VIRAL AND NON-VIRAL NANOPLASMID VECTORS WITH IMPROVED PRODUCTION
20210010021 ยท 2021-01-14
Inventors
Cpc classification
C12N2740/15052
CHEMISTRY; METALLURGY
C12N2750/14152
CHEMISTRY; METALLURGY
C12N2740/15043
CHEMISTRY; METALLURGY
C12N2750/14143
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
C12N2800/24
CHEMISTRY; METALLURGY
International classification
Abstract
A method for improving the replication of a covalently closed circular plasmid is provided. The method includes providing a covalently closed circular plasmid having a Pol I-dependent origin of replication, and an insert including a structured DNA sequence selected from the group consisting of inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication or eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is located at a distance of less than 1000 by from the Pol I-dependent origin of replication in the direction of replication. The method also includes modifying the covalently closed circular recombinant molecule such that the Poi I-dependent origin of replication is replaced with a Pol III-dependent origin of replication, whereby the resultant Pol III-dependent origin of replication covalently closed circular plasmid has improved replication. An antibiotic marker free covalently closed circular recombinant DNA molecule is also provided.
Claims
1. A method for replicating a covalently closed circular plasmid comprising the following steps: a. providing a cell containing a covalently closed circular plasmid, the covalently closed circular plasmid comprising: i. a backbone comprising a Pol III-dependent R6K origin of replication and an RNA-OUT RNA selectable marker, wherein the backbone is less than 1000 bp, and ii. an insert comprising a structured DNA sequence; and b. subjecting the cell to a fermentation process.
2. The method of claim 1, wherein the cell is an engineered Rep protein-expressing E. coli strain.
3. The method of claim 2, wherein the cell comprises a chromosomally-integrated arabinose inducible CI857ts gene.
4. The method of claim 3, wherein Rep protein includes the following mutations: P42L; P106I; F107S; and P113S.
5. The method of claim 1, wherein the structured DNA sequence is selected from the group consisting of an inverted repeat sequence, a direct repeat sequence, a homopolymeric repeat sequence, an eukaryotic origin of replication, and an eukaryotic promoter enhancer sequence.
6. The method of claim 1, wherein the insert is a transposon vector.
7. The method of claim 6, wherein the structured DNA sequence is an inverted repeat sequence, a direct repeat sequence, or an eukaryotic promotor enhancer sequence.
8. The method of claim 1, wherein the insert is a transposase vector.
9. The method of claim 1, wherein the insert is an AAV vector.
10. The method of claim 9, wherein the structured DNA sequence is an inverted repeat sequence.
11. The method of claim 1, wherein the insert is a lentiviral vector.
12. The method of claim 11, wherein the structured DNA sequence is a direct repeat sequence or an eukaryotic origin of replication,
13. The method of claim 1, wherein the Pol III-dependent R6K origin of replication possesses at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18.
14. The method of claim 1, wherein the RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5, and SEQ NO: 7.
15. The method of claim 1, wherein the structured DNA sequence is selected from the group consisting of a polyA repeat, a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, an AAV a CMV enhancer, and a SV40 enhancer.
16. The method of claim 1, wherein the fermentation process comprises growing the cells in media containing arabinose.
17. The method of claim 1, wherein the cell is an engineered Rep protein-expressing E. coli strain and comprises a chromosomally-integrated arabinose inducible CI857ts gene, wherein the structured DNA sequence is selected from the group consisting of a polyA repeat, a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer, wherein the Pol III-dependent R6K origin of replication possesses at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18, wherein the RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5, and SEQ ID NO: 7.
18. The method of claim 17, wherein the yield of the covalently closed circular plasmid following the fermentation process is in excess of 0.5 g/L.
19. An engineered E. Coli cell comprising one or more copies of a chromosomally-integrated arabinose-inducible CI857ts gene.
20. The engineered E. Coli cell of claim 19 further comprising a mutated Rep protein, wherein the mutated Rep protein comprises the following mutations: P42L; P106I; F107S; and P113S.
21. The engineered E. Coli cell of claim 20 further comprising a covalently closed circular plasmid, the covalently closed circular plasmid comprising a backbone comprising a Pol III-dependent R6K origin of replication and an RNA-OUT RNA selectable marker, wherein the backbone is less than 1000 bp.
22. The engineered E. Coli cell of claim 19 further comprising a covalently closed circular plasmid, the covalently closed circular plasmid comprising a backbone comprising a Pol III-dependent R6K origin of replication and an RNA-OUT RNA selectable marker, wherein the backbone is less than 1000 bp.
23. The engineered E. Coli cell of claim 22, wherein the Pol III-dependent R6K origin of replication possesses at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18.
24. The engineered E. Coli cell of claim 22, wherein the RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5, and SEQ ID NO: 7.
25. A covalently closed circular plasmid comprising a backbone and an insert, the backbone comprising a Pol III-dependent R6K origin of replication and an RNA-OUT RNA selectable marker, wherein the backbone is less than 1000 bp, and the insert comprising a structured DNA sequence.
26. The covalently closed circular plasmid of claim 25, wherein the Pol III-dependent R6K origin of replication possesses at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3. SEQ ID NO: 4, and SEQ ID NO: 18.
27. The covalently closed circular plasmid of claim 25, wherein the RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5, and SEQ ID NO: 7.
28. The covalently closed circular plasmid of claim 25, wherein the structured DNA sequence is selected from the group consisting of a polyA repeat, a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer.
29. The covalently closed circular plasmid of claim 25, wherein the insert is a transposon vector.
30. The covalently closed circular plasmid of claim 29, wherein the structured DNA sequence is an inverted repeat sequence, a direct repeat sequence, or an eukaryotic promotor enhancer sequence.
31. The covalently closed circular plasmid of claim 25, wherein the insert is a transposase vector.
32. The covalently closed circular plasmid of claim 25, wherein the insert is an AAV vector.
33. The covalently closed circular plasmid of claim 32, wherein the structured DNA sequence is an inverted repeat sequence.
34. The covalently closed circular plasmid of claim 25, wherein the insert is a lentiviral vector.
35. The covalently closed circular plasmid of claim 34, wherein the structured DNA sequence is a direct repeat sequence or an eukaryotic origin of replication.
36. The covalently closed circular plasmid of claim 25, wherein the Pol III-dependent R6K origin of replication possesses at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3. SEQ ID NO: 4, and SEQ ID NO: 18, and wherein the RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5, and SEQ ID NO: 7.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0103]
[0104]
[0105]
[0106]
[0107] Table 1: pVAX1 mammalian expression vector spacer region (SR) derivatives expression level
[0108] Table 2: NTC8685 mammalian expression vector spacer region (SR) derivatives expression level
[0109] Table 3: Minicircle applications with various viral and non-viral vector platforms
[0110] Table 4: pNTC multiple cloning site flanked R6K Origin-RNA-OUT selection marker vectors
[0111] Table 5: SV40 origin Lentiviral vectors: pUC versus R6K origin shake flask production yields/quality
[0112] Table 6: Sleeping Beauty Transposon vectors: pUC versus R6K origin shake flask production yields/quality
[0113] Table 7: AAV vectors: pUC versus R6K origin shake flask production yields/quality
[0114] Table 8: mRNA vectors: pUC versus R6K origin DH5 HyperGRO fermentation yields/quality
[0115] Table 9: AAV helper vectors: pUC versus R6K origin plasmid production yields/quality
[0116] SEQ ID NO:1: R6K gamma origin
[0117] SEQ ID NO:2: 1 CpG R6K gamma origin
[0118] SEQ ID NO:3: CpG free R6K gamma origin
[0119] SEQ ID NO:4: Extended R6K gamma origin
[0120] SEQ ID NO:5: RNA-OUT Selectable Marker
[0121] SEQ ID NO:6: RNA-OUT antisense repressor RNA
[0122] SEQ ID NO:7: 2 CpG RNA-OUT Selectable Marker
[0123] SEQ ID NO:8: R6K gamma origin-RNA-OUT bacterial region flanked by NheI and KpnI restriction sites
[0124] SEQ ID NO:9: 1 CpG R6K gamma origin-2 CpG RNA-OUT bacterial region flanked by NheI and KpnI restriction sites
[0125] SEQ ID NO:10: pNTC-NP1 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI/HindIII
[0126] SEQ ID NO:11: pNTC-NP2 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI/HindIII
[0127] SEQ ID NO:12: pNTC-NP3 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI/HindIII
[0128] SEQ ID NO:13: pNTC-NP4 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI/HindIII
[0129] SEQ ID NO:14: pNTC-NP5 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: KasI/HindIII
[0130] SEQ ID NO:15: pNTC-NP6 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI/SacI
[0131] SEQ ID NO:16: pNTC-NP7 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: BssHII-BssHII
[0132] SEQ ID NO:17: pNTC-3xCpG NP1 polylinker R6K-RNA-OUT polylinker cloning cassette: HindIII-EcoRI
[0133] SEQ ID NO:18: R6K gamma origin (7 iteron)
[0134] SEQ ID NO:19: R6K gamma origin 22 bp iteron repeat
[0135] SEQ ID NO:20: R6K gamma origin 22 bp iteron repeat
[0136] SEQ ID NO:21: R6K gamma origin 22 bp iteron repeat
[0137] SEQ ID NO:22: R6K gamma origin 22 bp iteron repeat
[0138] SEQ ID NO:23: R6K gamma origin 22 bp iteron repeat
DEFINITION OF TERMS
[0139] AAV vector: Adeno-associated virus vector, an episomal viral vector. Includes self-complementary (sc) Adeno-associated virus vectors (scAAV) and single-stranded (ss) Adeno-associated virus vectors (ssAAV)
[0140] AF: Antibiotic-free
[0141] amp: Ampicillin
[0142] ampR: Ampicillin Resistance gene
[0143] Antibiotic selectable marker: A gene that confers resistance to an antibiotic, e.g. ampicillin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene
[0144] Approximately: As used herein, the term approximately or about, as applied to one or more values of interest, refers to a value that is the same or similar to a stated reference value [0145] Bacterial region: Region of a plasmid vector required for propagation and selection in the bacterial host
[0146] bp: basepairs
[0147] ccc: Covalently Closed Circular
[0148] cI: Lambda repressor
[0149] cITs857: Lambda repressor further incorporating a C to T (Ala to Thr) mutation that confers temperature sensitivity. cITs857 is a functional repressor at 28-30 C. but is mostly inactive at 37-42 C. Also called cI857
[0150] Cat.sup.R: Chloramphenicol resistance gene
[0151] cmv: Cytomegalovirus
[0152] dem methylation: E. coli methyltransferase that methylated the sequences CC(A/T)GG at the C5 position of the second cytosine
[0153] DNA replicon: A genetic element that can replicate under its own control; examples include plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof
[0154] E. coli: Escherichia coli, a gram negative bacteria
[0155] EGFP: Enhanced green fluorescent protein
[0156] EP: Electroporation
[0157] Eukaryotic expression vector: A vector for expression of mRNA, protein antigens, protein therapeutics, shRNA, RNA or microRNA genes in a target eukaryotic organism using RNA Polymerase I, II or III promoters
[0158] Eukaryotic region: The region of a plasmid that encodes eukaryotic sequences and/or sequences required for plasmid function in the target organism. This includes the region of a plasmid vector required for expression of one or more transgenes in the target organism including RNA Pol II enhancers, promoters, transgenes and polyA sequences. This also includes the region of a plasmid vector required for expression of one or more transgenes in the target organism using RNA Pol I or RNA Pol II promoters, RNA Pol I or RNA Pol III expressed transgenes or RNAs. The eukaryotic: region may optionally include other functional sequences, such as eukaryotic transcriptional terminators, supercoiling-induced DNA duplex destabilized (SIDD) structures, S/MARs, boundary elements, etc. In a Lentiviral or Retroviral vector, the eukaryotic region contains flanking direct repeat LTRs, in a AAV vector the eukaryotic region contains flanking inverted terminal repeats, while in a Transposon vector the eukaryotic region contains flanking transposon inverted terminal repeats or IR/DR termini (e.g. Sleeping Beauty). In genome integration vectors, the eukaryotic region may encode homology arms to direct targeted integration
[0159] Exon: A nucleotide sequence encoded by a gene that is transcribed and present within a mature mRNA product after RNA splicing to remove introns has been completed
[0160] Expression vector: A vector for expression of mRNA, protein antigens, protein therapeutics, shRNA, RNA or microRNA genes in a target organism.
[0161] g: Gram, kg for kilogram
[0162] gene of interest: gene to be expressed in the target organism. Includes mRNA genes that encode protein or peptide antigens, protein or peptide therapeutics, and mRNA, shRNA, RNA or microRNA that encode RNA therapeutics, and mRNA, shRNA, RNA or microRNA that encode RNA vaccines, etc.
[0163] Hr(s): Hour(s)
[0164] ID: Intradermal
[0165] IM: Intramuscular
[0166] immune response: Antigen reactive cellular (e.g. antigen reactive T cells) or antibody (e.g. antigen reactive IgG) responses
[0167] Introit A nucleotide sequence encoded by a gene that is transcribed and subsequently removed from a mature mRNA product by RNA splicing
[0168] IR/OR: Inverted Repeats which are each Directly Repeated twice, For example, Sleeping Beauty transposon IR/DR repeats
[0169] Iteron: Directly repeated DNA sequences in a origin of replication that are required for replication initiation. R6K origin iteron repeats are 22 bp
[0170] ITR: Inverted Terminal Repeat
[0171] kan: Kanamycin
[0172] kanR: Kanamycin Resistance gene
[0173] Kd: Kilodalton
[0174] kozak sequence: Optimized consensus DNA sequence gccRccATG (R=G or A) immediately upstream of an ATG start codon that ensures efficient translation initiation. A SalI site (GTCGAC) immediately upstream of the ATG start codon (GTCGACATG) is an effective kozak sequence
[0175] Lentiviral vector: Integrative viral vector that can infect dividing and non-dividing cells. Also call Lentiviral transfer plasmid. Plasmid encodes Lentiviral LTR flanked expression unit. Transfer plasmid is transfected into production cells along with Lentiviral envelope and packaging plasmids required to make viral particles
[0176] Lentiviral envelope vector: Plasmid encoding envelope glycoprotein
[0177] Lentiviral packaging vector: One or two plasmids that express gag, pol and Rev functions required to package the Lentiviral transfer vector
[0178] minicircle: Covalently closed circular plasmid derivatives in which the bacterial region has been removed from the parent plasmid by in vivo or in vitro site-specific recombination or in vitro restriction digestion/ligation. Minicircle vectors are replication incompetent in bacterial cells
[0179] mRNA: Messenger RNA
[0180] mSEAP: Murine secreted alkaline phosphatase
[0181] NA: Not Applicable
[0182] Nanoplasmid vector: Vector combining an RNA selectable marker with a R6K, ColE2. or ColE2 related replication origin. For example, NTC9385C, NTC9685C, NTC9385R, NTC9685R vectors and modifications described in Williams, Supra, 2014 and included herein by reference
[0183] NTC8385: NTC8385, NTC8485 and NTC8685 plasmids are antibiotic-free pUC origin vectors that contain a short RNA (RNA-OUT) selectable marker instead of an antibiotic resistance marker such as kanR. The creation and application of these RNA-OUT based antibiotic-free vectors are described in Williams, J A 2008 World Patent Application WO2008153733 and included herein by reference
[0184] NTC8485: NTC8485 is an antibiotic-free pUC origin vector that contains a short RNA (RNA-OUT) selectable marker instead of an antibiotic resistance marker such as kanR. The creation and application of NTC8485 is described in Williams, J A. 2010 US Patent Application 20100184158 and included herein by reference
[0185] NTC8685: NTC8685 is an antibiotic-free pUC origin vector that contains a short RNA (RNA-OUT) selectable marker instead of an antibiotic resistance marker such as kanR. The creation and application of NTC8685 is described in Williams, Supra, 2010 and included herein by reference [0186] NTC9385R: The NTC9385R Nanoplasmid vector described in Williams, Supra, 2014 included herein by reference has a spacer region encoded NheI-trpA terminator-R6K origin RNA-OUT-KpnI bacterial region (SEQ ID NO:8) linked through the flanking NheI and KpnI sites to the eukaryotic region.
[0187] OD.sub.600: optical density at 600 nm
[0188] PAS: Primosomal assembly site, Priming of DNA synthesis on a single stranded. DNA ssi site. X174 type PAS: DNA hairpin sequence that binds priA, which, in turn, recruits the remaining proteins to form the preprimosome [priB, dnaT, recruits dnaB (delivered by dnaC)], which then also recruits primase (dnaG), which then, finally, makes a short RNA substrate for DNA polymerase I. ABC type PAS: DNA hairpin binds dnaA, recruits dnaB (delivered by dnaC) which then also recruits primase (dnaG), which then, finally, makes a short RNA substrate for DNA polymerase I. For example, the R6K plasmid CpG free ssiA primosomal assembly site or alternative XL74 type or ABC type primosomal assembly sites
[0189] PAS-BH: Primosomal assembly site on the heavy (leading) strand
[0190] PAS-BH region: pBR322 origin region between ROP and PAS-BL (approximately pBR322 2067-2351)
[0191] PAS-BL: Primosomal assembly site on the light (lagging) strand
[0192] PBS: Phosphate buffered Saline
[0193] PCR: Polymerase Chain Reaction
[0194] pDNA: Plasmid DNA
[0195] PiggyBac Transposon: PB transposon. A transposon system that integrates an ITR flanked PB transposon into the genome by a simple cut and paste mechanism mediated by PB transposase. The transposon vector typically contains a promoter-transgene-polyA expression cassette between the PB LTRs which is excised and integrated into the genome
[0196] pINT pR pL vector: The pINT pR pL att.sub.HK022 integration expression vector is described in Luke et al., 2011 Mol Biotechnol 47:43 and included herein by reference. The target gene to be expressed is cloned downstream of the pL promoter. The vector encodes the temperature inducible cI857 repressor, allowing heat inducible target gene expression
[0197] P.sub.L promoter: Lambda promoter left. P.sub.L is a strong promoter that is repressed by the cI repressor binding to OL1, OL2 and OL3 repressor binding sites. The temperature sensitive cI857 repressor allows control of gene expression by heat induction since at 30 C. the cI857 repressor is functional and it represses gene expression, but at 37-42 C. the repressor is inactivated so expression of the gene ensues
[0198] P.sub.L (OL1 G to T) promoter: Lambda promoter left. P.sub.L is a strong promoter that is repressed by the cI repressor binding to OL1, OL2 and OL3 repressor binding sites. The temperature sensitive cI857 repressor allows control of gene expression by heat induction since at 30 C. the cI857 repressor is functional and it represses gene expression, but at 37-42 C. the repressor is inactivated so expression of the gene ensues. The cI repressor binding to OL1 is reduced by the OL1 G to T mutation resulting in increased promoter activity at 30 C. and 37-42 C. as described in Williams, Supra, 2014.
[0199] Plasmid: An extra chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently from the chromosomal DNA
[0200] Plasmid copy number: the number of copies of a plasmid per cell. Increases in plasmid copy number increase plasmid production yield
[0201] Pol: Polymerase
[0202] Pol I: Escherichia coli DNA Polymerase
[0203] Pol I dependent origin of replication: A replication origin that requires Pol I, for example the pMB1, ColE1 or pBR322 or derivatives such as the high copy pUC origin. For these origins the RNAII primer forms an RNA: DNA R-loop that is cleaved by RNase H to create a primer for DNA pol I directed DNA synthesis. DNA synthesis then converts to DNA pol III. Numerous additional Pol I dependent replication origins are known in the art, many of which are summarized in del Solar et al., 1998 Microbiol. Mol. Biol. Rev 62:434-464 which is included herein by reference
[0204] Pol III: Escherichia coli DNA Polymerase Ill
[0205] Pol III dependent origin of replication: A replication origin that doesn't require Pol I, for example the rep protein dependent R6K gamma replication origin. Numerous additional Pol III dependent replication origins are known in the art, many of which are summarized in del Solar et al., Supra, 1998 which is included herein by reference
[0206] polyA: Polyadenylation signal or site. Polyadenylation is the addition of a poly(A) tail to an RNA molecule. The polyadenylation signal contains the sequence motif recognized by the RNA cleavage complex. Most human polyadenylation signals contain an AAUAAA motif and conserved sequences 5 and 3 to it. Commonly utilized polyA signals are derived from the rabbit globin, bovine growth hormone, SV40 early, or SV40 late polyA signals
[0207] pUC origin: pBR322-derived replication origin, with G to A transition that increases copy number at elevated temperature and deletion of the ROP negative regulator
[0208] pUC free: Plasmid that does not contain the pUC origin. Non-replicative fragments of the pUC origin may be included, for example the RNAI selectable marker
[0209] pUC plasmid: Plasmid containing the pUC origin
[0210] R6K plasmid: 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 modifications and alternative vectors containing a R6K replication origin that were described in Williams, Supra, 2014 and included herein by reference. Alternative R6K vectors known in the art including, but not limited to, pCOR vectors (Gencell), pCpGfree vectors (Invivogen), and CpG free University of Oxford vectors including pGM169
[0211] R6K replication origin: a region which is specifically recognized by the R6K Rep protein to initiate DNA replication. Includes but not limited to R6K gamma replication origin sequence disclosed as SEQ ID NO:1, SEQ ID NO:2 SEQ ID NO:4, and SEQ ID NO:18. Also includes CpG free versions (e.g. SEQ ID NO:3) as described in Drocourt et al., U.S. Pat. No. 7,244,609 and incorporated herein by reference
[0212] R6K replication origin-RNA-OUT bacterial region: Contains a R6K replication origin for propagation and the RNA-OUT selectable marker (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)
[0213] Rep: Replication
[0214] Replication intermediates: Linear DNA fragments resulting from premature termination of plasmid replication
[0215] Rep protein dependent plasmid: A plasmid in which replication is dependent on a replication (Rep) protein provided in Trans. For example, R6K replication origin, ColE2-P9 replication origin and ColE2 related replication origin plasmids in which the Rep protein is expressed from the host strain genome. Numerous additional Rep protein dependent plasmids are known in the art, many of which are summarized in del Solar et al., Supra, 1998 which is included herein by reference
[0216] Retroviral vector: Integrative viral vector that can infect dividing cells. Also call transfer plasmid. Plasmid encodes Retroviral LTR flanked expression unit. Transfer plasmid is transfected into production cells along with envelope and packaging plasmids required to make viral particles
[0217] Retroviral envelope vector: Plasmid encoding envelope glycoprotein
[0218] Retroviral packaging vector: Plasmid that encodes Retroviral gag, pol genes required to package the Retroviral transfer vector
[0219] RNA-IN: Insertion sequence 10 (IS10) encoded RNA-IN, an RNA complementary and antisense to a portion of RNA RNA-OUT. When RNA-IN is cloned in the untranslated leader of a mRNA, annealing of RNA-IN to RNA-OUT reduces translation of the gene encoded downstream of RNA-IN
[0220] RNA-IN regulated selectable marker: A genomically expressed RNA-IN regulated selectable marker. In the presence of plasmid borne RNA-OUT antisense repressor RNA (SEQ ID NO:6), expression of a protein encoded downstream of RNA-IN is repressed. An RNA-IN regulated selectable marker is configured such that RNA-IN regulates either 1) a protein that is lethal or toxic to said cell per se or by generating a toxic substance (e.g. SacB), or 2) a repressor protein that is lethal or toxic to said bacterial cell by repressing the transcription of a gene that is essential for growth of said cell (e.g. murA essential gene regulated by RNA-IN tetR repressor gene). For example, genomically expressed RNA-IN-SacB cell lines for RNA-OUT plasmid selection/propagation are described in Williams, Supra, 2008 and included herein by reference. Alternative selection markers described in the art may be substituted for SacB
[0221] RNA-OUT: Insertion sequence 10 (IS10) encoded RNA-OUT, an antisense RNA that hybridizes to, and reduces translation of, the transposon gene expressed downstream of RNA-IN. The sequence of the RNA-OUT RNA (SEQ ID NO:6) and complementary RNA-IN SacB genomically expressed 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. Nat Chem Biol 8:447, including, but not limited to, the RNA-OUT A08/RNA-IN S49 pair, the RNA-OUT A08/RNA-IN S08 pair, and CpG free modifications of RNA-OUT A08 that modify the CG in the RNA-OUT 5 TTCGC sequence to a non-CpG sequence. An example of a CpG free RNA-OUT selection marker, in which the two CpG motifs in the RNA-OUT RNA (one of which is present in the RNA-IN complementary region) are removed, was described in Williams 2015. Replicative minicircle vectors with improved expression. US Patent Application US 2015/0275221 and included herein by reference. A multitude of alternative substitutions to remove the two CpG motifs (mutating each CpG to either CpA, CpC, CpT, ApG, GpG, or TpG) may be utilized to make a CpG free RNA-OUT
[0222] RNA-OUT Selectable marker: An RNA-OUT selectable marker DNA fragment including E. coli transcription promoter and terminator sequences flanking an RNA-OUT RNA. An RNA-OUT selectable marker, utilizing the RNA-OUT promoter and terminator sequences, that is flanked by DraIII and KpnI restriction enzyme sites, and designer genomically expressed RNA-IN-SacB cell lines for RNA-OUT plasmid propagation, are described in Williams, Supra, 2008 and included herein by reference. The RNA-OUT promoter and terminator sequences in SEQ ID NO: 5 that flank the RNA-OUT RNA (SEQ ID NO:6;
[0223] RNA polymerase II promoter: Promoter that recruits RNA Polymerase II to synthesize mRNAs, most small nuclear RNAs and microRNAs. For example, constitutive promoters such as the human or murine CMV promoter, elongation factor 1 (EF1) promoter, the chicken -actin promoter, the -actin promoter from other species, the elongation factor-1 (EF1 ) promoter, the phosphoglycerokinase (PGK) promoter, the Rous sarcoma virus (RSV) promoter, the human serum albumin (SA) promoter, the spleen focus-forming virus (SFFV) promoter, the -1 antitrypsin (AAT) promoter, the thyroxine binding globulin (TBG) promoter, the cytochrome P450 2E1 (CYP2E1) promoter, etc. The vectors may also utilize combination promoters such as the chicken -actin/CMV enhancer (CAG) promoter, the human or murine CMV-derived enhancer elements combined with the elongation factor 1 (EF1) promoters, CpG free versions of the human or murine CMV-derived enhancer elements combined with the elongation factor 1 (EF1) promoters the albumin promoter combined with an -fetoprotein MERII enhancer, etc., or the diversity of tissue specific or inducible promoters know in the art such as the muscle specific promoters muscle creatine kinase (MCK), and C5-12 or the liver-specific promoter apolipoprotein A-I (ApoAI), etc.
[0224] RNA polymerase III promoter: Promoter that recruits RNA Polymerase III to synthesize tRNAs, 5S ribosomal RNA, and other small RNAs. For example, Class I promoters such as the 5s rRNA promoter, Class II promoter such as tRNA promoters, Class III promoters such as the U6 small nuclear RNA promoter or the H1 nuclear RNase P promoter, etc.
[0225] RNA selectable marker: An RNA selectable marker is a plasmid borne expressed non-translated RNA that regulates a chromosomally expressed target gene to afford selection. This may be a plasmid borne nonsense suppressing tRNA that regulates a nonsense suppressible selectable chromosomal target as described by Crouzet J and Soubrier F 2005 U.S. Pat. No. 6,977,174 included herein by reference. This may also be a plasmid borne antisense repressor RNA, a non limiting list included herein by reference includes RNA-OUT that represses RNA-IN regulated targets (Williams, Supra, 2008), pMB1 plasmid origin encoded RNAI that represses RNAII regulated targets (Grabherr R, Pfaffenzeller I. 2006 US patent application US20060063232; Cranenburgh R M. 2009; U.S. Pat. No. 7,611,883), IncB plasmid pMU720 origin encoded RNAI that represses RNA II regulated targets (Wilson I W, Siemering K R, Praszkier J, Pittard A J. 1997. J Bacteriol 179:742-53), ParB locus Sok of plasmid R1 that represses Hok regulated targets, Flm locus FlmB of F plasmid that represses flmA regulated targets (Morsey M A, 1999 U.S. Pat. No. 5,922,583). An RNA selectable marker may be another natural antisense repressor RNAs known in the art such as those described in Wagner E G H, Altuvia S, Romby P. 2002. Adv Genet 46:361-98 and Franch T, and Gerdes K. 2000. Current Opin Microbial 3:159-64. An RNA selectable marker may also be an engineered repressor RNAs such as synthetic small RNAs expressed SgrS, MicC or MicF scaffolds as described in Na D, Yoo S M, Chung H, Park H, Park J H, Lee S Y. 2013. Nat Biotechnot 31:170-4. An RNA selectable marker may also be an engineered repressor RNA as part of a selectable marker that represses a target RNA fused to a target gene to be regulated such as SacB as described in Williams, Supra, 2015
[0226] ROP: Repressor of primer
[0227] RSM: RNA selectable marker
[0228] SacB: Structural gene encoding Bacillus subtilis levansucrase. Expression of SacB in gram negative bacteria is toxic in the presence of sucrose
[0229] SD: Standard deviation
[0230] SEAP: Secreted alkaline phosphatase
[0231] Selectable marker: A selectable marker, for example a kanamycin resistance gene or an RNA selectable marker
[0232] Selection marker: A selectable marker, for example a kanamycin resistance gene or an RNA selectable marker
[0233] SIDD: supercoiling-induced DNA duplex destabilized (SIDD) structures. These sites, when incorporated into a vector, may alter the susceptibility of other sequences within the vector to be destabilized. This can alter function. For example, addition of a SIDD site to an expression vector may reduce the helical destabilization of a promoter. This may increase or decrease promoter activity, depending on the promoter since some promoters have increased expression with promoter helical destabilization, while others will have reduced expression with promoter helical destabilization
[0234] shRNA: Short hairpin RNA
[0235] S/MAR: Scaffold/matrix attached region. Eukaryotic sequences that mediate DNA attachment to the nuclear matrix
[0236] Sleeping Beauty Transposon: SB transposon. A transposon system that integrates an IR/DR flanked SB transposon into the genome by a simple cut and paste mechanism mediated by SB transposase. The transposon vector typically contains a promoter-transgene-polyA expression cassette between the IR/DRs which is excised and integrated into the genome
[0237] Spacer region: As used herein, spacer region is the region linking the 5 and 3 ends of the eukaryotic region sequences. The eukaryotic region 5 and 3 ends are typically separated by the bacterial replication origin and bacterial selectable marker in plasmid vectors (bacterial region so many spacer regions consist of the bacterial region. In Pol III dependent origin of replication vectors of the invention, this spacer region preferably is less than 1000 bp
[0238] SR: Spacer region.
[0239] ssi: Single stranded initiation sequences
[0240] Structured DNA sequence: As used herein, a DNA sequence that is capable of forming replication inhibiting secondary structures (Mirkin and Mirkin, 2007. Microbiology and Molecular Biology Reviews 71:13-35). This includes but is not limited to inverted repeats, palindromes, direct repeats, IR/DRs, homopolymeric repeats or repeat containing eukaryotic promoter enhancers, or repeat containing eukaryotic origin of replications.
[0241] SV40 origin: Simian Virus 40 genomic DNA that contains the origin of replication
[0242] SV40 enhancer: Simian Virus 40 genomic DNA that contains the 72 bp and optionally the 21 bp enhancer repeats
[0243] target antigen: Immunogenic protein or peptide epitope, or combination of proteins and epitopes, against which an immune response can be mounted. Target antigens may by derived from a pathogen for infectious disease or allergy applications or derived from a host organism for applications such as cancer, allergy, or autoimmune diseases. Target antigens are well defined in the art. Some examples are described in Williams, Supra, 2008 and are included herein by reference
[0244] TE buffer: A solution containing approximately 10 mM Tris pH 8 and 1 mM EDTA
[0245] TetR: Tetracycline resistance gene
[0246] Tol2 Transposon: A transposon system that integrates an ITR flanked Tol2 transposon into the genome by a simple cut and paste mechanism mediated by Tol2 transposase. The transposon vector typically contains a promoter-transgene-polyA expression cassette between the Tol2 ITRs which is excised and integrated into the genome
[0247] Transcription terminator: Bacterial: A DNA sequence that marks the end of a gene or operon for transcription. This may be an intrinsic transcription terminator or a Rho-dependent transcriptional terminator. For an intrinsic terminator, such as the trpA terminator, a hairpin structure forms within the transcript that disrupts the mRNA-DNA-RNA polymerase ternary complex. Alternatively, Rho-dependent transcriptional terminators require Rho factor, an RNA helicase protein complex, to disrupt the nascent mRNA-DNA-RNA polymerase ternary complex. Eukaryotic: PolyA signals are not terminators, instead internal cleavage at PolyA sites leaves an uncapped 5 end on the 3 UTR RNA for nuclease digestion. Nuclease catches up to RNA Pol II and causes termination. Termination can be promoted within a short region of the poly A site by introduction of RNA Pol II pause sites (eukaryotic transcription terminator). Pausing of RNA Pol II allows the nuclease introduced into the 3 UTR mRNA after PolyA cleavage to catch up to RNA Pol II at the pause site. A nonlimiting list of eukaryotic transcription terminators know in the art include the C2x4 and the gastrin terminator. Eukaryotic transcription terminators may elevate mRNA levels by enhancing proper 3-end processing of mRNA
[0248] transfection: Method to deliver nucleic acids into cells [e.g. poly(lactide-co-glycolide) (PLGA), ISCOMs, liposomes, niosomes, virosomes, block copolymers, Plutonic block copolymers, chitosan, and other biodegradable polymers, microparticles, microspheres, calcium phosphate nanoparticles, nanoparticles, nanocapsules, nanospheres, poloxamine nanospheres, electroporation, nucleofection, piezoelectric permeabilization, sonoporation, iontophoresis, ultrasound, SQZ high speed cell deformation mediated membrane disruption, corona plasma, plasma facilitated delivery, tissue tolerable plasma, laser microporation, shock wave energy, magnetic fields, contactless magneto-permeabilization, gene gun, microneedles, microdermabrasion, hydrodynamic delivery, high pressure tail vein injection, etc] as known in the art and included herein by reference
[0249] Transgene: Gene of interest that is cloned into a vector for expression in a target organism
[0250] Transposase vector: A vector which encodes a transposase
[0251] Transposon vector: A vector which encodes a transposon which is a substrate for transposase mediated gene integration
[0252] ts: Temperature sensitive
[0253] g: Microgram
[0254] l: Microliter
[0255] UTR: Untranslated region of a mRNA (5 or 3 to the coding region)
[0256] Vector: A gene delivery vehicle, including viral (e.g. Alphavirus, Poxvirus, Lentivirus, Retrovirus, Adenovirus, Adenovirus related virus, etc.) and non-viral (e.g. plasmid, MIDGE, transcriptionally active PCR fragment, minicircles, bacteriophage, etc.) vectors. These are well known in the art and are included herein by reference
[0257] Vector backbone: Eukaryotic region and bacterial region of a vector, without the transgene or target antigen coding region
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0258] The current technology relates generally to short <1 kb bacterial region plasmid DNA vector methods and compositions that improve plasmid manufacture yield and quality, reduce transfection associated toxicity, and increase transgene expression. The current technology can be practiced to improve expression and manufacturing of vectors such as non-viral vectors (transposon vector, transposase vector, Sleeping Beauty transposon vector, Sleeping Beauty transposase vector, PiggyBac transposon vector, PiggyBac transposase vector, expression vector, etc.) and viral vectors (e.g. AAV vector, AAV rep cap vector, AAV helper vector, Ad helper vector, Lentivirus vector, Lentiviral envelope vector, Lentiviral packaging vector, Retroviral vector, Retroviral envelope vector, Retroviral packaging vector, etc.).
[0259] Improved plasmid expression is defined herein as improved transgene expression level and/or expression duration in vitro or in vivo compared to a transgene encoding plasmid containing a bacterial region encoding the pUC replication origin. It is to be understood that all references cited herein are incorporated by reference in their entirety.
[0260] The methods of plasmid modification of the present current technology have been surprisingly found to provide a solution to provide short spacer region vectors containing structured DNA sequences with efficient high yield manufacture.
[0261] As used herein, the term sequence identity refers to the degree of identity between any given query sequence, e.g. SEQ ID NO: 2, and a subject sequence. A subject sequence may, for example, have at least 90 percent, at least 95 percent, or at least 99 percent sequence identity to a given query sequence. To determine percent sequence identity, a query sequence (e.g. a nucleic acid sequence) is aligned to one or more subject sequences using any suitable sequence alignment program that is well known in the art, for instance, the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid sequences to be carried out across their 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 the best match between a query and one or more subject sequences, and aligns them so that identities, similarities, and differences can be determined. Gaps of one or more nucleotides can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pair-wise alignments of nucleic acid sequences, suitable default parameters can be selected that are appropriate for the particular alignment program. The output is a sequence alignment that reflects the relationship between sequences. To further determine percent identity of a subject nucleic acid sequence to a query sequence, the sequences are aligned using the alignment program, the number of identical matches in the alignment is divided by the length of the query sequence, and the result is multiplied by 100. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
[0262] Turning now to the drawings,
[0263]
[0264]
[0265]
EXAMPLES
[0266] The methods of the current technology are further illustrated by the following examples. These are provided by way of illustration and are not intended in any way to limit the scope of the disclosure.
Example 1: pUC, and R6K Replication Origin Plasmid Replication and Production
[0267] pUC origin vector replication and production background: The vast majority of therapeutic plasmids use the pUC origin which is a high copy derivative of the pMBI 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 up derivative of the pMB1 origin that deletes the accessory ROP (rom) protein and has an additional temperature sensitive mutation that destabilizes the RNAI/RNAII interaction. Shifting of a culture containing these origins from 30 to 42 C. leads to an increase in plasmid copy number. pUC plasmids can be produced in a multitude of E. coli cell lines.
[0268] RNA-OUT antibiotic free selectable marker background: Antibiotic-free selection is performed in E. coli strains containing phage lambda attachment site chromosomally integrated pcAH63-CAT RNA-IN-SacB (P5/6 6/6) as described in Williams, Supra, 2008. SacB (Bacillus subtilis levansucrase) is a counterselectable marker which is lethal to E. coli cells in the presence of sucrose. Translation of SacB from the RNA-IN-SacB transcript is inhibited by plasmid encoded RNA-OUT (
[0269] R6K origin vector replication and production background: The R6K gamma plasmid replication origin requires a single plasmid replication protein that binds as a replication initiating monomer to multiple repeated iteron sites (seven core repeats containing TGAGNG consensus) and as a replication inhibiting dimer to repressive sites (TGAGNG) and to iterons with reduced affinity. Replication requires multiple host factors including IHF, DnaA, and primosomal assembly proteins DnaB, DnaC, DnaG (Abhyankar et al., 2003 J Biol Chem 278:45476-45484). The R6K core origin contains binding sites for DnaA and IHF that affect plasmid replication since , IHF and DnaA interact to initiate replication.
[0270] Different versions of the R6K gamma replication origin have been utilized in various eukaryotic expression vectors, for example pCOR vectors (Soubrier et al., 1999, Gene Therapy 6:1482-88) and a CpG free version in pCpGfree vectors (Invivogen, San Diego Calif.), and pGM169 (University of Oxford). Incorporation of the R6K replication origin per se does not improve transgene expression levels compared to an optimized pUC origin vector (Soubrier et al., Supra, 1999). However, use of a conditional replication origin such as R6K gamma that requires a specialized cell line for propagation adds a safety margin since the vector will not replicate if transferred to a patient's endogenous flora.
[0271] A highly minimalized 6 iteron R6K gamma derived replication origin (SEQ ID NO:1;
[0272] Typical R6K production strains express from the genome the protein derivative PIR116 that contains a P106L substitution that increases copy number (by reducing dimerization; monomers activate while dimers repress). Fermentation results with pCOR (Soubrier et al., Supra, 1999) and pCpG plasmids (Hebei H L, Cai Y. Davies L A, Hyde S C, Pringle I A, Gill D R. 2008. Mal Ther 16: S110) were low, around 100 mg/L in PIR116 cell lines.
[0273] Mutagenesis of the pir-116 replication protein and selection for increased copy number has been used to make new production strains. For example, the TEX2pir42 strain contains a combination of P106L and P42L. The P42L mutation interferes with DNA looping replication repression. The TEX2pir42 cell line improved copy number and fermentation yield with pCOR plasmids with reported yields of 205 mg/L (Soubrier F. 2004. World Patent Application WO2004033664).
[0274] Other combinations of copy number mutants that improve copy number include P42L and P113S and P42L, P106L and F107S (Ahhvankar et al., 2004, J Biol Chem 279:6711-6719).
[0275] Williams, Supra, 2014 describes host strains expressing phage HK022 attachment site integrated pL promoter heat inducible P42L, P106L and F107S high copy mutant replication (Rep) protein for selection and propagation of R6K origin Nanoplasmid vectors. This is an additional Nanoplasmid safety factor since R6K origin vectors can only replicate within the engineered Rep protein-expressing E. coli host strain.
[0276] RNA-OUT selectable marker-R6K plasmid propagation and fermentations described in Williams, Supra, 2014 were performed using heat inducible P42L, P106L and F107S copy number mutant cell lines such as DH5 host strain NTC711772=DH5, dcm-att.sub.::P.sub.5/6 6/6-RNA-IN-SacB, catR; att.sub.HK022::pL (OL1-G to T) P42L-P106L-F107S (P3-), SpecR StrepR. Production yields up to 695 mg/L were reported.
[0277] Additional cell lines were created and disclosed herein including:
[0278] NTC821601 DH5 att.sub.::P.sub.5/6 6/6-RNA-IN-SacB, catR; att.sub.HK022::pL (OL1-G to T) P42L-P106L-F107S (P3-), SpecR StrepR=dem+version of NTC711772
[0279] NTC940211 DH5 att.sub.::P.sup.5/6 6/6-RNA-IN-SacB, catR; att.sub.HK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR=high copy substitution of P106I for P106L combined with P113S to create a quadruple copy number increasing mutant rep protein derivative of NIC821601
[0280] NTC1050811 DH5, att.sub.::P.sub.5/6 6/6-RNA-IN-SacB, catR; att.sub.HK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; att.sub.80::pARA-CI857ts, tetR=pARA-CI857ts derivative of NTC940211. This strain contains a phage 80 attachment site chromosomally integrated copy of a arabinose inducible CI857ts gene. Addition of arabinose to plates or media (e.g. to 0.2-0.4% final concentration) induces pARA mediated CI857ts repressor expression which reduces copy number at 30 C. through CI857ts mediated downregulation of the Rep protein expressing promoter [i.e. additional CI857ts mediates more effective downregulation of the pL (OL1-G to T) promoter at 30 C.]. Copy number induction after temperature shift to 37-42 C. is not impaired since the CI857ts repressor is inactivated at these elevated temperatures. A dcm-derivative (NTC1050811 dcm-) is used in cases where dcm methylation is undesirable.
[0281] NTC1011641; Stbl4 att.sub.::P5/6 6/6-RNA-IN-SacB, catR; att.sub.HK022::pL P42L-P106L-F107S (P3-) SpecR StrepR=Stbl4 version of NTC661135 (XL1 Blue-dcm-att.sub.::P.sub.5/6 6/6-RNA-IN-SacB, catR; att.sub.HK022::pR pL P42L-P106L-F107S (P3-) SpecR StrepR described in Williams, Supra, 2014
[0282] Nanoplasmid production yields are improved with the quadruple mutant heat inducible pL (OL1-G to T) P42L-P106I-F107S P113S (P3-) compared to the triple mutant heat inducible pL (OL1-G to T) P42L-P106L-F107S (P3-) described in Williams, Supra, 2014. Yields in excess of 2 g/L Nanoplasmid have been obtained with the quadruple mutant NTC1050811 cell line (e.g. 2240 mg/L with NTC9 T7 A99 pA, Table 8)
[0283] Use of a conditional replication origin such as these R6K origins that requires a specialized cell line for propagation adds a safety margin since the vector will not replicate if transferred to a patient's endogenous flora.
Example 2: pUC and R6K Origin Vector Production
[0284] Shake flask production : Shake flask production was performed using proprietary Plasmid+ shake culture medium. The seed cultures were started from glycerol stocks or colonies and streaked onto LB medium agar plates containing 50 g/mL antibiotic (for ampR or kanR selection plasmids) or 6% sucrose Tor RNA-OUT selection plasmids). The plates were grown at 30-32 C.; cells were resuspended in media and used to provide approximately 2.5 OD.sub.600 inoculums for the 500 mL Plasmid+ shake flasks that contained 50 g/mL antibiotic for ampR or kanR selection plasmids or 0.5% sucrose to select for RNA-OUT plasmids. Flask were grown with shaking to saturation at the growth temperatures as indicated in Tables 5, 6, 7, and 9.
[0285] Fermentation production: Fermentations were performed using proprietary fed-batch media (NTC3019, HyperGRO media) in New Brunswick BioFlo 110 bioreactors as described (Carnes and Williams, Supra, 2011). The seed cultures were started from glycerol stocks or colonies and streaked onto LB medium agar plates containing 50 g/mL antibiotic (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 media and used to provide approximately 0.1% inoculums for the fermentations that contained 50 g/mL antibiotic for ampR or kanR selection plasmids or 0.5% sucrose for RNA-OUT plasmids. HyperGRO temperature shifts were as indicated in Tables 8 and 9.
[0286] Production hosts: pUC origin AmpR or KanR plasmid fermentations were performed in E. coli strain DH5 [F-80lacZM15 (lacZYA-argF) U169 recA1 endA1 hsdR17 (rK-, mK+) phoA supE44-thi-1 gyrA96 relA1] (Invitrogen, Carlsbad Calif.) or Stbl4.
[0287] Antibiotic-free pUC origin RNA-OUT plasmid fermentations were performed in E. coli strain DH5 containing phage lambda attachment site chromosomally integrated pCAH63-CAT RNA-IN-SacB (P5/6 6/6) as described in Williams, Supra, 2008. The production strain is NTC4862=DH5 att::P5/6 6/6-RNA-IN-SacB, catR.
[0288] Antibiotic-free R6K gamma origin RNA-OUT plasmid propagation and fermentations were performed using E. coli RNA-OUT selection hosts further encoding phage HK022 attachment site integrated pL promoter heat inducible copy number mutant cell line lines, methods for the creation of which are described in Williams, Supra, 2014 and included herein by reference.
Production Strains:
pUC Origin-AmpR or KanR Antibiotic Selection Hosts
[0289] DH5
[0290] Stbl4
pUC Origin-RNA-OUT Sucrose Selection Hosts
[0291] NTC4862 DH5 att.sub.::P.sub.5/6 6/6-RNA-IN-SacB, catR
[0292] NTC1011592 Stbl4 att::P5/6 6/6-RNA-IN-SacB, catR
R6K Origin-RNA-OUT Sucrose Selection Nanoplasmid hosts
[0293] NTC1050811 DH5 att.sub.::P.sub.5/6 6/6-RNA-IN-SacB, catR; att.sub.HK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; att.sub.80::pARA-CI857ts, tetR.
[0294] NTC1011641 Stbl4 att:P5/6 6/6-RNASacB, catR; att.sub.HK022::pL P42L-P106L-F107S (P3-) SpecR StrepR
[0295] Analytical Methods: Culture samples were taken at key points and at regular intervals during all fermentations, Samples were analyzed immediately for biomass (OD600) and for plasmid yield. Plasmid yield was determined by quantification of plasmid obtained from Qiagen Spin Miniprep Kit preparations as described (Carries and Williams, Supra, 2011), Briefly, cells were alkaline lysed, clarified, plasmid was column purified, and eluted prior to quantification. Plasmid quality was determined by agarose gel electrophoresis analysis (AGE) and was performed on 0.8-1% Tris/acetate/EDTA (TAE) gels as described in Carnes and Williams, Supra, 2011.
Example 3: pUC and R6K Origin Structured Vector Construction and Manufacturing
[0296] The R6K gamma origin (SEQ ID NO:1;
[0297] The R6K gamma origin (SEQ ID NO:1) is an engineered 6 iteron R6K origin (
[0298] SEQ ID NO:1 6 iteron 3203 bpR6K origin vector: a biomass of 120 OD.sub.600; plasmid titer of 1363 mg/L; plasmid specific yield of 11.3 mg plasmid/L/OD.sub.600
[0299] SEQ ID NO:18 7 iteron 3225 bp R6K origin vector: a biomass of 137 OD.sub.600; plasmid titer of 1503 mg/L; plasmid specific yield of 11.0 mg plasmid/L/OD.sub.600
[0300] The 7 iteron R6K gamma origin in SEQ ID NO:18 is a tandem duplication of iteron 5 (
TABLE-US-00004 TABLE 4 pNTC multiple cloning site flanked R6K Origin-RNA-OUT selection marker vectors R6K 5 flanking trpA R6K Linker RNA OUT RNA-OUT 3 flanking Vector restriction sites term origin site Selection marker restriction site pNTC-NP1 EcoRI, SacI, KpnI, Yes SEQ ID DraIII.sup.a SEQ ID NheI BamHI, XmaI, (SEQ ID NruI, NsiI, XmaIII, NO: 1 NO: 5 ApaI, SalI, HincII, NO: 10) NotI, NheI PstI, StuI, AatI, SphI, HindIII (in R6K) pNTC-NP2 EcoRI, SacI, KpnI, Yes SEQ ID DraIII.sup.a SEQ ID SpeI, XmaI, SspI (SEQ ID NruI, NsiI, XmaIII, NO: 1 NO: 5 BamHI, XmaI, ApaI, NO: 11) NotI, NheI SalI, HincII, PstI, StuI, AatI, SphI, HindIII (in R6K) pNTC-NP3 EcoRI, SacI, KpnI, Yes SEQ ID DraIII.sup.a SEQ ID KpnI, SacI BamHI, (SEQ ID NruI, NsiI, XmaIII, NO: 1 NO: 5 XmaI, ApaI, SalI, NO: 12) NotI, NheI HincII, PstI, StuI, AatI, SphI, HindIII (in R6K) pNTC-NP4 NheI, XmaIII, NotI, Yes SEQ ID DraIII.sup.a SEQ ID EcoRI, SacI, KpnI (SEQ ID NsiI, NruI, KpnI, NO: 1 NO: 5 NO: 13) SacI BamHI, XmaI, ApaI, SalI, HincII, SfcI, PstI, StuI, AatI, SphI, HindIII (in R6K) pNTC-NP5 KasI, NheI Yes SEQ ID DraIII.sup.a SEQ ID KpnI AflIII PstI, (SEQ ID NO: 1 NO: 5 AatI, SphI, HindIII NO: 14) (in R6K) pNTC-NP6 EcoRI, PstI, EcoRV, Yes SEQ ID DraIII.sup.a SEQ ID KpnI, ApaI, PvuI, (SEQ ID BstXI, NotI, NheI NO: 1 NO: 5 SalI, SacI NO: 15) pNTC-NP7 BssHII PacI NheI Yes SEQ ID DraIII.sup.a SEQ ID KpnI PacI BssHII (SEQ ID NO: 1 NO: 5 NO: 16) pNTC-3x XhoI, XbaI, ApaI, No SEQ ID BsrGI SEQ ID EcoRI, SacI, KpnI, CpG NP1 SalI, HincII, PstI, NO: 2 NO: 7 NruI, NsiI, XmaIII, (SEQ ID StuI, AatI, SphI, NotI, NheI, KpnI NO: 17) HindIII (in R6K) .sup.aNon-palindromic unique 3 bp NNN sticky end DraIII site (CACNNNGTG) separating R6K and RNA-OUT of sequence CACGTTGTG can be used to assemble R6K and RNA-OUT from separate pNTC vectors in directional multi-fragment ligation reactions
[0301] Viral and non-viral vector pUC origin-antibiotic selection bacterial backbone retrofits to R6K-RNA-OUT were performed by:
1) selecting restriction sites that flank the pUC origin and antibiotic selection marker region in the target viral and non-viral vector;
2) identifying a pNTC-NP compatible polylinker -R6K-RNA-OUT polylinker cassette (either pNTC-NP1, 2, 3, 4, 5, 6, or 7; Table 4);
3) Excising the pUC origin antibiotic selection marker region and replacing with the selected R6K origin RNA-OUT region using the selected restriction digestion approach and standard ligase mediated cloning.
[0302] In some cases, the R6K origin and RNA-OUT units were assembled in multi-fragment ligations from separate restriction fragments using the non-palindromic DraIII linker site (see Table 4). In the case of the fd6 Ad helper retrofit (Table 9), a 3-fragment ligation was performed using a short 500 bp synthetic gene DraIII RNA-OUT-Ad helper-AvrII to link RNA-OUT to a unique AvrII site in the fd6 Ad helper eukaryotic region in a 12 kb AvrII-SalI restriction fragment, and to a R6K origin-DraIII fragment from pNTC-NP4.
[0303] Example vector maps and vector characteristics of the original pUC origin-antibiotic selection marker vector and the retrofitted R6K origin-RNA-OUT antibiotic free selection marker vectors are shown for Sleeping Beauty (
[0304] In all cases, the bacterial backbone size was <1 kb in the R6K origin-RNA-OUT antibiotic free selection marker retrofitted vectors (460-610 bp). This is well below the 1,1 kb bacterial backbone size limit required to improve vector expression level (Tables 1-2) and duration (Quiviger et al., Supra, 2014). In all cases, the original pUC origin-antibiotic selection bacterial backbone prior to retrofit was >1.2 kb (2340-2750 bp) as were the pUC origin-RNA-OUT retrofits (1210-1500 bp). Thus, these AAV, AAV helper, Sleeping Beauty, and Lentiviral R6K origin-RNA-OUT antibiotic free selection marker retrofit vectors meet the short spacer region requirement for improved expression level and duration compared to the original pUC origin-antibiotic selection marker vector. Additionally, these AAV, AAV helper, Sleeping Beauty, and Lentiviral R6K origin-RNA-OUT antibiotic free selection marker retrofit vectors have no chance of antibiotic marker gene transfer by transduction (AAV, Lentiviral vectors) or transposition (Sleeping Beauty vectors) due to removal of the Kara or ampR antibiotic resistance selection marker in the parent vector. Additionally, the vectors of the current technology do not require the complicated difficult to scale expensive additional manufacturing steps required to remove the large bacterial region between the eukaryotic polyA and promoter with minicircle vectors (Kay et al.. Supra, 2010).
[0305] However, in a Lentiviral vector the eukaryotic region contains flanking direct repeal LTRs, in an AAV vector the eukaryotic region contains flanking inverted terminal repeats, while in a Sleeping Beauty transposon vector the eukaryotic region contains flanking transposon IR/DR termini. These flanking sequences are all structured DNA sequences.
[0306] Levy, Supra, 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, but not when the high copy replication origin is >1.5 kb. Consistent with this, Replication intermediates were formed in all pUC origin-RNA-OUT marker vectors in which the pUC origin was <1 kb from a Lentiviral vector LTR (Table 5: 400 bp) or a pUC origin-antibiotic resistance marker vector in which the pUC origin was <1 kb from a Sleeping Beauty IR/DR (Table 6; 280 bp). For AAV and mRNA vectors, the original pUC origin-antibiotic selection marker vectors have the pUC origin 0 bp from an ITR (AAV vector; Table 7) or 170 bp from a A99 repeat (mRNA vector, Table 8) which may make a replication intermediate that is too small to detect on an agarose gel. However, in these cases production yields were very low, indicative of low plasmid copy number due to replication blockage. By contrast, as expected, in the case where the original pUC origin-antibiotic selection marker vector pUC origin was >1.5 kb from a structured DNA sequence (A60 repeat), high plasmid production yields were obtained (Table 8: snRNA vector pGEM4Z T7 A60).
[0307] Williams, Supra, 2017 reported that pUC origin vector production yield is improved with a PAS-BH extended pUC origin when the pUC origin is >1.5 kb from a homopolymeric A64C31 repeat. However, production yields were low when a PAS-BH extended pUC origin is orientated <400 bp from the A64C31 repeat (Table 8, see footnotes d and e). This teaches that addition of a PAS-BH primosomal assembly site does not overcome the poor pUC origin directed replication of closely positioned structured DNA sequences.
[0308] Since the pUC origin itself is 1 kb, there is no configuration to make a <1.1 kb bacterial region AAV, Lentiviral, Retroviral or transposon vector containing the pUC origin which is not predicted to produce replication intermediates as seen above and predicted by Levy, Supra, 2004 and poor plasmid yields as reported herein.
[0309] Surprisingly, replication intermediates were not observed in any R6K origin-RNA-OUT antibiotic free selection marker retrofitted vectors, include those in which the R6K origin was <1 kb from a Lentiviral vector LTR (Table 5: 400 bp) or a Sleeping Beauty IR/DR (Table 6; <40 bp). Further, for AAV vectors, while the original pUC origin-antibiotic selection marker vectors with the pUC origin 0 bp from the ITR had very poor production yields, the two R6K origin-RNA-OUT antibiotic free selection marker retrofitted vectors with the R6K origin 40 bp from the ITR had much higher production yields (Table 7). This improved production is specific to R6K and not RNA-OUT, since the two AAV pUC-RNA-OUT retrofits with the pUC origin 50 bp from the ITR had equally poor plasmid production yields as the original pUC antibiotic marker vector (Table 7); as well the direct comparison of pUC-RNA-OUT with R6K-RNA-OUT retrofits positioned 400 bp from an LTR repeat in a Lentiviral backbone showed replication intermediates with all three pUC-RNA-OUT backbones but none of the three R6K-RNA-OUT backbones (Table 5),
[0310] This surprising improvement in plasmid copy number (plasmid production yields) and quality (eliminated replication intermediates) with the R6K origin vector implies that the R6K origin can replicate through a structured DNA sequence more effectively than the pUC origin. While Levy, Supra, 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, but not when the high copy replication origin is >1.5 kb away, the examples provided by Levy, Supra, 2004 were all with pUC origin plasmids.
[0311] A fundamental difference between these replication origins 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. With the pUC origin the RNAII primer forms an RNA: DNA R-loop that is cleaved by RNase H to create a primer for DNA Pol I directed DNA synthesis during initial leading strand synthesis. DNA synthesis then converts from slow DNA Pol I to the highly processive DNA Pol III from 400 bp to up to 1.3 kb downstream of the origin (Alien et al., 2011. Nucleic Acids Research 39:7020-33). The R6K gamma replication origin rep protein interacts with dnaB helicase and dnaG primase which creates short RNA primers for DNA Pol III replication without requirement for DNA Pol I (Abhyankar et al., Supra. 2003). The pUC origin DNA Pol I replication zone of up to 1.3 kb from the origin corresponds closely with the Levy, Supra, 2004 defined upper limit of replication intermediate formation (between 1 and 1.5 kb from the origin). We propose that the observed surprisingly improved replication of structured DNA when in close proximity to the R6K but not the pUC origin is due to an unexpected improvement of replication of structured DNA sequences by DNA Pol III compared to DNA Pol I.
[0312] The vector methods and compositions disclosed herein demonstrate that a Pol III-dependent origin of replication such as the R6K origin can be used to replicate structured DNA sequences which are poorly replicated by a Pol I-dependent origin of replication such as the pUC origin.
[0313] These results demonstrate the vectors of the invention are useful for improving viral and non-viral vector manufacturing yield and quality.
Example 4: Improved Performance of R6K Origin Structured Vector
[0314] The vectors of the invention are additionally useful for eliminating antibiotic resistance marker gene transfer by viral and non-viral vectors; reducing transfection associated toxicity; improving transposition from non-viral transposon vectors; improving packaging titers from viral vectors; improving expression of viral and non-viral vector encoded transgenes, etc.
[0315] As an example, R6K origin third generation lentiviral vectors [4 vectors: Table 5 transfer plasmid, gag pol packaging plasmid; env plasmid; REV plasmid (not shown) with R6K origin and <1 kb bacterial backbone]of the invention showed reduced toxicity and improved viral packaging titers compared to pUC origin vector comparators with >1.5 kb bacterial backbone. Transfection of Lenti-X 293 T cell line (Takara Bio Mountain View, Calif.) with Table 5 R6K origin third generation lentiviral vectors with <1 kb bacterial backbone or original pUC origin-antibiotic selection marker vector with >1.5 kb bacterial backbone control in 24 well plates using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, Mass.) as recommended by the manufacturer resulted in higher titer lentivirus production (>1.5 kb bacterial backbone pUC origin control vectors: 1.00x0.32; <1 kb bacterial backbone R6K origin vectors 1.45x0.42) as measured using the Lenti-X p24 Rapid Titer Kit (Takara Bio Mountain View, Calif.). Transfection of Lenti-X 293 T cell line in 24 well plates with Table 5 third generation lentiviral vectors (>1.5 kb bacterial backbone pUC origin control or <1 kb bacterial backbone R6K origin) using Calcium Phosphate transfection as described (Marino M P, Luce M J, Reiser J. 2003, Methods Mol Biol 229:43-55) resulted in higher titer lentivirus production (>1.5 kb bacterial backbone pUC origin control: 1.00x0.30; <1 kb bacterial backbone R6K origin vectors: 1.32x0.19) as measured using the Lenti-X p24 Rapid Titer Kit (Takara Bio Mountain View, Calif.). Significantly, Calcium Phosphate transfection of the >1.5 kb bacterial backbone pUC origin third generation Lentiviral vectors resulted in extensive transfection associated toxicity (>80% cell death) compared to low toxicity with the R6K origin <1 kb bacterial backbone R6K origin third generation Lentiviral vectors in this 24 well plate transfection. This reduced transfection associated toxicity should result in dramatically improved viral titers in larger manufacturing scale transfections. These results demonstrate the <1 kb bacterial backbone R6K origin Nanoplasmid vectors of the invention reduce transfection associated toxicity and improve packaging titers from viral vectors compared to >1.5 kb bacterial backbone vectors.
SUMMARY
[0316] While the above description contains many examples, these should not be construed as limitations on the scope of the disclosure, but rather should be viewed as an exemplification of preferred embodiments thereof. Many other variations are possible.
[0317] For example, in the vectors of the current technology various orientations of the Pol III dependent replication origin, and the RNA selectable marker, may be utilized. For example, any of the eight orientations of the Pol III dependent replication origin, and the RNA selectable marker in vectors of the current technology may be used (i.e. Pol III replication origin RSM.fwdarw.; Pol III replication originRSM; Pol III replication origin.fwdarw.RSM.fwdarw.; Pol III replication origin.fwdarw.RSM; RSM Pol III replication origin.fwdarw.; RSMPol III replication origin; RSM.fwdarw.Pol III replication origin.fwdarw.; RSM.fwdarw.Pol III replication origin).
[0318] Further, a variety of RNA selectable markers know in the art may be substituted for RNA-OUT.
[0319] Further, an antibiotic resistance maker may be substituted for RNA-OUT, for example in the case where a simple retrofit of the pUC origin to the R6K origin is desired to improve plasmid production yield and or quality.
[0320] Thus, the reader will see that the improved Pol III dependent replication origin vectors of the current technology provide for an approach to reduce transfection associated toxicity, improve transposition from non-viral transposon vectors, improve packaging titers from viral vectors, improve expression of viral and non-viral vector encoded genes, and eliminate viral vector and non-viral vector mediated antibiotic selection marker gene transfer (i.e. through incorporation of a bacterial region preferably less than 1000 bp) while dramatically improving manufacture compared to alterative vectors such as pUC plasmids and minicircles.
[0321] Accordingly, the scope of the disclosure should be determined not by the embodiments illustrated, but by the appended claims.
TABLE-US-00005 TABLE 5 Lentiviral vectors: pUC origin-RNA-OUT versus R6K origin-RNA-OUT shake flask production yields/quality Transfer plasmid Replication Harvest bacterial origin Replication spec backbone spacing origin yield Harvest Lentiviral Replication Vector (LTR from SV40 spacing Cell Production Harvest (mg/L/ yield Production Vector origin size spacing) .sup.a Ori .sup.b front LTR.sup.c line .sup.d culture .sup.e OD.sub.600 OD.sub.600) (mg/L) quality.sup.f Transfer pUC origin 9.9 kb 1210 bp <50 bp 400 bp DH5 30 to 37 C. 7.5 3.4 26 CCC plasmid 1 (1210 bp) Monomer + with SV40 Replicate Ori Intermediate R6K origin 9.2 kb 460 bp <50 bp 400 bp DH5 30 to 37 C. 5.0 4.7 24 CCC (460 bp) Monomer Transfer pUC origin 7.3 kb 1210 bp <50 bp 400 bp DH5 30 to 37 C. 9.0 3.9 35 CCC plasmid 2 (1210 bp) Monomer + with SV40 Replicate Ori Intermediate R6K origin 6.5 kb 460 bp <50 bp 400 bp DH5 30 to 37 C. 8.4 7.2 61 CCC (460 bp) Monomer Transfer pUC origin 8.3 kb 1210 bp <50 bp 400 bp DH5 30 to 37 C. 16.8 3.0 51 CCC plasmid 3 (1210 bp) Monomer + with SV40 Replicate Ori Intermediate R6K origin 7.5 kb 460 bp <50 bp 400 bp DH5 30 to 37 C. 11.9 7.7 92 CCC (460 bp) Monomer Gag pol pUC origin 8.4 kb 1210 bp NA NA DH5 30 to 37 C. 10.2 4.3 44 CCC packaging Monomer plasmid R6K origin 7.5 kb 460 bp NA NA DH5 30 to 37 C. 9.7 7.6 73 CCC Monomer Envelope pUC origin 5.4 kb 1210 bp NA NA DH5 30 to 37 C. 17.0 5.7 96 CCC plasmid Monomer R6K origin 4.4 kb 460 bp NA NA DH5 30 to 37 C. 10.8 6.0 65 CCC Monomer .sup.a All vectors are antibiotic free RNA-OUT selection retrofits. Original pUC origin-antibiotic selection marker vector bacterial backbone size were all >1.5 kb .sup.b Distance in bp from 3 end of replication origin to SV40 origin in direction of replication .sup.cDistance in bp from 3 end of replication origin to nearest LTR in direction of replication .sup.d DH5 = NTC4862 for pUC origin, NTC1050811 for R6K origin .sup.e 500 mL Plasmid + Shake Flask Culture temperature shifted from 30 to 37 C. .sup.fHarvest plasmid preparations with detectable replication intermediates indicated. Levy, Supra, 2004 teach that replication intermediates form when any high copy number prokaryotic origin of replication is less than 1000 bp from a promoter/enhancer or LTR promoter or DNA repeat or a complex secondary structure such as an IRES, while replication intermediates do not form if the origin of replication is a distance greater than about 1.5 kb.
TABLE-US-00006 TABLE 6 Sleeping Beauty Transposon and transposase vector: pUC versus R6K origin shake flask production yields/quality bacterial Replica- Harvest Replica- Vector backbone tion spec tion IR/DR size origin Produc- yield Harvest Produc- origin/ Bacterial Configura- Vector (IR/DR spacing from Cell tion Harvest (mg/L/ yield tion vector selection tion size spacing) IR/DR.sup.a line.sup.b culture .sup.c OD.sub.600 OD.sub.600) (mg/L) quality.sup.d Transposon KanR IR DR(R) 5.5 kb 2600 bp 280 bp DH5 30 to 37 C. 1.9 2.1 4 CCC vector <pUC<kanR (2600 bp) Monomer + pUC origin IR DR(L) Replicate (pUC57-kan- Promoter> Inter- SB1: see Transgene> mediate FIG. 2) Transposon RNA- IR DR(R) < 3.4 kb 475 bp <40 bp DH5 30 to 37 C. 11.2 1.0 11 CCC vector OUT AF R6K R-OUT > (475 bp) Monomer R6K origin IR DR(L) (NTC9-SB1: Promoter> see FIG. 2) Transgene> Transposon RNA- IR DR(R) < 3.6 kb 475 bp <40 bp DH5 30 to 37 C. 4.1 6.2 25 CCC vector OUT AF R6K R-OUT > (475 bp) Monomer R6K origin IR DR(L) (NTC9-SB2) Promoter> Transgene> Transposase KanR Not 4.7 kb 2600 bp Not DH5 30 to 37 C. 21.6 1.8 39 CCC vector Applicable (NA) Applicable Monomer pUC origin Transposase RNA- Not 2.5 kb 475 bp Not DH5 30 to 37 C. 10.9 4.7 51 CCC vector OUT AF Applicable (NA) Applicable Monomer R6K origin .sup.aDistance in bp from 3 end of replication origin to nearest Sleeping Beauty IR/DR in direction of replication .sup.bDH5 = DH5 for pUC57-kanR pUC origin, NTC1050811 for R6K origin .sup.c 500 mL Plasmid + Shake Flask Culture temperature shifted from 30 to 37 C. .sup.dHarvest plasmid preparations with detectable replication intermediates indicated. Levy, Supra, 2004 teach that replication intermediates form when any high copy number prokaryotic origin of replication is less than 1000 bp from a promoter/enhancer or LTR promoter or DNA repeat or a complex secondary structure such as an IRES, while replication intermediates do not form if the origin of replication is a distance greater than about 1.5 kb.
TABLE-US-00007 TABLE 7 AAV vectors: pUC versus R6K origin shake flask production yields/quality Replica- tion Harvest bacterial origin spec Replica- Vector backbone spacing Produc- yield Harvest Produc- AAV tion Bacterial Configura- Vector size (ITR Bacterial from Cell tion Harvest (mg/L/ yield tion Vector origin selection tion size spacing) .sup.e selection ITR.sup.a line.sup.b culture .sup.c OD.sub.600 OD.sub.600) (mg/L) quality .sup.d Back- pUC AmpR ITR <F1Ori- 5.4 kb 2600 bp AmpR 0 bp Stbl4 30-37 C. 17.8 0.4 8 CCC bone 1.sup.e origin AmpR>pUC> (2600 bp) shake Monomer (pAAV: ITR CMV- see transgene- FIG. 3) pA pUC RNA- ITR <pUC 4.0 kb 1240 bp RNA- 50 bp Stbl4 30-37 C. 10.4 0.7 7 CCC origin OUT AF R-OUT> (1240 bp) OUT AF shake Monomer (NTC8- ITR CMV- AAV: transgene- see pA FIG. 3) R6K RNA- ITR <R6K 3.3 kb 490 bp RNA- 40 bp Stbl4 30-37 C. 11.8 3.5 42 CCC origin OUT AF R-OUT> (490 bp) OUT AF shake Monomer (NTC9- ITR CMV- AAV: transgene- see pA FIG. 3) R6K RNA- ITR <R6K 7.7 kb 490 bp RNA- 40 bp Stbl4 30-37 C. 11.0 2.1 23 CCC origin OUT AF R-OUT> (4940 bp) OUT AF shake Monomer Rep> Cap> ITR CMV- transgene- pA Back- pUC RNA- ITR <pUC 5.5 kb 1250 bp RNA- 50 bp Stbl4 30-37 C. 11.7 0.4 5 CCC bone 2 origin OUT AF R-OUT> (NA) OUT AF shake Monomer (NTC8) ITR CAG- transgene- WPRE-pA R6K RNA- ITR <R6K 4.8 kb 490 bp RNA- 40 bp Stbl4 30-37 C. 11.1 1.6 18 CCC origin OUT AF R-OUT> (NA) OUT AF shake Monomer (NTC9) ITR CAG- transgene- WPRE-pA .sup.a Distance in bp from 3 end of replication origin from nearest AAV ITR in direction of replication .sup.bStbl4 = Stbl4 for pUC origin-ampR, NTC1011592 for pUC origin-RNA-OUT, NTC1011641 for R6K origin-RNA-OUT .sup.c Plasmid + Shake Flask Culture temperature shifted from 30 to 37 C. .sup.d High quality CCC monomer with no detectable replication intermediate and low amounts of recombination mediated ITR deletion. May not see replication intermediates on gel with pUC origin since <50 from DNA structure, but probably formed since plasmid yield lower than R6K origin .sup.e A R6K origin-RNA-OUT MIP intron version of this vector with 6 bp ITR spacing was unclonable, presumably due to toxicity of juxtaposing the 2 AAV ITR immediately adjacent to each other. A R6K origin-RNA-OUT MIP intron version in a second AAV vector backbone (10 bp ITR spacing) was also unclonable
TABLE-US-00008 TABLE 8 mRNA vector: pUC versus R6K origin DH5.sup.f HyperGRO .sup.g Fermentation Yields/quality Replica- Harvest Harvest polyA tion spec spec mRNA Replica- Vector Bacterial origin yield yield Harvest Harvest Produc- expression tion Selec- Configura- Vector backbone bp from Harvest (mg/L/ (mg/ yield yield tion vector origin tion tion size size polyA OD.sub.600 OD.sub.600) gWCW) (mg/L) (g).sup.b quality pT3/T7 pUC origin AmpR A99<pUC< 7.2 kb 2660 bp 170 bp 113 3.8 1.6 430 .sup.a 43 CCC A99 pA.sup.b (pT3/T7 AmpR T7> Monomer .sup.i mRNA A99 pA) Transgene expression pUC origin + kanR A99<KanR 7.2 kb 2660 bp 5000 bp 140 8.9 4.1 1250 .sup.a 12.5 CCC vector and PAS-BH.sup.d pUC>PAS- Monomer retrofit (NTC7 T7 BH> T7> derivatives A99 pA) Transgene R6K origin RNA- A99<R-OUT 5.2 kb 610 bp 4600 bp 147 15.3 6.7 2240 .sup.a 22.4 CCC (NTC9 T7 OUT AF R6K> T7> Monomer A99 pA) AmpR Transgene pGEM4Z pUC origin A60 AmpR> 6.8 kb 2500 bp 4400 bp 112 8.3 4.4 929.sup. 9.3 CCC A60 pA.sup.c (pGEM4Z pUC> T7> Monomer mRNA T7 A60 pA) Transgene expression pUC origin + RNA- A60<R-OUT 5.9 kb 1500 bp 4600 bp 146 11.5 4.9 1673 .sup.a 16.7 CCC vector and PAS-BH.sup.e OUT AF pUC> PAS- Monomer retrofit (NTC8 T7 BH> T7> derivatives A60 pA) Transgene R6K origin RNA- A60<R-OUT 4.9 kb 460 bP 4300 bp 113 13.1 5.0 1483 .sup.a 14.8 CCC (NTC9 T7 OUT AF R6K> T7> Monomer A60 pA) Transgene .sup.a PolyA region correct by sequencing .sup.bgram plasmid/10 L fermentor .sup.cSee FIG. 4 for vector maps .sup.d KanR-pUC + PASBH vector backbone described in Williams 2017 WO2017025447. This pUC + PASBH backbone dramatically improved plasmid yield when oriented >1.5 kb away from polyA64-polyC31 compared to a pUC origin oriented <200 bp away from the polyA64-polyC31 repeat or a pUC + PASBH origin oriented <400 bp away from the polyA64-polyC31 repeat (Williams, Supra, 2017, P1140-K2 versus P1140 and P1140-K1, Table 1). Thus PAS-BH does not dramatically improve pUC origin replication into a repeat structure <400 bp from the pUC origin. .sup.eRNA-OUT-pUC + PASBH vector backbone described in Williams, Supra, 2017. This pUC + PASBH backbone dramatically improved plasmid yield when oriented >1.5 kb away from polyA64-polyC31 compared to a pUC origin oriented <200 bp away from the polyA64-polyC31 repeat or a pUC + PASBH origin oriented <400 bp away from the polyA64-polyC31 repeat. (Williams, Supra, 2017, Table 1: P1140-AF2 versus P1140 and P1140-AF1). Thus PAS-BH does not dramatically improve pUC origin replication into a repeat structure <400 bp from the pUC origin. .sup.fDH5 = NTC4862 for NTC8; NTC1050811 for NTC9: NTC1050811 DH5 att.sub.::P.sub.5/6 6/6-RNA-IN-SacB, catR; att.sub.HK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3), SpecR StrepR; att.sub..sub.80::pARA-CI857ts, TetR. Arabinose induces pARA-CI857ts expression which reduces copy number through CI857ts mediated downregulation of the Rep protein expressing pL promoter .sup.g HyperGRO fermentation 30-42 C. temperature shift at 55 OD.sub.600. For NTC9 vectors fermentation media contained 0.2% arabinose .sup.h Elango et al., 2005. Biochemical and Biophysical Research Communications 330: 958-66 teach polyA100 mRNA vectors are unstable in DH5 and XL1Blue and cannot be produced in mg quantities. See FIG. 4 for vector maps .sup.i May not see replication intermediates on gel with pUC origin since <170 bp from DNA structure, but probably formed since plasmid yield lower than with R6K origin vectors
TABLE-US-00009 TABLE 9 AAV helper vectors: pUC versus R6K origin plasmid production yields/quality Harvest spec AAV bacterial yield Harvest Helper Replication Vector backbone Cell Production Harvest (mg/L/ yield Production Vector origin Selection size size line.sup.b culture .sup.a OD.sub.600 OD.sub.600) (mg/L) quality AAV2 Rep/ pUC origin RNA- 5.7 kb 1220 bp DH5 30 to 37 C. .sup.a 16.5 2.6 43 CCC AAV9 Cap OUT AF Monomer (Rep Cap helper) R6K origin RNA- 4.9 kb 470 bp DH5 30 to 37 C. .sup.a 17.5 5.5 96 CCC OUT AF Monomer Fd6 pUC origin ampR 15.4 kb 2750 bp Stbl4.sup.c 30 C. .sup.a, c 20 .sup.0.8 .sup.d .sup.16 .sup.d CCC (Ad Helper) Monomer and deletion productions R6K origin RNA- 12.9 kb 540 bp DH5 30 to 37 C. .sup.a 17.8 1.3 23 CCC OUT AF Monomer pHelper Rep R6K origin RNA- 14.2 kb 470 bp DH5 30 to 37 C. .sup.a 6.8 4.8 33 CCC Cap NP (Ad OUT AF Monomer Helper + Rep Cap single helper plasmid) pHelper pUC origin ampR 11.6 kb 2340 bp DH5 30-42 C. 129 14.2 1833 CCC (Ad Helper) HyperGRO Monomer shift pHelper-NP R6K origin RNA- 9.8 kb 460 bp DH5 30-42 C. 84.5 18.1 1530 CCC (Ad Helper) OUT AF HyperGRO Monomer RAMP .sup.e .sup.a 500 mL Plasmid + Shake Flask Culture; .sup.bDH5 = NTC1050811 for NTC9: NTC1050811 DH5 att.sub.::P.sub.5/6 6/6-RNA-IN-SacB, catR; att.sub.HK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3), SpecR StrepR; att.sub..sub.80::pARA-CI857ts, TetR. Arabinose induces pARA-CI857ts expression which reduces copy number through CI857ts mediated downregulation of the Rep protein expressing pL promoter. Retrofits were obtained using Sucrose + 0.2% Arabinose plates to reduce copy number .sup.cFd6 Stbl4 best cell line, Fd6/Stbl3 see deletions, Fd6/DH5 and Fd6/XL1Blue cell lines are unstable, used 30 C. throughout since plasmid lost in 30-37 C. process .sup.d Actual yield lower due to high gDNA in minipreps .sup.e HyperGRO fermentation, inoculated from sucrose + 0.2% arabinose culture grown at 30 C., arabinose added to batch medium to 0.2% final concentration. 30-42 C. ramp temperature shift