engineered GSDME with customized protease recognition sequence

Stages

1. 1.
   Design of rewired inducible pyroptosis circuit(library_design)

   This stage creates a GSDME effector that can be activated on demand by an engineered, drug-regulated protease rather than by its native proteolytic trigger.

   Selection: Replace the native GSDME activation motif with a customized protease recognition sequence and pair it with inducible protease variants regulated by a small molecule.

2. 2.
   Organoid functional characterization(functional_characterization)

   This stage tests whether the engineered control logic produces the intended pyroptotic mechanism in a disease-relevant ex vivo model before in vivo testing.

   Selection: Assess whether inducer administration produces rapid GSDME cleavage, pore formation, and cell lysis in patient-derived organoids.

3. 3.
   Xenograft confirmatory validation(in_vivo_validation)

   This stage evaluates whether externally controlled pyroptosis can translate into tumor growth inhibition in vivo using oral drug administration.

   Selection: Test whether oral treatment with the approved drug suppresses tumor growth in a xenograft model.

Steps

1. 1.
   Replace native GSDME activation motif with a customized protease recognition sequenceengineered effector construct

   Rewire GSDME so its activation can be controlled by a chosen protease input.

   The effector must first be made responsive to an orthogonal protease before small-molecule control can be imposed through inducible protease variants.

2. 2.
   Engineer small-molecule-regulated inducible protease variantscontrol module

   Create a drug-gated trigger that activates the rewired GSDME construct on demand.

   After rewiring the GSDME cleavage site, a matching inducible protease is needed to provide external temporal control using the approved small molecule.

3. 3.
   Administer inducer in patient-derived organoid models and assess pyroptosis outputsengineered system under test

   Determine whether the inducible circuit produces the intended mechanistic outputs of pyroptosis in a disease-relevant model.

   Organoid testing provides functional evidence of GSDME cleavage, pore formation, and cell lysis before moving to in vivo tumor studies.

4. 4.
   Treat xenograft-bearing animals orally with the approved drug and measure tumor growth responseengineered system under in vivo validation

   Test whether oral drug induction of the circuit yields tumor suppression in vivo.

   In vivo validation follows organoid mechanistic success to determine whether the externally controlled pyroptosis strategy translates into tumor growth inhibition.