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Developing and characterizing a two-layered safety switch for cell therapies
Filippo Rossignoli1,2, Danielle Hoffman1,2, Emaan Atif1,2
1Center for Stem Cell and Translational Immunotherapy (CSTI), Harvard Medical School, Boston, MA, USA.
Abstract:
Gene edited and engineered cell-based therapies are a promising approach for treating a variety of disorders, including cancer. However, the ability of engineered cells to persist for prolonged periods along with possible toxicity raises concerns over the safety of these approaches. Although a number of different one-dimensional suicide systems have been incorporated into therapeutic cell types, the incorporation of a two-layered suicide system that allows controlled killing of therapeutic cells at different time points is needed. In this study, we engineered a variety of therapeutic cells to express two different kill switches, RapaCasp9 and HSV-TK and utilized Rapamycin and Ganciclovir respectively to activate these kill switches. We show that the function of both RapaCasp9 and HSV-TK molecules is preserved and can be activated to induce apoptosis detected early (24 h) and late (48 h) post-activation respectively, with no toxicity. In vivo, we show the eradication of a majority of cells after treatment in subcutaneous and orthotopic models. Furthermore, we demonstrate how both suicide switches work independently and can be activated sequentially for an improved killing, thus ensuring a failsafe mechanism in case the activation of a single one of them is not sufficient to eliminate the cells. Our findings highlight the reliability of the double suicide system, effective on a variety of cells with different biological characteristics, independent of their anatomic presence.
Insights
Engineered cells for therapy can be made safer with a novel double suicide system. This system uses two kill switches, RapaCasp9 and HSV-TK, to control cell elimination and ensure therapeutic safety.
Area of Science:
- Biotechnology
- Cellular Therapy
- Cancer Research
Background:
- Gene-edited cell therapies offer therapeutic potential but raise safety concerns due to cell persistence and toxicity.
- Existing one-dimensional suicide systems lack comprehensive control over therapeutic cell elimination.
Purpose of the Study:
- To engineer a robust two-layered suicide system for enhanced safety and controlled elimination of therapeutic engineered cells.
- To validate the efficacy and reliability of a dual-kill switch system in various cell types and preclinical models.
Main Methods:
- Engineered therapeutic cells to express two distinct suicide genes: RapaCasp9 and HSV-TK.
- Activated suicide systems using specific drugs: Rapamycin for RapaCasp9 and Ganciclovir for HSV-TK.
- Assessed apoptosis induction, cell eradication in vitro and in vivo (subcutaneous and orthotopic models), and sequential activation efficacy.
Main Results:
- Both RapaCasp9 and HSV-TK demonstrated preserved function and induced apoptosis at distinct time points (24h and 48h) post-activation without observed toxicity.
- Significant eradication of therapeutic cells was achieved in vivo across different models.
- Sequential activation of both suicide switches provided improved cell killing, establishing a failsafe mechanism.
Conclusions:
- The developed double suicide system offers a reliable and effective method for controlling engineered cell populations.
- This failsafe mechanism enhances the safety profile of cell-based therapies, applicable across diverse cell types and anatomical locations.
