Gene editing technologies such as CRISPR/Cas9 have greater flexibility and high efficiency. CRISPR technology enables targeted insertion of transgenes at the desired locus and has the ability to multiplex knocking out of multiple genes. These features provide a great opportunity to use this technology to develop next-generation CAR T cells. The popular method for the generation of CART cell drug product is delivery of CAR and its integration into the T cell genome using viral vectors. Lentivirus or gamma retrovirus-based CAR delivery was used in multiple CART cell productions. CART cell production using viral vectors to deliver the transgene results in the random insertion of CAR sequence across the whole genome. In addition to random CAR insertion viral vectors also deliver partial viral sequences into the cell genome. These things together could disrupt essential gene functions and affect surrounding functional gene expression. CRISPR/Cas9 gene-editing technology can achieve targeted insertion of CAR construct sequence in the desired locus such as T cell receptor locus (TRAC) or any genomic safe harbor sites. The transition from viral vectors to CRISPR/Cas9 based gene editing requires multiple reagents that are GMP compatible. A lot of progress was achieved in the commercial availability of CRISPR/Cas9 reagents in recent years for the generation of cGMP CART drug products. However, there are multiple issues that require careful attention in process development to achieve a reliable cGMP process for CRISPR/Cas9 based cell therapy drug product. These include gene knockout and knock-in efficiency, off target and on target effects, and the ability to achieve the desired quantity of the drug product. A study on the feasibility of using CRISPR/cas9 gene-editing technology for autologous CART drug production and the relevance of this technology for the generation of safe drugs for the patients will be discussed.

Learning Objectives:

1. Describe CART cell production process

2. Explain CRISPR/Cas9 gene editing