RESUMO
phiC31 integrase is a sequence-specific phage recombinase that can recombine two short DNA sequences called attB and attP. The enzyme can also promote genomic integration of plasmids carrying attB into native mammalian sequences having partial identity to attP. To increase the efficiency of integration, we mutated the phiC31 integrase gene and screened the mutants in human cells in an assay for higher recombination frequency between attB and attP. We report in this article the isolation of a mutant, P2 that has twice the chromosomal integration frequency of wild-type phiC31 integrase, at both a preintegrated chromosomal attP site and at endogenous pseudo attP sequences in cultured human cells. In mouse liver, P2-mediated integration provided therapeutic long-term levels of human factor IX that were double those generated by wild-type phiC31 integrase. We also describe an additional mutant, P3 that combines the mutations of P2 with further changes and possesses an elevated specificity for integration at a chromosomally placed attP site in human cells. Forty-four percent of colonies carrying integration events mediated by P3 have integration at the placed attP site. These mutant integrases are useful for gene therapy and genome modification, and they demonstrate the feasibility of engineering phiC31 integrase toward more desirable properties.
Assuntos
Integrases/genética , Integrases/metabolismo , Recombinação Genética/genética , Sítios de Ligação Microbiológicos/genética , Linhagem Celular , Humanos , Mutação , Especificidade por Substrato/genéticaRESUMO
Gene therapy for hemophilia A has fallen short of success despite several clinical trials conducted over the past decade. Challenges to its success include vector immunogenicity, insufficient transgene expression levels of Factor VIII, and inhibitor antibody formation. Gene therapy has been dominated by the use of viral vectors, as well as the immunogenic and oncogenic concerns that accompany these strategies. Because of the complexity of viral vectors, the development of nonviral DNA delivery methods may provide an efficient and safe alternative for the treatment of hemophilia A. New types of nonviral strategies, such as DNA integrating vectors, and the success of several nonviral animal studies, suggest that nonviral gene therapy has curative potential and justifies its clinical development.
Assuntos
Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos , Hemofilia A/terapia , Transgenes , Ensaios Clínicos como Assunto , Fator VIII/biossíntese , Fator VIII/genética , Terapia Genética/tendências , Hemofilia A/genética , HumanosRESUMO
Certain genes from Lactococcus lactis and Pseudomonas aeruginosa, including the nfxB gene, generate a mutator phenotype in Escherichia coli. The results of this study, together with those of a previous study, support conservation of regulatory sequences in E. coli and P. aeruginosa and suggest that some efflux pumps prevent mutagenicity by exporting mutagenic products of metabolism.
