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Updated: Jun 5, 2025

Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates

Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates

Published on: May 9, 2025

Efficient non-viral immune cell engineering using circular single-stranded DNA-mediated genomic integration.

Keqiang Xie¹, Jakob Starzyk², Ishita Majumdar²

¹Full Circles Therapeutics, Cambridge, MA, USA. keqiang.xie@fullcirclestx.com.

Nature Biotechnology

|December 11, 2024

Summary

This summary is machine-generated.

This study introduces a novel non-viral genome writing catalyst (GATALYST) system using circular single-stranded DNA (cssDNA) donors for precise genome engineering. This method achieves high knock-in efficiency and improved safety, overcoming limitations of viral vectors and other non-viral methods.

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Area of Science:

Molecular Biology
Gene Editing Technologies
Biotechnology

Background:

Adeno-associated viruses (AAVs) face limitations in homology-directed repair (HDR)-mediated genome engineering due to safety, manufacturing, and packaging constraints.
Current non-viral methods using double-stranded DNA (dsDNA) exhibit low efficiency and high cytotoxicity, while linear single-stranded DNA (lssDNA) presents manufacturing scalability challenges.

Purpose of the Study:

To develop a novel non-viral system for efficient and precise genome engineering using circular single-stranded DNA (cssDNA) as donor templates.
To overcome the limitations associated with AAVs and existing non-viral DNA donors for gene integration.

Main Methods:

Development of the genome writing catalyst (GATALYST) system for producing cssDNA donors up to approximately 20 kilobases.
Testing cssDNA donor efficiency for transgene integration in induced pluripotent stem cells (iPSCs) and primary immune cells (including CAR-T and NK cells).
Evaluation of cssDNA performance with various nuclease editor systems at clinically relevant genomic loci.

Main Results:

Achieved knock-in efficiency of up to 70% in iPSCs using cssDNA donors.
Demonstrated improved efficiency in multiple primary immune cell types and at various genomic loci.
Showcased high precision and efficiency in chimeric antigen receptor (CAR)-T and natural killer (NK) cells.

Conclusions:

The cssDNA donor system offers a promising non-viral alternative for precision genome engineering, surpassing limitations of current viral and non-viral approaches.
The GATALYST system exhibits enhanced safety, payload flexibility, and scalable manufacturability, indicating significant potential for future clinical genome engineering applications.