Synthetic biology approaches to engineer T cells

Wu, Chia-Yung; Rupp, Levi J.; Roybal, Kole T.; Lim, Wendell A.

doi:10.1016/j.coi.2015.06.015

Cited by 35 publications

(24 citation statements)

References 50 publications

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“…The use of our method is not restricted only to these genes, and we feel that the synthetic biology field will benefit from this application. Control of biosynthetic pathways for production of useful secondary metabolites49, antibiotics50 or recombinant antibodies51, as well as introduction of controllable retrosynthetic and fully engineered pathways52 or ultimate control of metabolic pathways in the modelling of diseases are just a few among the multiple possible applications of this method in the future.…”

Section: Discussionmentioning

confidence: 99%

Rapid generation of hypomorphic mutations

et al. 2017

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Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.

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Section: Discussionmentioning

confidence: 99%

Rapid generation of hypomorphic mutations

et al. 2017

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“…To completely eliminate this risk, the nanoparticle-delivered CAR transgenes could be expressed under the control of a T-cell-specific promoter 23,24 . Compared with the ubiquitous EF-1 alpha promoter we chose for our studies, cell-specific promoters have a weaker transcriptional activity—but thanks to the emergence of adoptive T-cell therapy, improved vector systems that enable tighter control of gene expression in T cells are in development 25 .…”

Section: Discussionmentioning

confidence: 99%

In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers

et al. 2017

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An emerging approach for treating cancer involves programming patient-derived T cells with genes encoding disease-specific chimeric antigen receptors (CARs), so that they can combat tumour cells once they are reinfused. Although trials of this therapy have produced impressive results, the in vitro methods they require to generate large numbers of tumour-specific T cells are too elaborate for widespread application to treat cancer patients. Here, we describe a method to quickly program circulating T cells with tumour-recognizing capabilities, thus avoiding these complications. Specifically, we demonstrate that DNA-carrying nanoparticles can efficiently introduce leukaemia-targeting CAR genes into T-cell nuclei, thereby bringing about long-term disease remission. These polymer nanoparticles are easy to manufacture in a stable form, which simplifies storage and reduces cost. Our technology may therefore provide a practical, broadly applicable treatment that can generate anti-tumour immunity ‘on demand’ for oncologists in a variety of settings.

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“…Genome modification of the cellular product can repair genetic defects before subsequent autologous transplantation and allows equipping the cell with designer features to increase safety and efficacy (33), (34). Thus, protocols to efficiently introduce precise and targeted genetic modifications in hematopoietic cells, particularly T cells, are key to the success of adoptive T cell therapy.…”

Section: Discussionmentioning

confidence: 99%

Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells

Kornete

Marone

Jeker

2018

The Journal of Immunology

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Adoptive cell transfer (ACT) is an important approach for basic research and emerges as an effective treatment for various diseases including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. Here, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms (SNP) in up to 25% of the transfected primary T cells. We used gene editing-mediated allele switching to quantify homology directed repair (HDR), systematically optimize experimental parameters and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Both repairing the cause of the scurfy syndrome, a 2bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells.

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Synthetic biology approaches to engineer T cells

Cited by 35 publications

References 50 publications

Rapid generation of hypomorphic mutations

Rapid generation of hypomorphic mutations

In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers

Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells

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