A versatile CRISPR/Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cells
Abstract/Contents
- Abstract
- Decades of research in synthetic biology and immunology have endowed us with the blueprints for a new therapeutic modality: the engineered immune cell. Specifically, Chimeric Antigen Receptor (CAR) T cell therapy has proven to be a breakthrough in the treatment of relapsed or refractory hematologic cancers. Furthermore, recent advances in synthetic biology, such as the discovery and development of CRISPR/Cas9 technologies that facilitate precision genome engineering and control of molecular-cellular processes, offer innovative solutions to major challenges that limit the use of CAR T cells in solid tumors and other cancers. However, current CRISPR/Cas9 gene editing tools are limited in their safety, efficacy, and scope. To address these challenges, we present MEGA (Multiplexed Effector Guide Arrays), a versatile and multi-functional platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR/Cas13d. MEGA enables quantitative, reversible, and massively-multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell dysfunction, we demonstrate robust multiplexed suppression of canonical exhaustion-associated genes, perform a first-in-class combinatorial CRISPR/Cas13d screen to uncover paired regulators of T cell proliferation, and utilize these findings to enhance the anti-tumor activity of dysfunctional CAR T cells. We also establish a novel method to link the expression of multiple endogenous genes to the dosage of an FDA-approved drug, which we use to regulate CAR activation strength in a receptor-independent manner. Lastly, we generate MEGA CAR T cells with a diverse range of gene-set perturbations -- with up to 10 genes targeted at once -- and functionally disrupt an entire metabolic pathway to counteract adenosine-driven T cell immunosuppression. Compared to conventional genome editing, MEGA provides a unique synthetic immunology toolkit with applications in cancer immunotherapy, next-generation CAR T cell therapies, and beyond.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2023; ©2023 |
| Publication date | 2023; 2023 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Tieu, Victor |
|---|---|
| Degree supervisor | Qi, Lei, (Professor of Bioengineering) |
| Thesis advisor | Qi, Lei, (Professor of Bioengineering) |
| Thesis advisor | Mackall, Crystal |
| Thesis advisor | Majzner, Robbie |
| Degree committee member | Mackall, Crystal |
| Degree committee member | Majzner, Robbie |
| Associated with | Stanford University, School of Engineering |
| Associated with | Stanford University, Department of Bioengineering |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Victor Tieu. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis Ph.D. Stanford University 2023. |
| Location | https://purl.stanford.edu/my475nn8754 |
Access conditions
- Copyright
- © 2023 by Victor Tieu
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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