synNotch CAR-T cells

Stages

1. 1.
   Engineering of FUS-inducible CRISPR toolbox(library_design)

   This stage establishes the core inducible CRISPR systems needed for downstream functional and therapeutic testing.

   Selection: Creation of inducible CRISPR-based tools controllable by focused ultrasound.

2. 2.
   Functional demonstration of genome and epigenome modulation(functional_characterization)

   This stage verifies that the engineered ultrasound-inducible tools perform the intended regulatory functions before therapeutic deployment.

   Selection: Demonstration of FUS-inducible CRISPR, CRISPRa, and CRISPRee capabilities in modulating the genome and epigenome.

3. 3.
   Tumour priming by FUS-CRISPR telomere disruption(secondary_characterization)

   This stage tests whether the genomic intervention creates a therapeutically useful tumour state for downstream cell therapy.

   Selection: Assessment of whether FUS-CRISPR-mediated telomere disruption primes solid tumours for CAR-T therapy.

4. 4.
   In vivo AAV delivery and FUS-triggered training-center activation(in_vivo_validation)

   This stage validates that the inducible CRISPR system can be delivered in vivo and used to create localized tumour-cell training centers for downstream immunotherapy.

   Selection: In vivo delivery of FUS-CRISPR using AAVs followed by FUS-induced telomere disruption and induced antigen expression in a tumour-cell subpopulation.

Steps

1. 1.
   Engineer inducible CRISPR-based tools controllable by focused ultrasoundengineered system

   Create CRISPR-based tools that can be activated noninvasively by focused ultrasound.

   The inducible toolbox must be built before its genome, epigenome, and therapeutic functions can be tested.

2. 2.
   Demonstrate genome and epigenome modulation by FUS-inducible CRISPR systemsengineered system under test

   Verify that the ultrasound-inducible CRISPR toolbox can modulate genomic and epigenomic states.

   Functional capability is demonstrated after engineering and before therapeutic application to establish that the toolbox works as intended.

3. 3.
   Apply FUS-CRISPR-mediated telomere disruption to prime solid tumours for CAR-T therapytherapeutic genomic intervention

   Test whether localized telomere disruption creates a tumour state more amenable to CAR-T therapy.

   After establishing core CRISPR functionality, the authors test a specific therapeutic mechanism relevant to cancer immunotherapy.

4. 4.
   Deliver FUS-CRISPR in vivo using AAVsdelivered inducible CRISPR system and delivery harness

   Deploy the FUS-CRISPR system in vivo for localized tumour reprogramming.

   In vivo delivery is required before ultrasound-triggered tumour-cell reprogramming and downstream synNotch CAR-T activation can occur.

5. 5.
   Use focused ultrasound to induce telomere disruption and antigen expression in a tumour-cell subpopulationinducible tumour-cell reprogramming system

   Generate localized tumour-cell training centers that can activate synNotch CAR-T cells.

   This follows in vivo delivery because the tumour cells must first contain the inducible CRISPR system before FUS can trigger localized reprogramming.

6. 6.
   Activate synNotch CAR-T cells to produce CARs against a universal tumour antigen and kill neighboring tumour cellscell therapy responder

   Translate localized training-center induction into broader tumour-cell killing.

   synNotch CAR-T activation depends on prior creation of tumour-cell training centers expressing the induced antigen.