A Genetically Engineered Primary Human Natural Killer Cell Platform for Cancer Immunotherapy

Emily J Pomeroy; John T Hunzeker; Mitchell G Kluesner; Walker S Lahr; Branden A Smeester; Margaret R Crosby; Cara-Lin Lonetree; Kenta Yamamoto; Laura Bendzick; Jeffrey S Miller; Melissa A Geller; Bruce Walcheck; Martin Felices; Beau R Webber; Timothy K Starr; Branden S Moriarity

doi:10.1016/j.ymthe.2019.10.009

A Genetically Engineered Primary Human Natural Killer Cell Platform for Cancer Immunotherapy

Mol Ther. 2020 Jan 8;28(1):52-63. doi: 10.1016/j.ymthe.2019.10.009. Epub 2019 Oct 15.

Authors

Emily J Pomeroy¹, John T Hunzeker¹, Mitchell G Kluesner¹, Walker S Lahr², Branden A Smeester¹, Margaret R Crosby², Cara-Lin Lonetree¹, Kenta Yamamoto³, Laura Bendzick⁴, Jeffrey S Miller⁵, Melissa A Geller⁶, Bruce Walcheck⁷, Martin Felices⁸, Beau R Webber¹, Timothy K Starr⁹, Branden S Moriarity¹⁰

Affiliations

¹ Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
² Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.
³ Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA.
⁴ Department of Obstetrics, Gynecology, and Women's Health, University of Minnesota, Minneapolis, MN 55455, USA.
⁵ Department of Medicine, Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis, MN 55455, USA.
⁶ Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Department of Obstetrics, Gynecology, and Women's Health, University of Minnesota, Minneapolis, MN 55455, USA.
⁷ Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, MN 55455, USA.
⁸ Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Department of Medicine, Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis, MN 55455, USA.
⁹ Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA; Department of Obstetrics, Gynecology, and Women's Health, University of Minnesota, Minneapolis, MN 55455, USA.
¹⁰ Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address: mori0164@umn.edu.

Abstract

Enhancing natural killer (NK) cell cytotoxicity by blocking inhibitory signaling could lead to improved NK-based cancer immunotherapy. Thus, we have developed a highly efficient method for editing the genome of human NK cells using CRISPR/Cas9 to knock out inhibitory signaling molecules. Our method efficiently edits up to 90% of primary peripheral blood NK cells. As a proof-of-principle we demonstrate highly efficient knockout of ADAM17 and PDCD1, genes that have a functional impact on NK cells, and demonstrate that these gene-edited NK cells have significantly improved activity, cytokine production, and cancer cell cytotoxicity. Furthermore, we were able to expand cells to clinically relevant numbers, without loss of activity. We also demonstrate that our CRISPR/Cas9 method can be used for efficient knockin of genes by delivering homologous recombination template DNA using recombinant adeno-associated virus serotype 6 (rAAV6). Our platform represents a feasible method for generating engineered primary NK cells as a universal therapeutic for cancer immunotherapy.

Keywords: ADAM17; CRISPR; NK cells; PD1; immunotherapy.

Publication types

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

MeSH terms

ADAM17 Protein / genetics
Adoptive Transfer / methods*
Animals
CRISPR-Cas Systems
Cell Engineering / methods*
Cytotoxicity, Immunologic / genetics
Dependovirus
Female
Gene Knockout Techniques
Genetic Engineering / methods*
Healthy Volunteers
Humans
K562 Cells
Killer Cells, Natural / immunology*
Mice
Mice, Inbred NOD
Mice, SCID
Ovarian Neoplasms / pathology
Ovarian Neoplasms / therapy*
Parvovirinae / genetics
Programmed Cell Death 1 Receptor / genetics
Treatment Outcome
Xenograft Model Antitumor Assays

A Genetically Engineered Primary Human Natural Killer Cell Platform for Cancer Immunotherapy

Authors

Affiliations

Abstract

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MeSH terms

Substances

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