Flossie Wong-Staal

Transduction of human macrophages using a stable HIV-1/HIV-2-derived gene delivery system.

We have previously established a stable HIV-1 packaging cell line, ψ422, which yielded high titers of an HIV-1 vector capable of efficiently transducing CD4+ cells. In order to increase the safety of this gene delivery system, we have now replaced the HIV-1 vector with an HIV-2 vector to abolish any risk of homologous recombination between the packaging cells and the vector. The HIV-2 vector was also modified by insertion of a cis-acting constitutive transport element which confers Rev independence. The supernatant of ψ422 cells stably transfected with this new vector was capable of transducing CD4+ cells with a titer of 104 transducing units per milliliter. This result shows that cross-packaging of HIV-2 vectors with the HIV-1 packaging cells is quite efficient. Using this new stable HIV-1/HIV-2 gene delivery system, we were able to transduce human monocyte-derived primary macrophages, which are refractory to murine retrovirus-mediated transduction. The availability of a stable HIV-based gene delivery system for macrophages, a key target cell in HIV infection; is an important advance in gene therapy for AIDS.

Novel replication competent human immunodeficiency virus type 2 (hiv-2) proviral clone designated hiv-2kr

A full-length infectious molecular clone, designated HIV-K, was obtained from a recombinant bacteriophage library constructed from HI-2PIE-infected MOLT-4/8 genomic DNA. Nucleotide sequence analysis of this clone demonstrated that the rev coding region extends for nearly 69 amino acid residues past the end of most other HIV-2/SIV rev genes. In addition, the long terminal repeat (LTR) contains a deletion of 9-10 bp, depending upon the nucleotide sequence alignment parameters, upstream from the SpI binding sites. This deletion is not present in other HIV-2/SIV isolates and is not similar to previously disclosed NFkB duplications in this region. The HIV-2KR LTR displays higher levels of basal transcriptional acitivty as compared to prototypical HIV-2 isolates. HIV-2KR is capable of infecting macaque peripheral blood lymphocytes (PBMCs) in vitro and produces a productive and persistant, albeit attenuated, infection in Macacca nemestrina. These proviral constructs are useful, inter alia, for the generation of in vitro diagnostic reagents, cell transcuction vectors, and the generation of HIV-2 based packaging cell lines.