Introduction: Lipid nanoparticles (LNPs) are a promising nonviral alternative for delivering CRISPR-Cas9 components to difficult-to-transfect cell types like human primary T cells and CD34+ hematopoietic stem and progenitor cells (HSCs). We developed a library of novel ionizable lipids and LNP compositions for ex vivo applications. This study presents the screening and optimization of Cas9 mRNA and sgRNA delivery for gene knockouts in T cells and HSCs and explores protocols for scale-up and clinical translation. 

Methods: Novel LNPs were formulated using the NxGen™ mixing platform and tested on human primary T cells or CD34+ HSCs from peripheral blood. Various RNAs, including GFP, ɑCD19-CAR mRNA, Cas9 mRNA, and sgRNAs targeting TCR/CD52 (T cells) and CD33/CD45 (HSCs) were encapsulated into LNPs. Culture media, supplements, and transfection kinetics were optimized for gene delivery efficiency. Viability, proliferation, and colony-forming potential were measured post-editing.

Results: We achieved up to 90% TCR–/CD52–double knockout in T cells with > 90% viability. In HSCs, 80% and 90% knockouts (CD45 and CD33, respectively) were achieved, with > 95% viability and strong proliferative capacity. Colony-forming unit assays showed no significant changes in multilineage differentiation. Media optimization was crucial, with some commercial media yielding higher editing efficiencies. Treatment kinetics and media supplements significantly impacted editing efficiency in long-term HSCs. We successfully scaled-up LNPs and optimized conditions from well-plates to G-Rex bioreactors.

Conclusions: We present a versatile and scalable LNP platform with minimal cytotoxicity for efficient gene delivery and editing in difficult-to-transfect cell types. The method has potential for clinical translation in T cells and HSCs.