Novel bovine lentiviral vectors based on Jembrana disease virus

Background Safety is a concern that must be addressed prior to any clinical use of human immunodeficiency virus (HIV)-based lentiviral vectors in huma n patients. Unfortunately, efforts to examine the biosafety of the vectors in preclinical animal models are hampered due to the lack of animal models for HIV infection. We have developed new lentiviral vectors based on the re cently characterised Jembrana Disease Virus (JDV), which infects a specific species of cattle naturally in Ball, Indonesia. Methods Sequences from the JDV genome were amplified by splicing overlap ex tension polymerase chain reaction (PCR) for the construction of transfer ve ctors as well as a packaging construct. Co-transfection of these two plasmi ds into 293T cells with a third encoding a G glycoprotein of vesicular stom atitis virus produced pseudotyped, disabled, replication defective JDV vect or particles. Viral titre was obtained by transducing the cells with the su pernatant harvested from transfectants and determining the number of cells expressing the transgene. PCR and Southern blotting were used to detect the presence of potential replication-competent viruses as well as transgene i ntegration. Results Bicistronic JDV vectors encoding the green fluorescent protein (GFP ) and the neomycin phosphotransferase were harvested with a titre range of 0.4-1.2 x 10(6) colony forming units/ml from vector-producing cells and wer e further concentrated by ultracentrifugation to the high titre of approxim ately 10(7) CFU/ml. Vectors encoding GFP were shown to transduce and integr ate efficiently into the chromosomes of a range of primary and transformed cells of different origins in different differentiation status, including g rowth-arrested cells, with an efficiency of 25-75%. Exhaustive testing with a marker gene transfer assay in combination with a reverse transcriptase a ssay and PCR amplification of samples of serially passaged, transduced cell s showed that no detectable amount of replication competent lentivirus (RCL ) was produced. Conclusions We showed the feasibility of the development of gene transfer v ectors based on a non-primate bovine lentivirus, which will provide the opp ortunity for examination of the efficacy and biosafety of lentiviral vector -mediated gene transfer in vivo in animal models. JDV-based vectors may be applicable and more readily acceptable than those from HIV for human gene t herapy. Copyright (C) 2000 John Wiley & Sons, Ltd.