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Updated: Jan 3, 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

Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.

Beau R Webber^1,2,3,4, Cara-Lin Lonetree^1,2,3, Mitchell G Kluesner^1,2,3

¹Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.

Nature Communications

|November 21, 2019

Summary

This summary is machine-generated.

Base editors enable multiplex gene editing in human T cells for cancer therapy, offering a safer alternative to Cas9 nucleases by minimizing DNA damage and improving cell expansion for enhanced adoptive cellular therapy.

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Area of Science:

Genomic medicine
Immunotherapy
Gene editing technologies

Background:

Adoptive cellular therapy and genome engineering show promise for treating genetic diseases and cancer.
Multiplex genome engineering enhances therapy efficacy but poses risks like unintended genomic alterations and genotoxicity.

Purpose of the Study:

To apply base editor technology for multiplex gene modification in primary human T cells.
To support the development of an allogeneic CAR-T cell therapy platform.
To evaluate the safety and efficacy of base editing compared to Cas9 nuclease.

Main Methods:

Utilized base editor technology for multiplex gene disruption in primary human T cells.
Compared base editing outcomes with Cas9 nuclease-mediated editing in T cells.
Assessed double-strand break induction, genomic alterations, and cell expansion.

Main Results:

Base editors achieved highly efficient multiplex gene disruption with minimal double-strand break induction.
Multiplex base-edited T cells demonstrated improved expansion compared to Cas9-edited cells.
Base editing lacked the double-strand break-induced translocations observed with Cas9 nuclease.

Conclusions:

Base editor technology is a powerful platform for precise genetic modification of primary human T cells.
Base editing offers a safer approach for multiplex gene editing in therapeutic cell types, reducing genotoxicity.
This technology supports the advancement of allogeneic CAR-T platforms for cancer treatment.