Professional antigen-presenting cells, such as dendritic cells (DCs) and macrophages, are target cells for gene therapy of infectious disease and cancer. However, transduction of DCs and macrophages has proved difficult by most currently available gene transfer methods. Several recent studies have shown that lentiviral vector systems can efficiently transduce many nondividing and differentiated cell types. In this study, we examined the gene transfer to DCs and macrophages using a lentiviral vector system. Human DCs were propagated from the adherent fraction of peripheral blood mononuclear cells (PBMCs) by culture in medium containing GM-CSF, IL-4, and TNF-alpha. Human macrophages were propagated from adherent PBMCs in medium containing GM-CSF. High titers of a replication-defective vesicular stomatitis virus glycoprotein G pseudotyped HIV-1-based vector encoding the enhanced yellow fluorescent protein were produced. In immature DCs (culture days 3 and 5), transduction efficiencies of 25 to 35% were achieved at a multiplicity of infection of 100. However, the transduction efficiency was decreased in more mature DCs (culture day 8 or later). Furthermore, monocyte-derived macrophages were also transduced by the lentiviral vector system. In addition, Alu-LTR PCR demonstrated the integration of the HIV-1 provirus into the cellular genome of the transduced DCs and macrophages. Allogeneic mixed lymphocyte reactions revealed similar antigen-presenting functions of untransduced and lentivirally transduced DCs. Thus, the results of this study demonstrate that both PBMC-derived DCs and macrophages can be transduced by lentiviral vectors.
It is possible, that CD34+ cells in the megadose transplants-perhaps by their inability to provide costimulatory molecules-are actively reducing the frequency of CTL-p directed against their antigens, and thereby help to overcome allogeneic rejection, and enhance their own engraftment.
Replication-competent lentivirus (RCL) may be generated during the production phase or subsequently after introduction of a lentiviral vector into target cells, potentially by homologous or nonhomologous recombination. Because most gene transfer of HIV-based vectors involves the use of high-titer vesicular stomatitis virus (VSV) G-pseudotyped particles, one particular concern would be the generation of an RCL of altered host range, i.e., one that has incorporated the VSV G envelope in cis configuration. We report here on the artificial generation and properties of such a virus, including its detection after biological amplification. Viral spread, beginning with a very low inoculum, takes several weeks in culture and is characterized by "autoinfection," resulting in multiple proviral copies per cell, higher levels of viral gene expression, and eventual cell death. After this initial amplification step, the RCL is easily detectable by standard p24 assay or by "marker-rescue" assay. For the latter, a 293T-based cell line that has an integrated replication-defective provirus encoding alkaline phosphatase (AP) was used and mobilization of AP-containing virus was detected by transduction of naïve cells. Replication-defective virus was not amplified nor detected, demonstrating assay specificity. These results suggest that these artificial RCLs of broad host range have slightly different biological properties compared to wild-type HIV but still spread and are readily detectable.
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