Adenine Base Editing Improves Erythropoiesis in Diamond-Blackfan Anemia Syndrome Patient-Derived Induced Pluripotent Stem Cells

• Published in: Blood 2024-11, Vol.144 (Supplement 1), p.4088-4088
• Main Authors: Suryaprakash, Shruthi, Han, Lei, Manquen, Garret, Katta, Varun, Krzyzanowski, Damian, Prasad, Jayaram, Nimmagada, Nikitha, Mayberry, Kalin, Liu, Nana, Ju, Yan, Yao, Yu, Ray, Kelsey, Wlodarski, Marcin, Tsai, Shengdar Q, Yen, Jonathan S, Weiss, Mitchell J, Bhoopalan, Senthil Velan
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2024-209312

Diamond-Blackfan anemia syndrome (DBAS) is an inherited bone marrow failure (BMF) disorder characterized by hypoplastic anemia that presents in infancy. Over time, some patients develop multilineage cytopenias and bone marrow hypocellularity. DBAS is caused by heterozygous loss-of-function mutations in one of 24 ribosomal protein genes, most commonly RPS19. Current therapies such as corticosteroids and red blood cell transfusions are partially effective but have considerable side effects. Hematopoietic stem cell transplantation is curative although many patients lack a suitable donor and/or develop toxicities such as graft-versus-host disease. We and others have shown preclinical feasibility of lentiviral vector (LV) gene replacement therapy for RPS19-mutated DBAS. However, LVs cause TP53 activation in hematopoietic stem/progenitor cells (HSPCs), leading to reductions in cell viability and reduce clonal complexity during gene therapy. Base editors (BEs) consisting of a Cas9 nickase fused to a deaminase domain, can introduce precise nucleotide transitions (A·G or C·T) with minimal DNA double-stranded breaks or TP53 activation. This could be advantageous in RPS19-mutated DBAS, which is associated with reduced HSPC reserve and increased TP53 activity. Here we demonstrate the correction of a pathogenic, DBAS-associated RPS19 mutation using adenine (A) BE as proof-of-concept. Induced pluripotent stem cells (iPSCs) are a robust model to study hematopoietic defects of DBAS (Garcon et al, Blood. 2013). We recently established iPSCs from peripheral blood mononuclear cells (PBMC) of a DBAS patient with the pathological RPS19c.184C>T; p.Arg62Trp mutation. To create isogenic control iPSCs, we corrected the mutation via CRISPR/Cas9-mediated homology director repair (Osuna et al, Stem Cell Res, 2024). We performed in vitro differentiation of the iPSCs to generate CD34+ hematopoietic progenitor cells (HPCs). After seeding equal numbers of HPCs into erythroid cytokines (erythropoietin and stem cell factor) the DBAS lines generated 2-fold fewer erythroblasts compared to the isogenic control HPCs (PT mutation. Next-generation sequencing (NGS) showed roughly equal amounts of wildtype (c.184C) and mutant (c.184T) alleles in unedited cells, consistent with a heterozygous mutation. After adenine base editing, the fra