# Decoding and reprogramming T cells through synthetic biology for cancer immunotherapy

> **NIH NIH R01** · J. DAVID GLADSTONE INSTITUTES · 2023 · $771,499

## Abstract

ABSTRACT
Engineered T cell-based cancer therapies are a major advancement in cancer treatment; however the majority
of cancers still do not respond to adoptive cellular therapy. We need to “design” new T cell therapies with
increased potency, and we need to overcome cell dysfunction that occurs as T cells face chronic tumor antigen
stimulation. We and others have screened for genes that can be “knocked out” in antigen-specific T cells to
enhance their functions, but enormous opportunities still remain to “knock-in” new synthetic DNA sequences at
targeted genome sites. This proposal is focused on detailed evaluation of genes and inducible gene programs
that will enable next-generation cellular therapies for cancer. We have developed several complementary
technologies to discover synthetic gene programs that can be “inserted” into T cell genomes to enhance
therapeutic functions. We developed a CRISPR technology for high throughput pooled knock-ins to
accelerate discovery of synthetic knock-in programs (Roth et al., Cell, 2020), and have now have conducted
two screens with ~100-member libraries that include transcription factors and synthetic chimeric
receptors (“switch receptors”) to discover programs that make chronically stimulated T cells resistant to
dysfunction. In addition, we have optimized a complementary robust platform for genome-wide CRISPR
activation (CRISPRa) gain-of-function forward genetic screens in human T cells, and have already completed
systematic discovery of factors that regulate stimulation-dependent cytokine production (Schmidt and
Steinhart et al., Science, 2022). We propose to translate insights from these high-throughput discovery
efforts into preclinical testing of novel knock-in designs with screen hits in vivo using xenotransplanted mouse
models. In this proposal, we will test validated candidates from gain-of-function CRISPR PoKI (Aim 1) and
CRISPRa (Aim 2) screens to discover new components of knock-in constructs that improve cell-based T cell
therapies. We also recognize that these genetic components may be more beneficial if they are not expressed
constitutively. In Aim 3, we draw on the power of synthetic biology to engineer synthetic circuits that can
induce or repress genetic programs in response to antigen stimulation. This precise and dynamic
regulation of genetic elements has great potential to further enhance efficacy and safety of next-generation
immune cell therapies. Taken together, we present a proposal that leverages recent discoveries from
CRISPR discovery platforms and deep expertise in synthetic biology to engineer powerful “knock-in” circuits
that we will validate and study in preclinical cancer models. We leverage functional genomics, CRISPR
engineering and synthetic cell program design expertise to address insufficient T cell potency and T cell
dysfunction, which remain significant barriers to adoptive cell therapy for cancer.

## Key facts

- **NIH application ID:** 10568704
- **Project number:** 1R01CA276368-01
- **Recipient organization:** J. DAVID GLADSTONE INSTITUTES
- **Principal Investigator:** Alexander Marson
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $771,499
- **Award type:** 1
- **Project period:** 2023-01-01 → 2027-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10568704

## Citation

> US National Institutes of Health, RePORTER application 10568704, Decoding and reprogramming T cells through synthetic biology for cancer immunotherapy (1R01CA276368-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10568704. Licensed CC0.

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