T cell engineering by targeted small extracellular vesicles delivering CRISPR/Cas9 system for PD-1 knock-out.
7 affiliations
- 1Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, I.R, Jalal Ale Ahmad Highway, P.O. Box 14115-331, Tehran, Iran.
- 2Department of Animal Biotechnology, Faculty of Agriculture Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
- 3Department of Medical Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
- 4Asbestos and Dust Diseases Research Institute (ADDRI), Sydney, NSW, Australia.
- 5BC Children's Hospital Research Institute, Vancouver, British Columbia, Canada.
- 6Department of Urology, Faculty of Medicine, North Khorasan University of Medical Sciences, Bojnord, Iran.
- 7Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, I.R, Jalal Ale Ahmad Highway, P.O. Box 14115-331, Tehran, Iran. [email protected].
Abstract
Among the emerging cancer therapies, immunotherapy using T cell engineering stands out as one of the most promising approaches. While the clustered regularly interspaced palindromic repeats (CRISPR/Cas) system is widely recognized as one of the most common gene-editing system tools, the main challenge for in vivo delivery applications remains the lack of an efficient and safe intracellular carrier. Small extracellular vesicles (s-EVs), as natural nanoparticles with low immunogenicity, emerge as a promising candidate for delivering the CRISPR/Cas system. Two guide RNAs (gRNAs) targeting the programmed cell death (PD-1) gene were cloned into the PX-459 vector, and these CRISPR/Cas9 plasmids were loaded into targeted s-EVs by electroporation. Subsequently, T cells were exposed to these s-EVs, and the disruption to the PD-1 gene was examined using flow cytometry and sequencing. Flow cytometry results indicated that newly produced targeted s-EVs exhibited a higher uptake by T cells compared to non-targeted s-EVs. The successful loading of PX-459 plasmids into targeted s-EVs and their functional delivery to T cells were confirmed. Subsequent carboxyfluorescein succinimidyl ester (CFSE) staining and enzyme-linked immunosorbent assay (ELISA) analyses demonstrated that the knockout (KO) of the PD-1 gene in T cells significantly enhanced both their cytotoxicity and cytokine release compared to untreated T cells. Targeted s-EVs were used in the current study as a means to deliver the CRISPR/Cas9 system into T cells, enabling genetic modification of target cells. This approach holds the potential to improve and simplify immunotherapy. This study demonstrates a novel approach to genetic modification in T cells using targeted s-EVs containing the CRISPR/Cas9 gene-editing system. KEY POINTS: •CRISPR/Cas9 is a kind of powerful gene editing system in cancer therapeutics. •s-EVs can be applied as a biological carrier for CRISPR/Cas9 system. • T cell engineering-based immunotherapy is a promising form of anticancer therapy.
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BibTeX
@article{farahani2026cell,
title = {{T cell engineering by targeted small extracellular vesicles delivering CRISPR/Cas9 system for PD-1 knock-out.}},
author = {Mahboube Shahrabi Farahani and Mehdi Shamsara and Seyed Mohammad Moazzeni and Saeed Khalili and Ben Johnson and Leila Darzi and Mohammad Shoae and Mahlegha Ghavami and Abdolah Razi and Elham Hosseini-Beheshti and Mehdi Forouzandeh Moghadam},
journal = {Applied microbiology and biotechnology},
year = {2026},
month = aug,
doi = {10.1007/s00253-026-13976-x},
url = {https://europepmc.org/article/MED/42957818},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - T cell engineering by targeted small extracellular vesicles delivering CRISPR/Cas9 system for PD-1 knock-out.
AU - Mahboube Shahrabi Farahani
AU - Mehdi Shamsara
AU - Seyed Mohammad Moazzeni
AU - Saeed Khalili
AU - Ben Johnson
AU - Leila Darzi
AU - Mohammad Shoae
AU - Mahlegha Ghavami
AU - Abdolah Razi
AU - Elham Hosseini-Beheshti
AU - Mehdi Forouzandeh Moghadam
PY - 2026
DA - 2026/08/03
JO - Applied microbiology and biotechnology
DO - 10.1007/s00253-026-13976-x
UR - https://europepmc.org/article/MED/42957818
AB - Among the emerging cancer therapies, immunotherapy using T cell engineering stands out as one of the most promising approaches. While the clustered regularly interspaced palindromic repeats (CRISPR/Cas) system is widely recognized as one of the most common gene-editing system tools, the main challenge for in vivo delivery applications remains the lack of an efficient and safe intracellular carrier. Small extracellular vesicles (s-EVs), as natural nanoparticles with low immunogenicity, emerge as a promising candidate for delivering the CRISPR/Cas system. Two guide RNAs (gRNAs) targeting the programmed cell death (PD-1) gene were cloned into the PX-459 vector, and these CRISPR/Cas9 plasmids were loaded into targeted s-EVs by electroporation. Subsequently, T cells were exposed to these s-EVs, and the disruption to the PD-1 gene was examined using flow cytometry and sequencing. Flow cytometry results indicated that newly produced targeted s-EVs exhibited a higher uptake by T cells compared to non-targeted s-EVs. The successful loading of PX-459 plasmids into targeted s-EVs and their functional delivery to T cells were confirmed. Subsequent carboxyfluorescein succinimidyl ester (CFSE) staining and enzyme-linked immunosorbent assay (ELISA) analyses demonstrated that the knockout (KO) of the PD-1 gene in T cells significantly enhanced both their cytotoxicity and cytokine release compared to untreated T cells. Targeted s-EVs were used in the current study as a means to deliver the CRISPR/Cas9 system into T cells, enabling genetic modification of target cells. This approach holds the potential to improve and simplify immunotherapy. This study demonstrates a novel approach to genetic modification in T cells using targeted s-EVs containing the CRISPR/Cas9 gene-editing system. KEY POINTS: •CRISPR/Cas9 is a kind of powerful gene editing system in cancer therapeutics. •s-EVs can be applied as a biological carrier for CRISPR/Cas9 system. • T cell engineering-based immunotherapy is a promising form of anticancer therapy.
SN - 0175-7598
SN - 1432-0614
ER -