Development of a Nonintegrating Rev-dependent Lentiviral Vector Carrying Diphtheria Toxin A Chain and Human TRAF6 to Target HIV Reservoirs

Persistence of human immunodeficiency virus (HIV) despite highly active antiretroviral therapy (HAART) is a lasting challenge to virus eradication. To develop a strategy complementary to HAART, we constructed a series of Rev-dependent lentiviral vectors carrying diphtheria toxin A chain (DT-A) and its attenuated mutants, as well as human tumor necrosis factor receptor-associated factor 6 (TRAF6). Expression of these suicide genes following delivery through viral particles is dependent on Rev, which exists only in infected cells. Among these toxins, DT-A has been known to trigger cell death with as little as a single molecule, whereas two of the attenuated mutants in this study, DT-A(176) and DT-A(Delta N), were well tolerated by cells at low levels. TRAF6 induced apoptosis only with persistent overexpression. Thus, these suicide genes, which induce cell death at different expression levels, offer a balance between efficacy and safety. To minimize possible mutagenesis introduced by retroviral integration in nontarget cells, we further developed a nonintegrating Rev-dependent (NIRD) lentiviral vector to deliver these genes. In addition, we constructed a DT-A-resistant human cell line by introducing a human elongation factor 2 mutant into HEK293T cells. This allowed us to manufacture the first high-titer NIRD lentiviral particles carrying DT-A to target HIV-positive cells.