Viruses and non-viral nanoparticle plasmid vectors with improved production performance

Abstract

This invention provides a method for improving the replication of covalently closed circular plasmids. The method includes providing a covalently closed circular plasmid having a Pol I-dependent origin of replication and an insert comprising a structured DNA sequence selected from: inverted repeat sequences, homoplastic repeat sequences, homopolymeric repeat sequences, eukaryotic origin of replication, or eukaryotic promoter enhancer sequences, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the replication direction. The method further includes modifying a covalently closed circular recombinant molecule such that the Pol I-dependent origin of replication is replaced by a Pol III-dependent origin of replication, thereby improving the replication of the resulting Pol III-dependent origin of replication covalently closed circular plasmid. An antibiotic-free covalently closed circular recombinant DNA molecule is also provided. A recombinant DNA molecule and an associated method are also provided, the recombinant DNA molecule comprising one or more primosome sites and / or selective markers oriented to transcribe away from the structured DNA sequence.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 625,050, filed January 25, 2024, entitled "Viral and Non-viral Nanoplasmid Vectors with Improved Production," the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list that has been submitted electronically in XML format, which is hereby incorporated by full reference. The XML copy was created on January 9, 2025, named 0798038_SL, and has a size of 55,919 bytes.

[0005] Incorporate by reference

[0006] International Patent Application Publication No. WO 2019 / 183248 (PCT / US2019 / 023209), filed on March 20, 2019, is incorporated herein by reference in its entirety. All patents, patent applications and publications mentioned herein are incorporated by reference. Technical Field

[0007] This invention relates to recombinant DNA molecules, i.e. vectors, which can be used in viral and nonviral gene therapy, viral and nonviral cell therapy, and more particularly, for improving the production yield and quality of viral and nonviral vectors, reducing transfection-related toxicity, improving transposition from nonviral transposon vectors, improving the packaging titer of viral vectors, improving the expression of gene encoding viral and nonviral vectors, and for eliminating transfer of antibiotic selection marker genes mediated by viral and nonviral vectors.

[0008] These recombinant DNA molecules can be used in biotechnology, in vitro gene therapy, transgenic organisms, gene therapy, therapeutic vaccination, agriculture, and DNA vaccines. Background Technology

[0009] 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 therapy drugs and DNA vaccines, enter clinical trials and eventually reach the pharmaceutical market. Plasmid DNA vaccines can be used as preventative vaccines for viral, bacterial, or parasitic diseases; immunizing agents for the preparation of high-immunoglobulin products; 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 a patient. Plasmids are also used as in vitro transcription template vectors for mRNA vaccines and therapeutics. 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 integration into the genome. Plasmids are also used in viral vectors for gene therapy or gene replacement applications, where the desired gene product is packaged in transduced viral particles after transfection of production cell lines, and then expressed from the virus in target cells after viral transduction.

[0010] Numerous researchers have confirmed that microcircular vectors are superior to plasmid vectors in the production of AAV vectors (improved transduction unit titers, see Table 1) and transposon vectors (increased transposons, see Table 1). Performance improvements resulting from the improved expression duration of short-skeleton microcircular vectors should also be observed when using short bacterial skeleton plasmid vectors of up to 1.1 kb. In fact, it has been reported that the 1.1 kb bacterial backbone pFAR4 SB transposon vector / SB100x transposase vector combination increases the efficiency of transposing Sleeping Beauty into human cells by 2 times 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).

[0011] However, viral vectors, such as AAV, lentiviruses, and retroviral vectors, as well as transposon vectors, all contain structured DNA sequences at their ends. For example, the Sleeping Beauty transposon vector contains flanked IR / DR sequences, the AAV vector contains flanked ITR sequences, and lentiviruses and retroviral vectors contain flanked LTR sequences.

[0012] The close proximity of the pUC origin to the structured DNA sequence leads to aberrant replication termination, resulting in replication intermediates that unacceptably degrade plasmid quality (Levy J. 2004, US Patent 6709844). Levy taught that replication intermediates form when any high-copy origin of replication is < 1 kb from a structured DNA sequence (such as an enhancer, LTR, or IRES), but not when it is > 1.5 kb. Since the pUC origin itself is 1 kb, no configuration exists that can produce AAV, lentivirus, retrovirus, or transposon vectors containing a pUC origin, a bacterial region < 1.1 kb, and predicted not to produce replication intermediates.

[0013] Lu J, Williams JA, Luke J, Zhang F, Chu K, and Kay MA. 2017. *Human Gene Therapy* 28:125-34 discloses antibiotic-free mini intron plasmid (MIP) AAV vectors and demonstrates that the vector backbone of MIP intron AAV vectors can be removed to produce short backbone AAV vectors. Attempts to create a microloop with a 6 or 10 bp spacer region in a mini intron plasmid AAV vector are toxic (see footnote e in Table 5), likely due to the long palindromic structure resulting from this close juxtaposition of AAV ITRs. While MIP vectors with longer spacer regions (< 1 kb) can be prepared, a drawback of the MIP intron strategy is that it requires cloning replication and selection-encoding introns into eukaryotic regions, which is impossible or undesirable for many vectors.

[0014] The drawback of microcircular strategies for producing short bacterial AAV, lentivirus, retrovirus, or transposon vectors is the high cost and difficulty in scaling up the manufacturing process. For microcircular vectors, *E. coli*-based manufacturing systems have been developed, in which, after plasmid generation, the bacterial and eukaryotic regions are separated and circularized into microcircles (eukaryotic region) and bacterial loops by the action of phage recombinases on recognition sequences within the plasmid. In some methods, restriction enzymes are then used to digest the bacterial loop at specific sites to eliminate this persistent contaminant. These production processes are extremely inefficient. For example, optimal manufacturing of microcircular vectors yields only 5 mg of microcircles per liter of culture (Kay MA, He CY, Chen ZY. 2010. *Nature Biotechnol* 28:1287-1289).

[0015] No method has been reported for high-yield production of pFAR vectors; this system utilizes a repressive tRNA gene carried by a plasmid to complement the TAG amber nonsense mutation in the thyA gene, thereby complementing the thymidine auxotroph and allowing cells to grow on basal medium (Marie et al., see above, 2010).

[0016] A solution is needed to develop mRNA, AAV, lentivirus, retrovirus, or transposon vectors containing short spacer regions, preferably less than 1000 bp, which can be efficiently manufactured without replication intermediates or poor production. In some embodiments, the vector does not encode protein-based selection markers. In other embodiments, the vector is minimized to eliminate all non-essential sequences. Summary of the Invention

[0017] In the embodiments, vectors suitable for viral and nonviral gene therapies are disclosed.

[0018] In the embodiments, vectors suitable for viral and non-viral cell therapies are disclosed.

[0019] In the embodiments, vectors for improving the yield and quality of viral and non-viral vector manufacturing are disclosed.

[0020] In the embodiments, a vector for reducing transfection-related toxicity is disclosed.

[0021] In the embodiments, vectors for improving transposition from non-viral transposon vectors are disclosed.

[0022] In the embodiments, a carrier for improving the packaging titer of a viral vector is disclosed.

[0023] In the embodiments, vectors for improving the expression of viral and non-viral vector-encoded transgenes are disclosed.

[0024] In the embodiments, vectors for eliminating antibiotic resistance marker genes transferred via viral and nonviral vectors are disclosed.

[0025] An improved vector method and composition are disclosed that utilize Pol III-dependent replication origins to replicate structured DNA sequences.

[0026] An improved vector method and composition are disclosed, which utilize Pol III-dependent replication origins to replicate inverted repeat DNA sequences.

[0027] An improved vector method and composition are disclosed, which utilizes Pol III-dependent replication origins to replicate homologous DNA sequences.

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

[0029] An improved vector method and composition are disclosed, which utilizes Pol III-dependent replication origins to replicate enhancer-structured DNA sequences.

[0030] An improved vector method and composition are disclosed, which utilizes Pol III-dependent replication origins to replicate polyadenylated repetitive DNA sequences.

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

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

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

[0034] An improved vector method and composition are disclosed, which utilizes a Pol III-dependent replication origin to replicate viral LTRDNA sequences.

[0035] An improved vector method and composition are disclosed, which utilizes Pol III-dependent replication origins to replicate AAV ITRDNA sequences.

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

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

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

[0039] An improved vector method and composition are disclosed, which utilizes a Pol III-dependent replication origin to replicate CMV enhancer DNA sequences.

[0040] An improved vector method and composition are disclosed, which utilizes a Pol III-dependent replication origin to replicate the SV40 enhancer DNA sequence.

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

[0042] Methods and compositions for using improved lentiviral vectors, lentiviral envelope vectors, and lentiviral packaging vectors that utilize Pol III-dependent replication origins are disclosed.

[0043] Methods and compositions for using improved retroviral vectors, retroviral envelope vectors, and retroviral packaging vectors utilizing Pol III-dependent replication origins are disclosed.

[0044] An improved AAV vector and AAV helper vector method and composition utilizing Pol III-dependent replication origin are disclosed.

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

[0046] An improved method and composition for using nonviral transposons and transposase vectors utilizing Pol III-dependent replication origins are disclosed.

[0047] An improved method and composition for using nonviral Sleeping Beauty transposons and transposase vectors utilizing Pol III-dependent replication origins are disclosed.

[0048] Methods and compositions for using improved nonviral PiggyBac transposons and transposase vectors utilizing Pol III-dependent replication origins are disclosed.

[0049] An improved method and composition for using a nonviral Tol2 transposon and transposase vector utilizing a Pol III-dependent replication origin are disclosed.

[0050] An improved method and composition for nonviral polyadenylated mRNA vectors utilizing Pol III-dependent replication origins are disclosed.

[0051] An improved viral vector production method and composition with an improved viral transduction unit is disclosed, which utilizes the PolIII-dependent replication origin.

[0052] A method and composition for producing improved lentiviral vectors with improved viral transduction units are disclosed, which utilize Pol III-dependent replication origins.

[0053] Methods and compositions for producing improved retroviral vectors with improved viral transduction units are disclosed, which utilize Pol III-dependent origin of replication.

[0054] Methods and compositions for producing improved AAV vectors and AAV helper vectors with improved viral transduction units are disclosed, which utilize Pol III-dependent replication origins.

[0055] Methods and compositions for improving nonviral transposons and transposase vectors with improved transposation are disclosed, which utilize Pol III-dependent origin of replication.

[0056] Methods and compositions for using improved nonviral Sleeping Beauty transposons and transposase vectors with improved transposation are disclosed, which utilize Pol III-dependent replication origins.

[0057] Methods and compositions for using improved nonviral PiggyBac transposons and transposase vectors with improved transposation are disclosed, which utilize Pol III-dependent origin of replication.

[0058] Methods and compositions for using improved nonviral Tol2 transposons and transposase vectors with improved transposation are disclosed, which utilize Pol III-dependent origin of replication.

[0059] An improved viral vector method and composition with improved expression are disclosed, which utilizes the Pol III-dependent replication origin.

[0060] Methods and compositions for improving lentiviral vectors with enhanced expression, utilizing Pol III-dependent replication origins, are disclosed.

[0061] Methods and compositions for improving retroviral vectors with enhanced expression, utilizing Pol III-dependent origin of replication, are disclosed.

[0062] Methods and compositions for improving AAV vectors and AAV helper vectors with improved expression, utilizing PolIII-dependent replication origins, are disclosed.

[0063] Methods and compositions for improving nonviral transposons and transposase vectors with improved expression, utilizing Pol III-dependent origin of replication, are disclosed.

[0064] Methods and compositions for improving nonviral Sleeping Beauty transposons and transposase vectors with improved expression are disclosed, which utilize Pol III-dependent origin of replication.

[0065] Methods and compositions for expressing improved nonviral PiggyBac transposons and transposase vectors with improved expression, utilizing Pol III-dependent origin of replication, are disclosed.

[0066] Methods and compositions for improving nonviral Tol2 transposons and transposase vectors with improved expression, utilizing Pol III-dependent origin of replication, are disclosed.

[0067] An improved viral vector method and composition that eliminates the risk of antibiotic resistance marker gene transfer, utilizing a Pol III-dependent replication origin, are disclosed.

[0068] Improved lentiviral vectors, lentiviral envelope vectors, and lentiviral packaging vector methods and compositions that do not carry the risk of antibiotic resistance marker gene transfer are disclosed, utilizing Pol III-dependent replication origins.

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

[0070] An improved AAV vector and AAV helper vector method and composition that eliminate the risk of antibiotic resistance marker gene transfer are disclosed, utilizing a Pol III-dependent replication origin.

[0071] An improved method and composition for nonviral transposons and transposase vectors without the risk of antibiotic resistance marker gene transfer are disclosed, which utilizes Pol III-dependent replication origins.

[0072] An improved method and composition for nonviral Sleeping Beauty transposons and transposase vectors without the risk of antibiotic resistance marker gene transfer are disclosed, utilizing Pol III-dependent replication origins.

[0073] An improved nonviral PiggyBac transposon and transposase vector method and composition that eliminates the risk of antibiotic resistance marker gene transfer, utilizing a Pol III-dependent origin of replication, is disclosed.

[0074] An improved nonviral Tol2 transposon and transposase vector method and composition that eliminates the risk of antibiotic resistance marker gene transfer, utilizing a Pol III-dependent origin of replication, is disclosed.

[0075] An improved viral vector method and composition with reduced transfection-related toxicity are disclosed, which utilizes the PolIII-dependent replication origin.

[0076] An improved lentiviral vector method and composition with reduced transfection-related toxicity are disclosed, which utilizes the PolIII-dependent replication origin.

[0077] An improved retroviral vector method and composition with reduced transfection-related toxicity is disclosed, which utilizes the Pol III-dependent origin of replication.

[0078] An improved AAV vector and AAV helper vector method and composition with reduced transfection-related toxicity are disclosed, which utilize Pol III-dependent replication origin.

[0079] Improved nonviral transposon and transposase vector methods and compositions with reduced transfection-related toxicity are disclosed, which utilize Pol III-dependent replication origins.

[0080] An improved method and composition for nonviral Sleeping Beauty transposons and transposase vectors with reduced transfection-related toxicity are disclosed, which utilize Pol III-dependent replication origins.

[0081] An improved nonviral PiggyBac transposon and transposase vector method and composition with reduced transfection-related toxicity is disclosed, which utilizes the Pol III-dependent origin of replication.

[0082] An improved nonviral Tol2 transposon and transposase vector method and composition with reduced transfection-related toxicity is disclosed, which utilizes the Pol III-dependent origin of replication.

[0083] An improved vector method and composition are disclosed, having one or more primosome assembly sites downstream of the origin of replication, particularly the Pol III-dependent origin of replication, and including a structured DNA sequence insert.

[0084] An improved vector method and composition are disclosed, having an RNA-selective marker oriented to be transcribed in reverse of a structured DNA sequence, and comprising a structured DNA sequence insert.

[0085] By way of example and not limitation, each of the improvements described above and below is relative to implementation under similar or identical conditions, but using different recombinant DNA molecules that do not include Pol-III-dependent replication origins and / or one or more primosome assembly sites and / or markers, such as RNA-selective markers, which are directed to transcription in the reverse direction of the structured DNA sequence.

[0086] In the embodiments, the disclosed vector provides improved viral and non-viral vector manufacturing yields.

[0087] In the embodiments, the disclosed vectors provide improved manufacturing quality for both viral and non-viral vectors.

[0088] In the embodiments, the disclosed vector provides a viral vector with improved packaging titer.

[0089] In the embodiments, the disclosed vector provides a nonviral transposon vector with improved transposons.

[0090] In the embodiments, the disclosed vectors provide viral and nonviral vectors with improved encoding transgenic expression.

[0091] In the embodiments, the disclosed vectors provide viral and non-viral vectors that eliminate the transfer of antibiotic resistance marker genes.

[0092] In the embodiments, the disclosed vectors provide viral and non-viral vectors with reduced transfection-related toxicity.

[0093] In one embodiment, a covalently closed circular recombinant DNA molecule is provided, which may include a backbone and an insert, wherein the backbone may include a Pol III-dependent origin of replication, a selectable marker, and a first priming assembly site, wherein the first priming assembly site is located downstream of the Pol III-dependent origin of replication in the replication direction, and wherein the insert contains a structured DNA sequence.

[0094] In another embodiment, an antibiotic-free, covalently closed circular recombinant DNA molecule is provided, which may include a backbone and an insert, wherein the backbone may include a replication origin and an RNA-selective marker, wherein the insert may include a structured DNA sequence, and wherein the RNA-selective marker is transcribed in a direction opposite to the structured DNA sequence. Alternatively, the RNA-selective marker may be replaced by an antibiotic-selective marker, and the recombinant DNA molecule may be a covalently closed circular recombinant DNA molecule, wherein the antibiotic-selective marker is transcribed in a direction opposite to the structured DNA sequence. It should be understood that "opposite" can refer to a direction away from the structured DNA sequence approaching either end of the insert.

[0095] In any of the foregoing embodiments, the structured DNA sequence may be less than 1000 bp from the origin of replication. In any of the foregoing embodiments, the backbone may be less than 1000 bp. It should be understood that in any of the foregoing embodiments, the backbone and the insert may be operatively linked such that there are no intercalation sequences between the backbone and the insert. It should also be noted that in any embodiment of this disclosure, the component may be located on either strand of a covalently closed circular recombinant DNA molecule. By way of example and not limitation, the first primer assembly site may be on one strand, while the second primer assembly site may be on the opposite strand. For example, SEQ ID NO: 29 encodes two primer assembly sites, but the provided sequence has one in the sense direction and one in the antisense direction. It should also be understood that in some embodiments, the backbone may be the bacterial replication selection region of this disclosure.

[0096] In any of the foregoing embodiments, the origin of replication can be a Pol III-dependent origin of replication, such as, but not limited to, an R6K origin, a ColE2 origin, or a ColE2-related origin. In any of the foregoing embodiments, the Pol III-dependent origin of replication does not require Pol 1. In any of the foregoing embodiments, the Pol III-dependent origin of replication can be an R6K origin of replication, such as, but not limited to, an R6K γ origin. In any of the foregoing embodiments, the R6K origin of replication can be a sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24. In any of the foregoing embodiments, the Pol III dependency start point can be an R6K start point, which is a 6-iteron R6K γ start point or a 7-iteron R6K γ start point (e.g., but not limited to SEQ ID NO: 18) or has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with it. It should be understood that in some embodiments, the repeat sequence of the repeater can be selected from any of SEQ ID NO: 19-23.

[0097] In any of the foregoing embodiments, the selective marker may be an RNA selective marker. Alternatively, in embodiments where the selective marker is not an RNA selective marker, the selective marker may be an antibiotic selective marker. In any of the foregoing embodiments, the RNA selective marker may be an RNA-OUT RNA selective marker. In any of the foregoing embodiments, the RNA-OUT RNA selective marker may be an RNA-IN regulatory RNA-OUT functional variant having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5 or SEQ ID NO: 7. In other embodiments, the RNA selective marker may include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 6.

[0098] In any of the foregoing embodiments, the first primor assembly site may include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33. In any of the foregoing embodiments, the backbone may further include a second primor assembly site located downstream of the replication origin in the replication direction. In any of the foregoing embodiments, the second primor assembly site may include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33. In any of the foregoing embodiments, the backbone may include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29, such that the first and second primor assembly sites are located downstream of the replication origin in the replication direction.

[0099] In some embodiments, the recombinant DNA molecule may be antibiotic-free.

[0100] In any of the foregoing embodiments, the skeleton may include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with a sequence selected from 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.

[0101] In any of the foregoing embodiments, the structured DNA sequence can be any structured DNA sequence described in the embodiments of this disclosure. By way of example and not limitation, the structured DNA sequence can be selected from inverted repeat sequences, homoduplex repeat sequences, eukaryotic origin of replication, and eukaryotic promoter-enhancer sequences.

[0102] In any of the foregoing embodiments, the insert can be any type of vector described in this disclosure. By way of example and not limitation, the insert can be a transposon vector, a transposase vector, an mRNA vector, an AAV vector, or a lentiviral vector.

[0103] In any of the foregoing embodiments, when the insert is a transposon vector, the structured DNA sequence can be an inverted repeat sequence, a homologous repeat sequence, or a eukaryotic promoter-enhancer sequence.

[0104] In any of the foregoing embodiments, when the insert is an AAV vector, the structured DNA sequence may be an inverted repeat sequence. In any of the foregoing embodiments, when the insert is an AAV vector, the AAV vector may encode an AAV ITR. By way of example and not limitation, the AAV vector may include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 35 (5' inverted terminal repeat sequence) and a sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 36 (3' inverted terminal repeat sequence). In some embodiments, the insert may include one or more inverted repeat sequences of an AAV ITR. In some embodiments, the recombinant DNA molecule is a non-viral vector containing an AAV ITR.

[0105] In any of the foregoing embodiments, when the insert is a lentiviral vector, the structured DNA sequence may be a homologous repeat sequence or a eukaryotic origin of replication.

[0106] In any of the foregoing embodiments, the structured DNA sequence may be selected from: homopolymeric repeat sequences, such as polyadenylated repeat sequences; SV40 origin of replication; viral LTR; lentiviral LTR; retroviral LTR; transposon IR / DR repeat sequences; Sleeping Beauty transposon IR / DR repeat sequences; AAV ITR; CMV enhancer; and SV40 enhancer.

[0107] In any of the foregoing embodiments, where the structured DNA sequence includes a homopolymeric sequence, the homopolymeric sequence may be a polyadenylated repeat sequence. In any of the foregoing embodiments, the homopolymeric sequence, such as, but not limited to, a polyadenylated repeat sequence, may include about 3 to about 500 residues or more. In some embodiments, the polyadenylated repeat sequence may include the sequence of any one of SEQ ID NO: 37-39. In some embodiments, the recombinant DNA molecule may be an mRNA vector including one or more homopolymeric sequences.

[0108] In any of the foregoing embodiments, the selective marker may be an RNA selective marker that is transcribed in the reverse direction of the structured DNA sequence.

[0109] In any of the foregoing embodiments, the recombinant DNA molecule is selected from viral vectors, lentiviral vectors, retroviral vectors, AAV vectors, Ad vectors, nonviral transposon vectors, Sleeping Beauty transposon vectors, PiggyBac transposon vectors, Tol2 transposon vectors, and polyadenylated mRNA vectors.

[0110] It should be understood that the recombinant DNA molecules of the foregoing embodiments or any embodiment of this disclosure can be replicated by providing cells containing the recombinant DNA molecules and subjecting the cells to fermentation, wherein the cells and fermentation process are any of the embodiments described herein.

[0111] It should also be understood that existing recombinant DNA molecules can be prepared by replacing the origin of replication and / or selective markers to obtain the recombinant DNA molecule of any of the embodiments of this disclosure and having its components.

[0112] In any of the foregoing embodiments, if the replication origin is a Pol I-dependent replication origin, it can be a pUC origin, a pMB1 origin, or a ColE1 origin.

[0113] In any of the foregoing embodiments, within their applicable scope, the primosome assembly site, the selectivity marker, and the origin of replication can be in any order relative to the structured DNA sequence and within the backbone. By way of example, and not limitation, the primosome assembly site can be between the origin of replication and the selectivity marker, or at either end of the origin of replication and the selectivity marker. Similarly, the origin of replication and the selectivity marker can be in any order relative to the structured DNA sequence or the insert. By way of another example, and not limitation, the following configurations represent some embodiments of transcription with RNA selectivity markers in a direction opposite to the structured DNA sequence:

[0114] 1) Structured DNA sequence RNA-OUT > origin >

[0115] 2) Structured DNA sequence RNA-OUT > origin > PAS

[0116] 3) Structured DNA sequence origin > RNA-OUT >

[0117] 4) Structured DNA sequence origin > PAS RNA-OUT >

[0118] In any of the foregoing embodiments, the R6K starting point may include multiple repeaters, such as, but not limited to, 6 or 7 repeaters.

[0119] It should be understood that known equivalents and alternatives may be used in any embodiment of this disclosure, and such modifications will not depart from the spirit of this disclosure.

[0120] In one embodiment, the present invention provides a method for improving the replication of covalently closed circular plasmids, comprising the steps of: a) providing a covalently closed circular plasmid comprising: i) a Pol I-dependent origin of replication, and ii) an insert comprising a structured DNA sequence selected from inverted repeat sequences, homoplastic repeat sequences, homopolymeric repeat sequences, eukaryotic origins of replication, and eukaryotic promoter enhancer sequences, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the replication direction; b) modifying the covalently closed circular recombinant molecule such that a) the Pol I-dependent origin of replication is replaced by a Pol III-dependent origin of replication, thereby improving the replication of the resulting Pol III-dependent origin of replication covalently closed circular plasmid. In another embodiment, the Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMB1 origin, and ColE1 origin. In another embodiment, the Pol III-dependent origin of replication is an R6K γ origin of replication. In another embodiment, the Pol III-dependent origin of replication is an R6Kγ origin of replication having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24. In another embodiment, the structured DNA sequence is selected from polyadenylated repeat sequences, SV40 origin of replication, viral LTRs, lentiviral LTRs, retroviral LTRs, transposon IR / DR repeat sequences, Sleeping Beauty transposon IR / DR repeat sequences, AAV ITRs, CMV enhancers, and SV40 enhancers. In another embodiment, the improved replication is selected from reduced replication intermediate production and increased plasmid copy number.

[0121] In another embodiment, the present invention provides a method for improving the replication of covalently closed circular plasmids, comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a bacterial replication selector region comprising a Pol I-dependent origin of replication and an antibiotic-selective marker, and ii) an insert comprising a structured DNA sequence selected from inverted repeat sequences, homoplastic repeat sequences, homopolymeric repeat sequences, eukaryotic origin of replication, and eukaryotic promoter enhancer sequences, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the replication direction; b) modifying the covalently closed circular recombinant molecule of a) such that the antibiotic-selective marker is replaced by an RNA-selective marker, and the Pol I-dependent origin of replication is replaced by a Pol III-dependent origin of replication, thereby improving the replication of the resulting Pol III-dependent origin of replication covalently closed circular plasmid. In another embodiment, the Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMB1 origin, and ColE1 origin. In another embodiment, the Pol III-dependent origin of replication is an R6K γ origin of replication. In another embodiment, the Pol III-dependent origin of replication is an R6K γ origin of replication having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24. In another embodiment, the RNA-selectivity marker is an RNA-IN regulatory RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the RNA-selectivity marker is an RNA-OUT RNA-selectivity marker encoding an RNA-IN regulatory RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6.In another embodiment, the bacterial replication selector region containing the Pol I-dependent origin of replication and the antibiotic-selective marker is replaced with a Pol III-dependent R6K origin-RNA-OUT RNA-selective marker bacterial replication selector region having at least 95% sequence identity with sequences selected from the following: 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28. In another embodiment, the structured DNA sequence is selected from polyadenylated repeat sequences, SV40 origin of replication, viral LTRs, lentiviral LTRs, retroviral LTRs, transposon IR / DR repeat sequences, Sleeping Beauty transposon IR / DR repeat sequences, AAVITR, CMV enhancers, and SV40 enhancers. In another embodiment, the improved replication is selected from reducing the generation of replication intermediates and increasing the plasmid copy number.

[0122] In one embodiment, the present invention provides an antibiotic-free covalently closed circular recombinant DNA molecule comprising: a) an antibiotic-free insert comprising a structured DNA sequence selected from inverted repeat sequences, homopolymeric repeat sequences, eukaryotic origin of replication, and eukaryotic promoter enhancer sequences; b) a Pol III-dependent origin of replication comprising an R6Kγ origin of replication having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24; and c) an RNA-OUT RNA-selective marker comprising an RNA-IN regulatory RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the R6K γ origin of replication and the RNA-OUT RNA selectivity marker comprise an R6K origin-RNA-OUT RNA selectivity marker bacterial replication select region having at least 95% sequence identity with sequences selected from the following: 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28. In another embodiment, the structured DNA sequence is selected from polyadenylated repeat sequences, SV40 origin of replication, viral LTRs, lentiviral LTRs, retroviral LTRs, transposon IR / DR repeat sequences, Sleeping Beauty transposon IR / DR repeat sequences, AAV ITRs, CMV enhancers, and SV40 enhancers. In another embodiment, the recombinant DNA molecule is selected from viral vectors, lentiviral vectors, retroviral vectors, AAV vectors, Ad vectors, nonviral transposon vectors, Sleeping Beauty transposon vectors, PiggyBac transposon vectors, Tol2 transposon vectors, and polyadenylated mRNA vectors.

[0123] In one embodiment, the present invention provides a method for improving the production of AAV vector viral transduction units from a covalently closed circular plasmid, comprising the steps of: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication selector region comprising a Pol I-dependent origin of replication and an antibiotic-selective marker, and ii) an insert comprising a eukaryotic region selected from AAV vectors, AAV repcap vectors, Ad helper vectors, and Ad helper repcap vectors; b) modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication selector region comprising a Pol I-dependent origin of replication and an antibiotic-selective marker is replaced with a Pol III-dependent R6K origin-RNA-OUT RNA-selective marker bacterial replication selector region less than 1 kb, thereby resulting in a Pol III-dependent origin of replication covalently closed circular plasmid exhibiting improved AAV viral transduction unit production upon transfection into mammalian cells. In another embodiment, the Pol I-dependent origin of replication 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 an R6K γ replication origin having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24. In another embodiment, the RNA-OUT RNA-selectivity marker is an RNA-IN regulatory RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the RNA-OUT RNA-selectivity marker is an RNA-OUT RNA-selectivity marker encoding an RNA-IN regulatory RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6.In another embodiment, the less than 1 kb Pol III-dependent R6K origin-RNA-OUT RNA selectively marks the bacterial replication select region with at least 95% sequence identity to a sequence selected from the following: 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0124] In one embodiment, the present invention provides a method for improving the production of retroviral or lentiviral vector viral transduction units from covalently closed circular plasmids, comprising the steps of: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication selector region comprising a Pol I-dependent origin of replication and an antibiotic-selective marker, and ii) an insert comprising a eukaryotic region selected from retroviral vectors, lentiviral vectors, retroviral envelope vectors, lentiviral envelope vectors, retroviral packaging vectors, and lentiviral packaging vectors; and b) modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication selector region comprising a Pol I-dependent origin of replication and an antibiotic-selective marker is replaced with a Pol III-dependent R6K origin-RNA-OUT RNA-selective marker bacterial replication selector region of less than 1 kb, thereby resulting in a Pol III-dependent origin of replication covalently closed circular plasmid exhibiting improved viral transduction unit production when transfected into mammalian cells. In another embodiment, the Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMB1 origin, and ColE1 origin. In another embodiment, the Pol III-dependent R6K origin of replication is an R6K γ origin of replication having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24. In another embodiment, the RNA-OUT RNA-selectivity marker is an RNA-IN regulatory RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the RNA-OUT RNA-selectivity marker is an RNA-OUT RNA-selectivity marker encoding an RNA-IN regulatory RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6.In another embodiment, the less than 1 kb Pol III-dependent R6K origin-RNA-OUT RNA selectively marks the bacterial replication select region with at least 95% sequence identity to a sequence selected from the following: 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0125] In one embodiment, a method for improving transposition from a covalently closed circular nonviral transposon plasmid is disclosed, comprising the steps of: a) providing a covalently closed circular plasmid comprising i) a 1 kb or larger bacterial replication selector region comprising a Pol I-dependent origin of replication and an antibiotic-selective marker, and ii) an insert comprising a nonviral eukaryotic region selected from transposon vectors, Sleeping Beauty transposon vectors, Sleeping Beauty transposase vectors, PiggyBac transposon vectors, PiggyBac transposase vectors, Tol2 transposon vectors, and Tol2 transposase vectors; b) modifying the covalently closed circular recombinant molecule of (a) such that the 1 kb or larger bacterial replication selector region comprising a Pol I-dependent origin of replication and an antibiotic-selective marker is replaced with a Pol III-dependent R6K origin-RNA-OUT RNA-selective marker bacterial replication selector region less than 1 kb, thereby resulting in a Pol III-dependent origin of replication covalently closed circular plasmid exhibiting improved transposition upon transfection into mammalian cells. In another embodiment, the Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMB1 origin, or ColE1 origin. In another embodiment, the Pol III-dependent R6K origin of replication is an R6K γ origin of replication having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24. In another embodiment, the RNA-OUT RNA-selectivity marker is an RNA-IN regulatory RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the RNA-OUT RNA-selectivity marker is an RNA-OUT RNA-selectivity marker encoding an RNA-IN regulatory RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6.In another embodiment, the less than 1 kb Pol III-dependent R6K origin-RNA-OUT RNA selectively marks the bacterial replication select region with at least 95% sequence identity to a sequence selected from the following: 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0126] In one embodiment, the present invention provides a method for improving expression from a covalently closed circular viral vector or a non-viral transposon plasmid, comprising the steps of: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication selector region comprising a Pol I-dependent origin of replication and an antibiotic-selective marker, and ii) an insert comprising a eukaryotic region selected from lentiviral vectors, retroviral vectors, and AAV vectors or non-viral transposon vectors; and b) modifying the covalently closed circular recombinant molecule of (a) such that the 1 kb or larger bacterial replication selector region comprising a Pol I-dependent origin of replication and an antibiotic-selective marker is replaced with a Pol III-dependent R6K origin-RNA-OUT RNA-selective marker bacterial replication selector region less than 1 kb, thereby resulting in a Pol III-dependent origin of replication covalently closed circular plasmid exhibiting improved expression when transfected into mammalian cells. In another embodiment, the Pol I-dependent origin of replication 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 an R6K γ replication origin having at least 95% sequence identity with a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, and SEQ ID NO:24. In another embodiment, the RNA-OUT RNA-selectivity marker is an RNA-IN regulatory RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from SEQ ID NO:5 and SEQ ID NO:7. In another embodiment, the RNA-OUT RNA-selectivity marker is an RNA-OUT RNA-selectivity marker encoding an RNA-IN regulatory RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO:6. In another embodiment, the less than 1 kb Pol III-dependent R6K origin-RNA-OUT RNA selectively marks the bacterial replication select region with at least 95% sequence identity to a sequence selected from the following: 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0127] In one embodiment, the present invention provides a method for eliminating antibiotic resistance marker genes transferred from a covalently closed circular viral vector plasmid, comprising the steps of: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication selectivity region comprising a Pol I-dependent origin of replication and an antibiotic resistance marker, and ii) an insert comprising an antibiotic resistance marker-free eukaryotic region selected from viral vectors, lentiviral vectors, lentiviral packaging vectors, lentiviral envelope vectors, retroviral vectors, retroviral envelope vectors, retroviral packaging vectors, AAV vectors, AAV repcap vectors, Ad helper vectors, and Ad helper repcap vectors; and b) modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication selectivity region comprising a Pol I-dependent origin of replication and an antibiotic resistance marker is replaced with a Pol III-dependent R6K origin-RNA-OUT RNA-selective bacterial replication selectivity region less than 1 kb, thereby obtaining a Pol I-dependent origin of replication and an antibiotic resistance marker-free eukaryotic region. The Pol I-dependent origin of replication covalently closed circular plasmid does not possess an antibiotic resistance marker that can be packaged into lentivirus, retrovirus, or AAV transduced viral particles when transfected into mammalian cells. In another embodiment, the Pol I-dependent origin of replication is selected from the group consisting of pUC origin, pMB1 origin, and ColE1 origin. In another embodiment, the Pol III-dependent R6K origin of replication is an R6K γ origin of replication with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24. In another embodiment, the RNA-OUT RNA selectivity marker is an RNA-IN regulatory RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the RNA-OUT RNA-selective marker is an RNA-OUT RNA-selective marker that encodes an RNA-IN regulatory RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6.In another embodiment, the less than 1 kb Pol III-dependent R6K origin-RNA-OUT RNA selectively marks the bacterial replication select region with at least 95% sequence identity to a sequence selected from the following: 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0128] In one embodiment, the present invention provides a method for eliminating antibiotic resistance marker genes transferred from a covalently closed circular nonviral transposon plasmid, comprising the steps of: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication selector region comprising a Pol I-dependent origin of replication and an antibiotic resistance marker, and ii) an insert comprising an antibiotic resistance marker-free eukaryotic region selected from nonviral transposon vectors, nonviral transposase vectors, Sleeping Beauty transposon vectors, Sleeping Beauty transposase vectors, PiggyBac transposon vectors, PiggyBac transposase vectors, Tol2 transposon vectors, and Tol2 transposase vectors; and b) modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication selector region comprising a Pol I-dependent origin of replication and an antibiotic resistance marker is replaced with a Pol III-dependent R6K origin-RNA-OUTRNA selectively marker-bearing bacterial replication selector region less than 1 kb, thereby obtaining a Pol I-dependent origin of replication and an antibiotic resistance marker-bearing gene. The Pol I-dependent origin of replication covalently closed circular plasmid does not possess an antibiotic resistance marker that can be transposable into the genome when transfected into mammalian cells. In another embodiment, the Pol I-dependent origin of replication is selected from the group consisting of pUC origin, pMB1 origin, and ColE1 origin. In another embodiment, the Pol III-dependent R6K origin of replication is an R6K γ origin of replication having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24. In another embodiment, the RNA-OUT RNA-selectivity marker is an RNA-IN regulatory RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the RNA-OUT RNA-selectivity marker is an RNA-OUT RNA-selectivity marker encoding an RNA-IN regulatory RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6.In another embodiment, the less than 1 kb Pol III-dependent R6K origin-RNA-OUT RNA selectively marks the bacterial replication select region with at least 95% sequence identity to a sequence selected from the following: 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0129] In one embodiment, the present invention provides an antibiotic-free covalently closed circular recombinant DNA molecule comprising: a) an antibiotic-free insert comprising a eukaryotic region selected from lentiviral vectors, lentiviral envelope vectors, lentiviral packaging vectors, retroviral vectors, retroviral envelope vectors, retroviral packaging vectors, AAV vectors, AAV repcap vectors, Ad helper vectors, Ad helper repcap vectors, nonviral transposon vectors, and nonviral transposase vectors; b) a Pol III-dependent origin of replication comprising an R6Kγ origin of replication having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24; and c) an RNA-OUT RNA-selective marker comprising an RNA-IN regulatory RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7. In another embodiment, the R6K γ origin of replication and the RNA-OUT RNA selectivity marker comprise an R6K origin-RNA-OUT RNA selectivity marker bacterial replication select region having at least 95% sequence identity with sequences selected from the following: 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0130] In one embodiment, the present invention provides a method for reducing transfection-related toxicity of covalently closed circular viral vectors or non-viral transposon plasmids, comprising: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication selector region comprising a Pol I-dependent origin of replication and an antibiotic-selective marker, and ii) an insert comprising a eukaryotic region selected from lentiviral vectors, retroviral vectors, AAV vectors, and non-viral transposon vectors; and modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication selector region comprising a Pol I-dependent origin of replication and an antibiotic-selective marker is replaced with a Pol III-dependent R6K origin-RNA-OUT RNA-selective marker bacterial replication selector region less than 1 kb, thereby resulting in a Pol III-dependent origin of replication covalently closed circular plasmid exhibiting reduced toxicity when transfected into mammalian cells via a transfection-related pathway. In another embodiment, the Pol I-dependent origin of replication 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 an R6K γ replication origin having at least 95% sequence identity with a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, and SEQ ID NO:24. In another embodiment, the RNA-OUT RNA-selectivity marker is an RNA-IN regulatory RNA-OUT functional variant having at least 95% sequence identity with a sequence selected from SEQ ID NO:5 and SEQ ID NO:7. In another embodiment, the RNA-OUT RNA-selectivity marker is an RNA-OUT RNA-selectivity marker encoding an RNA-IN regulatory RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO:6. In another embodiment, the less than 1 kb Pol III-dependent R6K origin-RNA-OUT RNA selectively marks the bacterial replication select region with at least 95% sequence identity to a sequence selected from the following: 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0131] The resulting Pol III-dependent origin of replication plasmids exhibited surprisingly improved manufacturing quality and yield compared to origin of replication expression plasmid vectors derived from parental pMB1, ColE1, or pBR322.

[0132] Other objects and advantages of the invention will become apparent from consideration of the accompanying drawings and the following description. Attached Figure Description

[0133] Figure 1A-1F The starting point of R6K is depicted ( Figure 1A , 1E and 1F), RNA-OUT selective markers ( Figure 1B ) and 14 and 3CpG R6K-RNA-OUT bacterial skeleton ( Figure 1C and 1D );

[0134] Figure 2A-2B The Sleeping Beauty transposon vector, which describes the Pol I-dependent pUC initiation point, is described. Figure 2A ) and Sleeping Beauty transposon vectors with Pol III-dependent R6K initiation ( Figure 2B );

[0135] Figures 3A-3C AAV vectors that depict the Pol I-dependent pUC initiation point ( Figure 3A and 3B ) and AAV vectors with Pol III-dependent R6K initiation ( Figure 3C );as well as

[0136] Figures 4A-4F The A60 polyadenylated repeat sequence encoding the mRNA vector depicted by the Pol I-dependent pUC initiation point is described. Figures 4A-4B ), Pol III-dependent R6K origin A60 polyadenylated repeat sequence encoding mRNA vector ( Figure 4C ), Pol I-dependent pUC initiation A99 polyadenylated repeat sequence encoding mRNA vector ( Figure 4D-4E ) and the A99 polyadenylated repeat sequence encoding the Pol III-dependent R6K origin mRNA vector ( Figure 4F ).

[0137] Figures 5A-5C The R6K-RNA-OUT bacterial skeletons from SEQ ID NO: 25 (A), SEQ ID NO: 27 (B), SEQ ID NO: 28 (C), and SEQ ID NO: 34 were depicted.

[0138] Figures 6A-6BDepicts an AAV vector encoding AAV ITRs of the Pol III-dependent R6K origin ( Figure 6A ), an mRNA vector encoding the A100 polyadenylate repeat sequence ( Figure 6B ).

[0139] Figures 7A-7B Is a BspQ1 linearization map of purified plasmid DNA from the following fermentation harvests: mRNA vector - NP (polyadenylate 100 <ROUT R6K origin>) and mRNA vector - NP 7 repeaters PAS R6K> ROUT> (polyadenylate 100R6K origin>PAS ROUT>).

[0140] Figure 8 Depicts Table 1: Microcircular applications in combination with various viral and non-viral vector platforms.

[0141] Figure 9 Depicts Table 2: R6K origin - RNA - OUT selection marker vector flanked by the pNTC multiple cloning site.

[0142] Figure 10 Depicts Table 3: Lentiviral vector of the SV40 origin: Shake flask production yield / quality of pUC and R6K origin.

[0143] Figure 11 Depicts Table 4: Sleeping Beauty transposon vector: Shake flask production yield / quality of pUC and R6K origin.

[0144] Figure 12 Depicts Table 5: AAV vector: Shake flask production yield / quality of pUC and R6K origin.

[0145] Figure 13 Depicts Table 6: mRNA vector: DH5α HyperGRO fermentation yield / quality of pUC and R6K origin.

[0146] Figure 14 Depicts Table 7: AAV helper vector: Plasmid production yield / quality of pUC and R6K origin.

[0147] Figure 15 Depicts Table 8: AAV vector: Shake flask production yield / quality of R6K origin and R6K origin + primosome assembly site.

[0148] Figure 16 Depicts Table 9: AAV vector: Shake flask production yield / quality of R6K origin and R6K origin + primosome assembly site.

[0149] Figure 17Table 10 illustrates the yield / quality of HyperGRO fermentation with and without the AAV ITR vector and R6K initiation with and without PAS.

[0150] Figure 18 Table 11 illustrates the HyperGRO fermentation yield / quality of mRNA polyadenylation vectors with and without PAS R6K origins.

[0151] Description of Serial ID Number

[0152] SEQ ID NO:1: R6K γ origin

[0153] SEQ ID NO:2:1 CpGR6K γ origin

[0154] SEQ ID NO:3: CpG-free R6K gamma origin

[0155] SEQ ID NO:4: Extended R6K γ origin

[0156] SEQ ID NO:5: RNA-OUT selective marker

[0157] SEQ ID NO:6: RNA-OUT antisense repressor RNA

[0158] SEQ ID NO:7:2 CpG RNA-OUT selective labeling

[0159] SEQ ID NO:8: R6K γ origin-RNA-OUT bacterial region with NheI and KpnI restriction sites laterally attached

[0160] SEQ ID NO:9: A bacterial region with 1 CpGR6K γ origin-2 CpGRNA-OUT sites laterally attached to NheI and KpnI restriction sites.

[0161] SEQ ID NO:10: pNTC-NP1 multi-adaptor trpA R6K-RNA-OUT multi-adaptor cloning cassette: EcoRI / HindIII

[0162] SEQ ID NO:11: pNTC-NP2 multi-adaptor trpA R6K-RNA-OUT multi-adaptor cloning cassette: EcoRI / HindIII

[0163] SEQ ID NO:12: pNTC-NP3 multi-adaptor trpA R6K-RNA-OUT multi-adaptor cloning cassette: EcoRI / HindIII

[0164] SEQ ID NO:13: pNTC-NP4 multi-adaptor trpA R6K-RNA-OUT multi-adaptor cloning kit: EcoRI / HindIII

[0165] SEQ ID NO:14: pNTC-NP5 multi-adaptor trpA R6K-RNA-OUT multi-adaptor cloning cassette: KasI / HindIII

[0166] SEQ ID NO:15: pNTC-NP6 multi-adaptor trpA R6K-RNA-OUT multi-adaptor cloning cassette: EcoRI / SacI

[0167] SEQ ID NO:16: pNTC-NP7 multi-adaptor trpA R6K-RNA-OUT multi-adaptor cloning kit: BssHII-BssHII

[0168] SEQ ID NO:17: pNTC-3xCpG NP1 multi-adaptor R6K-RNA-OUT multi-adaptor cloning cassette: HindIII-EcoRI

[0169] SEQ ID NO:18: R6K γ origin (7 repeaters)

[0170] SEQ ID NO:19: 22 bp repeat sub-repetition sequence starting at R6K γ

[0171] SEQ ID NO:20: 22 bp repeat sub-repetition sequence starting at R6K γ

[0172] SEQ ID NO:21: 22 bp repeat sub-repetition sequence starting at R6K γ

[0173] SEQ ID NO: 22: 22 bp repeat sub-repetition sequence starting at R6K γ

[0174] SEQ ID NO:23: 22 bp repeat sub-repetition sequence starting at R6K γ

[0175] SEQ ID NO:24:1 CpGR6K γ origin (7 repeaters)

[0176] SEQ ID NO:25: R6K γ origin (7 repeats) - RNA-OUT bacterial region

[0177] SEQ ID NO:26: 1 CpGR6K γ origin (7 repeaters) - 2 CpGRNA-OUT bacterial region

[0178] SEQ ID NO:27: NP 7 repeat + PAS = R6K γ origin (7 repeats) + PAS-RNA-OUT bacterial region

[0179] SEQ ID NO:28: NP 7 repeat PAS R6K>ROUT>= R6K γ origin (7 repeats) + PAS-RNA-OUT bacterial region

[0180] SEQ ID NO:29: PAS

[0181] SEQ ID NO:30:PAS-BH

[0182] SEQ ID NO:31:PAS-BL

[0183] SEQ ID NO:32: TrpA terminator

[0184] SEQ ID NO:33: R6K plasmid lacks CpG ssiA primer assembly site

[0185] SEQ ID NO:34: NP 7 repeat + R6K PAS RNA-OUT bacterial region

[0186] SEQ ID NO:35: AAV2 ITR

[0187] SEQ ID NO:36: AAV2 ITR

[0188] SEQ ID NO:37: 80 bp polyadenylate repeat sequence

[0189] SEQ ID NO:38: 100 bp polyadenylate repeat sequence

[0190] SEQ ID NO:39: 120 bp polyadenylate repeat sequence

[0191] Definition of terminology

[0192] AAV vector: Adeno-associated virus vector, a type of augmentative viral vector. It includes self-complementary (sc) adeno-associated virus vector (scAAV) and single-stranded (ss) adeno-associated virus vector (ssAAV).

[0193] AF: Antibiotic-free

[0194] AMP: Ampicillin

[0195] ampR: Ampicillin resistance gene

[0196] Antibiotic-selective markers: Genes that confer resistance to antibiotics, such as ampicillin resistance genes, kanamycin resistance genes, chloramphenicol resistance genes, and tetracycline resistance genes.

[0197] Approximately: As used herein, when applied to one or more values ​​of interest, the term “approximately” or “about” refers to a value that is the same as or similar to the stated reference value.

[0198] Bacterial region: The region in the bacterial host where the desired plasmid vector is propagated and selected.

[0199] bp: base pairs

[0200] ccc: covalently closed ring

[0201] cI: λ repressor

[0202] cITs857: The λ repressor further incorporates a temperature-sensitive C-to-T (Ala-to-Thr) mutation. cITs857 is a functional repressor at 28℃-30℃, but is mostly inactive at 37℃-42℃. Also known as cI857.

[0203] Cat R Chloramphenicol resistance gene

[0204] CMV: Cytomegalovirus

[0205] dcm methylation: an E. coli methyltransferase that methylates the C5 position of the second cytosine in the sequence CC(A / T)GG.

[0206] DNA replicons: genetic elements that can replicate under their own control; examples include plasmids, granules, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors, and their hybrids.

[0207] Escherichia coli: A Gram-negative bacterium.

[0208] EGFP: Enhanced Green Fluorescent Protein

[0209] EP: Electroporation

[0210] Eukaryotic expression vectors: Vectors that express mRNA, protein antigens, protein therapeutics, shRNA, RNA, or microRNA genes in target eukaryotic organisms using RNA polymerase I, II, or III promoters.

[0211] Eukaryotic region: A region of a plasmid that encodes eukaryotic sequences and / or sequences required for plasmid function in the target organism. This includes plasmid vector regions required for expression of one or more transgenes in the target organism, comprising RNA Pol II enhancers, promoters, transgenes, and polyadenylated nucleotide sequences. It also includes plasmid vector regions required for expression of one or more transgenes in the target organism using RNA Pol I or RNA Pol III promoters, RNA Pol I or RNA Pol III promoters, or RNA. The eukaryotic region may optionally include other functional sequences, such as eukaryotic transcription terminators, supercoil-induced DNA double-strand destabilization (SIDD) structures, S / MARs, boundary elements, etc. In lentiviral or retroviral vectors, the eukaryotic region contains a flanked homologous repeat (LTR); in AAV vectors, the eukaryotic region contains a flanked inverted terminal repeat (IR) sequence; and in transposon vectors, the eukaryotic region contains a flanked transposon IR or IR / DR end (e.g., Sleeping Beauty). In genome integration vectors, the eukaryotic region may encode homologous arms to guide targeted integration.

[0212] Exons: Nucleotide sequences encoded by genes that are present in the mature mRNA product after transcription and RNA splicing to remove introns.

[0213] Expression vector: A vector used to express mRNA, protein antigens, protein therapeutics, shRNA, RNA or microRNA genes in target organisms.

[0214] g: gram, kg: kilogram

[0215] Genes of interest: Genes that will be expressed in the 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, etc.

[0216] Homopolymeric repeat sequences: Simple DNA repeat sequences consisting of polyadenylated, polyguanine (polyG), polycytidine (polyC), or polythymidine (polyT). They can range from a few base pairs to hundreds of consecutive base pairs.

[0217] Hr: hours

[0218] ID: Pi Nei

[0219] IM: intramuscular

[0220] Immune response: an antigen-reactive cell (e.g., antigen-reactive T cells) or antibody (e.g., antigen-reactive IgG) response.

[0221] Introns: Nucleotide sequences encoded by genes that are transcribed and subsequently removed from mature mRNA products via RNA splicing.

[0222] IR / DR: Inverted repeat sequences that are repeated twice in the same direction. For example, the Sleeping Beauty transposon IR / DR repeat sequence.

[0223] Repeatants: DNA sequences that repeat in the same direction at the origin of replication, required for replication initiation. The repeatant sequence of the R6K origin is 22 bp.

[0224] ITR: Inverted Terminal Repeat

[0225] Kanamycin

[0226] kanR: Kanamycin resistance gene

[0227] Kd: Thousand Daltons

[0228] Kozak sequence: An optimized shared DNA sequence, gccRccATG (R = G or A), immediately upstream of the ATG start codon, ensures efficient translation initiation. The SalI site (GTCGAC) immediately upstream of the ATG start codon is the efficient kozak sequence (GTCGACATG).

[0229] Lentiviral vectors: Integrative viral vectors that can infect both dividing and non-dividing cells. Also known as lentiviral transfer plasmids. The plasmid encodes a lentiviral LTR side expression unit. The transfer plasmid, along with the lentiviral envelope and packaging plasmids required for virus particle preparation, is transfected into production cells.

[0230] Lentiviral envelope vector: plasmid encoding envelope glycoprotein

[0231] Lentiviral packaging vector: one or two plasmids that express the gag, pol, and Rev functions required for packaging lentiviral transfer vectors.

[0232] Microcircles: Covalently closed circular plasmid derivatives in which the bacterial region has been removed from the parent plasmid via site-specific recombination in vivo or in vitro, or via in vitro restriction digestion / ligation. Microcircle vectors are not capable of replication in bacterial cells.

[0233] mRNA: messenger RNA

[0234] mRNA vectors: Vectors used as in vitro transcription templates for mRNA vaccines and therapeutics. Typically, the in vitro transcription template is a bacterial transcription unit containing a bacterial promoter (usually the T7 RNA polymerase promoter), followed by a 5' UTR, a Kozak sequence, a transgene coding region, a 3' UTR, and a polyadenylated homopolymeric repeat sequence typically 60 to 120 bp in length, ending at a unique restriction site (such as a BspQI site for vector linearization after the polyadenylated segment). Ideally, the BspQI site is positioned so that the linear DNA terminates at the polyadenylated repeat sequence without any "non-A" bases. The linear DNA is used in in vitro transcription reactions to prepare therapeutic or vaccine mRNAs.

[0235] mSEAP: Mouse secretory alkaline phosphatase

[0236] NA: Not applicable

[0237] Nanoplasmid TM Vector: A vector that combines RNA selective markers with R6K, ColE2, or ColE2-associated origins of replication. Examples include NTC9385C, NTC9685C, NTC9385R, and NTC9685R vectors and modifications, described in Williams, see above, 2014, and incorporated herein by reference.

[0238] NTC8385: NTC8385, NTC8485, and NTC8685 plasmids are antibiotic-free pUC origin vectors containing short RNA (RNA-OUT) selective markers instead of antibiotic resistance markers (such as kanR). The creation and application of these RNA-OUT-based antibiotic-free vectors are described in Williams, JA 2008 World Patent Application WO2008153733, which is incorporated herein by reference.

[0239] NTC8485: NTC8485 is an antibiotic-free pUC origin vector containing a short RNA (RNA-OUT) selective marker instead of an antibiotic resistance marker (such as kanR). The creation and use of NTC8485 are described in Williams, JA 2010, U.S. Patent Application 20100184158, which is incorporated herein by reference.

[0240] NTC8685: NTC8685 is an antibiotic-free pUC origin vector containing a short RNA (RNA-OUT) selective marker instead of an antibiotic resistance marker (such as kanR). The creation and application of NTC8685 are described in Williams, see above, 2010, and are incorporated herein by reference.

[0241] NTC9385R: Williams, see above, NTC9385R Nanoplasmid described in 2014. TM The vector (included herein by reference) has a spacer region encoding a NheI-trpA terminator-R6K starter RNA-OUT-KpnI bacterial region (SEQ ID NO:8), which is linked to the eukaryotic region via lateral NheI and KpnI sites.

[0242] OD 600 Optical density at 600 nm

[0243] PAS: Initiator assembly site. Initiates DNA synthesis at the ssi site of single-stranded DNA. ØX174 PAS The DNA hairpin sequence of priA binds to DNA, which in turn recruits the remaining proteins to form a preprimosome [priB, dnaT, recruiting dnaB (delivered by dnaC)], which then also recruits a primase (dnaG), which ultimately prepares a short RNA substrate for DNA polymerase I. Examples include PAS-BH and PAS-BL from plasmid pBR322. ABC type PAS DNA hairpins bind to DNAA, recruiting DNAB (delivered by DNAC), which in turn recruits a primase (DNAG). The primase then ultimately prepares a short RNA substrate for DNA polymerase I. For example, the R6K plasmid lacks a CpG ssiA primosome assembly site, or alternative ØX174 or ABC primosome assembly sites. Further examples demonstrate that the primosome assembly site can be used for DnaA-dependent or PriA-dependent primosomals, as described in Masai, Hisao, and Arai, Ken-ichi, *Frontiers in Bioscience* 1 (1996):d48-d58, which is incorporated herein by reference in its entirety.

[0244] PAS-BH: Initiator assembly site on the heavy (leader) chain of pBR322

[0245] PAS-BH region: The starting region of pBR322 between ROP and PAS-BL (approximately pBR322 2067-2351).

[0246] PAS-BL: Initiator assembly site on the light (hysteresis) chain of pBR322

[0247] PBS: Phosphate-buffered saline

[0248] PCR: Polymerase chain reaction

[0249] pDNA: plasmid DNA

[0250] PiggyBac transposon: PB transposon. A transposon system that integrates a PB transposon with an ITR side into the genome via a simple cleavage and paste mechanism mediated by PB transposase. Transposon vectors typically contain a promoter-transgene-polyadenylate expression cassette between the PB ITR, which is excised and integrated into the genome.

[0251] pINT pR pL vector: pINT pR pL att HK022 The integrated expression vector is described in Luke et al., 2011, *Molecular Biotechnol*, 47:43, and is included hereby cited. The target gene to be expressed is cloned downstream of the pL promoter. The vector encodes a temperature-inducible cI857 repressor, thereby allowing heat-inducible target gene expression.

[0252] P L Promoter: λ-left promoter. P L It is a strong promoter that is repressed by the cI repressor, which binds to the OL1, OL2, and OL3 repressor binding sites. The temperature-sensitive cI857 repressor allows for control of gene expression via heat induction, as it is functional and inhibits gene expression at 30°C, but inactivated between 37°C and 42°C, thus inhibiting gene expression.

[0253] P L (OL1 G to T) Promoter: λ left-handed promoter. P L It is a strong promoter that is repressed by the cI repressor, which binds to the OL1, OL2, and OL3 repressor binding sites. The temperature-sensitive cI857 repressor allows for control of gene expression via heat induction, as the cI857 repressor is functional and represses gene expression at 30°C, but is inactivated at 37°C–42°C, thus gene expression occurs. As described by Williams (see above, 2014), reducing the binding of the cI repressor to OL1 via an OL1 G-to-T mutation results in increased promoter activity at 30°C and 37°C–42°C.

[0254] Plasmid: An extrachromosomal DNA molecule isolated from chromosomal DNA that can replicate independently of chromosomal DNA.

[0255] Plasmid copy number: The number of plasmid copies per cell. An increase in plasmid copy number increases plasmid production yield.

[0256] Pol: Polymerase

[0257] Pol I: Escherichia coli DNA polymerase I

[0258] Pol I-dependent replication origins: These are replication origins that require Pol I, such as pMB1, ColE1, or pBR322 or derivatives, such as the high-copy pUC origin. For these origins, RNAII primers form an RNA:DNA R loop, which is cleaved by ribonuclease H, thereby generating primers for DNA pol I-guided DNA synthesis. DNA synthesis then transitions to DNA pol III. Many other Pol I-dependent replication origins are known in the art, many of which are summarized in del Solar et al., 1998 *Microbiology and Molecular Biology Reviews* 62:434-464, which are included herein by reference.

[0259] Pol III: Escherichia coli DNA polymerase III

[0260] Pol III-dependent origins of replication: Origins of replication that do not require Pol I, such as the rep protein-dependent R6Kγ origin of replication. Many other Pol III-dependent origins of replication are known in the art, many of which are outlined in del Solar et al., above, 1998, and are incorporated herein by reference.

[0261] Polyadenylation: A polyadenylation signal or site. Polyadenylation is the addition of a polyadenylation tail to an RNA molecule. Polyadenylation signals contain a sequence motif recognized by the RNA cleavage complex. Most human polyadenylation signals contain the AAUAAA motif and conserved sequences at its 5' and 3'. Commonly used polyadenylation signals are derived from rabbit β-globulin, bovine growth hormone, early SV40 polyadenylation signals, or late SV40 polyadenylation signals.

[0262] Polyadenylated repeat sequences: simple sequences of polyadenylated DNA repeat sequences ranging from a few base pairs to hundreds of consecutive base pairs.

[0263] pUC origin: A replication origin derived from pBR322, which exhibits a G-to-A transition that increases copy number at high temperatures and lacks the negative regulator of ROP.

[0264] pUC-free: Plasmids that do not contain a pUC origin. This may include non-replicating fragments containing a pUC origin, such as RNAi-selective markers.

[0265] pUC plasmid: a plasmid containing the pUC origin.

[0266] R6K plasmids: NTC9385R, NTC9685R, NTC9385R2-O1, NTC9385R2-O2, NTC9385R2a-O1, NTC9385R2a-O2, NTC9385R2b-O1, NTC9385R2b-O2, NTC9385Ra-O1, NTC9385Ra-O2, NTC9385RaF, and NTC9385RbF vectors, as well as modified and alternative vectors containing R6K origins of replication, are described in Williams, see above, 2014, and are 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 from the University of Oxford (including pGM169).

[0267] R6K Origin of Replication: A region specifically recognized by the R6K Rep protein to initiate DNA replication. This includes, but is not limited to, the R6K γ origin of replication sequences disclosed in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:18, and SEQ ID NO:24. It also includes CpG-free versions (e.g., SEQ ID NO:3), such as those described in Drocourt et al., U.S. Patent 7,244,609, which are incorporated herein by reference.

[0268] R6K Origin of Replication-RNA-OUT Bacterial Region: Contains the R6K origin of replication and RNA-OUT selectivity markers for reproduction (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; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28).

[0269] Rep: Copy

[0270] Replication intermediates: linear DNA fragments resulting from premature termination of plasmid replication.

[0271] Rep protein-dependent plasmids: A type of plasmid in which replication depends on a replication (Rep) protein provided in the Trans domain. Examples include the R6K origin of replication, the ColE2-P9 origin of replication, and the ColE2-associated origin of replication plasmids, in which the Rep protein is expressed from the host strain genome. Many other Rep protein-dependent plasmids are known in the art, many of which are summarized in del Solar et al., above, 1998, and are incorporated herein by reference.

[0272] Retroviral vectors: Integrative viral vectors that can infect dividing cells. Also known as transfer plasmids. The plasmid encodes a retroviral LTR side expression unit. The transfer plasmid, along with the envelope and packaging plasmids required for viral particle preparation, is transfected into production cells.

[0273] Retroviral envelope vector: plasmid encoding envelope glycoprotein

[0274] Retroviral packaging vectors: plasmids encoding the retroviral gag and pol genes required for packaging retroviral transfer vectors.

[0275] RNA-IN: Encoded by Insertion Sequence 10 (IS10), RNA-IN is a complementary and antisense RNA to a portion of RNA-OUT. When RNA-IN is cloned into an untranslated leader sequence of mRNA, annealing RNA-IN to RNA-OUT reduces the translation of downstream genes encoded by RNA-IN.

[0276] Selective Marker for RNA-IN Regulation: A selective marker for RNA-IN regulation of genome expression. In the presence of an RNA-OUT antisense repressor RNA (SEQ ID NO: 6) carried by a plasmid, the expression of a protein encoded downstream of RNA-IN is suppressed. The selective marker for RNA-IN regulation is configured such that RNA-IN regulates 1) a protein that is lethal or toxic to the cell itself, or by generating a toxic substance (e.g., SacB), or 2) a repressor protein that is lethal or toxic to the bacterial cell by inhibiting the transcription of genes essential for the cell's growth (e.g., the murA essential gene regulated by the RNA-IN tetR repressor gene). For example, the RNA-IN-SacB cell line for genome expression used for RNA-OUT plasmid selection / propagation is described in Williams, see above, 2008, and is incorporated herein by reference. Alternative selectable markers described in the art may replace SacB.

[0277] RNA-OUT: Insertion sequence 10 (IS10) encodes RNA-OUT, an antisense RNA that hybridizes with transposon genes expressed downstream of RNA-IN and reduces their translation. The sequences of RNA-OUT RNA (SEQ ID NO:6) and complementary RNA-IN SacB genome-expressing RNA-IN-SacB cell lines can be modified to incorporate alternative functional RNA-IN / RNA-OUT binding pairs, such as those described in Mutalik et al., 2012 *Nature Chemical Biology* 8:447, including but not limited to RNA-OUT A08 / RNA-IN S49 pairs, RNA-OUT A08 / RNA-IN S08 pairs, and a CpG-free version of RNA-OUT A08, which will insert RNA-OUT 5' TT CG The CG in the C sequence is modified to a non-CpG sequence. An example of a CpG-free RNA-OUT selective marker, wherein two CpG motifs (one of which is located in the RNA-IN complementary region) in the RNA-OUT RNA are removed, is described in Williams 2015, “Replicative minicircle vectors with improved expression,” U.S. Patent Application US 2015 / 0275221, which is incorporated herein by reference. Multiple substitutions can be used to remove the two CpG motifs (mutating each CpG to CpA, CpC, CpT, ApG, GpG, or TpG) to prepare CpG-free RNA-OUT.

[0278] RNA-OUT Selective Marker: An RNA-OUT selectively marked DNA fragment comprising an E. coli transcription promoter and terminator sequence with RNA-OUT RNA side-attached. An RNA-OUT selective marker utilizing the RNA-OUT promoter and terminator sequences, side-attached with DraIII and KpnI restriction enzyme sites, and an RNA-IN-SacB cell line designed for genome expression for RNA-OUT plasmid propagation, is described in Williams, see above, 2008, and is incorporated herein by reference. SEQ ID NO: 5 with side-attached RNA (SEQ ID NO: 6; Figure 1BThe RNA-OUT promoter and terminator sequences 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 the I-EC2K promoter or the P5 / 6 5 / 6 or P5 / 6 6 / 6 promoter, described in Williams, see above, 2008 and incorporated herein by reference. 2 CpG RNA-OUT selective markers, wherein two CpG motifs in the RNA-OUT promoter are removed, are given as SEQ ID NO: 7. Examples of CpG-free RNA-OUT transcription units, wherein two CpG motifs (one of which is located in the RNA-IN complementary region) in the RNA-OUT RNA and two CpG motifs in the RNA-OUT promoter are removed, described in Williams, see above, 2015 and incorporated herein by reference. Vectors incorporating CpG-selective RNA-OUT markers can be used for sucrose resistance selection using RNA-IN-SacB cell lines used for RNA-OUT plasmid propagation and described in Williams, see above, 2008. Alternatively, the RNA-IN sequence in these cell lines can be modified to incorporate a 1 bp variation to perfectly match the CpG-free RNA-OUT region complementary to RNA-IN.

[0279] RNA polymerase II promoters: Promoters that recruit RNA polymerase II to synthesize mRNA, most small nuclear RNAs, and microRNAs. Examples include constitutive promoters such as the human or murine CMV promoter, elongation factor 1 (EF1) promoter, chicken β-actin promoter, β-actin promoters from other species, elongation factor-1 α (EF1 α) promoter, phosphoglycerate kinase (PGK) promoter, Rous sarcoma virus (RSV) promoter, human serum albumin (SA) promoter, spleen focal formation virus (SFFV) promoter, α-1 antitrypsin (AAT) promoter, thyroxine-binding globulin (TBG) promoter, and cytochrome P450 2E1 (CYP2E1) promoter. Vectors can also utilize combinatorial promoters, such as the chicken β-actin / CMV enhancer (CAG) promoter, human or mouse CMV-derived enhancer elements combined with the elongation factor 1α (EF1α) promoter, CpG-free versions of human or mouse CMV-derived enhancer elements combined with the elongation factor 1α (EF1α) promoter, albumin promoters combined with the α-fetoprotein MERII enhancer, or a variety of tissue-specific or inducible promoters known in the art, such as the muscle-specific promoters muscle creatine kinase (MCK) and C5-12, or the liver-specific promoters apolipoprotein AI (ApoAI).

[0280] RNA polymerase III promoters: Promoters that recruit RNA polymerase III to synthesize tRNA, 5S ribosomal RNA, and other small RNAs. Examples include class I promoters such as the 5S rRNA promoter, class II promoters such as the tRNA promoter, and class III promoters such as the U6 small nuclear RNA promoter or the H1 RNase P promoter.

[0281] RNA-selective markers: RNA-selective markers are plasmid-carried, expressed non-translated RNAs that regulate the expression of target genes on chromosomes to provide selection. This can be a plasmid-carried nonsense repressor tRNA that regulates nonsense repressor-selective chromosomal targets, as described in Crouzet J and Soubrier F, U.S. Patent 6,977,174, 2005, which is incorporated herein by reference. This could also be an antisense repressive RNA carried by a plasmid, and the non-restrictive list included herein by reference includes: RNA-OUT, which represses RNA-IN regulatory targets (Williams, see above, 2008); RNAI encoded by the pMB1 plasmid origin, which represses RNAII regulatory targets (Grabherr R, Pfaffenzeller I. 2006 US Patent Application US20060063232; Cranenburgh RM. 2009; US Patent 7,611,883); RNAI encoded by the IncB plasmid pMU720 origin, which represses RNAII regulatory targets (Wilson IW, Siemering KR, Praszkier J, Pittard AJ. 1997. Journal of Bacteriology). The R1 plasmid contains the ParB locus Sok, which represses the Hok regulatory target; the F1 plasmid contains the FlmB locus FlmA, which represses the flmA regulatory target (Morsey MA, 1999 US Patent US5922583). RNA-selective markers can be other naturally occurring antisense repressive RNAs 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 Opinion on Microbiology 3:159-64. RNA-selective markers can also be engineered repressive RNAs, such as synthetic small RNAs expressed from SgrS, MicC, or MicF scaffolds, as described in Na D, Yoo SM, Chung H, Park H, Park JH, Lee SY. 2013. *Nature Biotechnology* 31:170-4. RNA-selective markers can also be engineered repressive RNAs that are part of a selectivity marker, repressing target RNAs fused to the target gene to be regulated (such as SacB), as described in Williams, see above, 2015.

[0282] ROP: Primer repressor

[0283] RSM: RNA-selective marker

[0284] SacB: The structural gene encoding levansucrase, a glycosaminoglycan in Bacillus subtilis. Expression of SacB in Gram-negative bacteria is toxic in the presence of sucrose.

[0285] SD: Standard Deviation

[0286] SEAP: Secretory alkaline phosphatase

[0287] 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 the subject sequence. The subject sequence may, for example, have at least 90%, at least 95%, or at least 99% sequence identity with the given query sequence. To determine the percentage of sequence identity, the query sequence (e.g., a nucleic acid sequence) is aligned with one or more subject sequences using any suitable sequence alignment program well known in the art, such as the computer program ClustalW (version 2.1, default parameters), which allows for nucleic acid sequence alignment over its entire length (global alignment). Chema et al., 2003 Nucleic Acids Res., 31:3497-500. In a preferred method, the sequence alignment program (e.g., ClustalW) calculates and aligns the best match between the query sequence and one or more subject sequences, allowing identity, similarity, and differences to be determined. Gaps of one or more nucleotides may be inserted into the query sequence, the subject sequence, or both to maximize the sequence alignment. For rapid alignment of nucleic acid sequences, appropriate default parameters can be selected for the specific alignment program. The output is a sequence alignment reflecting the relationship between sequences. To further determine the percentage of identity between the subject nucleic acid sequence and the query sequence, the sequences are aligned using the alignment program, the number of identical matches is divided by the length of the query sequence, and the result is multiplied by 100. Note that the percentage of identity can be rounded to the nearest decimal place. 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.

[0288] Selectable markers: Selective markers, such as kanamycin resistance genes or RNA selectable markers.

[0289] Selection marker: A selective marker, such as a kanamycin resistance gene or an RNA selection marker.

[0290] SIDD: Supercoil-induced DNA double-strand destabilization (SIDD) structures. These sites, when incorporated into a vector, may alter the sensitivity of other sequences within the vector to destabilization. This can alter function. For example, adding SIDD sites to an expression vector can reduce promoter helical destabilization. This may increase or decrease promoter activity, depending on the promoter, as some promoters show increased expression with promoter helical destabilization, while others show decreased expression.

[0291] shRNA: short hairpin RNA

[0292] S / MAR: Scaffold / Matrix Attachment Region. The eukaryotic sequence that mediates DNA attachment to the nuclear matrix.

[0293] Sleeping Beauty transposons: SB transposons. A transposon system that integrates SB transposons with IR / DR sides into the genome via a simple cleavage and paste mechanism mediated by SB transposases. Transposon vectors typically contain a promoter-transgene-polyadenylate expression cassette between the IR and DR sides, which is excised and integrated into the genome.

[0294] Spacer region: As used herein, a spacer region is a region connecting the 5' and 3' ends of a eukaryotic region sequence. The 5' and 3' ends of the eukaryotic region are typically separated by a bacterial origin of replication and a bacterial selectivity marker in a plasmid vector (bacterial region), therefore many spacer regions consist of bacterial regions. In the Pol III-dependent origin of replication vector of this invention, this spacer region is preferably less than 1000 bp.

[0295] SR: Interval zone.

[0296] SSI: Single-stranded start sequence

[0297] Structured DNA sequences: DNA sequences capable of forming secondary structures that repress replication, as used herein (Mirkin and Mirkin, 2007. Microbiology and Molecular Biology Reviews 71:13-35). This includes, but is not limited to, inverted repeats, palindromic repeats, homologous repeats, IR / DR repeats, homopolymeric repeats, or repeats containing eukaryotic promoter enhancers or eukaryotic origins of replication.

[0298] SV40 origin: primate DNA of simian virus 40 containing the origin of replication.

[0299] SV40 enhancer: simian virus 40 genomic DNA containing a 72 bp and an optional 21 bp enhancer repeat sequence.

[0300] Target antigen: An immunogenic protein or peptide epitope, or a combination of a protein and an epitope, against which an immune response can be elicited. Target antigens can be derived from pathogens used for infectious diseases or allergic applications, or from host organisms used for applications such as cancer, allergies, or autoimmune diseases. Target antigens are well-defined in the art. Some examples are described in Williams, see above, 2008, and are incorporated herein by reference.

[0301] TE buffer: A solution containing approximately 10 mM Tris pH 8 and 1 mM EDTA.

[0302] TetR: Tetracycline resistance gene

[0303] Tol2 transposons: A transposon system that integrates a Tol2 transposon with an ITR side into the genome via a simple cleavage and paste mechanism mediated by the Tol2 transposase. Transposon vectors typically contain a promoter-transgene-polyadenylate expression cassette between the Tol2 ITRs, which is then excised and integrated into the genome.

[0304] Transcription terminator: bacteria This refers to the DNA sequence at the transcriptional end of a marker gene or operon. This can be an intrinsic transcription terminator or a Rho-dependent transcription terminator. For intrinsic terminators, such as the trpA terminator, a hairpin structure forms within the transcript, disrupting the mRNA-DNA-RNA polymerase ternary complex. Alternatively, Rho-dependent transcription terminators require a Rho factor (an RNA helicase protein complex) to disrupt the nascent mRNA-DNA-RNA polymerase ternary complex. eukaryotes The polyadenylation signal is not a 'terminator', but rather a cleavage within the polyadenylation site, leaving an uncapped 5' end on the 3' UTR RNA for nuclease digestion. The nuclease catches up with RNA Pol II and causes termination. Termination can be promoted within a short region of the polyadenylation site by introducing an RNA Pol II pause site (eukaryotic transcription terminator). The pause of RNA Pol II allows nucleases introduced into the 3' UTR mRNA after polyadenylation 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 the C2x4 and gastrin terminators. Eukaryotic transcription terminators can enhance mRNA levels by strengthening appropriate 3' end processing of the mRNA.

[0305] Transfection: Methods for delivering nucleic acids into cells [e.g., poly(lactide-co-glycolic acid) (PLGA), ISCOM, liposomes, nonionic surfactant vesicles (niosomes), virions, block copolymers, Pluronic block copolymers, chitosan, and other biodegradable polymers, microparticles, microspheres, calcium phosphate nanoparticles, nanoparticles, nanocapsules, nanospheres, poloxamine nanospheres, electroporation, nuclear transfection, piezoosmosis, acoustic perforation, iontophoresis, ultrasound, SQZ high-speed cell deformation-mediated membrane rupture, corona plasma, plasma-enhanced delivery, tissue-tolerant plasma, laser microperforation, shock wave energy, magnetic field, non-contact magnetic permeation, gene gun, microneedles, microcrystal grinding, hydrodynamic delivery, high-pressure tail vein injection, etc.], as known in the art and incorporated herein by reference.

[0306] Transgenic: The gene of interest is cloned into a vector for expression in a target organism.

[0307] Transposase vector: a vector encoding a transposase

[0308] Transposon vector: A vector encoding a transposon, which is a substrate for transposase-mediated gene integration.

[0309] ts: Temperature sensitivity

[0310] μg: micrograms

[0311] μl: microliters

[0312] UTR: The untranslated region of mRNA (5' or 3' of the coding region).

[0313] Vectors: Gene delivery agents, including viral (e.g., alpha virus, poxvirus, lentivirus, retrovirus, adenovirus, adenovirus-associated virus, etc.) and non-viral (e.g., plasmids, MIDGE, transcriptionally active PCR fragments, microcircles, bacteriophages, etc.) vectors. These are well known in the art and are incorporated herein by reference.

[0314] Vector backbone: The eukaryotic and bacterial regions of the vector, containing no transgenic or target antigen coding regions. Detailed Implementation

[0315] Current technologies largely involve methods and compositions for short bacterial plasmid DNA vectors (<1kb), which improve plasmid production yield and quality, reduce transfection-related toxicity, and increase transgenic expression. These technologies can be implemented to improve the expression and fabrication of vectors, such as non-viral vectors (mRNA vectors, transposon vectors, transposase vectors, Sleeping Beauty transposon vectors, Sleeping Beauty transposase vectors, PiggyBac transposon vectors, PiggyBac transposase vectors, expression vectors, etc.) and viral vectors (e.g., AAV vectors, AAV repcap vectors, AAV helper vectors, Ad helper vectors, lentiviral vectors, lentiviral envelope vectors, lentiviral packaging vectors, retroviral vectors, retroviral envelope vectors, retroviral packaging vectors, etc.).

[0316] Improved plasmid expression, compared to transgene-coding plasmids containing bacterial regions encoding the pUC origin of replication, is defined in this paper as improved transgene expression levels and / or duration of expression, either in vitro or in vivo. It should be understood that all references cited herein are incorporated herein by reference in their entirety.

[0317] Surprisingly, the plasmid modification method of the present invention provides a solution for producing short-spacer vectors containing structured DNA sequences, which are manufactured efficiently and in high yield.

[0318] Compositions containing Pol III-dependent replication origins

[0319] In the embodiments, compositions comprising DNA molecules are disclosed, said DNA molecules comprising a backbone containing a Pol III-dependent origin of replication.

[0320] In one embodiment, a covalently closed circular recombinant DNA molecule is disclosed, comprising a backbone and an insert, wherein the backbone comprises a Pol III-dependent replication origin, a selective marker, and a first priming assembly site, wherein the first priming assembly site is located downstream of the Pol III-dependent replication origin in the replication direction, and wherein the insert comprises a structured DNA sequence.

[0321] In this embodiment, the structured DNA sequence is less than 1000 bp away from the Pol III-dependent replication origin.

[0322] In this embodiment, the skeleton is less than 1000 bp.

[0323] In one embodiment, the skeleton includes a bacterial replication selection region.

[0324] In this embodiment, the Pol III-dependent replication origin does not require Pol I.

[0325] In this embodiment, the Pol III-dependent replication origin is the Pol III-dependent R6K replication origin.

[0326] In the embodiments, the Pol III-dependent R6K replication origin has at least 80% sequence identity with sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24.

[0327] In this embodiment, the selective marker is an RNA selective marker.

[0328] In this embodiment, the RNA-selective marker is an RNA-OUT RNA-selective marker.

[0329] In the embodiments, the RNA-OUT RNA-selective marker is an RNA-IN regulatory RNA-OUT functional variant that has at least 80% sequence identity with sequences selected from SEQ ID NO: 5 and SEQ ID NO: 7.

[0330] In this embodiment, the RNA selective marker comprises a sequence having at least 80% sequence identity with SEQ ID NO: 6.

[0331] In an embodiment, the first primor assembly site comprises a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 33.

[0332] In the embodiments, the covalently closed circular recombinant DNA molecule also includes a second primoron assembly site located downstream of the Pol III-dependent replication origin in the replication direction.

[0333] In an embodiment, the second primer assembly site comprises a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 33.

[0334] In the embodiments, the covalently closed circular recombinant DNA molecule contains a sequence having at least 80% sequence identity with SEQ ID NO: 29, such that the first primordial assembly site and the second primordial assembly site are located downstream of the PolIII-dependent replication origin in the replication direction.

[0335] In an embodiment, the covalently closed circular recombinant DNA molecule according to any one of claims 13 to 14 comprises the sequence of SEQ ID NO: 29, such that the first primoron assembly site and the second primoron assembly site are located downstream of the Pol III-dependent replication origin in the replication direction.

[0336] In the examples, the covalently closed circular recombinant DNA molecules were not labeled with antibiotics.

[0337] In an embodiment, the backbone comprises a sequence having at least 80% sequence identity with a sequence selected from the following: 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0338] In an embodiment, the skeleton comprises a sequence selected from 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0339] In the embodiments, the structured DNA sequence is selected from inverted repeat sequences, homologous repeat sequences, homopolymeric repeat sequences, eukaryotic origin of replication, and eukaryotic promoter enhancer sequences.

[0340] In this embodiment, the insert is a transposable carrier.

[0341] In the embodiments, the structured DNA sequence is an inverted repeat sequence, a homologous repeat sequence, or a eukaryotic promoter enhancer sequence.

[0342] In this embodiment, the insert is a transposase vector.

[0343] In this embodiment, the insert is an mRNA vector.

[0344] In this embodiment, the insert is an AAV carrier.

[0345] In this embodiment, the structured DNA sequence is an inverted repeat sequence.

[0346] In this embodiment, the AAV carrier is encoded as AAV2 ITR.

[0347] In an embodiment, the insert comprises a 5' inverted terminal repeat sequence having a sequence having at least 80% sequence identity with SEQ ID NO: 35; and a 3' inverted terminal repeat sequence having a sequence having at least 80% sequence identity with SEQ ID NO: 36.

[0348] In this embodiment, the insert is a lentiviral vector.

[0349] In the embodiments, the structured DNA sequence is a homologous repeat sequence or a eukaryotic origin of replication.

[0350] In the embodiments, the structured DNA sequence is selected from: homopolymeric repeat sequences, such as polyadenylated repeat sequences; SV40 origin of replication; viral LTR; lentiviral LTR; retroviral LTR; transposon IR / DR repeat sequences; Sleeping Beauty transposon IR / DR repeat sequences; AAV ITR; CMV enhancer; and SV40 enhancer.

[0351] In this embodiment, the structured DNA sequence comprises homopolymeric repeat sequences.

[0352] In the examples, the homopolymer repeat sequence is a polyadenylate repeat sequence.

[0353] In the embodiments, the homopolymer repeat sequence contains about 3 to about 500 residues.

[0354] In this embodiment, the selective marker is an RNA selective marker and is transcribed in the reverse direction of the structured DNA sequence.

[0355] In one embodiment, an antibiotic-free covalently closed circular recombinant DNA molecule is disclosed, the molecule comprising a backbone and an insert, wherein the backbone comprises a replication origin and an RNA-selective marker, wherein the insert comprises a structured DNA sequence, and wherein the RNA-selective marker is oriented to be transcribed in the opposite direction to the structured DNA sequence.

[0356] In the embodiments, the structured DNA sequence of the antibiotic-free covalently closed circular recombinant DNA molecule is less than 1000 bp away from the origin of replication.

[0357] In the examples, the backbone of the antibiotic-free covalently closed circular recombinant DNA molecule is less than 1000 bp.

[0358] In the embodiments, the backbone of the antibiotic-free covalently closed circular recombinant DNA molecule contains a bacterial replication selection region.

[0359] In the examples, the replication origin of the antibiotic-free covalently closed circular recombinant DNA molecule is a Pol I-dependent replication origin.

[0360] In the examples, the replication origin of the antibiotic-free covalently closed circular recombinant DNA molecule is a Pol III-dependent replication origin.

[0361] In the examples, the Pol III-dependent replication origin of antibiotic-free covalently closed circular recombinant DNA molecules does not require Pol I.

[0362] In the examples, the Pol III-dependent replication origin of the antibiotic-free covalently closed circular recombinant DNA molecule is a Pol III-dependent R6K replication origin.

[0363] In the examples, the Pol III-dependent R6K replication origin of the antibiotic-free covalently closed circular recombinant DNA molecule has at least 80% sequence identity with sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24.

[0364] In this embodiment, the RNA-selective labeling of the antibiotic-free covalently closed circular recombinant DNA molecule is an RNA-OUT RNA-selective labeling.

[0365] In the embodiments, the RNA-OUT RNA-selective marker of the antibiotic-free covalently closed circular recombinant DNA molecule is an RNA-IN regulatory RNA-OUT functional variant having at least 80% sequence identity with sequences selected from SEQ ID NO: 5 and SEQ ID NO: 7.

[0366] In the embodiments, the RNA-selective marker of the antibiotic-free covalently closed circular recombinant DNA molecule comprises a sequence having at least 80% sequence identity with SEQ ID NO: 6.

[0367] In the embodiments, the backbone of the antibiotic-free covalently closed circular recombinant DNA molecule comprises a sequence having at least 80% sequence identity with a sequence selected from the following: 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0368] In the embodiments, the backbone of the antibiotic-free covalently closed circular recombinant DNA molecule comprises sequences selected from 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0369] In the embodiments, the structured DNA sequence of the antibiotic-free covalently closed circular recombinant DNA molecule is selected from reverse repeat sequences, homologous repeat sequences, homopolymer repeat sequences, eukaryotic origin of replication, and eukaryotic promoter enhancer sequences.

[0370] In the embodiments, the insert for the antibiotic-free covalently closed circular recombinant DNA molecule is a transposon vector.

[0371] In the embodiments, the structured DNA of the antibiotic-free covalently closed circular recombinant DNA molecule sequence is an inverted repeat sequence, a homologous repeat sequence, or a eukaryotic promoter enhancer sequence.

[0372] In the embodiments, the insert for the antibiotic-free covalently closed circular recombinant DNA molecule is a transposase vector.

[0373] In the embodiments, the insert for the antibiotic-free covalently closed circular recombinant DNA molecule is an mRNA vector.

[0374] In the embodiments, the insert for the antibiotic-free covalently closed circular recombinant DNA molecule is an AAV vector.

[0375] In the embodiments, the structured DNA sequence of the antibiotic-free covalently closed circular recombinant DNA molecule is an inverted repeat sequence.

[0376] In the examples, the AAV vector of the antibiotic-free covalently closed circular recombinant DNA molecule encodes AAV2ITR.

[0377] In the embodiments, the insert of the antibiotic-free covalently closed circular recombinant DNA molecule comprises a 5' inverted terminal repeat sequence having at least 80% sequence identity with SEQ ID NO: 35; and a 3' inverted terminal repeat sequence having at least 80% sequence identity with SEQ ID NO: 36.

[0378] In this embodiment, the insert for the antibiotic-free covalently closed circular recombinant DNA molecule is a lentiviral vector.

[0379] In the embodiments, the structured DNA sequence of the antibiotic-free covalently closed circular recombinant DNA molecule is a homologous repeat sequence or a eukaryotic replication origin.

[0380] In the embodiments, the structured DNA sequence of the antibiotic-free covalently closed circular recombinant DNA molecule was selected from polyadenylation repeat sequences, SV40 origin of replication, viral LTR, lentiviral LTR, retroviral LTR, transposon IR / DR repeat sequences, Sleeping Beauty transposon IR / DR repeat sequences, AAV ITR, CMV enhancer, and SV40 enhancer.

[0381] In the embodiments, the structured DNA sequence of the antibiotic-free covalently closed circular recombinant DNA molecule contains homopolymeric repeat sequences.

[0382] In the examples, the homopolymer repeat sequence of the antibiotic-free covalently closed circular recombinant DNA molecule is a polyadenylate repeat sequence.

[0383] In the examples, the homopolymer repeat sequence of the antibiotic-free covalently closed circular recombinant DNA molecule contains about 3 to about 500 residues.

[0384] In the embodiments, the antibiotic-free covalently closed circular recombinant DNA molecule also includes a first primoron assembly site located downstream of the origin of replication in the replication direction.

[0385] In the embodiments, the antibiotic-free covalently closed circular recombinant DNA molecule also includes a second primoron assembly site located downstream of the origin of replication in the replication direction.

[0386] In the embodiments, the first primordium assembly site of the antibiotic-free covalently closed circular recombinant DNA molecule contains a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 33.

[0387] In an embodiment, the first primordium assembly site of the antibiotic-free covalently closed circular recombinant DNA molecule comprises a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 33, and wherein the second primordium assembly site comprises a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 33.

[0388] In the embodiments, the antibiotic-free covalently closed circular recombinant DNA molecule contains a sequence having at least 80% sequence identity with SEQ ID NO: 29, such that the first primordium assembly site is located downstream of the replication origin in the replication direction.

[0389] In the embodiments, the antibiotic-free covalently closed circular recombinant DNA molecule contains a sequence having at least 80% sequence identity with SEQ ID NO: 29, such that the first primordium assembly site and the second primordium assembly site are located downstream of the origin of replication in the replication direction.

[0390] Methods for replicating DNA molecules

[0391] In the embodiments, a method for replicating a structured DNA sequence that is poorly replicated by a Pol I-dependent replication origin (such as a pUC origin) is disclosed.

[0392] In the embodiments, a method for improving the manufacture of viral vectors is disclosed.

[0393] In the embodiments, a method for improving the manufacture of non-viral vectors is disclosed.

[0394] In the embodiments, methods for increasing the production yield of viral and / or non-viral vectors are disclosed.

[0395] In the embodiments, methods for improving the quality and yield of viral and / or non-viral vector manufacturing are disclosed.

[0396] In the embodiments, a method for eliminating antibiotic resistance marker genes transferred via non-viral and / or viral vectors is disclosed.

[0397] In the embodiments, a method for reducing transfection-related toxicity is disclosed.

[0398] In the embodiments, a method for improving transposition from non-viral transposon vectors is disclosed.

[0399] In the embodiments, methods for improving the packaging titer of viral vectors and improving the expression of transgenes encoded by viral and non-viral vectors are disclosed.

[0400] In the embodiments, a method for improving R6K origin-mediated replication of tightly localized structured DNA sequences is disclosed.

[0401] In one embodiment, a method for replicating circular recombinant DNA molecules is disclosed. In another embodiment, a method for replicating closed circular recombinant DNA molecules is disclosed. In yet another embodiment, a method for replicating covalently closed circular recombinant DNA molecules is disclosed.

[0402] In one embodiment, the method includes providing cells containing recombinant DNA molecules as disclosed herein, and subjecting the cells to a fermentation process.

[0403] In this embodiment, the cells are engineered Escherichia coli strains expressing the Rep protein.

[0404] In this embodiment, the cell contains a chromosome-integrated arabinose-inducible CI857ts gene.

[0405] In one embodiment, the Rep protein contains at least one of the following mutations: P42L; P106I; F107S; and P113S. In another embodiment, the Rep protein contains at least two of the following mutations: P42L; P106I; F107S; and P113S. In yet another embodiment, the Rep protein contains three of the following mutations: P42L; P106I; F107S; and P113S. In yet another embodiment, the Rep protein contains all four of the following mutations: P42L; P106I; F107S; and P113S.

[0406] In one embodiment, the fermentation process involves growing cells in a culture medium containing arabinose.

[0407] In the examples, the yield of covalently closed circular plasmids exceeded 0.5 g / L after the fermentation process.

[0408] Now turn to the attached image. Figure 1A-1F The following labeled diagram is shown: Figure 1A The R6K start point has the following positions: 22 bp repeat sub-repetition sequence, DnaA boxes 1 and 2, and the regions included in the R6K start point of SEQ ID NO: 1, 2, 3 and 4; Figure 1BSEQ ID NO: 5 RNA-OUT selective marker having the following positions: RNA-OUT promoter -35 and -10 elements, SEQ ID NO: 6 RNA-OUT antisense RNA having RNA-IN complementary homologous region and RNA-OUT terminator 3' hairpin; Figure 1C )14 CpG R6K-RNA-OUT bacterial backbone, which consists of SEQ ID NO: 1 R6K origin of replication and SEQ ID NO: 5 RNA-OUT selectivity marker, including the trpA bacterial terminator located upstream of the R6K origin and side-attached NheI and KpnI cloning sites; Figure 1D )3 CpG R6K-RNA-OUT bacterial backbone, which consists of SEQ ID NO: 2 1x CpG R6K replication origin and SEQ ID NO: 7 2x CpG RNA-OUT selective marker, with NheI and KpnI cloning sites attached to the sides; Figure 1E The sequence originates from the R6K origin of SEQ IDNO: 1, with the positions of the six repeats highlighted. A separate 22 bp repeat sequence is shown below the origin map; and... Figure 1F The R6K start point from SEQ ID NO: 18 is shown, with the positions of the 7 repeats highlighted. The individual 22 bp repeat sequence is shown below the start diagram. In this example of a 7-repeat vector, repeat 5 has been tandemly copied; however, the 7-repeat vector of the present invention can be obtained by tandemly copying any one of repeats 1, 2, 3, 4, 5 or 6.

[0409] Figure 2A-2B The following labeled diagram is shown: Figure 2A )Pol I-dependent pUC origin-kanamycin-selective Sleeping Beauty transposon vector pUC57-Kan SB1 (see Table 4); and Figure 2B ) Pol III-dependent R6K origin-RNA-OUT antibiotic-free Sleeping Beauty transposon vector NTC9 SB1 (see Table 4). The positions of the left and right Sleeping Beauty IR / DRs relative to the bacterial skeleton replication origin and selection markers are shown.

[0410] Figures 3A-3C The following labeled diagram is shown: Figure 3A )Pol I-dependent pUC initiator-ampicillin-selective AAV vector pAAV (see Table 7); Figure 3B )Pol I-dependent pUC initiation-RNA-OUT antibiotic-free AAV vector NTC8-AAV (see Table 7); and Figure 3C)Pol III-dependent R6K origin-RNA-OUT antibiotic-free AAV vector NTC9-AAV (see Table 7). The positions of the left and right AAV ITRs relative to the bacterial backbone replication origin and selection markers are shown.

[0411] Figures 4A-4F The following labeled diagram is shown: Figure 4A )Pol I-dependent pUC origin-ampicillin-selective A60 polyadenylate repeat sequence encoding mRNA vector pGEM4Z T7 A60 pA (see Table 6); Figure 4B )Pol I-dependent pUC origin-RNA-OUT antibiotic-free A60 polyadenylate repeat sequence encoding mRNA vector NTC8-T7 A60 pA (see Table 6); Figure 4C )Pol III-dependent R6K origin-RNA-OUT antibiotic-selective A60 polyadenylated repeat sequence encoding mRNA vector NTC9-T7 A60 pA (see Table 6); Figure 4D )Pol I-dependent pUC origin - ampicillin-selective A99 polyadenylate repeat sequence encoding mRNA vector pT3 / T7 A99 pA (see Table 6); Figure 4E )Pol I-dependent pUC origin-kanR selective A99 polyadenylated repeat sequence encoding mRNA vector NTC7-T7 A99 pA (see Table 6); and Figure 4F The Pol III-dependent R6K origin-RNA-OUT antibiotic-selective A99 polyadenylated repeat sequence encodes the mRNA vector NTC9-T7 A99 pA (see Table 6). The positions of the A60 or A99 polyadenylated repeat sequences relative to the bacterial backbone replication origin and selection marker are shown.

[0412] Figures 5A-5D The diagram shows the labeled bacterial skeletons of Pol III-dependent R6K origin R6K-RNA-OUT from SEQ ID NO: 25 (A), SEQ ID NO: 27 (B), SEQ ID NO: 28 (C), and SEQ ID NO: 34 (D).

[0413] Figures 6A-6B An exemplary Pol III-dependent R6K starting point AAV ITR encoded AAV vector is shown. Figure 6A ) or A100 polyadenylated repeat sequence encoding mRNA vector ( Figure 6B (The labeled diagram)

[0414] Figures 7A-7B Here is a BspQ1 linearization plot of purified plasmid DNA from the following fermentation harvest: Figure 7A )mRNA carrier - NP (polyadenylate 100 <ROUT R6K origin>); and ( Figure 7B )Two different fermentation harvest batches of mRNA carrier - NP 7 - repeat PAS R6K> ROUT> (polyadenylate 100 R6K origin>PAS ROUT>). One microgram of purified DNA was digested overnight with BspQI at 50 °C, separated on an agarose gel, and post - stained with SybrII that recognizes DNA and RNA. The BspQI units per microgram of DNA are shown above each lane. The plasmid DNA preparation of the purified mRNA carrier - NP ( Figure 7A )had RNA contamination (low - molecular - weight bands diffusing on the gel). Since the concentration was determined by A260, the actual total amount of plasmid DNA in these reactions was less than the expected one microgram per lane.

[0415] Examples

[0416] The methods of the current technology are further illustrated by the following examples. These examples are provided by way of illustration and are not intended to limit the scope of the present disclosure in any way.

[0417] Example 1: Replication and production of plasmids with pUC and R6K replication origins

[0418] Background of replication and production of pUC - origin vectors:

[0419] 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 copy derivative of the pMB1 origin that lacks the accessory ROP (rom) protein and has additional temperature - sensitive mutations that destabilize the RNAI / RNAII interaction. Transferring cultures containing these origins from 30 °C to 42 °C results in an increase in plasmid copy number. pUC plasmids can be produced in a variety of Escherichia coli cell lines.

[0420] Background of RNA - OUT without antibiotic selection marker: Antibiotic - free selection was carried out in Escherichia coli strains containing the chromosomally integrated pCAH63 - CAT RNA - IN - SacB (P5 / 6 6 / 6) with the bacteriophage λ attachment site, as described in Williams, supra, 2008. SacB (Bacillus subtilis levansucrase) is a counter - selection marker that is lethal to Escherichia coli cells in the presence of sucrose. Plasmid - encoded RNA - OUT inhibits the translation of SacB from the RNA - IN - SacB transcript ( Figure 1BThis facilitates plasmid selection in the presence of sucrose by inhibiting SacB-mediated lethality.

[0421] Background of R6K origination vector replication and production: The R6K γ plasmid replication origin requires a single plasmid replication protein. It acts as a replication initiation monomer, binding to multiple repeating 'repeater' sites (seven core repeat sequences containing the TGAGNG shared sequence) and as a replication repressor dimer, binding to the repressor site (TGAGNG) and repeaters with reduced affinity. Replication requires multiple host factors, including IHF, DnaA, and primosome assembly proteins DnaB, DnaC, and DnaG (Abhyankar et al., 2003, *Journal of Biol Chem* 278:45476-45484). The R6K core origin contains DnaA and IHF binding sites that influence plasmid replication because... IHF and DnaA interact to initiate replication.

[0422] Different versions of the R6K γ origin of replication 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, the pCpG-free vector (Invivogen, San Diego, CA) and pGM169 (University of Oxford). The incorporation of the R6K origin of replication itself does not improve transgene expression levels compared to optimized pUC origin vectors (Soubrier et al., see above, 1999). However, the use of conditional origins of replication (such as R6K γ) requires specialized cell lines for propagation, which increases the safety margin because the vector will not replicate if transferred to the patient's endogenous microbiota.

[0423] Highly minimized R6Kγ-derived origin of replication of the 6-repeater (SEQ ID NO: 1); Figure 1E It contains the core sequence required for replication (including the DnaA box and stb 1-3 sites; Wu et al., 1995. Journal of Bacteriology. 177: 6338-6345), but upstream Dimer repressor binding site and downstream The promoter is missing (by removing a copy of the repeater), as described in Williams, see above, 2014 and incorporated herein by reference. This R6K start point contains 6 tandem, unidirectional repeaters ( Figure 1E NTC9385R Nanoplasmid TMThe vector, which includes this minimized R6K origin and spacer region of RNA-OUT AF selectivity marker, is described in Williams, see above, 2014 and is incorporated herein by reference.

[0424] Typical R6K producing strains express from the genome The protein derivative PIR116 contains a component that increases the copy number (by reducing...) Dimerization is achieved; Monomers have an activating effect, while The P106L was replaced with a dimer that has an inhibitory effect. Fermentation results using the pCOR plasmid (Soubrier et al., see above, 1999) and the pCpG plasmid (Hebel HL, Cai Y, Davies LA, Hyde SC, Pringle IA, Gill DR. 2008. Molecular Therapy 16: S110) were lower, approximately 100 mg / L in the PIR116 cell line.

[0425] Mutagenesis and copy number selection of the pir-116 replication protein have been used to prepare new production strains. For example, the TEX2pir42 strain contains a combination of P106L and P42L. The P42L mutation interferes with DNA circularization and inhibits replication. The TEX2pir42 cell line improved the copy number and fermentation yield of the pCOR plasmid, with a reported yield of 205 mg / L (Soubrier F. 2004. World Patent Application WO2004033664).

[0426] Improved copy number Other combinations of copy number mutants include 'P42L and P113S' and 'P42L, P106L and F107S' (Abhyankar et al., 2004. Journal of Biochemistry 279:6711-6719).

[0427] Williams, see above, 2014, describes a thermally inducible pL promoter expressing phage HK022 attachment site integration. Host strains containing high-copy mutant replication (Rep) proteins of P42L, P106L, and F107S for use with the R6K origin Nanoplasmid. TM Vector selection and propagation. This is additional Nanoplasmid. TM Safety factor, because the R6K origin vector can only replicate in engineered E. coli host strains that express the Rep protein.

[0428] The propagation and fermentation of the RNA-OUT selective marker R6K plasmid described in Williams, see above, 2014, uses heat-inducible 'P42L, P106L, and F107S' plasmids. Copy number mutant cell lines (such as DH5α host strain NTC711772 = DH5α dcm- att) λ ::P 5 / 6 6 / 6 -RNA-IN- SacB, catR; att HK022 The reaction was carried out using ::pL (OL1-G to T) P42L-P106L-F107S (P3-), SpecR StrepR). Production yields have been reported to be as high as 695 mg / L.

[0429] This article created and published additional cell lines, including:

[0430] NTC821601DH5α att 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

[0431] NTC940211DH5α att λ ::P 5 / 6 6 / 6 -RNA-IN- SacB, catR; att HK022 ::pL (OL1-G to T)P42L-P106I-F107S P113S (P3-), SpecR StrepR = High copy number substitution of P106I for P106L binds to P113S to produce a quadruple mutant rep protein derivative with increased copy number of NTC821601.

[0432] NTC1050811DH5α att λ ::P 5 / 6 6 / 6 -RNA-IN- SacB, catR; att HK022 ::pL (OL1-G to T)P42L-P106I-F107S P113S (P3-), SpecR StrepR; att φ80::pARA-CI857ts, tetR =NTC940211, is a pARA-CI857ts derivative. This strain contains a phage φ80 attachment site chromosomal integrated copy of the arabinose-inducible CI857ts gene. Adding arabinose to the plate or culture medium (e.g., to a final concentration of 0.2%–0.4%) induces pARA-mediated expression of the CI857ts repressor, thereby reducing copy number at 30°C through CI857ts-mediated downregulation of the pL promoter expressing Rep protein [i.e., additional CI857ts mediates a more effective downregulation of the pL (OL1-G to T) promoter at 30°C]. Copy number induction does not diminish upon temperature transition to 37°C–42°C, due to the inactivation of the CI857ts repressor at these high temperatures. A dcm-derived product (NTC1050811 dcm-) is used for cases where dcm methylation is not required.

[0433] NTC1011641; Stbl4 attλ::P5 / 6 6 / 6-RNA-IN- SacB, catR; att HK022 ::pL P42L-P106L-F107S (P3-) SpecR StrepR = NTC661135's Stbl4 version (XL1Blue-dcm-att) λ ::P 5 / 6 6 / 6 -RNA-IN- SacB, catR; att HK022 ::pR pL P42L-P106L-F107S (P3-) SpecR StrepR, described in Williams, see above, 2014

[0434] Compared to the triple mutant heat-inducible pL(OL1-G to T)P42L-P106L-F107S (P3-) described in Williams, see above, 2014, the quadruple mutant heat-inducible pL(OL1-G to T)P42L-P106L-F107S P113S (P3-) increases nanoplasmid. TM Production yield. Over 2 g / L of Nanoplasmid has been obtained using the quadruple mutant NTC1050811 cell line. TM (For example, the pA of NTC9 T7 A99 is 2240 mg / L, Table 6)

[0435] Using conditional origins of replication (such as these R6K origins) requires specialized cell lines for propagation, which increases the safety margin because the vector will not replicate if transferred to the patient's endogenous flora.

[0436] Example 2: Production of pUC and R6K initiation carriers

[0437] Shake-flask production: Shake-flask production is performed using proprietary plasmid + shake-flask medium. Seed cultures are started from glycerol stock solutions or colonies and streaked onto LB agar plates containing 50 μg / mL antibiotics (for ampR or kanR selection plasmids) or 6% sucrose (for RNA-OUT selection plasmids). The plates are incubated at 30–32°C; cells are resuspended in the medium and used to provide approximately 2.5 OD for 500 mL plasmid + shake flasks. 600 The inoculum, in the shake flask, contains 50 μg / mL of antibiotic for ampR or kanR selection plasmids or 0.5% sucrose for RNA-OUT selection plasmids. The flask is then grown with shaking at the growth temperatures shown in Tables 5, 6, 7, and 9 until saturation.

[0438] Fermentation Production: Fermentation was carried out in a New Brunswick BioFlo 110 bioreactor using proprietary fed-batch medium (NTC3019, HyperGRO medium), as described (Carnes and Williams, see above, 2011). Seed cultures were started from glycerol stock solutions or colonies and streaked onto LB agar plates containing 50 μg / mL antibiotics (for ampR or kanR selection plasmids) or 6% sucrose (for RNA-OUT selection plasmids). The plates were grown at 30–32°C; cells were resuspended in the medium and used to provide approximately 0.1% inoculum for fermentation containing 50 μg / mL antibiotics for ampR or kanR selection plasmids or 0.5% sucrose for RNA-OUT plasmids. HyperGRO temperature transitions are shown in Tables 8 and 9.

[0439] Production host: fermentation of pUC origin AmpR or KanR plasmid in Escherichia coli strain DH5α [F- Φ80lacZΔM15 Δ(lacZYA-argF) U169recA1 endA1 hsdR17 (rK-, mK+) phoA supE44 λ- thi-1 gyrA96 relA1] (Invitrogen, Carlsbad CA) or Stbl4.

[0440] Antibiotic-free fermentation of pUC-origin RNA-OUT plasmids was performed on *E. coli* strain DH5α, which contains pCAH63-CAT RNA-IN-SacB (P5 / 6 6 / 6) with phage λ attachment site chromosomal integration, as described in Williams, see above, 2008. The producing strain was NTC4862 = DH5α attλ::P5 / 6 6 / 6-RNA-IN-SacB, catR.

[0441] Using *E. coli* RNA-OUT as a host for antibiotic-free propagation and fermentation of an R6K γ origin RNA-OUT plasmid, the host further encodes a heat-inducible pL promoter integrated with the bacteriophage HK022 attachment site. The method for generating copy number mutant cell lines is described in Williams, see above, 2014, and is incorporated herein by reference.

[0442] Production strain:

[0443] pUC origin - AmpR or KanR antibiotics select the host

[0444] DH5α

[0445] Stbl4

[0446] pUC origin - RNA - OUT sucrose selects host

[0447] NTC4862 DH5α att λ ::P 5 / 6 6 / 6 -RNA-IN- SacB, catR

[0448] NTC1011592 Stbl4 attλ::P5 / 6 6 / 6-RNA-IN- SacB, catR

[0449] R6K origin - RNA - OUT sucrose selection Nanoplasmid TM Host

[0450] NTC1050811 DH5α att λ ::P 5 / 6 6 / 6 -RNA-IN- SacB, catR; att HK022 ::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; att φ80 ::pARA-CI857ts, tetR

[0451] NTC1011641 Stbl4 attλ::P5 / 6 6 / 6-RNA-IN- SacB, catR; att HK022 ::pL P42L-P106L-F107S (P3-) SpecR StrepR

[0452] Analytical Methods: Culture samples were collected at key points and regular intervals throughout all fermentation periods. The biological quality (OD) of the samples was analyzed immediately. 600 Plasmid yield was determined by quantifying the plasmids obtained from the Qiagen Spin Miniprep kit formulation, as described in (Carnes and Williams, see above, 2011). In short, cells were lysed in an alkaline environment, clarified, and the plasmid column was purified and eluted before quantification. Plasmid quality was determined by agarose gel electrophoresis (AGE) analysis on 0.8%–1% Tris / acetate / EDTA (TAE) gels, as described in Carnes and Williams, see above, 2011.

[0453] Example 3: Construction and fabrication of pUC and R6K origin-based structured carriers

[0454] The R6K γ starting point (SEQ ID NO:1; Figure 1E )-RNA-OUT (SEQ ID NO:5; Figure 1B Selective region for bacterial replication (SEQ ID NO:8; Figure 1C The pNTC-NP1, pNTC-NP2, pNTC-NP3, pNTC-NP4, pNTC-NP5, pNTC-NP6, and pNTC-NP7 vectors were cloned into multiple adapter regions of various pUC57-based vectors. Each vector has a different side-linking restriction site, which can be used to modify the target vector for R6K replication RNA-OUT selection. The 5' and 3' multiple adapter sequences of the R6K-RNA-OUT inserts in the pNTC-NP1–7 vectors are shown in Table 2. A 1 CpG R6K γ origin-2 CpG RNA-OUT bacterial replication selection region (SEQ ID NO: 9) was also created. Figure 1D The pUC57-based version (pNTC-3xCpG NP1) is shown in Table 2.

[0455] The R6K γ initiation (SEQ ID NO:1) is an engineered 6-repeating R6K initiation ( Figure 1E It also created a 7-repeater R6K γ origin (SEQ ID NO:18; Figure 1F )-RNA-OUT (SEQ ID NO:5; Figure 1B A pUC57-based version of the bacterial replication selection region was used to construct and evaluate the utility of additional repeaters in manufacturing. Similarly, high-quality, high-yield manufacturing was achieved using vectors differing only in the presence of either the SEQ ID NO:18 heptodecanone R6Kγ origin or the hexaodecanone R6Kγ origin (SEQ ID NO:1). For example, the following harvest production yields were obtained during 10-hour temperature-uniform HyperGRO fermentation at 30°C–42°C:

[0456] SEQ ID NO:1 6-repeater 3203 bp R6K origin vector: 120 OD biomass 600 The plasmid titer was 1363 mg / L; the plasmid specific yield was 11.3 mg plasmid / L / OD. 600

[0457] SEQ ID NO:18 7 repeat 3225 bp R6K origin vector: biomass 137 OD 600 The plasmid titer was 1503 mg / L; the plasmid specific yield was 11.0 mg plasmid / L / OD. 600

[0458] The 7-repeater R6K γ origin in SEQ ID NO:18 is a tandem replication of repeater 5. Figure 1F ; SEQ ID NO:18), but the 7-repeater R6K γ origin carrier of the present invention can be SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 ( Figure 1E Tandem replications of any of the repeaters shown, or a 7-repeater R6K origination composition randomly combined from SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, or repeater repeat sequence variants retaining the TGAGNG concordant sequence. Additional repeater derivatives (e.g., 8, 9, or 10 repeater vectors) are also contemplated for practicing the invention.

[0459] The viral and non-viral vector pUC origin-antibiotic-selective bacterial backbone was modified to R6K-RNA-OUT using the following steps:

[0460] 1) Select restriction sites for lateral pUC origin and antibiotic selection marker regions in target viruses and non-viral vectors;

[0461] 2) Identify pNTC-NP compatible multi-adaptor-R6K-RNA-OUT multi-adaptor cassettes (pNTC-NP1, 2, 3, 4, 5, 6, or 7; Table 2);

[0462] 3) Using the selected restriction digestion method and standard ligase-mediated cloning, the pUC origin antibiotic selection marker region was excised and replaced with the selected R6K origin RNA-OUT region.

[0463] In some cases, non-palindromic DraIII adapter sites are used to assemble R6K origin and RNA-OUT units from individual restriction fragments via multi-fragment ligation (see Table 2). In the case of fd6 Ad helper plasmid modification (Table 7), a short 500 bp synthetic gene DraIII RNA-OUT-Ad helper plasmid-AvrII is used for 3-fragment ligation to ligate RNA-OUT to the unique AvrII site in the eukaryotic region of the fd6 Ad helper plasmid within the 12 kb AvrII-SalI restriction fragment, and then to the SalI-R6K origin-DraIII fragment from pNTC-NP4.

[0464] For Sleeping Beauty (Fig. 2; Table 4), AAV (Fig. 3; Table 7), and mRNA (Fig. 4; Table 6) vectors, exemplary vector diagrams and vector characteristics of the original pUC initiation-antibiotic selection marker vector and the modified R6K initiation-RNA-OUT antibiotic-free selection marker vector are shown. For AAV helper vectors (Table 6), vector characteristics of the original pUC initiation-antibiotic selection marker vector and the modified R6K initiation-RNA-OUT antibiotic-free selection marker vector are shown. For lentiviral vectors (Table 3) and AAV vectors (Table 5), vector characteristics of the pUC initiation-RNA-OUT antibiotic-free selection marker vector and the modified R6K initiation-RNA-OUT antibiotic-free selection marker vector are shown.

[0465] In all cases, the bacterial backbone size in the R6K origin-RNA-OUT antibiotic-selective marker-free modified vectors was <1 kb (460–610 bp). This is well below the 1.1 kb bacterial backbone size limit required to improve vector expression levels (Tables 1–2) and duration (Quiviger et al., see above, 2014). In all cases, the original pUC origin-antibiotic-selective bacterial backbone before modification was >1.2 kb (2340–2750 bp), the same as that of the pUC origin-RNA-OUT modified vectors (1210–1500 bp). Therefore, these AAV, AAV helper plasmid, Sleeping Beauty, and lentiviral R6K origin-RNA-OUT antibiotic-selective marker-free modified vectors meet the short-spacer requirement, thereby improving expression levels and duration, compared to the original pUC origin-antibiotic-selective marker vectors. Furthermore, these AAV, AAV helper plasmids, Sleeping Beauty, and lentiviral R6K origin-RNA-OUT antibiotic resistance marker-free modified vectors, due to the removal of the KanR or ampR antibiotic resistance selector from the parent vector, do not offer the opportunity for antibiotic marker gene transfer via transduction (AAV, lentiviral vectors) or transposition (Sleeping Beauty vector). Additionally, current vector technologies do not require complex, difficult-to-scale, and costly additional manufacturing steps necessary for removing the large bacterial region between the eukaryotic polyadenylate and the promoter when using microcircular vectors (Kay et al., see above, 2010).

[0466] However, in lentiviral vectors, the eukaryotic region contains side-linked same-direction repeats (LTRs), in AAV vectors, the eukaryotic region contains side-linked inverted terminal repeats (IR / DRs), and in Sleeping Beauty transposon vectors, the eukaryotic region contains side-linked transposon IR / DR ends. These side-linked sequences are all structured DNA sequences.

[0467] Levy, see above, 2004 teaching, states that a replication intermediate will form 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), but will not form when the high copy number replication origin is more than 1.5 kb away. Consistent with this, replication intermediates formed in all pUC origin-RNA-OUT marker vectors (where the pUC origin is <1 kb from the LTR of the lentiviral vector) (Table 3: 400 bp) or pUC origin-antibiotic resistance marker vectors (where the pUC origin is <1 kb from the Sleeping Beauty IR / DR) (Table 4: 280 bp). For AAV and mRNA vectors, the original pUC origin-antibiotic selection marker vectors with a pUC origin 0 bp from the ITR (AAV vectors; Table 7) or 170 bp from the A99 repeat sequence (mRNA vectors, Table 6) may produce replication intermediates that are too small to be detected on agarose gel. However, in these cases, production yields were very low, indicating low plasmid copy numbers due to replication blockade. In contrast, as expected, high plasmid production yields were obtained when the original pUC origin-antibiotic selection marker vector pUC origin was >1.5 kb from the structured DNA sequence (A60 repeat sequence) (Table 6: mRNA vector pGEM4Z T7 A60).

[0468] Williams, see above, reported in 2017 that pUC origin vector production yield was improved when the pUC origin was >1.5 kb from the homopolymeric A64C31 repeat sequence when using PAS-BH extended pUC origins. However, production yield was lower when the PAS-BH extended pUC origin was oriented <400 bp from the A64C31 repeat sequence (Table 6, see footnotes d and e). This indicates that adding a PAS-BH primosome assembly site does not overcome the poor pUC origin oriented replication of tightly localized structured DNA sequences.

[0469] Since the pUC origin itself is 1 kb, there is no configuration that can produce AAV, lentivirus, retrovirus, or transposon vectors containing a pUC origin and a bacterial region <1.1 kb, which are predicted not to produce replication intermediates as seen above and predicted by Levy, see above, 2004, as well as poor plasmid yields as reported in this paper.

[0470] Surprisingly, no replication intermediates were observed in any of the R6K origin-RNA-OUT marker-free modified vectors, including those where the R6K origin was <1 kb from the lentiviral vector LTR (Table 3: 400 bp) or the Sleeping Beauty IR / DR (Table 4: <40 bp). Furthermore, for AAV vectors, while the original pUC origin-antibiotic selectable marker vector with a pUC origin 0 bp from the ITR had very poor production yields, the two R6K origin-RNA-OUT marker-free modified vectors with an R6K origin 40 bp from the ITR had much higher production yields (Table 5). This improved production is R6K-specific, not RNA-OUT-specific, because the two AAV pUC-RNA-OUT modifiers with a pUC origin 50 bp from the ITR had plasmid production yields comparable to the original pUC antibiotic-labeled vector (Table 5); and a direct comparison of pUC-RNA-OUT with an R6K-RNA-OUT modifier 400 bp 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 3).

[0471] Not wanting to be limited by theory, this surprising improvement in plasmid copy number (plasmid production yield) and quality (elimination of replication intermediates) of R6K origin vectors means that R6K origins can replicate more efficiently through structured DNA sequences than pUC origins. While Levy (see above, 2004) teaches that replication intermediates form when any high copy number prokaryotic origin of replication is < 1 kb from a structured DNA sequence (such as an enhancer, LTR, or IRES), and not when the distance is > 1.5 kb, the examples provided by Levy (see above, 2004) are all pUC origin plasmids.

[0472] The fundamental difference between these origins of replication is that the pUC origin is a Pol I-dependent origin of replication, while the R6K origin is a Pol III-dependent origin of replication. In the case of the pUC origin, the RNAII primer forms an RNA:DNA R loop, which is cleaved by ribonuclease H, thereby generating primers for DNA Pol I-guided DNA synthesis during initial leader strand synthesis. Then, from 400 bp downstream of the origin to a maximum of 1.3 kb, DNA synthesis transitions from the slow DNA Pol I to the highly persistent DNA Pol III (Allen et al., 2011. Nucleic Acids Research 39:7020-33). The R6K γ origin of replication rep protein interacts with dnaB helicase and dnaG primase to produce short RNA primers for DNA Pol III replication without the need for DNA Pol I (Abhyankar et al., see above, 2003). The pUC origin DNA Pol I replication region, located at a maximum distance of 1.3 kb from the origin, closely corresponds to the upper limit of replication intermediate formation (between 1 and 1.5 kb from the origin) defined by Levy, see above, 2004. Not wanting to be limited by theory, it is proposed that the surprising improvement in structured DNA replication observed when adjacent to R6K rather than the pUC origin is due to the unexpected improvement in structured DNA sequence replication by DNA Pol III compared to DNA Pol I.

[0473] The vector methods and compositions disclosed herein demonstrate that Pol III-dependent replication origins (such as R6K origins) can be used to replicate structured DNA sequences that are poorly replicated by Pol I-dependent replication origins (such as pUC origins).

[0474] These results demonstrate that the vector of the present invention can be used to improve the yield and quality of viral and non-viral vector manufacturing.

[0475] Example 4: Improved performance of R6K origin-based structured carriers

[0476] The vector of the present invention can also be used to eliminate the transfer of antibiotic resistance marker genes via viral and nonviral vectors; reduce transfection-related toxicity; improve transposition from nonviral transposon vectors; improve the packaging titer of viral vectors; and improve the expression of transgenes encoded by viral and nonviral vectors.

[0477] For example, compared to pUC origin vectors with a bacterial backbone of >1.5 kb, the third-generation lentiviral vectors of the present invention with an R6K origin [4 vectors: transfer plasmids (Table 53), gag pol packaging plasmids; env plasmids; REV plasmids with an R6K origin and a bacterial backbone of <1 kb (not shown)] showed reduced toxicity and improved viral packaging titers. As recommended by the manufacturer, lenti-X 293 T cell lines (Takara Bio, Mountain View, CA) were transfected in 24-well plates with a third-generation lentiviral vector with a <1 kb bacterial backbone and a Table 3 R6K initiation or a pUC initiation-antibiotic selection marker vector with a >1.5 kb bacterial backbone control using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA) to result in higher titers of lentiviral production (>1.5 kb bacterial backbone pUC initiation control vector: 1.00 x ± 0.32; <1 kb bacterial backbone R6K initiation vector: 1.45 x ± 0.42), as measured using the Lenti-X p24 rapid titer kit (Takara Bio, Mountain View, CA). As described in (Marino MP, Luce MJ, Reiser J. 2003, Methods in Molecular Biology 229:43-55), transfection of Lenti-X 293 T cell lines in 24-well plates with third-generation lentiviral vectors (>1.5 kb bacterial backbone pUC origin control or <1 kb bacterial backbone R6K origin) using calcium phosphate resulted in higher titers of lentiviral production (>1.5 kb bacterial backbone pUC origin control: 1.00x ± 0.30; <1 kb bacterial backbone R6K origin vector: 1.32x ± 0.19), as measured using the Lenti-X p24 rapid titration kit (Takara Bio Inc., Mountain View, California). Notably, compared to the low virulence of third-generation lentiviral vectors with an R6K originating in a <1 kb bacterial backbone, calcium phosphate transfection with third-generation lentiviral vectors with a pUC originating in a >1.5 kb bacterial backbone resulted in widespread transfection-related virulence (>80% cell death) in 24-well plate transfection. This reduced transfection-related virulence should lead to a significant improvement in viral titers in larger-scale manufacturing transfections.These results demonstrate that, compared to bacterial backbone vectors with a >1 kb bacterial backbone and an R6K initiation, the present invention’s <1 kb bacterial backbone nanoparticle vectors reduce transfection-related toxicity and improve viral vector packaging titers.

[0478] Example 5: Construction and fabrication of R6K-based PAS structured AAV carriers

[0479] The replication mediated by the R6K origin of a tightly localized structured DNA sequence was tested to see if it could be improved by inserting a sequence between the R6K origin and the structured DNA sequence in the replication direction. For this evaluation, an AAV ITR vector with an ITR structured DNA sequence having a lateral bacterial region was used.

[0480] The standard 6-repeater R6K γ origin (SEQ ID NO: 1) in the AAV ITR vector is extended to the R6K γ origin (7 repeaters) (SEQ ID NO: 4); Figure 1A Replacement. This region contains gyrase-binding sites that can improve R6K initiation activity (see [link]). Figure 1A A 7-repeater R6K γ origin without extension was also constructed as a control. Figure 5A ).

[0481] Although the addition of the PAS-BH primosome assembly site, as described by Williams (see above, 2017), did not overcome the poor pUC origin-directed replication of tightly localized structured DNA sequences (see Example 3), the effect of adding the primosome assembly site was tested here in the context of the R6K origin as follows.

[0482] Two ØX174 type priming site sequences from plasmid pBR322 (SEQ ID NO: 29), namely PAS-BL (SEQ ID NO: 31) and PAS-BH (SEQ ID NO: 30), were inserted between the R6Kγ origin of the 7-repeater without extension (SEQ ID NO: 18) and the ITR structured DNA sequence, wherein each PAS is located on a different strand of the vector. Figure 5B ).

[0483] Finally, the ABC-type PASR6K plasmid without the CpG ssiA primer assembly site (SEQ ID NO: 33) was inserted between the R6K γ origin of the 7-repeater without extension (SEQ ID NO: 18) and the ITR structured DNA sequence. Figure 5D ).

[0484] As described in Example 2, the yield and quality of the above-described vectors were tested in shake-flask production. The results are shown in Table 8. Compared with the standard 6-repeater R6K γ origin (SEQ ID NO: 1) and other tested vectors, the vectors with two ØX174-type PAS sequences inserted between the R6K origin and the ITR structured DNA sequence, namely PAS-BL and PAS-BH (SEQ ID NO: 29), showed improved yield.

[0485] Additional vectors were constructed with ØX174-type PAS sequences inserted between the R6Kγ origin of the 7-repeater without extension (SEQ ID NO: 18) and the ITR structured DNA sequence, namely PAS-BL (SEQ ID NO: 31) and PAS-BH (SEQ ID NO: 30) (SEQ ID NO: 29), and the original PAS vector (ITR PAS).<R6K R-OUT> ITR; Figure 5B Compared to the bacterial region elements (ITR R6K > PAS R-OUT > ITR), the bacterial region elements (ITR R6K > PAS R-OUT > ITR) are... Figure 5C The orientations differ. As described in Example 2, shake-flask production yields and quality of this new vector, as well as the other vectors mentioned above, were tested. Results from independent production operations are shown in Table 9. Compared to the standard 6-repeater R6K γ origin (SEQ ID NO: 1) and other tested vectors, both vectors with two ØX174-type PAS sequences inserted between the R6K origin and the ITR structured DNA sequence, namely PAS-BL and PAS-BH (SEQ ID NO: 29), showed improved yields.

[0486] As described in Example 2, two AAV ITR vectors with the R6K origin of the PAS-BL and PAS-BH 7-repeater sequence were evaluated in HyperGRO fermentation compared to the standard AAV ITR vector with the R6K γ origin of the 6-repeater sequence. The results are shown in Table 10 and demonstrate that both vectors significantly improved AAV ITR vector fermentation yields compared to the standard AAV ITR vector with the R6K γ origin of the 6-repeater sequence. This indicates that, in the configurations of SEQ ID NO: 27 and SEQ ID NO: 28, including the PAS region between the R6K origin and the structured DNA repeat sequence improves shake-flask and fermentation production of vectors containing structured DNA sequences with flanking bacterial regions. This region introduces ØX174 type primer assembly sites on both the heavy (leading) strand (PAS-BH) and the light (lagging) strand (PAS-BL), either of which can surprisingly improve R6K origin-mediated replication. Interestingly, including ABC-type primosome assembly sites between the R6K origin and the structured DNA repeat sequence did not improve production.

[0487] Example 6: Construction and Manufacture of an R6K Origin PAS-Structured mRNA Vector

[0488] The antibiotic-free R6K origin RNA-OUT vector for mRNA production disclosed in Example 3 ( Figure 4C ; Figure 4F ) was configured to replicate away from the polyadenylate homopolymeric segment structured DNA repeat sequence in the <RNA-OUT R6K origin> orientation of the polyadenylate repeat sequence. This configuration has an RNA-OUT tag transcribed towards the polyadenylate repeat sequence. Surprisingly, in the case of having some mRNA transgenic inserts, double-stranded RNA (mRNA vector-NP Table 11; Figure 7A ) that was not completely digested by RNaseA or removed in the standard plasmid DNA column purification process was formed. The basis for the formation of the problematic double-stranded RNA is unknown, but it may be due to the annealing of the forward strand transcription of the cryptic mRNA transgenic insert with the reverse strand transcription read-through of the mRNA terminator sequence in the RNA-OUT gene.

[0489] It was speculated that the production of the problematic double-stranded RNA could be eliminated by reversing the orientation of the RNA-OUT transcription unit so that it is not transcribed towards the polyadenylate repeat sequence. This was tested by replacing the polyadenylate repeat sequence <RNA-OUT R6K origin> backbone in the mRNA vector-NP with (Constructs 1-3 below, and Constructs 4-8 provide alternative configurations):

[0490] 1) Polyadenylate repeat sequence RNA-OUT > R6K origin> (mRNA vector-ROUT OPP-NP)

[0491] 2) Polyadenylate repeat sequence R6K origin > RNA-OUT> (mRNA vector-ROUT OPP-NP2)

[0492] 3) Polyadenylate repeat sequence R6K origin (7 repeats)>PAS RNA-OUT> (mRNA vector-NP 7 repeats PAS R6K > ROUT>) Figure 6B = SEQ ID NO: 28 Bacterial backbone configuration RNA-OUT > R6K origin > PAS

[0493] 4) Polyadenylate repeat sequence R6K origin>PAS RNA-OUT>

[0494] I5) Polyadenylate repeat sequence RNA-OUT > R6K origin (7 repeats)>

[0495] 6) Polyadenylate repeat sequence R6K origin (7 repeats)> RNA-OUT>

[0496] 7) Polyadenylated repeat sequence RNA-OUT > R6K origin (7 repeats) > PAS

[0497] 8) Polyadenylated repeat sequence RNA-OUT > R6K origin > PAS

[0498] All three configurations (1-3) combine high-yield production with the elimination of double-stranded RNA (Table 11); Figure 7B This indicates that the optimal nanoplasmid backbone orientation for mRNA vectors combines the orientation of both R6K origin replication and RNA-OUT transcription away from polyadenylated structured DNA repetitive sequences. An exemplary composition for a nanoplasmid backbone mRNA vector is:

[0499] R6K origin of polyadenylation repeat sequence > RNA-OUT >;

[0500] Polyadenylated repeat sequence R6K origin > PAS RNA-OUT > ( Figure 6B = SEQ ID NO: 28 R6K origin > PASRNA-OUT > bacterial skeleton configuration

[0501] Polyadenylation repeat sequence RNA-OUT> R6K origin>;

[0502] Polyadenylation repeat sequence RNA-OUT > R6K origin > PAS;

[0503] The above configuration can have 6 or 7 repeating R6K start points.

[0504] While the above description contains many examples, these should not be construed as limiting the scope of this disclosure, but rather as illustrative of preferred embodiments. Many other variations are possible.

[0505] For example, various orientations of Pol III-dependent origin of replication and RNA-selective markers can be utilized in current technology vectors. For example, any one of the eight orientations of Pol III-dependent origin of replication and RNA-selective markers in current technology vectors can be used. For example, in one embodiment, the orientation is ←Pol III origin of replication RSM→. For example, in one embodiment, the orientation is ←Pol III origin of replication ← RSM. For example, in one embodiment, the orientation is Pol III origin of replication → RSM→. For example, in one embodiment, the orientation is Pol III origin of replication → ← RSM. For example, in one embodiment, the orientation is ← RSM Pol III origin of replication→. For example, in one embodiment, the orientation is ← RSM ← Pol III origin of replication. For example, in one embodiment, the orientation is RSM → Pol III origin of replication→. For example, in one embodiment, the orientation is RSM → ← Pol III origin of replication.

[0506] In addition, a variety of RNA-selective markers known in the art can replace RNA-OUT.

[0507] In addition, antibiotic resistance markers can replace RNA-OUT, for example, in situations where it is desirable to simply modify the pUC origin to an R6K origin to improve plasmid production yield and / or quality.

[0508] Therefore, readers will see that the improved Pol III-dependent origin of replication vectors of the present technology provide a method for reducing transfection-related toxicity, improving transposition from non-viral transposon vectors, improving packaging titers of viral vectors, improving expression of viral and non-viral vector-encoded genes, and eliminating viral and non-viral vector-mediated transfer of antibiotic selection marker genes (i.e., by incorporating bacterial regions preferably less than 1000 bp), while significantly improving manufacturing compared to alternative vectors such as pUC plasmids and microcircles.

[0509] Therefore, the scope of this disclosure should not be determined by the illustrated embodiments, but by the appended claims.

Claims

1. A covalently closed circular recombinant DNA molecule comprising a backbone and an insert, wherein the backbone comprises a Pol III-dependent origin of replication, a selectivity marker, and a first priming assembly site, wherein the first priming assembly site is located downstream of the Pol III-dependent origin of replication in the replication direction, and wherein the insert comprises a structured DNA sequence.

2. The covalently closed circular recombinant DNA molecule according to claim 1, wherein the structured DNA sequence is less than 1000 bp away from the Pol III-dependent replication origin.

3. The covalently closed circular recombinant DNA molecule according to any one of claims 1 to 2, wherein the backbone is less than 1000 bp.

4. The covalently closed circular recombinant DNA molecule according to any one of claims 1 to 3, wherein the backbone comprises a bacterial replication selection region.

5. The covalently closed circular recombinant DNA molecule according to any one of claims 1 to 4, wherein the Pol III-dependent replication origin does not require Pol I.

6. The covalently closed circular recombinant DNA molecule according to any one of claims 1 to 5, wherein the Pol III-dependent origin of replication is a Pol III-dependent R6K origin of replication.

7. The covalently closed circular recombinant DNA molecule according to claim 6, wherein the Pol III-dependent R6K replication origin has at least 80% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO:

24.

8. The covalently closed circular recombinant DNA molecule according to any one of claims 1 to 7, wherein the selective marker is an RNA selective marker.

9. The covalently closed circular recombinant DNA molecule according to claim 8, wherein the RNA-selective marker is an RNA-OUT RNA-selective marker.

10. The covalently closed circular recombinant DNA molecule of claim 9, wherein the RNA-OUT RNA-selective marker is an RNA-IN regulatory RNA-OUT functional variant having at least 80% sequence identity with a sequence selected from SEQ ID NO: 5 and SEQ ID NO:

7.

11. The covalently closed circular recombinant DNA molecule of claim 9, wherein the RNA selective marker comprises a sequence having at least 80% sequence identity with SEQ ID NO:

6.

12. The covalently closed circular recombinant DNA molecule according to any one of claims 1 to 11, wherein the first primoron assembly site comprises a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO:

33.

13. The covalently closed circular recombinant DNA molecule according to any one of claims 1 to 12, further comprising a second primosome assembly site located downstream of the Pol III-dependent replication origin in the replication direction.

14. The recombinant DNA molecule of claim 13, wherein the second primoron assembly site comprises a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO:

33.

15. The covalently closed circular recombinant DNA molecule according to any one of claims 13 to 14, comprising a sequence having at least 80% sequence identity with SEQ ID NO: 29, such that the first primor assembly site and the second primor assembly site are located downstream of the Pol III-dependent replication origin in the replication direction.

16. The covalently closed circular recombinant DNA molecule according to any one of claims 13 to 14, comprising the sequence of SEQ ID NO: 29, such that the first primer assembly site and the second primer assembly site are located downstream of the Pol III-dependent replication origin in the replication direction.

17. The covalently closed circular recombinant DNA molecule according to any one of claims 1 to 16, wherein the covalently closed circular recombinant DNA molecule is free of antibiotic labeling.

18. The covalently closed circular recombinant DNA molecule according to any one of claims 1 to 3, wherein the backbone comprises a sequence having at least 80% sequence identity with a sequence selected from 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO:

28.

19. The covalently closed circular recombinant DNA molecule according to any one of claims 1 to 3, 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, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO:

28.

20. The covalently closed circular recombinant DNA molecule according to any one of claims 1 to 19, wherein the structured DNA sequence is selected from inverted repeat sequences, homologous repeat sequences, homopolymeric repeat sequences, eukaryotic origin of replication, and eukaryotic promoter enhancer sequences.

21. The covalently closed circular recombinant DNA molecule of claim 20, wherein the insert is a transposon vector.

22. The covalently closed circular recombinant DNA molecule of claim 21, wherein the structured DNA sequence is an inverted repeat sequence, a homologous repeat sequence, or a eukaryotic promoter enhancer sequence.

23. The covalently closed circular recombinant DNA molecule of claim 20, wherein the insert is a transposase vector.

24. The covalently closed circular recombinant DNA molecule of claim 20, wherein the insert is an mRNA vector.

25. The covalently closed circular recombinant DNA molecule of claim 20, wherein the insert is an AAV vector.

26. The covalently closed circular recombinant DNA molecule of claim 25, wherein the structured DNA sequence is an inverted repeat sequence.

27. The covalently closed circular recombinant DNA molecule according to any one of claims 25 to 26, wherein the AAV vector encodes AAV2 ITR.

28. The covalently closed circular recombinant DNA molecule according to any one of claims 25 to 26, wherein the insert comprises a 5' inverted terminal repeat sequence having a sequence having at least 80% sequence identity with SEQ ID NO: 35; and a 3' inverted terminal repeat sequence having a sequence having at least 80% sequence identity with SEQ ID NO:

36.

29. The covalently closed circular recombinant DNA molecule of claim 20, wherein the insert is a lentiviral vector.

30. The covalently closed circular recombinant DNA molecule of claim 29, wherein the structured DNA sequence is a homologous repeat sequence or a eukaryotic origin of replication.

31. The covalently closed circular recombinant DNA molecule of claim 20, wherein the structured DNA sequence is selected from: homopolymeric repeat sequences, such as polyadenylated (polyA) repeat sequences; SV40 origin of replication; viral LTR; lentiviral LTR; retroviral LTR; transposon IR / DR repeat sequences; Sleeping Beauty transposon IR / DR repeat sequences; AAVITR; CMV enhancer; and SV40 enhancer.

32. The covalently closed circular recombinant DNA molecule according to any one of claims 1 to 19, wherein the structured DNA sequence comprises a homopolymer repeat sequence.

33. The covalently closed circular recombinant DNA molecule according to claim 32, wherein the homopolymer repeat sequence is a polyadenylated repeat sequence.

34. The covalently closed circular recombinant DNA molecule according to any one of claims 32 to 33, wherein the homopolymer repeat sequence comprises about 3 to about 500 residues.

35. The covalently closed circular recombinant DNA molecule according to any one of claims 1 to 34, wherein the selective marker is an RNA selective marker and is transcribed in a direction opposite to the structured DNA sequence.

36. An antibiotic-free, covalently closed circular recombinant DNA molecule comprising a backbone and an insert, wherein the backbone comprises a replication origin and an RNA-selective marker, wherein the insert comprises a structured DNA sequence, and wherein the RNA-selective marker is oriented to be transcribed in the reverse direction of the structured DNA sequence.

37. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 36, wherein the structured DNA sequence is less than 1000 bp away from the origin of replication.

38. The antibiotic-free covalently closed circular recombinant DNA molecule according to any one of claims 36 to 37, wherein the backbone is less than 1000 bp.

39. The antibiotic-free covalently closed circular recombinant DNA molecule according to any one of claims 36 to 38, wherein the backbone comprises a bacterial replication selection region.

40. The antibiotic-free covalently closed circular recombinant DNA molecule according to any one of claims 36 to 39, wherein the origin of replication is a Pol I-dependent origin of replication.

41. The antibiotic-free covalently closed circular recombinant DNA molecule according to any one of claims 36 to 39, wherein the origin of replication is a Pol III-dependent origin of replication.

42. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 41, wherein the PolIII-dependent replication origin does not require Pol I.

43. The antibiotic-free covalently closed circular recombinant DNA molecule according to any one of claims 41 to 42, wherein the Pol III-dependent origin of replication is a Pol III-dependent R6K origin of replication.

44. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 43, wherein the PolIII-dependent R6K replication origin has at least 80% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO:

24.

45. The covalently closed circular recombinant DNA molecule according to any one of claims 36 to 45, wherein the RNA-selective marker is an RNA-OUT RNA-selective marker.

46. ​​The antibiotic-free covalently closed circular recombinant DNA molecule of claim 45, wherein the RNA-OUT RNA-selective marker is an RNA-IN regulatory RNA-OUT functional variant having at least 80% sequence identity with a sequence selected from SEQ ID NO: 5 and SEQ ID NO:

7.

47. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 45, wherein the RNA selective marker comprises a sequence having at least 80% sequence identity with SEQ ID NO:

6.

48. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 36, wherein the backbone comprises a sequence having at least 80% sequence identity with a sequence selected from 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO:

28.

49. The antibiotic-free covalently closed circular recombinant DNA molecule according to claim 36, The skeleton comprises a sequence selected from 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, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO:

28.

50. The antibiotic-free covalently closed circular recombinant DNA molecule according to any one of claims 36 to 49, wherein the structured DNA sequence is selected from inverted repeat sequences, homologous repeat sequences, homopolymeric repeat sequences, eukaryotic origin of replication, and eukaryotic promoter enhancer sequences.

51. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 50, wherein the insert is a transposon vector.

52. The antibiotic-free covalently closed circular recombinant DNA molecule according to claim 51, wherein the structured DNA sequence is an inverted repeat sequence, a homologous repeat sequence, or a eukaryotic promoter enhancer sequence.

53. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 50, wherein the insert is a transposase vector.

54. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 50, wherein the insert is an mRNA vector.

55. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 50, wherein the insert is an AAV vector.

56. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 55, wherein the structured DNA sequence is an inverted repeat sequence.

57. The antibiotic-free covalently closed circular recombinant DNA molecule according to any one of claims 55 to 56, wherein the AAV vector encodes AAV2 ITR.

58. The antibiotic-free covalently closed circular recombinant DNA molecule according to any one of claims 55 to 56, wherein the insert comprises a 5' inverted terminal repeat sequence having a sequence having at least 80% sequence identity with SEQ ID NO: 35; and a 3' inverted terminal repeat sequence having a sequence having at least 80% sequence identity with SEQ ID NO:

36.

59. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 50, wherein the insert is a lentiviral vector.

60. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 59, wherein the structured DNA sequence is a homologous repeat sequence or a eukaryotic origin of replication.

61. The antibiotic-free covalently closed circular recombinant DNA molecule according to claim 50, wherein the structured DNA sequence is selected from polyadenylated repeat sequences, SV40 origin of replication, viral LTR, lentiviral LTR, retroviral LTR, transposon IR / DR repeat sequences, Sleeping Beauty transposon IR / DR repeat sequences, AAV ITR, CMV enhancer, and SV40 enhancer.

62. The antibiotic-free covalently closed circular recombinant DNA molecule according to any one of claims 36 to 49, wherein the structured DNA sequence comprises a homopolymer repeat sequence.

63. The antibiotic-free covalently closed circular recombinant DNA molecule according to claim 62, wherein the homopolymer repeat sequence is a polyadenylated repeat sequence.

64. The antibiotic-free covalently closed circular recombinant DNA molecule according to any one of claims 62 to 63, wherein the homopolymer repeat sequence comprises about 3 to about 500 residues.

65. The antibiotic-free covalently closed circular recombinant DNA molecule according to any one of claims 36 to 64, further comprising a first primordium assembly site located downstream of the origin of replication in the direction of replication.

66. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 65, further comprising a second primosome assembly site located downstream of the origin of replication in the replication direction.

67. The antibiotic-free covalently closed circular recombinant DNA molecule according to any one of claims 65 to 66, wherein the first primoron assembly site comprises a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO:

33.

68. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 66, wherein the first priming assembly site comprises a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 33, and wherein the second priming assembly site comprises a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO:

33.

69. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 65, comprising a sequence having at least 80% sequence identity with SEQ ID NO: 29, such that the first primordium assembly site is located downstream of the origin of replication in the replication direction.

70. The antibiotic-free covalently closed circular recombinant DNA molecule of claim 66, comprising a sequence having at least 80% sequence identity with SEQ ID NO: 29, such that the first primoron assembly site and the second primoron assembly site are located downstream of the origin of replication in the replication direction.

71. A method for replicating a covalently closed circular recombinant DNA molecule, comprising the following steps: a. Providing a cell containing a recombinant DNA molecule according to any one of claims 1 to 70; and b. subject the cells to a fermentation process.

72. The method of claim 71, wherein the cell is an engineered strain of *Escherichia coli* expressing the Rep protein.

73. The method according to any one of claims 71 to 72, wherein the cell contains a chromosome-integrated arabinose-inducible CI857ts gene.

74. The method according to any one of claims 71 to 73, wherein the Rep protein comprises the following mutations: P42L; P106I; F107S; and P113S.

75. The method according to any one of claims 71 to 74, wherein the fermentation process comprises growing the cells in a culture medium containing arabinose.

76. The method according to any one of claims 71 to 75, wherein after the fermentation process, the yield of the covalently closed circular plasmid exceeds 0.5 g / L.

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