Generation of improved viral and non-viral nanoplasmid vectors

Abstract

A method for improving the replication of a covalently closed circular plasmid is provided. The method includes providing a covalently closed circular plasmid, the plasmid having a Pol I-dependent replication origin and an insert, the insert comprising a structured DNA sequence selected from the group consisting of: an inverted repeat sequence, a forward repeat sequence, a homopolymeric repeat sequence, a eukaryotic replication origin, or a eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is located at a distance of less than 1000bp from the Pol I-dependent replication origin along the replication direction. The method also includes modifying the covalently closed circular recombinant molecule so that the Pol I-dependent replication origin is replaced by the Pol III-dependent replication origin, and the Pol III-dependent replication origin covalently closed circular plasmid thus obtained has improved replication. A covalently closed circular recombinant DNA molecule without an antibiotic marker is also provided.

Description

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] not applicable.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Patent Application No. 62 / 645,892, filed on March 21, 2018, entitled “Generating Improved Viral and Non-Viral Nanoplasmid Vectors,” which is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to recombinant DNA molecules (i.e., vectors), which can be used for viral and non-viral gene therapy, viral and non-viral cell therapy, and more specifically, for improving the yield and quality of viral and non-viral vectors, reducing transfection-related toxicity, improving the transposition of non-viral transposon vectors, improving the packaging titer of viral vectors, improving the expression of viral and non-viral vector-encoded genes, and for eliminating viral and non-viral vector-mediated antibiotic selection marker gene transfer.

[0006] Such recombinant DNA molecules can be used in biotechnology, ex vivo gene therapy, transgenic organisms, gene therapy, therapeutic vaccination, agriculture, and DNA vaccines. Background Art

[0007] Escherichia coli (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, such as gene medicines and DNA vaccines, enter clinical trials and ultimately enter the pharmaceutical market. Plasmid DNA vaccines can be used as preventive vaccines for viral, bacterial, or parasitic diseases; as immunizing agents for the preparation of hyperimmune globulin products; as therapeutic vaccines for infectious diseases; or as 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 transducing virus particles after transfection of the producer cell line, and then the desired gene product is expressed from the virus in the target cell after viral transduction.

[0008] Non-viral and viral vector plasmids usually contain replication origins 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, which have a second site mutation that increases copy number (such as pMB1 pUC G to A point mutation, or ColE1 pMM1). Higher temperatures (42°C) can be used to induce selective amplification of plasmids with pUC and pMM1 replication origins.

[0009] Carnes, AE and Williams, JA 2011 U.S. Patent 7943377 describes a method of fed-batch fermentation, wherein the E. coli cells containing the plasmid are grown at a reduced temperature during part of the fed-batch phase, during which the growth rate is limited, and then the temperature is increased and growth continues at the increased temperature to accumulate the plasmid; the temperature change under the limited growth rate improves the plasmid yield and purity. Other fermentation processes for plasmid production are described in Carnes AE 2005 BioProcess Intl 3: 36-44, the entire contents of which are incorporated herein by reference.

[0010] One of the limitations of plasmid therapy and plasmid vaccine applications taught in the art is the safety concerns of regulatory agencies (e.g., Food and Drug Administration, European Medicines Agency) regarding: 1) plasmid transfer and replication in endogenous bacterial flora, or 2) expression of plasmid-encoded selection markers in human cells or endogenous bacterial flora. In addition, regulatory agency guidelines recommend the removal of all non-essential sequences in the vector. Plasmids containing replication origins derived from pMB1, ColE1, or pBR322 can replicate promiscuously in E. coli hosts. This raises a safety issue, i.e., plasmid therapeutic genes or antigens will be transferred to and replicated in the patient's endogenous flora. Ideally, therapeutic or vaccine plasmids will not replicate in endogenous E. coli strains. This requires replacing the replication origin derived from pMB1, ColE1, or pBR322 with a conditional replication origin that requires a specialized cell line for propagation. Additionally, regulatory agencies (such as EMEA and FDA) are concerned about the use of antibiotic resistance markers or surrogate protein markers in gene therapy and gene vaccine vectors due to concerns that the gene (antibiotic resistance marker or protein marker) may be expressed in the patient's cells. Ideally, plasmid therapies and plasmid vaccines would: 1) be unable to replicate in endogenous E. coli strains, 2) not encode a protein-based selection marker, and 3) be minimized to eliminate all non-essential sequences.

[0011] The art further teaches that one of the limitations of plasmid vector applications is the reduction in the duration of transgenic expression of plasmid vectors due to promoter inactivation mediated by the bacterial region of the vector (i.e., the region encoding the bacterial replication origin and the selection marker) (Chen ZY, He CY, Meuse L, Kay MA. 2004. Gene Ther 11: 856-864; Suzuki M, Kasai K, Saeki Y. 2006. J Virol 80: 3293-3300). This results in insufficient duration of transgenic expression. A strategy to improve the duration of transgenic expression is to remove the bacterial region of the plasmid. For example, a minicircle vector without a bacterial region has been developed. The removal of the bacterial region in the minicircle vector improves the duration of transgenic expression (Chen et al., Supra, 2004). In the minicircle vector, the eukaryotic region polyadenylation signal is covalently linked to the eukaryotic region promoter through a short spacer region, which is usually less than 200 bp and consists of a recombination connection site. This junction (spacer) can tolerate longer spacer sequences because, while long spacers ≥1 kb in length lead to silencing of transgene expression in vivo, short spacers ≤500 bp show a transgene expression pattern similar to conventional minicircle DNA vectors (Lu J, Zhang F, Xu S, Fire AZ, Kay MA. 2012. Mol Ther. 20:2111-9).

[0012] Williams, 2014. DNA plasmids with improved expression. World patent application WO2014035457 discloses a minimized Nanoplasmid TM Vector, which utilizes RNA-OUT without antibiotic selection and replaces the large 1000 bp pUC replication origin with a new 300 bp R6K origin. As expected from the teaching of Lu et al., Supra, 2012, the spacer connecting the 5' and 3' ends of the transgenic expression cassette is reduced to <500 bp with the R6K origin-RNA-OUT backbone, which improves the expression duration compared to conventional minicircle DNA vectors.

[0013] Compared with the 2.2 kb pUC origin-kanR antibiotic selection marker spacer, the 1.1 kb pFAR4 vector pUC origin tRNA antibiotic selection marker-free spacer showed improved expression duration (Quiviger, M, Arfi A, Mansard D, Delacotte L, Pastor M, Scherman D, Marie C. 2014. Gene Therapy 21: 1001-1007). This teaches that improved expression duration can be obtained with some bacterial regions up to 1.1 kb.

[0014] Improvements in expression levels compared to plasmid vectors were also observed for some spacers ≤ 1.1 kb. For example, pVAX1 derivatives (whose 2 kb bacterial backbone was reduced to 1.2, 1.1, or 0.7 kb) showed ≥ 2-fold improved expression compared to the parental pVAX1 vector (Table 1). NTC8685 derivatives (whose 1.5 kb bacterial backbone was reduced to 0.9 kb, 466 bp, or 281 bp (Nanoplasmid TM Vector)) showed ≥2-fold improved expression (Table 2).

[0015] This teaches that improved expression levels can be obtained with short bacterial regions of up to 1.2 kb.

[0016] Table 1: Expression levels of pVAX1 mammalian expression vector spacer (SR) derivatives

[0017]

[0018] a Carnes et al., (2010) J. Gene Medicine 12:818-31

[0019] b Ribeiro et al., (2011) Cell Reprogram 14:130

[0020] c Marie et al.,(2010)J.Gene Medicine 12:323-32

[0021] d Wang et al.,(2014)J Virology 88:1924-34

[0022] Table 2 Expression levels of NTC8685 mammalian expression vector spacer region (SR) derivatives

[0023]

[0024] a Williams, Supra, 2014

[0025] Various researchers have determined that minicircle vectors are superior to plasmid vectors in producing AAV vectors (improved transducing unit titers - Table 3) and transposon vectors (increased transposition - Table 3). The improved performance due to the extended duration of expression of short backbone minicircle vectors should also be observed for short bacterial backbone plasmid vectors up to 1.1 kb.

[0026] Table 3 Applications of microcircles with various viral and non-viral vector platforms

[0027]

[0028] a et al., (2016) Mol Ther-Nucleic Acids 5 e355. This reference, along with Chadeuf et al., (2005) Molecular Therapy 12:744-53 and Gray, 2017. WO2017066579 also reported that AAV helper plasmid antibiotic resistance markers were packaged into viral particles, demonstrating the need to remove antibiotic markers from AAV helper plasmids as well as AAV vectors. Interestingly, compared to plasmids, linear Doggybone TM No improvement in viral titer was observed with DNA single-stranded (ss) AAV vector modifications (Karbownickez et al., 2017. Cell and Gene Therapy Insights 731-8). This suggests that improvements in short-backbone minicircle vectors require short-backbone minicircle vectors, rather than linear vectors (such as Doggybone TM vector).

[0029] b Gray, Supra, 2017 describe the addition of a large spacer to move the selectable marker away from the ITRs. The resulting vector, pAAV CMV GFP, is 11 kb compared to the 5-6 kb of standard plasmid AAV vectors, thus reducing transduction units compared to the parental vector.

[0030] c Sharma et al., (2013) Molecular Therapy Nucleic Acids 2:e74

[0031] d Nucleofection: Monjezi et al., (2017) Leukemia 31:186-94

[0032] e Nucleofection: Holstein et al., (2018) Molecular Therapy epub Jan 17, 2018

[0033] In fact, it was reported that the 1.1 kb bacterial backbone pFAR4 SB transposon vector / SB100x transposase vector combination improved the transposition of Sleeping Beauty into human cells by 2-fold compared to the 2.8 kb bacterial backbone pT2 plasmid SB transposon vector / SB100x transposase vector combination (Pastor, M, Johnen S, Harmening N, Quiviger M, Pailloux J, Kropp, M, Walter P, Ivics Z, Izsvak Z, Thumann G, Scherman D. 2018 Molecular Therapy 11:57-67).

[0034] However, viral vectors such as AAV, lentiviral and retroviral vectors, and transposon vectors contain structured DNA sequences at their ends. For example, the Sleeping Beauty transposon vector contains flanking IR / DR sequences, AAV vectors contain flanking ITRs, and lentiviral and retroviral vectors contain flanking LTRs.

[0035] The close proximity of the pUC origin to a structured DNA sequence results in abnormal replication termination, resulting in replication intermediates that unacceptably reduce plasmid quality (Levy J. 2004. US Patent 6709844). Levy teaches that replication intermediates are formed when any high copy replication origin is less than 1 kb from a structured DNA sequence (such as an enhancer, LTR, or IRES), but replication intermediates are not formed when the high copy replication origin is greater than 1.5 kb from the structured DNA sequence. Since the pUC origin itself is 1 kb, there is no construction of an AAV, lentiviral, retroviral, or transposon vector containing a pUC origin that would not be expected to produce replication intermediates in a <1.1 kb bacterial region.

[0036] Lu J, Williams JA, Luke J, Zhang F, Chu K, and Kay MA. 2017. Human Gene Therapy 28: 125-34 discloses antibiotic-free small intron plasmid (MIP) AAV vectors and suggests that the MIP intron AAV vector can remove the vector backbone to produce a short backbone AAV vector. Attempts to create a minicircular structure similar to a 6 or 10 bp spacer in a small intron plasmid AAV vector are toxic (see Table 7, footnote e), probably because such a close juxtaposition of AAV ITRs produces a long palindrome. Although MIP vectors with a spacer length of <1 kb can be prepared, the disadvantage of the MIP intron strategy is that the introns encoding replication and selection need to be cloned into eukaryotic regions, which is impossible or undesirable for many vectors.

[0037] The disadvantage of the minicircle strategy of creating short bacterial region AAV, lentivirus, retrovirus or transposon vectors is that the method of producing minicircle vectors is expensive and not easy to expand. For minicircle vectors, a production system based on Escherichia coli has been developed, in which after plasmid production, the bacterial region and the eukaryotic region are separated and circularized into a minicircle (eukaryotic region) and a bacterial region ring by the action of a phage recombinase on a recognition sequence in the plasmid. In some methods, restriction enzymes are then used to digest the bacterial region ring at a unique site to eliminate this difficult-to-remove contaminant. These production methods are very inefficient. For example, the best production method for minicircle vectors produces only 5 mg of minicircles per liter of culture (Kay MA, He CY, Chen ZY. 2010. Nat Biotechnol 28: 1287-1289).

[0038] There are no reports of high-yield production methods for pFAR vectors; this system uses a plasmid-borne inhibitory tRNA gene to complement the TAG amber nonsense mutation of the thyA gene to complement thymidine auxotrophy and allow cells to grow on minimal media (Marie et al., Supra, 2010).

[0039] A solution is needed to develop AAV, lentiviral, retroviral or transposon vectors containing short spacers, preferably less than 1000 bp, that can be efficiently produced without replication intermediates or low yields. These vectors should not encode protein-based selection markers and should be minimized to eliminate all non-essential sequences. Summary of the invention

[0040] The present invention relates to vectors for viral and non-viral gene therapy, viral and non-viral cell therapy, and more specifically, for improving the production yield and quality of viral and non-viral vectors, for reducing transfection-related toxicity, for improving the transposition of non-viral transposon vectors, for improving the packaging titer of viral vectors, for improving the expression of transgenes encoded by viral and non-viral vectors, and for eliminating the transfer of antibiotic resistance marker genes by viral and non-viral vectors.

[0041] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate structured DNA sequences.

[0042] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate inverted repeat DNA sequences.

[0043] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate directly repeated DNA sequences.

[0044] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate homopolymeric repetitive DNA sequences.

[0045] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate enhancer structured DNA sequences.

[0046] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate polyA repeat DNA sequences.

[0047] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate the SV40 origin of replication DNA sequence.

[0048] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate lentiviral LTR DNA sequences.

[0049] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate retroviral LTR DNA sequences.

[0050] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate viral LTR DNA sequences.

[0051] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate AAV ITR DNA sequences.

[0052] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate transposon IR / DR DNA sequences.

[0053] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate the Sleeping Beauty IR / DR DNA sequence.

[0054] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate PiggyBac ITR DNA sequences.

[0055] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate CMV enhancer DNA sequences.

[0056] Disclosed are methods and compositions for improved vectors that utilize a Pol III-dependent origin of replication to replicate the SV40 enhancer DNA sequence.

[0057] Disclosed are methods and compositions for improved viral vectors utilizing a Pol III-dependent origin of replication.

[0058] Disclosed are methods and compositions for improved lentiviral vectors, lentiviral envelope vectors, and lentiviral packaging vectors utilizing a Pol III-dependent origin of replication.

[0059] Disclosed are methods and compositions for improved retroviral vectors, retroviral envelope vectors, and retroviral packaging vectors utilizing a Pol III-dependent origin of replication.

[0060] Disclosed are methods and compositions for improved AAV vectors and AAV helper vectors utilizing a Pol III-dependent origin of replication.

[0061] Disclosed are methods and compositions for improved adenoviral vectors utilizing a Pol III-dependent origin of replication.

[0062] Disclosed are methods and compositions for improved non-viral transposon and transposase vectors utilizing a Pol III-dependent origin of replication.

[0063] Disclosed are methods and compositions of improved non-viral Sleeping Beauty transposon and transposase vectors utilizing a Pol III-dependent origin of replication.

[0064] Disclosed are methods and compositions of improved non-viral PiggyBac transposon and transposase vectors utilizing a Pol III-dependent origin of replication.

[0065] Disclosed are methods and compositions of improved non-viral Tol2 transposon and transposase vectors utilizing a Pol III-dependent origin of replication.

[0066] Disclosed are methods and compositions for improved non-viral polyA-containing mRNA vectors utilizing a Pol III-dependent origin of replication.

[0067] Disclosed are methods and compositions for improved viral vectors utilizing a Pol III-dependent origin of replication with improved yields of viral transduction units.

[0068] Disclosed are methods and compositions for improved lentiviral vectors with improved yields of viral transduction units utilizing a Pol III-dependent origin of replication.

[0069] Disclosed are methods and compositions for improved retroviral vectors with improved yields of viral transduction units utilizing a Pol III-dependent origin of replication.

[0070] Disclosed are methods and compositions for improved AAV vectors and AAV helper vectors with improved yields of viral transduction units utilizing a Pol III-dependent origin of replication.

[0071] Disclosed are methods and compositions for improved non-viral transposons and transposase vectors with improved transposition utilizing a Pol III-dependent origin of replication.

[0072] Disclosed are methods and compositions of improved non-viral Sleeping Beauty transposons and transposase vectors with improved transposition utilizing a Pol III-dependent origin of replication.

[0073] Disclosed are methods and compositions for improved non-viral PiggyBac transposons and transposase vectors with improved transposition utilizing a Pol III-dependent origin of replication.

[0074] Disclosed are methods and compositions for improved non-viral Tol2 transposon and transposase vectors with improved transposition utilizing a Pol III-dependent origin of replication.

[0075] Disclosed are methods and compositions for improved viral vectors with improved expression utilizing a Pol III-dependent origin of replication.

[0076] Disclosed are methods and compositions for improved lentiviral vectors with improved expression utilizing a Pol III-dependent origin of replication.

[0077] Disclosed are methods and compositions for improved retroviral vectors with improved expression utilizing a Pol III-dependent origin of replication.

[0078] Disclosed are methods and compositions for improved AAV vectors and AAV helper vectors with improved expression utilizing a Pol III-dependent origin of replication.

[0079] Disclosed are methods and compositions for improved non-viral transposon and transposase vectors with improved expression utilizing a Pol III-dependent origin of replication.

[0080] Disclosed are methods and compositions for improved non-viral Sleeping Beauty transposon and transposase vectors with improved expression utilizing a Pol III-dependent origin of replication.

[0081] Disclosed are methods and compositions for improved non-viral PiggyBac transposon and transposase vectors with improved expression utilizing a Pol III-dependent origin of replication.

[0082] Disclosed are methods and compositions for improved non-viral Tol2 transposon and transposase vectors with improved expression utilizing a Pol III-dependent origin of replication.

[0083] Disclosed are methods and compositions for improved viral vectors utilizing a Pol III-dependent replication origin without the risk of antibiotic resistance marker gene transfer.

[0084] Disclosed are methods and compositions for improved lentiviral vectors, lentiviral envelope vectors, and lentiviral packaging vectors utilizing a Pol III-dependent replication origin without the risk of antibiotic resistance marker gene transfer.

[0085] Disclosed are methods and compositions for improved retroviral vectors, retroviral envelope vectors, and retroviral packaging vectors that utilize a Pol III-dependent replication origin without the risk of antibiotic resistance marker gene transfer.

[0086] Disclosed are methods and compositions for improved AAV vectors and AAV helper vectors utilizing a Pol III-dependent replication origin without the risk of antibiotic resistance marker gene transfer.

[0087] Disclosed are methods and compositions for improved non-viral transposon and transposase vectors utilizing a Pol III-dependent replication origin without the risk of antibiotic resistance marker gene transfer.

[0088] Disclosed are methods and compositions for improved non-viral Sleeping Beauty transposon and transposase vectors utilizing a Pol III-dependent replication origin without the risk of antibiotic resistance marker gene transfer.

[0089] Disclosed are methods and compositions for improved non-viral PiggyBac transposon and transposase vectors utilizing a Pol III-dependent replication origin without the risk of antibiotic resistance marker gene transfer.

[0090] Disclosed are methods and compositions for improved non-viral Tol2 transposon and transposase vectors utilizing a Pol III-dependent replication origin without the risk of antibiotic resistance marker gene transfer.

[0091] Disclosed are methods and compositions for improved viral vectors utilizing a Pol III-dependent origin of replication with reduced transfection-associated toxicity.

[0092] Disclosed are methods and compositions for improved lentiviral vectors utilizing a Pol III-dependent origin of replication with reduced transfection-associated toxicity.

[0093] Disclosed are methods and compositions for improved retroviral vectors utilizing a Pol III-dependent origin of replication with reduced transfection-associated toxicity.

[0094] Disclosed are methods and compositions for improved AAV vectors and AAV helper vectors with reduced transfection-associated toxicity utilizing a Pol III-dependent origin of replication.

[0095] Disclosed are methods and compositions of non-viral transposon and transposase vectors with reduced transfection-associated toxicity utilizing a Pol III-dependent origin of replication.

[0096] Disclosed are methods and compositions of non-viral Sleeping Beauty transposon and transposase vectors with reduced transfection-associated toxicity utilizing a Pol III-dependent origin of replication.

[0097] Disclosed are methods and compositions of non-viral PiggyBac transposons and transposase vectors with reduced transfection-associated toxicity utilizing a Pol III-dependent origin of replication.

[0098] Disclosed are methods and compositions of non-viral Tol2 transposon and transposase vectors with reduced transfection-associated toxicity utilizing a Pol III-dependent origin of replication.

[0099] Each of the above improvements and the improvements described below are, for example, improvements achieved relative to a different plasmid under similar or identical circumstances but which does not include a Pol III-dependent origin of replication.

[0100] It is an object of the present invention to provide improved production yields of viral and non-viral vectors.

[0101] Another object of the present invention is to provide improved production quality of viral and non-viral vectors.

[0102] Another object of the present invention is to provide viral vectors with improved packaging titers.

[0103] Another object of the present invention is to provide a non-viral transposon vector with improved transposition.

[0104] Another object of the present invention is to provide viral and non-viral vectors with improved expression of encoded transgenes.

[0105] Another object of the present invention is to provide viral and non-viral vectors that eliminate antibiotic resistance marker gene transfer.

[0106] Another object of the present invention is to provide viral and non-viral vectors with reduced transfection-related toxicity.

[0107] In one embodiment, the present technology provides a method for improving the replication of a covalently closed circular plasmid, comprising the following steps: a) providing a covalently closed circular plasmid, which comprises: i) a Pol III-dependent replication origin, and ii) an insert comprising a structured DNA sequence, wherein the structured DNA sequence is selected from an inverted repeat sequence, a forward repeat sequence, a homopolymeric repeat sequence, a eukaryotic replication origin, and a eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is less than 1000bp away from the Pol III-dependent replication origin along the replication direction; b) modifying the covalently closed circular recombinant molecule of a) so that the Pol I-dependent replication origin is replaced by the Pol III-dependent replication origin, and the covalently closed circular plasmid of the Pol III-dependent replication origin thus obtained has improved replication. In another embodiment, the Pol I-dependent replication origin is selected from the group consisting of: a pUC origin, a pMB1 origin, and a ColE1 origin. In another embodiment, the Pol III-dependent replication origin is an R6Kγ replication origin. In another embodiment, the Pol III-dependent replication origin is a R6Kγ replication origin having at least 95% sequence identity to 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 another embodiment, the structured DNA sequence is selected from the group consisting of polyA repeats, SV40 replication origin, viral LTR, lentiviral LTR, retroviral LTR, transposon IR / DR repeats, Sleeping Beauty transposon IR / DR repeats, AAV ITR, CMV enhancer, and SV40 enhancer. In another embodiment, the improved replication is selected from the group consisting of reducing the generation of replication intermediates and increasing plasmid copy number.

[0108] In one embodiment, the present technology provides a method for improving the replication of a covalently closed circular plasmid, comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a Pol III-dependent replication origin, and ii) an insert comprising a structured DNA sequence, wherein the structured DNA sequence is selected from an inverted repeat sequence, a forward repeat sequence, a homopolymeric repeat sequence, a eukaryotic replication origin, and a eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is less than 1000bp from the Pol III-dependent replication origin along the replication direction; b) modifying the covalently closed circular recombinant molecule of a) so that the antibiotic selection marker is replaced by an RNA selection marker, and the Pol I-dependent replication origin is replaced by a Pol III-dependent replication origin, and the covalently closed circular plasmid of the Pol III-dependent replication origin thus obtained has improved replication. In another embodiment, the Pol I-dependent replication origin is selected from the group consisting of: a pUC origin, a pMB1 origin, and a ColE1 origin. In another embodiment, the Pol III-dependent replication origin is an R6Kγ replication origin. In another embodiment, the Pol III-dependent replication origin is a R6Kγ replication origin having at least 95% sequence identity to 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 another embodiment, the RNA selection marker is a RNA-IN regulating RNA-OUT functional variant having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the RNA selection marker is an RNA-OUT RNA selection marker encoding an RNA-IN regulating RNA-OUT RNA having at least 95% sequence identity to SEQ ID NO: 6. In another embodiment, the bacterial replication selection region comprises replacing the Pol I-dependent replication origin and the antibiotic selection marker with a Pol III-dependent R6K start point-RNA-OUT RNA selection marker bacterial replication selection region, and the Pol III-dependent R6K start point-RNA-OUT RNA selection marker bacterial replication selection region 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.In another embodiment, the structured DNA sequence is selected from the group consisting of: polyA repeats, SV40 replication origin, viral LTR, lentiviral LTR, retroviral LTR, transposon IR / DR repeats, Sleeping Beauty transposon IR / DR repeats, AAV ITR, CMV enhancer, and SV40 enhancer. In another embodiment, the improved replication is selected from the group consisting of: reducing the generation of replication intermediates and increasing plasmid copy number.

[0109] In one embodiment, the present technology provides an antibiotic-marker-free covalently closed circular recombinant DNA molecule, comprising: a) an antibiotic-marker-free insert, which comprises a structured DNA sequence selected from the group consisting of: an inverted repeat sequence, a forward repeat sequence, a homopolymeric repeat sequence, a eukaryotic replication origin, and a eukaryotic promoter enhancer sequence; b) a PolIII-dependent replication origin, which includes a R6Kγ replication origin, and the R6Kγ replication origin has 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) an RNA-OUT RNA selection marker, which comprises an RNA-IN regulating RNA-OUT functional variant, which has at least 95% sequence identity with a sequence selected from the group consisting of: SEQ ID NO:5 and SEQID NO:7. In another embodiment, the R6Kγ replication origin and the RNA-OUT RNA selection marker comprise a R6K origin-RNA-OUT RNA selection marker bacterial replication selection region having at least 95% sequence identity to 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. In another embodiment, the structured DNA sequence is selected from the group consisting of: polyA repeats, SV40 replication origin, viral LTR, lentiviral LTR, retroviral LTR, transposon IR / DR repeats, Sleeping Beauty transposon IR / DR repeats, AAVITR, CMV enhancer, and SV40 enhancer. In another embodiment, the recombinant DNA molecule is selected from the group consisting of a viral vector, a lentiviral vector, a retroviral vector, an AAV vector, an Ad vector, a non-viral transposon vector, a Sleeping Beauty transposon vector, a PiggyBac transposon vector, a Tol2 transposon vector, and a polyA-containing mRNA vector.

[0110] In one embodiment, the present technology provides a method for improving the yield of AAV vector viral transduction units starting from a covalently closed circular plasmid, comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a bacterial replication selection region of 1 kb or greater, comprising a Pol I-dependent replication origin and an antibiotic selection marker, and ii) an insert comprising a eukaryotic region selected from the group consisting of: an AAV vector, an AAV rep cap vector, an Ad helper vector, and an Ad helper repcap vector; b) modifying the covalently closed circular recombinant molecule of a) so that the bacterial replication selection region of 1 kb or greater comprising a Pol I-dependent replication origin and an antibiotic selection marker is replaced by a Pol III-dependent R6K origin-RNA-OUTRNA selection marker bacterial replication selection region of less than 1 kb, whereby when transfected into mammalian cells, the resulting Pol III-dependent replication origin covalently closed circular plasmid has improved AAV viral transduction unit production. In another embodiment, the Pol I-dependent replication origin is selected from the group consisting of: a pUC origin, a pMB1 origin, and a ColE1 origin. In another embodiment, the Pol III-dependent R6K origin of replication is a R6Kγ origin of replication having at least 95% sequence identity to 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 another embodiment, the RNA-OUT RNA selection marker is a RNA-IN regulatory RNA-OUT functional variant having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the RNA-OUT RNA selection marker is a RNA-IN regulatory RNA-OUT RNA selection marker encoding a sequence having at least 95% sequence identity to SEQ ID NO: 6. In another embodiment, the Pol III-dependent R6K start point-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 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.

[0111] In one embodiment, the present technology provides a method for improving the yield of retroviral or lentiviral vector viral transduction units starting from a covalently closed circular plasmid, comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a bacterial replication selection region of 1 kb or larger, which comprises a Pol I-dependent replication origin and an antibiotic selection marker, and ii) an insert comprising a eukaryotic region selected from the group consisting of: a retroviral vector, a lentiviral vector, a retroviral envelope vector, a lentiviral envelope vector, a retroviral packaging vector, and a lentiviral packaging vector; and b) modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication selection region comprising a Pol I-dependent replication origin and an antibiotic selection marker is replaced by a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication selection region of less than 1 kb, whereby the resulting Pol III-dependent replication origin covalently closed circular plasmid has improved viral transduction unit production when transfected into mammalian cells. In another embodiment, the Pol I-dependent replication origin is selected from the group consisting of: pUC origin, pMB1 origin and ColE1 origin. In another embodiment, the PolIII-dependent R6K replication origin is a R6Kγ replication origin, which has 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 another embodiment, the RNA-OUT RNA selection marker is an RNA-IN regulatory RNA-OUT functional variant, which has at least 95% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the RNA-OUT RNA selection marker is a RNA-IN regulatory RNA-OUT RNA selection marker encoding a sequence having at least 95% sequence identity with SEQ ID NO: 6. In another embodiment, the Pol III-dependent R6K start point-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 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.

[0112] In one embodiment, the present invention contemplates a method for improving the transposition of a covalently closed circular non-viral transposon plasmid, comprising the following steps: a) providing a covalently closed circular plasmid comprising i) a bacterial replication selection region of 1 kb or larger, the bacterial replication selection region comprising a Pol I-dependent replication origin and an antibiotic selection marker, and ii) an insert comprising a non-viral eukaryotic region, the non-viral eukaryotic region being selected from a transposon vector, a Sleeping Beauty transposon vector, a Sleeping Beauty transposase vector, a PiggyBac transposon vector, a PiggyBac transposase vector, a Tol2 transposon vector, a Tol2 transposase vector; b) modifying the covalently closed circular recombinant molecule of (a) such that the bacterial replication selection region of 1 kb or larger comprising a Pol I-dependent replication origin and an antibiotic selection marker is replaced by a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication selection region of less than 1 kb, whereby the resulting Pol III-dependent replication origin covalently closed circular plasmid has improved transposition when transfected into mammalian cells. In another embodiment, the Pol I-dependent replication origin is selected from the group consisting of: pUC origin, pMB1 origin, or ColE1 origin. In another embodiment, the Pol III-dependent R6K replication origin is a R6Kγ replication origin, which has 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 another embodiment, the RNA-OUT RNA selection marker is an RNA-IN regulatory RNA-OUT functional variant, which has at least 95% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 7. In another embodiment, the RNA-OUT RNA selection marker is a RNA-IN regulatory RNA-OUT RNA selection marker encoding a sequence having at least 95% sequence identity with SEQ ID NO: 6. In another embodiment, the Pol III-dependent R6K start point-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 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.

[0113] In one embodiment, the present technology provides a method for improving the expression of a covalently closed circular viral vector or a non-viral transposon plasmid, comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a bacterial replication selection region of 1 kb or greater comprising a Pol I-dependent replication origin and an antibiotic selection marker, and ii) an insert comprising a eukaryotic region selected from the group consisting of a lentiviral vector, a retroviral vector, and an AAV vector or a non-viral transposon vector; and b) modifying the covalently closed circular recombinant molecule of (a) so that the bacterial replication selection region of 1 kb or greater comprising a Pol I-dependent replication origin and an antibiotic selection marker is replaced by a Pol III-dependent R6K origin-RNA-OUTRNA selection marker bacterial replication selection region of less than 1 kb, whereby the resulting Pol III-dependent replication origin covalently closed circular plasmid has improved expression when transfected into mammalian cells. In another embodiment, the Pol I-dependent replication origin is selected from the group consisting of a pUC origin, a pMB1 origin, and a ColE1 origin. In another embodiment, the Pol III-dependent R6K replication origin is a R6Kγ replication origin having at least 95% sequence identity to 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 another embodiment, the RNA-OUT RNA selection marker is a RNA-IN regulatory RNA-OUT functional variant having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the RNA-OUT RNA selection marker is a RNA-IN regulatory RNA-OUT RNA selection marker encoding a sequence having at least 95% sequence identity to SEQ ID NO: 6. In another embodiment, the Pol III-dependent R6K start point-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 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.

[0114] In one embodiment, the present technology provides a method for eliminating antibiotic resistance marker gene transfer from a covalently closed circular viral vector plasmid, comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a bacterial replication selection region of 1 kb or greater comprising a Pol I-dependent replication origin and an antibiotic resistance marker, and ii) an insert comprising a eukaryotic region without an antibiotic resistance marker selected from the group consisting of a viral vector, a lentiviral vector, a lentiviral packaging vector, a lentiviral envelope vector, a retroviral envelope vector, a retroviral packaging vector, an AAV vector, an AAV repcap vector, an Ad helper vector, and an Ad helper rep cap vector; and b) modifying the covalently closed circular recombinant molecule of a) such that the bacterial replication selection region of 1 kb or greater comprising a Pol I-dependent replication origin and an antibiotic selection marker is replaced by a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication selection region of less than 1 kb, whereby when transfected into mammalian cells, the resulting Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication selection region is replaced by a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication selection region. The Pol III-dependent replication origin covalently closed circular plasmid has no antibiotic resistance marker that can be packaged into lentiviral, retroviral or AAV transduction virus particles. In another embodiment, the Pol I-dependent replication origin is selected from the group consisting of: pUC origin, pMB1 origin and ColE1 origin. In another embodiment, the Pol III-dependent R6K replication origin is a R6Kγ replication origin, which has 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 another embodiment, the RNA-OUT RNA selection marker is an RNA-IN regulatory RNA-OUT functional variant, which has at least 95% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the RNA-OUT RNA selection marker is a RNA-IN regulatory RNA-OUT RNA selection marker encoding a sequence having at least 95% sequence identity with SEQ ID NO: 6. In another embodiment, the Pol III-dependent R6K start point-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 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.

[0115] In one embodiment, the present technology provides a method for eliminating antibiotic resistance marker gene transfer from a covalently closed circular non-viral transposon plasmid, comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a bacterial replication selection region of 1 kb or larger comprising a Pol I-dependent replication origin and an antibiotic resistance marker, and ii) an insert comprising a eukaryotic region without an antibiotic resistance marker selected from the group consisting of: a non-viral transposon vector, a non-viral transposase vector, a Sleeping Beauty transposon vector, a Sleeping Beauty transposase vector, a PigyBac transposon vector, a PigyBac transposase vector, a Tol2 transposon vector, a Tol2 transposase vector; and b) modifying the covalently closed circular recombinant molecule of a) so that the bacterial replication selection region of 1 kb or larger comprising a Pol I-dependent replication origin and an antibiotic selection marker is replaced by a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication selection region of less than 1 kb, whereby when transfected into mammalian cells, the resulting Pol The Pol I-dependent replication origin covalently closed circular plasmid has no antibiotic resistance marker that can be transposed into the genome. In another embodiment, the Pol I-dependent replication origin is selected from the group consisting of: pUC origin, pMB1 origin and ColE1 origin. In another embodiment, the Pol III-dependent R6K replication origin is a R6Kγ replication origin, which has 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 another embodiment, the RNA-OUT RNA selection marker is an RNA-IN regulatory RNA-OUT functional variant, which has at least 95% sequence identity with a sequence selected from the group consisting of: SEQ ID NO:5 and SEQ ID NO:7. In another embodiment, the RNA-OUT RNA selection marker is a RNA-IN regulatory RNA-OUT RNA selection marker encoding a sequence having at least 95% sequence identity with SEQ ID NO:6. In another 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 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.

[0116] In one embodiment, the present technology provides an antibiotic-marker-free covalently closed circular recombinant DNA molecule comprising: a) an antibiotic-marker-free insert comprising a eukaryotic region selected from the group consisting of a lentiviral vector, a lentiviral envelope vector, a lentiviral packaging vector, a retroviral vector, a retroviral envelope vector, a retroviral packaging vector, an AAV vector, an AAV rep cap vector, an Ad helper vector, an Ad helper rep cap vector, a non-viral transposon vector, and a non-viral transposase vector; b) a Pol III-dependent replication origin comprising a R6Kγ replication origin having at least 95% sequence identity to 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) an RNA-OUT RNA selection marker comprising an RNA-IN regulating RNA-OUT functional variant having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the R6Kγ replication origin and the RNA-OUT RNA selection marker comprise a R6K origin-RNA-OUT RNA selection marker bacterial replication selection region, which 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.

[0117] In one embodiment, the present technology provides a method for reducing transfection-related toxicity from a covalently closed circular viral vector or a non-viral transposon plasmid, comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a bacterial replication selection region of 1 kb or greater comprising a Pol I-dependent replication origin and an antibiotic selection marker, and ii) an insert comprising a eukaryotic region selected from the group consisting of: a lentiviral vector, a retroviral vector, an AAV vector, and a non-viral transposon vector; and b) modifying the covalently closed circular recombinant molecule of (a) so that the bacterial replication selection region of 1 kb or greater comprising a Pol I-dependent replication origin and an antibiotic selection marker is replaced by a Pol III-dependent R6K origin-RNA-OUT RNA selection marker bacterial replication selection region of less than 1 kb, whereby the resulting Pol III-dependent replication origin covalently closed circular plasmid has reduced toxicity when transfected by transfecting relevant mammalian cells. In another embodiment, the Pol I-dependent replication origin is selected from the group consisting of: a pUC origin, a pMB1 origin, and a ColE1 origin. In another embodiment, the Pol III-dependent R6K replication origin is a R6Kγ replication origin having at least 95% sequence identity to 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 another embodiment, the RNA-OUT RNA selection marker is a RNA-IN regulatory RNA-OUT functional variant having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the RNA-OUT RNA selection marker is a RNA-IN regulatory RNA-OUT RNA selection marker encoding a sequence having at least 95% sequence identity to SEQ ID NO: 6. In another embodiment, the Pol III-dependent R6K start point-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 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.

[0118] The resulting Pol III-dependent replication origin plasmid has surprisingly improved production quality and yield compared to the parental pMB1, ColE1 or pBR322 derived replication origin expression plasmid vector.

[0119] Other objects and advantages of the present invention will become apparent by reference to the drawings and ensuing description. Sequence Listing <110> Nature Technologies <120> Generation of improved viral and non-viral nanoplasmid vectors <130> 17699-000004-WO-POA <150> 62645892 <151> 2018-03-21 <160> twenty three <170> PatentIn version 3.5 <210> 1 <211> 281 <212> DNA <213> Artificial sequence <220> <223> R6K γStarting Point <400> 1 ggcttgttgt ccacaaccgt taaaccttaa aagctttaaa agccttatat attctttttt 60 ttcttataaa acttaaaacc ttagaggcta tttaagttgc tgatttatat taattttatt 120 gttcaaacat gagagcttag tacgtgaaac atgagagctt agtacgttag ccatgagagc 180 ttagtacgtt agccatgagg gtttagttcg ttaaacatga gagcttagta cgttaaacat 240 gagagcttag tacgtactat caacaggttg aactgctgat c 281 <210> 2 <211> 281 <212> DNA <213> Artificial sequence <220> <223> 1 CpG R6K γ origin <400> 2 ggcttgttgt ccacaaccat taaaccttaa aagctttaaa agccttatat attctttttt 60 ttcttataaa acttaaaacc ttagaggcta tttaagttgc tgatttatat taattttatt 120 gttcaaacat gagagcttag tacgtgaaac atgagagctt agtacattag ccatgagagc 180 ttagtacatt agccatgagg gtttagttca ttaaacatga gagcttagta cattaaacat 240 gagagcttag tacatactat caacaggttg aactgctgat c 281 <210> 3 <211> 260 <212> DNA <213> Artificial Sequence <220> <223> R6K γ origin without CpG <400> 3 aaaccttaaa acctttaaaa gccttatata ttcttttttt tcttataaaa cttaaaacct 60 tagaggctat ttaagttgct gatttatatt aattttattg ttcaaacatg agagcttagt 120 acatgaaaca tgagagctta gtacattagc catgagagct tagtacatta gccatgaggg 180 tttagttcat taaacatgag agcttagtac attaaacatg agagcttagt acatactatc 240 aacaggttga actgctgatc 260 <210> 4 <211> 389 <212> DNA <213> Artificial sequence <220> <223> Extended R6K γ start point <400> 4 tgtcagccgt taagtgttcc tgtgtcactg aaaattgctt tgagaggctc taagggcttc 60 tcagtgcgtt acatccctgg cttgttgtcc acaaccgtta aaccttaaaa gctttaaaag 120 ccttatatat tctttttttt cttataaaac ttaaaacctt agaggctatt taagttgctg 180 atttatatta attttattgt tcaaacatga gagcttagta cgtgaaacat gagagcttag 240 tacgttagcc atgagagctt agtacgttag ccatgagggt ttagttcgtt aaacatgaga 300 gcttagtacg ttaaacatga gagcttagta cgtgaaacat gagagcttag tacgtactat 360 caacaggttg aactgctgat cttcagatc 389 <210> 5 <211> 139 <212> DNA <213> Artificial sequence <220> <223> RNA-OUT selection marker <400> 5 gtagaattgg taaagagagt cgtgtaaaat atcgagttcg cacatcttgt tgtctgatta 60 ttgatttttg gcgaaaccat ttgatcatat gacaagatgt gtatctacct taacttaatg 120 attttgataa aaatcatta 139 <210> 6 <211> 69 <212> DNA <213> Artificial sequence <220> <223> RNA-OUT antisense repressor RNA <400> 6 tcgcacatct tgttgtctga ttattgattt ttggcgaaac catttgatca tatgacaaga 60 tgtgtatct 69 <210> 7 <211> 139 <212> DNA <213> Artificial sequence <220> <223> 2 CpG RNA-OUT selection markers <400> 7 gtagaattgg taaagagagt tgtgtaaaat attgagttcg cacatcttgt tgtctgatta 60 ttgatttttg gcgaaaccat ttgatcatat gacaagatgt gtatctacct taacttaatg 120 attttgataa aaatcatta 139 <210> 8 <211> 466 <212> DNA <213> Artificial sequence <220> <223> R6Kγ origin-RNA-OUT bacterial replication selection region, flanked by NheI and KpnI restriction enzyme sites <400> 8 gctagcccgc ctaatgagcg ggcttttttt tggcttgttg tccacaaccg ttaaacctta 60 aaagctttaa aagccttata tattcttttt tttcttataa aacttaaaac cttagaggct 120 atttaagttg ctgattttata ttaattttat tgttcaaaca tgagagctta gtacgtgaaa 180 catgagagct tagtacgtta gccatgagag cttagtacgt tagccatgag ggtttagttc 240 gttaaacatg agagcttagt acgttaaaca tgagagctta gtacgtacta tcaacaggtt 300 gaactgctga tccacgttgt ggtagaattg gtaaagagag tcgtgtaaaa tatcgagttc 360 gcacatcttg ttgtctgatt attgattttt ggcgaaacca tttgatcata tgacaagatg 420 tgtatctacc ttaacttaat gattttgata aaaatcatta ggtacc 466 <210> 9 <211> 439 <212> DNA <213> Artificial sequence <220> <223> 1 CpG R6K γ origin - 2 CpG RNA-OUT bacterial replication selection region flanked by NheI and KpnI restriction enzyme sites <400> 9 gctagctggc ttgttgtcca caaccattaa accttaaaag ctttaaaagc cttatatatt 60 cttttttttc ttataaaact taaaacctta gaggctattt aagttgctga tttatattaa 120 ttttattgtt caaacatgag agcttagtac gtgaaacatg agagcttagt acattagcca 180 tgagagctta gtacattagc catgagggtt tagttcatta aacatgagag cttagtacat 240 taaacatgag agcttagtac atactatcaa caggttgaac tgctgatctg tacagtagaa 300 ttggtaaaga gagttgtgta aaatattgag ttcgcacatc ttgttgtctg attattgatt 360 tttggcgaaa ccatttgatc atatgacaag atgtgtatct accttaactt aatgattttg 420 ataaaaatca ttaggtacc 439 <210> 10 <211> 565 <212> DNA <213> Artificial Sequence <220> <223> pNTC-NP1 Multiple Cloning Site trpA R6K-RNA-OUT Multiple Cloning Cassette: EcoRI-HindIII <400> 10 gaattcgagc tcggtacctc gcgaatgcat ctaggggacg gccgctagcc cgcctaatga 60 gcgggctttt ttttggcttg ttgtccacaa ccgttaaacc ttaaaagctt taaaagcctt 120 atatattctt ttttttctta taaaacttaa aaccttagag gctatttaag ttgctgattt 180 atattaattt tattgttcaa acatgagagc ttagtacgtg aaacatgaga gcttagtacg 240 ttagccatga gagcttagta cgttagccat gagggtttag ttcgttaaac atgagagctt 300 agtacgttaa acatgagagc ttagtacgta ctatcaacag gttgaactgc tgatccacgt 360 tgtggtagaa ttggtaaaga gagtcgtgta aaatatcgag ttcgcacatc ttgttgtctg 420 attattgatt tttggcgaaa ccatttgatc atatgacaag atgtgtatct accttaactt 480 aatgattttg ataaaaatca ttaggagcta gcattgggtc atcggatccc gggcccgtcg 540 actgcagagg cctgcatgca agctt 565 <210> 11 <211> 584 <212> DNA <213> Artificial Sequence <220> <223> pNTC-NP2 Multiple Cloning Site trpA R6K-RNA-OUT Multiple Cloning Cassette: EcoRI-HindIII <400> 11 gaattcgagc tcggtacctc gcgaatgcat ctaggggacg gccgctagcc cgcctaatga 60 gcgggctttt ttttggcttg ttgtccacaa ccgttaaacc ttaaaagctt taaaagcctt 120 atatattctt ttttttctta taaaacttaa aaccttagag gctatttaag ttgctgattt 180 atattaattt tattgttcaa acatgagagc ttagtacgtg aaacatgaga gcttagtacg 240 ttagccatga gagcttagta cgttagccat gagggtttag ttcgttaaac atgagagctt 300 agtacgttaa acatgagagc ttagtacgta ctatcaacag gttgaactgc tgatccacgt 360 tgtggtagaa ttggtaaaga gagtcgtgta aaatatcgag ttcgcacatc ttgttgtctg 420 attattgatt tttggcgaaa ccatttgatc atatgacaag atgtgtatct accttaactt 480 aatgattttg ataaaaatca ttaggactag tcccgggcgc tagttattaa tattgggtca 540 tcggatcccg ggcccgtcga ctgcagaggc ctgcatgcaa gctt 584 <210> 12 <211> 557 <212> DNA <213> Artificial Sequence <220> <223> pNTC-NP3 Multiple Cloning Site trpA R6K-RNA-OUT Multiple Cloning Cassette: EcoRI-HindIII <400> 12 gaattcgagc tcggtacctc gcgaatgcat ctaggggacg gccgctagcc cgcctaatga 60 gcgggctttt ttttggcttg ttgtccacaa ccgttaaacc ttaaaagctt taaaagcctt 120 atatattctt ttttttctta taaaacttaa aaccttagag gctatttaag ttgctgattt 180 atattaattt tattgttcaa acatgagagc ttagtacgtg aaacatgaga gcttagtacg 240 ttagccatga gagcttagta cgttagccat gagggtttag ttcgttaaac atgagagctt 300 agtacgttaa acatgagagc ttagtacgta ctatcaacag gttgaactgc tgatccacgt 360 tgtggtagaa ttggtaaaga gagtcgtgta aaatatcgag ttcgcacatc ttgttgtctg 420 attattgatt tttggcgaaa ccatttgatc atatgacaag atgtgtatct accttaactt 480 aatgattttg ataaaaatca ttaggtaccg agctcggatc ccgggcccgt cgactgcaga 540 ggcctgcatg caagctt 557 <210> 13 <211> 557 <212> DNA <213> Artificial Sequence <220> <223> pNTC-NP4 Multiple Cloning Site trpA R6K-RNA-OUT Multiple Cloning Cassette: HindIII-EcoRI <400> 13 aagcttgcat gcaggcctct gcagtcgacg ggcccgggat ccgagctcgg tacctcgcga 60 atgcatctag gggacggccg ctagcccgcc taatgagcgg gctttttttt ggcttgttgt 120 ccacaaccgt taaaccttaa aagctttaaa agccttatat attctttttt ttcttataaa 180 acttaaaacc ttagaggcta tttaagttgc tgatttatat taattttatt gttcaaacat 240 gagagcttag tacgtgaaac atgagagctt agtacgttag ccatgagagc ttagtacgtt 300 agccatgagg gtttagttcg ttaacatga gagcttagta cgttaaacat gagagcttag tacgtactat caacaggttg aactgctgat ccacgttgtg gtagaattgg taaagagagt cgtgtaaaat atcgagttcg cacatcttgt tgtctgatta ttgatttttg gcgaaaccat 480 ttgatcatat gacaagatgt gtatctacct taacttaatg attttgataa aaatcattag gtaccgagct cgaattc 557 <210> 14 <211> 503 <212> DNA <213> The snowstorm <220> <223> pNTC-NP5-induced trpA R6K-RNA-OUT promoter:KasI-HindIII <400> 14 ggcgccgcta gcccgcctaa tgagcgggct tttttttggc ttgttgtcca caaccgttaa accttaaaag ctttaaaagc cttatatatt cttttttttc ttataaact taaaacctta gaggctattt aagttgctga tttatattaa ttttattgtt caaacatgag agcttagtac gtgaaacatg agagcttagt acgttagcca tgagagctta gtacgttagc catgagggtt tagttcgtta aacatgagag cttagtagt taaacatgag agcttagtac gtactatcaa caggttgaac tgctgatcca cgttgtggta gaattggtaa agagagtcgt gtaaaatatc 360 gagttcgcac atcttgttgt ctgattattg atttttggcg aaaccatttg atcatatgac 420 aagatgtgta tctaccttaa cttaatgatt ttgataaaaa tcattaggta ccacatgtcc 480 tgcagaggcc tgcatgcaag ctt 503 <210> 15 <211> 539 <212> DNA <213> Artificial Sequence <220> <223> pNTC-NP6 Multiple Cloning Site trpA R6K-RNA-OUT Multiple Cloning Cassette: EcoRI-SacI <400> 15 gaattctgca gatatccatc acactggcgg ccgctagccc gcctaatgag cgggcttttt 60 tttggcttgt tgtccacaac cgttaaacct taaaagcttt aaaagcctta tatattcttt 120 tttttcttat aaaacttaaa accttagagg ctatttaagt tgctgattta tattaatttt 180 attgttcaaa catgagagct tagtacgtga aacatgagag cttagtacgt tagccatgag 240 agcttagtac gttagccatg agggtttagt tcgttaaaca tgagagctta gtacgttaaa 300 catgagagct tagtacgtac tatcaacagg ttgaactgct gatccacgtt gtggtagaat 360 tggtaaagag agtcgtgtaa aatatcgagt tcgcacatct tgttgtctga ttattgattt 420 ttggcgaaac catttgatca tatgacaaga tgtgtatcta ccttaactta atgattttga 480 taaaaatcat taggtaccgg gccccccctc gatcgaggtc gacggtatcg ggggagctc 539 <210> 16 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> pNTC-NP7 Multiple Cloning Site trpA R6K-RNA-OUT Multiple Cloning Cassette: BssHII-BssHII <400> 16 gcgcgcagcc ttaattaagc tagcccgcct aatgagcggg cttttttttg gcttgttgtc 60 cacaaccgtt aaaccttaaa agctttaaaa gccttatata ttcttttttt tcttataaaa 120 cttaaaacct tagaggctat ttaagttgct gatttatatt aattttattg ttcaaacatg 180 agagcttagt acgtgaaaca tgagagctta gtacgttagc catgagagct tagtacgtta 240 gccatgaggg tttagttcgt taaacatgag agcttagtac gttaaacatg agagcttagt 300 acgtactatc aacaggttga actgctgatc cacgttgtgg tagaattggt aaagagagtc 360 gtgtaaaata tcgagttcgc acatcttgtt gtctgattat tgatttttgg cgaaaccatt 420 tgatcatatg acaagatgtg tatctacctt aacttaatga ttttgataaa aatcattagg 480 taccttaatt aactgcgcgc 500 <210> 17 <211> 530 <212> DNA <213> Artificial sequence <220> <223> pNTC-3xCpG NP1 polylinker R6K-RNA-OUT polylinker cloning cassette: HindIII-EcoRI <400> 17 aagcttgcat gcaggcctct gcagtcgacg ggccctctag actcgagctg gcttgttgtc 60 cacaaccatt aaaccttaaa agctttaaaa gccttatata ttcttttttt tcttataaaa 120 cttaaaacct tagaggctat ttaagttgct gatttatatt aattttatattg ttcaaacatg 180 agagcttagt acgtgaaaca tgagagctta gtacattagc catgagagct tagtacatta 240 gccatgaggg tttagttcat taaacatgag agcttagtac attaaacatg agagcttagt 300 acatactatc aacaggttga actgctgatc tgtacagtag aattggtaaa gagagttgtg 360 taaaatattg agttcgcaca tcttgttgtc tgattattga tttttggcga aaccatttga 420 tcatatgaca agatgtgtat ctaccttaac ttaatgattt tgataaaaat cattaggtac 480 cgctagcggc cgtcccctag atgcattcgc gaggtaccga gctcgaattc 530 <210> 18 <211> 303 <212> DNA <213> Artificial Sequence <220> <223> R6K γ origin -7 iteron <400> 18 ggcttgttgt ccacaaccgt taaaccttaa aagctttaaa agccttatat attctttttt 60 ttcttataaa acttaaaacc ttagaggcta tttaagttgc tgatttatat taattttatt 120 gttcaaacat gagagcttag tacgtgaaac atgagagctt agtacgttag ccatgagagc 180 ttagtacgtt agccatgagg gtttagttcg ttaaacatga gagcttagta cgttaaacat 240 gagagcttag tacgttaaac atgagagctt agtacgtact atcaacaggt tgaactgctg 300 atc 303 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> R6K γ origin iteron <400> 19 aaacatgaga gcttagtacg tg 22 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> R6K γ origin iteron <400> 20 aaacatgaga gcttagtacg tt 22 <210> twenty one <211> twenty two <212> DNA <213> Artificial sequence <220> <223> R6K γStarting point iteron <400> twenty one agccatgaga gcttagtacg tt 22 <210> twenty two <211> twenty two <212> DNA <213> Artificial sequence <220> <223> R6K γStarting point iteron <400> twenty two agccatgagg gtttagttcg tt 22 <210> twenty three <211> twenty two <212> DNA <213> Artificial sequence <220> <223> R6K γStarting point iteron <400> twenty three aaacatgaga gcttagtacg ta 22 BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figures 1A-1F Describes the R6K starting point ( Figure 1A , 1E and 1F), RNA-OUT selective marker ( Figure 1B ) and 14 and 3CpGR6K-RNA-OUT bacterial backbone ( Figure 1C and 1D );

[0121] Figure 2A-2B The Sleeping Beauty transposon vector with a Pol I-dependent pUC origin is described ( Figure 2A ) and the Sleeping Beauty transposon vector with PolIII-dependent R6K origin ( Figure 2B );

[0122] Figures 3A-3CAAV vector depicting the Pol I-dependent pUC origin ( Figure 3A and 3B ) and Pol III-dependent R6K origin AAV vectors ( Figure 3C );and

[0123] Figures 4A-4F A60 polyA repeat encoding mRNA vector with Pol I-dependent pUC origin is described ( Figure 4A-4B ), A60 polyA repeat encoding mRNA vector with Pol III-dependent R6K origin ( Figure 4C ), Pol I-dependent pUC origin A99 polyA repeat encoding mRNA vector ( Figure 4D-4E ), and A99 polyA repeat encoding mRNA vectors with Pol III-dependent R6K origin ( Figure 4F ).

[0124] Table 1: Expression levels of pVAX1 mammalian expression vector spacer (SR) derivatives

[0125] Table 2: Expression levels of NTC8685 mammalian expression vector spacer (SR) derivatives

[0126] Table 3: Microcircle applications using various viral and non-viral vector platforms

[0127] Table 4: pNTC multiple cloning sites flanking the R6K origin-RNA-OUT selection marker vector

[0128] Table 5: SV40 origin lentiviral vector: pUC origin vs. R6K origin shake flask production yield / quality

[0129] Table 6: Sleeping Beauty transposon vector: pUC origin vs. R6K origin shake flask production yield / quality

[0130] Table 7: AAV vectors: pUC origin vs. R6K origin shake flask production yield / quality

[0131] Table 8: mRNA vectors: DH5α HyperGRO fermentation yield / quality with pUC origin vs. R6K origin

[0132] Table 9: AAV helper vectors: pUC origin vs. R6K origin plasmid production yield / quality

[0133] SEQ ID NO: 1: R6Kγ start point

[0134] SEQ ID NO:2: 1 CpG R6Kγ start point

[0135] SEQ ID NO: 3: R6Kγ start point without CpG

[0136] SEQ ID NO:4: Extended R6Kγ start point

[0137] SEQ ID NO:5: RNA-OUT selection marker

[0138] SEQ ID NO:6: RNA-OUT antisense suppressor RNA

[0139] SEQ ID NO:7: 2 CpG RNA-OUT selection marker

[0140] SEQ ID NO:8: R6Kγ Origin-RNA-OUT bacterial region flanked by NheI and KpnI restriction sites

[0141] SEQ ID NO:9: 1 CpG R6Kγ start-2 CpG RNA-OUT bacterial region flanked by NheI and KpnI restriction sites

[0142] SEQ ID NO: 10: pNTC-NP1 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI / HindIII

[0143] SEQ ID NO: 11: pNTC-NP2 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI / HindIII

[0144] SEQ ID NO: 12: pNTC-NP3 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI / HindIII

[0145] SEQ ID NO: 13: pNTC-NP4 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI / HindIII

[0146] SEQ ID NO: 14: pNTC-NP5 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: KasI / HindIII

[0147] SEQ ID NO: 15: pNTC-NP6 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI / SacI

[0148] SEQ ID NO: 16: pNTC-NP7 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: BssHII-BssHII

[0149] SEQ ID NO: 17: pNTC-3xCpG NP1 polylinker R6K-RNA-OUT polylinker cloning cassette: HindIII-EcoRI

[0150] SEQ ID NO:18: R6Kγ start point (7-iteron)

[0151] SEQ ID NO: 19: R6Kγ start point 22 bp repeat subrepeat sequence

[0152] SEQ ID NO:20: R6Kγ start point 22 bp repeat subrepeat sequence

[0153] SEQ ID NO:21: R6Kγ start point 22 bp repeat subrepeat sequence

[0154] SEQ ID NO:22: R6Kγ start point 22 bp repeat subrepeat sequence

[0155] SEQ ID NO:23: R6Kγ start point 22 bp repeat subregion repeat sequence

[0156] Definition of terms

[0157] AAV vector: adeno-associated virus vector, an additional viral vector. Including self-complementary (sc) adeno-associated virus vector (scAAV) and single-stranded (ss) adeno-associated virus vector (ssAAV)

[0158] AF: Antibiotic-free

[0159] amp: ampicillin

[0160] ampR: ampicillin resistance gene

[0161] Antibiotic selection markers: Genes that confer antibiotic resistance, such as ampicillin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene

[0162] Approximately: As used herein, the term "approximately" or "about" is applied to one or more values ​​of interest and refers to a value that is the same or similar to the reference value.

[0163] Bacterial Region: The region of the plasmid vector required for propagation and selection in a bacterial host

[0164] bp: base pair

[0165] ccc: covalently closed ring

[0166] cI:Lambda repressor

[0167] cITs857: The Lambda repressor has further incorporated a C to T (Ala to Thr) mutation that confers temperature sensitivity. cITs857 is a functional repressor at 28-30°C but is largely inactive at 37-42°C. Also known as cI857.

[0168] Cat R Chloramphenicol resistance gene

[0169] cmv: cytomegalovirus

[0170] dcmmethylation: E. coli methyltransferase, which methylates the C5 position of the second cytosine in the sequence CC(A / T)GG

[0171] DNA replicon: A genetic element capable of replicating under its own control; examples include plasmids, cosmids, bacterial artificial chromosomes, bacteriophages, viral vectors, and hybrids thereof

[0172] E.coli: Escherichia coli, a Gram-negative bacterium

[0173] EGFP: Enhanced green fluorescent protein

[0174] EP: electroporation

[0175] Eukaryotic expression vectors: vectors that use RNA polymerase I, II, or III promoters to express mRNA, protein antigens, protein therapeutics, shRNA, RNA, or microRNA genes in target eukaryotic organisms

[0176] Eukaryotic region: A region of a plasmid that encodes eukaryotic sequences and / or sequences required for the plasmid to function in a target organism. This includes regions of the plasmid vector required to express one or more transgenes in a target organism, including RNA PolII enhancers, promoters, transgenes, and polyA sequences. This also includes regions of the plasmid vector required to express one or more transgenes in a target organism using RNA Pol I or RNA Pol III promoters, transgenes expressed by RNA Pol I or RNA Pol III, 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, and the like. In lentiviral or retroviral vectors, the eukaryotic region contains flanking forward repeats LTRs, in AAV vectors, the eukaryotic region contains flanking reverse terminal repeats, and in transposon vectors, the eukaryotic region contains flanking transposon reverse terminal repeats or IR / DR ends (such as Sleeping Beauty). In genomic integration vectors, the eukaryotic region may encode homology arms to guide targeted integration.

[0177] Exon: A nucleotide sequence encoded by a gene that is transcribed and present in the mature mRNA product after RNA splicing to remove introns.

[0178] Expression vector: A vector that expresses mRNA, protein antigen, protein therapeutic, shRNA, RNA or microRNA genes in a target organism.

[0179] g: gram, kg stands for kilogram

[0180] Target gene: A gene expressed in a target organism. This includes mRNA genes encoding protein or peptide antigens, protein or peptide therapeutics, as well as mRNA, shRNA, RNA or microRNA encoding RNA therapeutics, and mRNA, shRNA, RNA or microRNA encoding RNA vaccines.

[0181] Hr(s): hours

[0182] ID:Intradermal

[0183] IM: intramuscular

[0184] Immune response: Antigen-reactive cells (such as antigen-reactive T cells) or antibodies (such as antigen-reactive IgG) response

[0185] Intron: A nucleotide sequence encoded by a gene that is transcribed and subsequently removed from the mature mRNA product by RNA splicing.

[0186] IR / DR: reverse repeat sequences that are repeated twice in the forward direction, such as the IR / DR repeat sequence of the Sleeping Beauty transposon.

[0187] Iteron: A DNA sequence that is repeated in the replication origin and is required for replication initiation. The iteron repeat sequence at the R6K origin is 22 bp

[0188] ITR: Inverted terminal repeat

[0189] kan:kanamycin

[0190] kanR: kanamycin resistance gene

[0191] Kd: kilodaltons

[0192] Kozak sequence: An optimized consensus DNA sequence gccRccATG (R = G or A) upstream of the ATG start codon that ensures efficient translation initiation. A SalI site (GTCGAC) upstream of the ATG start codon is an effective kozak sequence (GTCGACATG)

[0193] Lentiviral vector: An integrative viral vector that can infect both dividing and non-dividing cells. Also called a lentiviral transfer plasmid. The plasmid encodes the lentiviral LTR flanking expression unit. The transfer plasmid is transfected into production cells along with the lentiviral envelope and packaging plasmids required to make viral particles.

[0194] Lentiviral envelope vector: plasmid encoding envelope glycoprotein

[0195] Lentiviral packaging vector: one or two plasmids that express the functional proteins gag, pol, and Rev required for packaging of the lentiviral transfer vector

[0196] Minicircle: A covalently closed circular plasmid derivative in which the bacterial region has been removed from the parent plasmid by in vivo or in vitro site-specific recombination or in vitro restriction digestion / ligation. Minicircle vectors are not capable of replication in bacterial cells.

[0197] mRNA: messenger RNA

[0198] mSEAP: mouse secreted alkaline phosphatase

[0199] NA: Not applicable

[0200] Nanoplasmid TM Vector: A vector that combines an RNA selective marker with an R6K, ColE2, or ColE2-related replication origin. For example, NTC9385C, NTC9685C, NTC9385R, NTC9685R vectors and modifications described in Williams, Supra, 2014, and incorporated herein by reference.

[0201] NTC8385: NTC8385, NTC8485 and NTC8685 plasmids are antibiotic-free pUC origin vectors that contain a short RNA (RNA-OUT) selection marker instead of an antibiotic resistance marker (such as kanR). The creation and use of these RNA-OUT-based antibiotic-free vectors are described in Williams, JA 2008 International Patent Application WO2008153733 and are incorporated herein by reference.

[0202] NTC8485: NTC8485 is an antibiotic-free pUC origin vector that contains a short RNA (RNA-OUT) selection marker instead of an antibiotic resistance marker (such as kanR). The creation and use of NTC8485 is described in Williams, JA 2010 US Patent Application 20100184158 and is incorporated herein by reference.

[0203] NTC8685: NTC8485 is an antibiotic-free pUC origin vector that contains a short RNA (RNA-OUT) selection marker instead of an antibiotic resistance marker (such as kanR). The creation and use of NTC8685 is described in Williams, Supra, 2010, and is incorporated herein by reference.

[0204] NTC9385R: NTC9385R Nanoplasmid described in Williams, Supra, 2014 TM The vector, incorporated herein by reference, has a spacer encoding the NheI-trpA terminator-R6K origin RNA-OUT-KpnI bacterial region (SEQ ID NO: 8) linked to the eukaryotic region via flanking NheI and KpnI sites.

[0205] OD 600 : Optical density at 600nm

[0206] PAS: Protomer assembly site. Initiates DNA synthesis at the SSI site of single-stranded DNA. PAS : Binds to the DNA hairpin sequence of priA, which in turn recruits the remaining proteins to form the precursor [priB, dnaT, which recruits dnaB (delivered by dnaC)], which then also recruits the primase (dnaG), which then ultimately makes a short RNA substrate for DNA polymerase I. ABC type PAS : The DNA hairpin binds dnaA, recruits dnaB (transferred by dnaC), which then also recruits the primase (dnaG), which then ultimately makes a short RNA substrate for DNA polymerase I. For example, the R6K plasmid has no CpG ssiA protomer assembly site or alternatively Type or ABC type protomer assembly site.

[0207] PAS-BH: Protomer assembly site on the heavy chain (leading chain)

[0208] PAS-BH region: pBR322 origin region between ROP and PAS-BL (approximately pBR322 2067-2351)

[0209] PAS-BL: Protomer assembly site on the light chain (lagging chain)

[0210] PBS: Phosphate buffered saline

[0211] PCR: polymerase chain reaction

[0212] pDNA: plasmid DNA

[0213] PiggyBac transposon: PB transposon. A transposon system that integrates ITR-flanked PB transposons into the genome via a simple cut-and-paste mechanism mediated by PB transposase. The transposon vector typically contains a promoter-transgene-polyA expression cassette between the PB ITRs, which is excised and integrated into the genome.

[0214] pINT pR pL vector: pINT pR pL att HK022 The integration expression vector is described in Luke et al., 2011 Mol Biotechnol 47:43 and is incorporated herein by reference. The target gene to be expressed is cloned downstream of the pL promoter. The vector encodes a temperature-inducible cI857 repressor, allowing heat-inducible target gene expression.

[0215] P L Promoter: Lambda promoter. L is a strong promoter that is repressed by the cI repressor that binds to the OL1, OL2 and OL3 repressor binding sites. The temperature-sensitive cI857 repressor allows for control of gene expression by heat induction, because at 30°C the cI857 repressor is active and represses gene expression, but at 37-42°C the repressor is inactivated and gene expression ensues.

[0216] P L (OL1 G to T) Promoter: Lambda promoter. P L Is a strong promoter that is inhibited by the cI repressor that binds to the OL1, OL2 and OL3 repressor binding sites. The temperature-sensitive cI857 repressor allows for heat-induced control of gene expression because at 30°C the cI857 repressor is active and inhibits gene expression, but at 37-42°C the repressor is inactivated and gene expression ensues. As described in Williams, Supra, 2014, the binding of the cI repressor to OL1 is reduced by the OL1 G to T mutation, resulting in increased promoter activity at 30°C and 37-42°C.

[0217] Plasmid: An extrachromosomal DNA molecule that is separated from chromosomal DNA and is capable of replicating independently of the chromosomal DNA.

[0218] Plasmid copy number: The number of copies of a plasmid per cell. An increase in plasmid copy number increases plasmid production yield

[0219] Pol: polymerase

[0220] Pol I: Escherichia coli DNA polymerase I

[0221] Pol I dependent replication origin: A replication origin that requires Pol I, such as pMB1, ColE1 or pBR322 or derivatives, such as the high copy pUC origin. For these origins, the RNAII primer forms an RNA:DNA R loop that is cut by RNase H, thereby creating a primer for DNA synthesis directed by DNA pol I. DNA synthesis is then transferred to DNA pol III. Many other Pol I-dependent replication origins are known in the prior art, many of which are summarized in del Solar et al., 1998 Microbiol. Mol. Biol. Rev 62: 434-464, which is incorporated herein by reference.

[0222] Pol III: Escherichia coli DNA polymerase III

[0223] Pol III-dependent replication origin: An origin of replication that does not require Pol I, such as the rep protein-dependent R6Kγ replication origin. Many additional Pol III-dependent replication origins are known in the art, many of which are summarized in delSolar et al., Supra, 1998, which is incorporated herein by reference.

[0224] polyA: polyadenylation signal or site. Polyadenylation is the addition of a poly(A) tail to an RNA molecule. The polyadenylation signal contains a sequence motif that is recognized by the RNA cleavage complex. Most human polyadenylation signals contain the AAUAAA motif and conserved sequences 5' and 3' to it. Commonly used polyA signals come from rabbit beta globin, bovine growth hormone, SV40 early or SV40 late polyA signals.

[0225] pUC origin: pBR322-derived origin of replication with a G to A substitution that increases copy number at elevated temperatures and lacks the ROP negative regulator.

[0226] No pUC: A plasmid without the pUC origin. A non-replicating fragment of the pUC origin may be included, such as an RNAI selection marker.

[0227] pUC plasmid: a plasmid containing the pUC origin.

[0228] R6K plasmid: NTC9385R, NTC9685R, NTC9385R2-O1, NTC9385R2-O2, NTC9385R2a-O1, NTC9385R2a-O2, NTC9385R2b-O1, NTC9385R2b-O2, NTC9385Ra-O1, NTC9385Ra-O2, NTC9385RaF and NTC9385RbF vectors and modified and alternative vectors comprising the R6K origin of replication are described in Williams, Supra, 2014, which is incorporated herein by reference. Alternative R6K vectors known in the art include, but are not limited to, pCOR vectors (Gencell), pCpG-free vectors (Invivogen) and CpG-free vectors (including pGM169) of the University of Oxford.

[0229] R6K origin of replication: A region specifically recognized by the R6K Rep protein to initiate DNA replication. Including but not limited to the R6Kγ origin of replication sequence, published as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 18. Also included are versions without CpG (e.g., SEQ ID NO: 3) as described in U.S. Pat. No. 7,244,609 to Drocourt et al., and incorporated herein by reference.

[0230] R6K replication origin-RNA-OUT bacterial region: contains the R6K replication origin and RNA-OUT selection marker for replication (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).

[0231] Rep: Copy

[0232] Replication intermediates: linear DNA fragments resulting from premature termination of plasmid replication

[0233] Rep protein dependent plasmid: A plasmid whose replication is dependent on the replication (Rep) protein provided in Trans. For example, the R6K replication origin, ColE2-P9 replication origin, and ColE2-related replication origin plasmids, wherein the Rep protein is expressed from the host strain genome. Many additional Rep protein dependent plasmids are known in the art, many of which are summarized in delSolar et al., Supra, 1998, which is incorporated herein by reference.

[0234] Retroviral vector: An integrative viral vector that can infect dividing cells. Also called a transfer plasmid. The plasmid encodes the retroviral LTR flanking expression units. The transfer plasmid is transfected into producer cells along with the packaging plasmids required for envelope and production of viral particles.

[0235] Retroviral envelope vector: a plasmid encoding the envelope glycoprotein

[0236] Retroviral packaging vector: a plasmid encoding the retroviral gag and pol genes required for packaging retroviral transfer vectors

[0237] RNA-IN: Insertion sequence 10 (IS10) encodes RNA-IN, an RNA that is partially complementary and antisense to RNA RNA-OUT. When RNA-IN is cloned in the untranslated leader sequence of mRNA, annealing of RNA-IN to RNA-OUT reduces translation of genes encoded downstream of RNA-IN.

[0238] Selective markers regulated by RNA-IN: Selective markers regulated by RNA-IN of a genome expression. In the presence of RNA-OUT antisense repressor RNA (SEQ ID NO:6) carried by plasmid, the expression of the protein encoded by RNA-IN downstream is suppressed. The selective markers regulated by RNA-IN are configured to make RNA-IN regulate 1) protein, which is lethal or toxic to the cell itself or by producing toxic substances (such as SacB), or 2) repressor proteins, which are lethal or toxic to the bacterial cells by suppressing the transcription of the gene necessary for the cell growth (such as murA essential gene regulated by RNA-IN tetR repressor gene). For example, the RNA-IN-SacB cell line for the genome expression of RNA-OUT plasmid selection / replication is described in Williams, Supra, 2008, and is incorporated herein by reference. Alternative selection markers described in the art can replace SacB.

[0239] RNA-OUT: RNA-OUT encoded by insertion sequence 10 (IS10), an antisense RNA that hybridizes with the transposon gene expressed downstream of RNA-IN and reduces its translation. RNA-OUT RNA (SEQ ID NO: 6) and complementary RNA-IN SacB genome expressed RNA-IN SacB cell line sequence can be modified to bind to alternative functional RNA-IN / RNA-OUT binding pairs, such as those described in Mutalik et al., 2012 Nat Chem Biol 8: 447, including but not limited to RNA-OUT A08 / RNA-IN S49 pairs, RNA-OUT A08 / RNA-IN S08 pairs, and RNA-OUT A08 modifiers without CpG, which can modify the CG in the RNA-OUT 5'TTCGC sequence to a non-CpG sequence. Williams 2015. Replicative minicircle vectors with improved expression. An example of a CpG-free RNA-OUT selection marker is described in US patent application US2015 / 0275221, in which two CpG motifs in the RNA-OUT RNA are removed (one of which is present in the RNA-IN complementary region), and is incorporated herein by reference. A large number of substitutions can be used to remove two CpG motifs (mutating each CpG to CpA, CpC, CpT, ApG, GpG or TpG) to generate CpG-free RNA-OUT.

[0240] RNA-OUT selection marker: An RNA-OUT selection marker DNA fragment comprising an E. coli transcription promoter and a terminator sequence flanked by RNA-OUT RNA. A RNA-OUT selectable marker is described in Williams, Supra, 2008, which utilizes RNA-OUT promoter and terminator sequences flanked by DraIII and KpnI restriction enzyme sites, and an RNA-IN-SacB cell line for genomic expression of RNA-OUT plasmid replication is designed, and is incorporated herein by reference. The RNA-OUT promoter and terminator sequences in SEQ ID NO:5 flanked by RNA-OUT RNA (SEQ ID NO:6; FIG.1B) can be replaced with heterologous promoter and terminator sequences. For example, the RNA-OUT promoter can be replaced with a CpG-free promoter known in the art, such as an I-EC2K promoter or a P5 / 6 5 / 6 or P5 / 6 6 / 6 promoter, which are described in Williams, Supra, 2008, and incorporated herein by reference. A 2-CpG RNA-OUT selective marker, in which two CpG motifs in the RNA-OUT promoter are removed, is designated as SEQ ID NO: 7. Williams, Supra, 2015 describes an example of a CpG-free RNA-OUT transcription unit, in which two CpG motifs in the RNA-OUT RNA (one of which is present in the RNA-IN complementary region) and two CpG motifs in the RNA-OUT promoter are removed, and is incorporated herein by reference. Vectors incorporating a CpG-free RNA-OUT selective marker can be selected for sucrose resistance using the RNA-IN-SacB cell line, which is used for RNA-OUT plasmid replication and described in Williams, Supra, 2008. Alternatively, the RNA-IN sequence in these cell lines can be modified to include a 1-base change to perfectly match the CpG-free RNA-OUT region complementary to RNA-IN.

[0241] RNA polymerase II promoter: Promoters that recruit RNA polymerase II to synthesize mRNA, most small nuclear RNAs, and microRNAs. For example, constitutive promoters such as human or mouse CMV promoter, elongation factor 1 (EF1) promoter, chicken β-actin promoter, β-actin promoter from other species, elongation factor 1 alpha (EF1α) promoter, phosphoglycerol kinase (PGK) promoter, Rous sarcoma virus (RSV) promoter, human serum albumin (SA) promoter, spleen focus forming virus (SFFV) promoter, alpha-1 antitrypsin (AAT) promoter, thyroxine binding globulin (TBG) promoter, cytochrome P450 2E1 (CYP2E1) promoter, etc. The vector can also utilize a combination of promoters, such as the chicken β-actin / CMV enhancer (CAG) promoter, a human or mouse CMV-derived enhancer element combined with an elongation factor 1α (EF1α) promoter, a CpG-free version of a human or mouse CMV-derived enhancer element combined with an elongation factor 1α (EF1α) promoter, an albumin promoter combined with an alpha-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 C5-12 or liver-specific promoter apolipoprotein AI (ApoAI), etc.

[0242] RNA polymerase III promoter: A promoter that recruits RNA polymerase III to synthesize tRNAs, 5S ribosomal RNA, and other small RNAs. For example, class I promoters such as 5S rRNA promoters, class II promoters such as tRNA promoters, class III promoters such as U6 small nuclear RNA promoters or H1 nuclear RNase P promoters, etc.

[0243] RNA selection marker: An RNA selection marker is a plasmid-borne expressed non-translated RNA that regulates a chromosomally expressed target gene to provide selection. This may be a plasmid-borne nonsense suppressor tRNA that regulates a nonsense suppressible selectable chromosomal target as described in Crouzet J and Soubrier F 2005 US Patent 6,977,174, incorporated herein by reference. This can also be a plasmid-borne antisense repressor RNA, a non-limiting list of which is incorporated herein by reference including RNA-OUT that suppresses RNA-IN regulatory targets (Williams, Supra, 2008), RNA I encoded by the pMB1 plasmid origin that suppresses RNAII regulatory targets (Grabherr, Pfaffenzeller I. 2006 US patent application US20060063232; Cranenburgh RM. 2009; US patent 7,611,883), RNAI encoded by the IncB plasmid pMU720 origin that suppresses RNA II regulatory targets (Wilson IW, Siemering KR, Praszkier J, Pittard AJ. 1997. J Bacteriol 179:742-53), ParB locus Sok of the R1 plasmid that suppresses Hok regulatory targets, Flm locus FlmB of the F plasmid that suppresses flmA regulatory targets (Morsey MA, 1999 US Patent US5922583). The RNA selection marker can be another natural antisense repressor RNA known in the art, such as those described in Wagner EGH, Altuvia S, Romby P. 2002. Adv Genet 46: 361-98 and Franch T, and Gerdes K. 2000. Current Opin Microbiol 3: 159-64. The RNA selection marker can also be an engineered repressor RNA, such as the SgrS, MicC or MicF scaffold expressed by synthetic small RNA described in Na D, Yoo SM, Chung H, Park H, Park JH, Lee SY. 2013. Nat Biotechnol 31: 170-4. The RNA selection marker can also be an engineered repressor RNA that, as part of a selection marker, can suppress a target RNA (e.g., SacB) fused to a target gene to be regulated, as described in Williams, Supra, 2015.

[0244] ROP: Repressor of primers

[0245] RSM: RNA selection marker:

[0246] SacB: Structural gene encoding the L-sucrase of Bacillus subtilis. SacB is toxic when expressed in Gram-negative bacteria in the presence of sucrose.

[0247] SD: Standard deviation

[0248] SEAP: Secreted alkaline phosphatase

[0249] Selectable marker: A selectable marker, such as a kanamycin resistance gene or an RNA selectable marker.

[0250] Selection marker: A selection marker, such as a kanamycin resistance gene or an RNA selection marker.

[0251] SIDD: Supercoil-induced destabilization of DNA duplexes (SIDD) structures. When these sites are incorporated into a vector, they may alter the sensitivity of other sequences in the vector to being disrupted. This can alter function. For example, adding SIDD sites to an expression vector can reduce the helical instability of a promoter. Depending on the promoter, this may increase or decrease promoter activity, as some promoters increase expression with promoter helical destabilization and others decrease expression with promoter helical destabilization.

[0252] shRNA: short hairpin RNA

[0253] S / MAR: Scaffold / matrix attachment region. Eukaryotic sequences that mediate DNA attachment to the nuclear matrix

[0254] Sleeping Beauty Transposon SB Transposon: A transposon system that integrates IR / DR flanked by SB transposons into the genome by a simple cut and paste mechanism mediated by SB transposase. The transposon vector typically contains a promoter-transgene-polyA expression cassette between the IR / DR that is excised and integrated into the genome.

[0255] Spacer: As used herein, a spacer is a region connecting the 5' and 3' ends of a eukaryotic region sequence. The 5' and 3' ends of the eukaryotic region are usually separated by a bacterial replication origin and a bacterial selection marker (bacterial region) in a plasmid vector, so many spacers consist of bacterial regions. In the Pol III-dependent replication origin vector of the present invention, the spacer is preferably less than 1000 bp.

[0256] SR: spacer region.

[0257] ssi: single-stranded initiation sequence

[0258] Structured DNA sequence: As used herein, a DNA sequence that can form a secondary structure that inhibits replication (Mirkin and Mirkin, 2007. Microbiology and Molecular Biology Reviews 71: 13-35). This includes, but is not limited to, inverted repeats, palindromic repeats, direct repeats, IR / DR, homopolymeric repeats, or repeats containing eukaryotic promoter enhancers, or repeats containing eukaryotic replication origins.

[0259] SV40 origin: Simian virus 40 genomic DNA containing the replication origin

[0260] SV40 enhancer: Simian virus 40 genomic DNA containing 72 bp and optional 21 bp enhancer repeat sequences

[0261] Target antigen: An immunogenic protein or peptide epitope, or a combination of a protein and an epitope, against which an immune response can be generated. The target antigen can be derived from a pathogen for infectious diseases or allergy applications, or from a host organism for applications such as cancer, allergies, or autoimmune diseases. Target antigens are well defined in the art. Some examples are described in Williams, Supra, 2008, and are incorporated herein by reference.

[0262] TE buffer: A solution containing approximately 10 mM Tris pH 8 and 1 mM EDTA

[0263] TetR: tetracycline resistance gene

[0264] Tol2 transposon: A transposon system in which the ITR-flanked Tol2 transposon is integrated into the genome by a simple cut-and-paste mechanism mediated by the Tol2 transposase. The transposon vector typically contains a promoter-transgene-polyA expression cassette between the Tol2 ITRs, which is excised and integrated into the genome.

[0265] Transcription terminator: bacteria: A DNA sequence that marks the end of a gene or operon for transcription. This may be an intrinsic transcription terminator or a Rho-dependent transcription terminator. For intrinsic terminators, such as the trpA terminator, a hairpin structure is formed in the transcript that disrupts the mRNA-DNA-RNA polymerase ternary complex. Alternatively, Rho-dependent transcription terminators require Rho factor, an RNA helicase protein complex, to disrupt the nascent mRNA-DNA-RNA polymerase ternary complex. Eukaryotic thingThe polyA signal is not a "terminator", but rather cuts inside the polyA site, leaving an unblocked 5' end on the 3'UTR RNA for nuclease digestion. The nuclease catches up with RNA Pol II and causes termination. By introducing an RNA Pol II pause site (eukaryotic transcription terminator), termination can be promoted within a short region of the poly A site. The pause of RNA Pol II allows the nuclease introduced into the 3'UTR mRNA after PolyA cleavage to catch up with RNA Pol II at the pause site. A non-limiting list of eukaryotic transcription terminators known in the art includes C2x4 and gastrin terminators. Eukaryotic transcription terminators can increase mRNA levels by enhancing the appropriate 3' end processing of mRNA.

[0266] Transfection: Methods for delivering nucleic acids into cells [e.g., poly(lactide-co-glycolide) (PLGA), ISCOMs, liposomes, nonionic surfactant vesicles (niosomes), virosomes, 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 high-speed cell deformation-mediated membrane disruption, corona plasma, plasma-enhanced delivery, tissue-tolerant plasma, laser microporation, shock wave energy, magnetic field, non-contact magnetic permeation, gene gun, microneedle, microabrasion, hydrodynamic delivery, high-pressure tail vein injection, etc.], as known in the art and incorporated herein by reference

[0267] Transgene: A gene of interest that is cloned into a vector for expression in a target organism

[0268] Transposase vector: vector encoding transposase

[0269] Transposon vector: A vector encoding a transposon, which is a substrate for transposase-mediated gene integration

[0270] ts: temperature sensitive

[0271] μg: microgram

[0272] μl: microliter

[0273] UTR: untranslated region of mRNA (5' or 3' of the coding region)

[0274] Vector: Gene delivery vectors, including viral (such as alpha virus, pox virus, lentivirus, retrovirus, adenovirus, adenovirus-associated virus, etc.) and non-viral (such as plasmid, MIDGE, transcriptionally active PCR fragment, minicircle, phage, etc.) vectors. These are well known in the art and are incorporated herein by reference.

[0275] Vector backbone: the eukaryotic and bacterial regions of the vector, without the transgene or target antigen coding regions

[0276] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0277] Current technology generally relates to methods and compositions for short <1 kb bacterial region plasmid DNA vectors that improve plasmid production yield and quality, reduce transfection-related toxicity, and increase transgene expression. Current technology can be used to improve expression and preparation of vectors, such as non-viral vectors (transposon vectors, transposase vectors, Sleeping Beauty transposon vectors, Sleeping Beauty transposase vectors, PigyBac transposon vectors, PigyBac transposase vectors, expression vectors, etc.) and viral vectors (such as 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.).

[0278] Improved plasmid expression is defined herein as an improvement in the level and / or duration of transgene expression in vitro or in vivo compared to a transgene encoding plasmid containing a bacterial region encoding a pUC origin of replication. It should be understood that all references cited herein are incorporated herein by reference.

[0279] It has now surprisingly been found that the current state of the art plasmid modification methods provide a solution for the efficient and high yield production of short spacer vectors containing structured DNA sequences.

[0280] As used herein, the term "sequence identity" refers to the degree of identity between any given query sequence (e.g., SEQ ID NO: 2) and a subject sequence. A subject sequence may, for example, have at least 90%, at least 95%, or at least 99% sequence identity with a given query sequence. In order to determine the sequence identity percentage, the query sequence (e.g., nucleic acid sequence) is aligned with one or more subject sequences using any suitable sequence alignment program known in the art (e.g., computer program ClustalW (version 1.83, default parameters)), which allows alignment of nucleic acid sequences over the entire length of the nucleic acid sequence (global alignment). Chema et al., 2003 Nucleic Acids Res., 31: 3497-500. In a preferred method, the sequence alignment program (e.g., ClustalW) calculates the best match between the query sequence and one or more subject sequences, and aligns them, so that identity, similarity, and diversity can be determined. One or more nucleotide gaps may be inserted into the query sequence, the subject sequence, or both, to maximize sequence alignment. For rapid pairwise alignment of nucleic acid sequences, appropriate default parameters can be selected for a particular alignment program. The output is a sequence alignment reflecting the relationship between the sequences. To further determine the percent identity of the subject nucleic acid sequence to the query sequence, the sequences are aligned using an 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 percent identity values ​​may be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.

[0281] Now turning to the attached figure, Figures 1A-1F Figure showing annotations: Figure 1A ) the R6K start point, the position of the 22 bp iteron repeat sequence, DnaA boxes 1 and 2, and the region included in SEQ ID NOs: 1, 2, 3 and 4 R6K start point; Figure 1B ) an RNA-OUT selectable marker of SEQ ID NO: 5 having positions of -35 and -10 elements of the RNA-OUT promoter, an RNA-OUT antisense RNA of SEQ ID NO: 6 having a region of complementary homology to RNA-IN and a 3' hairpin of the RNA-OUT terminator; Figure 1C ) a 14 CpG R6K-RNA-OUT bacterial backbone consisting of the R6K replication origin of SEQ ID NO: 1 and the RNA-OUT selection marker of SEQ ID NO: 5, which includes the trpA bacterial terminator upstream of the R6K origin and is flanked by NheI and KpnI cloning sites; Figure 1D) 3 CpG R6K-RNA-OUT bacterial backbone consisting of SEQ ID NO:2 1x CpG R6K replication origin and SEQ ID NO:7 2x CpG RNA-OUT selection marker, flanked by NheI and KpnI cloning sites; Figure 1E ) R6K origin from SEQ ID NO: 1, with the locations of the six ITERs highlighted. A single 22 bp sub-repeat sequence is shown below the origin; and Figure 1F ) from the R6K start point of SEQ ID NO: 18, highlighting the positions of the 7 ITERs. The single 22 bp repeat sequence is shown below the start map. In this example, repeater 5 of the 7-repeater vector has been replicated in tandem; however, the 7-repeater vector of the present invention can be obtained by tandemly replicating any one of repeaters 1, 2, 3, 4, 5 or 6.

[0282] Figure 2A-2B Figure showing annotations: Figure 2A ) Pol I-dependent pUC origin-kanamycin selective Sleeping Beauty transposon vector pUC57-Kan SB1 (see Table 6); and Figure 2B ) Pol III-dependent R6K origin-RNA-OUT Sleeping Beauty transposon vector NTC9 SB1 without antibiotic selection (see Table 6). The positions of the left and right Sleeping Beauty IR / DR relative to the bacterial backbone replication origin and selection marker are shown.

[0283] Figures 3A-3C Figure showing annotations: Figure 3A ) Pol I-dependent pUC origin-ampicillin selective AAV vector pAAV (see Table 7); Figure 3B ) Pol I-dependent pUC origin-RNA-OUT AAV vector NTC8-AAV without antibiotic selection (see Table 7); and Figure 3C ) Pol III-dependent R6K origin-RNA-OUT AAV vector NTC9-AAV without antibiotic selection (see Table 7). The positions of the left and right AAV ITRs relative to the bacterial backbone replication origin and selection marker are shown.

[0284] Figures 4A-4F Figure showing annotations: Figure 4A ) Pol I-dependent pUC origin-ampicillin-selective A60 polyA repeat encoding mRNA vector pGEM4Z T7 A60 pA (see Table 8); Figure 4B ) Pol I-dependent pUC origin-RNA-OUT A60 polyA repeat encoding mRNA vector NTC8-T7 A60 pA without antibiotic selection (see Table 8); Figure 4C ) Pol III-dependent R6K origin-RNA-OUT A60 polyA repeat encoding mRNA vector NTC9-T7 A60 pA without antibiotic selection (see Table 8); Figure 4D ) Pol I-dependent pUC origin-ampicillin-selected A99 polyA repeat encoding mRNA vector pT3 / T7A99 pA (see Table 8); Figure 4E ) Pol I dependent pUC origin-kanR selected A99 polyA repeat encoding mRNA vector NTC7-T7 A99 pA (see Table 8); and Figure 4F ) Pol III-dependent R6K origin-RNA-OUT A99 polyA repeat encoding mRNA vector NTC9-T7 A99 pA without antibiotic selection (see Table 8). The position of the A60 or A99 polyA repeat sequence relative to the bacterial backbone replication origin and selection marker is shown. Example

[0285] The following examples further illustrate the methods of the current technology. These are provided in an exemplary manner and are not intended to limit the scope of the present disclosure in any way.

[0286] Example 1: pUC and R6K origin of replication plasmid replication and production

[0287] pUC origin vector replication and production background: The vast majority of therapeutic plasmids use the pUC origin, which is a high-copy derivative of the pMB1 origin (closely related to the ColE1 origin). For pMB1 replication, plasmid DNA synthesis is unidirectional and does not require a plasmid-borne initiator protein. The pUC origin is a high-copy derivative of the pMB1 origin that has the auxiliary ROP (rom) protein deleted and has an additional temperature-sensitive mutation that destabilizes the RNAI / RNAII interaction. Shifting cultures containing these origins from 30°C to 42°C results in an increase in plasmid copy number. pUC plasmids can be produced in many E. coli cell lines.

[0288] RNA-OUT without antibiotic selection marker background: Antibiotic-free selection was performed in an E. coli strain containing pCAH63-CAT RNA-IN-SacB (P5 / 66 / 6) with a chromosomal integration of the bacteriophage lambda attachment site as described in Williams, Supra, 2008. SacB (Bacillus subtilis L-sucrase) is a counter-selectable marker that is lethal to E. coli cells in the presence of sucrose. The plasmid-encoded RNA-OUT represses translation of SacB from the RNA-IN-SacB transcript ( Figure 1B). This promotes plasmid selection in the presence of sucrose by inhibiting SacB-mediated lethality.

[0289] R6K origin vector replication and production background: The R6Kγ plasmid replication origin requires a single plasmid replication protein It is attached to multiple repeated " repeat sub " sites (seven core repeat sequences comprising TGAGNG consensus sequence) as replication initiation monomer, and is attached to inhibition site (TGAGNG) and repeat sub with the affinity that reduces as replication inhibition dimer.Replication requires multiple host factors, including IHF, DnaA and protosome assembly protein DnaB, DnaC, DnaG (Abhyankar et al., 2003J Biol Chem 278:45476-45484).Described R6K core starting point comprises the binding site of DnaA and IHF, and DnaA and IHF affect plasmid replication, because IHF and DnaA interact to initiate replication.

[0290] Different versions of the R6Kγ replication origin have been used in various eukaryotic expression vectors, such as the pCOR vector (Soubrier et al., 1999, Gene Therapy 6: 1482-88) and the CpG-free version of the pCpGfree vector (Invivogen, San Diego CA) and pGM169 (Oxford University). Incorporation of the R6K replication origin itself does not increase transgene expression levels compared to the optimized pUC origin vector (Soubrier et al., Supra, 1999). However, the use of a conditional replication origin such as R6Kγ requires specialized cell lines for replication, which increases the safety margin because the vector will not replicate if transferred into the patient's endogenous flora.

[0291] Highly minimized 6-repeat R6Kγ-derived origin of replication (SEQ ID NO: 1; Figure 1E ) contains the core sequences required for replication (including the DnaA box and stb 1-3 sites; Wu et al., 1995. J Bacteriol. 177: 6338-6345), but the upstream Dimeric repressor binding site and downstream The promoter was deleted (by removing one copy of the repeats) as described in Williams, Supra, 2014, and incorporated herein by reference. This R6K origin contains 6 tandem direct repeats ( Figure 1E ). NTC9385R Nanoplasmid TMThe vector includes this minimized R6K origin and the RNA-OUT AF selectable marker in the spacer region and is described in Williams, Supra, 2014, and incorporated herein by reference.

[0292] Typical R6K production strains express from the genome Protein derivative PIR116, which contains the P106L substitution that increases the copy number (by reducing dimerization; The monomer is activated, and Dimer inhibition). Fermentation results using pCOR (Soubrier et al., Supra, 1999) and pCpG plasmids (Hebel HL, Cai Y, Davies LA, Hyde SC, Pringle IA, Gill DR. 2008. Mol Ther 16: S110) were lower, about 100 mg / L in the PIR116 cell line.

[0293] Mutagenesis of the pir-116 replication protein and selection for increased copy number have been used to make new production strains. For example, the TEX2pir42 strain contains a combination of P106L and P42L. The P42L mutation interferes with DNA circular replication repression. The TEX2pir42 cell line with the pCOR plasmid increases copy number and fermentation yield, with a reported yield of 205 mg / L (Soubrier F. 2004. International Patent Application WO2004033664).

[0294] Can increase the copy number Other combinations of copy number mutants include "P42L and P113S", and "P42L, P106L, and F107S" (Abhyankar et al., 2004. J Biol Chem 279:6711-6719).

[0295] Williams, Supra, 2014 describes a heat-inducible pL promoter that expresses the integration of the bacteriophage HK022 attachment site Host strains for high-copy mutant replication (Rep) proteins P42L, P106L, and F107S, for Nanoplasmids with an R6K origin TM Vector selection and replication. This is an additional Nanoplasmid TM Safety factor, because vectors with the R6K origin can only replicate in engineered E. coli host strains that express the Rep protein.

[0296] Replication and fermentation of the RNA-OUT selection marker R6K plasmid described in Williams, Supra, 2014 was performed using heat-inducible 'P42L, P106L and F107S' Copy number mutant cell lines (e.g. DH5α host strain NTC711772 = DH5αdcm-att λ ::P 5 / 6 6 / 6 -RNA-IN-SacB,catR;att HK022 ::pL(OL1-G to T)P42L-P106L-F107S(P3-),SpecR StrepR). The yield was reported to be as high as 695 mg / L.

[0297] Other cell lines created and disclosed herein include:

[0298] NTC821601DH5αatt λ ::P 5 / 6 6 / 6 -RNA-IN-SacB,catR;att HK022 ::pL(OL1-G to T)P42L-P106L-F107S(P3-),SpecR StrepR=dcm+NTC711772 version

[0299] NTC940211DH5αatt λ ::P 5 / 6 6 / 6 -RNA-IN-SacB,catR;att HK022 ::pL(OL1-G to T)P42L-P106I-F107S P113S(P3-),SpecR StrepR=P106I high copy number substitution of P106L combined with P113S to generate a quadruple copy number increase mutant rep protein derivative of NTC821601

[0300] NTC1050811DH5αatt λ ::P 5 / 6 6 / 6 -RNA-IN-SacB,catR;att HK022 ::pL(OL1-G to T)P42L-P106I-F107S P113S(P3-),SpecR StrepR; ::pARA-CI857ts, tetR = pARA-CI857ts derivative of NTC940211. This strain contains the arabinose-inducible CI857ts gene. Attachment site chromosomal integration copy. Addition of arabinose to the plate or medium (e.g., to 0.2-0.4% final concentration) induces pARA-mediated expression of the CI857ts repressor, thereby reducing the copy number at 30°C by CI857ts-mediated downregulation of the Rep protein expressing the pL promoter [i.e., additional CI857ts-mediated more efficient downregulation of the pL (OL1-G to T) promoter at 30°C]. Copy number induction is not affected after the temperature is raised to 37-42°C because the CI857ts repressor is inactivated at these elevated temperatures. The dcm derivative (NTC1050811dcm-) is used in cases where dcm methylation is not required.

[0301] 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 is described in Williams, Supra, 2014

[0302] Compared with the triple mutant heat-inducible pL(OL1-G to T)P42L-P106L-F107S(P3-) described in Williams, Supra, 2014, the quadruple mutant heat-inducible pL(OL1-G to T)P42L-P106L-F107S P113S(P3-) increased the Nanoplasmid TM More than 2 g / L of Nanoplasmid was obtained using the quadruple mutant NTC1050811 cell line. TM Yield (e.g., 2240 mg / L for NTC9 T7 A99pA, Table 8).

[0303] The use of a conditional origin of replication, such as the R6K origin, requires a specialized cell line for replication, which increases the safety margin because the vector will not replicate if transferred into the patient's endogenous flora.

[0304] Example 2: Production of pUC and R6K origin vectors

[0305] Shake flask production:Shake flask production is performed using proprietary plasmid+ shake flask medium. Seed cultures are started from glycerol stocks or colonies and streaked onto LB medium agar plates containing 50 μg / mL antibiotics (for ampR or kanR selection plasmids) or 6% sucrose (for RNA-OUT selection plasmids). Plates are grown at 30-32°C; cells are resuspended in medium and used to give approximately 2.5 OD to 500 mL plasmid+ shake flasks 600 The shake flasks contained 50 μg / mL of antibiotics for ampR or kanR selection plasmids or 0.5% sucrose for RNA-OUT selection plasmids. The flasks were grown to saturation with shaking at the growth temperatures shown in Tables 5, 6, 7, and 9.

[0306] Fermentation production: Fermentations were performed in a New Brunswick BioFlo 110 bioreactor using a proprietary fed-batch medium (NTC3019, HyperGRO medium) as described (Carnes and Williams, Supra, 2011). Seed cultures were started from glycerol stocks or colonies and streaked onto LB medium agar plates containing 50 μg / mL antibiotics (for ampR or kanR selection plasmids) or 6% sucrose (for RNA-OUT selection plasmids). Plates were grown at 30-32°C; cells were resuspended in medium and used to provide approximately 0.1% inoculum for fermentations containing 50 μg / mL antibiotics for ampR or kanR selection plasmids or 0.5% sucrose for RNA-OUT plasmids. Tables 8 and 9 show the HyperGRO temperature shifts.

[0307] Production host: Fermentations of AmpR or KanR plasmids with the pUC origin were performed in E. coli strains DH5α[F–Φ80lacZΔM15Δ(lacZYA-argF)U169 recA1endA1 hsdR17(rK–,mK+)phoAsupE44λ–thi-1gyrA96 relA1] (Invitrogen, Carlsbad CA) or Stbl4.

[0308] Fermentation of the RNA-OUT plasmid with antibiotic-free pUC origin was performed in the E. coli strain DH5α containing pCAH63-CAT RNA-IN-SacB (P5 / 66 / 6) with chromosomal integration of the bacteriophage lambda attachment site as described in Williams, Supra, 2008. The production strain is NTC4862 = DH5αattλ::P5 / 6 6 / 6-RNA-IN-SacB, catR.

[0309] The RNA-OUT plasmid without antibiotic R6Kγ origin was replicated and fermented using the E. coli RNA-OUT selection host, which further encodes the pL promoter heat-inducible type of the HK022 attachment site integration. Copy number mutant cell lines and methods for their generation are described in Williams, Supra, 2014, and are incorporated herein by reference.

[0310] Production strain:

[0311] pUC origin-AmpR or KanR antibiotic selection host

[0312] DH5α

[0313] Stbl4

[0314] pUC origin-RNA-OUT sucrose selection host

[0315] NTC4862 DH5αatt λ ::P 5 / 6 6 / 6 -RNA-IN-SacB,catR

[0316] NTC1011592Stbl4 attλ::P5 / 6 6 / 6-RNA-IN-SacB,catR

[0317] R6K Origin-RNA-OUT Sucrose Selected Nanoplasmid TM Host

[0318] NTC1050811DH5αatt λ ::P 5 / 6 6 / 6 -RNA-IN-SacB,catR;att HK022 ::pL(OL1-G to T)

[0319] P42L-P106I-F107S P113S(P3-),SpecR StrepR; ::pARA-CI857ts,tetR

[0320] NTC1011641 Stbl4 attλ::P5 / 6 6 / 6-RNA-IN-SacB,catR;att HK022 ::pL

[0321] P42L-P106L-F107S(P3-)SpecR StrepR

[0322] Analytical methods: During all fermentations, culture samples were collected regularly at key points. Samples were immediately analyzed for biomass (OD 600) and plasmid yield. Plasmid yield was determined by quantifying plasmid obtained from Qiagen Spin Miniprep kit preparations as described in (Carnes and Williams, Supra, 2011). Briefly, cells were alkaline lysed, clarified, plasmids were column purified, and eluted prior to quantification. Plasmid quality was determined by agarose gel electrophoresis analysis (AGE) and run on 0.8-1% Tris / acetate / EDTA (TAE) gels as described in Carnes and Williams, Supra, 2011.

[0323] Example 3: Construction and preparation of pUC and R6K origin structure vectors

[0324] The R6Kγ start point (SEQ ID NO: 1; Figure 1E )-RNA-OUT (SEQ ID NO: 5; Figure 1B ) bacterial replication selection region (SEQ ID NO: 8; Figure 1C ) were cloned into the polylinker regions of various pUC57-based vectors to generate pNTC-NP1, pNTC-NP2, pNTC-NP3, pNTC-NP4, pNTC-NP5, pNTC-NP6, and pNTC-NP7 vectors. Each vector has different flanking restriction enzyme sites that can be used to transform the target vector into an R6K replication-RNA-OUT selection. Table 4 shows the 5' and 3' polylinker sequences flanking the R6K-RNA-OUT insert fragments in the pNTC-NP1-7 vectors. A pUC57-based 1 CpG R6Kγ origin-2 CpG RNA-OUT bacterial replication selection region (SEQ ID NO: 9; Figure 1D ) version (pNTC-3xCpGNP1), as shown in Table 4.

[0325] The R6Kγ origin (SEQ ID NO: 1) is the modified R6K starting point of the 6-repeat subunit ( Figure 1E A pUC57-based 7-repeater R6Kγ origin (SEQ ID NO: 18; Figure 1F )-RNA-OUT (SEQ ID NO: 5; Figure 1B ) bacterial replication selection region and used it to construct and evaluate the utility of other repeaters in production. Similarly, high quality, high yield production was obtained using vectors that differed only in the inclusion of the 7-repeater R6Kγ origin of SEQ ID NO: 18 or the 6-repeater R6Kγ origin (SEQ ID NO: 1). For example, the following harvest yields were obtained in a 30-42°C 10 hr ramp temperature shift HyperGRO fermentation:

[0326] SEQ ID NO:1 6 repeats 3203bp R6K origin vector: biomass 120OD 600 ; Plasmid titer is 1363mg / L; Plasmid specific gravity is 11.3mg plasmid / L / OD 600

[0327] SEQ ID NO:18 7 repeats 3225bp R6K origin vector: biomass 137OD 600 ; Plasmid titer is 1503mg / L; Plasmid specific gravity is 11.0mg plasmid / L / OD 600

[0328] The starting point of the 7-repeater R6Kγ in SEQ ID NO: 18 is a tandem repeat of repeat 5 ( Figure 1F ; SEQ ID NO: 18), but the 7-repeater R6Kγ origin vector of the present invention can be a tandem repeat of any repeat, with SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23 ( Figure 1E ) is shown, or SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23 are randomly combined into a 7-repeater R6K starting point composition or a repeater repeat variant retaining the TGAGNG consensus sequence. Other repeater derivatives (e.g., 8, 9 or 10 repeater vectors) are also considered for use in practicing the present invention.

[0329] Table 4: pNTC multiple cloning site flanked by R6K origin-RNA-OUT selection marker vector

[0330]

[0331]

[0332]

[0333] a In a directional multi-fragment ligation reaction, the non-palindromic unique 3 bp NNN sticky-end DraIII site (CACNNNGTG) separates R6K and RNA-OUT of the sequence CACGTTGTG and can be used to assemble R6K and RNA-OUT from separate pNTC vectors

[0334] The viral and non-viral vector pUC origin-antibiotic selection bacterial backbone was transformed into R6K-RNA-OUT by the following method:

[0335] 1) Select restriction sites flanking the pUC origin and antibiotic selection marker region in target viral and non-viral vectors;

[0336] 2) determine the polylinker-R6K-RNA-OUT polylinker cassette compatible with pNTC-NP (pNTC-NP1, 2, 3, 4, 5, 6, or 7; Table 4);

[0337] 3) The antibiotic selection marker region of the pUC origin was excised and replaced with the R6K origin RNA-OUT region of choice using restriction digestion methods of choice and standard ligase-mediated cloning.

[0338] In some cases, the R6K origin and RNA-OUT units were assembled from separate restriction fragments in a multi-fragment ligation using a non-palindromic DraIII linker site (see Table 4). In the case of fd6 Ad helper modification (Table 9), a short 500 bp synthetic gene DraIII RNA-OUT-Ad helper-AvrII was used for a 3-fragment ligation to connect RNA-OUT to the unique AvrII site of the fd6 Ad helper eukaryotic region in the 12 kb AvrII-SalI restriction fragment and to the SalI-R6K origin-DraIII fragment from pNTC-NP4.

[0339] For Sleeping Beauty vectors (Figure 2; Table 6), AAV vectors (Figure 3; Table 7), and mRNA vectors (Figure 4; Table 8), exemplary vector maps and vector characteristics of the original pUC origin-antibiotic selection marker vector and the modified R6K origin-RNA-OUT antibiotic selection marker-free vector are shown. For AAV auxiliary vectors, vector characteristics of the original pUC origin-antibiotic selection marker vector and the modified R6K origin-RNA-OUT antibiotic selection marker-free vector are shown (Table 8). For lentiviral vectors (Table 5) and AAV vectors (Table 7), vector characteristics of the pUC origin-RNA-OUT antibiotic selection marker-free vector and the modified R6K origin-RNA-OUT antibiotic selection marker-free vector are shown.

[0340] In all cases, the bacterial backbone size in the R6K origin-RNA-OUT antibiotic-free selection marker modified vector is <1kb (460-610bp). This is far below the bacterial backbone size limit of 1.1kb required to improve the expression level (Table 1-2) and duration of the vector (Quiviger et al., Supra, 2014). In all cases, the original pUC origin-antibiotic selection bacterial backbone before modification is >1.2kb (2340-2750bp), and the same is true for the pUC origin-RNA-OUT modification (1210-1500bp). Therefore, compared with the original pUC origin-antibiotic selection marker vector, these AAV, AAV helper, Sleeping Beauty and lentiviral R6K origin-RNA-OUT antibiotic-free selection marker modified vectors meet the requirements of a short spacer region to improve the expression level and duration. In addition, these AAV, AAV helper, Sleeping Beauty and lentiviral R6K origin-RNA-OUT antibiotic selection marker-free modified vectors have no chance of antibiotic marker gene transfer by transduction (AAV, lentiviral vectors) or transposition (Sleeping Beauty vectors) due to the removal of KanR or ampR antibiotic resistance selection markers in the parental vectors. In addition, the vectors of this technology do not require complex and expensive additional manufacturing steps that are difficult to scale up, which require the use of minicircle vectors to remove a large bacterial region between the eukaryotic polyA and the promoter (Kay et al., Supra, 2010).

[0341] However, in lentiviral vectors, the eukaryotic region includes flanking direct repeats LTR, in AAV vectors, the eukaryotic region includes flanking inverted terminal repeats, and in Sleeping Beauty transposon vectors, the eukaryotic region includes flanking transposon IR / DR ends. These flanking sequences are all structured DNA sequences.

[0342] Levy, Supra, 2004 teaches that when the replication origin of any high copy number prokaryotic organism is <1 kb from a structured DNA sequence (such as an enhancer, LTR or IRES), replication intermediates will be formed, but when the high copy replication origin is >1.5 kb from a structured DNA sequence, replication intermediates will not be formed. Consistent with this, replication intermediates are formed in all pUC origin-RNA-OUT marker vectors (where the pUC origin is <1 kb from the lentiviral vector LTR (Table 5: 400 bp)) or pUC origin-antibiotic resistance marker vectors (where the pUC origin is <1 kb from the Sleeping Beauty IR / DR (Table 6; 280 bp)). For AAV and mRNA vectors, the original pUC origin-antibiotic selection marker vectors have pUC origins that are 0 bp from ITR (AAV vectors; Table 7) or 170 bp from A99 repeats (mRNA vectors, Table 8), which may produce replication intermediates that are too small to be detected on agarose gels. However, in these cases, the yields were very low, indicating low plasmid copy number due to replication blockage. In contrast, as expected, high plasmid yields were obtained in cases where the distance between the original pUC origin-antibiotic selection marker vector pUC origin and the structural DNA sequence (A60 repeats) was >1.5 kb (Table 8: mRNA vector pGEM4Z T7 A60).

[0343] Williams, Supra, 2017 reported that the yield of pUC origin vectors with PAS-BH extended pUC origin was improved when the pUC origin was >1.5 kb from the homopolymer A64C31 repeat sequence. However, when the directional distance of the PAS-BH extended pUC origin to the A64C31 repeat sequence was <400 bp, the yield was lower (Table 8, see footnotes d and e). This teaches that the addition of the PAS-BH protomer assembly site cannot overcome the directional replication of the poor pUC origin of tightly positioned structured DNA sequences.

[0344] Since the pUC origin itself is 1 kb, there are no constructs for AAV, lentiviral, retroviral or transposon vectors containing a pUC origin that would have a bacterial region <1.1 kb that would not produce replication intermediates as seen above and predicted by Levy, Supra, 2004, and would not have the low plasmid yields reported herein.

[0345] Surprisingly, no replication intermediates were observed in any R6K origin-RNA-OUT antibiotic selection marker-free modified vectors, including those in which the R6K origin was <1 kb from the lentiviral vector LTR (Table 5: 400 bp) or Sleeping Beauty IR / DR (Table 6; <40 bp). In addition, for AAV vectors, despite having a pUC origin with a distance of 0 bp from ITR, the yield of the original pUC origin-antibiotic selection marker vector was very low, while the two R6K origin-RNA-OUT antibiotic selection marker-free modified vectors with a R6K origin with a distance of 40 bp from ITR had much higher yields (Table 7). This improved yield was specific to R6K, not RNA-OUT, as the two AAV pUC-RNA-OUTs with a pUC origin 50 bp away from the ITR had the same poor plasmid yield as the original pUC antibiotic marker vector (Table 7); similarly, direct comparison of the pUC-RNA-OUT and R6K-RNA-OUT modifications (400 bp away from the LTR repeat sequence in the lentiviral backbone) showed that all three pUC-RNA-OUT backbones had replication intermediates, but none of the three R6K-RNA-OUT backbones had replication intermediates (Table 5).

[0346] This surprising improvement in plasmid copy number (plasmid yield) and quality (elimination of replication intermediates) of the R6K origin vector means that the R6K origin can replicate more efficiently through structured DNA sequences than the pUC origin. Although Levy, Supre, 2004 teaches that when any high copy number prokaryotic replication origin is less than 1 kb from a structured DNA sequence (such as an enhancer, LTR or IRES), replication intermediates will be formed, and when the high copy replication origin is >1.5 kb from the structured DNA sequence, replication intermediates will not be formed, the examples provided by Levy, Supre, 2004 are all pUC origin plasmids.

[0347] A fundamental difference between these replication origins is that the pUC starting point is a Pol I-dependent replication origin, while the R6K starting point is a Pol III-dependent replication origin. In the case of the pUC starting point, the RNAII primer forms an RNA:DNAR loop cut by RNase H, thereby creating a primer for the DNA synthesis guided by DNA Pol I in the initial leading strand synthesis process. Then, DNA synthesis is converted from the slow DNA Pol I to the highly processed DNA Pol III (Allen et al., 2011.Nucleic Acids Research 39:7020-33) from 400bp downstream of the starting point to a maximum of 1.3kb. The R6Kgamma replication origin rep protein interacts with dnaB helicase and dnaG priming enzyme to produce a short RNA primer for DNA Pol III replication without the need for DNA Pol I (Abhyankar et al., Supra, 2003). The DNA Pol I replication region of the pUC origin is at most 1.3 kb from the origin, which is very close to the upper limit of replication intermediate formation (1 to 1.5 kb from the origin) defined by Levy, Supra, 2004. We propose that the surprising improvement in structured DNA replication observed when very close to R6K rather than the pUC origin is due to the unexpected improvement of DNA Pol III in the replication of structured DNA sequences compared to DNA Pol I.

[0348] The vector methods and compositions disclosed herein demonstrate that a Pol III-dependent origin of replication (such as the R6K origin) can be used to replicate structured DNA sequences that are poorly replicated by a Pol I-dependent origin of replication (such as the pUC origin).

[0349] These results indicate that the vectors of the present invention can be used to improve the production yield and quality of viral and non-viral vectors.

[0350] Example 4: Improved performance of R6K starting point structured vector

[0351] The vectors of the present invention can also be used to eliminate the transfer of antibiotic resistance marker genes by viral and non-viral vectors; reduce transfection-related toxicity; improve the transposition of non-viral transposon vectors; increase the packaging titer of viral vectors; increase the expression of transgenes encoded by viral and non-viral vectors, etc.

[0352] Compared to vector comparators having a pUC origin with a bacterial backbone of >1.5 kb, for example, the third-generation lentiviral vectors of the present invention having an R6K origin [4 vectors: transfer plasmids in Table 5, gag pol packaging plasmids; env plasmids; REV plasmids having an R6K origin and a bacterial backbone of <1 kb (not shown)] show reduced toxicity and improved viral packaging titers. Lenti-X 293T cell line (Takara Bio Mountain View, CA) was transfected in 24-well plates with third generation lentiviral vectors with R6K origin of bacterial backbone <1 kb or antibiotic selection marker vectors with original pUC origin of bacterial backbone >1.5 kb (control) using Liofectamine 3000 (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's recommendations, resulting in higher titer lentiviral production (control vector with pUC origin of bacterial backbone >1.5 kb: 1.00x±0.32; vector with R6K origin of bacterial backbone <1 kb 1.45x±0.42), measured using Lenti-X p24 Rapid Titer Kit (TakaraBio Mountain View, CA). Lenti-X 293T cell line was transfected with third generation lentiviral vectors (pUC origin of >1.5 kb bacterial backbone (control) or R6K origin of <1 kb bacterial backbone) in 24-well plates using calcium phosphate as described by Marino MP, Luce MJ, Reiser J. 2003, Methods Mol Biol 229: 43-55, resulting in higher titer lentiviral production (pUC origin of >1.5 kb bacterial backbone (control): 1.00x±0.30; R6K origin vector of <1 kb bacterial backbone: 1.32x±0.19), measured using Lenti-X p24 Rapid Titer Kit (Takara Bio Mountain View, CA). It is noteworthy that calcium phosphate transfection of the third-generation lentiviral vector with a pUC origin of a bacterial backbone of >1.5kb resulted in extensive transfection-related toxicity (>80% cell death) in 24-well plate transfection, compared to the third-generation lentiviral vector with a R6K origin of a bacterial backbone of <1kb with low toxicity of the R6K origin. This reduced transfection-related toxicity should result in a significant increase in viral titer in large-scale production transfection. These results indicate that the nanoplasmid vector with a R6K origin of a bacterial backbone of <1kb of the present invention reduces transfection-related toxicity and increases the packaging titer of the viral vector compared to the bacterial backbone vector of >1.5kb.

[0353] Summarize

[0354] Although the above description contains many examples, these should not be construed as limitations on the scope of the disclosure, but rather should be viewed as exemplifications of preferred embodiments thereof. Many other variations are possible.

[0355] For example, in the vectors of the current technology, various directions of the Pol III-dependent replication origin and the RNA selection marker can be used. For example, any of the eight directions of the Pol III-dependent replication origin can be used, and the RNA selection marker in the vectors of the current technology (i.e., ←Pol III replication origin RSM→; ←Pol III replication origin←RSM; PolIII replication origin→RSM→; Pol III replication origin→←RSM; ←RSM Pol III replication origin→; ←RSM←PolIII replication origin; RSM→Pol III replication origin→; RSM→←Pol III replication origin).

[0356] In addition, various RNA selectable markers known in the art can be substituted for RNA-OUT.

[0357] Additionally, antibiotic resistance markers can be substituted for RNA-OUT, for example in cases where it is desirable to simply modify the pUC origin to an R6K origin to improve plasmid yield and / or quality.

[0358] Thus, the reader will see that the improved Pol III-dependent replication origin vectors of the current technology provide a means to reduce transfection-associated toxicity, improve transposition of non-viral transposon vectors, improve packaging titers of viral vectors, improve expression of viral and non-viral vector-encoded genes, and eliminate viral and non-viral vector-mediated transfer of antibiotic selectable marker genes (i.e., by introducing a bacterial region that is preferably less than 1000 bp), while significantly improving production compared to alternative vectors such as pUC plasmids and minicircles.

[0359] Therefore, the scope of the present disclosure should be determined not by the embodiments shown, but by the claims that follow.

[0360]

[0361]

[0362]

[0363]

[0364] .

Claims

1. A method for improving the replication of a covalently closed circular plasmid, comprising the following steps: a. Providing an engineered E. coli cell comprising a covalently closed circular plasmid, the covalently closed circular plasmid comprising: i. Pol III-dependent replication origins, including the R6Kγ replication origin, ii. RNA-OUT RNA selection marker, and iii. an insert comprising a structured DNA sequence selected from the group consisting of an inverted repeat sequence, a direct repeat sequence, or a eukaryotic origin of replication, wherein the structured The DNA sequence is located within 1000 m from the Pol III-dependent replication origin along the replication direction. bp; and b. The engineered E. coli cells are subjected to a fermentation process to replicate the covalently closed circular plasmid, whereby the resulting covalently closed circular plasmid has reduced production of replication intermediates and increased plasmid copy number compared to a covalently closed circular plasmid having a Pol I-dependent replication origin.

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

3. The method of claim 1, wherein the R6Kγ replication origin comprises 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.

4. A method for improving the replication of a covalently closed circular plasmid, comprising the following steps: a. providing an engineered E. coli cell expressing a Rep protein comprising a covalently closed circular plasmid, The covalently closed circular plasmid comprises: i. a backbone comprising a Pol III-dependent R6Kγ replication origin and an RNA-OUT RNA selection marker, and ii. an insert comprising a structured DNA sequence selected from the group consisting of: an SV40 replication origin, a viral LTR, a transposon IR / DR repeat, an AAV ITR, and a transposon ITR, wherein the structured DNA sequence is located less than 1000 bp from the PolIII-dependent replication origin along the direction of replication; and b. The engineered E. coli cells expressing the Rep protein are subjected to a fermentation process to replicate the covalently closed circular plasmid, thereby reducing the production of replication intermediates and increasing the plasmid copy number compared to the covalently closed circular plasmid with a Pol I-dependent replication origin.

5. The method of claim 4, wherein the R6Kγ replication origin comprises 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.

6. The method of claim 4, wherein the RNA-OUT RNA selection marker is an RNA-IN regulating RNA-OUT functional variant comprising a sequence selected from the group consisting of: SEQ ID NO: 5 and SEQ ID NO:

7.

7. The method of claim 4, wherein the RNA-OUT RNA selection marker encodes an RNA-IN regulatory RNA-OUT RNA comprising SEQ ID NO:

6.

8. The method of claim 4, wherein the backbone comprises 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.

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