Gene therapy vectors and methods of producing and using same

Abstract

Provided herein are improved DNA vectors (e.g., circular DNA vectors and / or non-viral DNA vectors) that can provide features including enhanced expression, persistence, safety, and manufacturability. Also provided herein are methods of producing such DNA vectors, host cells (e.g., engineered bacterial cells useful for producing such DNA vectors) containing such DNA vectors, methods of using such DNA vectors (e.g., engineered bacterial cells useful for producing such DNA vectors), and methods of using such DNA vectors. Methods of expressing therapeutic sequences in target cells by administering such DNA vectors and methods of treating diseases or conditions by administering such DNA vectors), as well as pharmaceutical compositions containing such DNA vectors.

Description

Technical Field

[0001] In various aspects and implementations, this disclosure provides therapeutic carriers and pharmaceutical compositions thereof, as well as related methods and engineered bacterial cells.

[0002] Cross-references to related applications

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 509,458, filed June 21, 2023, the entire contents of which are incorporated herein by reference.

[0004] sequence list

[0005] This application includes a sequence list in XML format, which was submitted electronically with the Patent Centre. The XML copy was created on June 11, 2024, named “IGT-014PC_138289-5014.xml”, and is 200,459 bytes in size. The sequence list is incorporated herein by reference in its entirety. Background Technology

[0006] Gene therapy is emerging as a promising approach for treating a wide range of diseases and conditions in human patients. Recombinant adeno-associated viral (rAAV) vectors have a proven track record of efficient gene transfer in human patients and various model systems. The genome of an rAAV vector is advantageous due to its ability to persist in vivo as a circular appendage throughout the life of the target cell. On the other hand, rAAV-based vectors have significant drawbacks, such as limited maximum payload, immunogenicity, and low manufacturing efficiency.

[0007] To address some of these challenges in rAAV technology, nonviral alternatives have gained attention in recent years. However, developing a scalable nonviral gene therapy platform that combines the efficiency and sustainability of rAAV has proven difficult. For example, while traditional bacterial plasmid DNA vectors represent a tool in gene delivery, their high content of bacterial components (such as antibiotic resistance genes and transcriptional regulatory elements) can lead to immunogenicity and loss of gene expression due to transcriptional silencing.

[0008] A non-viral vector with minimal bacterial components and a method for large-scale, efficient production are required. Summary of the Invention

[0009] This document provides circular DNA vectors (e.g., nonviral DNA vectors) that offer enhanced expression, persistence, safety, and manufacturability compared to previously described DNA vectors (e.g., AAV vectors, plasmids, and other circular DNA vectors lacking one or more backbone components). The DNA vectors described herein contain small (e.g., less than 50 base pairs) origins of replication and lack sequences encoding selectivity markers (e.g., antibiotic resistance genes), which can reduce the risk of introduction by exogenous sequences in the vector. In some embodiments, the DNA vectors disclosed herein have bacterial-derived nucleotide sequences of about 200 base pairs (bp) or less, or about 150 bp or less, or about 100 bp or less, or about 75 bp or less, or about 50 bp or less. Such DNA vectors can be efficiently and massively produced for therapeutic applications, including using the methods disclosed herein. Additionally, transcriptional silencing of circular DNA vectors can be reduced or eliminated by eliminating or reducing the bacterial plasmid DNA sequence in the vector, resulting in persistent presence of the vector sequence in the cells and tissues of the target. In some specific implementations, immunogenic components (e.g., bacterial endotoxins, DNA, or RNA, or bacterial signatures such as CpG motifs) are absent in the circular DNA vector or are present at very low levels suitable for pharmaceutical, clinical, or laboratory use; thus, the risk of stimulating a host immune response is reduced compared to certain conventional DNA vectors, such as plasmid DNA vectors.

[0010] This document also provides methods for producing DNA vectors, host cells containing such DNA vectors (e.g., engineered bacterial cells that can be used to produce such DNA vectors), methods for using such DNA vectors (e.g., methods for expressing therapeutic sequences in target cells by administering said DNA vectors, and methods for treating diseases or conditions by administering such DNA vectors), and pharmaceutical compositions containing such DNA vectors.

[0011] In one aspect, this disclosure provides a circular DNA vector or a pharmaceutical composition thereof, wherein the circular DNA vector comprises a therapeutic sequence and a sequence containing a bacterial origin of replication. In some embodiments, the sequence containing the bacterial origin of replication is less than 50 bp in length, and the circular DNA vector lacks a selectivity marker. In several embodiments, the vector also lacks a recombination site. In some embodiments, the vector contains a transposase scar.

[0012] In several implementations, the sequence containing the bacterial origin of replication directly links the 3' end of the therapeutic sequence to the 5' end of the therapeutic sequence.

[0013] In several embodiments, the circular DNA vector has a bacterial-derived sequence of about 200 base pairs (bp) or less, or about 150 bp or less, or about 100 bp or less, or about 75 bp or less, or about 50 bp or less.

[0014] In some embodiments, the origin of replication origins is derived from a ColE2-associated plasmid, and optionally, ColE2-P9. In some such embodiments, the origin of replication is recognized by the ColE2-P9 replication protein. An exemplary ColE2-P9 replication protein comprises the amino acid sequence of SEQ ID NO: 1.

[0015] In some embodiments, the origin of replication is 40 bp or less in length. In some embodiments, the origin of replication is 36 bp or less, or 34 bp or less, or 32 bp or less, or 30 bp or less, or 28 bp or less in length. For example, the origin of replication may have the nucleotide sequence of SEQ ID NO: 2, or it may be a functional variant or a truncated variant thereof.

[0016] In some implementations, the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication contains or is composed of the nucleotide sequence of SEQ ID NO: 12.

[0017] In some implementations, the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication contains or is composed of the nucleotide sequence of SEQ ID NO: 13.

[0018] In some implementations, the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication contains or is composed of the nucleotide sequence of SEQ ID NO: 14.

[0019] In some implementations, the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication contains or is composed of the nucleotide sequence of SEQ ID NO: 15.

[0020] In some implementations, the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication contains or is composed of the nucleotide sequence of SEQ ID NO: 16.

[0021] In some implementations, the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication contains or is composed of the nucleotide sequence of SEQ ID NO: 17.

[0022] In some implementations, the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication contains or is composed of the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0023] In some implementations, the replication origin includes The nucleic acid sequence or composition thereof, wherein each X is selected from A, T, C or G. In some implementations: X1 is A, T or C; X2 is A, T or C; X3 is A, T or G; X4 is A, T or C; X5 is A, T or G; X6 is C; X7 is A.

[0024] In some embodiments, the circular DNA vector is a non-integrative circular DNA vector. The therapeutic sequence may include a scaffold-matrix attachment region (S / MAR) to enhance cell persistence. In some embodiments, the therapeutic sequence is greater than 4.5 kb. In some embodiments, the circular DNA vector is monomeric and supercoiled.

[0025] In some embodiments, the therapeutic sequence includes an eye gene. In some embodiments, the eye gene is selected from... .

[0026] In some embodiments, the eye gene comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 19 or SEQ ID NO: 20, and the eye gene encodes a polypeptide having the amino acid sequence (CFH) of SEQ ID NO: 63.

[0027] In some embodiments, the eye gene comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 21, and the eye gene encodes a polypeptide having the amino acid sequence (ABCA4) of SEQ ID NO: 61. In some embodiments, the nucleic acid sequence has at least 90% identity with SEQ ID NO: 8, and optionally comprises the nucleic acid sequence of SEQ ID NO: 8.

[0028] In some embodiments, the eye gene comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 22 and encoding a polypeptide having the amino acid sequence (MYO7A) of SEQ ID NO: 62. In some embodiments, the nucleic acid sequence has at least 90% identity with SEQ ID NO: 23 and optionally comprises the nucleic acid sequence of SEQ ID NO: 23.

[0029] In several implementations, the therapeutic sequence includes a CAG promoter that controls the expression of a therapeutic gene (e.g., an eye gene).

[0030] In some embodiments, the therapeutic sequence further comprises a regulatory element comprising a sequence derived from intron 6 of ABCA4. For example, the regulatory element may be derived from the 5' half-region of intron 6 of ABCA4. In some embodiments, the regulatory element has at least 90% identity with at least 500 consecutive nucleotides within intron 6 of ABCA4. For example, the at least 500 consecutive nucleotides may comprise consecutive nucleotides within nucleotides 3,158 to 4,822 of intron 6. In some embodiments, the regulatory element comprises the nucleic acid sequence of SEQ ID NO: 24 or a functional variant thereof.

[0031] In other aspects, this disclosure provides methods for expressing therapeutic sequences in the eye cells of a subject. The methods include delivering a circular DNA vector encoding an ocular protein to eye cells (e.g., ocular cells of the retina). In some embodiments, the circular DNA vector is delivered to the eye cells via transocular administration. In some embodiments, expression in the eye cells persists for at least 12 months following administration of the circular DNA vector to the subject. In some embodiments, the circular DNA vector is administered at a frequency not exceeding approximately once every three months, approximately once every four months, approximately once every six months, approximately once every eight months, or approximately once a year. In several embodiments, the subject suffers from an eye disease, and the ocular gene may replace a mutated copy in the subject.

[0032] In other aspects, the therapeutic sequence comprises a respiratory system gene. In some embodiments, the respiratory system gene encodes a cystic fibrosis transmembrane receptor (CFTR), and the therapeutic sequence optionally comprises an elongation factor 1 (EF1A) promoter. In some embodiments, the therapeutic sequence also comprises a hypersensitivity sequence on the 5' side of the respiratory system gene. An exemplary hypersensitivity sequence comprises the nucleotide sequence of SEQ ID NO: 28 or a functional variant thereof.

[0033] In one aspect, this disclosure provides a method for expressing a therapeutic sequence in the airway cells of a subject. The method includes delivering a circular DNA vector (expressing a respiratory gene) of this disclosure into the airway cells of the subject. In some embodiments, the subject suffers from a respiratory disease, such as cystic fibrosis.

[0034] In another aspect, this disclosure provides circular DNA vectors (as disclosed) wherein the therapeutic sequence comprises a regulatory protein-encoding gene, which may be an immunomodulatory protein-encoding gene. In some embodiments, the immunomodulatory protein-encoding gene is a dendritic cell chemoattractant-encoding gene, a dendritic cell growth factor or activator-encoding gene, or a lymphocyte signaling protein-encoding gene. In some embodiments, the dendritic cell chemoattractant-encoding gene is XCL1, XCL2, CCL5, or CCL4; the dendritic cell growth factor or activator-encoding gene is FLT3L, GMCSF, or CD40; or the lymphocyte signaling protein-encoding gene is IL-12, IL-15, CXCL9, or CXCL10. In some embodiments, the circular DNA vector is a bicistronic or polycistronic vector encoding two, three, or more regulatory protein-encoding genes.

[0035] In one related aspect, this disclosure provides a method for expressing a therapeutic sequence comprising a regulatory protein-coding gene in target cells of a subject. The method includes delivering a circular DNA vector of this disclosure to the target cells of the subject (in some embodiments, which comprise a tumor). In some embodiments, the subject has cancer, and the method modulates the tumor microenvironment in the subject.

[0036] In other aspects and implementation schemes, therapeutic sequences encode antigen-binding proteins, enzymes, growth factors, hormones, interleukins, interferons, cytokines, anti-apoptotic factors, anti-diabetic factors, coagulation factors, anti-tumor factors, liver-secreting proteins, or neuroprotective factors.

[0037] In other aspects, the therapeutic sequence comprises a transcriptional knockdown sequence, and it may be selected from microRNA-coding sequences, short hairpin RNA-coding sequences, zinc finger nuclease-coding sequences, TALEN-coding sequences, dCAS9-coding sequences, guide RNA-coding sequences, or combinations thereof. In some embodiments, the transcriptional knockdown sequence comprises one or more guide RNA-coding sequences and a dCAS9-coding sequence. For example, the dCAS9-coding sequence may be fused to one or more repressor proteins (e.g., transcriptional repressor proteins). In some embodiments, the therapeutic sequence further comprises a therapeutic protein-coding sequence, wherein the transcriptional knockdown sequence is configured to silence the transcription of endogenous expression of a target gene, and wherein the therapeutic sequence encodes a therapeutic alternative protein encoded by the functional form of the target gene. In some related aspects, this disclosure provides a method for knocking down the transcription of a mutated target gene in the cells of a desired object. The method includes contacting a circular DNA vector of any of the present disclosure with a cell under conditions suitable for knocking down the transcription of the mutated target gene.

[0038] In another aspect, therapeutic sequences contain gene-editing sequences. Exemplary gene-editing sequences encode guide RNA and / or CRISPR-associated (Cas) endonucleases (e.g., Cas9).

[0039] In another aspect, this disclosure provides engineered bacterial cells for replicating circular DNA vectors. The engineered bacterial cells comprise: (a) the circular DNA vector of this disclosure, and (b) a Rep gene encoding a bacterial replication protein that binds to the bacterial origin of replication of the circular DNA vector, wherein the Rep gene replicates the circular DNA vector.

[0040] In some embodiments, the therapeutic sequence comprises a transposase overhang sequence, which may be (but is not limited to) TTAA. In some such embodiments, the bacterial cell contains a transposase protein, wherein the transposase protein hydrolyzes DNA adjacent to the transposase overhang sequence. In some embodiments, the transposase protein is encoded by a transposase gene expressed by the engineered bacterial cell, and the transposase gene may be integrated into the bacterial genome. In some embodiments, the engineered bacterial cell also contains an insertion sequence excision enhancer (IEE), which may be encoded by a gene integrated into the bacterial genome. In some embodiments, the engineered bacterial cell also contains a terminally closed linear DNA molecule containing a plasmid backbone. The plasmid backbone may contain selection markers (which may be antibiotic resistance genes and / or anti-selection markers).

[0041] In several embodiments, the origin of replication is a unique bacterial sequence within the circular DNA vector. In some embodiments, the engineered bacterial cells (e.g., in a culture) contain an average of at least 10 copies of the circular DNA vector. In several embodiments, the circular DNA vector is monolithic. In some embodiments, the bacterial cells in the culture contain an average of at least 10, at least 15, or at least 20 copies of the circular DNA vector per engineered bacterial cell.

[0042] In another aspect, this disclosure provides engineered bacterial cells for producing circular DNA vectors, the cells comprising: (a) a plasmid template, wherein the plasmid template comprises: (i) a first segment comprising a therapeutic sequence and a sequence containing a bacterial origin of replication, wherein the first segment is flanked by two transposase overhang sequences; and (ii) a second segment comprising a plasmid backbone, wherein the second segment is flanked by left-end (LE) repeats and right-end (RE) repeats, wherein the LE repeats and RE repeats are bindable to transposase proteins; and (b) a Rep gene encoding a bacterial replication protein that binds to the bacterial origin of replication. In several embodiments, the bacterial cells also comprise transposase proteins, wherein the transposase proteins hydrolyze DNA adjacent to the transposase overhang sequences. In some embodiments, the engineered bacterial cell also includes (e.g., produces): (c) a circular DNA vector containing a therapeutic sequence, a sequence containing a bacterial origin of replication, and one of two transposase overhang sequences; and / or (d) a linearly terminal closed DNA molecule containing a plasmid backbone flanked by LE and RE repeats.

[0043] In some embodiments, the transposase protein is encoded by a transposase gene expressed by the engineered bacterial cell, and said transposase gene may be integrated into the bacterial genome. In some embodiments, the engineered bacterial cell also expresses IEE, wherein the IEE may be encoded by a gene integrated into the bacterial genome.

[0044] In some embodiments, the sequence containing the bacterial origin of replication directly links the 3' end of the therapeutic sequence to the 5' end. In multiple embodiments, the sequence containing the bacterial origin of replication is less than 50 bp in length (e.g., as described herein). In multiple embodiments, the bacterial origin of replication is the ColE2-P9 origin of replication (e.g., as described herein).

[0045] In several implementations, the first segment and the circular DNA vector lack selection markers, while the second segment and the plasmid backbone contain selection markers. As described herein, selection markers include antibiotic resistance genes and anti-selection markers.

[0046] In several implementations, the therapeutic sequence is a eukaryotic sequence (e.g., as described herein).

[0047] In several implementations, the engineered bacterial cells do not contain any extragenomic circular DNA molecules other than a plasmid template, a second segment containing a plasmid backbone, or one or more copies of a circular DNA vector.

[0048] In some related aspects, this disclosure provides a method for preparing a circular DNA vector of the present disclosure. The method includes: (a) providing a bacterial cell containing a Rep gene encoding a bacterial replication protein that binds to a bacterial origin of replication and a plasmid template, wherein the plasmid template comprises: (i) a first segment containing a therapeutic sequence and a sequence containing a bacterial origin of replication; and (ii) a second segment containing a plasmid backbone; and (b) contacting the plasmid within the bacterial cell with an enzyme to circularize the first segment, thereby producing a circular DNA vector containing a therapeutic sequence and an origin of replication.

[0049] In several embodiments, the method further includes culturing bacterial cells under conditions suitable for the replication of the circular DNA vector, thereby preparing a bacterial cell culture for replicating the circular DNA vector. In some embodiments, the enzyme is encoded on a helper plasmid or bacterial artificial chromosome (BAC) within the bacterial cell. In some embodiments, the bacterial cell culture (replicating the circular DNA vector) is substantially free of helper plasmids or BACs. In several embodiments, the average copy number of the circular DNA vector per bacterial cell (e.g., in the culture) is at least 10, at least 15, or at least 20.

[0050] In some embodiments, the enzyme is a transposase. For example, the second segment is flanked by left-hand (LE) and right-hand (RE) repeats, wherein the LE and RE repeats are bound by a transposase protein. In some embodiments, the second segment contains a selection marker, and the first segment lacks a selection marker. In several embodiments, the selection marker is selected from one or more of antibiotic resistance genes and anti-selection markers. In still other embodiments, the bacterial cells express IEE.

[0051] In several implementations, the circular DNA vector is isolated from bacterial cells or their cultures.

[0052] Other aspects and embodiments of this disclosure will be apparent to those skilled in the art based on the following detailed description and accompanying drawings. Attached Figure Description

[0053] The features of this disclosure are set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description, which illustrates illustrative embodiments utilizing the principles of this disclosure and the following figures:

[0054] Figure 1This is a graph illustrating the results of such stability studies, which tested the ability of three ColE2-P9 origins of replication to confer plasmid stability during E. coli amplification. A 40-bp ColE2-P9 origin (SEQ ID NO:2) and its opposite strand sequence (SEQ ID NO: 43) are shown. SEQ ID NO: 44 and 45 represent truncated ColE2-P9 origins.

[0055] Figure 2 Examples of methods for assembling parent plasmids that can be used in the embodiments disclosed herein are illustrated.

[0056] Figure 3 An exemplary procedure for generating a test circular DNA vector according to an embodiment disclosed herein is illustrated, which involves a recombinase as an enzyme for removing the target sequence from the backbone and recirculating the circular DNA vector.

[0057] Figure 4 The results of agarose gel electrophoresis of extrachromosomal DNA purified from modified bacteria grown in rich media containing chloramphenicol (“Cm”; lanes 7 to 12) or without chloramphenicol (“Cm-free”; lanes 1 to 6) are shown. Lanes 2, 3, 5, 8, 9, and 11 show bands corresponding to the test circular DNA vector generated from the test parent plasmid recombination. Lanes 1, 4, 7, and 10 show bands corresponding to the test parent plasmid.

[0058] Figure 5 This is a graph showing the percentage of sfGFP-positive cells in a specified growth medium with or without chloramphenicol (“Cm”). ZB = Zymo Broth; TB = Terrific Broth; SB = Super Broth; SOB = Super Optimal Broth; SOC = Super Optimal Broth with Catabolite Repression; LB = Luria Broth.

[0059] Figure 6 This is a schematic diagram illustrating an exemplary process for generating a circular DNA vector of the present disclosure using inverse selection.

[0060] Figures 7A to 7F This is a schematic diagram illustrating the contents of a bacterial artificial chromosome (BAC)-based method for expressing Bxb1 to produce a circular DNA vector. Figure 7A It is the Rep gene that is integrated into the host genome. Figure 7B and 7C There are two optional BAC designs; Figure 7B Bxb1 is driven by a cumuloic acid-inducible promoter, while Figure 7C Bxb1 is driven by an arabinose-inducible promoter. Figure 7D It is a template plasmid, which becomes after being recombinated by Bxb1. Figure 7E Circular DNA vectors and Figure 7F The byproducts of the circular DNA vector. Because circular DNA vectors contain origins of replication, and (optionally) the byproducts contain the PheS anti-selection marker, circular DNA vectors become the dominant species during replication and amplification of host bacteria.

[0061] Figure 8A and 8B These are photographs showing the fluorescence of clones 24 hours and 72 hours after transformation using BAC 1696.

[0062] Figure 9A and 9B These are photographs showing the fluorescence of clones 24 hours and 72 hours after transformation using BAC 1696.

[0063] Figure 10 These are a set of photographs showing the fluorescence of clones 24 hours after contact with the cumene-induced agent.

[0064] Figure 11 These are a set of photographs showing the fluorescence of clones 24 hours after contact with the arabinose inducer.

[0065] Figure 12 This is a series of photographs showing the fluorescence of 1696 colonies that have been restretched and incubated overnight on LB agar plates under various conditions.

[0066] Figure 13 This is a series of photographs showing the fluorescence of 1697 colonies that have been restretched and incubated overnight on LB agar plates under various conditions.

[0067] Figure 14 This is a photograph of a gel electrophoresis experiment showing the presence of circular DNA vectors in the anti-selection cultures of 1696 and 1697. A digestion map showing the theoretical bands is shown on the left side of the photograph.

[0068] Figure 15A This is a plasmid map of the exemplary ABCA4 template plasmid.

[0069] Figure 15B It is by Figure 15A The plasmid map of the ABCA4 circular DNA vector generated from the template plasmid.

[0070] Figure 16A This is a theoretical gel diagram showing the band pattern of the digested circular DNA construct described in Example 7. Figure 16B It shows the corresponding Figure 16A A photograph of the actual strip-shaped gel.

[0071] Figure 17A This is a histogram showing long-read sequencing data from a purified ABCA4 circular DNA vector, generated using overnight Kan resistance incubation with a template plasmid. The main peaks are BAC and the dimeric circular DNA vector.

[0072] Figure 17B This is a histogram showing long-read sequencing data from the purified ABCA4 circular DNA vector, generated using a 2-hour Kan resistance incubation with the template plasmid. The main peaks are from the monomeric circular DNA vector and BAC.

[0073] Figure 18 This is a plasmid map showing the components that can be used to express Bxb1 in a bacterial host.

[0074] Figure 19 This is a photograph showing green fluorescent colonies (circled), which contain circular DNA vectors without backbone byproducts. This is achieved through... Figure 18 The results of Bxb1 expression using helper plasmids.

[0075] Figure 20 This is a set of diagrams depicting the process of integrating Bxb1 into the host cell genome.

[0076] Figure 21 This is a photograph of a gel showing two positive clones of Bxb1 integration. Triple copies of the 1696 plasmid control are shown in the lower left corner.

[0077] Figure 22 This is a schematic diagram illustrating an exemplary procedure for generating a test circular DNA vector containing GFP according to an embodiment disclosed herein, which involves a transposase as an enzyme for removing the target sequence from the backbone and recirculating the circular DNA vector.

[0078] Figure 23AThis is a table showing the results of a screening study in which the ColE2-P9 replication origin differs at its ends and colony size was tested. SEQ ID NO: 46 and 47 represent the two strands of a plasmid containing the ColE2-P9 origin. Partial plasmid sequences of plasmids 55002 (SEQ ID NO: 48), 55003 (SEQ ID NO: 49), 55004 (SEQ ID NO: 50), 55005 (SEQ ID NO: 51), 55006 (SEQ ID NO: 52), 55007 (SEQ ID NO: 53), 55008 (SEQ ID NO: 54), 55009 (SEQ ID NO: 55), 55010 (SEQ ID NO: 56), 55033 (SEQ ID NO: 57), 55034 (SEQ ID NO: 58), 55035 (SEQ ID NO: 59), and 55036 (SEQ ID NO: 60) are provided.

[0079] Figure 23B This indicates that the inoculation contains Figure 23A A series of photographs of carb plates after S1037 E. coli cells were transformed with circular DNA vectors of each variant of the ColE2-P9 replication origin.

[0080] Figure 24 This is a Western blot image showing the expression of ABCA4 protein in HEK293T cells transfected with a bacterial ABCA4 circular DNA vector. Detailed Implementation

[0081] Efforts have been made to improve plasmid DNA vectors (e.g., for gene therapy) by removing plasmid backbone components. For example, microcircles are prepared by using recombinant DNA in bacterial cells to remove the backbone from plasmids, resulting in microcircular vectors and circular backbone byproducts. However, microcircles are difficult to produce on a large scale, partly because it is difficult to remove backbone byproducts with similar properties to the microcircular products. Alternative vector types, such as nanoparticles, have been designed to facilitate easier purification through positive selection by including replication origins and selectivity markers within the vector. However, such foreign elements are relatively large (typically hundreds of base pairs in length) and are foreign substances to patients, which may introduce risks of immunogenicity and / or transgene silencing.

[0082] This document provides circular DNA vectors (e.g., nonviral DNA vectors) that can provide enhanced expression, persistence, safety, and manufacturability compared to previously described DNA vectors (e.g., AAV vectors, plasmids, and other circular DNA vectors lacking one or more backbone components). The DNA vectors described herein contain small (e.g., less than 50 base pairs) origins of replication and lack sequences encoding selectivity markers (e.g., antibiotic resistance genes), which can reduce the risk of introduction by exogenous sequences in the vector. In some embodiments, the DNA vectors disclosed herein have bacterial-derived sequences of about 200 base pairs (bp) or less, or about 150 bp or less, or about 100 bp or less, or about 75 bp or less, or about 50 bp or less. Such DNA vectors can be efficiently and on a large scale produced for therapeutic applications, including using the methods disclosed herein. Additionally, transcriptional silencing of circular DNA vectors can be reduced or eliminated by eliminating or reducing the bacterial plasmid DNA sequence, resulting in the persistence of the vector sequence in the target. In some specific embodiments, immunogenic components (e.g., bacterial endotoxins, DNA, RNA, and bacterial nucleic acid features (e.g., CpG motifs) are absent in the circular DNA vector of the present invention, or are present at very low levels suitable for pharmaceutical, clinical, or laboratory applications; thus, the risk of stimulating a host immune response is reduced compared to certain conventional DNA vectors, such as plasmid DNA vectors.

[0083] This article also provides methods for producing DNA vectors, host cells containing such DNA vectors (e.g., engineered bacterial cells that can be used to produce such DNA vectors), methods for using such DNA vectors (e.g., methods for expressing therapeutic sequences in target cells by administering such DNA vectors, and methods for treating diseases or conditions by administering such DNA vectors), and pharmaceutical compositions containing such DNA vectors.

[0084] I. Definition

[0085] Unless the context clearly specifies otherwise, as used in this specification and the appended claims, nouns not limited by quantifiers include plural references. It should also be noted that, unless the context explicitly states otherwise, the term "or" generally carries the meaning of "and / or". The terms "and / or" and "any combination thereof" and their grammatical equivalents are used interchangeably as used herein. These terms can indicate that any combination is explicitly contemplated. For illustrative purposes only, the phrases "A, B and / or C" or "A, B, C or any combination thereof" can mean "A alone; B alone; C alone; A and B; B and C; A and C; and A, B and C". The term "or" can be used in combination or separately unless the context specifically indicates separate use.

[0086] Unless otherwise stated or required by context, the term “about” as used herein means within ±10% variability relative to a reference value.

[0087] The terms “comprising” (and any variations thereof), “having” (and any variations thereof), “including” (and any variations thereof), or “containing” (and any variations thereof) as used in this specification and claims are inclusive or open-ended and do not exclude additional, unrecorded elements or method steps. It is contemplated that any embodiments discussed in this specification can be implemented with respect to any method or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure can be used to implement the methods of this disclosure.

[0088] References to “some embodiments,” “implementation,” “one embodiment,” or “other embodiments” in this specification mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some, but not necessarily all, embodiments of this disclosure. Certain specific details in this specification are set forth to provide a comprehensive understanding of the various embodiments. However, those skilled in the art will understand that this disclosure can be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0089] The term "promoter" refers to a regulatory element that controls the transcription of a gene operatively linked to it, and includes (a) one or more sequences sufficient to direct transcription and / or (b) recognition sites for RNA polymerase and other transcription factors required for transcription. In some embodiments, the promoter is operatively linked to the 5' end of the gene (e.g., operatively linked upstream of the gene). Some promoters can direct cell-specific expression.

[0090] As used herein, the term "operably linked" refers to an arrangement of elements in which the components described herein are configured to perform their usual or intended function. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter directs the transcription of one or more heterologous genes in a cell, then the promoter is operably linked to said one or more heterologous genes. Furthermore, control elements operably linked to a coding sequence can influence (e.g., enhance) the expression of the coding sequence. The control element does not need to be adjacent to the coding sequence, as long as it serves to direct the expression of the coding sequence. Therefore, for example, an inserted untranslated but transcribed sequence may exist between the promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0091] As used herein, the term "vector" refers to a nucleic acid molecule capable of delivering a therapeutic sequence to a target cell, where the therapeutic sequence can then be transcribed, replicated, processed, and / or expressed. One type of vector is a "plasmid," which is a circular double-stranded DNA loop containing a bacterial backbone to which additional DNA segments can be attached. Another type of vector is a bacteriophage vector. Yet another type of vector is a viral vector (e.g., an adeno-associated virus (AAV) vector), where additional DNA segments can be attached to a viral genome. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episome mammalian vectors). Other vectors (e.g., non-epithelial mammalian vectors) can integrate into the host cell's genome after being introduced into the host cell and thus replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are effectively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors" or "expression vectors"). Any nucleic acid vector described herein may be referred to as an "isolated nucleic acid vector."

[0092] As used herein, the term "circular DNA vector" refers to a DNA vector in a circular form. The term "circular DNA vector" may be used in conjunction with the terms "covalently closed and circular DNA vector" and "[other related terms]". "Used interchangeably. Those skilled in the art will understand that such circular vectors comprise covalently closed vectors with supercoiled and complex DNA topology. In some specific embodiments, the circular DNA vector is supercoiled (e.g., monomerically supercoiled). In other embodiments, the circular DNA vector is a relaxed open circular (covalently closed without supercoil). In some cases, the circular DNA vector is non-integrated (e.g., episome circular DNA vectors)."

[0093] As used herein, the term "therapeutic sequence" refers to a portion of a DNA molecule (e.g., a DNA vector) containing genetic material for expressing one or more therapeutic motifs in target cells (e.g., cells in an object, such as a mammalian or human object), which may include one or more protein-coding sequences or one or more RNAs. Other sequences that may be included within the term "therapeutic sequence" include promoters, terminators, introns, and / or other regulatory elements (e.g., S / MAR and / or intron-derived regulatory elements). Therapeutic motifs may be therapeutic proteins (e.g., substitute proteins, such as proteins that replace defective proteins in target cells) and / or therapeutic RNAs (e.g., microRNAs or siRNAs). Other therapeutic sequences are described herein. In circular DNA vectors having more than one transcription unit, the therapeutic sequence contains all transcription units. Therapeutic sequences may include one or more genes (e.g., heterologous genes or transgenes, such as eye genes) administered for therapeutic purposes. In some embodiments, the therapeutic sequence is a mammalian sequence (e.g., a human sequence).

[0094] As used in this article, “exogenous” means any substance introduced from or generated outside an organism, cell, tissue, or system.

[0095] As used herein, the terms “parental plasmid,” “plasmid template,” and “template plasmid” are used interchangeably to refer to a plasmid containing both a vector sequence (as defined below) and a “backbone sequence” (as defined below). The embodiments disclosed herein include a method for preparing a circular DNA vector, said method comprising removing the backbone sequence from a plasmid template.

[0096] As used herein, the term "vector sequence" of a parent plasmid refers to a portion of the plasmid DNA that includes the region of the parent plasmid that will be present in a vector (e.g., a circular DNA vector), namely (i) the origin of replication and (ii) a therapeutic sequence. In some embodiments, the vector sequence does not contain a selection marker (e.g., it does not contain a drug resistance gene). In some descriptions of embodiments herein, the vector sequence is referred to as the "first segment" of the plasmid.

[0097] As used herein, "backbone sequence" refers to the portion of plasmid DNA excluding the therapeutic sequence and origin of replication. The backbone sequence may contain bacterial-derived sequences. The backbone sequence contains selective markers, such as drug resistance genes or fragments thereof, and recombination sites. In several embodiments, a large portion of the backbone sequence is removed from the parental plasmid or plasmid template to produce a circular DNA vector. According to some embodiments, the circular DNA vector will contain a minimal bacterial sequence, such as about 200 bp or less, or about 150 bp or less, or about 100 bp or less, or about 75 bp or less, or about 50 bp or less.

[0098] As used in this article, "replication protein" refers to a protein essential for initiating replication at the replication origin sequence (e.g., where the replication protein binds to the replication origin to initiate replication). If the replication origin depends on the replication protein initiating replication at the replication origin sequence, then a specific replication origin sequence corresponds to a given replication protein. 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 essential for initiating DNA replication at the ColE2-P9 ori sequence.

[0099] As used herein, the term "recombination site" refers to a nucleic acid sequence that is the product of site-specific recombination and contains a first sequence corresponding to a portion of a first recombinase attachment site and a second sequence corresponding to a portion of a second recombinase attachment site. An example of a heterozygous recombination site is attR, which is the product of site-specific recombination and contains a first sequence corresponding to a portion of attP and a second sequence corresponding to a portion of attB. Alternatively, recombination sites can be generated by Cre / Lox recombination. Therefore, vectors generated by Cre / Lox recombination (e.g., vectors containing a LoxP site) contain recombination sites as used herein. Other site-specific recombination events that generate recombination sites involve, for example, λ integrase, FLP recombinase, and Kw recombinase. Nucleic acid sequences generated by non-site-specific recombination events (e.g., ITR-mediated intermolecular recombination) are not recombination sites as defined herein.

[0100] The “functional variants” of nucleic acid sequences used herein differ from a reference nucleic acid sequence (e.g., a naturally occurring nucleic acid sequence) by at least one nucleic acid residue, wherein the relevant functional activity of the variant is at least about 90% of the relevant functional activity level of the reference nucleic acid sequence (e.g., substantially similar in function to the reference nucleic acid sequence). In this context, the difference of at least one nucleic acid residue may be, for example, a mutation of a nucleic acid residue to another nucleic acid residue, a deletion of a nucleic acid residue, or an insertion of a nucleic acid residue. The variant may encode a homolog, isotype, or transcript variant of a therapeutic protein or fragment thereof encoded by the reference nucleic acid sequence, wherein the homolog, isotype, or transcript variant is characterized, respectively, by the degree of identity or homology, as defined herein.

[0101] In some cases, functional variants of polynucleotides or peptides contain at least one nucleic acid or amino acid substitution (e.g., 1 to 100, 1 to 50, 1 to 20, or 1 to 10 substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions). Nucleic acid substitutions that result in the expressed peptide having amino acid exchanges from the same class are referred to herein as conserved substitutions. Specifically, these are amino acids with aliphatic side chains, amino acids with positively or negatively charged side chains, amino acids with aromatic groups in their side chains, or amino acids with side chains capable of forming hydrogen bridges (e.g., side chains with hydroxyl functional groups). Through conservative substitution, for example, an amino acid with a polar side chain is replaced by another amino acid with a polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain can be replaced by another amino acid with a hydrophobic side chain (e.g., serine is replaced by threonine, or leucine is replaced by isoleucine).

[0102] To determine the percentage of identity between two sequences (e.g., nucleic acid sequences or amino acid sequences), the sequences can be aligned for subsequent comparison. For this purpose, gaps can be inserted into the first sequence, and the components at corresponding positions in 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, then the two sequences are identical at that position. The percentage of identity between the two sequences is a function of the number of identical positions divided by the total number of positions. The percentage of identity between the two sequences can be determined using a mathematical algorithm. Preferred but non-limiting examples of mathematical algorithms that can be used are those of 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, BLAST programs. Sequences that are partially identical to the sequences of this invention can be determined using this program.

[0103] As used herein, the term "flanking" and its variations refer to a pair of regions or sites on a nucleic acid molecule (e.g., a plasmid) outside a reference region of the nucleic acid molecule. In some embodiments, the pair of regions or sites flanking the reference region on the nucleic acid are adjacent to (i.e., adjacent to) the reference region (i.e., there are no intercalation bases between the reference site and the flanking site). In other embodiments, the pair of regions or sites flanking the reference region on the nucleic acid molecule are separated from the reference region by one or more intercalation bases (e.g., up to 1,000 intercalation bases). For example, if a first restriction site is 200 bases upstream of a given sequence and a second restriction site is 100 bases downstream of that sequence, the first and second restriction sites are said to be flanking the sequence. In some embodiments, no bacterial sequence is present in any intercalation sequence between the flanking region or flanking site and the reference region. In some such embodiments, no bacterial sequence other than the ori sequence is present in the circular DNA vector resulting from cleaving the vector sequence from the parent plasmid at the restriction site or recombination site flanking the vector sequence by self-ligation or circularization (e.g., by recombination or transposition). For example, in some such embodiments, an exogenous restriction enzyme that cleaves flanking sites of the vector sequence can produce a circular DNA vector with a sequence between the 5' and 3' ends having a therapeutic sequence; however, this region does not contain a bacterial sequence (e.g., a drug resistance gene). Such intercalation sequences can be artificial products derived from sticky end connections (e.g., corresponding to protruding bases produced by the exogenous restriction enzyme).

[0104] As used in this article, “eye gene” refers to a gene that is preferentially, selectively, or exclusively expressed in eye tissues or involved in eye functions such as vision.

[0105] As used in this article, the term "protein" refers to a group of amino acids linked together by peptide bonds (i.e., as the main structure).

[0106] As used herein, the term "therapeutic protein" refers to a protein that can treat a disease or condition in a subject. In some embodiments, a therapeutic protein is a therapeutic alternative protein administered to the subject to replace a defective (e.g., mutated) protein. In some embodiments, the therapeutic protein is identical or functionally similar to a defect-free natural protein in the subject.

[0107] As used herein, the term "therapeutic alternative protein" refers to a protein that is structurally similar (e.g., structurally identical) to a protein endogenously expressed by a normal (e.g., healthy) individual. Therapeutic alternative proteins can be administered to individuals suffering from a condition associated with dysfunction (or deficiency) of the protein to be replaced. In some embodiments, the therapeutic alternative protein corrects a protein defect caused by a mutation in the gene encoding the protein (e.g., a point mutation, insertion mutation, deletion mutation, or splicing variant mutation). Therapeutic alternative proteins do not include non-endogenous proteins, such as pathogen-associated proteins (e.g., as part of a vaccine). Therapeutic alternative proteins may include enzymes, growth factors, hormones, interleukins, interferons, cytokines, anti-apoptotic factors, anti-diabetic factors, coagulation factors, anti-tumor factors, liver-secreting proteins, or neuroprotective factors. In some cases, the therapeutic alternative protein is a single gene.

[0108] Unless otherwise stated, 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., native or synthetic variants), such as mutants, mutant proteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functional variants can be identified based on known ABCA4 signaling. ABCA4 encompasses full-length, unprocessed ABCA4, as well as any form of ABCA4 naturally produced in cells. Exemplary human ABCA4 sequences are provided as NCBI reference sequences: NG_009073 or NM_000350. The human ABCA4 amino acid sequence is provided herein as SEQ ID NO: 61.

[0109] As used herein, the term "ABCA4 intron 6" refers to a natural nucleic acid sequence that begins with a nucleotide immediately following the 3' end of exon 6 of ABCA4 (i.e., downstream) and terminates with a nucleotide immediately following the 5' end of exon 7 of ABCA4 (i.e., upstream). An exemplary sequence of natural human ABCA4 intron 6 is given by SEQ ID NO: 29. The nucleotide numbering of human ABCA4 intron 6 as used herein begins with the first nucleotide of intron 6 according to NG_009073; that is, the first nucleotide of ABCA4 intron 6 corresponds to position 94,564,349 of chromosome 1 (-) according to GRCh37 / hg19. For example, the 3158th nucleotide of ABCA4 intron 6 corresponds to position 94,561,192 of chromosome 1 (-) according to GRCh37 / hg19.

[0110] The terms “regulatory element” and “control element” are used interchangeably herein and each refers to a non-coding nucleic acid region, such as a promoter, enhancer, and silencer, that functions to influence gene expression (e.g., expression level and / or persistence of expression). In some embodiments, regulatory elements are not transcribed into mRNA. In other embodiments, regulatory elements are transcribed into mRNA but not translated into protein. Suitable regulatory elements are described in International Publication No. WO 2021 / 055760, which is incorporated herein by reference in its entirety.

[0111] When a regulatory element contains a functional sequence or a functional variant of a sequence included in a reference sequence (e.g., a functional sequence or a functional variant of a sequence having at least 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 nucleotide bases having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference sequence), the regulatory element is derived from the reference sequence (e.g., a natural intron). The regulatory element derived from the reference sequence does not need to have the same level or type of function as the reference sequence; however, the functional sequence of the regulatory element must confer a detectable function (e.g., increased expression level and / or persistence compared to an expression construct lacking the functional sequence of the regulatory element).

[0112] Unless otherwise stated, 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), as well as functional variants (e.g., natural or synthetic variants), such as mutants, mutant proteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functional variants can be identified based on known MYO7A signaling. MYO7A encompasses full-length, unprocessed MYO7A, as well as any form of MYO7A produced naturally in cells. An exemplary human MYO7A sequence is provided as National Center for Biotechnology Information (NCBI) gene ID: 4647. The human MYO7A amino acid sequence is provided herein as SEQ ID NO: 62.

[0113] As used herein, the terms “scaffold / nuclear matrix attachment region” and “S / MAR” each refer to a DNA sequence of at least 200 nucleotides that mediates the attachment of DNA to the nuclear matrix of a eukaryotic cell, wherein the DNA sequence has at least three sequence motifs ATTA per 100 nucleotides in a segment of up to 200 nucleotides. Exemplary S / MAR sequences are described in Liebich et al., Nucleic Acids Res. 2002, 30:312-374 and International Patent Publication No. WO 2019 / 060253, the respective S / MAR descriptions of which are incorporated herein by reference.

[0114] As used herein, the term "naked" refers to nucleic acid molecules (e.g., circular DNA carriers) that, when administered to an individual, are not encapsulated by a lipid membrane (e.g., liposomes) or a polymer matrix and are not physically associated (e.g., covalently or non-covalently) with a solid structure (e.g., particulate structure). In some aspects of the invention, pharmaceutical compositions comprise naked circular DNA carriers.

[0115] As used herein, the term "isolated" means artificially generated and not naturally integrated into the host genome. For example, isolated nucleic acid vectors include naked nucleic acid vectors, as well as those encapsulated in a lipid membrane (e.g., liposomes) or polymer matrix. In some embodiments, the term "isolated" refers to a DNA vector that is: (i) synthetic, for example, amplified in vitro (e.g., in a cell-free environment), such as by rolling circle amplification or polymerase chain reaction (PCR); (ii) produced by replication in host bacterial cells and recovered in purified or partially purified (e.g., cell-free) form; (iii) purified, such as by restriction endonuclease digestion and gel electrophoresis fractionation, or column chromatography; or (iv) synthesized by, for example, chemical synthesis. Isolated nucleic acid vectors can be readily manipulated using recombinant DNA techniques known in the art. Therefore, nucleotide sequences containing known 5' and 3' restriction enzyme sites or publicly disclosed PCR primer sequences are considered "isolated," but nucleic acid sequences present in the natural host in a natural state are not considered isolated. The isolated nucleic acid vectors can be largely purified, but this is not necessary.

[0116] The terms “individual” and “object” as used herein are used interchangeably and include any mammal requiring treatment or prevention, such as treatment or prevention via the circular DNA vector or its pharmaceutical composition described herein. In some embodiments, the individual or object is a human. In other embodiments, the individual or object is a non-human mammal (e.g., a non-human primate (e.g., a monkey), mouse, pig, rabbit, cat, dog, or horse). The individual or object can be male or female.

[0117] The term "effective amount" or "effective dose" for DNA vectors or pharmaceutical compositions thereof, as used herein, refers to an amount sufficient to achieve the desired biological, pharmacological, or therapeutic effect, for example, when administered to an individual according to a selected form, route, and / or schedule. As will be understood by those skilled in the art, the absolute amount of an effective particular composition can vary depending on factors such as the desired biological or pharmacological endpoint, the agent to be delivered, the target tissue, etc. Those skilled in the art will further understand that an "effective amount" can be administered to a subject through contact with cells or as a single dose or by using multiple doses. An effective dose of a composition used to treat a disease, relative to a control population (e.g., an untreated population or a placebo population, or a population receiving standard care), may slow or halt disease progression, or improve a partial or complete response.

[0118] As used in this article, “therapeutic effective amount” or “therapeutic effective dose” refers to the effective amount of DNA vector used to treat or prevent disease or symptom in subjects.

[0119] As used herein, “treatment” (and its grammatical variations, such as “treat” or “treating”) refers to a clinical intervention that attempts to alter the natural course of a treated individual, which may be performed for prevention or during a clinicopathological state. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or alleviating the disease state, and improving prognosis. In some embodiments, the circular DNA vector of the present invention is used to delay the development of disease or slow its progression.

[0120] The terms “level of expression” or “expression level” are used interchangeably and generally refer to the amount of polynucleotide or amino acid products or proteins in a biological sample (e.g., the retina). “Expression” generally refers to the process by which information encoded by a gene is translated into structures that are present and function in the cell. Therefore, according to the present invention, “expression” can refer to transcription into a polynucleotide, translation into a protein, or post-translational modification of a protein. Fragments of transcribed polynucleotides, translated proteins, or post-translational modified proteins should also be considered expressed, whether they originate from transcripts produced by alternative splicing or degraded transcripts, or from post-translational processing of proteins, such as through proteolysis. “Expressed genes” include those transcribed as mRNA into polynucleotides and subsequently translated into proteins, as well as those transcribed into RNA but not translated into proteins (e.g., miRNA or shRNA).

[0121] As used herein, "target cell" refers to a cell that expresses a therapeutic protein encoded by a therapeutic gene. In some embodiments, the target cell is a retinal cell. For example, in some specific embodiments, the target cell is a retinal pigment epithelial (RPE) cell or a photoreceptor cell. In other embodiments, the target cell is a respiratory system cell (e.g., lung epithelial cells). In many embodiments, the target cell is located in the subject to be treated.

[0122] As used herein, “delivering,” “to deliver,” and their grammatical variations refer to the delivery of a drug (e.g., a DNA vector) into target cells. A drug can be delivered by administration to an individual with target cells (e.g., systemic or local administration), enabling the drug to enter the organ or tissue containing the target cells. Alternatively or complementary, a drug can be delivered by stimulating a tissue or organ containing the drug, wherein the stimulation causes the drug to enter the target cells. Therefore, in some cases, the drug is delivered to target cells by transmitting an electric field into the tissue containing the drug under conditions suitable for electrotransferring the drug to target cells within the tissue.

[0123] As used herein, “electrotransfer” refers to the movement of molecules (e.g., nucleic acid carriers, such as naked nucleic acid carriers) across the membrane of a target cell (e.g., from the outside to the inside of a target cell, such as a retinal cell), induced by an electric field (e.g., a pulsed electric field) transmitted to the cell’s microenvironment (e.g., the retina). Electrotransfer can occur as a result of electrophoresis, i.e., the movement of molecules (e.g., nucleic acids, such as naked nucleic acids) along an electric field based on their charge. Electrophoresis can induce electrotransfer, for example, by moving molecules (e.g., nucleic acids, such as naked nucleic acids) near the cell membrane to allow biological transport processes (e.g., endocytosis, including pinocytosis or phagocytosis) or passive transport (e.g., diffusion or lipid partitioning) to transport molecules into the cell. As a complement or alternative, electrotransfer can occur as a result of electroporation, for example, the creation of pores in a target cell induced by the transmission of an electric field (e.g., a pulsed electric field), wherein the size, shape, and duration of the pores are suitable for accommodating the movement of molecules (e.g., nucleic acid carriers, such as naked nucleic acid carriers) from the outside to the inside of the target cell. Thus, in some cases, electrotransfer occurs as a result of a combination of electrophoresis and electroporation.

[0124] As used herein, “application” means a method of giving an individual or object a dose of the pharmaceutical agent (e.g., a DNA vector) or a combination thereof (e.g., a pharmaceutical composition).

[0125] As used herein, the term “expression persistence” refers to the duration for which a target sequence or a functional portion thereof (e.g., one or more coding sequences of a circular DNA vector) is expressed in its transfected cells (“intracellular persistence”) or in any progeny of its transfected cells (“transgenerational persistence”). A target sequence (e.g., a therapeutic sequence or a functional portion thereof) may be expressible if it is not silenced. Expression persistence can be assessed by detecting or quantifying: (i) mRNA transcribed from the sequence in the target cells or their progeny (e.g., by qPCR, RNA-seq, or any other suitable method) and (ii) protein translated from the sequence in the target cells or their progeny (e.g., by Western blotting, ELISA, or any other suitable method). In some cases, expression persistence is assessed by detecting or quantifying therapeutic DNA in the target cells or their progeny (e.g., the presence of a circular DNA vector in the target cells, e.g., by episome DNA copy number analysis) in combination with (i) mRNA transcribed from the therapeutic sequence in the target cells or their progeny and (ii) protein translated from the therapeutic sequence in the target cells or their progeny, or both. The persistence of expression of the target sequence or its functional portion can be quantified relative to a reference vector (e.g., a control vector having one or more bacterial backbone sequences not present in the vector of the present invention) using any gene expression characterization method known in the art. Expression persistence can be quantified at any given time point after vector administration. For example, in some embodiments, if the expression of the circular DNA vector of the present invention is detectable in target cells or their progeny two weeks after administration, then the expression of the circular DNA vector of the present invention persists for at least two weeks after administration. In some embodiments, gene expression is "persistent" in target cells if gene expression is detectable in target cells 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 longer after administration. In some embodiments, the expression of the therapeutic sequence persists in target cells (e.g., retinal cells) for at least four weeks, at least six weeks, or at least two months, or at least four months, or at least six months, or at least eight months, or at least one year. In some implementations, the expression of the therapeutic sequence persists in target cells (e.g., retinal cells) for more than a year, such as 18 months or longer, or two years or longer.

[0126] As used herein, "intracellular persistence" refers to the duration for which a sequence or its functional portion (e.g., one or more coding sequences of a circular DNA vector) can be expressed in its transfected cells (e.g., target cells, such as post-mitotic or resting cells). Intracellular persistence can be assessed by detecting or quantifying (i) mRNA transcribed from the sequence in the target cells and (ii) protein translated from the sequence in the target cells. In some cases, intracellular persistence is assessed by detecting or quantifying one or both of (i) mRNA transcribed from the sequence in the target cells and (ii) protein translated from the sequence in the target cells in combination with DNA in the target cells (e.g., the presence of a circular DNA vector in the target cells). 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).

[0127] As used herein, “transgenerational persistence” refers to the duration for which a sequence or a functional portion thereof (e.g., one or more coding sequences of a DNA vector) can be expressed in the gene-transfected cell progeny (e.g., the progeny of target cells, such as the first, second, third, or fourth generation progeny of the gene-transfected cells, for example, via a circular DNA vector). Transgenerational persistence accounts for any dilution of the gene during cell division and can therefore be used to measure the persistence of the vector in dividing tissue over time. In some embodiments, the circular DNA vector of the present invention exhibits 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 the vector sequence in the target cell progeny and (ii) protein translated from the vector sequence in the target cell progeny. In some cases, intracellular persistence is assessed by detecting or quantifying DNA in the target cell progeny (e.g., the presence of a circular DNA vector in the target cell progeny) in combination with one or both of (i) mRNA transcribed from the sequence in the target cell progeny and (ii) protein translated from the sequence in the target cell progeny. In some embodiments, the circular DNA vector of the present invention exhibits improved cross-generational persistence relative to a reference vector (e.g., a plasmid DNA vector).

[0128] The term “copy number” for DNA molecules, as used in this article, refers to the average number of copies of DNA molecules per cell in a given population of cells.

[0129] The term "medicinal" means that it is safe for use on mammals such as humans. In some embodiments, a pharmaceutically acceptable composition is one that has been approved by a federal or state regulatory agency or is listed in the United States Pharmacopeia or other recognized pharmacopoeia for use on animals and more specifically for use on humans.

[0130] The term "carrier" refers to a diluent, excipient, excipient, or carrier that is applied together with the carrier or composition of the present invention. Examples of suitable drug carriers are described in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA., 23rd edition, 2020.

[0131] In the event of any conflict of definitions among multiple sources or references, the definitions provided in this document shall prevail.

[0132] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although those similar to or equivalent to the methods and materials described herein may be used in practice or testing of this disclosure, suitable methods and materials are described below.

[0133] II. Generation of Circular DNA Vectors

[0134] The embodiments disclosed herein include a method for generating a circular DNA vector in engineered bacterial cells (e.g., engineered *E. coli* cells). The engineered bacterial cells disclosed herein can be used to generate a circular DNA vector from a parent plasmid. In some embodiments, the engineered bacterial cell comprises a Rep gene (e.g., directing replication from the ColE2-P9 origin) integrated into the bacterial genome and a parent plasmid. In some embodiments, the Rep gene is contained on an extrachromosomal DNA molecule such as a plasmid (e.g., a helper plasmid) or a bacterial artificial chromosome (“BAC”). In some embodiments, the Rep gene is contained on the parent plasmid. The parent plasmid comprises a vector sequence and a backbone sequence. The vector sequence contains a replication origin (ori) sequence corresponding to the Rep gene and does not include a selection marker. The backbone sequence contains a gene encoding a selection marker and does not contain the ori sequence contained in the vector sequence, but in some embodiments, it may contain a different ori sequence. The parent plasmid also possesses enzyme recognition sequences (e.g., restriction enzyme recognition sequences or transposase recognition sequences (e.g., transposase overhang sequences)) or site-specific recombination sequences flanking the vector sequence, arranged to allow the plasmid backbone sequence to be separated from the vector sequence within the cell via restriction digestion, transposition, or site-specific recombination. In the case of restriction digestion, a circular DNA vector is formed through self-ligation of the vector sequence. In the case of site-specific recombination or transposition, a circular DNA vector is formed when recombination or transposition is complete. Following vector sequence separation and circular DNA vector formation, Rep protein expression can maintain the circular DNA vector and keep it at a high copy number, despite the lack of selectivity markers. Conversely, by altering culture conditions to remove the selective pressure on selectivity markers, the maintenance of the plasmid backbone sequence in engineered bacterial cells after separation can be avoided. Cultures of bacterial cell populations carrying circular DNA vectors (which can have high copy numbers) can efficiently produce high-yield, high-purity circular DNA vectors without maintaining the parent plasmid.

[0135] A. Engineered bacterial cells

[0136] The methods for generating circular DNA vectors disclosed herein include using engineered bacterial cells, which may include, for example, engineered *Escherichia coli* cells or other suitable bacterial host cells. In some embodiments, the engineered bacterial cells contain a foreign *Rep* gene encoding a replication protein integrated into the bacterial genome and a parental 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 (e.g., a plasmid or BAC).

[0137] In some embodiments, the engineered bacterial cell has a foreign Rep gene integrated into the bacterial genome. Any suitable chromosome integration process (including integration cassettes and procedures known in the art) can be used to incorporate the Rep gene into the bacterial genome. In the case of using a short origin of replication (e.g., the ColE2-P9 origin of replication, or a functional variant thereof) in a circular DNA vector, the Rep gene may encode a ColE2-P9 replication protein or an associated protein. In some exemplary embodiments, the Rep gene encodes a ColE2-P9 replication protein having the amino acid sequence shown in SEQ ID NO: 1 (or a functional variant thereof, e.g., having at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity thereto). Other suitable replication proteins include replication proteins encoded by naturally occurring plasmids, including, for example, those associated with ColE2-P9, such as ColE3-CA38. The replication protein may be bound to its corresponding origin of replication sequence for use in the embodiments described herein.

[0138] The ori sequence included in the parent plasmid vector sequence is selected such that it corresponds to the Rep gene integrated into the genome of the engineered bacterial cell or otherwise present in the engineered bacterial cell (e.g., on a plasmid or BAC). In some exemplary embodiments, ori (e.g., one strand) comprises or is composed of the nucleotide sequence shown in SEQ ID NO: 2. Therefore, the engineered bacterial cell embodiments disclosed herein include functional pairs 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 the ColE2-P9 ori sequence or a functional variant thereof. In some embodiments, the ori sequence present in the vector sequence is a functional fragment of the ColE2-P9 ori sequence having the DNA sequence shown in SEQ ID NO: 2 (on one strand). The 40-base-pair functional fragment shown 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 their functional fragments, such as the ori sequence of ColE3-CA38. In some embodiments, ori is the ColE2-P9 origin and is no more than about 40 nucleotides, or no more than 38 nucleotides, no more than 37 nucleotides, or no more than 36 nucleotides, or no more than 34 nucleotides, or no more than 30 nucleotides in length. In many embodiments, the length of the ColE2-P9 origin is 30 to 40 nucleotides, or 35 to 40 nucleotides, or 36 to 40 nucleotides, thereby minimizing the bacterial-derived sequence in the circular vector. In some embodiments, the ori sequence is a naturally occurring ori sequence.

[0139] In some cases, the ori sequence is a functional variant of the naturally occurring ori, such as an ori sequence that has been modified to be shorter than the corresponding naturally occurring ori sequence while still retaining the ability to support replication initiation. Example 13 describes a study in which a functional truncated variant of ColE2-P9 was developed and characterized. Such functional variants of the ColE2-P9 replication origin include SEQ ID NOs: 3, 4, and 12 through 18. For convenience, such sequences are shown herein as single-stranded, but it should be understood that the origin will exist as double-stranded DNA in the vector. In some embodiments, the functional variant has 1, 2, 3, 4, or 5 nucleotide substitutions relative to the origin sequences of SEQ ID NOs: 3, 4, and 12 through 18.

[0140] In some cases, the Rep gene is operatively linked to an inducible promoter or a constitutive (e.g., non-inducible) promoter. Suitable inducible promoters include, but are not limited to, P promoters induced by T7 RNA polymerase. T7 Promoter, thermally induced P L Promoter, P that can be suppressed by Lacl tac The promoter (and therefore can be induced by the absence or removal of Lacl), or P that can be induced by arabinose bad Promoters. Other inducible promoters known in the art may also be used in the embodiments disclosed herein, including, for example, 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 according to this disclosure include, but are not limited to, positively regulated Escherichia coli promoters, such as positively regulated s1con, T3, T7, SP6, and bacterial promoters (e.g., PmgrB, pLlacO, Ptrc2, pLtetO, Plac / ara, Pm). 70 Promoters (e.g., inducible pBad / araC promoter, Lux box right promoter, plac Or2-62 (positive), pBad / AraC with additional REN sites, pBad, P(Las)TetO, P(Las)CIO, P(Rhl), Pu, FecA, pRE, cadC, hns, pLas, pLux), σ S Promoters (e.g., Psps), σ 32 Promoters (e.g., heat shock) and σ 54 Promoters (e.g., glnAp2); negatively regulated E. coli promoters, such as negatively regulated σ 70Promoters (e.g., PRM+, TetR-TetR-4C, P(Las)TetO, P(Las)CIO, P(Lac)IQ, RecA_DlexO_dLacO1, dapAp, FecA, Pspac-hy, pcl, plux-cl, plux-lac, CinR, CinL, glucose-controlled, modified Pr, modified Prm+, FecA, Pcya, rec A(SOS), Rec A (SOS), EmrR_regulatory, Betl_regulatory, pLac_lux, pTet_Lac, pLac / Mnt, pTet / Mnt, LsrA / cl, pLux / cl, LacI, LacIQ, pLacIQ1, pLas / cl, pLas / Lux, pLux / Las, pRecA with LexA binding site, reverse bBa_R0011, pLacl / ara-1, pLaclq, rrnB P1, cadC, hns, PfhuA, pBad / araC, nhaA, OmpF, RcnR), σ S Promoters (e.g., those with alternative σ factors) 38 Lutz-Bujard LacO), σ 32 Promoters (e.g., those with alternative σ factors) 32 Lutz-Bujard LacO), and σ 54 Promoters (e.g., glnAp2); negatively regulated Bacillus subtilis promoters, such as repressible Bacillus subtilis σ promoters. A Promoters (e.g., Gram-positive IPTG-inducible, Xyl, hyper-spank) and σ B Promoters. Other inducible bacterial promoters may be used according to this disclosure. For example, cumuloic acid-inducible promoters, such as pCymRC, may be used.

[0141] The expression level of the replication protein can affect the copy number of the parent plasmid or circular DNA vector containing the corresponding ori sequence. Therefore, when a relatively low copy number is desired (e.g., less than 5, 10, or 20 copies per cell on average), engineered bacterial cells can be maintained under conditions where the replication protein is expressed at a relatively low level. When a relatively high copy number is desired (e.g., more than 20, 50, or 100 copies per cell on average), engineered bacterial cells can be maintained under conditions where the replication protein is expressed at a relatively high level from a promoter (which may be an inducible promoter). In some embodiments, the Rep gene is operatively linked to an inducible promoter that provides a first level of expression under non-inducible conditions and can be induced to provide a second, higher level of expression, resulting in a higher copy number of the parent plasmid or circular DNA vector containing the corresponding ori sequence. In some embodiments, it is advantageous to maintain the parent plasmid at a low copy number in the engineered bacterial cells before isolating the vector and backbone sequences of the parent plasmid. In embodiments where the vector and backbone sequences are separated by restriction digestion, a relatively low copy number helps ensure that the linearized vector sequence self-ligates, rather than ligating to the backbone sequence or other copies of the vector sequence. After the vector and backbone sequences are separated and a circular DNA vector is formed, in some embodiments, it is advantageous to maintain the circular DNA vector containing the ori sequence at a relatively high copy number to produce a high yield of circular DNA vectors. Therefore, in some embodiments, after the circular DNA vector is formed in engineered bacterial cells, a higher expression level of the replication protein is induced, for example, by adding a molecule that induces higher expression of an inducible promoter operatively linked to the Rep gene. In some embodiments, the inducible promoter remains uninduced until after the vector and backbone sequences are separated. In some embodiments, the inducible promoter is induced after the vector and backbone sequences are separated. In some embodiments, the inducible promoter is induced simultaneously with the separation of the vector and backbone sequences. In some embodiments, the inducible promoter is induced before the vector and backbone sequences are separated.

[0142] The copy number of the parental plasmid may be maintained at at least about 5, or at least about 10, or at least about 20, or at least about 50 copies. In several embodiments, the parental plasmid is maintained at a copy number not exceeding about 400, or about 200, or about 100, or about 50 copies per cell. In some embodiments, the copy number of the circular DNA vector is maintained at or generated at a copy number of at least about 10, or at least about 20, or at least about 50, or at least about 75, or at least about 100, or at least about 150, or at least about 200 copies per cell.

[0143] In addition to parental plasmids or vector sequences separated from parental plasmids, engineered bacteria may also include other extrachromosomal DNA molecules, such as helper plasmids or BACs. Extrachromosomal DNA molecules may encode one or more of the following: exogenous recombinases, restriction enzymes, transposases, insertion sequence excision enhancers (IEEs), replication proteins, ligases, selection markers, anti-selection markers, or reporter genes. In some cases, transposases and IEEs encode the same or different extrachromosomal DNA molecules in the engineered bacterial cell.

[0144] In some cases, IEE corresponds to IS3 family insertion sequences, such as IS629 or IS2.

[0145] In some embodiments, extrachromosomal DNA molecules, other than the vector sequence, are removed from the engineered bacterial cells before purifying the circular DNA vector from the culture. In some embodiments, the extrachromosomal DNA molecules can be removed from the engineered bacterial cells by culturing the cells under conditions without applying selective pressure to maintain the extrachromosomal DNA molecules, or by culturing the cells under anti-selective conditions that reduce or eliminate the growth of cells containing extrachromosomal DNA molecules.

[0146] In some embodiments, the extrachromosomal DNA molecule contained in the engineered bacterial cell includes a reporter construct that can be used to track the presence of the extrachromosomal DNA molecule in the cell. For example, the backbone sequence of the parental plasmid or helper plasmid or BAC may contain a gene encoding a visually detectable protein (e.g., GFP or RFP). In this case, visual observation of colony color under UV light can reveal the presence of the extrachromosomal DNA molecule in the colony's cells. In this way, colonies 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 an engineered bacterial cell or colony contains a given extrachromosomal DNA molecule.

[0147] In some cases of engineered bacterial cells that use transposases to generate circular DNA vectors (e.g., non-integrative circular DNA vectors or episome circular DNA vectors), the engineered bacterial cells contain (a) a circular DNA vector with transposase overhangs, for example, wherein the circular DNA vector contains any one or more components of the therapeutic sequence described herein and any bacterial origin of replication described herein (e.g., ColE2-P9 origin of replication or a functional variant thereof), and (b) a Rep gene encoding a bacterial replication protein that binds to the bacterial origin of replication of the circular DNA vector (e.g., wherein the Rep gene is encoded in the genome of the engineered bacterial cell).

[0148] In embodiments of engineered bacterial cells that use transposases to generate circular DNA vectors (e.g., non-integrative circular DNA vectors or episome circular DNA vectors), the engineered bacterial cells comprise (a) a plasmid template for generating the circular DNA vector and (b) a Rep gene encoding a bacterial origin of replication protein. The plasmid template comprises: (i) a first segment containing a therapeutic sequence and a sequence containing any bacterial origin of replication described herein (e.g., the ColE2-P9 origin of replication or a functional variant thereof), wherein the first segment is flanked by two transposase overhang sequences; and (ii) a second segment containing a plasmid backbone, wherein the second segment is flanked by left-hand (LE) repeats and right-hand (RE) repeats, wherein the LE repeats and RE repeats are bindable to the transposase protein. In some embodiments, the bacterial replication protein binds to the bacterial origin of replication of the circular DNA vector (e.g., the Rep gene is encoded in the genome of the engineered bacterial cell). In some implementations, the engineered bacterial cell also includes (c) a circular DNA vector containing: a therapeutic sequence, a sequence containing a bacterial origin of replication, and one of two transposase overhang sequences.

[0149] The aforementioned engineered bacterial cells may also contain a transposase protein that generates a transposase overhang within a circular DNA vector. For example, the transposase protein may be the piggyBac transposase, in which case the transposase overhang may be TTAA (5'-TTAA). Alternatively, other known transposase-overhang pairs known in the art may be used in the engineered bacterial cells described herein. The transposase protein may be expressed by a non-integrating gene within the bacterial cell (e.g., a helper plasmid or BAC), or it may be integrated into the genome of the engineered bacterial cell. In either case, some embodiments of such engineered bacterial cells include an IEE, which may also be expressed by a non-integrating gene within the bacterial cell (e.g., a helper plasmid or BAC), or it may be integrated into the genome of the engineered bacterial cell. In some cases, the transposase and the IEE are encoded on the same helper plasmid or BAC within the engineered bacterial cell, and the helper plasmid or BAC contains an origin of replication (e.g., an origin of replication different from the origin of replication on the circular DNA vector). Such engineered bacterial cells that use transposases to generate circular DNA vectors may also contain other structures, such as byproducts of the transposition process. Such byproducts include terminally closed linear DNA molecules of plasmid backbone sequences (e.g., plasmid backbones present in leader template plasmids that produce circular DNA vectors). As products of transposition, such terminally closed linear DNA molecules may contain terminal hairpins with transposase overhang sequences, or left- and right-terminal (LE) repeats associated with (e.g., transposase-bound) transposase proteins in engineered bacterial cells. Such engineered bacterial cells are adapted to produce circular DNA vectors with small backbones (e.g., non-integrative circular DNA vectors, or episome circular DNA vectors), wherein a majority (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the sequence directly linking the 3' end of the therapeutic sequence to the 5' end of the therapeutic sequence is the origin of replication, which may be less than 50 bp (e.g., having a single nucleic acid sequence comprising or composed of any of SEQ ID NO: 2 to 4 or 12 to 18). In some cases, this feature can be achieved by superimposing a transposase overhang sequence onto a vector sequence (e.g., a therapeutic sequence or origin of replication), thereby effectively removing it from the backbone sequence. In such cases, the therapeutic sequence or origin of replication of the circular DNA vector contains a transposase overhang sequence associated with a transposase within the engineered bacterial cell.

[0150] Any engineered bacterial cells described herein may be isolated engineered bacterial cells (e.g., cultured engineered bacterial cells).

[0151] B. Affine granules

[0152] As part of the generation of any of the circular DNA vectors described herein, a host cell (e.g., engineered bacteria) may contain a parent plasmid (also called a template plasmid or plasmid template) containing a vector sequence and a backbone sequence separated from each other by two restriction sites (in the case of host cell expression of homologous restriction enzymes), a recombination site (in the case of host cell expression of homologous recombinases), or a transposase overhang (in the case of host cell expression of homologous transposases).

[0153] The vector sequence comprises an ori sequence and a target sequence, which in some embodiments is a therapeutic sequence. In some embodiments, the vector sequence comprises a reporter construct (e.g., a fluorescent protein-coding gene, such as GFP or RFP). The vector sequence may comprise any component of the circular DNA vector embodiments described herein. In some embodiments, the vector sequence does not contain any bacterial-derived sequence other than the ori sequence. In some embodiments, the parent plasmid contains restriction sites, recombination sites, or transposase sites immediately adjacent to the ori sequence and / or the therapeutic sequence or reporter construct, such that the vector sequence becoming a circular DNA vector contains little or no exogenous or nonfunctional DNA. In some embodiments, adjacent sequences are no more than about 50 bp, or no more than about 45 bp, or no more than about 40 bp.

[0154] In some embodiments, the backbone sequence contains a selection marker and does not contain an ori sequence corresponding to a foreign replication protein encoded by a foreign (e.g., integrated) Rep gene. In some embodiments, the backbone sequence contains an ori sequence that does not correspond to the integrated Rep gene, i.e., the sequence is orthologous to both the ori sequence in the vector sequence and the integrated Rep gene. In some embodiments, the selection marker contained in the backbone sequence helps ensure that the parental plasmid is maintained in an engineered bacterial cell population cultured under conditions where the selection marker is essential for cell growth or survival. For example, in some embodiments, the selection marker is an antibiotic resistance gene. Culturing engineered bacterial cells in the presence of the corresponding antibiotic applies selective pressure, causing the parental plasmid to be maintained in the engineered bacterial cell population. However, after the selective pressure is removed, for example by changing the growth medium to one lacking an antibiotic corresponding to the antibiotic resistance gene, the DNA molecule containing the antibiotic resistance gene may be lost from the population, especially if such a DNA molecule does not contain an ori sequence. Therefore, after the vector sequence separates from the backbone sequence, if the culture conditions do not apply selective pressure to maintain the backbone sequence, it may be lost from the population or fail to be maintained in large quantities.

[0155] In some embodiments, the backbone sequence includes an anti-selection marker. The anti-selection marker can provide a method for selectively culturing cells that do not contain the backbone sequence. In some embodiments, culturing cells under anti-selection conditions after the vector sequence has been separated from the backbone sequence can improve purity and reduce the amount of backbone sequence in the culture and / or in compositions containing the purified vector sequence. Suitable anti-selection markers are known in the art and may include, for example, pheS, sacB, thyA, lacY, gata-1, ccdB, rpsL, or tetAR.

[0156] The restriction sites, recombination sites, or transposase recognition sites located on the flanking side of the vector sequence in the parent plasmid can be selected from any suitable restriction sites, recombination sites, or transposase recognition sites that are not present in the vector sequence.

[0157] C. Restrictive Digestion & Connectivity

[0158] In some embodiments of bacterial production of circular DNA vectors, a restriction digestion step separates the vector sequence from the backbone sequence of the parent plasmid. In some embodiments, restriction digestion occurs within the engineered bacterial cell. In some embodiments, the restriction enzyme digesting 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 within the engineered bacterial cell. In some embodiments, it is necessary to suppress or delay the induction of restriction enzyme expression until it is desired to separate the vector sequence from the backbone sequence of the parent plasmid. Therefore, in some embodiments, the exogenous gene is operatively linked to an inducible promoter. When separation is desired, restriction enzyme expression can be induced, and separation can then proceed.

[0159] In some embodiments utilizing restriction enzymes, the restriction enzymes used to separate the vector sequence from the backbone sequence are exogenous restriction enzymes introduced into the engineered bacterial cells across the cell membrane. In some embodiments, this is achieved via electroporation. A non-limiting example of an electroporation and digestion procedure is as follows: Electrocompetent engineered *E. coli* carrying a parental plasmid are cultured in SOB at 30°C until an OD of 0.8 is reached. The bacteria are washed three times with ice-cold 10% glycerol and resuspended in 10% glycerol. 0.5 μl of each restriction enzyme and ligase is mixed with the electrocompetent cells, and 1 μg of DNA is digested with 10 units of the restriction enzyme. The mixture is transferred to cuvettes (1 mm slits) and electroporated using an electroporator (BTX) at 1800 volts. Cells are rescued by culturing in SOC at 37°C for 1 hour, and the cell plates are seeded onto antibiotic-free LB agar plates. Colonies are cultured and the DNA is purified using a QIAGEN miniprep kit.

[0160] After the vector sequence is isolated, a circular DNA vector can be formed by self-ligating the vector sequence. In some embodiments, ligation occurs within the engineered bacterial cell. In some embodiments, the ligase that ligates the ends of the 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 within the engineered bacterial cell. In some embodiments, ligase expression is suppressed or not induced until the vector sequence is completely separated from the backbone sequence of the parent plasmid. Therefore, in some embodiments, the exogenous ligase gene is operatively ligated to an inducible promoter.

[0161] In some cases, the ligase is an exogenous ligase introduced into the engineered bacterial cell across the cell membrane. In some embodiments, this is accomplished by electroporation, which may be performed according to the electroporation protocol described above. In some embodiments, the electroporation of the restriction enzyme and the ligase is completed in a single step, wherein both 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.

[0162] In some implementations, the self-ligation of the vector sequence is accomplished by an endogenous ligase produced by engineered bacterial cells.

[0163] In some embodiments, the exogenous restriction enzyme and the exogenous ligase are co-present in the engineered bacterial cell. In some embodiments, the exogenous restriction enzyme is introduced into the engineered bacterial cell (e.g., by electroporation of the exogenous restriction enzyme, by transformation with a DNA molecule encoding the exogenous restriction enzyme, or by inducing expression of the exogenous restriction enzyme gene under the control of an inducible promoter), and then the exogenous ligase is introduced into the engineered bacterial cell (e.g., by electroporation of the exogenous ligase, by transformation with a DNA molecule encoding the exogenous ligase, or by inducing expression of the exogenous ligase gene under the control of an inducible promoter). In some embodiments, the exogenous restriction enzyme is introduced into the engineered bacterial cell, and then the exogenous ligase is introduced into the engineered bacterial cell. In some embodiments, the exogenous restriction enzyme and the exogenous ligase are introduced into the engineered bacterial cell simultaneously.

[0164] D. Site-specific recombination

[0165] As an alternative to the aforementioned restriction digestion, site-specific recombination can be used to achieve bacterial production of circular DNA vectors with small backbones, which can be performed using a variety of systems that induce site-specific recombination between sequences. In some embodiments, site-specific recombination involves two specific sequences that are capable of recombinating with each other in the presence of a recombinase.

[0166] In some embodiments, the recombinase that separates the vector sequence from the plasmid sequence is an exogenous recombinase expressed by an exogenous recombinase gene introduced into the engineered bacterial cell. In some embodiments, the exogenous recombinase gene is integrated into the genome of the engineered bacterial cell. In some embodiments, the exogenous recombinase gene is encoded on a plasmid or BAC within the engineered bacterial cell. In some embodiments, it is necessary to suppress or delay the induction of recombinase expression until it is desired to separate the vector sequence from the backbone sequence of the parent plasmid. Therefore, in some embodiments, the exogenous recombinase gene is operatively linked to an inducible promoter, such as any of the inducible promoters disclosed herein. When separation is desired, the expression of the exogenous recombinase can be induced, and separation can be performed. 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 undergo recombination without inducing recombinase expression. In some embodiments, the recombinase is expressed at a relatively low level when the parent plasmid is introduced into the engineered bacterial cell. For example, in some embodiments, the engineered bacterial cells may contain a foreign recombinase gene (e.g., which may be contained on a plasmid or BAC present within the bacterial cell or integrated into the bacterial chromosome prior to the introduction of the parent plasmid into the engineered bacterial cells), operatively linked to an inducible promoter that provides low-level expression under non-inducible conditions. Introducing the parent plasmid into engineered bacterial cells with appropriately low levels of foreign recombinase expression allows the colonies to grow on media selected for selective markers on the backbone sequence of the parent plasmid, while also inducing sufficient recombination of the parent plasmid to generate a cell population within the colony with a vector sequence separable from the backbone sequence.

[0167] In some embodiments, the recombinase used to separate the vector sequence from the backbone sequence is an exogenous recombinase introduced into the engineered bacterial cells across the cell membrane. In some embodiments, this is accomplished by electroporation. A non-limiting example of an electroporation and recombination procedure is as follows: Electrocompetent engineered *E. coli* carrying a parental plasmid are cultured in SOB at 30°C until an OD of 0.8 is reached. The bacteria are washed three times with ice-cold 10% glycerol and resuspended in 10% glycerol. 1 μl of Cre (15 units, NEB, MO298M) is mixed with 50 μl of electrocompetent cells. The mixture is transferred to a cuvette (1 mm slit) and electroporated using an electroporator (BTX) at a setting of 1800 volts. Cells are rescued by culturing in SOC at 37°C for 1 hour, and the cell plates are seeded onto antibiotic-free LB agar plates. Colonies are cultured and DNA is purified using a QIAGEN miniprep kit. 1 μg of DNA is digested with 10 units of restriction enzyme.

[0168] The specific recombinant systems used in the embodiments disclosed herein may have different sources. Specifically, the specific sequences and recombinases used may belong to different structural classes, such as the integrase family of phage λ or the dissociation family of transposon Tn3.

[0169] Recombinases belonging to the phage λ integrase family include, for example, phage λ integrases (Landy et al., Science 197: 1147, 1977), P22 and φ80 integrases (Leong et al., J. Bid. Chem. 260:4468, 1985), Haemophilus influenzae HP1 (Hauser et al., J. Biol. Chem. 267 6859, 1992), phage P1 Cre integrase (which recognizes the LoxP site and induces recombination at that site), plasmid pSAM2 integrase (EP 350, 341), or optionally 2μ plasmid FLP recombinase. In embodiments of preparing circular DNA vectors via recombination using site-specific systems of the phage λ integrase family, the resulting circular DNA vector typically contains a sequence generated by recombination between two att attachment sequences of the corresponding phage or plasmid.

[0170] Recombinases belonging to the Tn3 transposon family include, for example, dissociation enzymes of transposon Tn3 or transposon Tn21 and Tn522 (Stark et al., Trends Genet, 8, 432-439, 1992); Gin convertase of bacteriophage mu; or optionally, dissociation enzymes of geoparticles, such as dissociation enzymes of the par fragment of RP4 (Albert et al., Mol. Microbiol. 12: 131, 1994). In embodiments of preparing circular DNA vectors via recombination using site-specific systems of the Tn3 transposon family, the resulting circular DNA vector typically contains a sequence generated by recombination between two recognition sequences of the dissociation enzyme of said transposon.

[0171] In some embodiments, the site-specific recombinant sequences on the parent plasmid are derived from bacteriophages. In some embodiments, the sequences are attachment sequences of bacteriophage integrase (attP and attB sequences) or sequences derived from such attachment sequences. These sequences are capable of specifically recombining with each other in the presence or absence of a recombinase called an integrase. The term "sequences derived from such attachment sequences" includes sequences obtained by modifying the attachment sequences of a bacteriophage, which retain the ability to specifically recombine in the presence of a suitable recombinase. Thus, such sequences can be shortened fragments of these sequences, or optionally extended fragments by adding other sequences (restriction sites, etc.). For example, they can also be variants obtained by mutation, particularly by point mutation, such as attP-GA and attB-GA attachment sequences.

[0172] In some implementations, the recognition sequence and recombinase used are derived from members of the tyrosine recombinase family, such as Flp, XerC, XerD, λ integrase, or HP1 integrase, or members of the serine recombinase family, such as φBT1, TP901, Bxb1, MR11, A118, φK38, φC31, or Wβ.

[0173] In some implementations, the recognition sequence and recombinase are derived from Bxb1 (e.g., the exogenous recombinase is Bxb1 and the recognition sequence is attP-GA and attB-GA).

[0174] E. Transposition

[0175] As an alternative to restriction digestion or site-specific recombination for separating the vector sequence from the parent plasmid backbone sequence, the enzyme used can be a transposase. One advantage of using a transposase-based system is the ability to further reduce the backbone size in circular DNA vectors. For example, site-specific recombinases can be used to generate recombination sites (e.g., attachment sites) within the vector, near or adjacent to the origin of replication. In contrast, using transposases allows the origin of replication to directly link the 5' end of the therapeutic sequence to the 3' end of the therapeutic sequence without the need for intercalation sequences.

[0176] In some cases, the use of transposases allows for the formation of a “trace-free” backbone by placing the transposable sequence (the transposase overhang) within a therapeutic sequence without altering its function. For example, the piggybac transposase produces a 4 bp transposase overhang for TTAA. By placing the plasmid backbone within the target sequence at the TTAA site, systems can be designed such that after transposase-mediated excision of the plasmid backbone from the target sequence, the original target sequence is restored, leaving only the original TTAA sequence as a transposase trace. This results in a transposase-free backbone in circular DNA vectors. Therefore, the plasmid backbone sequence in the vector can consist entirely of the origin of replication (e.g., less than 50 base pairs, e.g., the size of the origin of replication, including truncated functional variants described herein, e.g., in Example 13).

[0177] As a supplement or alternative, transposase traces may be located within the vector backbone (e.g., within a sequence containing the origin of replication). For example, if the parental plasmid contains inverted (left-handed) and (right-handed) repeats flanking the backbone, and / or transposase overhang sequences flanking the therapeutic sequence (similar to their presence in...). Figure 22 If the transposase trace is located between the 3' and 5' ends of the target sequence (e.g., immediately adjacent to the origin of replication), then the transposase trace will be located between the 3' and 5' ends of the target sequence. Figure 22 As shown in the image.

[0178] F. Amplification of circular DNA vectors through cell culture

[0179] After generating a circular DNA vector in engineered bacterial cells according to the embodiments described herein, the amount of the generated circular DNA vector can be increased by culturing a population of engineered bacterial cells containing the circular DNA vector. Culture conditions can be selected to maximize bacterial cell growth and the production of additional copies of the circular DNA vector. In some embodiments, culture conditions are selected to induce high levels of expression of replication proteins, thereby supporting high copy numbers of circular DNA vectors with the corresponding ori sequences. In some embodiments, culture conditions are selected to remove selection pressure on the backbone sequence containing selection markers, such that the backbone sequence is not maintained through multiple rounds of cell division. In some embodiments, culture conditions are selected to provide anti-selection pressure on anti-selection markers present on the backbone sequence, such that the growth potential of cells containing that backbone sequence is reduced or prevented from growing.

[0180] In some embodiments, culturing a population of engineered bacterial cells containing a circular DNA vector results in the circular DNA vector being maintained in the cultured cells for at least 5, 10, 15, 20, 25, 50, 75, or 100 rounds of cell division. In some embodiments, the cultured population of engineered bacterial cells maintains an average copy number of the circular DNA vector at at least 5, 10, 15, or 20 copies per cell after at least 10, 20, 25, 50, 75, or 100 doublings. In exemplary embodiments, there is at least 1 copy per cell after at least 10 doublings, or at least 5 copies per cell, or at least 10 copies per cell, or at least 20 copies per cell after at least 10 doublings. In some embodiments, each cell has at least 1 copy after at least 20 multiplications, or at least 5 copies, or at least 10 copies, or at least 20 copies after at least 20 multiplications. In some embodiments, each cell has at least 1 copy after at least 50 multiplications, or at least 5 copies, or at least 10 copies, or at least 20 copies after at least 50 multiplications. In some embodiments, each cell has at least 1 copy after at least 100 multiplications, or at least 5 copies, or at least 10 copies, or at least 20 copies after at least 100 multiplications. In some implementations, after the vector sequence is separated 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 per cell, or is undetectable after 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 multiplications. For example, in some implementations, each cell has less than 0.001 copies after at least one doubling, or less than 0.001 copies after at least 10 doublings, or less than 0.001 copies after at least 20 doublings, or less than 0.001 copies after at least 50 doublings, or less than 0.001 copies after at least 100 doublings.In some embodiments, the number of copies per cell is less than 0.01 after at least one doubling, or less than 0.01 copies per cell after at least 10 doublings, or less than 0.01 copies per cell after at least 20 doublings, or less than 0.01 copies per cell after at least 50 doublings, or less than 0.01 copies per cell after at least 100 doublings. In some embodiments, the number of copies per cell is less than 0.1 after at least one doubling, or less than 0.1 copies per cell after at least 10 doublings, or less than 0.1 copies per cell after at least 20 doublings, or less than 0.1 copies per cell after at least 50 doublings, or less than 0.1 copies per cell after at least 100 doublings. In some implementations, each cell contains less than 1 copy after at least 1 doubling, or less than 1 copy after at least 10 doublings, or less than 1 copy after at least 20 doublings, or less than 1 copy after at least 50 doublings, or less than 1 copy after at least 100 doublings.

[0181] In some implementations, the culture of engineered bacterial cells contains at least 10 5 One cell, or at least 10 6 One cell, or at least 10 7 One cell, or at least 10 8 One cell, or at least 10 9 One cell, or at least 10 10 One cell, or at least 10 11 One cell, or at least 10 12 Total cells. In some embodiments, the culture contains at least 10 4 10 cells / ml, or at least 10 5 10 cells / ml, or at least 10 6 10 cells / ml, or at least 10 7 10 cells / ml, or at least 10 8 10 cells / ml, or at least 10 9 10 cells / ml, or at least 10 10 Cells / ml

[0182] G. Recovery of circular DNA vectors

[0183] Circular DNA vectors generated by bacterial cells using the methods disclosed herein can be recovered from cultures of engineered bacteria using extraction and purification procedures known in the art. In some embodiments, at least about 1, or at least about 2, or at least about 3, or at least about 4, or at least about 5 mg of circular DNA vectors can be recovered per liter of cultured engineered bacterial cells. In some embodiments, the circular DNA vectors are subjected to purification procedures to reduce the amount of bacterial contaminants (e.g., endotoxins) to acceptable levels suitable for use in pharmaceutical compositions. Suitable purification procedures include chromatographic procedures, such as anion exchange chromatography and / or hydrophobic interaction chromatography. Purification procedures suitable for purifying the bacterial-generated circular DNA vectors described herein are readily available from purification procedures known in the art, such as procedures for purifying backbone byproducts from microcircular preparations, as described in, for example, Alves et al., Front. Chem. Eng., 2021 (doi:10.3389 / fceng.2020.612594), which is incorporated herein by reference in its entirety.

[0184] In some embodiments, the circular DNA vector is purified by gel electrophoresis to further avoid contamination by the backbone sequence that may be maintained in the engineered bacterial cell culture. In other embodiments, purification is not necessary to avoid detectable contamination of the circular DNA vector by the backbone sequence.

[0185] In some embodiments, circular DNA vectors can be purified from cultures of engineered bacterial cells described herein, with the purified product uncontaminated by the backbone sequence or any other extrachromosomal DNA molecules. In some embodiments, compositions of isolated circular DNA vectors purified from engineered bacterial cells disclosed herein contain backbone-containing DNA at concentrations of less than about 10 ng / ml, or less than about 1 ng / ml, or less than about 0.1 ng / ml, or less than about 0.01 ng / ml, or less than about 0.001 ng / ml, or less than about 0.0001 ng / ml. In some embodiments, the backbone-containing DNA is undetectable in the composition by quantitative PCR. In some embodiments, these purity levels are achieved after isolating the circular DNA vectors from engineered bacterial cells without gel purification or column purification steps.

[0186] In some implementations, the methods for preparing circular DNA vectors disclosed herein conform to current good manufacturing practice (GMP) as set forth in CFR Parts 210 and 211, issued and set forth by the U.S. Food and Drug Administration, which is incorporated herein by reference in its entirety.

[0187] III. DNA Vector

[0188] This document provides DNA vectors (e.g., bacterial-generated DNA vectors, such as circular DNA vectors), which contain a target sequence and a small backbone sequence containing the bacterial origin of replication (e.g., having fewer than 50 base pairs). Such DNA vectors can be generated without selectivity markers using the methods described herein (e.g., by anti-selection or by diluting backbone byproducts relative to the DNA vector). DNA vectors with a small amount of plasmid backbone sequence can be generated by any of the methods described herein and may be particularly useful for (e.g., in mammalian cells, e.g., in vitro or in vivo) expressing therapeutic genes.

[0189] DNA vectors with small bacterial replication origins (e.g., less than 50 base pairs) can be produced as plasmid DNA vectors according to known methods, such as nanoplasmid vectors (as described, for example, in International Patent Publications Nos. WO 2008 / 153733 and WO 2014 / 035457, which are incorporated herein by reference in their entirety), microcircular DNA vectors (as described, for example, in U.S. Patent Nos. 8,828,726 and 9,233,174, which are incorporated herein by reference), mini-intronic plasmids (as described, for example, in Lu et al., Mol. Ther. 2013, 21:954 and U.S. Patent No. 9,347,073, which are incorporated herein by reference), synthetic circular DNA vectors as described herein and in International Patent Publication No. WO 2019 / 178500 (which is incorporated herein by reference), and end-blocked DNA vectors (as described, for example, in U.S. Patent Publication No. 178500). The following are examples of DNA vectors: doggybone DNA vectors (as described in, for example, U.S. Patent Publication No. 2015 / 0329902 and U.S. Patent No. 9,499,847, which are incorporated herein by reference), or ministring DNA vectors (as described in, for example, U.S. Patent No. 9,290,778 and U.S. RE48908, which are incorporated herein by reference).

[0190] The circular DNA vectors described herein may exist intracellularly as episomes (e.g., in dividing or resting cells, such as post-mitotic cells), for example, in a manner similar to AAV vectors. In any of the embodiments described herein, the circular DNA vector may be a non-integrating (e.g., episome) vector. The circular DNA vectors provided herein may be naked DNA vectors, free of components inherent to viral vectors (e.g., viral proteins) and substantially free of bacterial plasmid DNA. In several embodiments, the circular DNA vectors are free of any significant immunogenic components (e.g., immunogenic bacterial features, such as CpG islands, CpG motifs, or CpG methylation) or other components associated with persistent degradation (e.g., CpG islands, CpG motifs, or CpG methylation). In some embodiments, the vector lacks bacterial methylation features, such as Dam methylation and Dcm methylation. For example, in some embodiments, the vector contains 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 which is unmethylated (e.g., unmethylated by Dam methyltransferase). Alternatively, the vector contains 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 and / or CCTGG sequences which are unmethylated (e.g., unmethylated by Dcm methyltransferase).

[0191] In some embodiments, the circular DNA vector is persistent in target cells in vivo, and the circular DNA vector exhibits improved expression persistence (e.g., intracellular persistence and / or cross-generation persistence) relative to a reference vector. The reference vector may contain additional backbone sequences or bacterial features as described herein, which may include, for example, antibiotic resistance genes or other selective markers.

[0192] In some embodiments, the circular DNA vector persists, and its expression persists for at least one month, or at least two months, or at least six months, or at least eight months, or at least one year, or longer, after administration. In some embodiments, the therapeutic persistence of the circular DNA vector ensures that administration is performed no more than four times per year, or no more than twice per year, or no more than once per year, or at a lower frequency. In some embodiments, the expression level of the circular DNA vector is reduced by no more than 50% within the first month, or the first two months, or the first six months after administration, compared to the expression level observed 1, 2, or 3 days after administration.

[0193] Circular DNA vectors can be monomers, dimers, trimers, tetramers, pentamers, hexamers, etc. In some preferred embodiments, the circular DNA vector is a monomer. In some embodiments, the DNA vector is supercoiled. The circular DNA vector may become supercoiled due to an endogenous process within an engineered bacterial cell or due to treatment with a topoisomerase (e.g., a helicase). In some embodiments, the circular DNA vector is a monomeric supercoiled circular DNA molecule. In some embodiments, the circular DNA vector is nicked. In some embodiments, the circular DNA vector is open-circular. In some embodiments, the circular DNA vector is double-stranded circular. In some embodiments, the composition comprising the circular DNA vector comprises at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% of supercoiled monomers, including embodiments that do not contain treatment with an exogenous topoisomerase.

[0194] A. Target sequence

[0195] The circular DNA vector of the present invention contains a target sequence (e.g., a therapeutic sequence), which preferably contains one or more eukaryotic (e.g., mammalian) coding sequences (e.g., transgenic or heterologous genes). The target sequence may contain one or more protein-coding domains and / or one or more non-protein-coding domains (e.g., therapeutic nucleic acids (e.g., miRNA, siRNA, shRNA, etc.)) or regulatory elements (e.g., S / MAR, intron sequences, etc.).

[0196] In specific embodiments involving therapeutic protein-coding therapeutic domains, the therapeutic sequence comprises, in a 5' to 3' orientation: a promoter and a single therapeutic protein-coding domain (e.g., a single transcription unit); a promoter and two or more therapeutic protein-coding domains (e.g., a polycistronic unit); or a first transcription unit and one or more additional transcription units (e.g., multiple transcription units). Any such protein-coding therapeutic sequence may also comprise non-protein-coding domains, such as polyadenylation sites, control elements, enhancers, sequences of marker 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, adapters, splicing sites, pre-mRNA-binding domains, regulatory sequences, and / or therapeutic nucleic acids (e.g., microRNA-coding sequences). The therapeutic protein-coding domain can be a full-length protein-coding domain (e.g., corresponding to a natural gene or its natural variant) or a functional portion thereof, such as a truncated protein-coding domain (e.g., a minigene).

[0197] In some embodiments, the therapeutic sequence encodes a monomeric protein (e.g., a monomeric protein having a secondary structure under physiological conditions, such as a monomeric protein having both secondary and tertiary structures under physiological conditions). As a supplement or alternative, the therapeutic sequence may encode a multimeric protein (e.g., a dimeric protein such as a homodimeric or heterodimeric protein, or a trimeric protein, etc.).

[0198] In certain cases, the therapeutic sequence includes an ocular gene. In some embodiments, the ocular gene is a gene expressed in ocular tissue, such as retinal tissue, which may include, for example, photoreceptor cells and / or retinal pigment epithelium (RPE) cells. In some embodiments, the coding sequence comprises a human ABCA4 or MYO7A gene sequence (e.g., encoding a human ABCA4 protein, as shown in SEQ ID NO: 61, or encoding a human MYO7A amino acid sequence, as shown in SEQ ID NO: 62). An exemplary human ABCA4 gene sequence is also provided as a 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 by NCBI gene ID: 4647. The amino acid sequence of an exemplary MYO7A protein is given by protein accession number Q13402. Eye genes (e.g., ABCA4 or MYO7A genes) can be any of those genes taught in PCT / 2022 / 0082078, which is incorporated herein by reference in its entirety.

[0199] In some cases, the eye gene is MYO7A, BEST1, CFH, CEP290, USH2A, ADGRV1, CDH23, CRB1, PCDH15, RPGR, ABCA4, ABCC6, RIMS1, LRPS, CC2D2A, TRPM1, C3, IFT172, COL11A1, TUBGCP6, KIAA1549, CACNA1F, SNRNF200, PRPF8, VCAN, USH2A, HMCN1, RPE65, NR2E3, NRL, RHO, RP1, RP2, or OFD1. For example, the eye gene may have at least 90% identity with SEQ ID NO: 19 (at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) and encode the CFH protein. In some cases, the eye gene contains or consists of SEQ ID NO: 19. In some cases, the nucleic acid sequence (e.g., a nucleic acid sequence in a circular DNA vector) has at least 90% identity with SEQ ID NO: 20 (at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) and encodes a CFH protein. In some cases, the nucleic acid sequence (e.g., a nucleic acid sequence in a circular DNA vector) contains or consists of SEQ ID NO: 20. In some cases, the eye gene may have at least 90% identity with SEQ ID NO: 21 (at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) and encode an ABCA4 protein (e.g., as shown in SEQ ID NO: 61). In some cases, the eye gene contains or consists of SEQ ID NO: 21. In some cases, the nucleic acid sequence (e.g., the nucleic acid sequence in a circular DNA vector) has at least 90% identity with SEQ ID NO:8 (at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) and encodes the ABCA4 protein (e.g., as shown in SEQ ID NO: 61), and contains the ColE2-P9 origin as described herein. In some cases, the nucleic acid sequence (e.g., the nucleic acid sequence in a circular DNA vector) comprises, or is composed of, SEQ ID NO: 8.

[0200] In some cases, the eye gene contains a nucleic acid sequence having at least 90% identity (at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) with SEQ ID NO: 22 and encodes the MYO7A protein (e.g., as shown in SEQ ID NO: 62). In some cases, the eye gene contains or is composed of the nucleic acid sequence of SEQ ID NO: 22. In some cases, the eye gene contains a nucleic acid sequence having at least 90% identity (at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) with SEQ ID NO: 23. In some cases, the eye gene contains or is composed of the nucleic acid sequence of SEQ ID NO: 23.

[0201] Therapeutic sequences containing any of the aforementioned eye genes may also contain promoters that drive eye gene expression (e.g., the CAG promoter). In some embodiments, the promoter contains a natural sequence derived from an endogenous promoter of an eye gene. In some embodiments, the promoter contains a natural sequence of the same gene to which it is operatively linked. For example, the ABCA4 coding sequence may be operatively linked to and controlled by a sequence derived from the natural ABCA4 locus (e.g., a sequence upstream of the ABCA4 transcription start site). As another example, the MYO7A coding sequence may be operatively linked to and controlled by a sequence derived from the natural MYO7A locus (e.g., a sequence upstream of the MYO7A transcription start site). In some embodiments, the promoter sequence and coding sequence are derived from natural sequences of the same species. For example, a therapeutic sequence may contain a natural ABCA4 promoter sequence from the human genome and an ABCA4 coding sequence from the human genome, or a functional variant thereof, or a natural MYO7A promoter sequence from the human genome and a MYO7A coding sequence from the human genome, or a functional variant thereof.

[0202] In some embodiments, the therapeutic sequence having an eye gene includes one or more of the following constructs, said constructs comprising sequences derived from natural promoter sequences: MYO7A promoter HS1 / 2_intron 1 (SEQ ID NO: 30), MYO7A promoter HS1-3 (SEQ ID NO: 31), MYO7A promoter Min (SEQ ID NO: 32), ABCA4 promoter exon_intron 1_short (SEQ ID NO: 33), ABCA4 promoter exon_intron 1_large (SEQ ID NO: 34), or ABCA4 promoter_long (SEQ ID NO: 35), ABCA4 promoter_short (SEQ ID NO: 36), or functional variants thereof. In some embodiments, the therapeutic sequence comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% sequence identity or 100% sequence identity with any of SEQ ID NO: 30 to 36. In some embodiments, the therapeutic sequence comprises at least 250 nucleotides, or at least 400 nucleotides, or at least 500 nucleotides, or at least 750 nucleotides, or at least 1000 nucleotides having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% sequence identity with any of SEQ ID NO: 30 to 36, and directs the expression of the therapeutic protein in photoreceptor cells and / or RFP cells.

[0203] In addition to sequences sufficient to guide transcription, the promoter sequences of the present invention may also include sequences of other regulatory elements involved in regulating transcription (e.g., enhancers, Kozak sequences, and introns). For example, in some embodiments disclosed herein, the therapeutic sequence comprises a sequence derived from the natural promoter of ABCA4 or MYO7A and a sequence derived from the natural intron 1 sequence of ABCA4 or MYO7A. In some embodiments, regulatory elements such as promoters, introns, insulators, enhancers, or other elements are derived from natural sequences of the same species as the gene operatively linked to in the therapeutic sequence.

[0204] In some embodiments, the promoters included in the therapeutic sequences disclosed herein are tissue-specific promoters because, under normal operation, they drive expression only when present in certain tissue types (e.g., ocular tissue). In some embodiments, the promoters used in the therapeutic sequences are non-tissue-specific but capable of driving expression in any tissue type. In some embodiments, the promoters are inducible promoters. In some embodiments, the promoters are constitutive promoters. In some embodiments, the constructs described herein comprise a cytomegalovirus (CMV) enhancer / β-actin (CAG) promoter (e.g., SEQ ID NO: 37 or a functional variant thereof), an elongation factor 1α (EF1A) promoter (e.g., SEQ ID NO: 38 or a functional variant thereof), an interphotoreceptor retinoid-binding protein (IBRP) promoter, a rhodopsin kinase (RK) promoter (e.g., a G protein-coupled receptor kinase 1 (GRK1) promoter), an SV40 promoter, a dihydrofolate reductase promoter, a β-actin promoter, a phosphoglycerol kinase (PGK) promoter, or a functional variant thereof. Inducible promoters allow for the regulation of gene expression and can be regulated by exogenous compounds, environmental factors such as temperature, or the presence of specific physiological states (e.g., acute phase, specific differentiation state of the cell, or only in replicating cells). Inducible promoters and inducible systems are available from a variety of commercial sources. Many other systems have been described and can be readily selected by those skilled in the art. Examples of inducible promoters regulated by exogenous promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone-inducible mouse mammary tumor virus promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline repression system, the tetracycline inducible system, the RU486 inducible system, and the rapamycin inducible system. Other types of inducible promoters that may be useful in this context are those regulated by specific physiological states (e.g., temperature, acute phase, specific differentiation state of the cell, or only in replicating cells).

[0205] In addition to promoters, the therapeutic sequences disclosed herein may also contain other regulatory elements, such as appropriate transcription initiation, termination, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance protein stability; and sequences that enhance the secretion of encoded products.

[0206] For therapeutic sequences containing genes encoding proteins, a polyadenylated (poly-A, or pA) sequence can be inserted after the gene (e.g., operatively linked to the 3' of the gene, e.g., directly linked to the 3' of the gene).

[0207] The exact nature of the regulatory sequences required for gene expression in host cells can vary between species, tissues, or cell types, but generally should include, as needed, 5' non-transcriptional and 5' non-translational sequences involved in transcription and translation initiation, respectively, such as TATA boxes, capping sequences, CAAT sequences, enhancer elements, etc. Such 5' non-transcriptional regulatory sequences may contain promoter regions containing promoter sequences for transcriptional control of operatively linked genes. Regulatory sequences may also contain enhancer sequences or upstream activator sequences. Vectors of this disclosure may optionally contain 5' leader sequences or signal sequences.

[0208] In some embodiments, the therapeutic sequences disclosed herein include a scaffold-nuclear matrix attachment region (S / MAR). Without being bound by theory, it is believed that S / MAR elements can assist in establishing long-term gene expression from a DNA vector through interactions between the S / MAR element and the nuclear matrix. Known S / MAR constructs include human IFN-γ S / MAR (SEQ ID NO:39) and human APOB S / MAR (NCBI gene ID 106632268). Other known S / MAR elements may be included in the therapeutic sequences disclosed herein, as well as their functional variants. In some embodiments, a variant of IFN-γ S / MAR containing a functionally partial tandem repeat of the IFN-γ S / MAR (SEQ ID NO: 40) is included in the therapeutic sequences provided herein. In some embodiments, the therapeutic sequence comprises a sequence having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5% sequence identity or having 100% sequence identity with SEQ ID NO: 39 or 40. The S / MAR element may be operatively connected to the 5' or 3' end of the coding sequence of the therapeutic sequence.

[0209] In some embodiments, the therapeutic sequences disclosed herein comprise chromatin insulator elements. In some embodiments, one or more chromatin insulator elements may comprise one or more chicken hypersensitive site 4 elements (cHS4; SEQ ID NO: 41), which are chromatin insulators derived from the chicken β-globin locus control region. In some embodiments, the therapeutic sequence comprises a sequence having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5% sequence identity or having 100% sequence identity with SEQ ID NO: 41.

[0210] In some embodiments, the therapeutic sequences disclosed herein comprise regulatory elements (e.g., a portion or variant thereof) derived from the natural sequence of intron 6 of ABCA4. Such regulatory elements (e.g., SEQ ID NO: 42) can enhance the persistence and expression level of genes operatively linked to them. Therefore, some embodiments of the invention are characterized by regulatory elements derived from the natural sequence of intron 6 of ABCA4, such as sequences in the 5' half region of ABCA4 intron 6 (i.e., sequences located upstream of the midpoint between the 5' and 3' ends of ABCA4 intron 6) or sequences in the 5' third region of ABCA4 intron 6 (i.e., sequences located within the last 33.3% of the 5' end of ABCA4 intron 6).

[0211] In some cases, the regulatory element derived from ABCA4 intron 6 has one or more (one, two, three, four, five or more) sequences that are identical to at least 100 consecutive nucleotides in natural human ABCA4 intron 6 (e.g., SEQ ID NO: 29) (e.g., at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500 or at least 1600 consecutive nucleotides in natural human ABCA4 intron 6 (e.g., SEQ ID NO: 29), such as 100 to 5000, 500 to 2500, 1000 to 2000 or 1500 to 1700 consecutive nucleotides in natural human ABCA4 intron 6 (e.g., SEQ ID NO: 29). In some cases, the sequence shared between the regulatory element and ABCA4 intron 6 contains all or part of the nucleotides located in positions 3158 to 4822 of ABCA4 intron 6 (numbered starting from the 5' end of ABCA4 intron 6), for example, SEQ ID NO: 42.

[0212] In some cases, the regulatory element is a functional variant of any of the regulatory elements derived from ABCA4 intron 6 described above. For example, in some cases, the regulatory element derived from ABCA4 intron 6 has one or more (one, two, three, four, five or more) sequences that correspond to at least 100 consecutive nucleotides in natural human ABCA4 intron 6 (e.g., SEQ ID NO: 29) (e.g., at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500 or at least 1600 consecutive nucleotides in natural human ABCA4 intron 6 (e.g., SEQ ID NO: 29)). 29) The 100 to 5000, 500 to 2500, 1000 to 2000, or 1500 to 1700 consecutive nucleotides within the ABCA4 intron 6 have at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%). In some cases, the sequence shared between the regulatory element and ABCA4 intron 6 comprises all or part of the nucleotides located in positions 3158 to 4822 of ABCA4 (numbered starting from the 5' end of ABCA4 intron 6), for example, SEQ ID NO: 42.

[0213] In some cases, the regulatory element derived from ABCA4 intron 6 has one or more (one, two, three, four, five or more) sequences that are identical to at least 100 consecutive nucleotides in the 5' third region of natural human ABCA4 intron 6 (e.g., SEQ ID NO: 29) (e.g., at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500 or at least 1600 consecutive nucleotides in the 5' third region of natural human ABCA4 intron 6 (e.g., SEQ ID NO: 29)). 29) The 5' third region containing 100 to 5000, 500 to 2500, 1000 to 2000, or 1500 to 1700 consecutive nucleotides has at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%). In some cases, the sequence shared between the regulatory element and ABCA4 intron 6 comprises all or part of the nucleotides located in positions 3158 to 4822 of ABCA4 intron 6 (numbered starting from the 5' end of ABCA4 intron 6), for example, SEQ ID NO: 42.

[0214] In some cases, the regulatory element derived from ABCA4 intron 6 has one or more (one, two, three, four, five or more) sequences that are identical to at least 100 consecutive nucleotides of nucleotides 3158 to 4822 of ABCA4 intron 6 (e.g., at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500 or at least 1600 consecutive nucleotides of nucleotides 3158 to 4822 of ABCA4 intron 6).

[0215] In some cases, the regulatory element derived from ABCA4 intron 6 has one or more (one, two, three, four, five or more) sequences that have at least 90% identity with at least 100 consecutive nucleotides from nucleotides 3158 to 4822 of ABCA4 intron 6 (e.g., at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500 or at least 1600 consecutive nucleotides from nucleotides 3158 to 4822 of ABCA4 intron 6) (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity).

[0216] In some of the foregoing cases, the regulatory element derived from ABCA4 intron 6 is mutated at one or more positions (e.g., one, two, three or more positions) relative to the native ABCA4 intron 6 sequence to remove the recognition site of a restriction enzyme (e.g., type IIs restriction enzyme (e.g., Bsal)). This can improve preparation efficiency by simplifying the cell-free production of the synthesized circular DNA vector using the methods described in the embodiments herein (e.g., by using a consolidating step of type IIs restriction enzymes). For example, nucleotide 3530 (G) of native human ABCA4 intron 6 (SEQ ID NO: 29) can be deleted to remove the Bsal recognition site in the regulatory element derived from ABCA4 intron 6, thereby facilitating an improved Bsal-based preparation process. For example, in some embodiments, the nucleotide sequence of nucleotides 3158 to 4822 of native ABCA4 intron 6 is modified to delete G3530, thereby producing the ABCA4 intron 6-derived regulatory element of SEQ ID NO: 42.

[0217] Promoters, coding sequences, and other elements may be included in the therapeutic sequences disclosed herein in any suitable order, providing effective expression and / or persistence. In some embodiments, the therapeutic sequence comprises a promoter, coding sequence, and polyadenylated sequence in a 5' to 3' orientation. In some embodiments, the therapeutic sequence comprises an enhancer sequence (e.g., S / MAR), promoter, coding sequence, and polyadenylated sequence in a 5' to 3' orientation. In some embodiments, the therapeutic sequence comprises an insulator sequence (e.g., cHS4), promoter, coding sequence, and polyadenylated sequence in a 5' to 3' orientation. In some embodiments, the therapeutic sequence comprises an insulator sequence (e.g., cHS4), promoter, coding sequence, polyadenylated sequence, and additional regulatory elements (e.g., regulatory elements derived from intron 6 of ABCA4) in a 5' to 3' orientation. In some embodiments, the therapeutic sequence comprises an insulator sequence (e.g., cHS4), promoter, coding sequence, polyadenylated sequence, and enhancer sequence (e.g., S / MAR) in a 5' to 3' orientation. In some embodiments, the therapeutic sequence comprises, in a 5' to 3' orientation, an insulator sequence (e.g., cHS4), a promoter, a coding sequence, an additional regulatory element (e.g., a regulatory element derived from intron 6 of ABCA4), and a polyadenylated sequence. In some embodiments, the therapeutic sequence comprises, in a 5' to 3' orientation, an insulator sequence (e.g., cHS4), a promoter, a coding sequence, an enhancer sequence (e.g., S / MAR), and a polyadenylated sequence. In some embodiments, the therapeutic sequence comprises, in a 5' to 3' orientation, a promoter, a regulatory element (e.g., an intron or a portion thereof, such as a portion of intron 6 of ABCA4, such as an intron 6 regulatory element), a coding sequence, and a polyadenylated sequence. In some embodiments, the therapeutic sequence comprises, in a 5' to 3' orientation, an enhancer sequence (e.g., S / MAR), a promoter, a regulatory element (e.g., an intron or a portion thereof, such as a portion of intron 6 of ABCA4, such as an intron 6 regulatory element), a coding sequence, and a polyadenylated sequence. In some embodiments, the therapeutic sequence comprises, in a 5' to 3' orientation, an insulator sequence (e.g., cHS4), a promoter, a regulatory element (e.g., an intron or a portion thereof, such as a portion of intron 6 of ABCA4, such as an intron 6 regulatory element), a coding sequence, a polyadenylated sequence, and additional regulatory elements (e.g., regulatory elements derived from intron 6 of ABCA4).In some embodiments, the therapeutic sequence comprises, in a 5' to 3' orientation, an insulator sequence (e.g., cHS4), a promoter, a regulatory element (e.g., an intron or a portion thereof, such as a portion of intron 6 of ABCA4, such as an intron 6 regulatory element), a coding sequence, a polyadenylated sequence, and an enhancer sequence (e.g., S / MAR). In some embodiments, the therapeutic sequence comprises, in a 5' to 3' orientation, an insulator sequence (e.g., cHS4), a promoter, a regulatory element (e.g., an intron or a portion thereof, such as a portion of intron 6 of ABCA4, such as an intron 6 regulatory element), a coding sequence, an additional regulatory element (e.g., a regulatory element derived from intron 6 of ABCA4), and a polyadenylated sequence. In some embodiments, the therapeutic sequence comprises, in a 5' to 3' orientation, an insulator sequence (e.g., cHS4), a promoter, a regulatory element (e.g., an intron or a portion thereof, such as a portion of intron 6 of ABCA4, such as an intron 6 regulatory element), a coding sequence, an enhancer sequence (e.g., S / MAR), and a polyadenylated sequence. The sequence elements disclosed herein can be arranged in other suitable combinations and orders.

[0218] Some embodiments of the therapeutic sequences disclosed herein include one or more coding sequences for one or more respiratory system genes, such as genes encoding CFTR. In some embodiments, the respiratory system gene (e.g., the gene encoding CFTR) is a gene expressed in airway tissue, such as airway epithelium (e.g., lung epithelium, nasal epithelium, tracheal epithelium, bronchial epithelium, bronchioles, or alveoli). In some embodiments, the respiratory system gene encodes a therapeutic protein, such as CFTR.

[0219] The coding sequence included in the therapeutic sequence (e.g., a therapeutic sequence encoding CFTR) may include a gene associated with a respiratory disease (e.g., cystic fibrosis). In some embodiments, the coding sequence is cDNA of CFTR. In some embodiments, the coding sequence is a codon-optimized CFTR sequence. In some cases, the coding sequence is a functional variant of a respiratory gene (e.g., a gene encoding CFTR). In some embodiments, the coding sequence of CFTR is the nucleic acid sequence of SEQ ID NO:25 or contains the nucleic acid sequence of SEQ ID NO:25. In some embodiments, the coding sequence of CFTR contains a sequence having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99%, or at least 99.5% sequence identity with SEQ ID NO:25. In some embodiments, the coding sequence may be a human sequence or a sequence of human origin.

[0220] The therapeutic sequences disclosed herein may contain one or more regulatory elements (upstream elements), such as promoter sequences and / or hypersensitive sequences, located upstream of the transcription start site of a protein-coding sequence. In some cases described herein, the therapeutic sequence comprises a hypersensitive sequence (e.g., containing a portion or variant thereof) derived from a natural intron sequence present upstream of the transcription start site of a CFTR (e.g., human CFTR). In some embodiments, the hypersensitive sequence is derived from a natural intron sequence present at its natural location (e.g., according to GRch37 / hg19) at least 5,000 nucleotides upstream of the transcription start site of a CFTR (e.g., human CFTR) (e.g., between 5,000 and 50,000 nucleotides upstream of the transcription start site of a CFTR (e.g., human CFTR)). In some embodiments, the hypersensitive sequence is derived from a natural intron sequence located at its natural position (e.g., according to GRch37 / hg19) at least 10,000 nucleotides upstream of the transcription start site of a CFTR (e.g., human CFTR) (e.g., between 10,000 and 50,000 nucleotides upstream of the transcription start site of a CFTR (e.g., human CFTR)). In some embodiments, the hypersensitive sequence is derived from a natural intron sequence located at its natural position (e.g., according to GRch37 / hg19) at at least 20,000 nucleotides upstream of the transcription start site of a CFTR (e.g., human CFTR) (e.g., between 20,000 and 50,000, 30,000 and 50,000, or 40,000 and 50,000 nucleotides upstream of the transcription start site of a CFTR (e.g., human CFTR)).

[0221] For example, a hypersensitive sequence may be derived from a natural human intron sequence located 40,000 to 50,000 nucleotides upstream of the human CFTR transcription start site, on chromosome 7, at positions 117,074,974 to 117,076,392 of Genome Reference Consortium Human Build 37 release 19 (GRCh37 / hg19; SEQ ID NO: 28). Such a hypersensitive site (e.g., SEQ ID NO: 28) can enhance the expression level of genes operatively linked to it (e.g., downstream operatively linked genes). Therefore, some embodiments of the present invention are characterized by a hypersensitive sequence derived from SEQ ID NO: 28 (e.g., containing a portion thereof or a variant thereof). In some cases, the hypersensitive sequence derived from SEQ ID NO: 28 has one or more sequences identical to at least 100 consecutive nucleotides in SEQ ID NO: 28, such as one, two, three, four, five, or more sequences identical to at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, or 1400 consecutive nucleotides in SEQ ID NO: 28. In some embodiments, the hypersensitive sequence derived from SEQ ID NO: 28 has one or more (e.g., one, two, three, four, five, or more) sequences identical to 100 to 1500, 200 to 1200, 300 to 1000, or 400 to 800 consecutive nucleotides in SEQ ID NO: 28.

[0222] In some embodiments, any of the above-mentioned hypersensitive sequences derived from a natural intron sequence located upstream of the transcription start site of a CFTR (e.g., human CFTR) have one or more (e.g., one, two, three, four, five or more) sequences that have at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with at least 100 consecutive nucleotides in SEQ ID NO: 28. For example, any of the above-mentioned hypersensitive sequences derived from a natural intron sequence present upstream of the transcription start site of a CFTR (e.g., human CFTR) has one or more (e.g., one, two, three, four, five or more) sequences that have at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) with at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300 or at least 1400 consecutive nucleotides in SEQ ID NO: 28.

[0223] In some cases, the hypersensitive sequence derived from a natural intron sequence present upstream of the transcription start site of a CFTR (e.g., human CFTR) has one or more (one, two, three, four, five or more) such sequences that are identical to at least 100 consecutive nucleotides of SEQ ID NO: 28 (e.g., at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, to 1200, at least 1300 or at least 1400 consecutive nucleotides of SEQ ID NO: 28).

[0224] In some embodiments, the therapeutic sequence comprises a sequence having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99%, or at least 99.5% sequence identity with SEQ ID NO: 28 or having 100% sequence identity.

[0225] The therapeutic sequences described herein may comprise promoter sequences derived from endogenous promoters of respiratory system genes (e.g., the CFTR gene). In some embodiments, the promoter sequence comprises a natural sequence of the same gene operatively linked to it. For example, the sequence encoding CFTR may be operatively linked to and controlled by a sequence derived from a natural CFTR gene site (e.g., a sequence upstream of the CFTR transcription start site). In some embodiments, the promoter sequence and the coding sequence are derived from natural sequences of the same species. For example, the expression construct may comprise a natural CFTR promoter sequence from the human genome and a sequence encoding CFTR from the human genome, or a functional variant thereof.

[0226] In some embodiments where the therapeutic sequence encodes CFTR, the promoter sequence is a tissue-specific promoter because, under normal operation, it drives expression only if it is present in certain tissue types (e.g., airway epithelium). In some embodiments, the promoter sequence used in the therapeutic sequence encoding CFTR is not tissue-specific but is capable of driving expression in any tissue type. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter sequence is a constitutive promoter. In some embodiments, the construct described herein comprises an extension factor 1α (EF1A) promoter sequence (e.g., SEQ ID NO: 43 or a functional variant thereof). In some cases, the constructs described herein include cytomegalovirus (CMV) enhancer / β-actin (CAG) promoters (e.g., SEQ ID NO: 44 or a functional variant thereof), interretinoid retinoid-binding protein (IBRP) promoters, rhodopsin kinase (RK) promoters (e.g., G protein-coupled receptor kinase 1 (GRK1) promoters), SV40 promoters, dihydrofolate reductase promoters, β-actin promoters, glycerol phosphokinase (PGK) promoters, or functional variants of any of the foregoing promoters.

[0227] In some cases, a hypersensitive sequence (e.g., any hypersensitive sequence described herein) is operatively linked (directly or indirectly) to a promoter (e.g., a natural promoter) of a natural intron sequence present upstream of the transcription start site of a CFTR (e.g., human CFTR). In some cases, the promoter is derived from a natural sequence that extends at its natural position (e.g., according to GRch37 / hg19) no more than 5,000 nucleotides upstream of the transcription start site of a CFTR (e.g., human CFTR) (e.g., no more than 4,500, 4,000, 3,500, or 3,000 nucleotides upstream of the transcription start site of a CFTR (e.g., human CFTR)).

[0228] In some cases, the promoter encoding the therapeutic sequence of CFTR is derived from positions 117,118,786 to 117,120,280 of human chromosome 7—GRch37 / hg19 (e.g., a promoter comprising SEQ ID NO: 26 or a functional variant thereof (e.g., SEQ ID NO: 27)). Such a promoter (e.g., SEQ ID NO: 26 or SEQ ID NO: 27) can enhance the expression level of a gene operatively linked to it (e.g., operatively linked downstream). Therefore, some embodiments of the present invention are characterized by a promoter derived from SEQ ID NO: 26 (e.g., comprising a portion of SEQ ID NO: 26 or a variant thereof). In some cases, the promoter derived from SEQ ID NO: 26 has one or more (e.g., one, two, three, four, five or more) sequences that are identical to at least 100 consecutive nucleotides in SEQ ID NO: 26, such as one or more (e.g., one, two, three, four, five or more) sequences that are identical to at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, or at least 1400 consecutive nucleotides in SEQ ID NO: 26, such as one or more (e.g., one, two, three, four, five or more) sequences that are identical to 100 to 1500, 200 to 1200, 300 to 1000, or 400 to 800 consecutive nucleotides in SEQ ID NO: 26.

[0229] In some embodiments, any of the aforementioned promoters derived from a natural intron sequence present upstream of the transcription start site in a CFTR (e.g., human CFTR) has one or more (e.g., one, two, three, four, five or more) sequences that have at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) with at least 100 consecutive nucleotides in SEQ ID NO: 26. For example, any of the aforementioned promoters derived from a natural intron sequence present upstream of the transcription start site in a CFTR (e.g., human CFTR) may have one or more (e.g., one, two, three, four, five or more) sequences that have at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) with at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, or at least 1400 consecutive nucleotides within SEQ ID NO: 26.

[0230] In some cases, the promoter sequence derived from a natural intron sequence present upstream of the transcription start site in a CFTR (e.g., human CFTR) has one or more (one, two, three, four, five or more) sequences that are identical to at least 100 consecutive nucleotides of SEQ ID NO: 26 (e.g., at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, or at least 1400 consecutive nucleotides of SEQ ID NO: 26).

[0231] In some cases, the promoter sequence derived from a natural intron sequence present upstream of the transcription start site of a CFTR (e.g., human CFTR) has one or more (one, two, three, four, five or more) sequences that have at least 90% identity (e.g., at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, or at least 1400) of at least 100 consecutive nucleotides of SEQ ID NO: 26 or SEQ ID NO: 27.

[0232] In some embodiments, the therapeutic sequence encoding CFTR includes a sequence having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99%, or at least 99.5% sequence identity with SEQ ID NO: 26 or SEQ ID NO: 27, or having 100% sequence identity.

[0233] In some embodiments, the therapeutic sequence encoding CFTR comprises any of the aforementioned hypersensitive sequences derived from a natural intron sequence (e.g., derived from SEQ ID NO: 28) present upstream of the transcription start site of a CFTR (e.g., human CFTR), operatively linked upstream of any of the aforementioned promoter sequences derived from a natural intron sequence present upstream of the transcription start site of a CFTR (e.g., human CFTR), such that the promoter sequence extends upstream of the transcription start site of a CFTR (e.g., human CFTR) by no more than 5,000 nucleotides (e.g., no more than 4,500, 4,000, 3,500, or 3,000 nucleotides) in its natural position (e.g., according to GRch37 / hg19) (e.g., derived from SEQ ID NO: 26). Therefore, in some cases, the expression construct of the present invention comprises a hypersensitive sequence derived from SEQ ID NO: 5 of 713, operatively linked upstream of the promoter sequence derived from SEQ ID NO: 26.

[0234] In some cases, the hypersensitive sequence (e.g., the hypersensitive sequence derived from SEQ ID NO: 28) is operatively linked to the promoter (e.g., the promoter derived from SEQ ID NO: 26, e.g., SEQ ID NO: 27) by an intercalation sequence (e.g., an inert intercalation sequence) of no more than 500 nucleotides (e.g., no more than 400, 300, 200 or 100 nucleotides) (e.g., 0 to 10, 2 to 20, 2 to 10 or 4 to 8 nucleotides).

[0235] In some cases, therapeutic sequences contain any of the characteristic structures described in International Patent Publication No. WO 2022 / 182795, which is incorporated herein by reference in its entirety. For example, therapeutic sequences may contain regulatory protein-coding genes, such as genes encoding immunomodulatory proteins, such as genes encoding dendritic cell chemical attractants (XCL1, XCL2, CCL5, or CCL4), genes encoding dendritic cell growth factors or activators (e.g., FLT3L, GMCSF, or CD40), or genes encoding lymphocyte signaling proteins (e.g., IL-12, IL-15, CXCL9, or CXCL10). Such vectors may be bicistronic or polycistronic vectors encoding two, three, or more regulatory protein-coding genes.

[0236] In some embodiments, the therapeutic sequence encodes an antibody, or a portion thereof, fragment, or variant thereof. Antibodies comprise fragments capable of binding to antigens, such as Fv, single-chain Fv (scFv), Fab, Fab', di-scFv, sdAb (single-domain antibody), (Fab')2 (including chemically linked F(ab')2), and nanobodies. Papain digestion of the antibody yields two identical antigen-binding fragments, referred to as "Fab" fragments, each having a single antigen-binding site, and a residual "Fc" fragment, the name reflecting its ease of crystallization. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites and still capable of cross-linking the antigen. Antibodies also include chimeric antibodies and humanized antibodies. Furthermore, variants with sequences from other organisms are considered for all antibody constructs provided herein. Therefore, if a human version of the antibody is disclosed, those skilled in the art will understand how to convert human-sequence-based antibodies into mouse, rat, cat, dog, horse, etc. sequences. Antibody fragments also include single-chain scFvs in any orientation, tandem di-scFvs, biantibodies, tandem tri-sdcFvs, microantibodies, nanobodies, etc. In some embodiments, for example when the antibody is an scFv, a single polynucleotide of the therapeutic gene sequence encodes a single polypeptide comprising a heavy chain and a light chain linked together. Antibody fragments also include nanobodies (e.g., sdAbs—antibodies having a single monomeric domain (e.g., a variable pair of domains of the heavy chain, without a light chain)). Multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.) are known in the art and are considered expression products of the therapeutic gene sequences of the present invention.

[0237] In some cases, the therapeutic sequence encodes one or more proteins (e.g., a single protein, two proteins, three proteins, four proteins, or more), each 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, or longer. In embodiments where such a therapeutic sequence encodes two or more proteins, the therapeutic sequence may be a polycistronic therapeutic sequence or a multi-transcriptional unit therapeutic sequence. Such a polycistronic therapeutic sequence may be, for example, a tricistronic cassette encoding Flt3L, IL-12, and XCL1.

[0238] In embodiments involving non-protein-coding therapeutic sequences, the therapeutic sequence lacks a protein-coding domain (e.g., a therapeutic protein-coding domain). For example, in some embodiments, the therapeutic sequence comprises a non-protein-coding therapeutic nucleic acid, such as microRNA, siRNA, short hairpin RNA (shRNA), or an immune-activating therapeutic nucleic acid (e.g., a TLR agonist).

[0239] In some cases, the therapeutic sequence comprises one or more regulatory elements, such as a transcriptional knockdown sequence (with or without a therapeutic protein-coding sequence as part of the same therapeutic sequence). In some embodiments of the invention, the transcriptional knockdown sequence is configured to silence the transcription of endogenous expression of a target gene, wherein the therapeutic sequence encodes a therapeutic alternative protein encoded by the functional form of the target gene. Suitable transcriptional knockdown sequences include zinc finger nuclease-coding sequences, TALEN-coding sequences, inactivated CAS9-coding sequences, guide RNA-coding sequences, or combinations thereof. For example, in some embodiments, the therapeutic sequence comprises a dCAS9-coding sequence fused to one or more repressor proteins, such as as described in U.S. Patent No. 11,629,342, which is incorporated herein by reference in its entirety.

[0240] In some cases, therapeutic sequences contain gene-editing sequences. Such gene-editing sequences suitable for incorporation into therapeutic sequences are known in the art and include, for example, sequences encoding guide RNA, reverse transcriptase, or CRISPR-associated (Cas) endonucleases.

[0241] In some embodiments, the therapeutic sequence or other target sequence (which may include non-therapeutic coding sequences, such as reporter genes used to measure expression or persistence) has a length of 0.1 Kb to 100 Kb, or 0.2 Kb to 90 Kb, or 0.5 Kb to 80 Kb, or 1.0 Kb to 70 Kb, or 1.5 Kb to 60 Kb, or 2.0 Kb to 50 Kb, or 2.5 Kb to 45 Kb, or 3.0 Kb to 40 Kb, or 4.0 Kb to 30 Kb, or 4.5 Kb to 25 Kb, or 5.0 Kb to 20 Kb, or 6.0 Kb to 17 Kb, or 7.0 Kb to 15 Kb, or 7.5 Kb to 14 Kb, or 8.0 Kb to 13 Kb, or 8.5 Kb... The therapeutic sequence is 1 kb to 12.5 kb, or 9.0 kb to 12.0 kb in length, or 9.5 kb to 11.5 kb in length, or 10.0 kb to 11.0 kb in length. In some embodiments, the therapeutic sequence is at least 1 kb, or at least 2 kb, or at least 3 kb, or at least 4 kb, or at least 4.5 kb, or at least 5 kb, or at least 10 kb, or at least 12 kb, or at least 15 kb.

[0242] In some implementations, the 3' end of the target sequence (e.g., a therapeutic or non-therapeutic coding sequence) is linked to the 5' end of the ori sequence in the circular DNA vector by a non-bacterial sequence (e.g., a recombination site, such as a recombination trace) of no more than 50 bp, or no more than 45 bp, or no more than 30 bp, or no more than 25 bp, or no more than 20 bp, or no more than 15 bp, or no more than 10 bp.

[0243] In some embodiments, the target sequence contained in the circular DNA vector described herein includes a reporter sequence in addition to a therapeutic protein-coding domain or a therapeutic non-protein-coding domain. In some embodiments, the therapeutic sequence lacks a reporter sequence. In some embodiments, the target sequence includes a reporter sequence but does not include a therapeutic sequence. The reporter sequence may be, for example, a reporter gene. Such a reporter gene can be used, for example, to validate the expression of a therapeutic gene sequence in specific cells and tissues. Reporter sequences that may be provided in the circular DNA vector include, but are not limited to, DNA sequences encoding β-lactamases, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art. When associated with regulatory elements driving their expression, reporter sequences provide detectable signals via conventional methods, including enzymatic, radiographic, colorimetric, fluorescence, or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry. For example, in the case of the marker sequence being the LacZ gene, the presence of the signal-carrying vector is detected by measuring β-galactosidase activity. In the case of the transgene being green fluorescent protein or luciferase, the signal-carrying vector can be visually measured by light generation in a colorimeter or photometer.

[0244] As part of a therapeutic or other target sequence, the circular DNA vector of the present invention may contain conventional control elements that regulate or improve transcription, translation, and / or expression in target cells. Suitable control elements are described in International Publication No. WO2021 / 055760, which is incorporated herein by reference in its entirety.

[0245] B. Copying Start Point

[0246] The DNA vectors of the present invention (e.g., circular DNA vectors) also include a replication origin (e.g., a bacterial replication origin). In embodiments of DNA vectors lacking selectivity markers, the replication origin may be selected from a Poll I-dependent replication origin, such as a pUC origin, a pMBI origin, or a ColE1 origin. In some cases where the DNA vector has a small backbone (e.g., less than 100 base pairs, or less than 50 base pairs), a small replication origin is necessary. The present invention is based in part on the unexpected discovery that a ColE2-P9 replication origin of less than 50 base pairs enables efficient fabrication of the circular DNA vectors of the present invention by maintaining a stable copy number during cell amplification. Furthermore, the inventors have developed functional variants of the ColE2-P9 replication origin that require even fewer base pairs to maintain the production of the circular DNA vectors of the present disclosure in bacterial cells, thereby allowing for even smaller segments of non-eukaryotic material in the DNA vector. Therefore, the DNA vectors described herein can reduce the risks associated with the entry of foreign DNA into the host object.

[0247] The ori sequence contained in the vector sequence of the parent plasmid was selected such that it corresponds to the Rep gene integrated into the genome of the modified bacterial cell or otherwise present in the modified bacterial cell (e.g., on a plasmid or BAC). In some exemplary embodiments, ori comprises the nucleotide sequence shown in SEQ ID NO: 2. Therefore, the embodiments of the modified bacterial cells disclosed herein include functional pairs of replication proteins and origins of replication 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 the ColE2-P9 ori sequence or a functional variant thereof. In some embodiments, the ori sequence present in the vector sequence is a functional fragment of the ColE2-P9 ori sequence having the DNA sequence shown in SEQ ID NO: 2. The 40-base-pair functional fragment shown 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 their functional fragments, such as the ori sequence of ColE3-CA38. In some embodiments, the ori sequence is a naturally occurring ori sequence.

[0248] In some cases, the ori sequence is a functional variant of the naturally occurring ori sequence, such as an ori sequence that has been modified to be shorter than the corresponding naturally occurring ori sequence while still retaining the ability to support replication initiation. Example 13 below details a study in which a functional truncated variant of ColE2-P9 was developed and characterized. Such functional variants of the ColE2-P9 replication origin comprise SEQ ID NO: 12 to 17.

[0249] For example, in some embodiments, the DNA vector (e.g., a circular DNA vector) contains a replication origin that is (in one strand) a nucleotide sequence containing SEQ ID NO: 12. This can be a truncated ColE2-P9 origin of replication, or a truncated ColE2-P9 origin of replication composed of such a truncated ColE2-P9 origin of replication, for example, where the truncated ColE2-P9 origin of replication lacks one or more of the nucleic acid bases 1 to 7 or 35 to 40 of SEQ ID NO: 2. In some cases, the truncated ColE2-P9 origin of replication (in one strand) contains the nucleotide sequence of SEQ ID NO: 13 ( ) or composed of. In some cases, the truncated ColE2-P9 origin of replication (in one strand) contains the nucleotide sequence of SEQ ID NO: 14 ( ) or composed of. In some cases, the truncated ColE2-P9 origin of replication (in one strand) contains the nucleotide sequence of SEQ ID NO: 15 ( ) or composed of. In some cases, the truncated ColE2-P9 origin of replication (in one strand) contains the nucleotide sequence of SEQ ID NO: 16 ( ) or composed of. In some cases, the truncated ColE2-P9 origin of replication (in one strand) contains the nucleotide sequence of SEQ ID NO: 17 ( ) or composed of. In some cases, the truncated ColE2-P9 origin of replication (in one strand) contains the nucleotide sequence of SEQ ID NO: 4 ( (or composed of) the origin of replication (in one strand) contains the nucleic acid sequence of SEQ ID NO: 18. X1 is A, T, C, or G. In some embodiments, X2 is A, T, or C; X3 is A, T, or G; X4 is A, T, or C; X5 is A, T, or G; or any combination thereof.

[0250] V. Pharmaceutical Composition

[0251] The improved production efficiency makes the methods of this invention particularly suitable for the scalable preparation of pharmaceutical compositions comprising circular DNA vectors (e.g., circular DNA vectors prepared by any of the methods described herein, such as circular DNA vectors having a replication origin (e.g., a replication origin less than 50 bp, such as the ColE2-P9 replication origin or a functional variant thereof) and lacking a selectivity marker). Any method described herein for producing circular DNA vectors is applicable to producing pharmaceutical compositions comprising circular DNA vectors in pharmaceutically acceptable carriers.

[0252] Downstream purification processes can be readily applied as part of any of the methods described herein. For example, multiple chromatographic steps are known in the art and can be suitably applied to remove bacterial byproducts, endotoxins, bacterial artificial chromosomes (BACs), helper plasmids, etc. In some cases, pharmaceutical compositions containing bacterial circular DNA vectors are purified by anion exchange chromatography or hydrophobic interaction chromatography.

[0253] In some embodiments, the pharmaceutical composition of the present invention contains at least about 0.1 mg of DNA carrier, or at least about 0.5 mg, or at least about 1.0 mg of DNA carrier, or at least about 2.0 mg of DNA carrier, or at least 5 mg of DNA carrier, or at least 10 mg of DNA carrier in a pharmaceutically acceptable carrier.

[0254] In some embodiments, the pharmaceutical compositions of the present invention are substantially free of impurities. For example, in some embodiments, the pharmaceutical formulation contains <2.0% by mass of protein (e.g., <1.5% by mass, <1.0% by mass, <0.5% by mass, <0.1% by mass, <0.05% by mass, or <0.01% by mass of protein). In some cases, the protein content is determined by dicaprin determination. Alternatively or supplementally, the protein content is determined by ELISA.

[0255] In some cases, the pharmaceutical composition contains <5.0% by mass of RNA (e.g., <4.0% by mass, <3.0% by mass, <2.0% by mass, <1.5% by mass, <0.5% by mass, <0.1% by mass, <0.05% by mass, or <0.01% by mass of RNA). In some embodiments, the RNA content is determined by agarose gel electrophoresis. In some embodiments, the RNA content is determined by quantitative PCR. In some embodiments, the RNA content is determined by fluorescence assay (e.g., Ribogreen).

[0256] In some embodiments, the pharmaceutical composition comprises <5.0% by mass of gDNA (e.g., <4.0% by mass, <3.0% by mass, <2.0% by mass, <1.5% by mass, <1.0% by mass, <0.5% by mass, <0.1% by mass, <0.05% by mass, or <0.01% by mass of gDNA). In some embodiments, the gDNA content is determined by agarose gel electrophoresis or capillary electrophoresis. In some embodiments, the gDNA content is determined by quantitative PCR. In some embodiments, the gDNA content is determined by Southern blotting.

[0257] In some embodiments, the pharmaceutical composition contains <40 EU / mg of endotoxin. In some embodiments, the pharmaceutical formulation contains <20 EU / mg of endotoxin. In some embodiments, the pharmaceutical formulation contains <10 EU / mg of endotoxin. In some embodiments, the pharmaceutical formulation contains <5 EU / mg of endotoxin (e.g., <4 EU / mg endotoxin, <3 EU / mg endotoxin, <2 EU / mg endotoxin, <1 EU / mg endotoxin, <0.5 EU / mg endotoxin), for example, as measured by Limulus ameobocyte lysate (LAL) assay.

[0258] In some embodiments, the pharmaceutical compositions disclosed herein comply with current good operating practices (GMP) as set forth in Parts 210 and 211 of Title 21 of the United States Food and Drug Administration (21C.FR Parts 210 and 211), which are incorporated herein by reference in their entirety.

[0259] The pharmaceutical compositions described herein may comprise one or more pharmaceutically acceptable carriers, such as excipients and / or stabilizers that are non-toxic to the individual receiving treatment (e.g., a human patient) at the doses and concentrations employed. In some embodiments, the pharmaceutically acceptable carrier is an aqueous pH buffer solution. Some examples of pharmaceutically acceptable carriers include: buffer solutions, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides; 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 Tween, polyethylene glycol (PEG), and Pluronic acid.

[0260] If the pharmaceutical composition is provided in liquid form, the pharmaceutically acceptable carrier may be water (e.g., pyrogen-free water), isotropic saline, or a buffered aqueous solution, such as a phosphate buffer or a citrate buffer. Injection of the pharmaceutical composition may be performed in water or a buffer solution, such as an aqueous buffer containing a sodium salt (e.g., at least 50 mM sodium), a calcium salt (e.g., at least 0.01 mM calcium), or a potassium salt (e.g., at least 3 mM potassium). According to a specific embodiment, the sodium, calcium, or potassium salt may be present as its halide, such as chloride, iodide, or bromide, or as its hydroxide, carbonate, bicarbonate, or sulfate. Some examples of sodium salts include, but are not limited to, NaCl, NaI, NaBr, Na₂CO₂, NaHCO₂, and Na₂SO₄. Some examples of potassium salts include, for example, KCl, KI, KBr, K₂CO₂, KHCO₂, and K₂SO₄. Some examples of calcium salts include, for example, CaCl2, CaI2, CaBr2, CaCO2, CaSO4, and Ca(OH)2.

[0261] Additionally, the organic anions of the aforementioned cations may be included in the buffer solution. According to a specific embodiment, the buffer solution suitable for injection purposes as defined above may contain a salt selected from sodium chloride (NaCl), calcium chloride (CaCl2), or potassium chloride (KCl), wherein additional anions may be present. CaCl2 may also be replaced by another salt, such as KCl. In some embodiments, the salts in the injection buffer are present at a concentration of at least 50 mM sodium chloride (NaCl), at least 3 mM potassium chloride (KCl), and at least 0.01 mM calcium chloride (CaCl2). The injection buffer may be hypertonic, isotonic, or hypotonic relative to a specific reference medium, i.e., the buffer may have a higher, same, or lower salt content relative to the specific reference medium, wherein preferably, the aforementioned salts at concentrations that do not cause cell damage due to osmosis or other concentration effects can be used. The reference medium may be a liquid, such as blood, lymph, cytoplasm, other body fluids, or a common buffer solution. Such common buffer solutions or liquids are known to those skilled in the art. Lactated Ringer's solution is particularly preferred as a liquid base.

[0262] One or more compatible solid or liquid fillers, diluents, or encapsulating compounds may be suitable for human administration. As defined herein, the components of the pharmaceutical compositions according to the invention can be mixed with the nucleic acid carrier according to the invention in such a way that no interaction occurs that would significantly reduce the pharmaceutical efficacy of the (pharmaceutical) compositions according to the invention under typical conditions of use. Pharmaceutically acceptable carriers, fillers, and diluents may have sufficiently high purity and sufficiently low toxicity to be suitable for administration to individuals receiving treatment. Some examples of compounds that can be used as pharmaceutically acceptable carriers, fillers, or components are: sugars, such as lactose, glucose, trehalose, and sucrose; starches, such as corn starch or potato starch; dextrose; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; animal fats; solid glidants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, and corn oil; polyols, such as polypropylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; or alginic acid.

[0263] The choice of pharmaceutically acceptable delivery vehicle can be determined based on the method of administration of the pharmaceutical composition.

[0264] Suitable unit dosage forms for injection include sterile aqueous solutions, physiological saline, and mixtures thereof. The pH of such solutions can be adjusted to approximately 7.4. Suitable delivery vehicles for injection include hydrogels, devices for controlled or delayed release, polylactic acid, and collagen matrices. Suitable pharmaceutically acceptable delivery vehicles for topical application include those suitable for lotions, creams, gels, etc. If the pharmaceutical composition is intended for oral administration, tablets, capsules, etc., are preferred unit dosage forms.

[0265] Other additives that may be included in a pharmaceutical composition are emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; drug carriers; stabilizers; antioxidants; and preservatives.

[0266] The pharmaceutical compositions according to the invention can be provided in liquid or dry (e.g., lyophilized) form. In one specific embodiment, the nucleic acid carrier of the pharmaceutical composition is provided in lyophilized form. The lyophilized composition containing the nucleic acid carrier of the invention can be reconstituted in a suitable buffer prior to administration, advantageously based on an aqueous carrier, such as lactated Ringer's solution, Ringer's solution, or phosphate buffer solution.

[0267] In some embodiments of the invention, any DNA vector of the invention may be complexed with one or more cationic or polycationic compounds, such as cationic or polycationic polymers, cationic or polycationic peptides or proteins (e.g., protamine), cationic or polycationic polysaccharides, and / or cationic or polycationic lipids.

[0268] According to one specific embodiment, the DNA carrier 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 DNA carrier.

[0269] Lipid-based formulations can be effective nucleic acid delivery systems due to their biocompatibility and ease of large-scale production. Cationic lipids have been extensively studied as synthetic materials for nucleic acid delivery. After mixing, nucleic acids are condensed through cationic lipids to form lipid / nucleic acid complexes, known as lipid complexes. These lipid complexes can protect genetic material from nucleases and deliver it into cells by interacting with negatively charged cell membranes. Lipid complexes can be prepared by directly mixing positively charged lipids at physiological pH with negatively charged nucleic acids.

[0270] Conventional liposomes comprise a lipid bilayer, which can be composed of cationic, anionic, or neutral phospholipids and cholesterol, encapsulating an aqueous core. Both the lipid bilayer and the aqueous space can contain hydrophobic or hydrophilic compounds. The in vivo behavior and characteristics of liposomes can be modified by adding a hydrophilic polymer coating (e.g., polyethylene glycol (PEG)) to the liposome surface, thereby imparting steric stability. Furthermore, liposomes can be used for specific targeting by linking ligands (e.g., antibodies, peptides, and carbohydrates) to their surface or to the ends of the attached PEG chains.

[0271] Liposomes are delivery systems based on colloidal lipids and surfactants, consisting of a phospholipid bilayer surrounding an aqueous compartment. They can exist as spherical vesicles and can range in size from 20 nm to several micrometers. Cationic lipid-based liposomes can complex with negatively charged nucleic acids via electrostatic interactions, creating complexes that offer the possibility of large-scale production for biocompatibility, low toxicity, and in vivo clinical applications. Liposomes can fuse with the plasma membrane for uptake; once inside the cell, they are processed via endocytosis, and genetic material is subsequently released from the endosome / transporter into the cytoplasm.

[0272] 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-trimethylammonium methyl sulfate), can form complexes or lipid complexes with negatively charged nucleic acids, forming nanoparticles through electrostatic interactions, providing high in vitro transfection efficiency. Furthermore, neutral lipid-based nanoliposomes for nucleic acid vector delivery, such as those based on neutral 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), are also available.

[0273] Therefore, in one embodiment of the present invention, the circular DNA carrier is complexed with cationic lipids and / or neutral lipids to form liposomes, lipid nanoparticles, lipid complexes or neutral lipid-based nanoliposomes in the pharmaceutical composition of the present invention.

[0274] In one specific embodiment, the pharmaceutical composition comprises the circular DNA carrier of the present invention, which is formulated together with a cationic or polycationic compound and / or with a polymeric carrier. Therefore, in another embodiment of the invention, the circular DNA carrier as defined herein is associated or compounded with a cationic or polycationic compound or a polymeric carrier, optionally at a weight ratio selected from about 5:1 (w / w) to about 0.25:1 (w / w) of the nucleic acid carrier to the cationic or polycationic compound and / or to the polymeric carrier, 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) to about 1:1 (w / w), for example about 3:1 (w / w) to about 2:1 (w / w). The weight ratio of the nucleic acid carrier to the cationic or polycationic compound and / or to the polymeric carrier (w / w); or optionally, the nitrogen / phosphate (N / P) ratio of the nucleic acid carrier to the cationic or polycationic compound and / or the polymeric carrier is from about 0.1 to 10, for example, from about 0.3 to 4 or 0.3 to 1, for example, from about 0.5 to 1 or 0.7 to 1, for example, from about 0.3 to 0.9 or 0.5 to 0.9. For example, the N / P ratio of the circular DNA carrier to one or more polycations is from about 0.1 to 10, including from about 0.3 to 4, from about 0.5 to 2, from about 0.7 to 2, and from about 0.7 to 1.5.

[0275] The nucleic acid vectors described herein can also be associated with carriers, transfection agents, or complexing agents to improve transfection efficiency and / or the expression of regulatory genes according to the present invention.

[0276] In some cases, the circular DNA vector according to the invention is complexed with one or more polycations, preferably with protamine or oligofectamine. Other cationic or polycationic compounds that can be used as transfection or complexing agents may comprise cationic polysaccharides, such as chitosan, polygluconamine, cationic polymers, such as polyethyleneimine (PEI), cationic lipids, such as DOTMA: [1-(2,3-diolenoyloxy)propyl]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPE, LEAP, DOPE: diolenoylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: bis(octadecylamido)glycyl succinate, DIMRI: dimyristoyl-oxypropyl dimethylhydroxyethylammonium bromide, MAP: Dioleoyloxy-3-(trimethylammonium)propane, DC-6-14: O,O-bistetradecanoyl-N-(α-trimethylammoniumacetyl)diethanolamine chloride, CLIP1: rac-[(2,3-bisoctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-bishexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium, CLIP9: rac-[2(2,3-bishexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers, such as modified polyamino acids (e.g., β-amino acid polymers or reversed polyamides), modified polyethylene (e.g., PVP (poly(N-ethyl-4-vinylpyridine))), etc. Brominated acrylates (e.g., pDMAEMA (poly(dimethylaminoethyl methacrylate))), modified amide amines (e.g., pAMAM (poly(amide amine))), modified poly(β-amino esters) (e.g., diamine-terminated 1,4-butanediol diacrylate-copoly-5-amino-1-pentanol polymers, or polymers described in U.S. Patent No. 8,557,231; PEGylated PBAEs, such as those described in U.S. Patent Application No. 2018 / 0112038; any suitable polymers disclosed in Green et al., Acc. Chem. Res. 2008, 41(6): 749-759, such as diamine-terminated 1,4-butanediol diacrylate-copoly-5-amino-1-pentanol polymers; such as International Patent Publication No. WO 2020 / 077159 or WO Any suitable modified PBAE described in 2019 / 070727; such as PBAE 457 disclosed in Shen et al., Sci. Adv. 2020, 6: eaba1606, etc.), dendritic macromolecules, such as polypropylene dendritic macromolecules or pAMAM-based dendritic macromolecules, etc., polyimides, such as PEI: poly(ethyleneimine), poly(propyleneimine), etc., polyallylamine, polymers based on sugar backbones, such as polymers based on cyclodextrin, polymers based on dextran, chitosan, etc., polymers based on silan backbones, such as PMOXA-PDMS copolymers, etc., block polymers consisting of a combination of one or more cationic blocks (e.g., selected from cationic polymers as described above) and a combination of one or more hydrophilic or hydrophobic blocks (e.g., polyethylene glycol); etc.

[0277] According to one specific embodiment, the pharmaceutical composition comprises a DNA carrier encapsulated within or linked to a polymeric carrier. The polymeric carrier used according to the invention may be a polymeric carrier formed from cationic components crosslinked by disulfide bonds. The cationic components crosslinked by disulfide bonds may be the same or different from each other. The polymeric carrier may also contain additional components. Also particularly preferred is that the polymeric carrier used according to the invention comprises a mixture of cationic peptides, proteins, or polymers, and optionally additional components as defined herein, which are crosslinked by disulfide bonds as described herein. In this context, the disclosure of WO 2012 / 013326 is incorporated herein by reference. In this context, the cationic components forming the basis of the polymeric carrier through disulfide bond crosslinking are generally selected from any suitable cationic or polycationic peptide, protein, or polymer suitable for this purpose, particularly any cationic or polycationic peptide, protein, or polymer capable of complexing a nucleic acid carrier as defined herein or other nucleic acids contained in the composition, and thus preferably condensing the nucleic acid carrier. The cationic or polycationic peptide, protein, or polymer may be linear molecules; however, branched cationic or polycationic peptides, proteins, or polymers may also be used.

[0278] In some embodiments, any DNA vector described herein may be compounded with or used to encapsulate any component, peptide, or particle taught in International Patent Application No. PCT / US2023 / 065763, which is incorporated herein by reference.

[0279] Each disulfide-crosslinked cationic or polycationic protein, peptide, or polymer that can be used to composite a DNA carrier according to the invention, which is contained as part of a pharmaceutical composition, may contain at least one SH moiety (e.g., at least one cysteine ​​residue or any other chemical group exhibiting an SH moiety), which is capable of forming a disulfide bond upon condensation with at least one additional cationic or polycationic protein, peptide, or polymer that is a cationic component of the polymer carrier as described herein.

[0280] Such polymeric carriers for the DNA vectors of the present invention can be formed from disulfide-crosslinked cationic (or polycationic) components. In particular, such cationic or polycationic peptides, proteins, or polymers comprising at least one SH moiety or modified to comprise at least one SH moiety can be selected from proteins, peptides, and polymers used as conjugates.

[0281] In other embodiments, the DNA vector according to the invention can be administered naked in a suitable buffer solution without associating with any other carrier, transfection or conjugation agent.

[0282] VI. How to Use

[0283] This document provides methods (e.g., as part of a gene therapy regimen) for inducing the expression (persistent expression) of a target sequence (e.g., a therapeutic sequence) in a subject by administering any vector (e.g., a circular DNA vector, such as a circular DNA vector prepared by any method described herein) or a pharmaceutical composition thereof to the subject. The target cells or tissues of the subject can be characterized by examining the nucleic acid sequence of the host cells (e.g., RNA sequence, such as mRNA sequence), for example by Southern blotting or PCR analysis or RNA sequencing, to detect or quantify the presence (e.g., persistence) of the delivered therapeutic sequence. Alternatively, the expression of the therapeutic sequence in the subject can be characterized (e.g., quantitative or qualitative) by monitoring the progression of a disease treated with the delivered therapeutic sequence (e.g., association with a defect or mutation targeted by the therapeutic sequence). In some embodiments, transcription or expression of the therapeutic sequence (e.g., persistent transcription or persistent expression) is determined by observing a reduction in one or more symptoms associated with the disease.

[0284] Therefore, some embodiments of the present invention include a method of treating a subject's disease by administering any circular DNA vector or pharmaceutical composition thereof described herein to the subject. Any circular DNA vector or pharmaceutical composition thereof described herein may be administered to the subject in the following doses: 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 DNA; e.g., about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, or about 1 ... μg, approximately 80 μg, approximately 90 μg, approximately 100 μg, approximately 150 μg, approximately 200 μg, approximately 250 μg, approximately 300 μg, approximately 350 μg, approximately 400 μg, approximately 450 μg, approximately 500 μg, approximately 600 μg, approximately 700 μg, approximately 750 μg, approximately 1.0 mg, approximately 2.0 mg, approximately 2.5 mg, approximately 5.0 mg, approximately 7.5 mg, or approximately 10 mg DNA).

[0285] In some implementations, administration of a circular DNA vector or its pharmaceutical composition is less likely to induce an immune response in the subject compared to administration of other gene therapy vectors (e.g., plasmid DNA vectors and viral vectors).

[0286] In some cases, the circular DNA vectors and pharmaceutical compositions thereof provided herein are suitable for repeated administration due to their ability to transfect target cells without triggering an immune response or inducing a reduced immune response relative to a reference vector (e.g., plasmid DNA vectors or AAV vectors discussed above). Therefore, the present invention provides a method for repeated administration of the circular DNA vectors and pharmaceutical compositions described herein. Any of the aforementioned dosages can be repeated at appropriate frequencies and durations. In some embodiments, the subject receives a dose at a frequency not exceeding about once a month, or at a frequency not exceeding about once every three months, or at a frequency not exceeding about once every four months, or at a frequency not exceeding about once every six months (e.g., not exceeding twice a year), or at a frequency not exceeding once a year. In some embodiments, the amount and frequency of the dose correspond to the turnover rate of the target cells. It will be understood that in long-lived post-mitotic target cells transfected using the vectors described herein, a single dose of the vector may be sufficient to maintain the expression of a heterologous gene within the target cells for a considerable period. Therefore, in other embodiments, the circular DNA vectors provided herein can be administered to the subject as a single dose. The number of times the circular DNA vector is delivered to the subject may be the number of times necessary to maintain clinical (e.g., therapeutic) benefit.

[0287] The method of the present invention includes administering a DNA vector or a pharmaceutical composition thereof via any suitable route. The circular DNA vector or a pharmaceutical composition thereof may be administered systemically or locally, for example, intravenously, via the eye (e.g., intravitreal, subretinal, via eye drops, intraocular, intraorbital), intramuscularly, intradermally, intrahepatically, intracerebrally, intramuscularly, percutaneously, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, intratumorally, subcutaneously, subconjunctivally, intracysticly, intramuscularly, via mucosa, intraperitoneally, intraumbilically, via oral, superficially, transdermally, via inhalation, via aerosolization, via injection (e.g., via jet injection), via electroporation, via implantation, via infusion (e.g., via continuous infusion), via direct local perfusion of bathing target cells, via catheter, via irrigation, in the form of an emulsion, or in the form of a lipid composition.

[0288] The DNA vectors described herein can be delivered into cells via in vivo electrotransfer (e.g., in vivo electroporation). In vivo electroporation has been demonstrated in certain tissues, such as skin, skeletal muscle, certain tumor types, and lung epithelium. Delivery of naked DNA into cells via in vivo electroporation involves applying DNA to a target tissue and then applying an electric field to temporarily increase the permeability of the cell membrane within the tissue by creating pores, allowing the DNA molecules to cross the cell membrane. As an example, delivery to skin using in vivo electroporation is described in Cha & Daud Hum. Vaccin. immunother. 2012, 8(1 1):1734-1738, which is incorporated herein by reference in its entirety; and in vivo electroporation of skeletal muscle is described in Sokolowska & Blachnio-Zabielska,Int. J. Molecular Sci. 2019, 20:2776, which is incorporated herein by reference in its entirety. Intratumoral delivery using in vivo electroporation is described in Aung et al. Gene Therapy 2009, 16:830-839, which is incorporated herein by reference in its entirety. In vivo electroporation of DNA into lung cells is described in Pringle et al. J. Gene Med. 2007, 9:369-380, which is incorporated herein by reference in its entirety. In vivo electrotransfer of circular DNA vectors to cells of the eye (e.g., retinal cells / photoreceptor cells) is described in International Patent Publication No. WO 2022 / 198138, which is incorporated herein by reference in its entirety. In some cases, after the circular DNA vector is applied to the eye, electrodes can be placed inside the eye (e.g., within approximately 1 mm of the retina), and an electric field can be transmitted to the target ocular tissue via the electrodes under conditions suitable for electrotransferring the circular DNA vector to the target cells (e.g., by applying six to ten pulses of 10 to 100 V each). Devices and systems having electrodes suitable for transmitting electric fields in mammalian tissues are commercially available and can be used in the methods disclosed herein. In some cases, the electric field is transmitted via electrodes comprising needles (e.g., needles placed in the vitreous body or subretinal cavity). Suitable needle electrodes include CLINIPORATOR® electrodes sold by IGEA® and needle electrodes sold by AMBU®. The methods of the present invention involve administering any circular DNA carrier or pharmaceutical composition thereof described herein to the skin, skeletal muscle, tumors (including, for example, melanoma), eye, and lung via in vivo electrotransfer.

[0289] In some embodiments, the DNA vector or pharmaceutical composition thereof carrying an ocular gene (e.g., ABCA4) described in International Patent Publication No. WO 2022 / 198138 is administered to the eye via electroporation. This International Patent Publication is incorporated herein by reference in its entirety, and the therapeutic product encoded by the ocular gene (e.g., ABCA4) is expressed in target ocular cells (e.g., retinal cells, such as RPE cells or photoreceptor cells). In some embodiments, expression of the ocular gene persists for at least 6 months, e.g., at least 12 months, following administration of the DNA vector. Such a method can be used to treat ocular diseases, such as retinal diseases or conditions (e.g., ABCA4-related retinal diseases).

[0290] In some embodiments, a DNA vector or pharmaceutical composition thereof carrying a respiratory system gene (e.g., the cystic fibrosis transmembrane receptor (CFTR) (e.g., human CFTR) described in this disclosure, administered to the airway (e.g., the lungs) according to the methods described in International Patent Application No. PCT / US2022 / 082365 or International Patent Publication No. WO 2023 / 064934, which is incorporated herein by reference in its entirety (e.g., via respiratory administration and / or in vivo electroporation), and the therapeutic product encoded by the respiratory system gene (e.g., CFTR) is expressed in target airway cells (e.g., respiratory epithelial cells). Such methods can be used to treat respiratory diseases, such as cystic fibrosis.

[0291] In some embodiments, a DNA vector or pharmaceutical composition thereof carrying a therapeutic sequence (e.g., a regulatory protein-coding sequence, such as an immunomodulatory protein-coding sequence, or a gene-coding sequence encoding a dendritic cell chemical attractor, a dendritic cell growth factor or activator, and / or a lymphocyte signaling protein) is delivered to a subject (e.g., at or around a tumor) via electroporation, according to the method described in International Patent Application No. PCT / US2022 / 017575, which is incorporated herein by reference in its entirety, and the therapeutic product encoded by the therapeutic sequence is expressed in the subject (e.g., in an amount sufficient to modulate an immune response (e.g., to the tumor)). Such a method can be used to treat diseases or conditions, such as cancer.

[0292] As a supplement or alternative, the DNA vector or its pharmaceutical composition can be administered ex vivo to host cells (e.g., eukaryotic, mammalian, or human cells), for example, by removing (or otherwise deriving from) cells from a subject (e.g., a human patient), followed by (e.g., re-implanting the host cells into the subject after selecting cells incorporating the vector). Thus, in some aspects, this disclosure provides transfected host cells, their cultures, their pharmaceutical compositions, and methods of administering them to treat a disease or ailment of a subject or to administer them to another subject, for example, via allogeneic transfer.

[0293] Therefore, the present invention includes a method for treating a subject suffering from a disease or condition by applying the DNA vector (or combination thereof) of the present invention to the subject (e.g., a human subject) using techniques described herein and known in the art.

[0294] The assessment of transfection efficiency (e.g., expression and / or persistence) of any DNA vector described herein can be performed using any method known in the art or described herein. Isolation of transfected cells can also be performed according to standard techniques. For example, cells containing a therapeutic gene may express a visible marker encoded by the sequence of the heterologous gene, such as a fluorescent protein (e.g., GFP) or other reporter protein, which helps in the identification and isolation of one or more cells containing the heterologous gene. Cells containing a therapeutic gene can also be characterized by examining the host cell's nucleic acid cellularity (e.g., RNA sequence, such as mRNA sequence), for example by Southern blotting or PCR analysis, to determine the presence of the heterologous gene contained in the vector.

[0295] Therefore, the method of the present invention includes administering a circular DNA vector encoding any target sequence (e.g., a therapeutic sequence) as described herein to a subject, followed by detection of gene expression in the subject. Expression can be detected one to four weeks after administration, one to four months after administration, four months to one year after administration, one 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). The persistence of the circular DNA vector (e.g., episome persistence) can be observed at any of these detection time points.

[0296] VII. Medicine boxes and products

[0297] In another aspect of the invention, articles or kits comprising any circular DNA vector or a pharmaceutical composition thereof are described herein. The article comprises a container and markings or instructions for use on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. Containers may be made of a variety of materials, such as glass or plastic. The container contains a composition that is effective, alone or in combination with another composition, for the treatment, prevention, and / or diagnosis of a condition, and may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle). At least one active agent in the composition is a circular DNA vector of the present invention or a pharmaceutical composition comprising such a circular DNA vector. The markings or instructions for use indicate that the composition is intended to treat a condition that can be treated through its contents. Furthermore, the article may comprise (a) a first container containing a composition comprising a circular DNA vector or a pharmaceutical composition thereof; and (b) a second container containing a composition comprising an additional therapeutic agent. The article may also include instructions for use indicating that the composition can be used to treat a specific condition. As an alternative or supplement, the article may also include a second (or third) container containing a pharmaceutically acceptable carrier, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, dextran solution, or any of the pharmaceutically acceptable carriers disclosed above. It may also contain other materials desired from a commercial and user perspective, including additional buffers, diluents, filters, needles, syringes, or other delivery devices.

[0298] Example

[0299] Example 1. ColE2-P9 replication origin confers stable plasmid maintenance

[0300] To test whether the ColE2-P9 origin of replication (ori) located within the plasmid could confer stable plasmid maintenance in *E. coli*, plasmids containing the R6K origin, a carbenicillin resistance marker, and one of three variants of ori (SEQ ID NO: 2 to 4) were constructed and prepared in pir+ cells. The plasmids were transformed into S1037 cells and selected on LB agar plates supplemented with carbenicillin. Colonies from each plate were cultured in antibiotic-free LB agar at 37°C. Overnight cultures were diluted 1000-fold in fresh antibiotic-free LB agar and cultured at 37°C. After five passages, each overnight culture was streaked again on antibiotic-free LB agar plates and cultured overnight at 37°C. Twenty colonies from each plate were inoculated onto LB agar plates with or without carbenicillin. After a total of six days (during which an estimated ≥210 population doublings occurred), all origin variants had normal colony sizes, and plasmids were detected in all 20 colonies of all three origin variants. Figure 1 These results indicate that a 40 bp origin of replication allows the plasmid to remain stable during multiple population doublings in E. coli.

[0301] Example 2. Generation of parental plasmids for circular DNA vectors

[0302] Figure 2 An example of a parent plasmid generation process for producing a circular DNA vector according to an embodiment disclosed herein is shown. Individual components of the transcription unit are assembled into a transcription unit via Golden Gate assembly. The transcription unit is then assembled into a parent plasmid via Golden Gate assembly. In this embodiment, the parent plasmid contains a LoxP recombination site sequence flanking the vector sequence (containing segments of the ori and MYO7A genes) and separating the vector sequence from the backbone sequence (containing segments of the SpecR, KanR, and RFP genes).

[0303] Example 3. Generation of circular DNA vectors from parental mitochondria

[0304] Figure 3The experimental procedure for generating a circular DNA vector in vivo is illustrated. A test parent plasmid containing a vector sequence flanked by LoxP recombination sites was generated using the Golden Gate assembly method. The test vector sequence of the test parent plasmid contains a ColE2-P9 ori sequence and a reporter gene (sfGFP). For the purposes of this experiment, the test vector sequence also contains a chloramphenicol resistance gene (CmR), although some embodiments of the circular DNA vector described herein lack an antibiotic resistance gene. The test parent plasmid also contains a test backbone sequence containing the antibiotic resistance genes SpecR and KanR, as well as a reporter gene (RFP). The test parent plasmid was transformed into modified *E. coli* cells possessing the ColE2-P9 Rep gene integrated into the genome under the control of a constitutive promoter (J23119). Additional versions of the test parent plasmid were prepared, which had two I-Ppol restriction sites, two I-Scel restriction sites, two PI-Scel restriction sites, two I-Ceul restriction sites, or one PI-Pspl restriction site and one Scel restriction site located flanking the test vector sequence, instead of the LoxP site located flanking the test vector sequence. The test parent plasmid with the LoxP site was named p1603, and the test parent plasmid with two PI-Scel restriction sites was named p1600.

[0305] Bacterial colonies containing the parental test plasmid were identified by positive RFP fluorescence. Cre recombinase was electroporated into cells containing p1603 to induce recombination at the LoxP site and generate a test circular DNA vector. The Cre electroporation procedure was as follows: Electrocompetent cells of *E. coli* containing the parental plasmid were cultured in SOB at 30°C until an OD of 0.8 was reached. The *E. coli* were washed three times with ice-cold 10% glycerol and resuspended in 10% glycerol. 1 μl of Cre (15 units, NEB, M0298M) was mixed with 50 μl of electrocompetent cells. The mixture was transferred to cuvettes (1 mm slit) and electroporated using a BTX electroporator at 1800 volts. Cells were rescued by incubation in SOC at 37°C for 1 hour, and the cells were seeded onto antibiotic-free LB agar plates. Colonies were allowed to grow, and DNA was purified using a QIAGEN miniprep kit. Electroporated cells were spread onto antibiotic-free LB plates. GFP-positive colonies on the LB plates were streaked onto LB plates containing kanamycin (Kan) and spectinomycin (Spec) to test for loss of backbone sequences (Kan / Spec-sensitive colonies).

[0306] Figure 4Agarose gel electrophoresis of purified extragenomic DNA from single-colony cultures is shown, and (1) test circular DNA vectors generated by Cre electroporation of cells containing p1603 are shown, and (2) test circular DNA vectors maintained in cells without selective pressure are shown. Lanes 2, 3, and 5 show extragenomic DNA derived from p1603-transformed cells that were GFP-positive and Kan / Spec-sensitive after Cre electroporation and grown in chloramphenicol-rich medium. The bands in these lanes migrated to approximately 1500 bp, which is the expected size of the test circular DNA vectors, indicating that Cre electroporation resulted in the generation of the test circular DNA vectors. Lanes 8, 9, and 11 correspond to lanes 2, 3, and 5, respectively, but were grown in chloramphenicol-rich medium. The DNA abundance in lanes 2, 3, and 5 was similar to that in lanes 8, 9, and 11, indicating that the circular DNA vectors were maintained in cells without selective pressure. Lane 4 shows extragenomic DNA derived from cells transformed with p1603 that were GFP-positive, RFP-positive, and Kan / Spec resistant after Cre electroporation. The band in this lane migrates to approximately 5000 bp, which is the expected size for testing the parent plasmid. Lane 10 corresponds to lane 4 but shows DNA from cells grown in a chloramphenicol-containing medium. Lane 1 shows extrachromosomal DNA purified from GFP-positive, RFP-positive, Kan / Spec resistant colonies derived from cells transformed with p1600. The band in this lane migrates to approximately 5000 bp, which is the expected size for testing the parent plasmid. Lane 7 corresponds to lane 1 but shows DNA from cells grown in a chloramphenicol-containing medium. Lane 6 shows extrachromosomal DNA purified from the same modified *E. coli* cells transformed with a plasmid lacking ColE2-P9ori and grown in a chloramphenicol-free medium. No detectable plasmids were recovered from this culture, which may indicate that ori is required to maintain the plasmids even without selective pressure. Lane 12 shows DNA purified from a culture of the same cells used in lane 6 but grown in the presence of chloramphenicol. Plasmids were recovered from these cells, which may indicate that selective pressure maintained the plasmids in the cells.

[0307] Example 4. Circular DNA vectors with ColE2-P9 ori maintained in modified cells expressing ColE2-P9 replication protein under no selective pressure.

[0308] To test the ability of a circular DNA vector containing 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) without selective pressure, cells containing the test circular DNA vector generated via Cre recombination as described in Example 2 were cultured in various broths containing and without chloramphenicol. The percentage of sfGFP-positive cells in each culture was quantified. Results were presented in... Figure 5 As shown in the figure. The tested circular DNA vector was maintained at a high level in all chloramphenicol-containing and non-chloramphenicol-containing media, except for SOC medium, which had a lower maintenance level in both chloramphenicol-containing and non-chloramphenicol-containing conditions.

[0309] The test circular DNA vectors were purified and quantified from chloramphenicol-free TB and ZB cultures. TB culture produced 0.33 mg / L of the test circular DNA vectors, and SB culture produced 0.45 mg / L. These results indicate that the circular DNA vectors were maintained regardless of selective stress.

[0310] Example 5. Process for generating circular DNA vectors in vivo using anti-selection

[0311] Figure 6 An exemplary process for generating the circular DNA vector of the present invention using anti-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 transgene cassettes. On day 0, competent modified bacterial cells expressing the Rep gene are prepared using any of the processes described herein (e.g., by transforming cells with a recombinase encoded on a bacterial artificial chromosome (BAC)). On day 1, cell plates are inoculated onto LB agar plates supplemented with Kan and a template plasmid is added. In this example, the template plasmid contains the origin of replication (ori) and the ABCA4 transgene downstream of the promoter. The flanking sides of this ori-ABCA4 cassette are recombination sites (attP-GA and attB-GA). The opposite side (backbone region) of the plasmid contains selection markers: the antibiotic resistance genes SpR and KanR, the anti-selection marker PheS, and the fluorescent marker RFP. On day 2, white colonies are picked from red colonies and grown in LB agar supplemented with 4 CP for anti-selection. On day 3, the circular DNA vector was purified.

[0312] Example 6. Testing the generation of circular DNA vectors by inducible Bxb1

[0313] To test whether Bxb1, as an exogenous recombinase, can efficiently generate circular DNA vectors, the Bxb1 recombinase was encoded on a bacterial artificial chromosome (BAC) and transformed into host *Escherichia coli*, which possesses a Rep gene (SEQ ID NO: 1) integrated into its genome and driven by a constitutive promoter. Figure 7A Two inducible Bxb1 BACs were tested: 1696 containing the kumaric acid inducible promoter and the chloramphenicol (Cm) resistance (CmR) gene. Figure 7B ; SEQ ID NO: 5), and 1697 (containing an arabinose inducible promoter and the CmR gene) Figure 7C (SEQ ID NO: 6). Each BAC was transformed into S1037 cells by electroporation and plate-seeded in the presence of chloramphenicol.

[0314] Next, the cells were transformed using a template plasmid carrying GFP as the reporter transgene. Figure 7D The ColE2-P9 origin of replication (ori) is located upstream of GFP and its promoter, and the recombination sites (attP-GA and attB-GA) are located on the flanks of the ori-GFP box. The contralateral side (backbone region) of the plasmid contains selection markers (antibiotic resistance genes SpR and KanR, anti-selection marker PheS, and fluorescent marker RFP). Therefore, cells containing the template plasmid are GFP+, RFP+, Kan-resistant, Spec-resistant, and 4CP-sensitive, while cells containing only the circular DNA vector (…) Figure 7E (No skeletal byproducts) Figure 7F It is GFP+, RFP-, Kan-sensitive, Spec-sensitive, and 4CP resistant.

[0315] The template plasmid was electroporated into S1037 cells without an inducer and plated on Cm+Kan plates. Results at 24 and 72 hours post-transformation are shown in Figures 8 and 9. Figure 8A and 8B As shown, most colonies containing 1696 BAC were green at 24 and 72 hours post-transformation, respectively. A few red colonies were observed. Green colonies were selected, and the presence and sequence of the circular DNA vector were determined by Sanger sequencing and gel electrophoresis. Compared to 1696, Figure 9A and 9B As shown, most colonies containing 1697 BAC were yellow at 24 and 72 hours post-transformation, while a few green colonies were observed at 72 hours. Figure 9B ).

[0316] To assess the effects of Cm and arabinose inducers, colonies of various colors were selected from each plate and incubated with Cm and either cumuloic acid or arabinose for 24 hours. Results were... Figure 10 (1696) and Figure 11 As shown in (1697). Next, each culture was diluted 500-fold and incubated overnight in LB agar supplemented with 4 CP, with or without an inducer, for anti-selection. The overnight cultures were then streaked onto plain LB agar plates and fluorescence was observed. Results are shown in Figure 12 (1696) and Figure 13 The results are shown in (1697). Single colonies from each plate were cultured overnight in LB supplemented with 4 CP, prepared in small batches, and mapped using digestion maps (Bsal). Gel electrophoresis results are shown in... Figure 14 The predicted bands for each expected category 1 to 4 are shown in Table 1 below:

[0317] Table 1. Predicted bands for each digestible species

[0318]

[0319]

[0320] For colonies containing either type of BAC (1696 or 1697), the addition of either inducer appeared to increase GFP-expressing colonies, indicating that Bxb1 expression occurs in the absence of an inducer. Indeed, Bxb1 recombination occurred after transformation of the template plasmid and subsequent inoculation onto Cm / Kan plates. In 1606 BAC containing bacteria, non-inducible Bxb1 produced >90% green colonies, indicating that over 90% contained circular DNA vectors with little or no template plasmid.

[0321] Example 7. Generation of a circular DNA vector containing therapeutic transgenes

[0322] In this study, circular DNA vectors containing several types of therapeutic transgenes were prepared: (1) ABCA4, (2) IL-12, and (3) tricistronic cassettes encoding Flt3L, IL-12, and XCL1. The ABCA4 and IL-12 constructs contained CAG promoters, and the tricistronic constructs contained upstream CAG promoters for each of the three genes. Exemplary sequences of the ABCA4 template plasmid and the resulting circular DNA vectors were obtained from [the study / study]. Figure 15A (SEQ ID NO: 7) and Figure 15B (SEQ IDNO: 8) is used to illustrate this.

[0323] First, S1037 cells were transformed with 1696 BAC and allowed to grow overnight. One day later, the cells were made competent and transformed with template plasmids encoding ABCA4, IL-12, or a tricistronic cassette. Cell plates were seeded on LB agar plates supplemented with Kans. After three days of culture, small white colonies were selected (leaving red colonies) and grown overnight in LB+4CP for anti-selection prior to purification.

[0324] Purified constructs were screened from individual colonies in each group using Bsal-mapped digestion maps. All colonies produced circular DNA vector bands of the expected size, such as... Figure 16A (Theoretical gel spectroscopy) and Figure 16B As shown in the (actual gel spectra). These results demonstrate that the circular DNA vector generation process described herein can be widely used to efficiently generate circular DNA vectors with a variety of sizes and structures, such as polycistronic transgenes.

[0325] Example 8. Stability of circular DNA vectors during bacterial growth and scale-up.

[0326] A significant advantage of the vector system described herein is its ability to allow bacterial cells containing circular DNA vectors to grow in the absence of selectivity markers (e.g., after selectivity markers and other bacterial scaffold elements have been removed from the culture). To this end, the applicant tested whether circular DNA vectors containing therapeutic transgenes could be stably expressed in bacterial cultures during multiple cell divisions associated with scale-up vector production.

[0327] As in Example 7, cells identified as containing a circular DNA vector encoding ABCA4 but lacking a backbone were cultured overnight for seven consecutive nights (14 to 16 hours each). After the seventh culture, the presence of the circular DNA vector was confirmed by sequencing. Based on an average division rate of three divisions per hour, the culture underwent at least 294 divisions, and unexpectedly, the expression of the circular DNA vector was maintained despite the absence of selection.

[0328] Taking advantage of this remarkable stability, the cells were scaled up to produce larger quantities of circular DNA vectors containing the ABCA4 transgene. Cells were re-stretched onto LB plates and grown to produce a 2.5 L formulation (prep). A glycerol stockpile was produced and re-stretched to produce a 25 L formulation. The circular DNA vector was purified from this formulation, which yielded 18 mg of circular DNA vector.

[0329] Example 9. Detection of monomeric circular DNA vectors.

[0330] In this study, circular DNA vectors containing the ABCA4 transgene, generated using 1696 BAC, were sequenced to determine that the circular DNA vectors were in monomeric form (unlike dimers, which can be generated by trans recombination with another template plasmid). S1037 bacteria were cultured at 37°C for 2 hours in LB broth containing 25 μg / mL Kan. Next, the cells were transferred to plates containing 4 CP and 10 ng DNA and incubated overnight at 37°C. Samples were analyzed using conventional methods via long-read sequencing (Oxford Nanopore). Figure 17A As shown, monomeric peaks were observed, and no dimers were observed. In contrast, when the culture was incubated overnight with Kan, the circular DNA vector was predominantly dimerized. Figure 17B ).

[0331] Example 10. Circular DNA vector prepared using Bxb1 helper plasmid

[0332] As an alternative strategy to BAC-Bxb1, a helper plasmid is used to prepare a circular DNA vector using Bxb1 transformed into host cells. An exemplary helper plasmid is shown in... Figure 18 As shown, it contains a cumate-inducible promoter (CuO). Additionally, the helper plasmid contains a temperature-sensitive backbone, allowing its removal after the generation of the circular DNA vector. The DNA sequence of this helper plasmid is given by SEQ ID NO: 11. The host cell used in this method is the same as in similar examples, the S1027 cell, which has a Rep (SEQ ID NO: 1) integrated into the host genome and driven by a constitutive promoter. The template plasmid is the same as that in Example 6 (…). Figure 7D ).

[0333] In this study, helper plasmids were transformed into S1037 cells and incubated overnight with 100 μg / mL carbenicillin (carb100). The plasmid templates were then transformed and seeded onto carb100 and 500 μM Cuma plates, and the cells were grown at 30°C. Using this helper plasmid method, several green colonies were observed. Figure 19 This indicates that cells containing circular DNA vectors without scaffold byproducts were successfully generated.

[0334] Example 11. Preparation of circular DNA vectors by integrating Bxb1 into the host genome

[0335] Another source of recombinases described in this article is through integration into the bacterial host genome. In this example, Bxb1 is integrated into... Figure 20The procedure was performed after the steps shown in the diagram. First, S1037 cells containing the λ-red recombinant helper plasmid were grown with 0.2% arabinose to make them electrocompetent. Then, 500 ng of linearized 1696 BAC was electroporated. After BAC 1696 was integrated into the rsd-thiC locus, the λ-red recombinant plasmid was removed.

[0336] Colony PCR was performed on the obtained colonies, and positive clones were identified by gel electrophoresis. Figure 21 The resulting cells were engineered to express Rep and Bxb1 through genome integration.

[0337] Example 12. Circular DNA vector generated by transposition

[0338] The method of generating circular DNA vectors using bacteria that separate plasmid backbone components from therapeutic sequences using recombinases produces recombination traces, which can result in dozens of additional base pairs within the backbone that are foreign to the host. For example, the method described herein using recombinases produces a recombination trace of 43 base pairs. Thus, the recombination trace, together with the ColE2-P9 origin of replication, produces a backbone such that more than 70 nucleotide bases directly link the 3' end of the therapeutic sequence to the 5' end of the therapeutic sequence. While this size is significantly smaller than the backbone size (more than 400 bases) taught in the generation of other circular DNA vectors containing origins of replication, the inventors sought to further reduce the backbone size to simplify the backbone structure, thereby reducing safety risks and the possibility of silenced expression. To this end, a transposase was used instead of a recombinase in the following method, which... Figure 22 As shown in the image.

[0339] The plasmid template contains a first segment containing ori (which corresponds to the Rep gene integrated into the host bacterial cell) and the target sequence (promoter and reporter gene (sfGFP) (the reporter gene is replaced with a therapeutic sequence to produce a therapeutic circular DNA vector)). The first segment is flanked by two transposase overhangs (To), adjacent to a left-hand repeat (inverted repeat left; IRL) and a right-hand repeat (inverted repeat right; IRR). On the other side of the IRR and IRL is the second segment of the plasmid backbone, containing the drug resistance genes SpecR and KanR, and the RFP reporter gene. Here, RFP is used only for biomarker purposes (it can be easily omitted to accommodate therapeutic vector production).

[0340] The host bacterial cell also contains a second circular construct that encodes a transposase and an IEE, and contains a second origin of replication (ts ori).

[0341] Bacterial cells were cultured under standard conditions that allowed for the expression of transposase and IEE. The transposase protein acted on the plasmid template to cleave the second segment (plasmid backbone) from the first segment (target sequence). As part of the transposition process, each of the first and second segments was recirculated to produce two sub-constructs: (1) a circular DNA vector containing the sfGFP target sequence, ori, and one of the transposase overhangs, and (2) a backbone byproduct containing an RFP reporter, two drug resistance genes, and two inverted repeats.

[0342] As bacterial cells grow, the Rep protein is expressed via the Rep gene in the genome, which in turn acts on ori to continuously replicate the circular DNA vector. As the circular DNA vector replicates, backbone byproducts are diluted from the cell culture. This process can be monitored using GFP and RFP marker genes to identify colonies containing only the target sequence without a backbone. With the maintenance of the culture and the selection of desired colonies, the final host cell population contains a pure population of circular DNA vectors without plasmid backbone byproducts.

[0343] Example 13. Identification of functional variants of the ColE2-P9 replication origin

[0344] This embodiment describes a systematic study of the effects of shortening the ColE2-P9 replication origin, starting from both sides of the 5' and 3' ends. The results presented herein provide information on many suitable functional variants of ColE2-P9 and describe points where further truncation is detrimental to function.

[0345] ColE2P-9 replication origin is at Figure 23A The top shows a series of missing variants, namely markers 55001 to 550036. Figure 23A The bottom portion is shown. The deletion (indicated by dashed lines) was introduced into a circular DNA vector, and S1037 *E. coli* cells were transformed with each variant and grown on carb plates. Colony size was observed. Colony size is shown on each culture plate. Figure 23B As shown in the figure. The size of the colony is visualized and characterized, and... Figure 23A The corresponding variant is labeled on the right-hand side.

[0346] Normal colony size was observed in cultures transfected with a DNA vector containing the origin of replication, where the origin of replication was truncated by two and three bases on the right side (55002 and 55003), and by four bases on the left side (55004) (“right side” and “left side” correspond to…). Figure 23A(The sequence orientation is shown in the figure). However, when only one more base was removed from the left side relative to 55004 (55005), mixed colony sizes were observed. Removing another base from the left side also resulted in mixed colony sizes (55006). And, unexpectedly, removing one more base from the left side completely terminated cell growth (55007), revealing a critical boundary for truncation while maintaining the ColE2-P9 replication origin. Variants 55004 to 55006 were then tested, in which three bases from the right side were removed from each (55008 to 55010). Such truncation had no effect on colony size relative to the corresponding variants (i.e., 55008 colony size was consistent with 55004 (normal), 55009 colony size was consistent with 55005 (mixed), and 55010 colony size was consistent with 55006 (mixed)). These tests indicated that the three bases on the right (CCT) are dispensable for the ColE2-P9 replication origin function. Next, the remaining bases on the right were removed and tested in cells 55033 to 55036, and all of these bases showed normal colony growth.

[0347] In summary, these results indicate that while certain bases at the ColE2-P9 replication origin can be omitted (thus enabling improved vector design by reducing backbone size), removal of the left-hand A, as performed in 55007, is not permissible and results in loss of replication origin function.

[0348] Example 14. ABCA4 protein expression via a circular DNA vector with ColE2-P9 ori.

[0349] To determine whether a circular DNA vector with a ColE2-derived origin of replication produced by bacteria could express proteins in human cells, an in vitro study was conducted in which such a circular DNA vector carrying the human ABCA4 gene (SEQ ID NO: 21) was transfected into HEK293T cells, and ABCA4 protein expression was evaluated by Western blotting.

[0350] HEK293T cells were seeded at 150,000 cells per well in 0.5 mL of standard medium in 24-well plates. The plates were incubated at 37°C for 24 hours. At transfection, the cells were 60% to 80% confluence. Cells were transfected using a circular DNA vector with Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol. The total amount of DNA added to each well was 500 ng. After incubation at 37°C for 24 hours, cells were harvested for Western blot analysis, using β-actin as a control. Western blot results are shown in [link to data]. Figure 24As shown in the table, each lane is identified as shown in Table 2 below.

[0351] Table 2: Sample Identification for Protein Expression Assays

[0352] swimming lane sample 1 & 2 Lipofectamine only (negative control) 3 & 4 Synthesized circular DNA (positive control) 5 to 14 Bacterial-derived circular DNA

[0353] These results demonstrate that a bacterial circular DNA vector with a ColE2-derived origin expresses its ABCA4 transgene in HEK293T human cells.

[0354] sequence

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[0380]

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Claims

1. A circular DNA vector or a pharmaceutical composition thereof, wherein the circular DNA vector comprises a therapeutic sequence and a sequence comprising a bacterial origin of replication, wherein the sequence comprising the bacterial origin of replication is less than 50 bp in length, and wherein the circular DNA vector lacks a selectable marker.

2. The circular DNA vector of claim 1, wherein the vector lacks a recombination site.

3. The circular DNA vector of claim 1 or 2, wherein the vector comprises a transposase footprint.

4. The circular DNA vector of claim 1, wherein the sequence comprising the bacterial origin of replication directly links the 3' end of the therapeutic sequence to the 5' end of the therapeutic sequence.

5. The circular DNA vector of any one of claims 1 to 4, wherein the vector has about 200 base pairs (bp) or less, or about 150 bp or less, or about 100 bp or less, or about 75 bp or less, or about 50 bp or less of sequence of bacterial origin.

6. The circular DNA vector of claim 5, wherein the origin of replication is from a ColE2-related plasmid, and which is optionally ColE2-P9.

7. The circular DNA vector of claim 6, wherein the origin of replication is recognized by a ColE2-P9 replication protein.

8. The circular DNA vector of claim 7, wherein the ColE2-P9 replication protein comprises the amino acid sequence of SEQ ID NO:

1.

9. The circular DNA vector of any one of claims 6 to 8, wherein the origin of replication is 40 bp or less in length.

10. The circular DNA vector of claim 9, wherein the origin of replication is 36 bp or less in length, or 34 bp or less in length, or 32 bp or less in length, or 30 bp or less in length, or 28 bp or less.

11. The circular DNA vector of claim 9 or 10, wherein the origin of replication has the nucleotide sequence of SEQ ID NO: 2, or a functional variant or a truncation variant thereof.

12. The circular DNA vector of claim 11, wherein the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication comprises or consists of the nucleotide sequence of SEQ ID NO:

12.

13. The circular DNA vector of claim 11, wherein the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication comprises or consists of the nucleotide sequence of SEQ ID NO:

13.

14. The circular DNA vector of claim 11, wherein the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication comprises or consists of the nucleotide sequence of SEQ ID NO:

14.

15. The circular DNA vector of claim 11, wherein the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication comprises or consists of the nucleotide sequence of SEQ ID NO:

15.

16. The circular DNA vector of claim 11, wherein the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication comprises or consists of the nucleotide sequence of SEQ ID NO:

16.

17. The circular DNA vector of claim 11, wherein the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication comprises or consists of the nucleotide sequence of SEQ ID NO:

17.

18. The circular DNA vector of claim 11, wherein the origin of replication is a truncated ColE2-P9 origin of replication, wherein one strand of the origin of replication comprises or consists of the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

19. The circular DNA vector of claim 11, wherein the origin of replication comprises the nucleic acid sequence of, or consisting of, SEQ ID NO: 18, wherein each X is selected from A, T, C, or G.

20. The circular DNA of claim 19, wherein: X1 is A, T, or C; X2 is A, T, or C; X3 is A, T, or G; X4 is A, T, or C; X5 is A, T, or G; X6 is C; X7 is A.

21. The circular DNA vector of any one of claims 1-20, wherein the circular DNA vector is a non-integrating circular DNA vector.

22. The circular DNA vector of claim 21, wherein the therapeutic sequence comprises a scaffold-matrix attachment region (S / MAR).

23. The circular DNA vector of any one of claims 1-22, wherein the therapeutic sequence is 4.5 kb or greater.

24. The circular DNA vector of any one of claims 1-23, wherein the circular DNA vector is monomeric and supercoiled.

25. The circular DNA vector of any one of claims 1-24, wherein the therapeutic sequence comprises an ocular gene.

26. The circular DNA vector of claim 25, wherein the ocular gene is MY07A, BEST1, CFH, CEP290, USH2A, ADGRV1, CDH23, CRB1, PCDH15, RPGR, ABCA4, ABCC6, RIMS1, LRPS, CC2D2A, TRPM1, C3, IFT172, COL11A1, TUBGCP6, KIAA1549, CACNA1F, SNRNF200, PRPF8, VCAN, USH2A, HMCN1, RPE65, NR2E3, NRL, RHO, RP1, RP2, or OFDI.

27. The circular DNA vector of claim 26, wherein the ocular gene comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 19 or SEQ ID NO:

20.

28. The circular DNA vector of claim 27, wherein the ocular gene encodes a polypeptide having the amino acid sequence of SEQ ID NO:

63.

29. The circular DNA vector of claim 26, wherein the ocular gene comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO:

21.

30. The circular DNA vector of claim 29, wherein the ocular gene encodes a polypeptide having the amino acid sequence of SEQ ID NO:

61.

31. The circular DNA vector of claim 29 or 30, comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 8, optionally comprising the nucleic acid sequence of SEQ ID NO:

8.

32. The circular DNA vector of claim 26, wherein the ocular gene comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO:

22.

33. The circular DNA vector of claim 32, wherein the ocular gene encodes a polypeptide having the amino acid sequence of SEQ ID NO:

62.

34. The circular DNA vector of claim 32 or 33, comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 23, optionally comprising the nucleic acid sequence of SEQ ID NO:

23.

35. The circular DNA vector of any one of claims 1 to 34, wherein the therapeutic sequence comprises a CAG promoter controlling expression of a therapeutic gene, optionally an ocular gene.

36. The circular DNA vector of any one of claims 26 to 35, wherein the therapeutic sequence further comprises a regulatory element comprising a sequence derived from ABCA4 intron 6.

37. The circular DNA vector of claim 36, wherein the regulatory element is derived from the 5' half-region of ABCA4 intron 6.

38. The circular DNA vector of claim 36, wherein the regulatory element has at least 90% identity to at least 500 contiguous nucleotides within ABCA4 intron 6, wherein the at least 500 contiguous nucleotides comprise any of nucleotides 3,158 to 4,822 of intron 6, and wherein the regulatory element optionally comprises the nucleic acid sequence of SEQ ID NO: 24 or a functional variant thereof.

39. A method of expressing a therapeutic sequence in an ocular cell of a subject, the method comprising delivering to the ocular cell the circular DNA vector of any one of claims 26 to 38.

40. The method of claim 39, wherein the circular DNA vector is delivered to the ocular cell by ocular administration to the subject.

41. The method of claim 39 or 40, wherein expression persists for at least 12 months after administration of the circular DNA vector to the subject.

42. The method of any one of claims 39 to 41, wherein the circular DNA is administered no more frequently than about once every three months, once every four months, once every six months, once every eight months, and once every year.

43. The method of any one of claims 39-42, wherein the subject has an ocular disease.

44. The circular DNA vector of any one of claims 1-24, wherein the therapeutic sequence comprises a respiratory gene.

45. The circular DNA vector of claim 44, wherein the respiratory gene encodes a cystic fibrosis transmembrane receptor (CFTR), and the therapeutic sequence optionally comprises an elongation factor I (EF1A) promoter.

46. The circular DNA vector of claim 44 or 45, wherein the therapeutic sequence comprises a hypersensitive sequence 5' to the respiratory gene, and wherein the hypersensitive sequence optionally comprises the nucleotide sequence of SEQ ID NO: 28 or a functional variant thereof.

47. A method of expressing a therapeutic sequence in an airway cell of a subject, the method comprising delivering to the airway cell the circular DNA vector of any one of claims 44-46.

48. The method of claim 47, wherein the respiratory disease is cystic fibrosis.

49. The circular DNA vector of any one of claims 1-24, wherein the therapeutic sequence comprises a regulatory protein-encoding gene, which is optionally an immunomodulatory protein-encoding gene.

50. The circular DNA vector of claim 49, wherein the immunomodulatory protein-encoding gene is a dendritic cell chemoattractant-encoding gene, a dendritic cell growth factor or activator-encoding gene, or a lymphocyte signaling protein-encoding gene.

51. The circular DNA vector of claim 50, wherein: (a) the dendritic cell chemoattractant-encoding gene is XCL1, XCL2, CCL5, or CCL4; (b) the dendritic cell growth factor or activator-encoding gene is FLT3L, GMCSF, or CD40; or (c) the lymphocyte signaling protein-encoding gene is IL-12, IL-15, CXCL9, or CXCL10.

52. The circular DNA vector of any one of claims 49-51, wherein the circular DNA vector is a bi- or multi-cistronic vector encoding two, three, or more regulatory protein-encoding genes.

53. A method of expressing a therapeutic sequence in a target cell of a subject, the method comprising delivering to the target cell the circular DNA vector of any one of claims 49-52.

54. The method of claim 53, wherein the subject has a cancer, and the method modulates a tumor microenvironment in the subject.

55. The circular DNA vector of any one of claims 1-24, wherein the therapeutic sequence encodes an antigen-binding protein.

56. The circular DNA vector of any one of claims 1-24, wherein the therapeutic sequence encodes an enzyme, a growth factor, a hormone, an interleukin, an interferon, a cytokine, an anti-apoptotic factor, an anti-diabetic factor, a coagulation factor, an anti-tumor factor, a liver-secreted protein, or a neuroprotective factor.

57. The circular DNA vector of any one of claims 1 to 24, wherein the therapeutic sequence comprises a transcriptional knockdown sequence, and which is optionally a microRNA coding sequence, a short hairpin RNA coding sequence, a zinc finger nuclease coding sequence, a TALEN coding sequence, a dCAS9 coding sequence, a guide RNA coding sequence, or a combination thereof.

58. The circular DNA vector of claim 57, wherein the transcriptional knockdown sequence comprises one or more guide RNA coding sequences and a dCAS9 coding sequence.

59. The circular DNA vector of claim 57 or 58, wherein the dCAS9 coding sequence is fused to one or more repressor proteins.

60. The circular DNA vector of any one of claims 57 to 59, wherein the therapeutic sequence further comprises a therapeutic protein coding sequence, wherein the transcriptional knockdown sequence is configured to silence transcription of endogenous expression of a target gene, and wherein the therapeutic sequence encodes a therapeutic replacement protein encoded by a functional variant of the target gene.

61. A method of knocking down transcription of a mutant target gene in a cell in a subject in need thereof, the method comprising contacting the circular DNA vector of any one of claims 57 to 60 with the cell under conditions suitable for knocking down transcription of the mutant target gene.

62. The circular DNA vector of any one of claims 1 to 22, wherein the therapeutic sequence comprises a gene editing sequence.

63. The circular DNA vector of claim 62, wherein the gene editing sequence encodes a guide RNA and / or a CRISPR-associated (Cas) endonuclease.

64. An engineered bacterial cell comprising: (a) the circular DNA vector of any one of claims 1 to 24, and (b) a Rep gene that encodes a bacterial replication protein that binds to the bacterial origin of replication of the circular DNA vector, wherein the Rep gene replicates the circular DNA vector.

65. The engineered bacterial cell of claim 64, wherein the therapeutic sequence comprises a transposase overhang sequence, which is optionally TTAA.

66. The engineered bacterial cell of claim 65, further comprising a transposase protein, wherein the transposase protein hydrolyzes DNA adjacent to the transposase overhang sequence.

67. The engineered bacterial cell of claim 66, wherein the transposase protein is encoded by a transposase gene expressed by the engineered bacterial cell.

68. The engineered bacterial cell of claim 67, wherein the transposase gene is integrated into the bacterial genome.

69. The engineered bacterial cell of any one of claims 64 to 68, wherein the engineered bacterial cell comprises an insertion sequence excision enhancer (IEE), which is optionally encoded by a gene integrated into the bacterial genome.

70. The engineered bacterial cell of any one of claims 64 to 69, wherein the engineered bacterial cell further comprises a terminally closed linear DNA molecule comprising a plasmid backbone.

71. The engineered bacterial cell of claim 70, wherein the plasmid backbone comprises a selectable marker.

72. The engineered bacterial cell of any one of claims 64-71, wherein the origin of replication is the only bacterial sequence in the circular DNA vector.

73. The engineered bacterial cell of any one of claims 64-72, wherein the engineered bacterial cell comprises an average of at least 10 copies of the circular DNA vector.

74. The engineered bacterial cell of any one of claims 64-73, wherein the circular DNA vector is monomeric.

75. The engineered bacterial cell of any one of claims 64-74, in culture, wherein the average number of copies of the circular DNA vector per engineered bacterial cell is at least 10, or at least 15, or at least 20.

76. An engineered bacterial cell comprising: (a) a plasmid template, wherein the plasmid template comprises: (i) a first segment comprising a therapeutic sequence and a sequence comprising a bacterial origin of replication, wherein the first segment is flanked by two transposase overhang sequences; and (ii) a second segment comprising a plasmid backbone, wherein the second segment is flanked by a left end (LE) repeat and a right end (RE) repeat, wherein the LE repeat and the RE repeat are bindable by a transposase protein; and (b) a Rep gene encoding a bacterial replication protein that binds to the bacterial origin of replication.

77. The engineered bacterial cell of claim 76, further comprising a transposase protein, wherein the transposase protein hydrolyzes DNA adjacent to a transposase overhang sequence.

78. The engineered bacterial cell of claim 76 or 77, further comprising: (c) a circular DNA vector comprising the therapeutic sequence, the sequence comprising a bacterial origin of replication, and one of the two transposase overhang sequences; and / or (d) a linear end-closed DNA molecule comprising a plasmid backbone flanked by a LE repeat and a RE repeat.

79. The engineered bacterial cell of any one of claims 76-78, wherein the transposase protein is encoded by a transposase gene expressed by the engineered bacterial cell, and the transposase gene is optionally integrated into the bacterial genome.

80. The engineered bacterial cell of any one of claims 76-79, wherein the engineered bacterial cell expresses an IEE, wherein the IEE is optionally encoded by a gene integrated into the bacterial genome.

81. The engineered bacterial cell of any one of claims 76-80, wherein the sequence comprising a bacterial origin of replication directly links the 3' end of the therapeutic sequence to the 5' end of the therapeutic sequence.

82. The engineered bacterial cell of any one of claims 76-81, wherein the sequence comprising a bacterial origin of replication is less than 50 bp in length.

83. The engineered bacterial cell of claim 82, wherein the bacterial origin of replication is a ColE2-P9 origin of replication.

84. The engineered bacterial cell of any one of claims 76-83, wherein the first segment and the circular DNA vector lack a selectable marker, and wherein the second segment and the plasmid backbone comprise a selectable marker.

85. The engineered bacterial cell of any one of claims 76-84, wherein the therapeutic sequence is a eukaryotic sequence.

86. The engineered bacterial cell of any one of claims 64-85, wherein the engineered bacterial cell does not comprise any extragenomic circular DNA molecule other than one or more copies of the plasmid template, the second segment comprising the plasmid backbone, or the circular DNA vector.

87. A method of making the circular DNA vector of any one of claims 1-24, the method comprising: (a) providing a bacterial cell comprising a Rep gene encoding a bacterial replication protein that binds to a bacterial origin of replication and a plasmid template, wherein the plasmid template comprises: (i) a first segment comprising a therapeutic sequence and a sequence comprising a bacterial origin of replication; and (ii) a second segment comprising a plasmid backbone; and (b) contacting the plasmid within the bacterial cell with an enzyme to circularize the first segment, thereby producing a circular DNA vector comprising the therapeutic sequence and the origin of replication.

88. The method of claim 87, wherein the method further comprises culturing the bacterial cell under conditions suitable for replication of the circular DNA vector, thereby producing a bacterial cell culture that replicates the circular DNA vector.

89. The method of claim 87 or 88, wherein the enzyme is encoded on a helper plasmid or a bacterial artificial chromosome (BAC) within the bacterial cell.

90. The method of any one of claims 87-89, wherein the bacterial cell culture is substantially free of a helper plasmid or a BAC.

91. The method of any one of claims 87-90, wherein the average copy number of the circular DNA vector per bacterial cell is at least 10, or is at least 15, or is at least 20.

92. The method of any one of claims 87-91, wherein the enzyme is a transposase.

93. The method of claim 92, wherein the second segment is flanked by a left end (LE) repeat and a right end (RE) repeat, wherein the LE repeat and the RE repeat are bindable by a transposase protein.

94. The method of any one of claims 87-93, wherein the second segment comprises a selectable marker and the first segment lacks a selectable marker.

95. The method of claim 94, wherein the selectable marker is selected from one or more of an antibiotic resistance gene and a counterselectable marker.

96. The method of any one of claims 87-95, wherein the bacterial cell expresses an IEE.

97. The method of any one of claims 87-96, further comprising isolating the circular DNA vector from the bacterial cell.

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