Correction of splicing, chain imbalance, and adult hemoglobin synthesis in IVS-2-745 β-thalassemia specimens using 2'-o-methoxyethyl splice-switching oligos

β-thalassemia is one of the most frequently-occurring disorders due to altered hemoglobin protein synthesis (Modell & Darlison, 2008). In this disease, the β-globin gene is mutated, causing severe anemia and ineffective erythropoiesis. Patients can present with a number of life-threatening co-morbidities, including iron overload, extramedullary erythropoiesis, hypogonadism and osteoporosis (Origa, 2016). Current standard of care for patients with severe anemia involves blood transfusion and iron chelation. Allogeneic bone marrow transplant is the only curative option, but is constrained by the availability of matching donors and the occurrence of graft-versus-host disease. (Breda et al., 2016) As this is a monogenic disease, it makes it an attractive setting for genetic therapy at the DNA and RNA level. Currently, there are a few clinical trials and preclinical tests underway to evaluate the curative potential of lentiviral vectors to correct the mutated β-globin or add back a functional copy of β-globin (Jorge Mansilla-Soto, Isabelle Riviere, Farid Boulad, 2016, Negre et al., 2016) One of the processes that affects β-globin synthesis in β-thalassemia is occurrence of aberrant splicing. IVS-2-745 is a splicing mutation that occurs in intron 2 of the β-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 IVS-2-745 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% adult hemoglobin (HbA) in erythroid cells from thalassemic patients. Furthermore, we demonstrated a restoration of the balance between β-like- and α-globin chains, and up to an 87% reduction in toxic α-heme aggregates. While next examining the potential benefit of 2’MOE SSOs in a sickle-thalassemic phenotypic setting, we found reduced HbS synthesis and sickle cell formation due to HbA induction, as a result of 2’MOE SSO treatment. In summary, 2’MOE-SSOs are a promising therapy for splicing forms of β-thalassemia. Their ability to functionally modulate the thalassemia and sickle cell anemia phenotype by correcting the underlying defect offers a pharmacological treatment that is both direct and specific