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Abstract <p>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 (<i>PD-1</i>) 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 <i>PD-1</i> 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 <i>PD-1</i> 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.</p> Key points <p>•<i>CRISPR/Cas9 is a kind of powerful gene editing system in cancer therapeutics.</i></p> <p>•<i>s-EVs can be applied as a biological carrier for CRISPR/Cas9 system.</i></p> <p>• <i>T cell engineering-based immunotherapy is a promising form of anticancer therapy.</i></p>

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T cell engineering by targeted small extracellular vesicles delivering CRISPR/Cas9 system for PD-1 knock-out

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年8月3日

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  • Mahboube Shahrabi Farahani,
  • Mehdi Shamsara,
  • Seyed Mohammad Moazzeni,
  • Saeed Khalili,
  • Ben Johnson,
  • Leila Darzi,
  • Mohammad Shoae,
  • Mahlegha Ghavami,
  • Abdolah Razi,
  • Elham Hosseini-Beheshti,
  • Mehdi Forouzandeh Moghadam

摘要

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.