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
Authors: Elena I. Marusich, Christine B. Mitchell, David S. Strayer
Journal: Molecular Therapy (2006)

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) transduced cultures showed progressively decreasing levels of HIV replication with sequential challenges, compared with mock-transduced cells or control cells transduced with an unrelated rSV40. Levels of anti- IN SFv expression remained relatively constant throughout almost 6 months of multiple round challenges. Thus, an anti-HIV-1 integrase single chain Fv antibody was efficiently delivered to human T-lymphocytes by SV40-based vectors, was expressed durably in those cells and blocked HIV-1 integration repeatedly at each round of HIV-1 challenge.