Evaluation of functional efficacy and safety of base editing systems correcting the sickle cell mutation

• Grant number: 23/00986-5
• Support Opportunities: Scholarships abroad - Research
• Start date: April 17, 2023
• End date: May 16, 2023
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Agreement: European Research Council
• Principal Investigator: Karina Tozatto Maio
• Grantee: Karina Tozatto Maio
• Host Investigator: Annarita Miccio
• Host Institution: Instituto Israelita de Ensino e Pesquisa Albert Einstein (IIEPAE). Sociedade Beneficente Israelita Brasileira Albert Einstein (SBIBAE). São Paulo , SP, Brazil
• Institution abroad: Institute of genetic diseases Imagine, France

Abstract

Sickle cell disease (SCD) is a severe disease caused by a single mutation in the beta-globin gene (HBB). The only curative therapy for SCD is allogeneic hematopoietic stem cell transplantation (alloHSCT), however most patients do not have a suitable donor. Gene therapy is a promising curative treatment for SCD. However, which is the best genome editing strategy remains an open question. Although genome editing based on CRISPR/Cas9 nuclease has shown good results in clinical settings, there are concerns on safety, as the double strand breaks induced by the Cas9 nuclease might lead to apoptosis and large genomic rearrangements. Base editing (BE) is a technology based on the chemical modification of target DNA bases using an inactivated Cas fused to a deaminase and it has been shown to be effective in preclinical models, although the risk of DNA and RNA off-targets (i.e., editing at genomic sites other than the target gene) exist. Recently, in the context of SCD, base editors that induce expression of fetal hemoglobin (HbF) and the benign G-Makassar hemoglobin (Hb G-Makassar) variant have been developed by several groups including ours. The aim of this study is to evaluate DNA and RNA off-target activity of published and novel base editing systems to induce the expression of Hb G-Makassar using in silico analysis, GUIDE-seq coupled to deep sequencing of the potential off-target candidates, whole exome sequencing (WES) and RNA-seq analyses. We will compare the off-target activity of the different base editing systems generating Hb G-Makassar to select the best-performing and safe tool. Furthermore, we will compare the off-target activity of base editing systems generating Hb G-Makassar or inducing HbF expression previously published by our group. Finally, we will compare the strategies generating Hb G-Makassar or inducing HbF in terms of efficacy in reversing the sickle cell phenotype. (AU)