Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV
Meisam Naeimi Kararoudi1,2, Shibi Likhite3, Ezgi Elmas1,4
1Center for Childhood Cancer and Blood Diseases, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH, USA.
Abstract:
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.
Insights
We developed an efficient CRISPR-AAV method for gene editing primary human natural killer (NK) cells for cancer immunotherapy. This approach enhances CAR-T cell therapy efficacy against AML targets.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- Natural killer (NK) cells are crucial for cancer immunotherapy.
- Existing gene editing methods for NK cells face challenges like low transduction efficiency, high cell death, and unstable transgene expression.
Purpose of the Study:
- To develop a highly efficient and site-specific gene insertion method for primary human NK cells.
- To optimize CRISPR-Cas9/RNP and adeno-associated virus (AAV) vector parameters for gene editing NK cells.
Main Methods:
- Utilized CRISPR-Cas9 ribonucleoprotein (RNP) complexes and AAV vectors for gene insertion into primary human NK cells.
- Compared different AAV vector designs, homology arm lengths, and concentrations to optimize gene insertion efficiency.
- Validated the method by site-directed insertion of a CD33-specific chimeric antigen receptor (CAR) into NK cells.
Main Results:
- Achieved highly efficient and site-specific gene insertion in primary human NK cells.
- Demonstrated stable CAR expression in NK cells after expansion.
- Showcased improved anti-leukemic efficacy against Acute Myeloid Leukemia (AML) targets.
Conclusions:
- The developed CRISPR-AAV method overcomes limitations of previous gene editing techniques for NK cells.
- This optimized approach enables efficient CAR-NK cell generation with sustained expression and enhanced anti-cancer activity, paving the way for improved cancer immunotherapy.
