Periodic Reporting for period 2 - DITSB (Development of Innovative Therapeutic Strategies for beta-hemoglobinopathies)
Berichtszeitraum: 2021-12-01 bis 2023-05-31
Beta-thalassemia and sickle cell disease (SCD) are caused by mutations affecting the synthesis or the structure of the adult hemoglobin (Hb) beta-chain. The only definitive cure is transplantation of allogeneic hematopoietic stem cells (HSCs) from a compatible donor, an option available to <30% of the patients. The clinical severity of beta-hemoglobinopathies is alleviated by the co-inheritance of mutations causing expression of fetal gamma-globin in adult life - a condition termed hereditary persistence of fetal hemoglobin (HPFH). Transplantation of autologous, genetically modified HSCs is an attractive therapeutic option for patients lacking a suitable donor. To this aim, genome editing approaches based on the use of site-specific nucleases have been explored by many groups, including ours. These approaches may either revert the single point mutation causing SCD or reactivate fetal globin expression, by mimicking HPFH mutations or by decreasing the level of BCL11A, a master repressor of fetal Hb synthesis. Site-specific nucleases, however, generate double-strand breaks (DSBs) in the genome and raise safety concerns for clinical applications, particularly when used in DSB-sensitive HSCs. In this proposal, we aim at exploiting targeted base-editing to develop novel, efficacious and safe strategies for beta-hemoglobinopathies without generating DSBs. This will be attempted by (i) correcting the SCD-causing mutation, (ii) mimicking HPFH mutations in the gamma-globin promoters, or (iii) modulating the activity of BCL11A erythroid-specific enhancers. These approaches will be tested in human adult erythroid cell lines and patient HSCs, differentiated in vitro and in vivo into mature red cells to evaluate editing efficiency, fetal Hb expression, phenotypic cell correction and biosafety. The ultimate goal of the project is to provide sufficient proof of efficacy and safety to enable the clinical development of base-edited HSCs for the therapy of beta-hemoglobinopathies.
Our approaches should ideally solve the limitations of the current gene therapy trials and result in a one-time curative treatment for these patients with two major benefits for the society: they will significantly improve the quality of life of the patients and they will significantly reduce the economic cost and the burden related to the treatment of these life-long diseases.
Our approaches should ideally solve the limitations of the current gene therapy trials and result in a one-time curative treatment for these patients with two major benefits for the society: they will significantly improve the quality of life of the patients and they will significantly reduce the economic cost and the burden related to the treatment of these life-long diseases.
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.
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.
In WP1, we developed a highly efficient BE strategy to correct the SCD mutation and generate the HbG functional hemoglobin variant. Current experiments aim to test the efficacy of our strategy in SCD patients’ hematopoietic stem/progenitor cells in vitro, the target population of gene therapy approaches. This work will also provide further characterization of the HbG structure and properties.
In WP2, our proof-of-concept study showed that generation of HPFH mutation in the HBG promoters is a novel and potent gene therapy approach to cure beta-thalassemia and SCD. Further studies aim at achieving high efficiency of base editing in long-term repopulating hematopoietic stem/progenitor cells in vivo.
In WP3, we identified the core nucleotides of the BCL11A 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. Future experiments aim at validating these results in hematopoietic stem/progenitor cells in vivo.
Overall the expected results of the project are to provide sufficient proof of efficacy and safety to enable the clinical development of base-edited HSPCs for the therapy of β-hemoglobinopathies.
In WP2, our proof-of-concept study showed that generation of HPFH mutation in the HBG promoters is a novel and potent gene therapy approach to cure beta-thalassemia and SCD. Further studies aim at achieving high efficiency of base editing in long-term repopulating hematopoietic stem/progenitor cells in vivo.
In WP3, we identified the core nucleotides of the BCL11A 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. Future experiments aim at validating these results in hematopoietic stem/progenitor cells in vivo.
Overall the expected results of the project are to provide sufficient proof of efficacy and safety to enable the clinical development of base-edited HSPCs for the therapy of β-hemoglobinopathies.