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Updated: Dec 9, 2025
Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells
Published on: February 14, 2025
Charge-altering releasable transporters enable phenotypic manipulation of natural killer cells for cancer
Aaron J Wilk1,2,3, Nancy Lynn-Benner Weidenbacher4, Rosemary Vergara1
1Division of Infectious Diseases and Geographic Medicine, Department of Medicine.
Abstract:
Chimeric antigen receptor (CAR) natural killer (NK) cells are an emerging cell therapy with promising results in oncology trials. However, primary human NK cells are difficult to transfect, hampering both mechanistic studies and clinical applications of NK cells. Currently, NK cell CAR modification relies on viral vectors or cell activation. The former raises cost and tolerability issues, while the latter alters NK cell biology. Here, we report that readily synthesized and inexpensive nonviral charge-altering releasable transporters (CARTs) efficiently transfect primary human NK cells with messenger RNA without relying on NK cell activation. Compared with electroporation, CARTs transfect NK cells more efficiently, better preserve cell viability, and cause minimal reconfiguration of NK cell phenotype and function. We use CARTs to generate cytotoxic primary anti-CD19 CAR NK cells, demonstrating this technology can drive clinical applications of NK cells. To our knowledge, CARTs represent the first efficacious transfection technique for resting primary human NK cells that preserves NK cell phenotype and can enable new biological discoveries and therapeutic applications of this understudied lymphocyte subset.
Insights
Nonviral charge-altering releasable transporters (CARTs) efficiently deliver mRNA to primary human natural killer (NK) cells. This novel method overcomes transfection challenges for CAR NK cell therapy, preserving cell function and viability.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Chimeric antigen receptor (CAR) natural killer (NK) cells show promise in oncology.
- Transfecting primary human NK cells is challenging, limiting research and clinical use.
- Current CAR NK modification methods (viral vectors, cell activation) have drawbacks.
Purpose of the Study:
- To develop an efficient, nonviral method for transfecting primary human NK cells with mRNA.
- To overcome limitations of existing NK cell modification techniques.
- To enable new discoveries and therapeutic applications of CAR NK cells.
Main Methods:
- Utilized nonviral charge-altering releasable transporters (CARTs) for mRNA delivery.
- Transfected resting primary human NK cells without requiring cell activation.
- Compared CART transfection with electroporation regarding efficiency, viability, and phenotype.
Main Results:
- CARTs efficiently transfected primary human NK cells with mRNA.
- CART transfection showed higher efficiency and better cell viability than electroporation.
- CARTs minimally altered NK cell phenotype and function.
- Generated anti-CD19 CAR NK cells using CART technology.
Conclusions:
- CARTs offer an effective, nonviral transfection method for primary human NK cells.
- This technique preserves NK cell phenotype and function, unlike other methods.
- CARTs facilitate the development of CAR NK cell therapies and biological studies.
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