Massively parallel knock-in engineering of human T cells

Area of Science:

• Gene editing and cell therapy
• Molecular biology and immunology

Background:

• Targeted gene knock-in for cell therapeutics often suffers from low efficiency and limited scalability.
• Current methods struggle to generate diverse, engineered cell populations for optimizing therapeutic outcomes.

Purpose of the Study:

• To develop a high-efficiency, high-throughput system for targeted gene knock-in engineering.
• To create a platform for pooled generation and selection of optimized CAR-T cell variants for cancer therapy.

Main Methods:

• Developed CLASH (Cas12a/Cpf1 mRNA combined with pooled adeno-associated viruses) for simultaneous gene editing and precise transgene knock-in via massively parallel homology-directed repair.
• Applied CLASH in primary human T cells, including CD3, CD8, and CD4 T cells, across blood cancer and solid tumor models.
• Utilized time-coursed experiments for pooled generation and unbiased selection of favorable CAR-T variants.

Main Results:

• CLASH enables efficient, high-throughput knock-in engineering, producing stably integrated mutant variants.
• A specific CRISPR RNA (crRNA) identified via CLASH induced an exon 3 skip mutant of PRDM1 in CAR-T cells.
• This PRDM1 mutation enhanced CAR-T cell proliferation, stem-like properties, central memory, and longevity, leading to superior in vivo efficacy in multiple cancer models.

Conclusions:

• CLASH is a versatile and powerful technology for engineering diverse cellular and therapeutic applications.
• The identified PRDM1 exon 3 skip mutant demonstrates significant potential for improving CAR-T cell therapy efficacy, particularly in solid tumors.
• This platform facilitates the discovery of beneficial genetic modifications for enhanced cell-based therapeutics.