Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy.

Area of Science:

• Immunology
• Gene Therapy
• Cellular Therapy

Background:

• Adoptive T-cell therapy using T-cells with transgenic T-cell receptors (TCRs) shows promise for treating infections and cancer.
• Current methods for generating these engineered T-cells often result in variable expression and unpredictable in vivo function, limiting their clinical application.

Purpose of the Study:

• To compare the efficacy of CRISPR/Cas9-mediated TCR replacement versus conventional viral transduction for generating TCR-transgenic T-cell products.
• To assess the impact of gene editing on T-cell product homogeneity and in vivo predictability.

Main Methods:

• Studied 51 different TCRs to evaluate gene engineering techniques.
• Utilized CRISPR/Cas9-mediated TCR replacement for targeted transgene insertion into the TCR gene locus.
• Compared CRISPR/Cas9 method with conventional viral transduction regarding transgene copy number, integration site, and TCR expression levels.

Main Results:

• Conventional viral transduction resulted in variable TCR expression and function due to non-specific integration and differing transgene copy numbers.
• CRISPR/Cas9-mediated TCR replacement achieved targeted insertion, leading to homogeneous TCR expression comparable to physiological T-cells.
• Enhanced homogeneity of T-cell products after targeted gene editing correlated with predictable in vivo T-cell responses.

Conclusions:

• CRISPR/Cas9-mediated TCR replacement offers a superior method for generating defined TCR-transgenic T-cell products.
• Targeted gene editing enhances T-cell product homogeneity, crucial for predictable in vivo function and clinical translation in adoptive T-cell immunotherapy.