Retroviral transfer of the glucocerebrosidase gene into CD34+ cells from patients with Gaucher disease: in vivo detection of transduced cells without myeloablation.

Retroviral gene transfer of the glucocerebrosidase gene to hematopoietic progenitor and stem cells has shown promising results in animal models and corrected the enzyme deficiency in cells from Gaucher patients in vitro. Therefore, a clinical protocol was initiated to explore the safety and feasibility of retroviral transduction of peripheral blood (PB) or bone marrow (BM) CD34+ cells with the G1Gc vector. This vector uses the viral LTR promoter to express the human glucocerebrosidase cDNA. Three adult patients have been entered with follow-up of 6-15 months. Target cells were G-CSF-mobilized and CD34-enriched PB cells or CD34-enriched steady state BM cells, and were transduced ex vivo for 72 hr. Patient 1 had PB cells transduced in the presence of autologous stromal marrow cells. Patient 2 had PB cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. Patient 3 had BM cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. At the end of transduction, the cells were collected and infused immediately without any preparative treatment of the patients. The transduction efficiency of the CD34+ cells at the end of transduction was approximately 1, 10, and 1 for patients 1, 2, and 3, respectively, as estimated by semiquantitative PCR on bulk samples and PCR analysis of individual hematopoietic colonies. Gene marking in vivo was demonstrated in patients 2 and 3. Patient 2 had vector-positive PB granulocytes and mononuclear bone marrow cells at 1 month postinfusion and positive PB mononuclear cells at 2 and 3 months postinfusion. Patient 3 had a positive BM sample at 1 month postinfusion but was negative thereafter. These results indicate that gene-marked cells can engraft and persist for at least 3 months postinfusion, even without myeloablation. However, the level of corrected cells (<0.02%) is too low to result in any clinical benefit, and glucocerebrosidase enzyme activity did not increase in any patient following infusion of transduced cells. Modifications of vector systems and transduction conditions, along with partial myeloablation to allow higher levels of engraftment, may be necessary to achieve beneficial levels of correction in patients with Gaucher disease.

Gene therapy for pediatric AIDS.

Gene therapy is an experimental treatment modality under investigation for applications to HIV-1 infection. We have developed retroviral vectors carrying anti-HIV-1 genes, demonstrated that these genes cause significant suppression of HIV-1 replication in cultures of primary hematopoietic cells, and performed a clinical trial in pediatric AIDS patients. Four HIV-1-infected children and adolescents underwent bone marrow harvest from which CD34+ cells were isolated and transduced by a retroviral vector carrying an RRE decoy gene. The cells were re-infused into the subjects, without complications, showing that gene transfer in pediatric AIDS patients in safe and feasible. However, gene-containing leukocytes in the peripheral blood were seen only at a low level and only in the first months following cell infusion. To attain some degree of efficacy, it will be necessary to achieve a higher level of gene transfer and to obtain sustained gene expression. We are currently developing new gene transfer methods and vectors designed to improve the results in future trials. If it becomes possible to reach the ideal goal of producing high percentages of T lymphocytes and monocytic cells that are resistant to HIV-1 infection, gene therapy could serve as a complement to antiretroviral drug therapy and help to sustain immunologic function.