2'-O-methoxyethyl splice-switching oligos correct splicing from IVS2-745 b-thalassemia patient cells restoring hemoglobin A production and chain rebalance

Abstract

B-thalassemia is a disorder caused by altered hemoglobin protein synthesis which affects individuals worldwide. Severe forms of the disease, left untreated, can result in death before the age of 3 years.1 The standard of care consists of chronic and costly palliative treatment by blood transfusion combined with iron chelation. This dual approach suppresses anemia and reduces iron-related toxicities in patients. Allogeneic bone marrow transplant is an option, but limited by the availability of a highly compatible hematopoietic stem cell donor. While gene therapy is being explored in several trials, its use is highly limited to developed regions with centers of excellence and well-established healthcare systems.2Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for b-thalassemia.3 Occurrence of aberrant splicing is one of the processes that affects b-globin synthesis in b-thalassemia. The (C>G) IVS2-745 is a splicing mutation within intron 2 of the b-globin (HBB) gene. It leads to an aberrantly spliced mRNA that incorporates an intron fragment. This results in an in-frame premature termination codon that inhibits b-globin production. Here, we propose the use of uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSO) to reverse this aberrant splicing in the pre-mRNA. With these SSO we show aberrant to wild-type splice switching. This switching leads to an increase of adult hemoglobin up to 80% in erythroid cells from patients with the IVS2-745 HBB mutation. Furthermore, we demonstrate a restoration of the balance between b-like- and α-globin chains, and up to an 87% reduction in toxic heme aggregates. While examining the potential benefit of 2'-MOE-SSO in a mixed sickle-thalassemic phenotypic setting, we found reduced sickle hemoglobin synthesis and sickle cell formation due to HbA induction. In summary, 2'-MOE-SSO are a promising therapy for forms of b-thalassemia caused by mutations leading to aberrant splicing.