Although genetically altered cells were detected in one individual up to 2 months after gene transfer, the recombinant gene was not detectable in two patients after 2 weeks. compared with 3 weeks for the previously Rabbit Polyclonal to ADCK3 reported nonviral vector delivery. These findings suggest that retroviral delivery of an antiviral gene may potentially contribute to immune reconstitution in AIDS and could provide a more effective vector to prolong survival of CD4+ cells in HIV?contamination. The inhibition of HIV replication by interference with essential structural or regulatory viral genes has been investigated to understand the molecular pathogenesis of HIV contamination and to explore its potential clinical applications (1C15). Among viral products that may be targeted genetically, Rev is usually a regulatory protein required for productive viral replication. Rev represents a 118-aa protein encoded by a fully spliced mRNA synthesized early after viral contamination of host cells, which facilitates the accumulation of unspliced and partially spliced viral mRNAs in the cytoplasm (1, 16C18) through its conversation with host cell factors (19C21) and plays an important role in the activation of computer virus in infected cells (22). Introduction of two point mutations in a highly conserved region of Rev gives rise to a defective protein, Rev M10, which acts as a potent inhibitor of computer virus replication that does not adversely impact a variety of normal T cell functions (1C3, 23C25). We previously have evaluated the potential of Rev M10 gene delivery to inhibit the replication of both cloned and clinical isolates of HIV in main T cells transduced with plasmid or retroviral vectors expressing Rev M10 protein (4). A human clinical study also has examined the potential of Rev M10 to prolong the survival of transduced CD4+ T cells after transduction, growth, and reinfusion into HIV-seropositive patients. By using plasmid vectors with platinum particle-mediated gene delivery, a 4- to 5-fold survival advantage was found in cells that expressed Rev M10 compared with unfavorable control transduced cells (26); however, the period of engraftment was limited. Although genetically altered cells were detected in one patient up to 2 months after gene transfer, the recombinant gene was not detectable in two patients after 2 weeks. To determine whether more durable engraftment could be achieved, option gene transfer vectors and protocols for T cell activation thus have been developed (27). We Etifoxine hydrochloride now have analyzed lymphocyte survival in HIV-seropositive individuals whose peripheral blood CD4+ lymphocytes were stimulated with anti-CD3 and interleukin 2 and transduced with retroviral vectors that express Rev M10 or a negative control frameshifted vector, which transcribes a similar mRNA but encodes no functional Rev M10 protein. We have found that genetically altered cells are detected for longer time periods with these?vectors. MATERIALS AND?METHODS Isolation and Culture of Human Peripheral Blood Lymphocytes (PBLs). Blood for these studies was obtained from normal or HIV-seropositive donors. Etifoxine hydrochloride PBLs were isolated by using Ficoll-Hypaque separation (28). The cells were stimulated in flasks coated with immobilized Etifoxine hydrochloride OKT3 mAb and soluble interleukin 2 (50 models/ml) for 48C72 hr. Cells were recovered and resuspended at 5 105/ml in X-Vivo-15 medium (BioWhittaker) made up of 50 models/ml of interleukin 2 and 5 M delavirdine. Cells were managed at 2 105 to 1 1.5 106/ml throughout the?experiments. Retroviral Transduction. Freshly isolated human PBLs from donors were purified by centrifugation on Ficoll gradients. Retroviral vectors were derived from the pLJ plasmid (29). After activation, cells were infected for 6C12 hr with -Crip supernatants made up of pLJ-Rev M10 or frameshift pLJ-Rev M10 retroviruses (1). Cells (1 106/ml) were inoculated in medium consisting of equivalent volumes of -Crip supernatant and X-Vivo.
Although genetically altered cells were detected in one individual up to 2 months after gene transfer, the recombinant gene was not detectable in two patients after 2 weeks