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通过由尤因肉瘤特异性GGAA启动子驱动的CRISPR/Cas9对尤因肉瘤中的EWSR1::FLI1基因进行靶向失活。

Targeted inactivation of EWSR1 : : FLI1 gene in Ewing sarcoma via CRISPR/Cas9 driven by an Ewing-specific GGAA promoter.

作者信息

Cervera Saint T, Martínez Selene, Iranzo-Martínez María, Notario Laura, Melero-Fernández de Mera Raquel M, Alonso Javier

机构信息

Unidad de Tumores Sólidos Infantiles. Instituto de Investigación de Enfermedades Raras. Instituto de Salud Carlos III. Majadahonda, Madrid, Spain.

Centro de Investigación Biomédica en Red de Enfermedades Raras. Instituto de Salud Carlos III (CB06/07/1009; CIBERER-ISCIII), Madrid, Spain.

出版信息

Cancer Gene Ther. 2025 Apr;32(4):437-449. doi: 10.1038/s41417-025-00887-8. Epub 2025 Mar 15.

DOI:10.1038/s41417-025-00887-8
PMID:40089636
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11976297/
Abstract

We have recently demonstrated that genetic inactivation of EWSR1 : : FLI1 by CRISPR/Cas9, successfully blocks cell proliferation in a cell model of Ewing sarcoma. However, CRISPR/Cas9-mediated gene editing can exhibit off-target effects, and thus, precise regulation of Cas9 expression in target cells is essential to develop gene-editing strategies to inactivate EWSR1 : : FLI1 in Ewing sarcoma cells. In this study, we demonstrate that Cas9 can be specifically expressed in Ewing sarcoma cells when located downstream a promoter consisting of GGAA repeats and a consensus TATA box (GGAAprom). Under these conditions, Cas9 is selectively expressed in Ewing sarcoma cells that express EWSR1 : : FLI1 oncoproteins, but not in cells expressing wild-type FLI1. Consequently, Ewing sarcoma cells infected with GGAAprom>Cas9 and a specific gRNA designed to inactivate EWSR1 : : FLI1, showed successful EWSR1 : : FLI1 inactivation and the subsequent blockade of cell proliferation. Notably, GGAAprom>Cas9 can be efficiently delivered to Ewing sarcoma cells via adenoviral vectors both in vitro and in vivo, highlighting the potential of this approach for Ewing sarcoma treatment. Our results demonstrate that the CRISPR/Cas9 machinery is safe and specific for Ewing sarcoma cells when driven under a GGAAprom, paving the way for the development of cancer gene therapies based on the selective expression of genes with therapeutic potential.

摘要

我们最近证明，通过CRISPR/Cas9对EWSR1::FLI1进行基因失活，可在尤因肉瘤细胞模型中成功阻断细胞增殖。然而，CRISPR/Cas9介导的基因编辑可能会出现脱靶效应，因此，精确调控靶细胞中Cas9的表达对于开发使尤因肉瘤细胞中EWSR1::FLI1失活的基因编辑策略至关重要。在本研究中，我们证明，当Cas9位于由GGAA重复序列和共有TATA框（GGAAprom）组成的启动子下游时，它可以在尤因肉瘤细胞中特异性表达。在这些条件下，Cas9在表达EWSR1::FLI1癌蛋白的尤因肉瘤细胞中选择性表达，但在表达野生型FLI1的细胞中不表达。因此，感染了GGAAprom>Cas9和设计用于使EWSR1::FLI1失活的特异性gRNA的尤因肉瘤细胞，显示出EWSR1::FLI1成功失活以及随后细胞增殖的阻断。值得注意的是，GGAAprom>Cas9可以通过腺病毒载体在体外和体内有效地递送至尤因肉瘤细胞，突出了这种方法在尤因肉瘤治疗中的潜力。我们的结果表明，当在GGAAprom驱动下时，CRISPR/Cas9机制对尤因肉瘤细胞是安全且特异的，为基于具有治疗潜力的基因选择性表达的癌症基因治疗的发展铺平了道路。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bd8/11976297/6c9ac1f0f8f0/41417_2025_887_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bd8/11976297/dc0ed23dfd51/41417_2025_887_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bd8/11976297/b847496a0aef/41417_2025_887_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bd8/11976297/2f525315070a/41417_2025_887_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bd8/11976297/87682a9319a9/41417_2025_887_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bd8/11976297/591d4b94c6ed/41417_2025_887_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bd8/11976297/6c9ac1f0f8f0/41417_2025_887_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bd8/11976297/dc0ed23dfd51/41417_2025_887_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bd8/11976297/b847496a0aef/41417_2025_887_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bd8/11976297/2f525315070a/41417_2025_887_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bd8/11976297/87682a9319a9/41417_2025_887_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bd8/11976297/591d4b94c6ed/41417_2025_887_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bd8/11976297/6c9ac1f0f8f0/41417_2025_887_Fig6_HTML.jpg

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