Journal Article

A highly efficient transgene knock-in technology in clinically relevant cell types

Nelson, Jacqueline D., Zhang, Deric, Huang, Yan, Shearman, Mark S., Izzo, Kaitlyn M., Pattali, Rithu, Nehil, Michael, Blaha, Laura, Sun, Xiaoqi, Zhao, Amy H., Cochran, Nadire R., Follit, John A., Scott, Sean N., Wilson, Christopher J., Lele, Swarali, Wu, Meng, Zuris, John A., Chang, Kai-Hsin, Margulies, Carrie M., Allen, Alexander G., Sherman, Stephen E., Jaskolka, Michael C., Viswanathan, Ramya, Giannoukos, Georgia, Khan, Samia Q., Mazzucato, Patrizia, Wilson, Aaron C., Marco, Eugenio, Holland, Carl S., Ciulla, Dawn M., Zhang, Peisheng, Gerew, Alexandra

2024

Overview

Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo. Transgene knock-ins into housekeeping genes lead to high efficiency of cell selection.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: 631/61/2300/1514 / 631/67/1059/2325 / Agriculture / Antigens / Bioinformatics / Biomedical and Life Sciences / Biomedical Engineering/Biotechnology / Biomedicine / Biotechnology / CD16 antigen / Cloning / CRISPR / CRISPR-Cas Systems - genetics / Efficiency / Gene Editing / Gene Knock-In Techniques / Good Manufacturing Practice / Life Sciences / Lymphocytes / Lymphocytes T / Natural killer cells / Nuclease / Pluripotency / Proteins / Stem cells / Transgenes / Transgenes - genetics