By Reka Geczy, PhD, Aruna Balgi, Stella Park, Rita Zhao, Ethan Watt, Maggie Wong, Cooper Webb, Nikita Jain, PhD, Angela Zhang, PhD, Anitha Thomas, PhD, Samuel Clarke, PhD
The expression of the chimeric antigen receptor (CAR) on T cells turns a patient’s cells into cell-based cancer therapies and has revolutionized cancer treatment today1. Despite its successes and high response rates, evidence suggests an increasing need for more complex genetic engineering enabled by CRISPR/Cas-mediated genome editing technologies. Such examples include the disruption of inhibitory pathways exploited by the tumor microenvironment2, 3, improvement of CAR T cell efficiency4, 5, and manufacturing of universal CAR T cells from allogeneic donors6, 7. The desire to achieve both gene editing and transgene expression in next-generation T cell therapies emphasizes the significance of the genetic material delivery method, which plays a critical role in cell function, cell yield, ease of production, and scale-up.
A promising new approach for T cell engineering is the use of RNA to express therapeutic proteins and gene editing nucleases. RNA is typically delivered to cells using electroporation; however, the sequential electrical pulses for multi-step gene engineering leads to a dramatic trade-off between efficiency and cell viability. This type of trade-off is not observed with lipid nanoparticles (LNPs), making it an attractive alternative for RNA delivery. LNPs are entirely synthetic lipid formulations designed to encapsulate and protect RNA before delivering it into cells. The production of LNPs is well-established and is scalable for large-scale gene delivery and gene editing, which are key to meeting clinical demand now and in the future. The RNA-LNP complex structurally resembles low density lipoproteins (LDL) and can co-opt the endogenous uptake pathway of LDL to enter cells using receptor-mediated endocytosis. This gentle uptake mechanism enables successful genome engineering of T cells while maintaining high cell viability.
Herein, we report a novel method for sequential genetic engineering of T cells using the GenVoy-ILM™ T Cell Kit for mRNA. We utilized a manufacturing workflow optimized to deliver various RNA cargoes. In this case study, we show CRISPR/Cas9-mediated knockouts (KO) of the T cell receptor (TCRɑβ) and explore multi-step LNP engineering to produce TCRɑβ KO CAR T cells, a promising approach towards allogeneic CAR T cell therapy6, 8, 9. We describe in detail LNP production and cell culture treatment protocols, as well as optimization strategies for T cell gene editing to ensure success with the GenVoy-ILM T Cell Kit for mRNA.