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FIG. 2. General scheme for construction of packaging and vector plasmids.

linked sequentially to the 5′ leader including the psi region, a short portion of the p17 region of the gag gene, the respective rev response element (RRE), an internally promoted marker gene and the 3′ LTR. Marker genes used in these studies included xanthine-guanine phosphoriboxyl transferase (gpt), neomycin phosphotransferase (neoR), Escherichia coli β-galactosidase (lacZ) green fluorescent protein (gfp) (48, 49), and the Streptomyces hindustanus phleomycin resistance gene (she ble). In some vectors an element from Mason-Pfizer monkey virus previously shown to substitute for Rev-RRE-mediated activity in HIV mRNAs nuclear transport was included instead of the RRE (50).

Vector Packaging and Transduction

HIV-1 and HIV-2 vectors were transfected into the producer cell lines and the cells were doubly selected for stable expression of viral proteins and vectors. Clones of these stable cell lines were also generated, and titers of the transducing vector in the supernatants were measured. As shown in Table 1, both HIV-1 and HIV-2 vectors were packaged in the HIV-1MN packaging cell line Ψ422. Titers of 10e4 to 10e5 were achievable. These vectors were able to transduce terminally differentiated primary macrophages, in contrast to murine retrovirus vectors, which failed to do so (P.C.G.Kraus, F.W.-S., unpublished work). With the HIV-2KR packaging line, transfection of an HIV-2 neoR vector yielded titers of 10e3 to 10e4 (Table 2). These titers are two to three logs higher than previously reported values of the stable HIV-1 packaging line. It is not clear whether it is the choice of the packaging constructs (both HIV-1MN and HIV-2KR contain coding sequences for all of the accessory genes, and both are duotropic for T cells and monocytes) or that of the producer cells which allowed expression of high titers of infectious vectors. Pseudotyped HIV-2 vectors were generated by transient triple cotransfection of a packaging construct with an additional deletion in the env gene, the vector plasmid, and a plasmid encoding VSV-G under control of the hCMV promoter (kindly supplied by T.Friedman, Uuniversity of California, San Diego). Production of 10e5 or higher titers of the pseudotyped vectors was observed. A vector expressing GFP as a reporter gene gave similar titers (E.P. and F.W.S., unpublished work).

Table 2. Transducing titer of HIV-2 neoR vectors produced from HIV-2KR packaging cell clone on U937 cells

Exp.

Titer, transducing units/ml

Mean

1

1.3×10e4

1.8×10e4

2

8.5×10e3

 

3

3.2×10e4

 

Experiments to determine if these vectors can transduce non-cycling CD34+/CD38− cells in culture, or long-term repopulating cells in in vivo animal models are in progress.

Perspectives

Our current understanding of AIDS pathogenesis affirms the central role of HIV in both disease initiation and progression. Recent studies on virus dynamics in patients under chemotherapy (1), as well as long-term prospective studies of plasma viral burden in patients that progress to disease at different rates (2) support a virus threshold hypothesis for disease progression. It is now also recognized that insidious damage inflicted by the virus upon the immune system occurs from the onset of infection, underscoring the importance of early intervention in infected individuals. Although recent clinical results from trials of combinations of antiviral agents, including the potent protease inhibitors, have been encouraging, whether such therapy can be sustained lifelong without recument problems of toxicity, viral resistance, and economics is unclear. Gene therapy has been considered by many to be an attractive strategy for conferring long-term therapeutic benefits.

Gene therapy for HIV infection, however, faces experimental obstacles common to gene therapy and genes that are intrinsic to the nature of HIV infection in particular. The extreme inefficiency of transducing hematopoietic progenitor cells that would give rise to long-term repopulation of multilineage progeny cells in animals is a general frustration. For HIV infection, the need to access the nonproliferative macrophage target cell reservoir, the uncertainty of whether bone marrow derived hematopoiesis may be impaired in adult AIDS patients, and the lack of high-titer vectors that allow in vivo targeting are additional concerns. The ability of HIV vectors to both target CD4+ cells in vivo and transduce non-cycling cells may help resolve some of these issues.

E.P. is a recipient of a National Institutes of Health Physician-Scientist Award. The work described here is supported by the National Institutes of Health SPIRAT award to F.W.-S. and the University of California, San Diego Center for AIDS Research.

1. Perelson, A.S., Neumann, A.S., Markowitz, M., Leonard, J.M. & Ho, D.D. (1996) Science 271, 1582–1586.

2. Mellors, J.W., Rinaldo, C.R., Gupta, P., White, M.R., Todd, J.A. & Kingsley, L.A. (1996) Science 272, 1167–1170.

3. Ho, D.D., Neumann, A.U., Perelson, A.S., Chen, W., Leonard, J.M. & Markowitz, M. (1995) Nature (London) 373, 123–126.

4. Wei, X.P., Ghosh, S.K., Taylor, M.E., Johnson, V.A., Emini, E. A., et al. (1995) Nature (London) 373, 117–123.

5. Pantaleo, G., Graziosi, C., Demarest, J.F., Butini, L., Montroni, M., et al. (1993) Nature (London) 362, 355–358.



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