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Red Cell Biology & its Disorders
2'-O-methoxyethyl splice-switching oligos correct splicing from IVS2-745 b-thalassemia patient cells restoring hemoglobin A production and chain rebalance
Alisa Dong,1,2* Valentina Ghiaccio,1* Irene Motta,3 Shuling Guo,4
Raechel Peralta,4 Susan M. Freier,4 Andy Watt,4 Sagar Damle,4
Yasuhiro Ikawa,1 Danuta Jarocha,1 Maxwell Chappell,1 Coralea Stephanou,5 Paola Delbini,3Connie Chen,2 Soteroula Christou,5 Marina Kleanthous,5
Kim Smith-Whitley,1 Deepa Manwani,6 Carla Casu,1 Osheiza Abdulmalik,1
Ferrata Storti Foundation
1Division of Hematology, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA; 2Graduate School of Medical Sciences, Weill Cornell Medical College of Cornell University, New York, NY, USA; 3University of Milan - Fondazione IRCCS Ca' Granda Ospedale Policlinico, Milan, Italy; 4Ionis Pharmaceuticals, Carlsbad, CA, USA; 5Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus and 6The Children’s Hospital at Montefiore, Bronx, New
Haematologica 2021 Maria Domenica Cappellini, Stefano Rivella and Laura Breda Volume 106(5):1433-1442
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ork, NY, USA
Yb
-thalassemia is a disorder caused by altered hemoglobin protein
*AD and VG contributed equally as co-first authors.
ABSTRACT
synthesis which affects individuals worldwide. Severe forms of the
disease, left untreated, can result in death before the age of 3 years. The standard of care consists of chronic and costly palliative treatment by blood transfusion combined with iron chelation. This dual approach sup- presses 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 sys- tems.2 Hence, 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-tha- lassemia. 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 phenotyp- ic setting, we found reduced sickle hemoglobin synthesis and sickle cell formation due to HbA induction. In summary, 2'-MOE-SSO are a prom- ising therapy for forms of b-thalassemia caused by mutations leading to aberrant splicing.
Introduction
b-thalassemia is caused by inheritance of one or more of over 400 different muta- tions in the b-globin (HBB) gene, which results in reduced (b+ allele) or absent (b0 allele) synthesis of the b-globin chains. The severity of b-thalassemia correlates to the level of imbalance between α- and b-like globin chains. The excess α-globin
1
Correspondence:
LAURA BREDA
bredal@email.chop.edu
Received: June 25, 2019. Accepted: April 28, 2020. Pre-published: May 21, 2020.
https://doi.org/10.3324/haematol.2019.226852
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