GENE THERAPY Lentivirus vectors expressing short hairpin RNAs against the U3-overlapping region of HIV nef inhibit HIV replication and infectivity in primary macrophages

Although successful attempts to inhibit HIV-1 replication in T cells using RNAi have been reported, the effect of HIV-specific RNAi on macrophages is not well known. Macrophages are key targets for anti–HIV-1 therapy because they are able to survive long after the initial infection with HIV and can spread the virus to T cells. In this study, we identified a puta-tive RNAi target of HIV, consisting of the portion of the nef gene overlapping the U3 region (Nef366), and generated a lenti-virus-based short hairpin RNA (shRNA) expression vector (Lenti shNef366). We show that Lenti shNef366 inhibits (1) HIV-1 replication in a monocytic cell line and in primary monocyte-derived macrophages (MDMs), (2) reactivation of latent HIV-1 infection, and (3) the production of sec-ondary HIV-1 from MDMs harboring a genomic copy of Nef366. Moreover, we found that the up-regulated production of macrophage inflammatory protein 1 (MIP-1), but not MIP-1, in MDMs by Nef expression was considerably suppressed by Lenti shNef366, which suggests that HIV-1 dissemination to T cells through its interaction with HIV-1–infected MDMs can also be controlled by Lenti shNef366. Thus, lentivirus-mediated shRNA expres-sion targeting the U3-overlapping region of HIV nef represents a feasible approach to genetic vaccine therapy for HIV-1