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This research aims to develop a novel approach for creating CAR-γδ T cells to enhance tumor therapy.

May 20, 2026Cancer Medicine0 citationsOpen Access

A Novel Strategy to Produce CAR ‐γδ T Cells via Site‐Directed Gene Integration by a Combination of CRISPR /Cas9 and AAV

RDRuoyu DongSir Run Run Shaw Hospital YZYixi ZhangGreat Ormond Street Hospital GYGuangxun YuanNational Clinical Research Center for Digestive Diseases

Key Points

This research aims to develop a novel approach for creating CAR-γδ T cells to enhance tumor therapy.
Utilized CRISPR/Cas9 to disrupt TCR delta chain constant region for TCR knockout.
Performed site-directed insertion of CAR sequence into TRDC locus via AAV gene delivery.
Optimized electroporation and infection conditions for efficient gene editing in γδ T cells.
Achieved effective TCR knockout and CAR insertion in γδ T cells.
Newly generated CAR-γδ T cells demonstrated strong cytokine production and tumor cell killing.
CAR-γδ T cells reduced tumor burden and improved survival in tumor-bearing mice.

Abstract

Chimeric antigen receptor (CAR)-αβ T cells are commonly employed in tumor therapy but hindered by some limitations. γδ T cells are promising substrates for CAR therapy for their major histocompatibility complex (MHC)-unrestricted recognition manner and innate immune function. Here, we established a novel method for generating CAR-γδ T cells. We utilized the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) method to interrupt the TCR delta chain constant region (TRDC) sequence, followed by the site-directed insertion of the CAR sequence into the TRDC locus via homologous complementation mediated by adeno-associated virus (AAV) gene delivery. We optimized electroporation parameters for Cas9/ribonucleoproteins (RNP) delivery and infection conditions for CAR-gene carrying AAV in γδ T cells. These optimizations facilitated efficient TCR knockout and site-directed CAR insertion, ultimately yielding functional CAR-γδ T cells. In vitro experiments demonstrated that these newly prepared CAR-γδ T cells could stimulate cytokine production, kill tumor cells as well as exhibit robust proliferative potential and memory-like phenotype. These state-of-the-art CAR-γδ T cells could reduce tumor burden and extend the survival period of tumor-bearing mice.

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