Background Ex vivo gene editing of T cells is widely used for various applications like allogeneic chimeric antigen receptor (CAR)-T cell and T cell receptor (TCR)-T cell generation, including TCR gene knockout (KO). The conventional method for gene editing in T cells, electroporation (EP), has high cytotoxicity and reduces cell yield. Therefore, there is an urgent need for a highly efficient and less cytotoxic delivery technique for primary T cells. In our previous work, we successfully developed ionizable lipids, a key material for lipid nanoparticles (LNPs) used in mRNA pharmaceuticals and applied them to our contract development and manufacturing organization business. Building on this, we aimed to explore the potential of our LNP technology as an alternative delivery method to EP for gene editing in T cells.

Methods We screened our proprietary lipid library to identify ionizable lipids capable of highly efficient mRNA delivery in human primary T cells. Additionally, we further optimized the LNP formulation for the highest transfection efficiency. Notably, we developed Ready-to-Use LNPs that can encapsulate Cas9 mRNA/sgRNA through simple mixing, without requiring any specialized instruments. To compare the transfection outcomes, we introduced Cas9 mRNA and sgRNA against TRAC using either our Ready-to-Use LNP or Cas9 Ribonucleoprotein (RNP) with EP into activated human primary T cells. Following the transfection treatment, we assessed cell viability, transfection efficiency, yield of TCR-KO cells, and memory phenotypes in the LNP and EP groups. Additionally, the γH2AX positivity rate of each treated cell was evaluated to assess chromosomal damage.

Results Our LNP transfection resulted in improved cell viability and proliferation compared to EP. Furthermore, LNP transfection exhibited a high KO efficiency, with a TCR-negative rate of approximately 80% and an improved TCR-negative cell yield compared to EP. TCR-negative cells treated using either method showed a high frequency of memory-type T cells. Cells treated with EP had a significantly higher rate of γH2AX positivity, whereas cells treated with LNP did not show any increase compared to untreated cells.

Conclusions Our study demonstrates the potential of our LNP technology as a low damage and efficient delivery method for gene editing in human primary T cells. LNPs deliver nucleic acids into cells through endocytic pathway, suggesting that they are less damaging than EPs, which are delivered by physical stimuli. Our LNP-based approach enables the effective delivery of mRNA into primary T cells, overcoming previous challenges and significantly advancing the development of gene-edited T cell therapies.

Ethics Approval This study obtained ethics approval of the Fujifilm Bioethics Review Committee Bio Laboratory Institute Office and the head of the institution.(approval number #2022–005) The human materials used in this study were declared by the distributor to have obtained the informed consent of the donor. (STEMCELL TECHNOLOGIES STATEMENT).