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Updated: Jun 30, 2025
Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates
Published on: May 9, 2025
Precision Enhancement of CAR-NK Cells through Non-Viral Engineering and Highly Multiplexed Base Editing
Minjing Wang1,2,3,4, Joshua B Krueger1,2,3, Alexandria K Gilkey1,2,3
1Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.
Abstract:
Natural killer (NK) cells' unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However, NK cells have demonstrated only moderate responses against cancer in clinical trials and likely require advanced genome engineering to reach their full potential as a cancer therapeutic. Multiplex genome editing with CRISPR/Cas9 base editors (BE) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here, we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations. We observed highly efficient single and multiplex base editing, resulting in significantly enhanced NK cell function. Next, we combined multiplex BE with non-viral TcBuster transposon-based integration to generate IL-15 armored CD19 CAR-NK cells with significantly improved functionality in a highly suppressive model of Burkitt's lymphoma both in vitro and in vivo. The use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated.
Insights
This study introduces base editors (BE) for enhancing natural killer (NK) cells, a promising immunotherapy. Engineered NK cells show improved cancer-killing ability, paving the way for more effective cancer treatments.
Area of Science:
- Immunology
- Genetics
- Biotechnology
Background:
- Natural killer (NK) cells are crucial for immunotherapy due to their ability to target cancer cells.
- Current NK cell therapies show moderate efficacy, necessitating advanced genome engineering for improved cancer treatment.
- CRISPR/Cas9 base editors (BE) have enhanced T cell function but haven't been applied to human NK cells.
Purpose of the Study:
- To report the first application of base editing (BE) in primary human NK cells.
- To demonstrate enhanced NK cell function through multiplex genome editing.
- To develop IL-15 armored CD19 CAR-NK cells using multiplex BE and non-viral transposon integration for improved cancer immunotherapy.
Main Methods:
- Utilized CRISPR/Cas9 base editors (BE) for multiplex genome editing in primary human NK cells.
- Achieved both loss-of-function and gain-of-function mutations efficiently.
- Combined multiplex BE with TcBuster transposon-based integration for CAR-NK cell generation.
Main Results:
- Demonstrated highly efficient single and multiplex base editing in human NK cells.
- Observed significantly enhanced NK cell function post-editing.
- Generated IL-15 armored CD19 CAR-NK cells with improved in vitro and in vivo functionality in a Burkitt's lymphoma model.
Conclusions:
- Multiplex base editing is effective in primary human NK cells, enhancing their function.
- The combination of non-viral transposon engineering and multiplex BE offers a versatile platform for CAR-NK cell generation.
- This approach allows tailored gene edits to maximize therapeutic effectiveness for various cancers.
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