A selectable bicistronic retroviral vector corrects the molecular defect in a cell line derived from a patient with leukocyte adhesion deficiency (2006)

Leukocyte adhesion deficiency (LAD) is an immunodeficiency disease resulting from defects in the integrin CD18. Children with LAD suffer severe, recurrent bacterial infections due to failure of leukocytes to adhere to endothelium and migrate to sites of infection. Approximately 75% of severe LAD patients die by two years of age. Allogeneic hematopoietic stem cell transplant after myeloablation can cure LAD, however regimen-related toxicity and graft-vs.-host disease (GvHD) limit the use of this approach. Genetic correction of autologous CD34+ cells represents an optimal therapy for LAD since no donor is required, nor is GvHD a risk. In this study we developed a retroviral vector that confers a selectable growth advantage for pre-clinical testing of gene therapy in the canine model of LAD (CLAD). This retroviral vector allows for correction of the CD18 defect in CLAD and selection of the corrected cells. The vector, MSCV(cCD18)IRES-cMGMTP144K, harbors the canine CD18 cDNA followed by an internal ribosome entry site (IRES) for expression of mutated canine methylguanine methyltransferase (MGMT). Mutant MGMT confers resistance to the combination of carmustine (BCNU) and O6-benzylguanine (BG). A high-titer producer line was identified using the PG13 packaging line, and supernatant from this producer line was used to transduce an EBV-transformed B cell line derived from a CD18- LAD patient. Transduced and mock-transduced cells were grown in 25 μM BG and increasing concentrations of BCNU. In one experiment, 5 μM BCNU + 25 μM BG allowed selection of CD18+ cells to > 99% purity, however a distinct CD18- population remained. In a second experiment, cells selected in 10 μM BCNU + 25 μM BG were ∼ 99.9% CD18+ with no distinct CD18− population. These studies demonstrate that a bicistronic retroviral vector leads to selection of a population of molecularly corrected LAD cells. Selection was achieved despite placement of MGMT after the IRES, where expression of the selectable gene product is expected to be decreased. Experiments with CD34+ cells from CLAD dogs will allow this approach to be tested in vivo. These studiesdemonstrate that mutant MGMT after an IRES facilitates the selection of cells expressing a gene that is therapeutic for a human disease. This model should be useful for gene therapy of human LAD, and may be a prototype for gene therapy for other single gene defects. Leukocyte adhesion deficiency (LAD) is an immunodeficiency disease resulting from defects in the integrin CD18. Children with LAD suffer severe, recurrent bacterial infections due to failure of leukocytes to adhere to endothelium and migrate to sites of infection. Approximately 75% of severe LAD patients die by two years of age. Allogeneic hematopoietic stem cell transplant after myeloablation can cure LAD, however regimen-related toxicity and graft-vs.-host disease (GvHD) limit the use of this approach. Genetic correction of autologous CD34+ cells represents an optimal therapy for LAD since no donor is required, nor is GvHD a risk. In this study we developed a retroviral vector that confers a selectable growth advantage for pre-clinical testing of gene therapy in the canine model of LAD (CLAD). This retroviral vector allows for correction of the CD18 defect in CLAD and selection of the corrected cells. The vector, MSCV(cCD18)IRES-cMGMTP144K, harbors the canine CD18 cDNA followed by an internal ribosome entry site (IRES) for expression of mutated canine methylguanine methyltransferase (MGMT). Mutant MGMT confers resistance to the combination of carmustine (BCNU) and O6-benzylguanine (BG). A high-titer producer line was identified using the PG13 packaging line, and supernatant from this producer line was used to transduce an EBV-transformed B cell line derived from a CD18- LAD patient. Transduced and mock-transduced cells were grown in 25 μM BG and increasing concentrations of BCNU. In one experiment, 5 μM BCNU + 25 μM BG allowed selection of CD18+ cells to > 99% purity, however a distinct CD18- population remained. In a second experiment, cells selected in 10 μM BCNU + 25 μM BG were ∼ 99.9% CD18+ with no distinct CD18− population. These studies demonstrate that a bicistronic retroviral vector leads to selection of a population of molecularly corrected LAD cells. Selection was achieved despite placement of MGMT after the IRES, where expression of the selectable gene product is expected to be decreased. Experiments with CD34+ cells from CLAD dogs will allow this approach to be tested in vivo. These studiesdemonstrate that mutant MGMT after an IRES facilitates the selection of cells expressing a gene that is therapeutic for a human disease. This model should be useful for gene therapy of human LAD, and may be a prototype for gene therapy for other single gene defects.