Two Is Better Than One: Fetal Hemoglobin Reactivation and Alpha-Globin Downregulation to Correct the β-Hemoglobinopathy Phenotype through Base Editing

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult hemoglobin β-chain. In β-thalassemia, the reduced (β⁺) or absent (β⁰) production of β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6^Glu→Val) in the adult hemoglobin (Hb) β^S-chain causes Hb polymerization with consequent RBC sickling, vaso-occlusive crises, organ damage and reduced life expectancy. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors or CRISPR/Cas9 nuclease are not equally effective in all the patients and/or raise safety concerns. Base editing (BE) allows the introduction of point mutations (C>T by cytidine base editors, CBEs; A>G by adenine base editors, ABEs) without generating dangerous double strand breaks, thus allowing the multiplexed editing of several targets.

Mutations leading to elevated fetal Hb (HbF) or reduced α-globin expression are associated with a better clinical phenotype in both β-thalassemia and SCD patients. Fetal β-like γ-globin compensates for adult β-globin deficiency and reduced α-globin levels impairs its precipitation and inhibit Hb polymerization in SCD. In this study, we have developed BE strategies aiming to (i) reactivate HbF by down-regulating BCL11A, a master repressor of γ-globin expression though inactivation of its enhancers, and (ii) reduce the α-globin levels through targeting of the MCS-R2 enhancer of the α-globin genes (HBA1/2).

First, we used CBEs and ABEs 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 (CFC) 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.

In parallel, we used CBEs and ABEs to target the GATA1 or NF-E2 activator BS within the MCS-R2 enhancer of HBA1/2. We screened single guide RNAs (sgRNAs) in an erythroid cell line (K562) that allowed editing of different nucleotides within the GATA1 and NF-E2 BS and we identified the most efficient sgRNAs diminishing α-globin expression. Next, selected sgRNAs were delivered to SCD HSPCs, achieving high editing efficiency (~85%). A CFC assay showed a good progenitor viability. RT-qPCR and Western Blot analyses in the erythroid progeny of edited SCD HSPCs confirmed α-globin downregulation at RNA and protein levels.

Lastly, we combined both strategies using ABEs to target simultaneously BS within the BCL11A and MCS-R2 enhancers in SCD HSPCs. The concomitant usage of two sgRNAs did not affect editing efficiency at the different targeted sites, which was ~80% at both BCL11A and MCS-R2 enhancers. The erythroid progeny of edited SCD HSPCs harboring mutations in both BCL11A and MCS-R2 enhancers showed downregulation of α-globin and up-regulation of γ-globin at RNA and protein expression level. We expect that this combined strategy will efficiently rescue the pathological phenotype of RBCs derived from both SCD and β-thalassemic HSPCs.

In conclusion, our study identified the core nucleotides of both the BCL11A and HBA1/2 MCS-R2 enhancers, which are crucial for regulating the expression of these genes and represent potential targets for scarless editing through BE to finely modulate globin expression. We also developed efficient BE strategies to simultaneously reactivate HbF and downregulate the α-globin expression in patient HSPCs. Validation of the above-described results in HSPCs in vivo will provide sufficient proof of efficacy and safety to enable the clinical development of base-edited HSPCs for the therapy of β-hemoglobinopathies.

Cavazzana:Noga: Consultancy; Cellectis: Consultancy; Smart-Immune: Current Employment, Current equity holder in private company. Miccio:Cellectis: Consultancy, Research Funding.