HIV-1 gene therapy offers a promising alternative to small molecule antiretroviral treatments and current vaccination strategies by transferring, into HIV-1-susceptible cells, the genetic ability to resist infection. The need for novel and innovative strategies to prevent and treat HIV-1 infection is critical due to devastating effects of the virus in developing countries, high cost, toxicity, generation of escape mutants from antiretroviral therapies, and the failure of past and current vaccination efforts. As a first step toward achieving this goal, an HIV-1-susceptible cell-specific targeting vector was evaluated to selectively transfer, into CCR5-positive target cells, an anti-HIV CCR5 shRNA gene for subsequent knockdown of CCR5 expression and protection from HIV-1 infection. Using a ZZ domain/monoclonal antibody-conjugated Sindbis virus glycoprotein pseudotyped lentiviral vector, here we demonstrate the utility of this strategy for HIV-1 gene therapy by specifically targeting HIV-1-susceptible cells and engineering these cells to resist HIV-1 infection. CCR5-positive human cells were successfully and specifically targeted in vitro and in vivo for transduction by a lentiviral vector expressing a highly potent CCR5 shRNA which conferred resistance to HIV-1 infection. Here we report the initial evaluation of this targeting vector for HIV-1 gene therapy in a preexposure prophylactic setting.