RESUMEN
The transduction efficiencies of immunoselected rhesus macaque (Macaca mulatta) CD34+ cells and colony-forming progenitor cells based on polymerase chain reaction (PCR) analysis were comparable for an amphotropic Moloney murine leukemia virus (MLV) retroviral vector and a retroviral vector derived from the gibbon ape leukemia virus (GaLV) packaging cell line, PG13. On performing autologous transplantation studies using immunoselected CD34+ cells transduced with the GaLV envelope (env) retroviral vector, less than 1% of peripheral blood (PB) contained provirus. This was true whether bone marrow (BM) or cytokine-mobilized PB immunoselected CD34+ cells were reinfused. This level of marking was evident in two animals whose platelet counts never fell below 50,000/microliter and whose leukocyte counts had recovered by days 8 and 10 after having received 1.7 x 10(7) or greater of cytokine-mobilized CD34+ PB cells/kg. Reverse transcriptase(RT)-PCR analysis of CD34+ subsets for both the GaLV and amphotropic receptor were performed. The expression of the GaLV receptor was determined to be restricted to CD34+ Thy-1+ cells, and both CD34+ CD38+ and CD34+ CD38dim cells, while the amphotropic receptor was present on all CD34+ cell subsets examined. Our findings suggest that, in rhesus macaques, PG13-derived retroviral vectors may only be able to transduce a subset of CD34+ cells as only CD34+ Thy-1+ cells express the GaLV receptor.
Asunto(s)
Antígenos CD34 , Antígenos CD , Vectores Genéticos/genética , Trasplante de Células Madre Hematopoyéticas , Virus de la Leucemia del Gibón/genética , Virus de la Leucemia Murina de Moloney/genética , Transfección/métodos , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Animales , Antígenos de Diferenciación , Expresión Génica , Células Madre Hematopoyéticas/inmunología , Macaca mulatta , NAD+ Nucleosidasa , Antígenos Thy-1RESUMEN
In an attempt to improve our gene transfer efficiency into hematopoietic stem cells and to evaluate the capacity of immunoselected CD34+Thy-1+(CDw90) cells to reconstitute hematopoiesis following myeloablation, bone marrow (BM) transplantation was performed using autologous, immunoselected CD34+Thy-1+ cells in rhesus macaques. BM samples were positively selected for cells that express CD34, further subdivided using high gradient immunomagnetic selection for cells that express Thy-1, and transduced using a 7-day supernatant transduction protocol with a replication-defective retroviral vector that contained the human glucocerebrosidase (GC) gene. Circulating leukocytes were evaluated using a semiquantitative polymerase chain reaction (PCR) assay for the human GC gene, with the longest surviving animal evaluated at day 558. Provirus was detected at all time points in both CD20+ B cells and CD2+ dim T cells, but long-term gene transfer was not observed in the granulocyte population. The CD2+ dim population was phenotypically identified as being CD8+ natural killer cells. By day 302 and day 330, both the CD2+ bright and dim cell populations and sorted CD4+ and CD8+ cells had detectable provirus. Vector-derived GC mRNA was detected by reverse transcriptase (RT)-PCR analysis as far out as day 588. Thus, CD34+Thy-1+ cells isolated using high gradient magnetic separation techniques can engraft, be transduced with a replication-defective retroviral vector, and contribute to CD20+ B lymphocytes, CD8+ T lymphocytes, and CD4+ T lymphocytes; making them a suitable cell population to target for gene therapies involving lymphocytes.
Asunto(s)
Células de la Médula Ósea , Técnicas de Transferencia de Gen , Glucosilceramidasa/genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Antígenos Thy-1/análisis , Adenovirus Humanos/genética , Adenovirus Humanos/aislamiento & purificación , Animales , Antígenos CD34/análisis , Antígenos CD2/análisis , Antígenos CD8/análisis , ADN Recombinante/análisis , Virus Defectuosos/genética , Virus Defectuosos/aislamiento & purificación , Vectores Genéticos/genética , Vectores Genéticos/aislamiento & purificación , Supervivencia de Injerto , Humanos , Separación Inmunomagnética , Subgrupos Linfocitarios/química , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/virología , Macaca mulatta , Reacción en Cadena de la Polimerasa , Provirus/genética , Provirus/aislamiento & purificación , Trasplante AutólogoRESUMEN
Successful gene transfer into stem cells would provide a potentially useful therapeutic modality for treatment of inherited and acquired disorders affecting hematopoietic tissues. Coculture of primate bone marrow cells with retroviral producer cells, autologous stroma, or an engineered stromal cell line expressing human stem cell factor has resulted in a low efficiency of gene transfer as reflected by the presence of 0.1-5% of genetically modified cells in the blood of reconstituted animals. Our experiments in a nonhuman primate model were designed to explore various transduction protocols that did not involve coculture in an effort to define clinically useful conditions and to enhance transduction efficiency of repopulating cells. We report the presence of genetically modified cells at levels ranging from 0.1% (granulocytes) to 14% (B lymphocytes) more than 1 year following reconstitution of myeloablated animals with CD34+ immunoselected cells transduced in suspension culture with cytokines for 4 days with a retrovirus containing the glucocerebrosidase gene. A period of prestimulation for 7 days in the presence of autologous stroma separated from the CD34+ cells by a porous membrane did not appear to enhance transduction efficiency. Infusion of transduced CD34+ cells into animals without myeloablation resulted in only transient appearance of genetically modified cells in peripheral blood. Our results document that retroviral transduction of primate repopulating cells can be achieved without coculture with stroma or producer cells and that the proportion of genetically modified cells may be highest in the B-lymphoid lineage under the given transduction conditions.