Successful treatment of murine beta-thalassemia using in vivo selection of genetically modified, drug-resistant hematopoietic stem cells.

Successful gene therapy of beta-thalassemia will require replacement of the abnormal erythroid compartment with erythropoiesis derived from genetically corrected, autologous hematopoietic stem cells (HSCs). However, currently attainable gene transfer efficiencies into human HSCs are unlikely to yield sufficient numbers of corrected cells for a clinical benefit. Here, using a murine model of beta-thalassemia, we demonstrate for the first time that selective enrichment in vivo of transplanted, drug-resistant HSCs can be used therapeutically and may therefore be a useful approach to overcome limiting gene transfer. We used an oncoretroviral vector to transfer a methylguanine methyltransferase (MGMT) drug-resistance gene into normal bone marrow cells. These cells were transplanted into beta-thalassemic mice given nonmyeloablative pretransplantation conditioning with temozolomide (TMZ) and O6-benzylguanine (BG). A majority of mice receiving 2 additional courses of TMZ/BG demonstrated in vivo selection of the drug-resistant cells and amelioration of anemia, compared with untreated control animals. These results were extended using a novel gamma-globin/MGMT dual gene lentiviral vector. Following drug treatment, normal mice that received transduced cells had an average 67-fold increase in gamma-globin expressing red cells. These studies demonstrate that MGMT-based in vivo selection may be useful to increase genetically corrected cells to therapeutic levels in patients with beta-thalassemia.