Periodic Reporting for period 2 - ReviveSTEM (Dissecting and targeting senescence programs to advance Hematopoietic Stem Cell-based gene therapies)
Reporting period: 2023-09-01 to 2025-02-28
Inherited diseases of blood lineages can be devastating, but gene-editing offers hope for treating them by precisely correcting genetic defects. One promising method involves editing specific genes in hematopoietic stem and progenitor cells (HSPCs), which are cells that can develop into various blood cell types. However, there have been challenges in making this technique efficient and safe for clinical use. Specifically, genetic manipulation may inadvertedly trigger cellular responses, including a premature aging pehnotype defined as seenscence, that could jeopardize the efficacy of the therapy and aggravate the risks of developing hematological malignancies in the long-term. My objective is twofold. First, I intend to elucidate the molecular determinants that promote senescence in HSPCs during ex-vivo manipulation as well as upon transplantation. Second, I plan to overcome senescence barriers and to design innovative hypothesis-based strategies for more effective and safer gene therapy approaches. To pursue these objectives, I will i) combine a unique know-how of senescence mechanisms and HSPC genetic engineering with pioneering technologies ; ii) take advantage of a drug discovery chemical screen and apply principles of mechanobiology; iii) tackle key ill-defined issues pertaining the functional properties of gene-manipulated HSPCs in state-of-the-art humanized mouse models of transplantation and in uniquely available cohorts of gene therapy patients.
In our previous work, we found that edited HSPCs undergo a process called senescence, which limits their ability to repopulate the blood system after transplantation. Additionally, we discovered that activating these cells outside the body (ex vivo) before editing can trigger DNA damage responses, further complicating the process. In our latest research, we made significant strides in addressing these challenges. We found that blocking a protein called p53 during gene editing can reduce senescence in edited HSPCs, leading to better long-term blood cell production. However, this approach also increased the risk of genetic mutations. Alternatively, we explored using anti-inflammatory treatments to modulate senescence, which improved the edited cells' ability to repopulate the blood system without adding to the risk of genotoxocity. Moreover, we investigated the effects of shortening the time HSPCs spend in ex vivo culture before gene editing. While this approach reduced the DNA damage response, it also lowered the efficiency of gene correction. We uncovered that ex vivo activation triggers a stress response in the cells, leading to DNA damage. By inhibiting a specific pathway involved in this stress response,driven by the MAPK p38, we were able to improve the edited cells' ability to produce various blood cell types and engraft more effectively when transplanted into animals. Importantly, 3D scaffolds can be implemented into emerging and existing gene therapy workflows to improve the efficacy and safety of HSPC-based gene therapy applictaions. Overall, our findings highlight the importance of understanding the cellular responses involved in gene editing HSPCs and provide valuable insights for developing safer and more effective gene therapy strategies for inherited diseases. By addressing key barriers such as senescence and stress-induced DNA damage, we aim to pave the way for better clinical applications of gene editing in treating genetic disorders.
While most efforts in the GT field focus on developing methods to enhance gene-editing precision and HDR efficiency, or to improve gene-transfer in the most primitive HSPC compartment, my distinctive contribution will be to investigate previously unexplored mechanisms of senescence induction and define their functional short and long-term consequences on the biology of engineered human hematopoiesis, a conceptual turning point for such translational applications. I trust that, over the past years, the identification of senescence determinants in HSPC gene therapies may have been overlooked because of the lack of ad hoc designed experimental approaches or available technologies and the natural compensation provided by the HSPC biology in transplantation experiments, for which even a single gene-corrected HSPC may repopulate a mouse, albeit at a significant cost for long-term resilience and safety. Thus this ERC proposal has the potential to transform the way we think about HSPC-gene therapy applictaions.