Abstract
A totally redesigned host/vector system with improved properties in terms of safety has been developed. The pCOR plasmids are narrow-host range plasmid vectors for nonviral gene therapy. These plasmids contain a conditional origin of replication and must be propagated in a specifically engineered E. coli host strain, greatly reducing the potential for propagation in the environment or in treated patients. The pCOR backbone has several features that increase safety in terms of dissemination and selection: (1) the origin of replication requires a plasmid-specific initiator protein, π protein, encoded by the pir gene limiting its host range to bacterial strains that produce this trans-acting protein; (2) the plasmid’s selectable marker is not an antibiotic resistance gene but a gene encoding a bacterial suppressor tRNA. Optimized E. coli hosts supporting pCOR replication and selection were constructed. High yields of supercoiled pCOR monomers were obtained (100 mg/l) through fed-batch fermentation. pCOR vectors carrying the luciferase reporter gene gave high levels of luciferase activity when injected into murine skeletal muscle.
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References
Yew N et al. Optimization of plasmid vectors for high-level expression in lung epithelial cells Hum Gene Ther 1997 8: 575–584
Lahijani R et al. High-yield production of pBR322-derived plasmids intended for human gene therapy by employing a temperature-controllable point mutation Hum Gene Ther 1996 7: 1971–1980
Gilligan P . Microbiology of airway disease in patients with cystic fibrosis Clin Microbiol Rev 1991 4: 35–51
Smith K, Shepherd A, Boyd J, Lees G . Gene delivery systems for use in gene therapy: an overview of quality assurance and safety issues Gene Therapy 1996 3: 190–200
Filutowicz M et al. Regulation of replication of an iteron-containing DNA molecule Prog Nucleic Acid Res Mol Biol 1994 48: 239–273
Greener A, Filutowicz M, McEachern M, Helinski D . N-terminal truncated forms of the bifunctional pi initiation protein express negative activity on plasmid R6K replication Mol Gen Genet 1990 224: 24–32
Herrero M, de LV, Timmis K . Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria J Bacteriol 1990 172: 6557–6567
Vieira J, Messing J . The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers Gene 1982 19: 259–268
Metcalf W, Jiang W, Wanner B . Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6K gamma origin plasmids at different copy numbers Gene 1994 138: 1–7
Ferrero L et al. Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones Mol Microbiol 1994 13: 641–653
Sherratt D et al. Site-specific recombination and circular chromosome segregation Philos Trans R Soc Lond B Biol Sci 1995 347: 37–42
Leung D, Chen E, Cachianes G, Goeddel D . Nucleotide sequence of the partition function of Escherichia coli plasmid ColE1 DNA 1985 4: 351–355
Simoes D et al. A sugar-inducible excretion system for the production of recombinant proteins with Escherichia coli Ann NY Acad Sci 1991 646: 254–258
Normanly J et al. Construction of two Escherichia coli amber suppressor genes: tRNAPheCUA and tRNACysCUA Proc Natl Acad Sci USA 1986 83: 6548–6552
Messing J, Vieira J . A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments Gene 1982 19: 269–276
Blum P et al. Gene replacement and retrieval with recombinant M13mp bacteriophages J Bacteriol 1989 171: 538–546
Wright M . Mutants of Escherichia coli lacking endonuclease I, ribonuclease I, or ribonuclease II J Bacteriol 1971 107: 87–94
Dürwald H, Hoffmann-Berling H . Endonuclease-I-deficient and ribonuclease I-deficient Escherichia coli mutants J Mol Biol 1968 34: 331–346
Taylor R, Walker D, McInnes R . E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing Nucleic Acids Res 1993 21: 1677–1678
Schoenfeld T et al. Effects of bacterial strains carrying the endA1 genotype on DNA quality isolated with Wizard(TM) Plasmid Purification Systems Promega Corporation: Promega Notes 1995 53
Jekel M, Wackernagel W . Location of the endA gene coding for endonuclease I on the physical map of the Escherichia coli K-12 chromosome J Bacteriol 1994 176: 1550–1551
Ananthaswamy H . The release of endonuclease I from Escherichia coli by a new cold shock procedure Biochem Biophys Res Commun 1977 76: 289–298
Frost L, Ippen-Ihler K, Skurray R . Analysis of the sequence and gene products of the transfer region of the F sex factor Microbiol Rev 1994 58: 162–210
Panicker M, Minkley EJ . DNA transfer occurs during a cell surface contact stage of F sex factor-mediated bacterial conjugation J Bacteriol 1985 162: 584–590
Clark A, Warren G . Conjugal transmission of plasmids Annu Rev Genet 1979 13: 99–125
Prentki P, Krisch H . In vitro insertional mutagenesis with a selectable DNA fragment Gene 1984 29: 303–313
Hartikka J et al. An improved plasmid DNA expression vector for direct injection into skeletal muscle Hum Gen Ther 1996 7: 1205–1217
del Solar G, Alonso J, Espinosa M, Diaz-Orejas R . Broad-host-range plasmid replication: an open question Mol Microbiol 1996 21: 661–666
Avila P, Nunez B, de lCF . Plasmid R6K contains two functional oriTs which can assemble simultaneously in relaxosomes in vivo J Mol Biol 1996 261: 135–143
Posfai G et al. In vivo excision and amplification of large segments of the Escherichia coli genome Nucleic Acids Res 1994 22: 2392–2398
Schmoll T, Ott M, Oudega B, Hacker J . Use of a wild-type gene fusion to determine the influence of environmental conditions on expression of the S fimbrial adhesin in an Escherichia coli pathogen J Bacteriol 1990 172: 5103–5111
Skrzypek E, Haddix P, Plano G, Straley S . New suicide vector for gene replacement in yersiniae and other gram-negative bacteria Plasmid 1993 29: 160–163
Azad A, Coote J, Parton R . Construction of conjugative shuttle and suicide vectors for Pasteurella haemolytica and P. multocida Gene 1994 145: 81–85
Penfold R, Pemberton J . An improved suicide vector for construction of chromosomal insertion mutations in bacteria Gene 1992 118: 145–146
Novick R, Hoppensteadt F . On plasmid incompatibility Plasmid 1978 1: 421–434
Filutowicz M et al. DNA and protein interactions in the regulation of plasmid replication J Cell Sci Suppl 1987 7: 15–31
Davies J, Smith D . Plasmid-determined resistance to antimicrobial agents Annu Rev Microbiol 1978 32: 469–518
Jacobson K . Reaction of aminoacyl-tRNA synthetases with heterologous tRNAs Prog Nucleic Acid Res Mol Biol 1971 11: 461–488
Drabkin H, Park H, RajBhandary U . Amber suppression in mammalian cells dependent upon expression of an Escherichia coli aminoacyl-tRNA synthetase gene Mol Cell Biol 1996 16: 907–913
Nakamura Y, Gojobori T, Ikemura T . Codon usage tabulated from the international DNA sequence databases Nucleic Acids Res 1997 25: 244–245
Valle R, Morch M, Haenni A . Novel amber suppressor tRNAs of mammalian origin EMBO J 1987 6: 3049–3055
Laski F, Belagaje R, RajBhandary U, Sharp P . An amber suppressor tRNA gene derived by site-specific mutagenesis: cloning and function in mammalian cells Proc Natl Acad Sci USA 1982 79: 5813–5817
Hudziak R et al. Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes Cell 1982 31: 137–146
Choisne N, Martin CA, Small I . Transactivation of a target gene using a suppressor tRNA in transgenic tobacco plants Plant J 1997 11: 597–604
Sato Y et al. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization Science 1996 273: 352–354
Schwartz D et al. CpG motifs in bacterial DNA cause inflammation in the lower respiratory tract J Clin Invest 1997 100: 68–73
Liang H et al. Activation of human B cells by phosphorothioate oligodeoxynucleotides J Clin Invest 1996 98: 1119–1129
Peterson D, Beifuss K, Morley K . Context-dependent gene expression: cis-acting negative effects of specific procaryotic plasmid sequences on eucaryotic genes Mol Cell Biol 1987 7: 1563–1567
Acknowledgements
We would like especially to thank JM Masson for providing sup Phe and XAC-1 and B Wanner for ori γ and uidA: :pir cassettes. W Wackernagel is acknowledged for the generous gift of the endA fragment. We would like to thank F Blanche and collaborators for topoisomerase experiments and T Ciora for oligonucleotide synthesis and sequencing.
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Soubrier, F., Cameron, B., Manse, B. et al. pCOR: a new design of plasmid vectors for nonviral gene therapy. Gene Ther 6, 1482–1488 (1999). https://doi.org/10.1038/sj.gt.3300968
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DOI: https://doi.org/10.1038/sj.gt.3300968
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