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Updated: Apr 10, 2026
Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates
Published on: May 9, 2025
Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA
Matthew J Johnson1, Anthony P DeFeo1, Nicholas J Slipek1
1Department of Pediatrics, University of Minnesota; Masonic Cancer Center, University of Minnesota; Center for Genome Engineering, University of Minnesota.
Abstract:
Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) or exogenous T cell receptor (TCR) to target a specific tumor-associated antigen. Reliance on viral vectors for the production of therapeutic T cells significantly increases the timeline, cost, and complexity of manufacturing while limiting the translation of new therapies, particularly in the academic setting. A process is presented for efficient non-viral engineering of T cells using CRISPR/Cas9 and homology-mediated end joining to achieve targeted integration of large, multicistronic DNA cargo. This approach has achieved integration frequencies comparable to those of viral vectors while yielding highly functional T cells capable of potent anti-tumor efficacy both in vitro and in vivo. Notably, this method is rapidly adaptable to current good manufacturing practices (cGMP) and clinical scale-up, providing a near-term option for the manufacturing of therapeutic T cells for use in clinical trials.
Insights
This study introduces a non-viral method using CRISPR/Cas9 for engineering T cells, offering a faster, cheaper alternative to viral vectors for cancer therapies. The new process yields functional T cells with potent anti-tumor effects, suitable for clinical trials.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- Current adoptive cellular therapies often use viral vectors (lentivirus, retrovirus) to engineer T cells for cancer treatment.
- Viral vector reliance increases manufacturing time, cost, and complexity, hindering therapeutic translation, especially in academia.
- Existing methods face challenges in efficient, large-scale T cell engineering for clinical applications.
Purpose of the Study:
- To develop an efficient non-viral method for engineering T cells for adoptive immunotherapy.
- To enable targeted integration of large DNA constructs into T cells for enhanced therapeutic potential.
- To provide a scalable and cost-effective manufacturing process for clinical T cell therapies.
Main Methods:
- Utilized CRISPR/Cas9 gene editing technology combined with homology-mediated end joining (HMEJ).
- Engineered T cells for expression of chimeric antigen receptors (CAR) or T cell receptors (TCR) targeting tumor antigens.
- Validated T cell function and anti-tumor efficacy in vitro and in vivo.
Main Results:
- Achieved T cell engineering with integration frequencies comparable to viral vectors.
- Produced highly functional T cells demonstrating potent anti-tumor efficacy.
- Demonstrated rapid adaptability to current good manufacturing practices (cGMP) and clinical scale-up.
Conclusions:
- The non-viral CRISPR/Cas9 and HMEJ approach offers an efficient alternative for T cell engineering.
- This method facilitates the manufacturing of therapeutic T cells, accelerating clinical translation.
- Presents a viable near-term option for producing T cells for clinical trials, overcoming limitations of viral vectors.
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