Improved generation of viral and nonviral nanoplasmid vectors

Abstract

A method for improving the replication of covalent closed circular plasmids is provided. [Solution] The method comprises the step of preparing a covalent closed circular plasmid having a Pol I-dependent origin of replication and an insert containing a structured DNA sequence selected from the group consisting of a reverse repeat sequence, a directional repeat sequence, a homopolymer repeat sequence, a eukaryotic origin of replication, or a eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction of replication. The method also comprises the step of modifying the covalent closed circular recombinant molecule so that the Pol I-dependent origin of replication is replaced with a Pol III-dependent origin of replication, thereby improving the replication of the covalent closed circular plasmid with the resulting Pol III-dependent origin of replication. A covalent closed circular recombinant DNA molecule without antibiotic markers is also provided.

Description

[Technical Field]

[0001] Statement on federally funded research and development Not applicable.

[0002] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 645,892, filed on 21 March 2018, entitled “Improved Generation of Viral and Nonviral Nanoplasmid Vectors,” the entire contents of which are incorporated herein by reference.

[0003] Field of Invention The present invention relates to recombinant DNA molecules, i.e., vectors useful for viral and nonviral gene therapy and viral and nonviral cell therapy, more specifically, to improve the production yield and quality of viral and nonviral vectors, reduce transfection-related toxicity, improve transposition from nonviral transposon vectors, improve packaging titer from viral vectors, improve the expression of genes encoded by viral and nonviral vectors, and eliminate antibiotic selection marker gene transfer mediated by viral and nonviral vectors.

[0004] Such recombinant DNA molecules are useful in biotechnology, ex vivo gene therapy, transgenic organisms, gene therapy, therapeutic vaccination, agriculture, and DNA vaccines. [Background technology]

[0005] E. coli plasmids have long been an important source of recombinant DNA molecules used by researchers and industry. Today, plasmid DNA is becoming increasingly important as next-generation biotechnology products (e.g., gene therapies and DNA vaccines) enter clinical trials and eventually enter the pharmaceutical market. Plasmid DNA vaccines can find applications as prophylactic vaccines for viral, bacterial, or parasitic diseases, immunizers for the preparation of high-immune globulin products, therapeutic vaccines for infectious diseases, or cancer vaccines. Plasmids are also used in gene therapy or gene replacement applications, where the desired gene product is expressed from the plasmid after administration to the patient. Plasmids are also used in non-viral transposon vectors for gene therapy or gene replacement applications, where the desired gene product is expressed from the genome after transposition from the plasmid and genomic integration. Plasmids are also used in viral vectors for gene therapy or gene replacement applications, where the desired gene product is packaged in transducible viral particles after transfection of the generating cell line and then expressed from the virus in target cells after viral transduction.

[0006] Non-viral and viral vector plasmids typically contain origins of replication derived from pMB1, ColE1, or pBR322. Common high-copy-number derivatives have mutations that affect copy number regulation, such as ROP (primer gene repressor) deletions, and second site mutations that increase copy number (e.g., pMB1 pUC G to A point mutation, or ColE1 pMM1). By employing higher temperatures (42°C), selective plasmid amplification with pUC and pMM1 origins of replication can be induced.

[0007] Carnes, AE and Williams, JA, 2011, U.S. Patent No. 7,943,377, describes a method for feed batch fermentation in which plasmid-containing E. coli cells are grown at low temperatures for a portion of the feed batch phase with a limited growth rate, and then subjected to a temperature increase shift and continuous growth at high temperatures to accumulate the plasmid. The temperature shift with a limited growth rate improved plasmid yield and purity. Other fermentation processes for plasmid generation are described in Carnes AE 2005, BioProcess Intl, Vol. 3: pp. 36-44, which are incorporated herein by reference in their entirety.

[0008] In this technical field, it has been taught that one of the limitations of plasmid therapy and plasmid vaccine application is the safety concerns of regulatory authorities (e.g., Food and Drug Administration, European Medicines Agency) regarding 1) plasmid transfer and replication in the endogenous microbiota, or 2) expression of plasmid-encoding select markers in human cells or the endogenous microbiota. Furthermore, regulatory guidance recommends the removal of all non-essential sequences in the vector. Plasmids containing pMB1, ColE1, or pBR322 origins of replication can replicate indiscriminately in E. coli hosts. This raises safety concerns that plasmid therapeutic genes or antigens may be transferred into and replicated within the patient's endogenous microbiota. Ideally, therapeutic or vaccine plasmids should be non-replicable in endogenous E. coli strains. This requires replacing the pMB1, ColE1, or pBR322 origins of replication with conditional origins that require specialized cell lines for growth. Similarly, regulatory authorities, such as EMEA and the FDA, are interested in the use of antibiotic resistance or alternative protein markers in gene therapy and gene vaccine vectors due to concerns that genes (antibiotic resistance markers or protein markers) may be expressed in patients' cells. Ideally, plasmid therapies and plasmid vaccines should be 1) non-replicable in endogenous E. coli strains, 2) not encode protein-based selection markers, and 3) minimized to eliminate all non-essential sequences.

[0009] In this field, it is further taught that one of the limitations of plasmid vector application is that promoter inactivation mediated by the bacterial region of the vector (i.e., the region encoding the bacterial origin of replication and selection marker) shortens the transgene expression period from plasmid vectors. This results in a shortened transgene expression period. A strategy to improve the transgene expression period is to remove the bacterial region of the plasmid. For example, minicircle vectors that do not contain the bacterial region have been developed. Removal of the bacterial region from minicircle vectors improved the transgene expression period (Chen et al., op. cit., 2004). In minicircle vectors, the polyadenylation signal in the eukaryotic region is covalently linked to the promoter in the eukaryotic region via a short spacer, typically less than 200 bp in length, which consists of a recombination binding site. While longer spacers (1 kb or longer) resulted in transgene expression silencing in vivo, shorter spacers (500 bp or less) exhibited a transgene expression pattern similar to conventional minicircle DNA vectors (Lu J, Zhang F, Xu S, Fire AZ, Kay MA. 2012. Mol Ther. Vol. 20: pp. 2111-2119), demonstrating that even very long spacer sequences can be tolerated.

[0010] Williams, 2014. Improved expression of DNA plasmids. World Patent Application WO2014 / 035457 discloses that a minimized Nanoplasmid™ vector utilizes RNA-OUT antibiotic-free selection to replace a large 1000 bp pUC origin with a novel 300 bp R6K origin. The reduction of the spacer region connecting the 5' and 3' ends of the transgene expression cassette to less than 500 bp with the R6K origin-RNA-OUT backbone improved the expression duration of conventional minicircle DNA vectors, as predicted from the teachings of Lu et al., op. cit., 2012.

[0011] A 1.1kb pFAR4 vector pUC origin-tRNA antibiotic-free selective spacer improved expression duration compared to a 2.2kb pUC origin-kanR antibiotic-selective marker spacer region (Quiviger, M, Arfi A, Mansard D, Delacotte L, Pastor M, Scherman D, Marie C. 2014. Gene Therapy vol. 21: pp. 1001-1007). This indicates that improved expression duration can be obtained in some bacterial regions up to 1.1kb.

[0012] Improvements in expression levels compared to plasmid vectors were also observed when some spacer regions were less than 1.1 kb. For example, pVAX1 derivatives with a 2 kb bacterial skeleton reduced to 1.2, 1.1, or 0.7 kb showed more than twofold improvement in expression compared to the parent pVAX1 vector (Table 1). NTC8685 derivatives with a 1.5 kb bacterial skeleton (Nanoplasmid® vector) were reduced to 0.9 kb, 466 bp, or 281 bp and showed more than twofold improvement in expression compared to the parent NTC8685 vector (Table 2). This teaches that improved expression levels can be obtained with short bacterial regions of up to 1.2 kb.

[0013] [Table 1]

[0014] [Table 2]

[0015] Mini - circle vectors have been confirmed by various researchers to be superior to plasmid vectors in the generation of AAV vectors (improvement of transduction unit titer - Table 3) and transposon vectors (increase in transfer - Table 3). Also, the improvement in performance by improving the expression duration using short - backbone mini - circle vectors needs to be observed in short bacterial - backbone plasmid vectors up to 1.1 kb.

[0016] [Table 3]

[0017] In fact, in the combination of a 1.1 - kb bacterial - backbone pFAR4 SB transposon vector / SB100x transposase vector, it has been reported that the transposition of Sleeping Beauty into human cells was improved two - fold compared to the combination of a 2.8 - kb bacterial - backbone pT2 plasmid SB transposon vector / SB100x transposase vector (Pastor, M, Johnen S, Harmening N, Quiviger M, Pailloux J, Kropp, M, Walter P, Ivics Z, Izsvak Z, Thumann G, Scherman D. Molecular Therapy 11: 57 - 67, 2018).

[0018] However, viral vectors, such as AAV, lentiviral vectors, and retroviral vectors, and transposon vectors contain structured DNA sequences at their termini. For example, the Sleeping Beauty transposon vector contains adjacent IR / DR sequences, the AAV vector contains adjacent ITRs, and lentiviral and retroviral vectors contain adjacent LTRs.

[0019] When a pUC origin is close to a structured DNA sequence, replication termination becomes abnormal, resulting in replication intermediates that unacceptably degrade plasmid quality (Levy J. 2004. U.S. Patent No. 6,709,844). Levy teaches that replication intermediates are formed when the high-copy origin is less than 1 kb from a structured DNA sequence, such as an enhancer, LTR, or IRES, but not when the high-copy origin is more than 1.5 kb away. Since the pUC origin itself is 1 kb, there are no configurations for constructing bacterial AAV, lentivirus, retrovirus, or transposon vectors containing a pUC origin of less than 1.1 kb that is not expected to generate replication intermediates.

[0020] Lu J, Williams JA, Luke J, Zhang F, Chu K, and Kay MA. 2017. Human Gene Therapy vol. 28: pp. 125-134 disclose an antibiotic-free miniintron plasmid (MIP) AAV vector, suggesting that MIP intron AAV vectors can have their vector skeleton removed to produce shorter skeleton AAV vectors. Attempts to create minicircles, such as 6 or 10 bp spacer regions, within miniintron plasmid AAV vectors have resulted in toxicity, likely due to the creation of long palindromes by such close juxtaposition of AAV ITRs (see Table 7, footnote e). While it is possible to create MIP vectors with longer spacer regions of less than 1 kb, a drawback of the MIP intron strategy is the need for cloning of the replication and selection of intron-coding regions into the eukaryotic region, which is impossible or undesirable for many vectors.

[0021] The drawbacks of the minicircle strategy for producing short bacterial region AAV vectors, lentiviral vectors, retroviral vectors, or transposon vectors are that the methods for producing minicircle vectors are expensive and not easily scalable. For minicircle vectors, an E. coli-based production system has been developed where, after plasmid generation, the bacterial and eukaryotic regions are separated and cyclically formed into minicircles (eukaryotic region) and bacterial region circles via the action of phagericombinase on the recognition sequence in the plasmid. Some methods then utilize restriction enzymes to digest the bacterial region circles at specific sites to eliminate the difficulty of removing contaminants. These production procedures are highly inefficient. For example, optimal production of minicircle vectors yields only about 5 mg of minicircles per liter of culture medium (Kay MA, He CY, Chen ZY. 2010. Nat Biotechnol vol. 28: pp. 1287-1289).

[0022] No high-yield production methods for pFAR vectors have been reported. This system utilizes a plasmid-mediated suppressor tRNA gene to complement the TAG amber nonsense mutation in the thyA gene to compensate for thymidine requirements and enable cell proliferation on minimal media (Marie et al., op. cit., 2010). [Overview of the Initiative] [Problems that the invention aims to solve]

[0023] A solution is needed to develop AAV, lentiviral, retroviral, or transposon vectors that can be efficiently manufactured without replication intermediates or poor production, preferably containing short spacer regions of less than 1000 bp. These vectors do not need to encode protein-based selection markers and should be minimal to eliminate all non-essential sequences. [Means for solving the problem]

[0024] The present invention relates to vectors useful for viral and nonviral gene therapy and viral and nonviral cell therapy, and more specifically, to vectors for improving the production yield and quality of viral and nonviral vectors, reducing transfection-related toxicity, improving transfer from nonviral transposon vectors, improving packaging titers from viral vectors, improving the expression of transgenes encoded by viral and nonviral vectors, and eliminating the transfer of antibiotic resistance marker genes by viral and nonviral vectors.

[0025] An improved vector method and composition that utilize a Pol III-dependent origin of replication to replicate a structured DNA sequence are disclosed.

[0026] An improved vector method and composition that utilize a Pol III-dependent origin of replication to replicate reverse repeat DNA sequences are disclosed.

[0027] An improved vector method and composition that utilize a Pol III-dependent origin of replication to replicate a direct repeat DNA sequence are disclosed.

[0028] An improved vector method and composition that utilize Pol III-dependent origins of replication to replicate homopolymer repeat DNA sequences are disclosed.

[0029] An improved vector method and composition that utilize a Pol III-dependent origin of replication to replicate an enhancer-structured DNA sequence are disclosed.

[0030] An improved vector method and composition that utilize a Pol III-dependent origin of replication to replicate polyA repeat DNA sequences are disclosed.

[0031] An improved vector method and composition are disclosed that utilizes a Pol III-dependent origin of replication to replicate the SV40 origin of a replicated DNA sequence.

[0032] An improved vector method and composition that utilize a Pol III-dependent origin of replication to replicate lentiviral LTR DNA sequences are disclosed.

[0033] An improved vector method and composition that utilize a Pol III-dependent origin of replication to replicate retroviral LTR DNA sequences are disclosed.

[0034] An improved vector method and composition that utilize a Pol III-dependent origin of replication to replicate a viral LTR DNA sequence are disclosed.

[0035] An improved vector method and composition that utilize a Pol III-dependent origin of replication to replicate AAV ITR DNA sequences are disclosed.

[0036] An improved vector method and composition that utilize a Pol III-dependent origin of replication to replicate transposon IR / DR DNA sequences is disclosed.

[0037] An improved vector method and composition that utilize a Pol III-dependent origin of replication to replicate the Sleeping Beauty IR / DR DNA sequence are disclosed.

[0038] An improved vector method and composition that utilize a Pol III-dependent origin of replication to replicate the PiggyBac ITR DNA sequence are disclosed.

[0039] An improved vector method and composition that utilize a Pol III-dependent origin of replication to replicate a CMV enhancer DNA sequence are disclosed.

[0040] An improved vector method and composition that utilize a Pol III-dependent origin of replication to directly replicate the SV40 enhancer DNA sequence are disclosed.

[0041] An improved viral vector method and composition utilizing Pol III-dependent replication origins are disclosed.

[0042] Improved lentiviral vectors, lentiviral envelope vectors, and lentiviral packaging vector methods and compositions utilizing Pol III-dependent replication origins are disclosed.

[0043] Improved retroviral vectors, retroviral envelope vectors, and retroviral packaging vector methods and compositions utilizing Pol III-dependent replication origins are disclosed.

[0044] Improved AAV vectors and AAV helper vector methods utilizing Pol III-dependent replication origins, as well as compositions, are disclosed.

[0045] An improved adenovirus vector method and composition utilizing Pol III-dependent replication origins are disclosed.

[0046] Methods and compositions for improved nonviral transposons and transposase vectors utilizing Pol III-dependent replication origins are disclosed.

[0047] Improved non-viral sleeping beauty transposons and transposase vector methods and compositions utilizing Pol III-dependent replication origins are disclosed.

[0048] Improved nonviral PiggyBac transposons and transposase vector methods and compositions utilizing Pol III-dependent replication origins are disclosed.

[0049] Methods and compositions for improved nonviral Tol2 transposons and transposase vectors utilizing Pol III-dependent replication origins are disclosed.

[0050] An improved non-viral polyA-containing mRNA vector method and composition utilizing a Pol III-dependent replication origin are disclosed.

[0051] An improved viral vector method utilizing Pol III-dependent replication origins and a composition for generating an improved viral transduction unit are disclosed.

[0052] An improved lentiviral vector method utilizing a Pol III-dependent replication origin and a composition for generating an improved viral transduction unit are disclosed.

[0053] An improved retroviral vector method utilizing a Pol III-dependent replication origin and a composition involving the generation of an improved viral transduction unit are disclosed.

[0054] Improved AAV vectors and AAV helper vector methods utilizing Pol III-dependent replication origins, as well as compositions involving improved viral transduction unit generation, are disclosed.

[0055] Improved nonviral transposons and transposase vector methods utilizing Pol III-dependent replication origins, as well as compositions having improved transpositions, are disclosed.

[0056] Improved non-viral sleeping beauty transposons and transposase vector methods utilizing Pol III-dependent replication origins, as well as compositions having improved transpositions, are disclosed.

[0057] An improved nonviral PiggyBac transposon and transposase vector method utilizing Pol III-dependent replication origins, as well as compositions having improved transpositions, are disclosed.

[0058] An improved nonviral Tol2 transposon and transposase vector method utilizing a Pol III-dependent replication origin, as well as compositions having improved transpositions, are disclosed.

[0059] An improved viral vector method utilizing Pol III-dependent replication origins and a composition having improved expression are disclosed.

[0060] An improved lentiviral vector method utilizing a Pol III-dependent origin of replication, and a composition having improved expression are disclosed.

[0061] An improved retroviral vector method utilizing a Pol III-dependent replication origin, and a composition having improved expression are disclosed.

[0062] Improved AAV vectors and AAV helper vector methods utilizing Pol III-dependent replication origins, as well as compositions having improved expression, are disclosed.

[0063] Improved nonviral transposons and transposase vector methods utilizing Pol III-dependent replication origins, as well as compositions having improved expression, are disclosed.

[0064] Improved non-viral sleeping beauty transposons and transposase vector methods utilizing Pol III-dependent replication origins, as well as compositions having improved expression, are disclosed.

[0065] An improved nonviral PiggyBac transposon and transposase vector method utilizing Pol III-dependent replication origins, as well as compositions having improved expression, are disclosed.

[0066] An improved nonviral Tol2 transposon and transposase vector method utilizing a Pol III-dependent replication origin, as well as compositions having improved expression, are disclosed.

[0067] An improved viral vector method and composition that utilize Pol III-dependent replication origins and eliminate the risk of antibiotic resistance marker gene transfer (tansfer) are disclosed.

[0068] Improved lentiviral vectors, lentiviral envelope vectors, and lentiviral packaging vector methods that utilize Pol III-dependent replication origins and eliminate the risk of antibiotic resistance marker gene transfer, as well as compositions, are disclosed.

[0069] Improved retroviral vectors, retroviral envelope vectors, and retroviral packaging vector methods that utilize Pol III-dependent replication origins and eliminate the risk of antibiotic resistance marker gene transfer, as well as compositions, are disclosed.

[0070] Improved AAV vectors and AAV helper vector methods that utilize Pol III-dependent replication origins and eliminate the risk of antibiotic resistance marker gene transfer, as well as compositions, are disclosed.

[0071] Methods and compositions for improved nonviral transposons and transposase vectors that utilize Pol III-dependent replication origins and eliminate the risk of antibiotic resistance marker gene transfer are disclosed.

[0072] An improved non-viral sleeping beauty transposon and transposase vector method, as well as compositions, that utilize Pol III-dependent replication origins and eliminate the risk of antibiotic resistance marker gene transfer are disclosed.

[0073] An improved nonviral PiggyBac transposon and transposase vector method, as well as compositions, that utilize Pol III-dependent replication origins and eliminate the risk of antibiotic resistance marker gene transfer are disclosed.

[0074] An improved nonviral Tol2 transposon and transposase vector method, and compositions that utilize Pol III-dependent replication origins and eliminate the risk of antibiotic resistance marker gene transfer are disclosed.

[0075] An improved viral vector method and composition with reduced transfection-related toxicity utilizing a Pol III-dependent replication origin are disclosed.

[0076] An improved lentiviral vector method and composition with reduced transfection-related toxicity utilizing a Pol III-dependent replication origin are disclosed.

[0077] An improved retroviral vector method and composition with reduced transfection-related toxicity utilizing a Pol III-dependent replication origin are disclosed.

[0078] Improved AAV vectors and AAV helper vector methods with reduced transfection-related toxicity utilizing Pol III-dependent replication origins, as well as compositions, are disclosed.

[0079] Improved nonviral transposons and transposase vector methods with reduced transfection-related toxicity utilizing Pol III-dependent replication origins, as well as compositions, are disclosed.

[0080] Improved non-viral sleeping beauty transposons and transposase vector methods, as well as compositions, are disclosed, which utilize Pol III-dependent replication origins and exhibit reduced transfection-related toxicity.

[0081] An improved nonviral PiggyBac transposon and transposase vector method, as well as compositions, are disclosed, which utilize Pol III-dependent replication origins and exhibit reduced transfection-related toxicity.

[0082] An improved nonviral Tol2 transposon and transposase vector method with reduced transfection-related toxicity utilizing a Pol III-dependent replication origin, as well as compositions, are disclosed.

[0083] Each of the improvements described above and below is relative to those achieved, for example, with different plasmids that do not contain a Pol III-dependent origin of replication, under similar or identical circumstances.

[0084] One object of the present invention is to provide improved production yields for viral vectors and non-viral vectors.

[0085] Another object of the present invention is to provide improved manufacturing quality for viral and non-viral vectors.

[0086] Another object of the present invention is to provide a viral vector having an improved packaging titer.

[0087] Another object of the present invention is to provide a nonviral transposon vector having improved transposition.

[0088] Another object of the present invention is to provide viral vectors and non-viral vectors in which the expression of encoded transgenes is improved.

[0089] Another object of the present invention is to provide viral and non-viral vectors that eliminate the transfer of antibiotic resistance marker genes.

[0090] Another object of the present invention is to provide viral and non-viral vectors with reduced transfection-related toxicity.

[0091] In one embodiment, the present technology provides a method for improving the replication of a covalent closed circular plasmid, comprising the steps of: a) providing a covalent closed circular plasmid comprising i) a Pol I-dependent origin of replication and ii) an insert comprising a structured DNA sequence selected from the group consisting of reverse repeat sequences, directional repeat sequences, homopolymer repeat sequences, eukaryotic origins of replication and eukaryotic promoter-enhancer sequences, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction of replication; and b) modifying the covalent closed circular recombinant molecule of a) to replace the Pol I-dependent origin of replication with a Pol III-dependent origin of replication, thereby improving the replication of the covalent closed circular plasmid with the resulting Pol III-dependent origin of replication. In a further embodiment, the Pol I-dependent origin of replication is selected from the group consisting of pUC origins, pMB1 origins, and ColE1 origins. In a further embodiment, the Pol III-dependent origin of replication is an R6K gamma origin of replication. In a further embodiment, the Pol III-dependent origin of replication is an R6K gamma origin of replication having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18. In a further embodiment, the structured DNA sequence is selected from the group consisting of poly(A) repeats, SV40 origin of replication, viral LTRs, lentiviral LTRs, retroviral LTRs, transposon IR / DR repeats, sleeping beauty transposon IR / DR repeats, AAV ITRs, CMV enhancers, and SV40 enhancers. In a further embodiment, the improved replication is selected from the group consisting of reduced generation of replication intermediates and increased plasmid copy number.

[0092] In another embodiment, the technique provides a method for improving the replication of a covalent closed circular plasmid, comprising: a) the step of providing a covalent closed circular plasmid comprising: i) a bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selection marker; and ii) an insert comprising a structured DNA sequence selected from the group consisting of reverse repeats, directional repeats, homopolymer repeats, eukaryotic origins of replication and eukaryotic promoter-enhancer sequences, wherein the structured DNA sequence is located less than 1000 bp away from the Pol I-dependent origin of replication in the direction of replication; and b) the step of modifying the covalent closed circular recombinant molecule of a) to replace the antibiotic selection marker with an RNA selection marker and replace the Pol I-dependent origin of replication with a Pol III-dependent origin of replication, thereby improving the replication of the covalent closed circular plasmid with the resulting Pol III-dependent origin of replication. In a further embodiment, the Pol I-dependent origin of replication is selected from the group consisting of pUC origins, pMB1 origins, and ColE1 origins. In a further embodiment, the Pol III-dependent origin of replication is an R6K gamma origin of replication. In a further embodiment, the Pol III-dependent origin of replication is an R6K gamma origin of replication having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18. In a further embodiment, the RNA selection marker is an RNA-IN regulated RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7. In a further embodiment, the RNA selection marker is an RNA-OUT RNA selection marker encoding an RNA-IN regulated RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6. In a further embodiment, the bacterial replication-selection region, which includes a Pol I-dependent replication origin and an antibiotic selection marker, is replaced with a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17.In further embodiments, the structured DNA sequence is selected from the group consisting of polyA repeats, SV40 origins of replication, viral LTRs, lentiviral LTRs, retroviral LTRs, transposon IR / DR repeats, sleeping beauty transposon IR / DR repeats, AAV ITRs, CMV enhancers, and SV40 enhancers. In further embodiments, the improved replication is selected from the group consisting of reduced generation of replication intermediates and increased plasmid copy number.

[0093] In one embodiment, the technology provides an antibiotic marker-free covalent closed circular recombinant DNA molecule, comprising: a) an antibiotic marker-free insert comprising a structured DNA sequence selected from the group consisting of reverse repeat sequences, directional repeat sequences, homopolymer repeat sequences, eukaryotic origins of replication, and eukaryotic promoter enhancer sequences; b) a Pol III-dependent origin of replication comprising an R6K gamma origin of replication having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 1, SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 4, and SEQ ID NOs. 18; and c) an RNA-OUT RNA selection marker comprising an RNA-IN regulated RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 5 and SEQ ID NOs. 7. In further embodiments, the R6K gamma replication origin and the RNA-OUT RNA selection marker include an R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17. In further embodiments, the structured DNA sequence is selected from the group consisting of poly(A) repeats, SV40 replication origins, viral LTRs, lentiviral LTRs, retroviral LTRs, transposon IR / DR repeats, sleeping beauty transposon IR / DR repeats, AAV ITRs, CMV enhancers, and SV40 enhancers. In further embodiments, the recombinant DNA molecule is selected from the group consisting of viral vectors, lentiviral vectors, retroviral vectors, AAV vectors, Ad vectors, nonviral transposon vectors, sleeping beauty transposon vectors, PiggyBac transposon vectors, Tol2 transposon vectors, and poly(A)-containing mRNA vectors.

[0094] In one embodiment, the present technology provides a method for improving the generation of AAV vector viral transduction units from a covalent closed circular plasmid, comprising the steps of: a) i) providing a covalent closed circular plasmid comprising a bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic selection marker, and ii) an insert comprising a eukaryotic region selected from the group consisting of AAV vectors, AAV rep cap vectors, Ad helper vectors, and Ad helper rep cap vectors; and b) modifying the covalent closed circular recombinant molecule of a) to replace the bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic selection marker with a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region of less than 1 kb, thereby improving the generation of AAV viral transduction units when the resulting covalent closed circular plasmid is transfected into mammalian cells. In a further embodiment, the Pol I-dependent origin of replication is selected from the group consisting of pUC origins, pMB1 origins, and ColE1 origins. In a further embodiment, the Pol III-dependent R6K replication origin is an R6K gamma replication origin having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18. In a further embodiment, the RNA-OUT RNA selection marker is an RNA-IN regulated RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7. In a further embodiment, the RNA-OUT RNA selection marker is an RNA-OUT RNA selection marker encoding an RNA-IN regulated RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6. In a further embodiment, the Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region less than 1kb has at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

[0095] In one embodiment, the present technology provides a method for improving the generation of retroviral or lentiviral vector viral transduction units from a covalent closed circular plasmid, comprising: a) i) providing a covalent closed circular plasmid comprising a bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic selection marker, and ii) an insert comprising a eukaryotic region selected from the group consisting of retroviral vectors, lentiviral vectors, retroviral envelope vectors, lentiviral envelope vectors, retroviral packaging vectors, and lentiviral packaging vectors; and b) modifying the covalent closed circular recombinant molecule of a) to replace the bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic selection marker with a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region of less than 1 kb, thereby improving the generation of viral transduction units when the resulting covalent closed circular plasmid is transfected into mammalian cells with the Pol III-dependent origin of replication. In a further embodiment, the Pol I-dependent replication origin is selected from the group consisting of pUC origins, pMB1 origins, and ColE1 origins. In a further embodiment, the Pol III-dependent R6K replication origin is an R6K gamma replication origin having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18. In a further embodiment, the RNA-OUT RNA selection marker is an RNA-IN regulated RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7. In a further embodiment, the RNA-OUT RNA selection marker is an RNA-OUT RNA selection marker encoding an RNA-IN regulated RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6.In a further embodiment, the Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region of less than 1 kb has at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17.

[0096] In one embodiment, the present invention is intended to provide a method for improving transposition from a covalent closed circular nonviral transposon plasmid, comprising: a) i) providing a covalent closed circular plasmid comprising a bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic selection marker, and ii) an insert comprising a nonviral eukaryotic region selected from the group consisting of transposon vectors, Sleeping Beauty transposon vectors, Sleeping Beauty transposase vectors, PiggyBac transposon vectors, PiggyBac transposase vectors, Tol2 transposon vectors, and Tol2 transposase vectors; and b) modifying the covalent closed circular recombinant molecule of a) to replace the bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic selection marker with a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region of less than 1 kb, thereby improving transposition when the resulting covalent closed circular plasmid is transfected into mammalian cells by the Pol III-dependent origin of replication. In a further embodiment, the Pol I-dependent replication origin is selected from the group consisting of pUC origins, pMB1 origins, or ColE1 origins. In a further embodiment, the Pol III-dependent R6K replication origin is an R6K gamma replication origin having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18. In a further embodiment, the RNA-OUT RNA selection marker is an RNA-IN regulated RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7. In a further embodiment, the RNA-OUT RNA selection marker is an RNA-OUT RNA selection marker encoding an RNA-IN regulated RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6.In a further embodiment, the Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region of less than 1 kb has at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17.

[0097] In one embodiment, the technology provides a method for improving expression from a covalent closed circular viral vector or a nonviral transposon plasmid, comprising the steps of: a) providing a covalent closed circular plasmid comprising an insert comprising a bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic selection marker, and ii) an insert comprising a eukaryotic region selected from the group consisting of lentiviral vectors, retroviral vectors, and AAV vectors or nonviral transposon vectors; and b) modifying the covalent closed circular recombinant molecule of a) to replace the bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic selection marker with a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region of less than 1 kb, thereby improving the expression of the covalent closed circular plasmid when transfected into mammalian cells with the resulting Pol III-dependent origin of replication. In a further embodiment, the Pol I-dependent origin of replication is selected from the group consisting of pUC origins, pMB1 origins, and ColE1 origins. In a further embodiment, the Pol III-dependent R6K replication origin is an R6K gamma replication origin having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, and SEQ ID NOs: 18. In a further embodiment, the RNA-OUT RNA selection marker is an RNA-IN regulated RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 5 and SEQ ID NOs: 7. In a further embodiment, the RNA-OUT RNA selection marker is an RNA-OUT RNA selection marker encoding an RNA-IN regulated RNA-OUT RNA having at least 95% sequence identity with SEQ ID NOs: 6. In a further embodiment, the Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region less than 1kb has at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16, and SEQ ID NOs: 17.

[0098] In one embodiment, the present technology provides a method for eliminating the transfer of an antibiotic resistance marker gene from a covalent closed circular viral vector plasmid, comprising: a) i) a bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic resistance marker; and ii) a covalent closed circular plasmid comprising an insert containing an antibiotic resistance marker-free eukaryotic region, selected from the group consisting of viral vectors, lentiviral vectors, lentiviral packaging vectors, lentiviral envelope vectors, retroviral vectors, retroviral envelope vectors, retroviral packaging vectors, AAV vectors, AAV rep cap vectors, Ad helper vectors, and Ad helper rep cap vectors; and b) a modification of the covalent closed circular recombinant molecule of a) to replace the bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic selection marker with a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region of less than 1 kb, thereby eliminating the transfer of an antibiotic resistance marker gene from a covalent closed circular recombinant molecule. Covalent closed circular plasmids with a Pol III-dependent replication origin do not have antibiotic resistance markers that can be packaged in lentiviral, retroviral, or AAV transduction virus particles when transfected into mammalian cells. In further embodiments, the Pol I-dependent replication origin is selected from the group consisting of pUC origins, pMB1 origins, and ColE1 origins. In further embodiments, the Pol III-dependent R6K replication origin is an R6K gamma replication origin having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 1, SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 4, and SEQ ID NOs. 18. In further embodiments, the RNA-OUT RNA selection marker is an RNA-IN regulated RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 5 and SEQ ID NOs. 7. In further embodiments, the RNA-OUT RNA selection marker is an RNA-OUT RNA selection marker encoding an RNA-IN regulated RNA-OUT RNA having at least 95% sequence identity with SEQ ID NOs. 6.In a further embodiment, the Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region of less than 1 kb has at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17.

[0099] In one embodiment, the technology provides a method for eliminating the transfer of an antibiotic resistance marker gene from a covalent closed circular nonviral transposon plasmid, comprising the steps of: a) providing a covalent closed circular plasmid containing an insertion comprising a bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic resistance marker, and ii) an insertion comprising an antibiotic resistance marker-free eukaryotic region selected from the group consisting of nonviral transposon vectors, nonviral transposase vectors, Sleeping Beauty transposon vectors, Sleeping Beauty transposase vectors, PiggyBac transposon vectors, PiggyBac transposase vectors, Tol2 transposon vectors, and Tol2 transposase vectors; and b) modifying the covalent closed circular recombinant molecule of a) to replace the bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic selection marker with a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region of less than 1 kb, thereby eliminating the transfer of an antibiotic resistance marker gene from a covalent closed circular recombinant molecule of a). A covalently bound closed circular plasmid with a Pol III-dependent replication origin does not have an antibiotic resistance marker that can be transferred into the genome when transfected into mammalian cells. In a further embodiment, the Pol I-dependent replication origin is selected from the group consisting of pUC origins, pMB1 origins, and ColE1 origins. In a further embodiment, the Pol III-dependent R6K replication origin is an R6K gamma replication origin having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, and SEQ ID NOs: 18. In a further embodiment, the RNA-OUT RNA selection marker is an RNA-IN regulated RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 5 and SEQ ID NOs: 7. In a further embodiment, the RNA-OUT RNA selection marker is an RNA-OUT RNA selection marker encoding an RNA-IN regulated RNA-OUT RNA having at least 95% sequence identity with SEQ ID NOs: 6.In a further embodiment, the Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region of less than 1 kb has at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17.

[0100] In one embodiment, the technology provides an antibiotic marker-free covalent closed circular recombinant DNA molecule, comprising: a) an antibiotic marker-free insert comprising a eukaryotic region selected from the group consisting of lentiviral vectors, lentiviral envelope vectors, lentiviral packaging vectors, retroviral vectors, retroviral envelope vectors, retroviral packaging vectors, AAV vectors, AAV rep cap vectors, Ad helper vectors, Ad helper rep cap vectors, nonviral transposon vectors, and nonviral transposase vectors; b) a Pol III-dependent origin of replication comprising an R6K gamma origin of replication having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 1, SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 4, and SEQ ID NOs. 18; and c) an RNA-OUT RNA selection marker comprising an RNA-IN regulated RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 5 and SEQ ID NOs. 7. In further embodiments, the R6K gamma replication origin and the RNA-OUT RNA selection marker include an R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17.

[0101] In one embodiment, the technology provides a method for reducing transfection-associated toxicity from a covalent closed-circular viral vector or a nonviral transposon plasmid, comprising the steps of: a) providing a covalent closed-circular plasmid comprising an insert containing a bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic selection marker, and ii) a eukaryotic region selected from the group consisting of lentiviral vectors, retroviral vectors, AAV vectors, and nonviral transposon vectors; and modifying the covalent closed-circular recombinant molecule of a) to replace the bacterial replication-selection region of 1 kb or more containing a Pol I-dependent origin of replication and an antibiotic selection marker with a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region of less than 1 kb, thereby reducing the toxicity of the covalent closed-circular plasmid when transfected by transfection associated with mammalian cells due to the resulting Pol III-dependent origin of replication. In a further embodiment, the Pol I-dependent origin of replication is selected from the group consisting of pUC origins, pMB1 origins, and ColE1 origins. In a further embodiment, the Pol III-dependent R6K replication origin is an R6K gamma replication origin having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, and SEQ ID NOs: 18. In a further embodiment, the RNA-OUT RNA selection marker is an RNA-IN regulated RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 5 and SEQ ID NOs: 7. In a further embodiment, the RNA-OUT RNA selection marker is an RNA-OUT RNA selection marker encoding an RNA-IN regulated RNA-OUT RNA having at least 95% sequence identity with SEQ ID NOs: 6. In a further embodiment, the Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region less than 1kb has at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16, and SEQ ID NOs: 17.

[0102] The resulting Pol III-dependent origin-of-replication plasmids exhibited remarkably improved manufacturing quality and yield compared to origin-of-replication expression plasmid vectors derived from parental pMB1, ColE1, or pBR322.

[0103] Further objectives and advantages of the present invention will become apparent from the drawings and the considerations in the following description. [Brief explanation of the drawing]

[0104] [Figure 1A-D] Figures 1A-1F show the R6K origin (Figures 1A, 1E, and 1F), the RNA-OUT selection marker (Figure 1B), and the 14 and 3 CpG R6K-RNA-OUT bacterial skeletons (Figures 1C and 1D). [Figure 1E-F] Figures 1A-1F show the R6K origin (Figures 1A, 1E, and 1F), the RNA-OUT selection marker (Figure 1B), and the 14 and 3 CpG R6K-RNA-OUT bacterial skeletons (Figures 1C and 1D). [Figure 2] Figures 2A and 2B show the Pol I-dependent pUC-originating Sleeping Beauty transposon vector (Figure 2A) and the Pol III-dependent R6K-originating Sleeping Beauty transposon vector (Figure 2B). [Figure 3] Figures 3A-3C show Pol I-dependent pUC-initiated AAV vectors (Figures 3A and 3B) and Pol III-dependent R6K-initiated AAV vectors (Figure 3C). [Figure 4] Figures 4A-4F show mRNA vectors encoding Pol I-dependent pUC-origin A60 polyA repeats (Figures 4A-4B), Pol III-dependent R6K-origin A60 polyA repeats (Figure 4C), Pol I-dependent pUC-origin A99 polyA repeats (Figures 4D-4E), and Pol III-dependent R6K-origin A99 polyA repeats (Figure 4F). [Modes for carrying out the invention]

[0105] Table 1: Expression levels of pVAX1 mammalian expression vector spacer region (SR) derivatives. Table 2: Expression levels of NTC8685 mammalian expression vector spacer region (SR) derivatives. Table 3. Minicircle applications using various viral and non-viral vector platforms. Table 4: R6K-starting RNA-OUT selection marker vectors with adjacent pNTC multicloning sites. Table 5: SV40-starting lentiviral vector: pUC vs. R6K-starting shaking flask yield / quality. Table 6: Sleeping Beauty transposon vector: pUC vs. R6K-starting shaken flask yield / quality. Table 7: AAV vector: pUC vs. R6K-starting shaken flask yield / quality. Table 8: mRNA vector: pUC vs. R6K-derived DH5α HyperGRO fermentation yield / quality. Table 9: AAV helper vector: pUC vs. R6K-based plasmid generation yield / quality.

[0106] Sequence ID 1: R6K Gamma Origin Sequence ID 2:1 CpG R6K gamma origin Sequence ID 3: CpG-free R6K gamma origin Sequence ID 4: Extended R6K Gamma Origin Sequence ID 5: RNA-OUT selection marker Sequence ID 6: RNA-OUT antisense repressor RNA Sequence ID 7: 2CpG RNA-OUT selection marker Sequence ID 8: R6K gamma-starting RNA-OUT bacterial region where NheI and KpnI restriction sites are adjacent Sequence ID 9: 1 CpG R6K gamma origin - 2 CpG RNA-OUT bacterial region adjacent to NpeI and KpnI restriction sites Sequence ID 10: pNTC-NP1 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI / HindIII Sequence ID 11: pNTC-NP2 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI / HindIII Sequence ID 12: pNTC-NP3 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI / HindIII Sequence ID 13: pNTC-NP4 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI / HinndIII Sequence ID 14: pNTC-NP5 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: KasI / HinndIII Sequence ID 15: pNTC-NP6 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI / SacI Sequence ID 16: pNTC-NP7 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: BssHII / BssHII Sequence ID 17: pNTC-3xCpG NP1 polylinker R6K-RNA-OUT polylinker cloning cassette: HindIII / EcoRI Sequence ID 18: R6K gamma origin (7 iterons) Sequence ID 19: R6K gamma-based 22bp iteron repeats Sequence ID 20: R6K gamma-based 22bp iteron repeats Sequence ID 21: R6K gamma-based 22bp iteron repeats Sequence ID 22: R6K gamma-based 22bp iteron repeats Sequence ID 23: R6K gamma-based 22bp iteron repeats

[0107] Definition of Terms AAV vectors: Adeno-associated virus vectors, episomal virus vectors. Includes self-complementary (sc) adeno-associated virus vectors (scAAV) and single-stranded (ss) adeno-associated virus vectors (ssAAV). AF: Antibiotic free. amp: ampicillin. ampR: Ampicillin resistance gene. Antibiotic selection markers: Genes that confer resistance to antibiotics, such as ampicillin resistance genes, kanamycin resistance genes, chloramphenicol resistance genes, and tetracycline resistance genes. Approximately: As used herein, the terms "approximately" or "about" refer to a value that is the same as or similar to the reference value described, when applied to one or more values ​​of interest. Bacterial region: The region of the plasmid vector required for amplification and selection in a bacterial host. bp: base pair

[0108] ccc: Covalent closed ring (a ring closed by covalent bonds). cI: Lambda Repressor. cITs857: A lambda repressor that incorporates a further mutation from C to T (Ala to Thr) to confer temperature sensitivity. cITs857 is a functional repressor at 28-30°C but is almost inactive at 37-42°C. Also known as cI857. Cat R Chloramphenicol resistance gene. CMV: Cytomegalovirus. dcm methylation: E. coli methyltransferase in which the sequence CC(A / T)GG at the C5 position of the second cytosine is methylated. DNA replicons: Genetic elements that can replicate under their own control. Examples include plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors, and hybrids thereof.

[0109] E. coli: Escherichia coli, a Gram-negative bacterium. EGFP: Enhanced green fluorescent protein. EP: Electroporation. Eukaryotic expression vectors: Vectors used to express mRNA, protein antigens, protein therapeutics, shRNA, RNA, or microRNA genes in target eukaryotes using RNA polymerase I, II, or III promoters. Eukaryotic region: A region of the plasmid that encodes a eukaryotic sequence and / or a sequence required for plasmid function in the target organism. This includes a plasmid vector region required for the expression of one or more transgenes in the target organism, including RNA Pol II enhancers, promoters, transgenes, and poly(A) sequences. This also includes a plasmid vector region required for the expression of one or more transgenes in the target organism using RNA Pol I or RNA Pol III promoters, RNA Pol I or RNA Pol III expressing transgenes, or RNA. The eukaryotic region may optionally include other functional sequences, such as eukaryotic transcription terminators, supercoil-induced DNA double-strand destabilization (SIDD) structures, S / MARs, boundary elements, etc. In lentivirus or retroviral vectors, the eukaryotic region includes adjacent directional repeats (LTRs); in AAV vectors, the eukaryotic region includes adjacent inverted terminal repeats; and in transposon vectors, the eukaryotic region includes adjacent transposon inverted terminal repeats or IR / DR terminals (e.g., Sleeping Beauty). In genome-integrated vectors, the eukaryotic region may encode homology arms to direct targeted integration. Exon: A sequence of nucleotides encoded by a gene, present in the mature mRNA product after transcription and the completion of RNA splicing to remove introns. Expression vector: A vector used to express mRNA, protein antigens, protein therapeutics, shRNA, RNA, or microRNA genes in a target organism.

[0110] g: grams, kilograms are equivalent to kg. Target gene: A gene expressed in a target organism. This includes mRNA genes encoding protein or peptide antigens, mRNA, shRNA, RNA, or microRNA encoding protein or peptide therapeutics, and RNA therapeutics, as well as mRNA, shRNA, RNA, or microRNA encoding RNA vaccines. Hr: Time (multiple hours possible). ID: Intradermal. IM: Intramuscular. Immune response: A reaction involving antigen-reactive cells (e.g., antigen-reactive T cells) or antibodies (e.g., antigen-reactive IgG). Introns: Nucleotide sequences encoded by genes that are transcribed and subsequently removed from mature mRNA products through RNA splicing. IR / DR: Inverted repeats where each is performed twice as a directly repeated repetition. For example, the Sleeping Beauty Transposon IR / DR repeat. Iteron: A directional repeat DNA sequence at the origin of replication, necessary for replication initiation. The iteron repeat at the R6K origin is 22 bp. ITR: Inverted terminal repeat.

[0111] kan: Kanamycin. kanR: Kanamycin resistance gene. Kd: Kilodalton. Kozak sequence: An optimized consensus DNA sequence gccRccATG (R=G or A) located immediately upstream of the ATG start codon to ensure efficient translation initiation. The SalI site (GTCGAC) immediately upstream of the ATG start codon (GTCGACATG) is the effective Kozak sequence.

[0112] Lentiviral vectors are embedded viral vectors that can infect both dividing and non-dividing cells. They are also called lentiviral transfer plasmids. The plasmid encodes a lentiviral LTR flanking expression unit. The transfer plasmid is transfected into production cells along with the lentiviral envelope and packaging plasmids necessary to produce viral particles. Lentiviral envelope vector: A plasmid that encodes an envelope glycoprotein. Lentiviral packaging vector: One or two plasmids expressing the gag, pol, and Rev functions necessary for packaging a lentiviral transfer vector. Minicircle: A covalently bound closed cyclic plasmid derivative in which the bacterial region has been removed from the parent plasmid by in vivo or in vitro site-directed recombination or in vitro restriction digestion / ligation. Minicircle vectors are incompatible with bacterial cell replication. mRNA: Messenger RNA. mSEAP: Mouse secreted alkaline phosphatase.

[0113] NA: Not applicable. Nanoplasmid® vectors: Vectors combining an RNA selection marker with R6K, ColE2, or a ColE2-associated origin of replication. For example, the NTC9385C, NTC9685C, NTC9385R, NTC9685R vectors, and modifications described in Williams, 2014 (which is incorporated herein by reference). The NTC8385, NTC8485, and NTC8685 plasmids are antibiotic-free pUC origin vectors that include an antibiotic resistance marker, such as a short RNA (RNA-OUT) selection marker instead of kanR. The construction and application of these RNA-OUT-based antibiotic-free vectors are described by reference in Williams, JA 2008, International Patent Application WO2008 / 153733, included herein. NTC8485: NTC8485 is an antibiotic-free pUC origin vector that includes an antibiotic resistance marker, such as a short RNA (RNA-OUT) selection marker instead of kanR. The preparation and application of NTC8485 are described by reference in Williams, JA 2010, U.S. Patent Application No. 2010 / 0184158, which is included herein by reference. NTC8685: NTC8685 is an antibiotic-free pUC-derived vector containing an antibiotic resistance marker, such as a short RNA (RNA-OUT) selection marker instead of kanR. The preparation and application of NTC8685 are described in Williams, op. cit., 2010, which is included herein by reference. NTC9385R: The NTC9385R nanoplasmid® vector, as incorporated herein by reference, described by Williams, op. cit., 2014, has an NheItrpA terminator R6K-origin RNA-OUT-KpNI bacterial region (SEQ ID NO: 8) encoding a spacer region linked to the eukaryotic region via adjacent NheI and KpNI sites.

[0114] OD600: Optical density at 600 nm. PAS: Primosome assembly site. Priming of DNA synthesis at the single-stranded DNA ssi site. φX174 type PAS: It is a DNA hairpin sequence that binds to priA, thereby recruiting the remaining proteins to form a preprimosome [priB, dnaT recruits dnaB (delivered by dnaC)], which in turn recruits primase (dnaG), ultimately generating a short RNA substrate for DNA polymerase I. ABC type PAS: A DNA hairpin binds to dnaA, recruits dnaB (delivered by dnaC), then recruits primase (dnaG), and finally creates a short RNA substrate for DNA polymerase I. For example, the R6K plasmid CpG free ssiA primosome assembly site, or alternative φX174 type or ABC type primosome assembly sites. PAS-BH: Primosome assembly site in the heavy (leading) chain. PAS-BH region: The pBR322 starting region between ROP and PAS-BL (approximately pBR322 2067~2351). PAS-BL: Primosome assembly site in the light (lagging) chain. PBS: Phosphate-buffered saline. PCR: Polymerase chain reaction. pDNA: Plasmid DNA. PiggyBac transposon: PB transposon: A transposon system in which an ITR incorporates an adjacent PB transposon into the genome via a simple cut-and-paste mechanism mediated by PB transposase. Transposon vectors typically contain a promoter-transgene-polyA expression cassette between PB ITRs that are excised and incorporated into the genome. pINT pR pL vector: pINT pR pL att HK022 The integrated expression vector is described by reference to Luke et al., 2011, Mol Biotechnol 47:43, included herein. The target gene to be expressed is cloned downstream of the pL promoter. The vector encodes a temperature-inducible cI857 repressor, enabling the expression of the heat-inducible target gene. P L Promoter: Lambda promoter left. P L This is a potent promoter that is suppressed when the cI repressor binds to the OL1, OL2, and OL3 repressor binding sites. The temperature-sensitive cI857 repressor allows for heat-induced control of gene expression, as it is functional at 30°C, suppressing gene expression, but is inactivated at 37-42°C, thus allowing gene expression to occur. P L (OL1 G→T) Promoter: Lambda promoter left. P LThis is a potent promoter that is repressed by the binding of cI repressors to the OL1, OL2, and OL3 repressor binding sites. The temperature-sensitive cI857 repressor allows for heat-induced regulation of gene expression, as it is functional at 30°C, repressing gene expression, but is inactivated at 37–42°C, thus allowing gene expression. The binding of cI repressors to OL1 is reduced by the OL1 G to T mutation, which results in increased promoter activity at 30°C and 37–42°C, as described by Williams, op. cit., 2014. Plasmid: An extra chromosomal DNA molecule that can replicate independently of chromosomal DNA and is distinct from chromosomal DNA. Plasmid copy number: The number of plasmid copies per cell. Increasing plasmid copy number reduces plasmid production yield. Pol: Polymerase. Pol I: Escherichia coli DNA polymerase I Pol I-dependent replication origins: Replication origins that require Pol I, e.g., pMB1, ColE1, or pBR322 or derivatives (e.g., high-copy pUC origins). At these origins, RNAII primers form an RNA:DNA R loop, which is cleaved by RNase H to form a primer for DNA pol I-directed DNA synthesis. DNA synthesis is then converted to DNA pol III. Numerous additional Pol I-dependent replication origins are known in the art, many of which are summarized in del Solar et al., 1998 Microbiol. Mol. Biol. Rev 62:434-464, which are incorporated herein by reference. Pol III: Escherichia coli DNA polymerase III. Pol III-dependent origins of replication: Origins of replication that do not require Pol I, e.g., the rep protein-dependent R6K gamma origin of replication. Numerous additional Pol III-dependent origins of replication are known in the art, many of which are summarized in del Solar et al., 1998 (op. cit.) (included herein by reference). Poly(A): Polyadenylation signal or site. Polyadenylation is the addition of a poly(A) tail to an RNA molecule. Polyadenylation signals contain sequence motifs recognized by RNA cleavage complexes. Most human polyadenylation signals contain the AAUAAA motif and its corresponding 5' and 3' conserved sequences. Commonly used poly(A) signals are derived from rabbit β-globin, bovine growth hormone, early SV40, or late SV40 poly(A) signals. pUC origin: A replication origin derived from pBR322, characterized by a G-to-A translocation that increases copy number at high temperatures, and the deletion of a ROP-negative regulator. pUC-free: Plasmids that do not contain a pUC origin. These may contain non-replicating fragments of the pUC origin, such as RNAI selection markers. pUC plasmid: A plasmid containing a pUC origin.

[0115] R6K plasmids: NTC9385R, NTC9685R, NTC9385R2-O1, NTC9385R2-O2, NTC9385R2a-O1, NTC9385R2a-O2, NTC9385R2b-O1, NTC9385R2b-O2, NTC9385Ra-O1, NTC9385Ra-O2, NTC9385RaF, and NTC9385RbF vectors, as well as modified and alternative vectors containing R6K replication origins as described herein by reference by Williams, cited above, 2014. Alternative R6K vectors known in the art include, but are not limited to, pCOR vectors (Gencell), pCpG-free vectors (Invivogen), and CpG-free Oxford University vectors containing pGM169. R6K replication origin: A region specifically recognized by the R6K Rep protein to initiate DNA replication. This includes, but is not limited to, the R6K gamma replication origin sequences disclosed as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 18. It also includes the CpG-free version described in Drocourt et al., U.S. Patent No. 7,244,609, which is incorporated herein by reference (e.g., SEQ ID NO: 3). R6K origin-of-replication-RNA-OUT bacterial region: Contains R6K origin-of-replication and RNA-OUT selection markers for amplification (e.g., SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17). Rep: Reproduction. Replication intermediates: Linear DNA fragments resulting from premature termination of plasmid replication. Rep protein-dependent plasmids: plasmids whose replication depends on the replication (Rep) protein provided to Trans. Examples include R6K origin of replication, ColE2-P9 origin of replication, and ColE2-related origin of replication plasmids, in which the Rep protein is expressed from the host strain genome. Numerous further Rep protein-dependent plasmids are known in the art, many of which are summarized by reference in del Solar et al., op. cit., 1998. Retroviral vectors: Embedded viral vectors that can infect dividing cells. Also called transfer plasmids. Plasmids encode adjacent expression units of retroviral LTRs. Transfer plasmids are transfected into producing cells along with envelope and packaging plasmids necessary to create viral particles. Retroviral envelope vectors: plasmids that encode envelope glycoproteins. Retroviral packaging vectors: Plasmids encoding retroviral gag and pol genes required for retroviral transfer vectors. RNA-IN: RNA-IN encoding insertion sequence 10 (IS10), complementary to a portion of RNA-OUT, and antisense RNA. When RNA-IN is cloned into an untranslating reader of mRNA, annealing of RNA-IN to RNA-OUT reduces the translation of genes encoded downstream of RNA-IN. RNA-IN regulatory selection markers: Genetically expressed RNA-IN regulatory selection markers. In the presence of plasmid-borne RNA-OUT antisense repressor RNA (SEQ ID NO: 6), the expression of proteins encoded downstream of RNA-IN is suppressed. RNA-IN regulatory selection markers are configured such that RNA-IN regulates either 1) proteins that are lethal or toxic to the cells themselves or by producing toxic substances (e.g., SacB), or 2) repressor proteins that are lethal or toxic to the bacterial cells by repressing the transcription of genes essential for the proliferation of the cells (e.g., the murA essential gene regulated by the RNA-IN tetR repressor gene). For example, a genetically expressed RNA-IN-SacB cell line for RNA-OUT plasmid selection / amplification is described in Williams, op. cit., 2008, which is incorporated herein by reference. Alternative selection markers described in the art may be used instead of SacB. RNA-OUT: An antisense RNA that hybridizes to a transposon gene expressed downstream of RNA-OUT and RNA-IN, encoding the insertion sequence 10 (IS10), thereby reducing its translation. RNA-IN-SacB cell lines that genetically express the RNA-OUT RNA sequence (SEQ ID NO: 6) and complementary RNA-IN SacB can be modified to incorporate alternative functional RNA-IN / RNA-OUT binding pairs, such as those described by Mutalik et al., 2012, Nat Chem Biol 8:447, which include, but are not limited to, the RNA-OUT A08 / RNA-IN S49 pair, the RNA-OUT A08 / RNA-IN S08 pair, and CpG-deprived modifications of RNA-OUT A08 that modify the CG in the RNA-OUT 5'TTCGC sequence to a non-CpG sequence. An example of a CpG-free RNA-OUT selection marker in which two CpG motifs (one of which is located in the RNA-IN complementary region) in the RNA-OUT RNA have been removed was described in Williams 2015. This is a replicating minicircle vector with improved expression. U.S. Patent Application No. 2015 / 0275221 is incorporated herein by reference. CpG-free RNA-OUT can be constructed using a number of alternative substitutions to remove the two CpG motifs (mutating each CpG to either CpA, CpC, CpT, ApG, GpG, or TpG). RNA-OUT selection markers: RNA-OUT selection marker DNA fragments containing Escherichia coli transcription promoter and terminator sequences adjacent to RNA-OUT RNA. RNA-OUT selection markers utilizing RNA-OUT promoter and terminator sequences adjacent to DraIII and KpnI restriction enzyme sites, as well as RNA-IN-SacB cell lines expressed genetically as designers for RNA-OUT plasmid amplification, are described in Williams, op. cit., 2008, which are incorporated herein by reference. The RNA-OUT promoter and terminator sequences in Sequence ID No. 5 adjacent to RNA-OUT RNA (SEQ ID No. 6, Figure 1B) may be replaced with heterologous promoter and terminator sequences. For example, the RNA-OUT promoter may be replaced with a CpG-free promoter known in the art, such as the I-EC2K promoter, or the P5 / 6 5 / 6 or P5 / 6 6 / 6 promoter described in Williams, op. cit., 2008, which are incorporated herein by reference. A 2CpG RNA-OUT selection marker, in which two CpG motifs are removed from the RNA-OUT promoter, is shown as Sequence ID No. 7. An example of a CpG-free RNA-OUT transcription unit in which two CpG motifs (one of which is located in the RNA-IN complementary region) and two CpG motifs in the RNA-OUT promoter are removed is described in Williams, op. cit., 2015, included herein by reference. Vectors incorporating the CpG-free RNA-OUT selection marker can be selected for sucrose tolerance using the RNA-IN-SacB cell line for RNA-OUT plasmid amplification described in Williams, op. cit., 2008. Alternatively, the RNA-IN sequence in these cell lines can be modified to incorporate a 1 bp change necessary to perfectly match the CpG-free RNA-OUT region complementary to RNA-IN. RNA polymerase II promoter: A promoter that recruits RNA polymerase II to synthesize mRNA, most micronuclear RNAs, and microRNAs. Examples include constitutive promoters such as the human or mouse CMV promoter, elongation factor 1 (EF1) promoter, chicken β-actin promoter, β-actin promoters from other species, elongation factor-1α (EF1α) promoter, phosphoglycerokinase (PGK) promoter, Roussarcoma virus (RSV) promoter, human serum albumin (SA) promoter, splenic fociforming virus (SFFV) promoter, α-1 antitrypsin (AAT) promoter, thyroxine-binding globulin (TBG) promoter, and cytochrome P450 2E1 (CYP2E1) promoter. The vector may also utilize combination promoters, such as the chicken β-actin / CMV enhancer (CAG) promoter, human or mouse CMV-derived enhancer elements combined with the elongation factor 1α (EF1α) promoter, CpG-free versions of human or mouse CMV-derived enhancer elements combined with the elongation factor 1α (EF1α) promoter, albumin promoters combined with α-fetoprotein MERII enhancer, or a variety of tissue-specific or inducible promoters known in the art, such as the muscle-specific promoter muscle creatine kinase (MCK), and the C5-12 or liver-specific promoter apolipoprotein A-1 (ApoAI). RNA polymerase III promoter: A promoter that recruits RNA polymerase III to synthesize tRNA, 5S ribosomal RNA, and other small RNA molecules. Examples include class I promoters, e.g., the 5s rRNA promoter; class II promoters, e.g., the tRNA promoter; and class III promoters, e.g., the U6 micronucleus RNA promoter or the H1 nuclear RNase P promoter. RNA selection markers: RNA selection markers are plasmid-retained non-coding RNAs that regulate and enable selection of target genes expressed on chromosomes. These may be plasmid-retained nonsense repressive tRNAs that regulate nonsense repressive selectable chromosomal targets, as described by reference herein by Crouzet J and Soubrier F 2005, U.S. Patent No. 6,977,174. This may also be plasmid-borne antisense repressor RNAs, and an unrestricted list included herein by reference includes: RNA-OUT (Williams, op. cit., 2008), which represses the RNA-IN regulatory target; pMB1 plasmid origin encoding RNAI, which represses the RNA-II regulatory target (Grabherr R, PfaffeNzeller I. 2006, U.S. Patent Application Publication No. 2006 / 0063232; CraNeNburgh RM. 2009; U.S. Patent No. 7,611,883); IncB plasmid pMU720 origin encoding RNAI, which represses the RNA-II regulatory target (Wilson IW, Siemering KR, Prazkier J, Pittard AJ. 1997. J Bacteriol 179: pp. 742-753); ParB locus Sok of plasmid R1, which represses the Hok regulatory target; and FlmB (Morsey) of plasmid F, which represses the flmA regulatory target. This includes MA, 1999, U.S. Patent No. 5,922,583). RNA selection markers may be other natural antisense repressor RNAs known in the art, e.g., those described in Wagner EGH, Altuvia S, and Romby P. 2002. Adv Genet 46:361-98, and Franch T and Gerdes K. 2000. Current Opin Microbiol 3:159-64. RNA selection markers may also be engineered repressor RNAs, e.g., synthetic small RNAs expressing SgrS, MicC, or MicF scaffolds, as described in Na D, Yoo SM, Chung H, Park H, Park JH, and Lee SY. 2013. Nat Biotechnol 31:170-174.RNA selection markers may also be engineered repressor RNAs as part of a selection marker that suppresses target RNA fused to the regulated target gene, e.g., SacB, described by Williams, op. cit., 2015. ROP: Primer replacer. RSM: RNA selection marker.

[0116] SacB: A structural gene encoding Bacillus subtilis levans sucrase. SacB expression in Gram-negative bacteria is toxic in the presence of sucrose. SD: standard deviation. SEAP: Secretory alkaline phosphatase. Selectable markers: Selective markers, such as kanamycin resistance genes or RNA selectable markers. Selection marker: A selection marker, such as a kanamycin resistance gene or RNA selection marker. SIDD: Supercoil-induced DNA double-strand destabilization (SIDD) structures. These sites, when incorporated into a vector, can alter the sensitivity of other sequences within the vector to destabilize them. This can alter their function. For example, adding SIDD sites to an expression vector can reduce promoter helical destabilization. This can increase or decrease promoter activity depending on the promoter, as some promoters may show increased expression due to promoter helical destabilization, while others show decreased expression. shRNA: Short hairpin RNA. S / MAR: Scaffold / matrix attachment region. A sequence in eukaryotes that mediates the attachment of DNA to the nuclear matrix. Sleeping Beauty transposons: SB transposons. A transposon system in which IR / DR incorporates adjacent SB transposons into the genome via a simple cut-and-paste mechanism mediated by SB transposases. Transposon vectors typically contain a promoter-transgene-polyA expression cassette between the IR / DR, which is excised and incorporated into the genome. Spacer region: As used herein, the spacer region is a region that ligates the 5' and 3' ends of a eukaryotic region sequence. The 5' and 3' ends of the eukaryotic region are typically separated by a bacterial origin and bacterial selection marker in the plasmid vector (bacterial region), and many spacer regions consist of a bacterial region. In the Pol III-dependent origin of the replication vector of the present invention, this spacer region is preferably less than 1000 bp. SR: Spacer area. SSI single-stranded start sequence. Structured DNA sequences: DNA sequences capable of forming replication-inhibiting secondary structures, as used herein (Mirkin and Mirkin, 2007. Microbiology and Molecular Biology Reviews 71:13-35). This includes, but is not limited to, reverse repeats, palindromes, directional repeats, IR / DR, homopolymer repeats, or repeats containing eukaryotic promoter enhancers, or repeats containing eukaryotic origins of replication. SV40 origin: Simian virus 40 genomic DNA including the origin of replication. SV40 enhancer: Simian virus 40 genomic DNA containing enhancer repeats of 72 bp and optionally 21 bp.

[0117] Target antigen: An immunogenic protein or peptide epitope, or a combination of protein and epitope, capable of eliciting an immune response. The target antigen may originate from a pathogen for an infectious or allergic application, or from a host organism for an application such as cancer, allergy, or autoimmune disease. Target antigens are well defined in the art. Several examples are described in Williams, op. cit., 2008, which are included herein by reference. TE buffer: A solution containing approximately 10 mM Tris (pH 8) and 1 mM EDTA. TetR: Tetracycline resistance gene. Tol2 transposons: A transposon system in which an ITR incorporates an adjacent Tol2 transposon into the genome via a simple cut-and-paste mechanism mediated by Tol2 transposase. Transposon vectors typically contain a promoter-transgene-polyA expression cassette between Tol2 ITRs that are excised and incorporated into the genome. Transfer Terminator: bacteria A DNA sequence that marks the end of a gene or operon for transcription. This can be an endogenous transcription terminator or a Rho-dependent transcription terminator. In the case of an endogenous terminator, e.g., the trpA terminator, a hairpin structure is formed within the transcript that disrupts the mRNA-DNA-RNA polymerase ternary complex. Alternatively, a Rho-dependent transcription terminator requires a Rho factor, an RNA helicase protein complex, to disrupt the nascent mRNA-DNA-RNA polymerase ternary complex. eukaryotesThe PolyA signal is not a "terminator"; instead, internal cleavage at the PolyA site leaves the 3'UTR RNA with an uncapped 5' end for nuclease digestion. The nuclease then catches up with RNA Pol II, causing termination. The introduction of an RNA Pol II rest site (a eukaryotic transcriptional terminator) can facilitate termination within a short region of the PolyA site. RNA Pol II rest allows the nuclease introduced into the 3'UTR mRNA after PolyA cleavage to catch up with RNA Pol II at the rest site. A non-exclusive list of eukaryotic transcriptional terminators known in the art includes the C2×4 and gastrin terminators. Eukaryotic transcriptional terminators can elevate mRNA levels by enhancing proper 3' end processing of mRNA. Transfection: Methods for delivering nucleic acids into cells that are known in the art and are included herein by reference [e.g., poly(lactide-co-glycolide) (PLGA), ISCOM, liposomes, niosomes, visomes, block copolymers, Pluronic® block copolymers, chitosan, and other biodegradable polymers, microparticles, microspheres, calcium phosphate nanoparticles, nanoparticles, nanocapsules, nanospheres, poloxamine nanospheres, electroporation, nucleofection, piezoelectric permeation, sonoporation, iontophoresis, ultrasound, SQZ rapid cell deformation-mediated membrane disruption, corona plasma, plasma-accelerated delivery, tissue-resistant plasma, laser microporation, shock wave energy, magnetic field, non-contact magnetic permeability, gene guns, microneedles, microdermabrasion, hydrodynamic delivery, high-pressure tail vein injection, etc.]. Transgene: The target gene cloned into a vector for expression in a target organism. Transposase vector: A vector that codes for transposases. Transposon vector: A vector that encodes a transposon, which is a substrate for gene integration mediated by a transposase. ts: temperature sensitive.

[0118] μg: microgram. μl: microliter. UTR: The untranslated region of mRNA (from 5' or 3' to the coding region). Vectors: Gene delivery vehicles including viral (e.g., alphaviruses, poxviruses, lentiviruses, retroviruses, adenoviruses, adenovirus-related viruses, etc.) and non-viral (e.g., plasmids, MIDGE, transcriptional-active PCR fragments, minicircles, bacteriophages, etc.) vectors. These are well known in the art and are incorporated herein by reference. Vector skeleton: The eukaryotic and bacterial regions of the vector, excluding the transgene or target antigen coding region.

[0119] [Detailed description of preferred embodiments] This technology generally relates to methods and compositions for short bacterial region plasmid DNA vectors of less than 1 kb, which improve plasmid production yield and quality, reduce transfection-related toxicity, and increase transgene expression. This technology can be implemented to improve the expression and production of vectors, such as non-viral vectors (transposon vectors, transposase vectors, Sleeping Beauty transposon vectors, Sleeping Beauty transposase vectors, PiggyBac transposon vectors, PiggyBac transposase vectors, expression vectors, etc.) and viral vectors (e.g., AAV vectors, AAV rep cap vectors, AAV helper vectors, Ad helper vectors, lentiviral vectors, lentiviral envelope vectors, lentiviral packaging vectors, retroviral vectors, retroviral envelope vectors, retroviral packaging vectors, etc.).

[0120] Improved plasmid expression is defined herein as an improved transgene expression level and / or in vitro or in vivo expression duration compared to a plasmid encoding a transgene that includes a bacterial region encoding a pUC origin of replication. All references cited herein should be understood to be incorporated in their entirety.

[0121] The plasmid modification method of this technology has been found to provide a solution for providing vectors containing short spacer regions with structured DNA sequences, which can be produced efficiently and in high yield.

[0122] As used herein, the term “sequence identity” refers to the degree of identity between any given query sequences, for example, between sequence number 2 and the target sequence. The target sequence may have, for example, at least 90 percent, at least 95 percent, or at least 99 percent sequence identity with a given query sequence. To determine the sequence identity percentage, any suitable sequence alignment program known in the art, e.g., the computer program ClustalW (version 1.83, default parameters), is used to align the query sequence (e.g., a nucleic acid sequence) to one or more target sequences. This allows the alignment of nucleic acid sequences to be performed over their entire length (global alignment). Chema et al., 2003. Nucleic Acids Res., Vol. 31: pp. 3497–500. In a preferred method, the sequence alignment program (e.g., ClustalW) calculates the best match between the query and one or more target sequences and aligns them so that identity, similarity, and difference can be determined. One or more nucleotide gaps can be inserted into the query sequence, target sequence, or both to maximize the sequence alignment. For fast pairwise alignment of nucleic acid sequences, appropriate default parameters can be selected for a specific alignment program. The output is a sequence alignment that reflects the relationships between sequences. To further determine the percentage of identity of the target nucleic acid sequence to the query sequence, the sequences are aligned using the alignment program, the number of identical matches in the alignment is divided by the length of the query sequence, and the result is multiplied by 100. Note that the percentage identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, and 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.

[0123] Refer to the diagrams here. Figures 1A-1F show the following annotated maps. Figure 1A) The R6K origin is shown with the location of the 22bp iteron repeat, DnaA boxes 1 and 2, and the region included in the R6K origin of SEQ ID NOs. 1, 2, 3 and 4; Figure 1B) The SEQ ID NO. 5 RNA-OUT selection marker is shown with the location of the SEQ ID NO. 6 RNA-OUT antisense RNA, which includes the RNA-OUT promoter elements -35 and -10, the RNA-IN complementary homology region, and the RNA-OUT terminator 3' hairpin. Figure 1C) Shows a 14-CpG R6K-RNA-OUT bacterial skeleton composed of SEQ ID NO: 1 R6K origin and SEQ ID NO: 5 RNA-OUT selection marker, including a trpA bacterial terminator upstream of the R6K origin and adjacent NheI and KpnI cloning sites; Figure 1D) Shows a 3-CpG R6K-RNA-OUT bacterial skeleton composed of SEQ ID NO: 2 1x CpG R6K origin and SEQ ID NO: 7 2x CpG RNA-OUT selection marker, with adjacent NheI and KpnI cloning sites; Figure 1E) R6K origin from SEQ ID NO: 1, with the positions of six iterons highlighted. Individual 22bp iteron repeat sequences are shown below the origin map; Figure 1F) R6K origin from SEQ ID NO: 18, with the positions of seven iterons highlighted. Individual 22bp iteron repeat sequences are shown below the origin map. In this example, the 7-iteron vector has tandem overlap of iteron 5, but the 7-iteron vector of the present invention can be obtained by tandem overlap of any of iterons 1, 2, 3, 4, 5, or 6.

[0124] Figures 2A and 2B show the following annotated maps: Figure 2A) Pol I-dependent pUC origin-kanamycin-selective sleeping beauty transposon vector pUC57-Kan SB1 (see Table 6); Figure 2B) Pol III-dependent R6K origin-RNA-OUT antibiotic-free selective sleeping beauty transposon vector NTC9 SB1 (see Table 6). The positions of the left and right sleeping beauty IR / DR relative to bacterial skeletal replication origins and selection markers are shown.

[0125] Figures 3A-3C show the following annotated maps: Figure 3A) Pol I-dependent pUC origin - ampicillin-selective AAV vector pAAV (see Table 7); Figure 3B) Pol I-dependent pUC origin - RNA-OUT antibiotic-free selective AAV vector NTC8-AAV (see Table 7); Figure 3C) Pol III-dependent R6K origin - RNA-OUT antibiotic-free selective AAV vector NTC9-AAV (see Table 7). The positions of the left and right AAV ITRs relative to bacterial skeleton replication origins and selection markers are shown.

[0126] Figures 4A-4F show the following annotated maps. Figure 4A) Pol I-dependent pUC origin - mRNA vector pGEM4Z T7 A60 pA encoding ampicillin-selective A60 polyA repeat (see Table 8); Figure 4B) Pol I-dependent pUC origin - RNA-OUT antibiotic-free-selective A60 polyA repeat encoding mRNA vector NTC8-T7 A60 pA (see Table 8); Figure 4C) Pol III-dependent R6K origin - RNA-OUT antibiotic-free-selective A60 polyA repeat encoding mRNA vector NTC8-T7 A60 pA (see Table 8); Figure 4D) Pol I-dependent pUC origin - mRNA vector pT3 / T7 A99 pA encoding ampicillin-selective A99 polyA repeat (see Table 8); Figure 4E) Pol I-dependent pUC origin - kanR-selective A99 polyA repeat encoding mRNA vector NTC7-T7 A99 pA (see Table 8); Figure 4F) Pol NTC9-T7 A99 pA mRNA vector encoding III-dependent R6K origin-RNA-OUT antibiotic-free selective A99 polyA repeats (see Table 8). The positions of the A60 or A99 polyA repeats relative to the bacterial skeletal origin and selection marker are shown. [Examples]

[0127] The methods of this technology are further illustrated by the following embodiments, which are provided as examples and are not intended to limit the scope of this disclosure in any way.

[0128] Example 1: Replication and generation of pUC and R6K origin plasmids Background to the duplication and generation of pUC origin vectors:Most therapeutic plasmids utilize pUC origins, which are high-copy derivatives of the pMB1 origin (closely related to the ColE1 origin). In pMB1 replication, plasmid DNA synthesis is unidirectional and does not require the initiation factor protein present in the plasmid. pUC origins are copy-up derivatives of pMB1 origins that lack the accessory ROP (rom) protein and have temperature-sensitive mutations that destabilize RNAI / RNAII interactions. When cultures containing these origins are changed from 30°C to 42°C, the plasmid copy number increases. pUC plasmids can be generated in a wide variety of E. coli cell lines.

[0129] Background of RNA-OUT antibiotic-free selective markers Antibiotic-free selection is performed in *E. coli* strains containing pCAH63-CAT RNA-IN-SacB(P5 / 6 6 / 6) with a phage-lambda attachment site incorporated into the chromosome, as described by Williams, op. cit., 2008. SacB (Bacillus subtilis levans sucrase) is a lethal counterselection marker against *E. coli* cells in the presence of sucrose. Translation of SacB from RNA-IN-SacB transcripts is inhibited by plasmid-encoded RNA-OUT (Figure 1B). This promotes plasmid selection in the presence of sucrose by inhibiting SacB-mediated lethality.

[0130] Background of R6K origin vector replication and generation The R6K gamma plasmid replication origin requires multiple repeat "iteron" sites (seven core repeats, including the TGAGNG consensus) as replication initiation monomers, and a single plasmid replication protein π that binds to a repression site (TGAGNG) and an iteron with reduced affinity as a replication inhibitory dimer. Replication requires multiple host factors, including IHF, DnaA, and primosome assembly proteins DnaB, DnaC, and DnaG (Abhyankar et al., 2003 J Biol Chem 278:45476-45484). The R6K core origin contains DnaA and IHF binding sites that affect plasmid replication, as π, IHF, and DnaA interact to initiate replication.

[0131] Different versions of the R6K gamma origin have been used in various eukaryotic expression vectors, such as the pCOR vector (Soubrier et al., 1999, Gene Therapy 6:1482-88), pGM169 (University of Oxford), and the CpG-free version of the pCpGfree vector (Invivogen, San Diego CA). Incorporating the R6K origin itself does not improve the expression level of the transgene compared to optimized pUC origin vectors (Soubrier et al., op. cit., 1999). However, the use of a conditional origin requiring a specific cell line for amplification, such as R6K gamma, improves the safety margin because the vector will not replicate if transferred to the patient's endogenous flora.

[0132] A highly minimized replication origin (SEQ ID NO: 1, Figure 1E) derived from 6 iterone R6K gamma, which contains the core sequence required for replication (including the DnaA box and stb 1-3 sites; Wu et al., 1995. J Bacteriol. 177: 6338-6345) but has a deleted upstream π-dimer repressor binding site and a downstream π-promoter (by removal of one copy of iterone), is described in Williams, op. cit., 2014, which is incorporated herein by reference. This R6K origin contains 6 tandem directional repeat iterones (Figure 1E). The NTC9385R nanoplasmid® vector, which contains this minimized R6K origin and an RNA-OUT AF selection marker in the spacer region, is described in Williams, op. cit., 2014, which is incorporated herein by reference.

[0133] Typical R6K-producing cell lines express the π protein derivative PIR1 16 from the genome, which contains a P106L substitution that increases the copy number (by reducing π dimerization, the π monomer is activated, but the π dimer is suppressed). Fermentation results using pCOR (Soubrier et al., op. cit., 1999) and pCpG plasmid (Hebel HL, Cai Y, Davies LA, Hyde SC, Pringle IA, Gill DR. 2008. Mol Ther 16: S110) showed a low concentration of approximately 100 mg / L in the PIR1 16 cell line.

[0134] Selection of pir-116 replication protein for mutagenesis and copy number increase has been used to create new production strains. For example, the TEX2pir42 strain contains a combination of P106L and P42L. The P42L mutation interferes with the suppression of DNA looping replication. The TEX2pir42 cell line showed improved copy number and fermentation yield using a pCOR plasmid with a reported yield of 205 mg / L (Soubrier F, 2004, International Patent Application WO2004033664).

[0135] Other combinations of π copy number mutants that improve copy number include "P42L and P113S" and "P42L, P106L and F107S" (Abhyankar et al., 2004. J Biol Chem 279:6711-6719).

[0136] Williams, op. cit., 2014, describes host strains expressing heat-inducible πP42L, P106L, and F107S high-copy mutant replication (Rep) proteins with a pL promoter incorporating the phage HK022 attachment site for the selection and amplification of R6K-origin nanoplasmid® vectors. This is a further nanoplasmid® safety factor because the R6K-origin vector can only replicate in E. coli host strains expressing genetically modified Rep proteins.

[0137] The amplification and fermentation of the RNA-OUT selection marker - R6K plasmid described in Williams, supra, 2014 was carried out using the DH5α host strain NTC711772 = DH5α dcm- att λ ::P 5 / 6 6 / 6 -RNA-IN- SacB, catR; att HK022 ::pL (OL1-G→T) P42L-P106L-F107S (P3-), SpecR StrepR using a thermoinducible "P42L, P106L and F107S" π copy number mutant cell line such as this. A maximum production yield of 695 mg / L was reported.

[0138] The following additional cell lines were generated and disclosed herein: NTC821601 DH5α att λ ::P 5 / 6 6 / 6 -RNA-IN- SacB, catR; att HK022 :: pL (OL1-G→T) P42L-P106L-F107S (P3-), SpecR StrepR = the dcm+ version of NTC711772. NTC940211 DH5α att λ ::P 5 / 6 6 / 6 -RNA-IN- SacB, catR; att HK022 ::pL (OL1-G→T) P42L-P106I-F107S P113S (P3-), SpecR StrepR = a quadruple copy number increased mutant rep protein derivative of NTC821601 was created by combining a high copy substitution of P106L for P106I with P113S. NTC1050811 DH5α att λ ::P 5 / 6 6 / 6 -RNA-IN- SacB, catR; att HK022 ::pL (OL1-G→T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; att φ80::pARA-CI857ts, tetR = NTC940211 is a pARA-CI857ts derivative. This strain contains a phage φ80 attachment site chromosome-integrated copy of the arabinose-inducible CI857ts gene. Addition of arabinose to the plate or culture medium (e.g., final concentration 0.2-0.4%) induces expression of the pARA-mediated CI857ts repressor, and the copy number at 30°C is reduced by CI857ts-mediated downregulation of the pL promoter expressing the Rep protein [i.e., additional CI857ts mediates a more effective downregulation of the pL(OL1-G→T) promoter at 30°C]. Copy number induction after temperature shifts to 37-42°C is not impaired because the CI857ts repressor is inactivated at these higher temperatures. If methylation of dcm is undesirable, a dcm- derivative (NTC1050811 dcm-) can be used. NTC1011641;Stbl4 attλ::P5 / 6 6 / 6-RNA-IN- SacB, catR; att HK022 ::pL P42L-P106L-F107S (P3-) SpecR StrepR = Stbl4 version of NTC661135 (XL1Blue- dcm- att λ ::P 5 / 6 6 / 6 -RNA-IN- SacB, catR; att HK022 ::pR pL P42L-P106L-F107S (P3-) SpecR StrepR (as described in Williams, op. cit., 2014).

[0139] Nanoplasmid® yield is improved by the quadruple mutant heat-inducible pL (OL1-G→T) P42L-P106I-F107S P113S (P3-) compared to the triple mutant heat-inducible pL (OL1-G→T) P42L-P106L-F107S (P3-) (Williams, op. cit., 2014). The yield of excess 2 g / L nanoplasmid® was obtained using the quadruple mutant NTC1050811 cell line (e.g., 2240 mg / L using NTC9 T7 A99 pA, Table 8).

[0140] The use of conditional origins of replication, such as these R6K origins, which require specific cell lines for amplification, adds a safety margin because the vector will not replicate if it is transferred into the patient's endogenous flora.

[0141] Example 2: Generation of pUC and R6K origin vectors Shaking Flask Preparation: Shaking flasks were prepared using our own Plasmid+ shaking culture medium. Seed culture was started from glycerol stocks or colonies and streaked onto LB agar plates containing 50 μg / mL antibiotic (for ampR or kanR selective plasmids) or 6% sucrose (for RNA-OUT selective plasmids). Plates were grown at 30-32°C. Cells were resuspended in medium and used to prepare a 500 mL plasmid+ shaking flask containing 50 μg / mL antibiotic for amp-R or kanR selective plasmids or 0.5% sucrose for RNA-OUT plasmid selection, with an OD of approximately 2.5. 600 Inoculation material was provided. The cells were grown in flasks at the growth temperatures shown in Tables 5, 6, 7, and 9, shaking until the flasks were saturated.

[0142] Fermentation process: Fermentation was carried out using a proprietary fed-batch medium (NTC3019, HyperGRO medium) in a New Brunswick BioFlo 110 bioreactor, as described (Carnes and Williams, op. cit., 2011). Seed culture was started from glycerol stocks or colonies and streaked onto LB agar plates containing 50 μg / mL antibiotic (for ampR or kanR selective plasmids) or 6% sucrose (for RNA-OUT selective plasmids). Plates were grown at 30–32°C; cells were resuspended in medium and used to provide approximately 0.1% inoculum for fermentation containing 50 μg / mL antibiotic for ampR or kanR selective plasmids or 0.5% sucrose for RNA-OUT plasmids. The HyperGRO temperature shifts were as shown in Tables 8 and 9.

[0143] Generation host: Fermentation of pUC-initiated AmpR or KanR plasmids was performed in E. coli DH5α strain [F- Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK-, mK+) phoA supE44 λ- thi-1 gyrA96 relA1] (Invitrogen, Carlsbad CA) or Stbl4 strain.

[0144] Fermentation of antibiotic-free pUC-origin RNA-OUT plasmids was performed in *E. coli* DH5α strain containing pCAH63-CAT RNA-IN-SacB (P5 / 6 6 / 6) with the phage λ attachment site integrated into the chromosome, as described by Williams, op. cit., 2008. The resulting strain was NTC4862 = DH5α attλ::P5 / 6 6 / 6-RNA-IN-SacB, catR.

[0145] Amplification and fermentation of antibiotic-free R6K gamma-origin RNA-OUT plasmids were performed using an E. coli RNA-OUT selective host that further encodes a phage HK022 attachment site-integrated pL promoter-thermally induced π copy number mutant cell line (the method for which this was prepared is described in Williams, op. cit., 2014, and is incorporated herein by reference).

[0146] Generating stocks: pUC-initiated AmpR or KanR antibiotic-selective host DH5α Stbl4

[0147] pUC-initiated RNA-OUT sucrose-selective host NTC4862 DH5α att λ ::P 5 / 6 6 / 6 -RNA-IN- SacB, catR NTC1011592 Stbl4 attλ::P5 / 6 6 / 6-RNA-IN- SacB, catR

[0148] R6K-based RNA-OUT sucrose-selective nanoplasmid™ host NTC1050811 DH5α att λ ::P5 / 6 6 / 6 -RNA-IN- SacB, catR; att HK022 ::pL (OL1-G→T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; att φ80 ::pARA-CI857ts, tetR NTC1011641 Stbl4 attλ::P5 / 6 6 / 6-RNA-IN- SacB, catR; att HK022 ::pL P42L-P106L-F107S (P3-) SpecR StrepR

[0149] Analysis method: Culture samples were taken at key points and regular intervals throughout all fermentation. The samples were immediately processed into biomass (OD). 600 Plasmid yield was analyzed. Plasmid yield was determined by quantification of plasmids obtained from preparations using the Qiagen Spin Miniprep Kit (Carnes and Williams, op. cit., 2011). Briefly, cells were lysed with alkali, clarified, and plasmids were column-purified and eluted before quantification. Plasmid quality was determined by agarose gel electrophoresis (AGE), performed on 0.8–1% Tris / acetic acid / EDTA (TAE) gel as described in Carnes and Williams, op. cit., 2011.

[0150] Example 3: Construction and production of pUC and R6K-based structured vectors The R6K gamma origin (SEQ ID NO: 1; Figure 1E)-RNA-OUT (SEQ ID NO: 5; Figure 1B) bacterial replication-selection region (SEQ ID NO: 8; Figure 1C) was cloned into the polylinker regions of various pUC57-based vectors to create the pNTC-NP1, pNTC-NP2, pNTC-NP3, pNTC-NP4, pNTC-NP5, pNTC-NP6, and pNTC-NP7 vectors. Each vector has various adjacent restriction sites that can be used to retrofit the target vector for R6K replication-RNA-OUT selection. The 5' and 3' polylinker sequences adjacent to the R6K-RNA-OUT insert in the pNTC-NP1-NP7 vectors are shown in Table 4. A pUC57-based version of the 1 CpG R6K gamma origin-2 CpG RNA-OUT bacterial replication-selection region (SEQ ID NO: 9; Figure 1D) was also created (pNTC-3xCpG NP1) and is shown in Table 4.

[0151] The R6K gamma origin (SEQ ID NO: 1) shows the manipulated 6-iteron R6K origin (Figure 1E). A pUC57-based version of the 7-iteron R6K gamma origin (SEQ ID NO: 18; Figure 1F)-RNA-OUT (SEQ ID NO: 5; Figure 1B) bacterial replication-selection region was also prepared and used to construct and evaluate the usefulness of additional iterone for production. Similarly, high-quality, high-yield production was obtained using vectors that differed only in containing either the 7-iteron R6K gamma origin of SEQ ID NO: 18 or the 6-iteron R6K gamma origin (SEQ ID NO: 1). For example, the following harvested yields were obtained with 10 hours of gradient temperature-shifted HyperGRO fermentation at 30–42°C.

[0152] Sequence ID 1: 6iteron 3203 bp R6K origin vector: Biomass 120 OD 600 Plasmid titer 1363 mg / L; Plasmid specific yield 11.3 mg plasmid / L / OD 600

[0153] Sequence ID 18: 7iteron 3225 bp R6K origin vector: Biomass 137 OD 600 Plasmid titer 1503 mg / L; Plasmid specific yield 11.0 mg plasmid / L / OD 600

[0154] The 7-iteron R6K gamma origin in SEQ ID NO: 18 is a tandem duplication of iterone 5 (Figure 1F; SEQ ID NO: 18), but the 7-iteron R6K gamma origin vector of the present invention may be a tandem duplication of any of the iterones shown as SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23 (Figure 1E), or a random combination of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23 to the 7-iteron R6K origin composition, or an iterone repeat mutant that maintains the TGAGNG consensus. Further iterone derivatives (e.g., 8, 9, or 10-iteron vectors) are also envisioned in the implementation of the present invention.

[0155] [Table 4] Viral and nonviral vector pUC-based antibiotic-selective bacterial scaffold retrofitting for R6K-RNA-OUT was performed as follows: 1) Select restriction sites adjacent to the pUC origin and antibiotic selection marker region in target viruses and nonviral vectors. 2) Identify the pNTC-NP compatible polylinker-R6K-RNA-OUT polylinker cassette (any of pNTC-NP1, 2, 3, 4, 5, 6, or 7, Table 4), 3) Using a selected restriction digestion approach and standard ligase-mediated cloning, the pUC-origin antibiotic selection marker region is excised and replaced with a selected R6K-origin RNA-OUT region.

[0156] In some cases, the R6K origin and RNA-OUT unit were assembled by multi-fragment ligation from separate restriction fragments using a non-palindromic DraIII linker site (see Table 4). In the case of the fd6 Ad helper retrofit (Table 9), a short 500 bp synthetic gene DraIII RNA-OUT-Ad helper-AvrII was used to ligate RNA-OUT to the specific AvrII site in the eukaryotic region of the 12 kb AvrII-SalI restriction fragment and to the pNTC-NP4-derived SalI-R6K origin-DraIII fragment, performing a three-fragment ligation.

[0157] Exemplary vector maps and vector characteristics of the original pUC-origin-antibiotic selection marker vectors and retrofitted R6K-origin-RNA-OUT antibiotic-free selection marker vectors are shown for Sleeping Beauty (Figure 2, Table 6), AAV (Figure 3, Table 7), and mRNA (Figure 4, Table 8) vectors. Vector characteristics of the original pUC-origin-antibiotic selection marker vectors and retrofitted R6K-origin-RNA-OUT antibiotic-free selection marker vectors are shown for the AAV helper vector (Table 8). Vector characteristics of the pUC-origin-RNA-OUT antibiotic-free selection marker vectors and retrofitted R6K-origin-RNA-OUT antibiotic-free selection marker vectors are shown for lentiviral vectors (Table 5) and AAV vectors (Table 7).

[0158] In all cases, the bacterial skeleton size was less than 1 kb in the R6K origin-RNA-OUT antibiotic-free selective marker retrofitted vectors (460–610 bp). This is well below the 1.1 kb bacterial skeleton size limit required to improve vector expression levels (Tables 1–2) and duration (Quiviger et al., op. cit., 2014). In all cases, the original pUC origin-antibiotic selective bacterial skeleton before retrofitting was greater than 1.2 kb (2340–2750 bp), similar to the pUC origin-RNA-OUT retrofit (1210–1500 bp). Therefore, these AAV, AAV helper, Sleeping Beauty, and lentivirus R6K origin-RNA-OUT antibiotic-free selective marker retrofitted vectors meet the short spacer region requirement for improving expression levels and duration compared to the original pUC origin-antibiotic selective marker vectors. Furthermore, these AAV, AAV Helper, Sleeping Beauty, and lentiviral R6K origin-RNA-OUT antibiotic-free selective marker retrofit vectors do not have the opportunity for antibiotic marker gene transfer by transduction (AAV, lentiviral vectors) or translocation (Sleeping Beauty vector) by removing the KanR or ampR antibiotic resistance selective marker in the parent vector. Moreover, the vectors of this technology do not require the complex difficulties of increasing the costly additional manufacturing steps necessary to remove the large bacterial region between the eukaryotic polyA and promoter in the minicircle vector (Kay et al., op. cit., 2010).

[0159] However, in lentiviral vectors, the eukaryotic region contains adjacent directional repeats (LTRs), in AAV vectors, the eukaryotic region contains adjacent reverse-terminal repeats, while in sleeping beauty transposon vectors, the eukaryotic region contains adjacent transposon IR / DR terminals. All of these adjacent sequences are structured DNA sequences.

[0160] Levy, op. cit., 2004, taught that replication intermediates are formed when the origin of replication of any high-copy-number prokaryote is less than 1 kb from a structured DNA sequence, such as an enhancer, LTR, or IRES, but not when the high-copy-number origin is greater than 1.5 kb. Consistent with this, replication intermediates were formed in all pUC origin-RNA-OUT marker vectors (Table 5: 400 bp) where the pUC origin is less than 1 kb from the lentiviral vector LTR, or in pUC origin-antibiotic resistance marker vectors (Table 6: 280 bp) where the pUC origin is less than 1 kb from the Sleeping Beauty IR / DR. For AAV and mRNA vectors, the original pUC origin-antibiotic selection marker vectors have pUC origins at 0 bp from the ITR (AAV vector, Table 7) or at 170 bp from the A99 repeat (mRNA vector, Table 8), which can produce replication intermediates that are too small to be detected on agarose gels. However, in these cases, the production yield was very low, indicating a low plasmid copy number due to replication blockade. In contrast, as expected, when the original pUC origin-antibiotic selection marker vector pUC origin exceeded 1.5 kb from the structured DNA sequence (A60 repeat), high plasmid production yields were obtained (Table 8: mRNA vector pGEM4Z T7 A60).

[0161] Williams, op. cit., 2017, reported that when the pUC origin exceeds 1.5 kb from the homopolymer A64C31 repeat, the PAS-BH elongated pUC origin improves the yield of pUC origin vectors. However, when the PAS-BH elongated pUC origin is oriented to less than 400 bp from the A64C31 repeat, the yield was low (see Table 8, footnotes d and e). This suggests that the addition of the PAS-BH primosome assembly site does not overcome pUC origin-directed replication with few closely spaced structured DNA sequences.

[0162] Since the pUC origin itself is 1 kb, as shown above and as predicted by Levy, op. cit., 2004, there are no configurations for producing bacterial AAV, lentivirus, retrovirus, or transposon vectors containing a pUC origin of less than 1.1 kb that is not expected to generate replication intermediates, and plasmid yields are poor as reported herein.

[0163] Surprisingly, replication intermediates were not observed in any of the R6K origin-RNA-OUT antibiotic-free selection marker retrofitted vectors, including those with R6K origins less than 1kb from the lentiviral vector LTR (Table 5: 400 bp) or Sleeping Beauty IR / DR (Table 6, <40 bp). Furthermore, for AAV vectors, the original pUC origin-antibiotic selection marker vector with a 0 bp pUC origin from the ITR showed very poor production yields, while two R6K origin-RNA-OUT antibiotic-free selection marker retrofitted vectors with a 40 bp R6K origin from the ITR showed much higher production yields (Table 7). This improved production is specific to R6K and not to RNA-OUT. This is because the two AAV pUC-RNA-OUT retrofits with a pUC origin 50 bp from the ITR produced plasmid yields comparable to those of the original pUC antibiotic marker vector (Table 7). Furthermore, a direct comparison of the pUC-RNA-OUT retrofit with the R6K-RNA-OUT retrofit located 400 bp from the LTR repeat of the lentiviral backbone showed replication intermediates with all three pUC-RNA-OUT backbones, but none of the three R6K-RNA-OUT backbones (Table 5).

[0164] This remarkable improvement in plasmid copy number (plasmid yield) and quality (elimination of replication intermediates) using R6K origin vectors means that R6K origins can replicate more effectively through structured DNA sequences than pUC origins. Levy, op. cit., 2004, taught that replication intermediates are formed when any high-copy-number prokaryotic replication origin is less than 1kb from a structured DNA sequence, such as an enhancer, LTR, or IRES, but not when the high-copy-number replication origin is more than 1.5kb away, although all examples provided by Levy, op. cit., 2004, were pUC origin plasmids.

[0165] The fundamental difference between these origins of replication is that the pUC origin is a Pol I-dependent origin, while the R6K origin is a Pol III-dependent origin. At the pUC origin, the RNAII primer forms an RNA:DNA R loop, which is cleaved by RNase H to create a primer for DNA Pol I-directed DNA synthesis during the initial leading strand synthesis. DNA synthesis then converts the slow DNA Pol I to the highly processable DNA Pol III, ranging from 400 bp downstream of the origin up to 1.3 kb (Allen et al., 2011. Nucleic Acids Research, Vol. 39: pp. 7020-7033). The R6K gamma origin of replication rep protein interacts with dnaB helicase and dnaG primase, which produce short RNA primers for DNA Pol III replication without requiring DNA Pol I (Abhyankar et al., op. cit., 2003). The pUC origin DNA Pol I replication zone, extending up to 1.3kb from the origin, closely corresponds to the upper limit of replication intermediate formation (1-1.5kb from the origin) as defined by Levy, op. cit., 2004. We propose that the remarkably improved replication of structured DNA observed near R6K, rather than the pUC origin, is due to an unexpected improvement in structured DNA sequence replication by DNA Pol III compared to DNA Pol I.

[0166] The vector methods and compositions disclosed herein demonstrate that structured DNA sequences that are not well replicated by Pol I-dependent replication origins, such as pUC origins, can be replicated using Pol III-dependent replication origins, such as R6K origins.

[0167] These results demonstrate that the vectors of the present invention are useful in improving the production yield and quality of viral and nonviral vectors.

[0168] Example 4: Improved performance of R6K-based structured vectors The vectors of the present invention are further useful for eliminating the transfer of antibiotic resistance marker genes by viral and non-viral vectors, reducing transfection-related toxicity, improving transfer from non-viral transposon vectors, improving packaging titers from viral vectors, and improving the expression of transgenes encoded by viral and non-viral vectors.

[0169] As an example, the R6K-based third-generation lentiviral vectors of the present invention [four vectors: Table 5, R6K-based and transfer plasmids with a bacterial skeleton of less than 1 kb, gag pol packaging plasmid, env plasmid, REV plasmid (not shown)] showed reduced toxicity and improved viral packaging titer compared to pUC-based vectors with a bacterial skeleton of more than 1.5 kb. Transfection of Lenti-X293 T cell line (Takara Bio Mountain View, CA) with third-generation lentiviral vectors with a bacterial skeleton of less than 1kb originating from R6K, or with the original pUC originating antibiotic selection marker vector with a bacterial skeleton control of more than 1.5kb, in 24-well plates using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA) as recommended by the manufacturer, resulted in higher lentiviral titer generation (pUC originating control vector with a bacterial skeleton of more than 1.5kb: 1.00 × ±0.32, R6K originating vector with a bacterial skeleton of less than 1kb: 1.45 × ±0.42) as measured using the Lenti-X p24 Rapid Titer Kit (Takara Bio Mountain View, CA). As described, transfection of the Lenti-X293 T cell line in 24-well plates using third-generation lentiviral vectors (bacterial skeleton pUC originating from a bacterial skeleton greater than 1.5kb or bacterial skeleton R6K originating from a bacterial skeleton less than 1kb) using calcium phosphate transfection (Marino MP, Luce MJ, Reiser J. 2003, Methods Mol Biol Vol. 229: pp. 43-55) as shown in Table 5 resulted in higher lentiviral titer generation (bacterial skeleton pUC originating from a bacterial skeleton greater than 1.5kb: 1.00 × ±0.30, bacterial skeleton R6K originating from a bacterial skeleton less than 1kb: 1.32 × ±0.19) when measured using the Lenti-X p24 Rapid Titer Kit (Takara Bio Mountain View, CA).Importantly, calcium phosphate transfection of third-generation lentiviral vectors originating from a bacterial skeleton pUC exceeding 1.5 kb resulted in widespread transfection-related toxicity (over 80% cell death) in this 24-well plate transfection compared to the low toxicity of R6K-originating third-generation lentiviral vectors originating from a bacterial skeleton of less than 1 kb. This reduced transfection-related toxicity should lead to a dramatic improvement in viral titer in transfections on a large production scale. These results demonstrate that the R6K-originating nanoplasmid vectors originating from a bacterial skeleton of less than 1 kb of the present invention reduce transfection-related toxicity and improve packaging titer from viral vectors compared to bacterial skeleton vectors exceeding 1.5 kb.

[0170] overview The above description includes numerous examples, which should not be interpreted as limiting the scope of the disclosure, but rather as illustrative examples of preferred embodiments. Many other variations are possible.

[0171] For example, the vector of this technology can utilize various orientations of Pol III-dependent origins of replication and RNA selection markers. For instance, any of the eight orientations of Pol III-dependent origins of replication and the RNA selection marker in the vector of this technology can be used (i.e., ←Pol III origin RSM→; ← Pol III origin ← RSM; Pol III origin → RSM →; Pol III origin → ← RSM; ← RSM Pol III origin →; ← RSM ← Pol III origin; RSM → Pol III origin →; RSM → ← Pol III origin).

[0172] Furthermore, various RNA selection markers known in the relevant technical field can be used instead of RNA-OUT.

[0173] Furthermore, for example, if a simple retrofit of a pUC origin to an R6K origin is desired to improve plasmid yield and / or quality, an antibiotic resistance marker may be used instead of RNA-OUT.

[0174] Therefore, readers will understand that the improved Pol III-dependent originating vector of this technology reduces transfection-associated toxicity, improves transposition from non-viral transposon vectors, improves packaging titers from viral vectors, improves the expression of viral and non-viral vector-encoding genes, and eliminates viral and non-viral vector-mediated antibiotic selection marker gene transfer (i.e., via the incorporation of bacterial regions less than 1000 bp), while providing an approach that dramatically improves production compared to alternative vectors, such as pUC plasmids and minicircles.

[0175] Therefore, the scope of the disclosure should be determined by the appended claims, not by the embodiments shown.

[0176] [Table 5]

[0177] [Table 6]

[0178] [Table 7]

[0179] [Table 8]

[0180] [Table 9]

Claims

1. A method for improving the replication of covalent closed circular plasmids, a. Below: i. Pol I dependency replication origin, and ii. An insert comprising a structured DNA sequence selected from the group consisting of reverse repeat sequences, directional repeat sequences, homopolymer repeat sequences, eukaryotic origins of replication, or eukaryotic promoter enhancer sequences, wherein the structured DNA sequence is located at a distance of less than 1000 bp from a Pol I-dependent origin of replication in the direction of replication. The steps include providing a covalent closed circular plasmid containing, and The method comprises the step of modifying a covalently bound closed-circular recombinant molecule of ba to replace a Pol I-dependent origin of replication with a Pol III-dependent origin of replication, thereby improving the replication of the covalently bound closed-circular plasmid with the resulting Pol III-dependent origin of replication.

2. The method according to claim 1, wherein the Pol I-dependent replication origin is selected from the group consisting of a pUC origin, a pMB1 origin, and a ColE1 origin.

3. The method according to claim 1, wherein the Pol III-dependent replication origin is an R6K gamma replication origin.

4. The method according to claim 1, wherein the Pol III-dependent replication origin is an R6K gamma replication origin having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO:

18.

5. The method according to claim 1, wherein the structured DNA sequence is selected from the group consisting of polyA repeats, SV40 origin of replication, viral LTRs, lentiviral LTRs, retroviral LTRs, transposon IR / DR repeats, sleeping beauty transposon IR / DR repeats, AAV ITRs, transposon ITRs, PiggyBac transposon ITRs, CMV enhancers, and SV40 enhancers.

6. The method according to claim 1, wherein the improved replication is selected from the group consisting of a reduction in the generation of replication intermediates and an increase in the plasmid copy number.

7. A method for improving the replication of covalent closed circular plasmids, a. Below: i. Bacterial replication-selection regions including Pol I-dependent origins of replication and antibiotic selection markers, and ii. An insert comprising a structured DNA sequence selected from the group consisting of reverse repeat sequences, directional repeat sequences, homopolymer repeat sequences, eukaryotic origins of replication, and eukaryotic promoter-enhancer sequences, wherein the structured DNA sequence is located at a distance of less than 1000 bp from a Pol I-dependent origin of replication in the direction of replication. The steps include providing a covalent closed circular plasmid containing, and The method comprises the steps of modifying a covalently bound closed circular recombinant molecule of ba to replace an antibiotic selection marker with an RNA selection marker and replacing a Pol I-dependent origin of replication with a Pol III-dependent origin of replication, thereby improving the replication of the covalently bound closed circular plasmid with the resulting Pol III-dependent origin of replication.

8. The method according to claim 7, wherein the Pol I-dependent replication origin is selected from the group consisting of a pUC origin, a pMB1 origin, and a ColE1 origin.

9. The method according to claim 7, wherein the Pol III-dependent replication origin is an R6K gamma replication origin.

10. The method according to claim 7, wherein the Pol III-dependent replication origin is an R6K gamma replication origin having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO:

18.

11. The method according to claim 7, wherein the RNA selection marker is an RNA-IN regulated RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO:

7.

12. The method according to claim 7, wherein the RNA selection marker is an RNA-OUT RNA selection marker that encodes an RNA-IN regulated RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO:

6.

13. The method according to claim 7, wherein the bacterial replication-selection region, which includes a Pol I-dependent replication origin and an antibiotic selection marker, is replaced with a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16, and SEQ ID NOs:

17.

14. The method according to claim 7, wherein the structured DNA sequence is selected from the group consisting of polyA repeats, SV40 origin of replication, viral LTRs, lentiviral LTRs, retroviral LTRs, transposon IR / DR repeats, sleeping beauty transposon IR / DR repeats, AAV ITRs, transposon ITRs, PiggyBac transposon ITRs, CMV enhancers, and SV40 enhancers.

15. The method according to claim 7, wherein the improved replication is selected from the group consisting of a reduction in the generation of replication intermediates and an increase in the plasmid copy number.

16. A covalent closed circular recombinant DNA molecule that does not contain antibiotic markers, a. Antibiotic marker-free inserts containing structured DNA sequences selected from the group consisting of reverse repeat sequences, directional repeat sequences, homopolymer repeat sequences, eukaryotic origins of replication, and eukaryotic promoter-enhancer sequences. b. A Pol III-dependent origin of replication, including an R6K gamma origin of replication having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18, and c. RNA-OUT RNA selection markers containing RNA-IN regulatory RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6 Recombinant DNA molecules containing this molecule.

17. The recombinant DNA molecule according to claim 16, wherein the RNA-OUT RNA selection marker is an RNA-IN regulated RNA-OUT functional mutant having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO:

7.

18. The recombinant DNA molecule according to claim 16, wherein the R6K gamma replication origin and the RNA-OUT RNA selection marker include an R6K origin-RNA-OUT RNA selection marker bacterial replication-selection region having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16, and SEQ ID NOs:

17.

19. The recombinant DNA molecule according to claim 16, wherein the structured DNA sequence is selected from the group consisting of polyA repeats, SV40 origin of replication, viral LTRs, lentiviral LTRs, retroviral LTRs, transposon IR / DR repeats, sleeping beauty transposon IR / DR repeats, AAV ITRs, transposon ITRs, PiggyBac transposon ITRs, CMV enhancers, and SV40 enhancers.

20. The recombinant DNA molecule according to claim 16, wherein the recombinant DNA molecule is selected from the group consisting of viral vectors, lentiviral vectors, retroviral vectors, AAV vectors, Ad vectors, nonviral transposon vectors, sleeping beauty transposon vectors, PiggyBac transposon vectors, Tol2 transposon vectors, and polyA-containing mRNA vectors.

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