71. Chimeric lentiviral vectors derived from HIV-1 and HIV-2

Abstract

While a source of great misery, lentiviruses can be modified and channeled toward the useful purpose of developing vectors for gene transfer in gene therapy, functional genomics, and animal transgenesis. Being retroviruses and by virtue of their integration into the target cell genome, they provide for long term transgene expression. The added advantage of lentiviral vectors is their ability to affect gene transfer in non-dividing as well as dividing cells, without any anticipated immune reaction. To make a compelling case for their use, specially in gene therapy, three interrelated issues need to be addressed - targeting, regulation and safety. To enhance their safety or to minimize the chance of recombination, we are creating chimeric vectors derived from HIV-1 and HIV-2. This is being done both for transfer vectors and packaging constructs. It is based on the premise that there is sufficient sequence diversity between HIV-1 and HIV-2 to curtail recombination. Vectors were produced by transient transfection of human embryo kidney 293T cells, using matching sets of transfer vector and packaging construct DNAs, and pseudotyped with VSV-G. For ease of detection, the transfer vectors carried indicator GFP gene. The resulting supernatants were titrated on 293T cells and their content of the core antigen (p24 or p27) determined by Eliza assays. Preliminary results show that the titer for the homologously packaged HIV-1 vector [HIV-1 × HIV-1p] was about 2.7 × 10e3 TU/ng and that of the homologously packaged HIV-2 vector [HIV-2 × HIV-2p] was about 0.2 × 10e3 TU/ng. The cross-packaged transfer vectors, HIV-1 × HIV-2p and HIV-2 × HIV-1p, had titer of about 0.12 × 10e3 TU/ug and 0.16 × l0e3 TU/ug, respectively. For the chimeric transfer vectors, the titer of the HIV-1 vector with the replacement of the HIV-1 leader-gag segment with that of the HIV-2 leader-gag segment was roughly the same as for the wild type vector, both in homologous and hetrologous packaging reaction. Initial experiments with the reverse transfer vector chimera gave titers lower than that for the corresponding wild type vector. Two of the packaging construct chimeras also have been tested. These chimeras consist of the exchanges of 5′ halves ('gag-pol') and 3′ halves ('tat-rev-nef') genes. When tested with HIV-2 transfer vectors, 3′ half chimeras appeared to be more like the wild type constructs than the 5′ half chimeras. This may reflect specificity of interaction between the leader sequence and the gag protein. Additional chimeric vectors are being created to test this hypothesis. The results show that the creation of chimeric vectors between HIV-1 and HIV-2 is feasible and suggest that an optimal combination of chimeric transfer vectors and packaging constructs with enhanced safety and minimal loss of efficiency can be designed.