Electroporation And Lipid Nanoparticles As Delivery Methods For Targeted CRISPR/Cas9 Gene Editing In Primary Human T-Cells

By Aaron Hoover and Ming Jin, Sharlene Amador, Chesney Michels, Jeff Cram, Mercedes Segura, and Mike Paglia

Chimeric Antigen Receptor (CAR)-T cell therapy has revolutionized the treatment of autoimmune diseases and cancers, particularly B-cell malignancies, with impressive clinical outcomes. However, the use of viral vectors for gene editing in CAR-T cell production presents several challenges, including high costs, stringent regulatory requirements, and safety concerns such as insertional mutagenesis, all of which can jeopardize both manufacturing success and patient safety. While alternative gene editing methods like transposon systems and mRNA-based transduction have been explored to mitigate these risks, they often face limitations such as random integration and suboptimal or transient CAR expression.

Recently, the CRISPR-Cas9 system has emerged as a promising alternative, enabling gene-specific CAR-T cell production through non-viral transfection techniques, such as electroporation (EP) and lipid nanoparticles (LNP), which allow for more precise and targeted gene editing.

In this study, we compared the effects of these non-viral transfection methods for gene knockout and insertion in primary human T-cells, demonstrating the feasibility of an LNP-based transfection process that encapsulates CRISPR/Cas9 nucleic acids and transgene HDR template DNA in a single LNP payload, providing a more efficient and controlled approach to CAR-T cell engineering.