Novel extragenic genomic safe harbors for precise therapeutic T-cell engineering

Area of Science:

• Immunology
• Gene Therapy
• Bioinformatics

Background:

• Current genetic engineering methods for cell therapies, including CAR therapy, often lead to unpredictable transgene expression and carry risks of insertional mutagenesis.
• Variegated transgene expression in CAR therapy can cause tonic signaling, T-cell exhaustion, and variable persistence, limiting therapeutic efficacy.
• There is a need for strategies that ensure uniform and controlled transgene expression for enhanced T-cell function and predictable patient outcomes.

Purpose of the Study:

• To develop and validate an algorithm for identifying extragenic genomic safe harbors (GSHs) for precise DNA integration.
• To enable sustained and predictable transgene expression, specifically for chimeric antigen receptor (CAR) therapy, in human peripheral blood T cells.
• To minimize genotoxicity and avoid transgene silencing through targeted integration.

Main Methods:

• Developed a 7-criterion algorithm to identify extragenic genomic safe harbors (GSHs).
• Utilized CRISPR/Cas9 for efficient and targeted DNA integration into identified GSHs.
• Engineered human peripheral blood T cells to express a CD19 CAR at a validated GSH (GSH6) and the TRAC locus for comparison.

Main Results:

• The algorithm successfully identified functional extragenic GSHs capable of supporting sustained and predictable CAR expression.
• T cells engineered with a CD19 CAR at GSH6 demonstrated curative potential at low cell doses in a mouse model of acute lymphoblastic leukemia.
• GSH6-engineered CAR T cells matched the potency of TRAC locus-engineered cells and resisted tumor rechallenge 100 days post-infusion.

Conclusions:

• The developed algorithm provides a method for identifying genomic safe harbors that enable precise and safe gene integration.
• Functional extragenic GSHs expand the available genomic landscape for therapeutic precision engineering in cell therapies.
• This approach offers a promising strategy for improving the safety and efficacy of CAR T-cell therapies by ensuring controlled transgene expression.