Senior Scientist Revvity Cambridgeshire, England, United Kingdom
Abstract: Hematopoietic stem and progenitor cells (HSPCs) are a foundational cell type for the development of engineered therapies. Since engineered HSPCs are meant to persist throughout a patient’s lifetime and are sensitive to DNA damage, it is important to utilize a technology which minimizes genotoxicity and allows engineered cells to retain functionality. Nuclease-induced double strand breaks (DSBs) trigger a DNA damage response which may lead to chromosomal aberrations, decreased viability and cell function, and ultimately reduce the efficacy and safety of engineered therapies. Base editors are not reliant on DSBs and represent an effective mitigation strategy but must be optimised for efficient delivery into HSPCs. Our proprietary Pin-pointTM platform is a flexible modular base editor typically comprising a DNA binding Cas, a DNA modifying deaminase, and a sequence-targeting guide RNA which assembles at the target locus, and demonstrates an advanced safety profile compared to nuclease-based technologies. This makes it well suited to the development of cell and gene therapies in sensitive cell types such as HSPCs. In this study we used a Pin-point base editing system composed of Rat APOBEC1 mRNA, SpCas9 nickase mRNA and a synthetic aptamer-containing gRNA to edit CD34+ HSPCs with 80% efficiency. Flow cytometry of edited cells revealed an unaltered frequency of hematopoietic stem cells (HSCs) compared to the control and high levels of gene knock out in both the bulk and HSC population which was retained over 5 weeks. Moreover, primitive hematopoietic cells edited with the Pin-point platform retain their stemness capacity as validated in a Long-Term Culture-Initiating Cell (LTC-IC) assay. Additionally, qPCR revealed no significant DNA damage response, further supporting the gentler activity on cells. Next, we validated the technology on two separate loci known to reactivate γ-globin expression and achieved high levels of base editing that corresponded with an increase in γ-globin mRNA and protein expression as a relevant therapeutic outcome. The ability to base edit HSPCs efficiently and safely, while maintaining their stemness and differentiation potential, demonstrates the strength of the Pin-point platform as a tool for the generation of advanced cell therapies using sensitive multipotent cell types.
