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.
