Integrated epigenetic and genetic programming of primary human T cells.

Area of Science:

• Molecular Biology
• Gene Editing
• Immunotherapy

Background:

• Traditional gene editing using double-strand breaks poses safety risks.
• Precise control of gene expression is crucial for developing advanced cell therapies.

Purpose of the Study:

• To develop a novel all-RNA platform for epigenetic engineering in primary human T cells.
• To achieve stable and multiplexed gene silencing (CRISPRoff) and activation (CRISPRon).

Main Methods:

• Utilized CRISPRoff and CRISPRon epigenetic editors for targeted gene manipulation.
• Demonstrated stable gene silencing through cell divisions, stimulations, and in vivo transfer.
• Combined genetic and epigenetic engineering using CRISPR Cas12a-dCas9 systems.

Main Results:

• Achieved efficient and durable epigenetic programming of endogenous genes without sustained CRISPR expression.
• CRISPRoff-mediated silencing was maintained across cell divisions and in vivo, avoiding cytotoxicity.
• Successfully integrated epigenetic silencing with chimeric antigen receptor (CAR) knock-in for enhanced CAR-T cell therapy.

Conclusions:

• The all-RNA epigenetic platform provides a safe and effective method for programming T cell phenotypes.
• This technology holds significant potential for improving CAR-T cell therapies and other cell-based treatments.
• Epigenetic engineering offers a promising alternative to double-strand break-based gene editing.