Improving CAR-T Therapy for Cancer: Using PROTAC to Decrease

Off-Target Effects of CRISPR-Cas9

Abstract (ca. 5 sentences)

The use of synthetic chimeric antigen receptor (CAR) T cells has shown promise in treating various types of cancer,
but their excessive activity can cause severe toxicity. In this project, we aim to address this issue by engineering
CARs with PROTAC, a chemical toolbox for degrading engineered proteins, which can switch off the continuous
activation of Cas9 enzyme in CARs. The goal is to precisely control the timing, location, and dosage of T cell activity,
thereby mitigating toxicity. We will study the effect of PROTAC on the potency of CAR-T cells using a mouse model.
Our findings could pave the way for safer and more effective CAR-T cell therapy.

Key words (5~6)

CAR-T cells, PROTAC, Gene Editing, Safety Switch, Toxicity, Mouse

Specific aims
Design and generate CAR-T cells with a PROTAC safety switch
Evaluate the functionality and safety of these cells in vitro
Assess the impact of PROTAC on the potency and persistence of CAR-T cells in vivo using a mouse model,
Explore the potential clinical translation of PROTAC-based safety switches for CAR-T cell therapy.
 
CRISPR-Cas9 Engineering of T cells in
cancer patients
Cas9WT
Cas9FCPF
Safety concerns of CRISPR based therapy
TWO Specific Aims to address

• To evaluate the possibility of using PROTAC to precisely

control the timing, location, and dosage of T cell activity in
CAR-T cell therapy, thereby mitigating toxicity.
• To investigate the effectiveness of PROTAC in reducing the
toxic side effect due to the off-target effect from CRISPR
gene editing in CARs.
Hypothesis

The use of PROTAC in CAR-T cell therapy can reduce the toxic side effect
due to the off target effect from CRISPR gene editing in CARs.
Expected Outcome & Contingency plans

• Expected Outcome: Reduce off-target effects from CRISPR gene editing

in CARs using PROTAC, thereby reducing autoimmune response.

• Contingency plans: Evaluate the function of T cells using ELISA, cytokine

assay, and proliferation assay, if the expected outcome is not achieved.
Experimental design

Cas9WT
Cas9FCPF
Measuring the potency of CAR-T cells
targeting cancer cells (WT vs PROTAC)

(+10 μM of PROTAC-
FCPF and incubate 8h)
References
• Lee SM, Kang CH, Choi SU, Kim Y, Hwang JY, Jeong HG, Park CH. A
Chemical Switch System to Modulate Chimeric Antigen Receptor T Cell
Activity through Proteolysis-Targeting Chimaera Technology. ACS Synth Biol.
2020 May 15;9(5):987-992. doi: 10.1021/acssynbio.9b00476. Epub 2020 Apr
30. PMID: 32352759.
• Li R, Liu M, Yang Z, Li J, Gao Y, Tan R. Proteolysis-Targeting Chimeras
(PROTACs) in Cancer Therapy: Present and Future. Molecules. 2022 Dec
12;27(24):8828. doi: 10.3390/molecules27248828. PMID: 36557960; PMCID:
PMC9785308.
• Pineda M, Lear A, Collins JP, Kiani S. Safe CRISPR: Challenges and Possible
Solutions. Trends Biotechnol. 2019 Apr;37(4):389-401. doi:
10.1016/j.tibtech.2018.09.010. Epub 2018 Oct 21. PMID: 30352704.
• Dickson I. Improved CAR T therapy for PDAC. Nat Rev Gastroenterol Hepatol.
2021 Jul;18(7):456. doi: 10.1038/s41575-021-00476-8. PMID: 34079103.