Adenine Base Editing of Gamma Globin Gene Promoters Shows No Detectable Off-Target RNA or DNA Editing

Sickle cell disease (SCD) and β-thalassemia are hemoglobinopathies that lead to severe disease complications across multiple organ systems and severe anemia. Patients harboring mutations in the gamma globin gene (HBG1/2) promoters often display elevated fetal hemoglobin (HbF) levels and have significantly fewer complications from SCD or b-thalassemia. This condition, known as hereditary persistence of fetal hemoglobin (HPFH), provides a unique opportunity of base editing to treat these diseases by recreating these naturally occurring mutations. We have previously demonstrated that the adenine base editor (ABE) ABEV2 can induce single base changes in HBG1/2 promotors in human CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro, resulting in >90% editing and >60% upregulation of gamma globin. This level of editing and gamma globin production was also retained in NBSGW mice at 16 weeks post-transplantation. To further characterize the specificity of ABEV2, we explored its potential to cause unintended single nucleotide changes in the genome by either guide-dependent or guide-independent mechanisms. To accomplish this, we employed a series of orthogonal biochemical assays, multiplex amplicon sequencing, single cell whole genome sequencing (scWGS), and transcriptome-wide RNA sequencing in primary human CD34+ hematopoietic stem and progenitor cells (HSPCs).

We used the highly sensitive biochemical assays ONE-seq and Digenome‑seq to interrogate thousands of in silico predicted off-target sites as well as the whole genome. Off-target sequences (>200) identified in these biochemical assays were validated for editing in CD34+ HSPCs using multiplex amplicon sequencing. These analyses revealed no off-target DNA editing in ABEV2 edited cells when compared to unedited HSPCs from three independent donors. We next used whole transcriptome sequencing and somatic variant calling to characterize guide-independent off-target RNA editing. Our results revealed that there was no significant variant allele frequency of A-to-I mutations in RNA between edited and unedited HSPCs, consistent with previous studies showing that off-target RNA editing is minimal or equivalent to background when base editors are delivered as mRNA. We finally investigated guide-independent DNA editing using scWGS in a clonal population of ABEV2 edited cells. WGS libraries were prepared from individual CFU clones, and somatic variant callers were used to identify the frequency of SNVs in edited cells compared to unedited cells. These results revealed that the fold change of A-to-G mutations compared to unedited controls was not significant, confirming previously reported results demonstrating that ABEs do not elevate genome-wide guide-independent DNA deamination.

Overall, our multi-faceted strategy revealed an absence of off-target RNA and DNA editing in primary human CD34+ HSPCs. Combined with the on-target editing efficiency and increase in gamma-globin expression, ABE presents a promising gene editing opportunity for the treatment of SCD and β-thalassemia.