1. Academic Validation
  2. Engineered Extracellular Vesicle-Delivered CRISPR/Cas9 for Radiotherapy Sensitization of Glioblastoma

Engineered Extracellular Vesicle-Delivered CRISPR/Cas9 for Radiotherapy Sensitization of Glioblastoma

  • ACS Nano. 2023 Aug 30. doi: 10.1021/acsnano.2c12857.
Xiao Liu 1 2 Zhengcong Cao 1 Weizhong Wang 2 Cheng Zou 1 Yingwen Wang 1 Luxiang Pan 1 Bo Jia 2 Kuo Zhang 1 Wangqian Zhang 1 Weina Li 1 Qiang Hao 1 Yingqi Zhang 1 Wei Zhang 1 Xiaochang Xue 3 Wei Lin 2 Meng Li 1 Jintao Gu 1
Affiliations

Affiliations

  • 1 State Key Laboratory of Cancer Biology, Biotechnology Center, School of Pharmacy, The Fourth Military Medical University, Xi'an, 710000, China.
  • 2 Department of Neurosurgery, Xijing Hospital, Xi'an, 710000, China.
  • 3 The Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, The Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi'an, 710000, China.
Abstract

Radiotherapy is a mainstay of glioblastoma (GBM) treatment; however, the development of therapeutic resistance has hampered the efficacy of radiotherapy, suggesting that additional treatment strategies are needed. Here, an in vivo loss-of-function genome-wide CRISPR screen was carried out in orthotopic tumors in mice subjected to radiation treatment to identify synthetic lethal genes associated with radiotherapy. Using functional screening and transcriptome analyses, glutathione synthetase (GSS) was found to be a potential regulator of radioresistance through Ferroptosis. High GSS levels were closely related to poor prognosis and relapse in patients with glioma. Mechanistic studies demonstrated that GSS was associated with the suppression of radiotherapy-induced Ferroptosis in glioma cells. The depletion of GSS resulted in the disruption of glutathione (GSH) synthesis, thereby causing the inactivation of GPX4 and iron accumulation, thus enhancing the induction of Ferroptosis upon radiotherapy treatment. Moreover, to overcome the obstacles to broad therapeutic translation of CRISPR editing, we report a previously unidentified genome editing delivery system, in which Cas9 protein/sgRNA complex was loaded into Angiopep-2 (Ang) and the trans-activator of the transcription (TAT) peptide dual-modified extracellular vesicle (EV), which not only targeted the blood-brain barrier (BBB) and GBM but also permeated the BBB and penetrated the tumor. Our encapsulating EVs showed encouraging signs of GBM tissue targeting, which resulted in high GSS gene editing efficiency in GBM (up to 67.2%) with negligible off-target gene editing. These results demonstrate that a combination of unbiased genetic screens, and CRISPR-Cas9-based gene therapy is feasible for identifying potential synthetic lethal genes and, by extension, therapeutic targets.

Keywords

CRISPR-Cas9; GSS; extracellular vesicle; glioblastoma; radiotherapy.

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