A non-human primate model has been established in rhesus monkeys to develop methods for gene insertion into hematopoietic stem cells. Bone marrow is harvested, and stem cells are purified by immunoselection based on expression of the CD34 antigen. The enriched hematopoietic stem and progenitor cells are cultured in vitro in hematopoietic growth factors that promote cell survival and stimulate cell division with retroviral vector particles designed for gene insertion into repopulating stem cells. The recipient animal receives high-dose irradiation to ablate the bone marrow and the cultured autologous cells are reinfused. In the initial series of experiments, a retroviral producer clone was utilized that generated a very high titer of recombinant vector particles and also generated replication-competent virus at a somewhat lower titer. Three of ten animals reconstituted with cells exposed to this virus preparation developed a rapidly progressive T-cell lymphoma. Animals that remained free of lymphoma had mounted a primary immune response to the replication-competent virus as reflected by serum antibody to viral proteins whereas the animals with lymphoma lacked antibody. In the absence of an effective immune response, a productive infection occurred. Multiple copies of the replication-competent proviral genome were found in tumor cells with probable proto-oncogene activation by the mechanism of insertional mutagenesis. The producer clone has subsequently been shown to generate a replication-competent xenotrophic virus that arose by recombination. This virus may also be implicated in the pathogenesis of the lymphomas. Recent experiments have utilized recombinant vector producer clones that are free of replication-competent virus. Successful gene insertion into repopulating hematopoietic cells has been achieved as reflected by the presence of the retroviral genome in circulating blood cells several weeks after bone marrow reconstitution.
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