Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV

• DOI: 10.1016/j.crmeth.2022.100236
• Publication Date: 2022-06-13T14:54:05Z
• Authors: Meisam Naeimi Kar...; Shibi Likhite; Ezgi Elmas; Kenta Yamamoto; Maura Schwartz; Kinnari Sorathia; Marcelo de Souza ...; Yasemin Sezgin; Raymond D. Devine; Justin M. Lyberger; Gregory K. Behbehani; Nitin Chakravarti; Branden S. Moriarity; Kathrin Meyer; Dean A. Lee
• Keywords: Killer Cells, Natural; Gene Editing; 0301 basic medicine; 03 medical and health sciences; Humans; Immunotherapy; Article; 3. Good health

Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets.