602. Effectiveness of Gene Delivery Targeting HIV-1 Integrase in Inhibiting HIV Integration: Lack of HIV-1 Genomes in T-Lymphocytes during Repeated Challenges with HIV-1

HIV-1 integration into cell genomes is a key step in the HIV-1 replicative cycle. We applied a gene therapy strategy designed to block HIV replication at this early stage. To do this, we used a single chain antibody (SFv) against the DNA-binding domain of HIV-1 integrase (IN). SV40-derived vectors were used for gene transfer to human T-lymphocytes because of their high transduction efficiency and durability of transgene expression. HIV-1 replication was measured by supernatant p24 antigen levels. Protection by the test vector, SV(Aw), compared with mock- and SV(HBS) transduced cells used as negative controls, was assessed during 3 successive rounds of HIV-1 NL4-3 challenge. HIV-1 infectious dose escalated from 400 infectious units (IU) to 4000 IU.SV(Aw)-transduced SupT1 cells were protected from HIV-1 replication at all stages of the study, and surviving cells became progressively more resistant with each challenge. Since the transgene target was HIV-1 IN, we measured HIV proviral DNA integration after each round of challenge. For these purposes, multiplex real- time PCR was used to quantify numbers of HIV-1 cDNA copies in SV(Aw) - transduced cells, compared to controls. Sequence-specific primers coupled with fluorescent probes were used to amplify either the most conserved catalytic domain of the HIV-1 integrase gene, or the human p53 gene. Probes were labeled with reporter dyes that emit light at different wavelengths to enable simultaneous detection and quantification of both genes in a single reaction. Plasmids containing the respective HIV and human genes were used to generate standard calibration curves.No HIV-1 cDNA in SV(Aw) was detected in the transduced SupT1 cells after the first HIV-1 challenge at 400 IU. HIV-1 cDNA was also undetectable in genomes of the highly HIV-resistantSV(Aw)-transduced SupT1 cells after a second challenge with 400 IU HIV, compared with 4.44 × 105 copies of p53. Similar results were observed after the third round of challenge with variable doses of HIV-1: HIV-1 provirus genomic DNA was undetectable at all challenge doses, in comparison with 3.6 × 105 p53 DNA copies at 80 IU challenge dose, 6.17 × 105 p53 copies at 400 IU challenge dose and 4.94 × 105 copies of p53 DNA at 4000 IU challenge dose. In contrast, control SupT1 cells challenged with 400 IU HIV-1 carried 2.55 × 105 copies of HIV integrase and 6.4 × 105 copies of p53. These findings were also correlated with HIV replication, as measured by p24 antigen ELISA. SV(Aw