592. Effective Transduction of Nondividing PBL, Monocytes and Monocyte-Derived Macrophages with SV40-Derived Vectors Protects from HIV-1 Challenge
Authors: Elena I. Marusich, David S. Strayer
Journal: Molecular Therapy (2006)

One of the main challenges in application of gene therapy to treatment of HIV-1 infection is the need to protect non-dividing lymphocytes and monocytes, as well as monocyte-derived macrophages (MDM), from HIV-1. Since monocyte-derived macrophages represent a major reservoir HIV-1 in vivo, and because at any given time most T cells, monocytes and macrophages in the body are quiescent, gene transfer to these types of cells is very important, yet very difficult, in considering gene delivery to protect from HIV-1. Recombinant SV40-derived vectors (rSV40s) transduce resting cells, especially lymphocytes, monocytes and macrophages, very efficiently. Therefore we tested the effectiveness of a series of anti-HIV-1 transgenes delivered to human PBL, monocytes and MDM by rSV40-derived vectors.We have engineered a series of constructs carrying: single chain Fv antibodies (SFvs) against the DNA-binding and catalytic regions of HIV-1 integrase (IN), SFvs binding two different regions of HIV-1 reverse transcriptase (RT) and the trans-dominant mutant of HIV-1 Rev, RevM10. In all cases, transgene expression was driven by CMV-IEP. Normal human PBMC were fractionated into lymphocytes (PBL) and monocytes by differential adherence. Monocytes were differentiated into macrophages using standard cytokine cocktails.Unstimulated PBL were transduced with these vectors by sequential exposure over 3 days at a cumulative MOI=16. Control cultures were mock-transduced or treated with control rSV40s. Transgene expression was measured by FACS. The quiescent state of the PBL at the time of transduction was verified by propidium iodide staining of their DNA, followed by flow cytometry to confirm that cells were in the G0/G1 phase of the cycle. After transduction, cultures were challenged using 80 IU and 400 infectious units (IU) HIV-1NL4-3, or with 400 IU and 4000 IU primary isolate HIV-1 92HT594. PBL were then stimulated (to maintain viability) with IL-2, and maintained in this condition for 1 month of challenge. At 80 IU challenge dose PBL treated with rSV40 vectors carrying RevM10, either of the anti-IN SFvs or either anti-RT SFvs were completely protected, compared with controls. Similarly, at the higher 400 IU challenge dose of HIV-1, virus replication was also completely inhibited for all test transduced infectious cultures. PBL transduced with the anti-HIV-1-integrase SFvs were better protected at highest 4000 IU dose HIV-1 primary isolate 92HT594, in comparison with other transduced cultures.Comparably high protective efficiency was also observed in challenge studies using monocytes and MDM transduced with all of the tested SV40-delivered constructs. HIV-1 replication was inhibited at least 90-95%, at all challenge doses of HIV-1, compared with controls. Thus, we verified the potency of an array of anti-HIV-1 transgenes delivered by SV40 based vectors to human non-dividing PBL, monocytes and macrophages. Anti-HIV-1 transgenes so transferred to all these cell types proved protective when tested against multiple strains of HIV-1. Therefore, rSV40 gene delivery to resting HIV-1-susceptible blood cells provides effective protection from even high dose HIV-1 challenge. One of the main challenges in application of gene therapy to treatment of HIV-1 infection is the need to protect non-dividing lymphocytes and monocytes, as well as monocyte-derived macrophages (MDM), from HIV-1. Since monocyte-derived macrophages represent a major reservoir HIV-1 in vivo, and because at any given time most T cells, monocytes and macrophages in the body are quiescent, gene transfer to these types of cells is very important, yet very difficult, in considering gene delivery to protect from HIV-1. Recombinant SV40-derived vectors (rSV40s) transduce resting cells, especially lymphocytes, monocytes and macrophages, very efficiently. Therefore we tested the effectiveness of a series of anti-HIV-1 transgenes delivered to human PBL, monocytes and MDM by rSV40-derived vectors. We have engineered a series of constructs carrying: single chain Fv antibodies (SFvs) against the DNA-binding and catalytic regions of HIV-1 integrase (IN), SFvs binding two different regions of HIV-1 reverse transcriptase (RT) and the trans-dominant mutant of HIV-1 Rev, RevM10. In all cases, transgene expression was driven by CMV-IEP. Normal human PBMC were fractionated into lymphocytes (PBL) and monocytes by differential adherence. Monocytes were differentiated into macrophages using standard cytokine cocktails. Unstimulated PBL were transduced with these vectors by sequential exposure over 3 days at a cumulative MOI=16. Control cultures were mock-transduced or treated with control rSV40s. Transgene expression was measured by FACS. The quiescent state of the PBL at the time of transduction was verified by propidium iodide staining of their DNA, followed by flow cytometry to confirm that cells were in the G0/G1 phase of the cycle. After transduction, cultures were challenged using 80 IU and 400 infectious units (IU) HIV-1NL4-3, or with 400 IU and 4000 IU primary isolate HIV-1 92HT594. PBL were then stimulated (to maintain viability) with IL-2, and maintained in this condition for 1 month of challenge. At 80 IU challenge dose PBL treated with rSV40 vectors carrying RevM10, either of the anti-IN SFvs or either anti-RT SFvs were completely protected, compared with controls. Similarly, at the higher 400 IU challenge dose of HIV-1, virus replication was also completely inhibited for all test transduced infectious cultures. PBL transduced with the anti-HIV-1-integrase SFvs were better protected at highest 4000 IU dose HIV-1 primary isolate 92HT594, in comparison with other transduced cultures. Comparably high protective efficiency was also observed in challenge studies using monocytes and MDM transduced with all of the tested SV40-delivered constructs. HIV-1 replication was inhibited at least 90-95%, at all challenge doses of HIV-1, compared with controls. Thus, we verified the potency of an array of anti-HIV-1 transgenes delivered by SV40 based vectors to human non-dividing PBL, monocytes and macrophages. Anti-HIV-1 transgenes so transferred to all these cell types proved protective when tested against multiple strains of HIV-1. Therefore, rSV40 gene delivery to resting HIV-1-susceptible blood cells provides effective protection from even high dose HIV-1 challenge.