Several approaches to improve the efficiency of gene transfer into reconstituting hematopoietic stem cells are presently being evaluated by the Simian Gene Transfer and Bone Marrow Transplantation Program of the National Heart, Lung, and Blood Institute. These involve methods for 1) expansion of populations of cycling progenitors and stem cells in vitro and in vivo through the use of cytokines and chemotherapeutic agents; and 2) isolation of cellular fractions having a high proliferative capacity from bone marrow, peripheral blood, and neonatal cord blood. Over the past year supernatants from cell lines that have been engineered to produce high titers of replication-defective retroviruses containing genes for either adenosine deaminase, multiple drug resistance, or glucocerebrosidase have been used in autologous bone marrow transplants using immunoselected CD34+ cells. CD34 is a transmembrane glycophosphoprotein of unknown function expressed on a population of early hematopoietic cells which contains the reconstituting hematopoietic stem cell. Immunoselected CD34+ cells have been cultured for 4-5 days or longer in medium conditioned by the retrovirus producer cell line and combinations of growth factors. Cells have been transduced using suspension culture, autologous stromal feeder layers, or a stromal cell line engineered to produce the membrane bound form of human Stem Cell Factor (SCF). Following total body gamma-irradiation (TBI) (6.5Gyx2), the transduced cells have been reinfused, and peripheral blood cells analyzed at regular intervals for evidence of gene transfer using polymerase chain reaction (PCR) based methodology. Longterm survival studies have demonstrated approximately 1-5% of the circulating peripheral blood cells continue to contain the provirus. In the study evaluating the expression of the adenosine deaminase gene after transduction into rhesus repopulating stem cells, granulocytes, B- lymphocytes, and T-lymphocytes of high purity contained the provirus suggesting that a multilineage stem cell was transduced with the vector. Presently, the program is attempting to further define the reconstituting stem cell, in an attempt to improve upon the 1-5% gene transfer efficiency observed in longterm reconstituting stem cells. With continued improvements in vector design, and continued advancements in the understanding of stem cell biology and gene regulation, studies utilizing primate models will prove of major importance in developing suitable protocols permitting safe and effective gene transfer into human subjects.
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