584. Enhanced Inhibition of HIV Using Combinatorial Genetic Therapy Against HIV-1 Integrase Delivered by SV40 Vectors
Authors: Elena I. Marusich, David S. Strayer
Journal: Molecular Therapy (2006)

Highly active anti-retroviral therapy, HAART, is a well-established combination pharmacotherapy. Its fundamental principle is to combat HIV-1 replication simultaneously at multiple points. However, the toxicity of HAART, its highly cost and rapid development of multiply resistant strains of HIV-1 all suggest that other therapeutic approaches are needed.We studied combinatorial anti-HIV-1 genetic therapy and hypothesized, that multiple transduction with several anti-HIV moieties might compensate for the limitations of any individual construct. To test this hypothesis we used SV40-derived vectors because of their highly efficient gene delivery to HIV-susceptible cells. We have established that these vectors can be used to deliver several transgenes in sequence to the same cells without selection. For these purposes we combined single chain variable fragment antibodies, SFvs, against HIV-1 integrase (IN). IN4 SFv inhibits the activity of the IN catalytic region, and Aw SFv targets the DNA- binding domain of IN. IN is used as a target because it is relatively invariant among the various strains of HIV-1.SupT1 cells were transduced with SV(Aw), then re-transduced 1 week later with SV(IN4). Control cultures were transduced with either vector alone or with control rSV40 vectors. To test whether combinatorial anti-IN gene transfer improved protection from higher doses HIV, cells were challenged with 40 to 1000 IU HIV-1NL4-3Sequential transduction with both rSV40 constructs gave simultaneous expression of both transgenes in > 95% of unselected cells, as detected by FACS. HIV-1 replication was measured in singly and multiply transduced cultures challenged with escalating doses of HIV-1. We found that individual constructs generally inhibited HIV-1 replication totally, or almost totally, at low challenge doses (e.g., 40 IU/culture). However, protection by individual constructs was less effective with increasing challenge dose. As input HIV-1 dose increased to 80, then to 400 and finally to 1000 IU/culture, single transgenes reduced HIV-1 replication by between 62% and 72%. By contrast, doubly transduced cultures were far more effectively protected from HIV: even at the highest challenge doses double transduction with both anti-IN SFv provided synergistic protection, reducing HIV-1 replication by 94-96%.The principle of combination therapy, i.e., simultaneously targeting multiple aspects of the disease process, is well established. Here we focused combination genetic therapy on HIV-1 integrase, using two different single chain Fv antibodies, one directed to IN catalytic domain and the other directed to IN DNA-binding domain. Sequential transduction with SV40-derived vectors readily lends itself to application in delivering flexible combination genetic therapies to unselected target cell populations. Pushing the limits of protection by escalating HIV-1 challenge doses, we found that combining two anti-IN SFv's gave supra-additive protection from HIV. Thus, combining multiple transgenes, whether delivered by rSV40s or other vectors, may be a potentially effective approach anti-HIV genetic therapy. Highly active anti-retroviral therapy, HAART, is a well-established combination pharmacotherapy. Its fundamental principle is to combat HIV-1 replication simultaneously at multiple points. However, the toxicity of HAART, its highly cost and rapid development of multiply resistant strains of HIV-1 all suggest that other therapeutic approaches are needed. We studied combinatorial anti-HIV-1 genetic therapy and hypothesized, that multiple transduction with several anti-HIV moieties might compensate for the limitations of any individual construct. To test this hypothesis we used SV40-derived vectors because of their highly efficient gene delivery to HIV-susceptible cells. We have established that these vectors can be used to deliver several transgenes in sequence to the same cells without selection. For these purposes we combined single chain variable fragment antibodies, SFvs, against HIV-1 integrase (IN). IN4 SFv inhibits the activity of the IN catalytic region, and Aw SFv targets the DNA- binding domain of IN. IN is used as a target because it is relatively invariant among the various strains of HIV-1. SupT1 cells were transduced with SV(Aw), then re-transduced 1 week later with SV(IN4). Control cultures were transduced with either vector alone or with control rSV40 vectors. To test whether combinatorial anti-IN gene transfer improved protection from higher doses HIV, cells were challenged with 40 to 1000 IU HIV-1NL4-3 Sequential transduction with both rSV40 constructs gave simultaneous expression of both transgenes in > 95% of unselected cells, as detected by FACS. HIV-1 replication was measured in singly and multiply transduced cultures challenged with escalating doses of HIV-1. We found that individual constructs generally inhibited HIV-1 replication totally, or almost totally, at low challenge doses (e.g., 40 IU/culture). However, protection by individual constructs was less effective with increasing challenge dose. As input HIV-1 dose increased to 80, then to 400 and finally to 1000 IU/culture, single transgenes reduced HIV-1 replication by between 62% and 72%. By contrast, doubly transduced cultures were far more effectively protected from HIV: even at the highest challenge doses double transduction with both anti-IN SFv provided synergistic protection, reducing HIV-1 replication by 94-96%. The principle of combination therapy, i.e., simultaneously targeting multiple aspects of the disease process, is well established. Here we focused combination genetic therapy on HIV-1 integrase, using two different single chain Fv antibodies, one directed to IN catalytic domain and the other directed to IN DNA-binding domain. Sequential transduction with SV40-derived vectors readily lends itself to application in delivering flexible combination genetic therapies to unselected target cell populations. Pushing the limits of protection by escalating HIV-1 challenge doses, we found that combining two anti-IN SFv's gave supra-additive protection from HIV. Thus, combining multiple transgenes, whether delivered by rSV40s or other vectors, may be a potentially effective approach anti-HIV genetic therapy.