A highly efficient transgene knock-in technology in clinically relevant cell types

Area of Science:

• CRISPR gene editing
• Cellular engineering
• Gene therapy

Background:

• Inefficient transgene knock-in hinders the development of advanced cell-based medicines.
• Current methods often lack the efficiency and specificity required for clinical applications.

Purpose of the Study:

• To develop a novel CRISPR-based technology for highly efficient and precise transgene knock-in.
• To enable the integration of therapeutic genes into essential gene loci without compromising cell function.

Main Methods:

• Utilized a CRISPR nuclease targeting an essential gene exon for precise genomic integration.
• Designed a cargo template ensuring retained essential gene function post-knock-in.
• Implemented a negative selection strategy for cells with non-productive insertions/deletions.

Main Results:

• Achieved over 90% knock-in efficiency in clinically relevant cell types using SeLection by Essential-gene Exon Knock-in (SLEEK).
• Demonstrated SLEEK's superior efficiency compared to AAV6-mediated TRAC knock-in in T cells, even with non-viral DNA.
• Engineered natural killer cells with SLEEK knock-in of CD16 and mbIL-15, showing enhanced tumor killing and in vivo persistence.

Conclusions:

• SLEEK technology significantly improves transgene knock-in efficiency and reliability for cell therapy development.
• This method maintains cell viability and expansion capacity, crucial for therapeutic applications.
• SLEEK offers a promising platform for engineering enhanced cell-based therapies, including improved cancer treatments.