Mehreen Hai

Successful treatment of canine leukocyte adhesion deficiency by foamy virus vectors

Recent successes in treating genetic immunodeficiencies have demonstrated the therapeutic potential of stem cell gene therapy. However, the use of gammaretroviral vectors in these trials led to insertional activation of nearby oncogenes and leukemias in some study subjects, prompting studies of modified or alternative vector systems. Here we describe the use of foamy virus vectors to treat canine leukocyte adhesion deficiency (CLAD). Four of five dogs with CLAD that received nonmyeloablative conditioning and infusion of autologous, CD34+ hematopoietic stem cells transduced by a foamy virus vector expressing canine CD18 had complete reversal of the CLAD phenotype, which was sustained more than 2 years after infusion. In vitro assays showed correction of the lymphocyte proliferation and neutrophil adhesion defects that characterize CLAD. There were no genotoxic complications, and integration site analysis showed polyclonality of transduced cells and a decreased risk of integration near oncogenes as compared to gammaretroviral vectors. These results represent the first successful use of a foamy virus vector to treat a genetic disease, to our knowledge, and suggest that foamy virus vectors will be effective in treating human hematopoietic diseases.

458. Reversal of Canine Leukocyte Adhesion Deficiency by Retroviral-Vector Mediated Gene Therapy with Non-Myeloablative Conditioning

Children with leukocyte adhesion deficiency or LAD suffer recurrent, life-threatening bacterial infections due to defective adherence and migration of their leukocytes. LAD is due to heterogenous molecular defects in the leukocyte integrin CD18 molecule. Dogs with the canine form of leukocyte adhesion deficiency or CLAD, like children with LAD, also experience severe bacterial infections and typically die within the first few months of life. CLAD represents a disease-specific, large animal model for testing new therapeutic approaches for the human disease LAD. We tested a retroviral-vector mediated gene therapy approach in CLAD using a non-myeloablative conditioning regimen. Seven CLAD dogs received autologous, CD34+ gene-corrected cells following 200 cGy total body irradiation (TBI). CLAD CD34+ 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 ranged from 15 to 33%. Following transduction, cells were re-infused (0.3 to 1.8 |[times]| 106 CD18+ cells per kg) after 200 cGy TBI. One group of 4 dogs received post-transplant immunosuppression consisting of cyclosporine and mycophenolate mofetil. A second cohort group of 3 dogs received no post-transplant immunosuppression. Peripheral blood samples were analyzed by flow cytometry for CD18 expression. At a mean time of 6 months post-gene transfer, the CD18+ gene-corrected leukocyte frequency in the peripheral blood ranged from 0.044% to a high of 3.15%. Five of the 7 CLAD dogs (3 with immunosuppression, 2 without immunosuppression) receiving CD18+ gene corrected cells have had significant improvement of their CLAD disease and are alive and well at 9 to13 months of age. 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. Our studies indicate that a non-myeloablative regimen of 200 cGy TBI regimen facilitates the engraftment of sufficient autologous, CD18-gene corrected cells to correct the disease phenotype in CLAD, and that post-transplant immunosuppression is not required for the persistence of CD18 gene-corrected cells. These results provide support for the use of a non-myeloablative conditioning regimen prior to the infusion of autologous, CD18 gene-corrected cells in gene therapy clinical trials for LAD.