Genetic Approaches To Treat Beta-Thalassemia

Abstract

Beta-thalassemia is one of the most common diseases related to the hemoglobin protein. In this disease, the beta-globin gene is mutated, causing severe anemia and ineffective erythropoiesis. Patients can additionally present with a number of life-threatening co-morbidities, such as anemia and iron overload. Current treatment involves transfusion and iron chelation; allogeneic bone marrow transplant is the only curative option, but is limited by the availability of matching donors and graft-versus-host disease. As this diseases is a monogenic disease, it makes an attractive setting for genetic therapy at the RNA and DNA level. At the DNA level, gene therapy aims to correct the mutated beta-globin or add back a functional copy of beta-globin. Initial gene therapy work was done with oncoretroviral vectors, but has since shifted to lentiviral vectors. Currently, there are a few clinical trials and preclinical tests underway to test the curative potential of some of these lentiviral vectors, including our new gene therapy vector described here with the ankyrin insulator. IVS2-745 is a splicing mutation that occurs in intron 2 of the beta-globin gene. It creates an aberrantly spliced form that incorporates an extra exon and leads to a premature stop codon. Here we report novel uniform 2’-O-methoxyethyl (2’-MOE) splice switching oligos (SSOs) to reverse this aberrant splicing. Lead 2’-MOE SSOs were generated to redirect splicing in the IVS2-745 pre-mRNA. With these lead SSOs we have demonstrated aberrant to wild type splice switching. This switching led to an increase from 3-6% to up to 80% HbA in erythroid cells from thalassemic patients. Furthermore, we have demonstrated a restoration of the balance between beta-like chains and alpha chains, and up to an 87% reduction in toxic alpha-heme aggregates. While next examining the potential benefit of 2’MOE SSOs to patients with a sickle-thalassemic genotype, we observed reduced sickling as a result of 2’MOE SSO induced HbA. In summary, 2’MOE-SSOs are a promising therapy for splicing forms of beta-thalassemia. Their ability to functionally modulate the thalassemia phenotype by correcting the underlying splicing cause offers a pharmacological treatment that is both direct and specific.