Periodic Reporting for period 1 - EDITSCD (Assessing efficacy and safety of genome EDITing approaches for Sickle Cell Disease)
Reporting period: 2022-09-01 to 2024-02-29
Sickle cell disease (SCD) is one of the most prevalent monogenic diseases in Europe. A single amino acid substitution in the beta-globin chain of the adult hemoglobin (Hb) drives red blood cell sickling and multi-organ damage. The clinical severity of SCD 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 hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option for SCD patients. To this end, genome editing approaches based on the use of site-specific nucleases or, more recently, base editors have been explored by many groups, including teams in our consortium. These approaches either correct the single point mutation causing SCD or reactivate fetal gamma-globin expression by mimicking HPFH mutations. On the other hand, (pre)clinical data from SCD patients or SCD mouse models, as well as preliminary data from our labs suggest that SCD HSPCs are characterized by a high mutational burden, oxidative stress and expression of inflammatory genes. This can alter HSPC properties as well as their interactions within the bone marrow niche. In the context of gene therapy, it is essential to understand the mechanisms underlying SCD HSPC dysfunction and assess the impact of genome editing approaches on SCD HSPCs. In this proposal, we have assembled a multidisciplinary team to: (i) understand the molecular and cellular mechanisms underlying SCD HSPC autonomous and non-cell-autonomous dysfunctions and (ii) evaluate the impact of established and novel genome editing approaches on SCD HSPC properties and genome integrity.
In particular, we will compare established and novel therapeutic approaches for SCD based on genome editing. In particular, we will test three therapeutic strategies:
1. A CRISPR/Cas9 nuclease approach to target the HBG promoters and disrupt the LRF BS by NHEJ to reactivate HbF.
2. A CRISPR/Cas9 approach to revert the SCD mutation by HDR.
3. A base editing approach either to generate HPFH mutations in the HBG promoters or to revert the SCD mutation by generating the Makassar β-globin variant. We recently developed a base editing approach to introduce HPFH mutations that either disrupt the LRF repressor BS using CBE or generate a KLF1 activator BS using ABE.Furthermore we have established a highly efficient ABE-based strategy to generate the Makassar variant in SCD hHSPCs.
This study will lay the foundation of an improved gene therapy strategy to treat SCD and provide best practice tools and protocols for genome editing-based therapies in HSPCs.
In particular, we will compare established and novel therapeutic approaches for SCD based on genome editing. In particular, we will test three therapeutic strategies:
1. A CRISPR/Cas9 nuclease approach to target the HBG promoters and disrupt the LRF BS by NHEJ to reactivate HbF.
2. A CRISPR/Cas9 approach to revert the SCD mutation by HDR.
3. A base editing approach either to generate HPFH mutations in the HBG promoters or to revert the SCD mutation by generating the Makassar β-globin variant. We recently developed a base editing approach to introduce HPFH mutations that either disrupt the LRF repressor BS using CBE or generate a KLF1 activator BS using ABE.Furthermore we have established a highly efficient ABE-based strategy to generate the Makassar variant in SCD hHSPCs.
This study will lay the foundation of an improved gene therapy strategy to treat SCD and provide best practice tools and protocols for genome editing-based therapies in HSPCs.
We have identified alterations in the human SCD HSPC properties potentially caused by chronic inflammation and oxidative stress and tested some pharmacological approaches to correct the SCD HSPC pathological phenotype. We have also started a deep characterization of non-hematopoietic bone marrow niche components, suggesting potential alterations of mesenchymal stem cells from SCD patients,
This work was paralleled by the study of the different bone marrow components in vivo in SCD murine models. Preliminary data showed alteration in murine HSPCs.
Furthermore, we have attempted to optimize genome editing approaches to correct the SCD mutation via homology-directed repair (HDR; strategy 2) through: (i) the modification of the current designer nuclease platforms; (ii) the use of novel chemical compounds and (large-scale) culture conditions to favour HDR over NHEJ in hHSPCs, and (iii) the use of different types of donor templates and delivery methods to increase the availability of the repair matrix during DNA repair.
This strategy (HDR-mediated gene correction; strategy 2) was compared to the other proposed strategies (1 and 3) in terms of unwanted on- and off-targets events using cutting-edge technologies such as GUIDE-seq, Discover-seq and CAST-seq.
This work was paralleled by the study of the different bone marrow components in vivo in SCD murine models. Preliminary data showed alteration in murine HSPCs.
Furthermore, we have attempted to optimize genome editing approaches to correct the SCD mutation via homology-directed repair (HDR; strategy 2) through: (i) the modification of the current designer nuclease platforms; (ii) the use of novel chemical compounds and (large-scale) culture conditions to favour HDR over NHEJ in hHSPCs, and (iii) the use of different types of donor templates and delivery methods to increase the availability of the repair matrix during DNA repair.
This strategy (HDR-mediated gene correction; strategy 2) was compared to the other proposed strategies (1 and 3) in terms of unwanted on- and off-targets events using cutting-edge technologies such as GUIDE-seq, Discover-seq and CAST-seq.
Our single-cell transcriptomic analysis performed on HSPCs from SCD patients showed the enrichment in inflammatory signatures. Therefore, our study provided the first evidence of the influence of the SCD pathophysiology on HSC properties. Transcriptomic analysis of HSPCs in SCD patients will aid in defining critical parameters for achieving successful outcomes in future gene therapy/genome editing clinical trials for SCD and other diseases characterized by inflammation. The first high-impact scientific publication is under review and patent filing is ongoing. Furthermore, a manuscript including data on the use of compounds to preserve stemness of SCD HSCs is under revision (https://www.biorxiv.org/content/10.1101/2024.01.14.575586v2(opens in new window)). Our work also showed for the first time preliminary evidences of alterations of mesenchymal stem cells from SCD patients
Regarding the assessment and optimization of our gene editing approaches for SCD, our data showed: (i) a substantial increase in the frequency of HDR-mediated gene correction events in hematopoietic cell lines and in primary HSPCs compared to state-of-the art strategies (ii(opens in new window) a detailed comparison of the potential on- and off-targets events associated with different therapeutic approaches to SCD.
Regarding the assessment and optimization of our gene editing approaches for SCD, our data showed: (i) a substantial increase in the frequency of HDR-mediated gene correction events in hematopoietic cell lines and in primary HSPCs compared to state-of-the art strategies (ii(opens in new window) a detailed comparison of the potential on- and off-targets events associated with different therapeutic approaches to SCD.