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

Meisam Naeimi Kararoudi; Shibi Likhite; Ezgi Elmas; Kenta Yamamoto; Maura Schwartz; Kinnari Sorathia; Marcelo de Souza Fernandes Pereira; Yasemin Sezgin; Raymond D Devine; Justin M Lyberger; Gregory K Behbehani; Nitin Chakravarti; Branden S Moriarity; Kathrin Meyer; Dean A Lee

doi:10.1016/j.crmeth.2022.100236

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

Cell Rep Methods. 2022 Jun 13;2(6):100236. doi: 10.1016/j.crmeth.2022.100236. eCollection 2022 Jun 20.

Authors

Meisam Naeimi Kararoudi^{1

2}, Shibi Likhite³, Ezgi Elmas^{1

4}, Kenta Yamamoto⁵, Maura Schwartz³, Kinnari Sorathia¹, Marcelo de Souza Fernandes Pereira¹, Yasemin Sezgin¹, Raymond D Devine⁶, Justin M Lyberger⁶, Gregory K Behbehani⁶, Nitin Chakravarti⁷, Branden S Moriarity⁵, Kathrin Meyer^{2

3}, Dean A Lee^{1

2}

Affiliations

¹ Center for Childhood Cancer and Blood Diseases, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH, USA.
² Department of Pediatrics, The Ohio State University, Columbus, OH, USA.
³ Center for Gene Therapy, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH, USA.
⁴ Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH, USA.
⁵ Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.
⁶ Department of Medicine, Division of Hematology, The Ohio State University Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.
⁷ Department of Medical Oncology, Thomas Jefferson University, Philadelphia, PA 19107, 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.

Keywords: AAV6; AML; Acute Myeloid Leukemia; CAR; CD33CAR-NK; CRISPR; CRISPaint; Cas9/RNP; Cas9/ribonucleoprotein; HDR; NK; adeno-associated virus 6; chimeric antigen receptor; clustered regularly interspaced short palindromic repeats; gene editing; homology directed repair; natural killer cell.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Gene Editing* / methods
Humans
Immunotherapy
Killer Cells, Natural* / metabolism

Grants and funding

U54 CA232561/CA/NCI NIH HHS/United States