WP 1. Correction of the SCD mutation
The SCD-causing A>T mutation cannot be reverted with current BE technology. However, using adenine base editors (ABE), we can convert A to G, generating the “Makassar” allele. This rare genetic variant leads to the production of the Makassar hemoglobin (HbG), which is non-pathogenic in both homozygous and heterozygous carriers. We used SCD-HUDEP2, a cell line carrying the SCD mutation, to select combinations of base editors and gRNAs able to generate the Makassar mutation. The SCD mutation was inaccessible with first generation base editors. Therefore, we engineered base editors and optimized the gRNA length in order increase the accessibility to the locus and base editing efficiency at the target base. We achieved >90% correction of the SCD mutation and production of HbG at both RNA and protein levels.
Of note, we have applied a similar strategy to beta-thalassemia causing mutations (Hardouin et al, Blood, 2022)
WP 2. Introduction of HPFH-like mutations into the γ-globin promoters
Mutations clustering ~200 nucleotides upstream of the gamma-globin (HBG) transcriptional start sites either reduce binding of the LRF repressor or recruit the KLF1 activator. Here, we used base editing to generate a variety of mutations in the -200 region of the HBG promoters, including potent combinations of four to eight gamma-globin-inducing mutations. Editing of beta-thalassemic and SCD patient hematopoietic stem/progenitor cells is safe, led to fetal hemoglobin reactivation and rescued the pathological phenotype. Creation of a KLF1 activator binding site was the most potent strategy - even in long-term repopulating hematopoietic stem/progenitor cells. Compared with a Cas9-nuclease approach, base editing avoids the generation of insertions, deletions and large genomic rearrangements and results in higher gamma-globin levels. Our results demonstrate that base editing of HBG promoters is a safe, universal strategy for treating beta-hemoglobinopathies (Antoniou et al, Nat Comm, 2022).
WP 3. Modulating the activity of an erythroid-specific BCL11A enhancer
We used base editors to target the GATA1 or ATF4 activator binding site (BS) within the erythroid-specific BCL11A enhancers (+58-kb and +55-kb regions) in SCD HSPCs through RNA delivery and achieved efficiencies of up to ~90%. A colony forming cell assay showed no impact on progenitors’ viability. RBCs derived from base-edited HSPCs exhibited high HbF expression levels that were sufficient to ameliorate the sickling phenotype. Samples carrying different mutations within the GATA1 or ATF4 BS were associated with different HbF levels, implying that some nucleotides are more essential for GATA1 and ATF4 binding and the consequent BCL11A gene activation.
