# A Cas13d-based screening approach to engineer exhaustion-resistant CAR T cells

> **NIH NIH R21** · STANFORD UNIVERSITY · 2022 · $182,863

## Abstract

ABSTRACT
Chimeric Antigen Receptor (CAR) T cell therapy has proven to be a breakthrough treatment with curative
potential in hematologic cancer patients as well as in aggressive preclinical models. However, recent studies
have shed light on major barriers to progress – many patients that initially respond completely to CAR T cell
therapy eventually relapse, and CAR T cells have demonstrated limited clinical efficacy in the treatment of solid
tumors. A key phenomenon that has been causally implicated in these failure modes is CAR T cell exhaustion,
where tonically-signaling CAR T cells are driven to a distinct and dysfunctional phenotype with restrained
antitumor activity. Previous genome-wide perturbation studies using CRISPR-Cas9 have identified a growing list
of single gene targets that, when knocked out, help mitigate exhaustion and modestly improve T cell function.
However, the resulting individual gene hits from these screens are often context-dependent and disparate across
different tumor and CAR models. Altogether, these studies indicate that the exhausted T cell phenotype is largely
driven by the upregulation of key gene programs rather than single genes, though the complex network of these
genetic interactions remains poorly defined.
To address these unmet needs, we propose a synthetic biology-driven approach using CRISPR-Cas13d
transcriptome engineering to develop potent, exhaustion-resistant CAR T cells. Cas13d is a small CRISPR RNA-
guided RNA endonuclease that can process a single guide RNA array to degrade multiple distinct target RNA
transcripts in a highly sequence-specific and robust manner. In AIM 1, we will develop a novel platform using
Cas13d to simultaneously downregulate multiple endogenous genes in primary human T cells, with a specific
focus on improving exhausted CAR T cell effector function. In AIM 2, we will use this technology to conduct a
double knockdown proliferation screen in exhausted CAR T cells targeting pairs of putative negative regulators
of T cell antitumor activity. We will utilize an established computational framework to identify highly enriched
gene pairings, to map genetic interactions (GI) between genes, and to define a network of exhaustion. We
hypothesize that our multimodal screening methodology can be used to identify new synergistic gene pairings
that outperform single knockdown phenotypes seen in prior studies.
Our proposed project will establish a new platform for multiplexed gene repression and screening in primary
human T cells that overcomes limitations faced by state-of-the-art CRISPR-Cas9 and RNAi technologies.
Furthermore, our studies will demonstrate a novel strategy to mitigate CAR T cell exhaustion, which will improve
upon current immune therapies and enhance their effectiveness. Ultimately, our work will address clinical unmet
needs as well as help the broader scientific community 1) better understand the complex network of T cell
exhaustion and 2) use this data to inform the developm...

## Key facts

- **NIH application ID:** 10431227
- **Project number:** 1R21CA270609-01
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Lei Stanley Qi
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $182,863
- **Award type:** 1
- **Project period:** 2022-03-01 → 2024-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10431227

## Citation

> US National Institutes of Health, RePORTER application 10431227, A Cas13d-based screening approach to engineer exhaustion-resistant CAR T cells (1R21CA270609-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10431227. Licensed CC0.

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