The purpose of our studies is to develop new methods of gene transfer into hematopoietic stem cells using the genetic disease leukocyte adhesion deficiency or LAD as a model. We are using the canine form of LAD to first to identify the number of hematopoietic stem cells required to reverse the disease phenotype and then to test new retroviral vectors, conditioning regimens, and immunosuppressive agents required 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 are likely to 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 in that the background genetic heterogeneity between non-inbred dogs is similar to the genetic diversity of affected human populations, and d) the presence of a human counterpart to CLAD, namely LAD, allows the results from the animal model to be directly extrapolated to humans. We have established a CLAD colony and utilized this model to test a non-myeloablative allogeneic transplant regimen for dogs with a matched littermate donor and a gene therapy approach for the dogs who lacked a matched littermate donor. Of the 15 CLAD animals who received an allogeneic hematopoietic stem cell transplants from a matched donor following a non-myeloablative conditioning regimen, 13 dogs had engraftment of sufficient CD18 positive cells to reverse the disease phenotype. The transplant studies in CLAD have formed the basis for a clinical trial involving allogeneic hematopoietic stem cell transplant for LAD. Five CLAD animals have had reversal of the CLAD phenotype after receiving infusions of autologous, CD18-gene corrected cells. These animals are being followed for the durability of the correction, and for possible genotoxicity from the vectors used for gene transfer. These studies are anticipated to form the basis for a clinical trial in LAD using autologous gene corrected hematopoietic stem cells.
