Enhancing the therapeutic potential of mesenchymal stem cell-based therapy via CRISPR/Cas9-based genome editing

Abstract

Background & Aim Mesenchymal stem cell (MSC) based therapy have been shown promising efficacy and safety in treating various diseases. However, transplanted MSCs last only short-term which may require frequent repeated administration. One of the potential reasons behind low viability of MSCs upon transplantation is un-balanced oxygen tension between culture condition and biological tissues. For example, bone marrow or umbilical cord which are considered promising sources of MSCs were shown to have low oxygen tension. MSCs grown under hypoxia condition has been shown to have increased survival and potency upon transplantation. One of the key factors driving these positive effects of hypoxia conditioning in MSCs suggested is HIF1a and we hypothesized that increasing HIF1a level would increase the therapeutic potential of MSCs. In this study, we employed CRISPR/Cas9-based non-viral gene editing to enhance HIF1a level to increase the survival and potency in a mouse model of Alzheimer's disease. Methods, Results & Conclusion CRISPR/Cas9 was designed to target genes involved in regulation of human HIF1A to enhance HIF1A level. Using Wharton's jelly-derived human MSCs, selected CRISPR/Cas9 components were treated in ribonucleoprotein complexes. Viability and growth factor release upon gene editing of different genes involved in HIF1a regulation were tested in the context of sub-lethal hypoxia and oxidative stress. Furthermore, survival and potency of MSCs upon gene editing were tested via intra-hippocampal injection into 5XFAD AD mouse model. Several genes involved in HIF1a regulation were successfully edited without alterations in viability and tri-lineage differentiation potentials and particularly, we found that HIF1AN gene edited MSCs showed significantly increased viability under sub-lethal hypoxic and oxidative stress condition. HIF1AN gene edited MSCs also showed better survival upon transplantation into hippocampus of 5XFAD mice when compared to control MSCs. More importantly, HIF1AN gene edited MSC transplanted 5XFAD mice showed less Aβ burden when compared to control MSC transplanted 5XFAD mice. We established a simple CRISPR/Cas9-based method to edit genome of human MSCs without compromising their physiology. Specifically, we mimicked hypoxia pre-conditioning of MSCs via targeted editing of HIF1AN which showed improved therapeutic potential of MSCs. Therefore, targeted gene editing of HIF1AN of MSCs prior to transplantation may hold promise to enhance the therapeutic potential of MSC-based therapy.