Engineering of Cas9-embedded cytosine base editors to improve safety of gene therapy for hemoglobinopathies

• Grant number: 24/14747-5
• Support Opportunities: Scholarships in Brazil - Master
• Start date: January 01, 2025
• End date: July 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Davi Coe Torres
• Grantee: João Victor José de Barros Dantas
• Host Institution: Instituto Israelita de Ensino e Pesquisa Albert Einstein (IIEPAE). Sociedade Beneficente Israelita Brasileira Albert Einstein (SBIBAE). São Paulo , SP, Brazil

Abstract

Sickle cell disease (SCD) and beta-thalassemia are hemoglobinopathies, a group of diseases defined by defects in the structure or synthesis of hemoglobin molecules, respectively. These diseases are considered a global health problem that requires measures to reduce their social and economic burden. Allogeneic hematopoietic stem cell transplantation (AHST) is the only available curative therapy for both diseases. However, it has limitations due to donor availability, as well as potential post-AHST complications. Elevated levels of fetal hemoglobin (HbF) can significantly reduce morbidity and mortality in individuals with SCD or beta-thalassemia, as previously observed in patients with hemoglobinopathies and hereditary persistence of HbF. Cytosine base editors (CBEs) are capable of editing the genome more efficiently and safely than the canonical CRISPR-Cas system. However, CBEs can cause stochastic deamination throughout the genome and transcriptome of a target cell. To mitigate this off-target effect, more precise base editors have been developed harboring novel mutations, deletions, or architectural features. Concerns about the clinical use of base editors prompted us in this project to seek the development of new gene editing tools based on a Cas-embedding base editor strategy with the latest generation deaminase CBE6. Therefore, the efficiency of the established tool will be assessed through the HbF reactivation strategy targeting clinically relevant targets for hemoglobinopathies. First, the different architectures of the CBE expression vectors will be screened in HEK 293T cells according to their editing efficacy at the target site and potential off-target effects. Subsequently, HUDEP-2 erythroid progenitor cells will be transfected with the selected CBE for gene editing analysis and assessment of genotoxicity. Lastly, the edited HUDEP-2 cells will undergo in vitro erythroid differentiation to assess the increase in HbF expression. By integrating diverse approaches to enhance CBE functionality, our group aims to develop a potentially safer curative strategy for individuals with DF or beta-thalassemia.