No abstract

Retroviral vectors derived from amphotropic murine leukemia viruses (MLV) mediate gene transfer into almost all human cells and are thus not suitable for in vivo applications in gene therapy in which cell-specific gene delivery is required. We and others recently reported the generation of MLV-derived vectors pseudotyped by variants of the envelope glycoproteins (Env) of human immunodeficiency virus type 1 (HIV-1), thus displaying the CD4-dependent tropism of the parental lentivirus (Mammano et al., 1997, J. Virol. 71, 3341-3345; Schnierle et al., 1997, Proc. Natl. Acad. Sci. USA 76, 8640-8645). However, because of their HIV-1-derived envelopes these vectors are neutralized by HIV-specific antibodies present in some infected patients. To circumvent this problem, we pseudotyped MLV capsid particles with variants of Env proteins derived from the apathogenic simian immunodeficiency virus (SIVagm) of African green monkeys (AGM; Chlorocebus pygerythrus). Truncation of the C-terminal domain of the transmembrane protein was found to be necessary to allow formation of infectious pseudotype vectors. These [MLV(SIVagm)] vectors efficiently transduced various human CD4-expressing cell lines using the coreceptors CCR5 and Bonzo to enter target cells. Moreover, they were resistant to neutralization by antibodies directed against HIV-1. Therefore, [MLV(SIVagm)] vectors will be useful to study the mechanisms of SIVagm cell entry and for the selective gene transfer into CD4+ T-cells of AIDS patients.

No abstract

As a single-gene defect in phagocytes, the X-linked form of chronic granulomatous disease (X-CGD) is a disorder potentially amenable to gene therapy by transfer of a functional copy of the gp91(phox) gene into hematopoietic stem cells (HSC). Although antimicrobial agents and interferon-gamma (IFN-gamma) have significantly improved its prognosis, CGD is still associated with high morbidity and mortality. The disease can be cured by bone marrow transplantation (BMT); however, BMT in CGD has been associated with unacceptably high rates of morbidity, mortality, and graft failure, except in very selected cases in which an HLA-identical donor is available. Prerequisites for a clinical gene therapy of CGD are an efficient mobilization of peripheral blood stem cells (PBSC) as well as the preservation of their viability and hematopoietic potential following transduction and ex vivo culture. We show that (i) mobilization and collection of CD34(+) cells after a 4-week IFN-gamma-free period by G-CSF results in sufficient numbers of cells for transplantation; (ii) the quality of collected stem cells is not altered in comparison to cells obtained from healthy volunteers as assessed by long-term culture initiating cells (LTC-IC) and progenitor cell expansion; (iii) retroviral transfer of the gp91(phox) gene under defined, serum-free conditions leads to high and stable reconstitution of the respiratory burst activity in X-CGD neutrophils derived from transduced CD34(+) progenitor and LTC-IC. Withdrawal of IFN-gamma in CGD patients may improve mobilization of CD34(+) stem cells by G-CSF. The gene transfer conditions established here are applicable to a clinical approach for gene therapy of X-CGD.