Non-Myeloablative Conditioning with Busulfan Prior to Allogeneic Hematopoietic Stem Cell Transplant Results in Mixed Chimerism and Reversal of the Phenotype in Canine Leukocyte Adhesion Deficiency with Minimal Regimen Related Toxicity and without Graft-vs.-Host Disease.

Leukocyte adhesion deficiency (LAD), a genetic disease caused by defects in the integrin CD18, results in a clinical syndrome in which the affected children suffer severe recurrent infections due to the inability of their leukocytes to adhere to endothelium and migrate to sites of infection. Approximately 75% of children with the severe deficiency phenotype of LAD die by two years of age. Allogeneic hematopoietic stem cell transplant (HSCT) preceded by a myeloablative conditioning regimen corrects the LAD phenotype, however this treatment carries significant regimen-related toxicity and mortality. Full donor chimerism after myeloablation is commonly accompanied by graft-vs.-host disease (GvHD). We utilized an animal model of LAD, canine leukocyte adhesion deficiency (CLAD), to test new transplant regimens for LAD. CLAD is due to a point mutation in canine CD18, and the phenotype closely resembles the severe deficiency phenotype of LAD. We have previously shown that treatment with 200 cGy total body irradiation (TBI), matched sibling HSCT, and immunosuppression with cyclosporine A (CsA) and mycophenolate mofetil (MMF) corrects the CLAD phenotype. Because TBI in children is associated with growth suppression, sterility and increased risk of cancer, translation of this approach to humans will be limited by concern for toxicity. To develop a non-TBI conditioning regimen for LAD transplants, we tested single-agent busulfan. CLAD pups were identified by absence of CD18 on leukocytes by fluorescence-activated cell sorting (FACS). Three dogs that lacked a DLA-matched littermate donor died at 2, 4 and 6 months of age, respectively. Three CLAD pups were given 10 mg/kg busulfan IV followed 2 days later by 6-17x10^6 CD34+ cells/kg from matched sibling donors and immunosuppression with CsA/MMF. Chimerism was assessed by CD18 FACS. Dogs were observed for evidence of infection, regimen-related toxicity, graft-versus-host disease and overall health. All three dogs engrafted, and two animals maintained CD18+ leukocyte levels of 17.0% and 19.0% at 16 and 4 months post-transplant respectively. In both dogs, blood counts are normal with no signs of CLAD. These dogs are clinically and behaviorally indistinguishable from their healthy littermates. The third dog has had decreasing CD18+ cells and at 12 months post-transplant has 0.2% CD18+ donor leukocytes. Despite requiring two courses of parenteral antibiotics since stopping immunosuppression, this dog has considerably exceeded the life expectancy of non-transplanted CLAD pups. Thus, two CLAD dogs have had complete correction of the phenotype, and the third has had partial correction. There has been no need for transfusion and no other regimen-related toxicity. Post-transplant fevers were easily managed with no cases of refractory infection. There has been no GvHD. Our data show that a regimen using a single, non-myeloablative dose of busulfan can lead to long-term engraftment, stable mixed chimerism and reversal of CLAD phenotype with minimal toxicity. This treatment may provide LAD patients with matched sibling donors with an alternative to either myeloablation with full donor chimerism and risk of GvHD or non-myeloabaltive transplant using TBI with its attendant complications.