Non-viral targeted integration at the CISH locus enables CAR-NK cell engineering with enhanced anti-tumor activity
Jiao Wang1, Yao Sun2,3, Jakob Starzyk1
1Full Circles Therapeutics, Inc. 625 Mt. Auburn St., Ste. 105, Cambridge, MA 02138, USA.
Abstract:
Natural killer (NK) cells hold promise for adoptive cell therapy due to their innate cytotoxicity. Early clinical trials confirm their safety and efficacy in cancer and autoimmune disease treatment. Engineering NK cells with chimeric antigen receptors (CARs) enhances target specificity and facilitates their development as off-the-shelf allogeneic therapies. However, both viral and non-viral engineering methods of NK cells present challenges. Here, we introduce CISH locus integrated CAR killer (CLICK), a novel non-viral approach using a mini-circular single-stranded DNA genome editing system. CLICK enables efficient integration of CD19CAR sequences into the metabolic checkpoint CISH locus, simultaneously disrupting CISH and driving stable, progressively increasing CAR expression in peripheral blood-derived NK cells. CLICK-engineered CAR-NK cells exhibit potent cytotoxicity, enhanced anti-tumor activity ex vivo and in vivo, extended persistence, and reduced exhaustion. Together, these findings highlight CLICK as a highly efficient and versatile platform for non-viral CAR-NK cell engineering, offering a scalable approach for next-generation allogeneic immune cell therapies.
Insights
A new non-viral method, CLICK, efficiently engineers Natural Killer (NK) cells with chimeric antigen receptors (CARs). This approach enhances NK cell anti-tumor activity and persistence, paving the way for advanced cell therapies.
Area of Science:
- Immunology
- Cell Therapy
- Genetic Engineering
Background:
- Natural Killer (NK) cells are crucial for innate immunity and show promise in adoptive cell therapy for cancer and autoimmune diseases.
- Engineering NK cells with chimeric antigen receptors (CARs) improves target specificity and enables allogeneic 'off-the-shelf' therapies.
- Current viral and non-viral methods for engineering NK cells face significant challenges.
Purpose of the Study:
- To introduce a novel non-viral method for engineering NK cells using a mini-circular single-stranded DNA genome editing system.
- To demonstrate the efficiency of integrating CAR sequences into the CISH locus for stable CAR expression.
- To evaluate the therapeutic potential of engineered NK cells.
Main Methods:
- Developed a non-viral approach named CISH locus integrated CAR killer (CLICK).
- Utilized a mini-circular single-stranded DNA genome editing system for gene integration.
- Engineered peripheral blood-derived NK cells by integrating CD19CAR sequences into the CISH locus.
Main Results:
- CLICK enabled efficient integration of CD19CAR sequences into the CISH locus, disrupting CISH and driving stable CAR expression.
- CLICK-engineered CAR-NK cells displayed potent cytotoxicity and enhanced anti-tumor activity both ex vivo and in vivo.
- These engineered NK cells showed extended persistence and reduced exhaustion compared to controls.
Conclusions:
- CLICK is a highly efficient and versatile platform for non-viral CAR-NK cell engineering.
- This approach offers a scalable strategy for developing next-generation allogeneic immune cell therapies.
- CLICK overcomes challenges associated with viral gene delivery and enhances NK cell-based cancer immunotherapy.
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