Mapping of the bovine immunodeficiency virus packaging signal and RRE and incorporation into a minimal gene transfer vector.
Gene transfer systems based on lentiviruses have emerged as promising gene delivery vehicles for human gene therapy due to their ability to efficiently transduce nondividing target cells. Both primate and nonprimate lentiviruses have been used for construction of lentiviral vectors. An early generation of gene transfer system based on bovine immunodeficiency virus (BIV) has been developed (R. D. Berkowitz, H. Ilves, W. Y. Lin, K. Eckert, A. Coward, S. Tamaki, G. Veres, and I. Plavec, 2001, J. Virol. 75, 3371-3382). In this study, we mapped the BIV Rev response element (RRE) to 312 bp of the Env coding region. Furthermore, we compared transduction efficiencies of vectors containing different portions of the BIV Gag coding region and found that the first 104 bp of gag contains a functional part of the BIV packaging signal. These findings enabled the generation of a minimal BIV-based lentiviral vector. The minimal transfer vector construct consists of a self-inactivating long terminal repeats (LTR), minimal packaging sequence, putative central polypurine tract, minimal RRE, an internal promoter driving the gene of interest, and a woodchuck hepatitis posttranscriptional regulatory element. In addition, we constructed a BIV packaging construct containing gag/pol, minimal Rev/RRE, and the accessory gene vpy. The regulatory gene tat and the accessory genes vif and vpw have been inactivated or truncated. The current system has significantly reduced regions of homologies between the transfer vector and the packaging constructs. The vectors generated from this system achieved a titer of greater than 1 x 10(6) transducing units per milliliter and are fully functional as indicated by their ability to efficiently transduce both dividing and nondividing cells. These modifications should provide improved safety features for the BIV-based gene transfer system.
Molina
Rene P
RP
Genetic Therapy, Inc., A Novartis Company, Gaithersburg, Maryland 20878, USA. tianci.luo@pharma.novartis.com
Matukonis
Meghan
M
Paszkiet
Brian
B
Zhang
Jingli
J
Kaleko
Michael
M
Luo
Tianci
T
D001483:Base Sequence; D002478:Cells, Cultured; D015683:Gene Products, gag / Q000235:genetics / Q000502:physiology; D018014:Gene Transfer Techniques; D015752:Genes, env / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016087:Immunodeficiency Virus, Bovine / Q000235:genetics*; D008969:Molecular Sequence Data; D014161:Transduction, Genetic; D019065:Virus Assembly / Q000235:genetics*
D015683:Gene Products, gag