Long-term persistence of canine hematopoietic cells genetically marked by retrovirus vectors.

In 1991 we reported gene transduction into autologous long-term repopulating marrow cells in dogs using amphotropic helper-free retrovirus vectors containing the bacterial neomycin phosphotransferase gene (neo) and the human adenosine deaminase gene (ADA). Two of the dogs are still alive and healthy now more than 5 years after transplantation of transduced autologous marrow cells. In one of the surviving dogs, polymerase chain reaction (PCR) analysis showed the neo and ADA genes to be present in peripheral blood granulocytes and lymphocytes up to the present time. The estimated percentage of neo-positive cells ranged from < 0.001% to 0.1%. ADA mRNA expression was detected by reverse transcriptase PCR (RT-PCR) in granulocytes 63 months after transplantation. The other surviving dog failed to show either persistence or expression of the transduced genes after 50 months. Three additional dogs have been transplanted according to the same transduction protocols and with the same retrovirus vectors, and persistence of the transduced neo gene has been documented in peripheral blood myeloid and lymphoid cells along with G418-resistant colony-forming unit-granulocyte/macrophage (CFU-GM) for now more than 2 years. These findings represent the longest follow-up of retrovirus-mediated gene transduction in any animal species. Long-term transduction efficiency, though, has remained low and will need to be improved for therapeutic application to be possible.