Yu-chen Gu

Haploidentical In Utero Hematopoietic Cell Transplantation Improves Phenotype and Can Induce Tolerance for Postnatal Same-Donor Transplants in the Canine Leukocyte Adhesion Deficiency Model

In the murine model, in utero hematopoietic cell transplantation (IUHCT) has been shown to achieve low levels of allogeneic chimerism and associated donor-specific tolerance permitting minimal conditioning postnatal hematopoietic stem cell transplantation (HSCT). In this pilot study, we investigated IUHCT in the canine leukocyte adhesion deficiency (CLAD) model. Haploidentical IUHCT resulted in stable low-level donor cell chimerism in all dogs that could be analyzed by sensitive detection methodology (4 of 10) through 18 months of follow-up. In the 2 CLAD recipients, low-level chimerism resulted in amelioration and complete reversal of the CLAD phenotype, respectively. Six recipients of IUHCT (5 carriers and 1 CLAD) subsequently received postnatal HSCT from the same haploidentical prenatal donor after minimal conditioning with busulfan 10 mg/kg. Chimerism in 2 of 5 CLAD carriers that underwent HSCT increased from < 1% pre-HSCT to sustained levels of 35% to 45%. Control animals undergoing postnatal haploidentical HSCT without IUHCT had no detectable donor chimerism. These results demonstrate that haploidentical IUHCT in the CLAD model can result in low-level donor chimerism that can prevent the lethal phenotype in CLAD dogs, and can result in donor-specific tolerance that can facilitate postnatal minimal conditioning HSCT.

Leukocyte Adhesion Deficiency in Children and Irish Setter Dogs

Children with the genetic immunodeficiency disease leukocyte adhesion deficiency, or LAD, develop life-threatening bacterial infections as a result of the inability of their leukocytes to adhere to the vessel wall and migrate to the sites of infection. Recently, the canine counterpart to LAD, known as canine leukocyte adhesion deficiency, or CLAD, has been described in Irish setter dogs. This review describes how the clinical phenotype of dogs with CLAD closely parallels that of children with the severe deficiency phenotype of LAD, thus enabling the CLAD dog to provide a disease-specific, large-animal model for testing novel hematopoietic stem cell and gene therapy strategies before their translation to children with LAD.

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.