# SYNTHETIC GENE CIRCUITS FOR MONITORING T-CELL EXHAUSTION

> **NIH NIH F30** · STANFORD UNIVERSITY · 2020 · $32,501

## Abstract

Chimeric antigen receptor (CAR) T-cell therapy has emerged as a promising cancer immunotherapy, but many
patients still fail to respond to therapy. T-cell exhaustion is hypothesized to be a major driver of therapy failure,
and strategies to non-invasively monitor this phenotype could (1) provide insight into the kinetics of its
emergence and persistence (2) be used for early prediction of therapy failure and (3) predict response to
interventions that reverse T-cell exhaustion. Existing strategies for blood-based monitoring can give a measure
of T-cell expansion and persistence, but exhaustion often emerges at the site(s) of disease and could vary
from site to site suggesting that these strategies are unlikely to provide accurate phenotypic information.
Here we propose using positron emission tomography (PET) reporter genes in combination with a synthetic
gene circuit for monitoring of CAR T-cell viability, location, and exhaustion in vivo. The PET reporter genes
herpes simplex virus type 1 thymidine kinase (HSV1-tk) and dopamine type 2 receptor (D2R) are useful tools
for imaging cell state since their expression can be non-invasively, repeatedly, and independently monitored by
PET upon administration of their cognate radiolabeled substrates [18F]FHBG and [18F]FESP. We thus propose
engineering CAR T-cells with both a constitutively expressed PET reporter gene (D2R) for monitoring of
viability and location and a second PET reporter gene (HSV1-tk) whose expression is linked to T-cell
exhaustion. Since there exists no single marker of T-cell exhaustion, we present a two-input gene circuit using
the Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system to express
HSV1-tk only if PD-1 is expressed and IL-2 expression is lost, a combination unique to T-cell exhaustion.
In Aim 1, we construct a doxycycline- and cumate-controlled CRISPRi circuit to test candidate guide RNAs for
efficient CRISPRi. In Aim 2, we replace the chemically inducible promoters with promoters for PD-1 and IL-2
and assess the circuit’s ability to mirror the exhaustion phenotype of mouse GD2-targeting CAR T-cells in vitro
using radiotracer cellular uptake studies. Thereafter we will perform repeated PET imaging of T-cell location
and exhaustion in mouse models of GD2+ osteosarcoma and melanoma to better understand the
spatiotemporal kinetics of exhaustion. In Aim 3, we assess whether monitoring of T-cell exhaustion enables
early prediction of therapy failure and/or predicts response to checkpoint blockade with anti-PD-1 antibody.
The training plan will provide the applicant technical skills in PET reporter genes and molecular imaging, multi-
input gene circuits, and immunotherapy models as well as professional skills in oral and written communication
to facilitate growth as an independent investigator. Training will take place in Stanford University’s highly
collaborative and well-resourced research environment. The applicant will be mentored primari...

## Key facts

- **NIH application ID:** 9993108
- **Project number:** 5F30CA236339-02
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Amin Aalipour
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $32,501
- **Award type:** 5
- **Project period:** 2019-09-02 → 2021-06-13

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9993108

## Citation

> US National Institutes of Health, RePORTER application 9993108, SYNTHETIC GENE CIRCUITS FOR MONITORING T-CELL EXHAUSTION (5F30CA236339-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/9993108. Licensed CC0.

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