Retroviral-Mediated Gene Transfer of CD18 into Hematopoietic Stem Cells in Dogs with Canine Leukocyte Adhesion Deficiency Reverses the Severe Deficiency Phenotype.

Abstract Background: Canine leukocyte adhesion deficiency (CLAD) represents a disease-specific, large-animal model for the human disease leukocyte adhesion deficiency (LAD). Puppies with CLAD, like children with LAD, experience recurrent life-threatening bacterial infections due to the inability of their leukocytes to adhere and migrate to sites of infection. Mutations in the gene encoding the leukocyte integrin CD18 are responsible for both CLAD and LAD. Allogeneic bone marrow or hematopoietic stem cell transplantation is currently the only curative therapy for LAD. We recently reported the results of non-myeloablative allogeneic transplants in CLAD dogs and showed that very low levels of CD18+ donor-derived neutrophils (less than 300/microliter) were sufficient to reverse the CLAD disease phenotype. These results indicated that CLAD dogs may be amenable to treatment using gene therapy, where there are frequently low numbers of transduced cells. We report the results of retroviral- mediated transduction in autologous hematopoietic stem cells with the canine CD18 gene. Method: Bone marrow was harvested and CD34+ selected from four dogs with CLAD at approximately 3–4 months of age. The purified CD34+ cells were either used immediately or were frozen and subsequently thawed. Cells were pre-stimulated with cSCF, hFlt3-L, hTPO and cIL-6 for approximately 24 hours, then exposed to two rounds of supernatant from the retroviral vector PG13/MSCV-cCD18 for 24 hours each on recombinant fibronectin. At the end of the transduction, the cells were infused back into the animal that had been conditioned with 200 cGy total body irradiation. Post-transplant immunosuppression consisted of cyclosporine given at a dose of 30 mg/kg from day -1 to day 35, then 15 mg/kg from day 36 to day 60, and mycophenolate mofetil at a dose of 20 mg/kg from day 0 to day 28. Peripheral blood samples, as well as pus samples from one animal, were analyzed by flow cytometry at designated time points post-transplant. Results: The four dogs who received autologous, gene-corrected cells have been followed for 7–12 weeks post-infusion. The number of CD18+ CD34+ cells infused per dog ranged from 0.2 to 0.55 x 106 cells/kg. The post-infusion percentage of CD18+ neutrophils in each dog was 0.09%, 0.13%, 0.62% and 0.02% at 12, 10, 8 and 6 weeks respectively. Clinically all four treated CLAD dogs are alive with marked improvement of their CLAD disease. These dogs are now 6–7 months of age. These results contrast with those seen in untreated CLAD dogs who uniformly die or are euthanized within the first few months of life. The reversal of the severe CLAD phenotype despite the very low levels of CD18+ neutrophils in the peripheral blood is likely due to the selective egress of CD18+ neutrophils into the tissue since one treated CLAD dog who had less than 1% CD18+ neutrophils in the blood had nearly 10% CD18+ neutrophils in pus collected from an inflammatory dental lesion. Conclusion: These data suggest that a non-myeloablative conditioning regimen coupled with a minimal immunosuppressive regimen may enable sufficient CD18+ autologous gene-corrected cells to engraft and result in reversal of the severe CLAD phenotype.