Enhancing the engraftment of CRISPR gene-edited HSPCs for the treatment of blood disorders using epigenome editing technologies

Abstract

Enhancing the homing and engraftment of haematopoietic stem and progenitor cells (HSPCs) after ex vivo gene editing holds significant promise for treating haematological disorders. However, challenges such as inefficient bone marrow homing and limited long-term engraftment after ex vivo manipulation remain. In this study, we employed an innovative epigenome editing platform utilising the CRISPRa system to upregulate key engraftment enhancer genes (CXCR4 and MLLT3) in ex vivo cultured human HSPCs. The CRISPRa system increased gene expression by up to 98% within 24 hours of electroporation. The migratory response of these edited cells was evaluated using an in vitro transwell migration assay, which established a chemoattractant gradient with either recombinant SDF-1α or conditioned media derived from primary bone marrow mesenchymal stem cells (MSC-CM). The MSC phenotype used for MSC-CM production was confirmed by flow cytometry, and SDF-1α and VEGF were quantified by ELISA. Our findings showed that upregulating CXCR4 and MLLT3 in HSPCs using CRISPRa enhanced the migration towards MSC-CM compared to recombinant SDF-1α alone. Conversely, overexpression of CXCR4 using mRNA encoding the main isoform reduced migration potential with MSC-CM. These findings suggest that leveraging CRISPRa-mediated epigenome editing to upregulate endogenous gene expression in HSPCs, in conjunction with bioactive components within MSC-CM, can synergistically enhance stem cell migration and potentially improve clinical outcomes. This study highlights the potential of epigenome editing technologies to enhance the clinical application of HSPCs in gene therapies.