The purpose of our studies is to develop new methods of gene transfer into hematopoietic stem cells using the canine genetic disease leukocyte adhesion deficiency or CLAD as a model. We are using the canine form of LAD to identify new vectors for gene transfer and conditioning regimens to enable sufficient numbers of gene modified hematopoietic stem cells to engraft and reverse the disease phenotype. The canine form of this disease is an optimal model for these studies since: a) the defect involves a membrane receptor on the surface of leukocytes, flow cytometry allows fascile detection and analysis of the number of gene corrected cells, b) low levels of gene corrected cells result in reversal of the disease phenotype, and c) studies in the canine model have been predictive of success in humans in the field of hematopoietic stem cell biology. The presence of a human counterpart to CLAD, namely LAD, allows the results from the animal model to be directly extrapolated to humans. The long-term objective of these studies is to develop strategies which will allow levels of expression of CD18 in hematopoietic cells of children with LAD which are sufficient to reverse the clinical phenotype. We have utilized this model to test both retroviral and foamy viral gene transfer into the bone marrrow celsl of dogs with CLAD. Six of 11 CLAD dogs who received an infusion of autologous, CD18-gene corrected cells which were transduced by a retroviral vector had reversal of the CLAD phenotype. These animals are being followed for the durability of the correction, and for possible genotoxicity from the vectors used for gene transfer. Although recent successes in treating genetic immunodeficiencies have demonstrated the therapeutic potential of stem cell gene therapy, the gammaretroviral vectors used in several of these trials have led to insertional activation of nearby oncogenes and leukemia in some patients, prompting studies of modified or alternative vector systems. Pre-clinical data obtained from mouse models may not have adequately assessed the genotoxic risks of these vectors. Re cently we have achieved positive results using a vector based on foamy virus (FV) to treat dogs with the canine leukocyte adhesion deficiency or CLAD. Four of five CLAD dogs receiving non-myeloablative conditioning and infusion of autologous CD34+ hematopoietic stem cells transduced by the FV vector expressing canine CD18 had complete reversal of the CLAD phenotype which was sustained one and one-half years following treatment. In vitro assays demonstrated correction of the lymphocyte proliferation and neutrophil adhesion defects that characterize CLAD. There were no genotoxic complications and integration site analysis demonstrated polyclonal marking by transduced cells. These results represent the first successful use of a FV vector to treat a genetic disease, and they suggest that FV vectors will be effective in treating human hematopoietic diseases such as LAD.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Intramural Research (Z01)
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National Cancer Institute Division of Clinical Sciences
United States
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