One approach to restore immune function in individuals with AIDS is to infuse autologous CD4+ T cells that have been genetically modified to resist HIV infection. Since many questions central to this approach can be appropriately addressed in the SIV/macaque model, we have analyzed the conditions necessary to achieve optimal retroviral transduction of rhesus CD4+ T cells. Transduction has assessed using a retroviral vector expressing the murine CD2 molecule, thereby allowing assessment of transduction efficiency by flow cytometry. We have observed a several fold increase in transduction efficiency using retroviral vectors produced in the PG13 packaging cell line (which expresses the gibbon ape leukemia virus envelope) as compared with the Psi crip cell line (which expresses the amphotropic murine retrovirus envelope). Conditions that have been found in increase transduction efficiency include harvesting of retroviral vector at 32 degrees C, phosphate depletion and centrifugation. This optimized retroviral transduction protocol will be used to examine the ability of different gene therapy constructs to inhibit SIV replication in primary rhesus CD4+ T cells.
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