Gates Package — page 818

Introduction Autologous HSC gene therapy for hemoglobinopathies has shown promising clinical efficacy 1-4. However, current protocols involving isolation of patient HSCs, their in vitro genetic modification with integrating vectors, and reinfusion of the modified HSCs following myelotoxic bone marrow conditioning, are technically complex and expensive. We are attempting to develop an in vivo HSC gene therapy approach that does not require myeloablation and integrating vectors, and that is technically easier. In this approach, we are using capsid-modified, helper-dependent HDAd5/35++ vectors (1, 2). These vectors target CD46, a receptor that is expressed on primitive HSCs (2, 3). HDAd5/35++ vectors are injected intravenously after mobilization of HSCs from the bone marrow by agents routinely used for HSC mobilization/harvesting. Mobilized HSCs are transduced while they are in the periphery. A large fraction of HSCs returns to the bone marrow. Mobilization of HSCs is critical for in vivo transduction because in the bone marrow they are surrounded by extracellular stroma proteins (4), and are not accessible to gene transfer vectors (2). To expand in vivo transduced HSCs, we currently use an in vivo selection mechanism based on a mutant O6-methylguanine-DNA methyltransferase (mgmtP140K) gene that confers resistance to O6-BG/BCNU (O6-Benzylguanine/Carmustine) given at doses that are 20-30-fold lower than used for cancer chemotherapy (5-7). We have demonstrated the safety (including the absence of clonal dominance (8-10)) and efficacy of the in vivo approach in mice (9-15) and rhesus macaques (3, 8, 16). One of our goals is to broadly apply this approach for the treatment of hemoglobinopathies, i.e. -thalassemia and Sickle Cell Disease (SCD). Because of its simplicity and lower costs, this method could be applicable in developing countries where the burden from these diseases is high (17). -thalassemia and Sickle Cell Disease (SCD) are the most common inherited diseases in humans worldwide. -thalassemia is caused by mutations in the -globin gene which result in absent (β0/β0) or deficient (β+/β+, β+/β0) β-globin chain synthesis. -thalassemia patients have a multitude of pathological symptoms and die prematurely. Most SCD patients are homozygous for a Glu6Val substitution resulting in the production of βS-globin. Clinically, SCD is characterized by recurrent episodes of severe bone pain, multi-organ failure, and early mortality. Individuals with Hereditary Persistence of Fetal Hemoglobin (HPFH) and thalassemic -globin mutations exhibit few or no pathological effects because HbF inhibits hemoglobin precipitation by reversing the globin chain imbalance and improving erythropoiesis (18). This led to a number of hematopoietic stem cell (HSC) gene therapy approaches aimed toward the reactivation of fetal/-globin, e.g. by targeting two transcriptional repressors, ZBTB7A and BCL11A, that participate in the developmental silencing of the two -globin genes (HBG1/G and HBG2/A) through