Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo.
Lentivirus-derived vectors are among the most promising viral vectors for gene therapy currently available, but their use in clinical practice is limited by the associated risk of insertional mutagenesis. We have overcome this problem by developing a nonintegrative lentiviral vector derived from HIV type 1 with a class 1 integrase (IN) mutation (replacement of the 262RRK motif by AAH). We generated and characterized HIV type 1 vectors carrying this deficient enzyme and expressing the GFP or neomycin phosphotransferase transgene (NEO) under control of the immediate early promoter of human CMV. These mutant vectors efficiently transduced dividing cell lines and nondividing neural primary cultures in vitro. After transduction, transient GFP fluorescence was observed in dividing cells, whereas long-term GFP fluorescence was observed in nondividing cells, consistent with the viral genome remaining episomal. Moreover, G418 selection of cells transduced with vectors expressing the NEO gene showed that residual integration activity was lower than that of the intact IN by a factor of 500-1,250. These nonintegrative vectors were also efficient in vivo, allowing GFP expression in mouse brain cells after the stereotactic injection of IN-deficient vector particles. Thus, we have developed a generation of lentiviral vectors with a nonintegrative phenotype of great potential value for secure viral gene transfer in clinical applications.
Philippe
Stéphanie
S
Laboratoire de Génétique Moléculaire de la Neurotransmission et des Processus Neurodégénératifs, Université Pierre et Marie Curie Paris 6, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7091, 83 bd de l'Hôpital, 75013 Paris, France.
Sarkis
Chamsy
C
Barkats
Martine
M
Mammeri
Hamid
H
Ladroue
Charline
C
Petit
Caroline
C
Mallet
Jacques
J
Serguera
Che
C
D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D016679:Genome, Viral; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D016086:Lentivirus / Q000201:enzymology / Q000235:genetics; D051379:Mice; D010957:Plasmids; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
D011993:Recombinant Fusion Proteins; D019426:Integrases