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Updated: Apr 18, 2026

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

Ultra-large targeted DNA integrations in primary human cells.

Courtney Kernick¹, Lauren Chow^2,3,4, Mikail Alejandro^5,6

¹Department of Pathology, Stanford University, Stanford, CA, USA.

Biorxiv : the Preprint Server for Biology

|April 17, 2026

Summary

This summary is machine-generated.

Researchers developed a novel non-viral method for ultra-large DNA integrations (>10kb) into human cells, overcoming previous size limitations for genetic engineering and cell therapies.

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Last Updated: Apr 18, 2026

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

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Published on: May 9, 2025

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Area of Science:

Molecular Biology
Gene Therapy
Cellular Engineering

Background:

Current gene editing methods like AAV and lentiviral vectors face limitations in DNA integration size, with efficiency dropping significantly beyond 5kb.
This constraint hinders the development of advanced genetic therapies and research requiring large DNA payloads.

Purpose of the Study:

To identify and optimize non-viral methods for efficient integration of large DNA sequences (>5kb) into human cells.
To enable the development of next-generation cellular therapies and accelerate genetic research.

Main Methods:

Systematic evaluation of circular single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) as non-viral delivery templates.
Optimization of delivery using helper plasmids, mRNA-encoded nucleases, and sequence design.
Testing integration efficiency in primary human T cells and induced pluripotent stem cells (iPSCs).

Main Results:

Circular DNA templates demonstrated capability for >5kb integrations, surpassing existing non-viral methods.
Optimized delivery strategies significantly improved efficiency and cell viability for large DNA integrations.
Achieved ultra-large DNA integrations (up to 10kb) at >20% efficiency in human T cells and >60% efficiency in iPSCs.
Clinically manufactured T cells with ultra-large integrations showed functionality in vitro and in vivo.

Conclusions:

Circular DNA templates, optimized delivery, and sequence design enable efficient ultra-large DNA integrations (>10kb) in human cells.
This breakthrough provides a scalable platform for genetic engineering and the development of advanced cellular therapies.
The findings offer crucial insights for both basic genetic research and clinical applications.