5613417 CO-TREATMENT OF ERYTHROID CELLS ISOLATED FROM Β039-THALASSEMIA PATIENTS WITH CRISPR-CAS9 GENE EDITING AND FETAL HEMOGLOBIN INDUCTION

Abstract

Background: Gene editing (GE) can be considered among the most promising strategies to correct hereditary mutations in a variety of monogenetic diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9 gene editing can be proposed for efficient correction of the β039-thalassemia mutation. GE is suitable for personalized approaches in precision medicine for β-thalassemia [1]. Aims: On the other hand, robust evidence demonstrate that increased production of fetal hemoglobin (HbF) can be beneficial for β-thalassemia patients. This can be obtained also using CRISPR-Cas9 based protocols. In this case, the objective of the gene editing strategy is (a) the silencing of transcriptional repressors of the γ-globin genes (such as BCL11A), (b) the disruption of their regulatory binding sites within the γ-globin genes, or (c) the induction of natural hereditary persistence of fetal hemoglobin mutations in the γ-globin gene or the b-like-globin gene cluster. Our study aimed to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols based on the use of repositioned drugs. Results: Gene Editing of the β039-thalassemia mutation was obtained by the CRISPR-Cas9 method; gene correction and transcription of the corrected gene have been analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; production of HbA and HbF was studied by HPLC and Western blotting. For HbF induction, the repurposed drug sirolimus (rapamycin) was used. The data obtained conclusively demonstrate that maximal production of HbA and HbF is obtained in GE-corrected, sirolimus-induced erythroid progenitors from β039-thalassemia patients. In conclusion, GE and HbF in-duction might be used in combination in order to achieve the de novo production of HbA together with increased production of induced HbF. Acknowledgements This study was sustained by the Wellcome Trust (innovator award 208872/Z/17/Z) and by AIFA (AIFA-2016-02364887). The research leading to these results has received funding also from the UE THALAMOSS Project (Thalassemia Modular Stratification System for Personalized Therapy of Βeta-Thalassemia; no. 306201-FP7-HEALTH-2012-INNOVATION-1). We thanks A.L.T. (Associazione per la lotta alla Talassemia) “Rino Vullo” - Ferrara, and A.V.L.T. (Associazione Veneta per la Lotta alla Talassemia) “Elio Zago” - APS – Rovigo and Association “Tutti per Chiara”, Montagnana, PD, Italy. Reference 1. Cosenza L.C., Gasparello J., Romanini N., Zurlo M., Zuccato C., Gambari R., Finotti A. Efficient CRISPR-Cas9-based genome editing of β-globin gene on erythroid cells from homozygous β039-thalassemia patients. Mol Ther Methods Clin Dev 2021;21:507−523. https://doi.org/10.1016/j.omtm.2021.03.025