacterial cell is maintained in the absence of cuminate upon said introduction, and the Bxb1 is expressed at a non-induced level upon said introduction.

114. A pharmaceutical composition comprising: (a) a circular DNA vector produced by the method of any one of claims 99 to 113; and (b) a suitable carrier for delivering the pharmaceutical composition to a subject.

115. A circular DNA vector comprising: (a) Eukaryotic promoter; (b) eukaryotic coding sequences; and (c) a bacterial origin of replication less than 50 bp in length, The circular DNA vector lacks a selectable marker.

116. The circular DNA vector of claim 115, wherein the 3' end of the eukaryotic coding sequence is linked to the 5' end of the promoter via a sequence comprising the bacterial origin of replication, wherein the sequence comprising the bacterial origin of replication is less than 100 bp in length.

117. A pharmaceutical composition comprising: (d) the circular DNA vector of any one of claims 115 or 116; and (e) a suitable carrier for delivering the pharmaceutical composition to a subject.

118. A host cell comprising the circular DNA vector of any one of claims 115 or 116.

119. The host cell of claim 118, wherein the host cell is a mammalian cell.

120. The host cell of claim 118 or 119, wherein the host cell is a human cell.

121. A host cell as described in any one of claims 117 to 120, wherein the host cell is isolated in vitro.

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