The isolation and characterization of a population of canine putative hematopoietic progenitor cells and their utilization as targets for retroviral mediated gene therapy

The treatment of choice for many congenital genetic diseases, such as severe combined immunodeficiency (SCID), is bone marrow transplantation (BMT) early in life using bone marrow obtained from HLA-identical relatives. Although the success of these transplants is high, HLA-identical marrow is not available for most recipients. This has led to the development of alternative strategies such as the use of HLA-haploidentical BMT using either T cell-depleted marrow or subsets of marrow which contain hematopoietic progenitor cell activity. These protocols have greatly reduced the incidence of post-BMT graft-versus-host-disease and overt graft rejection, although transplant recipients have not been completely immunolgically reconstituted in many cases. More recently, the technology for gene therapy of putative hematopoietic progenitor cells using viral vectors has been developed. The efficient transfer of foreign genetic information into autologous targets could eliminate the need for heterologous BMT. In mice, retroviral transduction efficiencies of up to 90% are routinely achieved, however results using human hematopoietic progenitors have been disappointing. Because of ethical considerations and the fact that human SCID patients are transplanted at an early age, alternative animal models have been exploited in an effort to not only increase transduction efficiencies, but also to examine the persistence of transferred genetic sequences and the long-term affects of viral expression. The canine X-linked SCID model, a homologue to human XSCID, is an excellent system to examine these issues. Issues which need to be addressed when using this model for gene therapy studies include (1) defining populations of canine bone marrow cells that contain hematopoietic progenitor activity, (2) determining if sufficient numbers of these cells can be harvested, (3) understanding the optimal ex vivo conditions for efficient retroviral transduction, (4) determining if these cells can be transduced with retroviral vectors and if the retroviral transduction protocols reduce the engraftment capabilities of the target cells, and (5) determining if retrovirally marked cells can be followed in vivo. This study was designed to answer these questions