CRISPRi-Mediated Transient Inhibition of DNMT3A Gene Expression For Expansion of Human HSPCs

Abstract

Hematopoietic stem and progenitor cells (HSPCs) are central targets of a regenerative therapy-based approach to the long-term treatment of inherited diseases of the blood and bone marrow. Allogeneic transplantation and autologous ex vivo gene correction strategies remain the only curative treatment options for inherited diseases of blood and bone marrow. However, these strategies are often restricted due to insufficient numbers of HSPCs derived from umbilical cord blood units or from marrow and mobilized peripheral blood of patients with impaired hematopoiesis. Hence, the successful treatment of inherited hematopoietic diseases would be greatly facilitated by the ability to expand HSPCs ex vivo. In this study, we investigated whether transient downregulation of DNMT3A gene expression in human HSPCs could promote their expansion while avoiding the long-term complications associated with permanent DNMT3A knockout. We developed a new approach based on the CRISPRi system for transient inhibition of targeted gene expression in human HSPCs. Furthermore, we showed that transfection CRISPRi components in the form of mRNA was a safe and efficient delivery approach in human HSPCs. In ex vivo experiments, the approach had minimal toxicity but we did not observe significant expansion of hematopoietic progenitors. Similarly, in primary and secondary transplantation experiments, HSPCs subjected to CRISPRi maintained their ability to engraft, suggesting limited cellular toxicity of this approach; however, no significant benefit was observed in overall levels of engraftment compared to untreated control groups. Overall, these studies established a novel platform for transient inhibition of targeted gene expression in human HSPCs but additional optimization will be required to ensure expansion of functional HSPCs.