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

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 (P<0.05). To correct the RPS19mutation we electroporated HPCs with ribonucleoprotein complex consisting of ABE8e and single-guide (sg) RNA targeting the RPS19 c.184C>T 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 fraction of wild type allele increased to approximately 90% (P<0.05). Erythroid differentiation of base-edited DBA HPCs showed a 3.5-fold increase in erythroblast count compared to unedited HPCs (P<0.05). We isolated PBMCs from the same patient and corrected approximately 80% of the mutant alleles (P<0.05), with negligible nonsynonymous bystander edits.

DBA patient bone marrow CD34+ cells are not easily available in large quantities to perform xenotransplantation studies. To address this limitation and to assess the editing efficiency in bone marrow repopulating hematopoietic stem cells (HSCs), we transduced CD34+ HSPCs from healthy donors with an LV encoding the mutant RPS19 c.184C>T cDNA and GFP sequence separated by self-cleaving P2A (RPS19^mut.P2A.GFP LV). This resulted in over 60% GFP+ cells. Studies to test base editing in these RPS19^mut.P2A.GFP LV-transduced HSPCs followed by xenotransplantation into immunodeficient mice are ongoing. Lastly, we performed off-target analysis using CHANGE-Seq-BE, a recently described unbiased approach to identify off-target editing by ABEs (Lazzarotto et al, bioRxiv, 2024). CHANGE-Seq-BE identified 196 candidate off-target sites, in comparison to 23 sites nominated by in silico analysis using Cas-OFFinder.

In summary, these studies show feasibility of using base editing approach to correct mutations and rescue the hematopoietic defects associated with DBAS. These studies could serve as a proof-of-concept to develop individualized, mutation-specific genetic therapies for BMF disorders. Further studies are needed to de-risk this BE-based approach to reduce off-target effects.