Expression of CD18 in Leukocytes Following Retroviral Mediated Gene Transfer Reverses the Clinical Phenotype in Canine Leukocyte Adhesion Deficiency.

Children with the severe deficiency phenotype of leukocyte adhesion deficiency (LAD-1) suffer recurrent, life-threatening bacterial infections due to defective adherence and migration of their leukocytes. LAD-1 is caused by heterogeneous molecular defects in the leukocyte integrin CD18 molecule. Dogs with the canine form of leukocyte adhesion deficiency (CLAD), like children with severe deficiency LAD-1, experience severe bacterial infections, and typically die within the first few months of life from infection. CLAD represents a disease-specific, large animal model for evaluating new therapeutic approaches for the human disease LAD. In these studies, we tested a retroviral-vector mediated gene therapy approach in CLAD. Autologous CLAD CD34+ bone marrow hematopoietic stem cells were pre-stimulated overnight with growth factors cIL-6, cSCF, hFlt3-L, and hTPO, then incubated with retroviral vector PG13/MSCV-cCD18 over 48 hours on recombinant fibronectin. Transduction of the CLAD CD34+ cells was measured by flow cytometry for CD18+ cells and ranged from 11% to 21%. The transduced cells were re-infused (0.26 − 1.49 x 106 CD18+ cells / kg) into the dogs following the administration of two different non-myeloablative conditioning regimens: 5 CLAD dogs received autologous, gene-corrected CD34+ cells following 200 cGy total body irradiation (TBI) and 2 CLAD dogs received autologous, gene-corrected CD34+ cells following 10 mg/kg busulfan. Peripheral blood samples were analyzed by flow cytometry for CD18 expression following the re-infusion of the transduced CD34+ cells. The frequency of CD18+ gene-corrected leukocytes in the peripheral blood ranged from 0.04% to a high of 4.44% at 6 – 11 months post-gene transfer. Two of the five dogs in the first group and one of the two dogs in the second group that received CD18+ gene-corrected cells are alive and well on no prophylactic treatment at 9 – 14 months of age. Of note, the CLAD dog receiving busulfan conditioning has the highest level of CD18+ gene-corrected cells (4.44% at 6 months post-infusion), with the levels increasing at monthly intervals since the second month following re-infusion. These results contrast markedly with those seen in untreated CLAD dogs that die or are euthanized within the first few months of life due to intractable infection. These studies indicate that a clinically applicable non-myeloablative regimen of either 200 cGy TBI or 10 mg/kg busulfan facilitates the engraftment of sufficient autologous, CD18-gene corrected cells to correct the lethal disease phenotype in CLAD. No evidence of monoclonality has been detected by LAM-PCR in any of the dogs with therapeutic levels of gene-corrected cells. In future studies we will optimize the transduction protocol in order to increase the number of CD34+ gene-corrected cells for infusion, as well as closely monitor the gene-corrected animals for any evidence of insertional mutagenesis or other complications related to the therapy. Together, these findings support the use of either of two clinically applicable, non-myeloablative conditioning regimens prior to the infusion of autologous, CD18 gene-corrected cells in gene therapy clinical trials for LAD.