Brain‐Targeted Cas12a Ribonucleoprotein Nanocapsules Enable Synergetic Gene Co‐Editing Leading to Potent Inhibition of Orthotopic Glioblastoma

• Published in: Advanced science 2024-09, Vol.11 (33), p.e2402178-n/a
• Main Authors: Ruan, Weimin, Xu, Sen, An, Yang, Cui, Yingxue, Liu, Yang, Wang, Yibin, Ismail, Muhammad, Liu, Yong, Zheng, Meng
• Format: Article
• Language: English
• Subjects: Animals; Bacterial Proteins; Brain cancer; Brain Neoplasms - genetics; Brain Neoplasms - metabolism; Brain Neoplasms - therapy; Cas12a; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell Line, Tumor; CRISPR; CRISPR-Associated Proteins - genetics; CRISPR-Associated Proteins - metabolism; CRISPR-Cas Systems - genetics; Disease Models, Animal; Drug resistance; Efficiency; Endodeoxyribonucleases; ErbB Receptors - genetics; ErbB Receptors - metabolism; Flow cytometry; Gene Editing - methods; Genes; gene‐editing; Genome editing; glioblastoma; Glioblastoma - genetics; Glioblastoma - metabolism; Glioblastoma - therapy; Glioma; Humans; Medical prognosis; Mice; Mice, Nude; Microscopy; nanocapsule; Nanocapsules; Particle size; Polo-Like Kinase 1; Protein Serine-Threonine Kinases - genetics; Protein Serine-Threonine Kinases - metabolism; Proteins; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Vectors (Biology)
• ISSN: 2198-3844; 2198-3844
• DOI: 10.1002/advs.202402178

Gene‐editing technology shows great potential in glioblastoma (GBM) therapy. Due to the complexity of GBM pathogenesis, a single gene‐editing‐based therapy is unlikely to be successful; therefore, a multi‐gene knockout strategy is preferred for effective GBM inhibition. Here, a non‐invasive, biodegradable brain‐targeted CRISPR/Cas12a nanocapsule is used that simultaneously targeted dual oncogenes, EGFR and PLK1, for effective GBM therapy. This cargo nanoencapsulation technology enables the CRISPR/Cas12a system to achieve extended blood half‐life, efficient blood‐brain barrier (BBB) penetration, active tumor targeting, and selective release. In U87MG cells, the combinatorial gene editing system resulted in 61% and 33% knockout of EGFR and PLK1, respectively. Following systemic administration, the CRISPR/Cas12a system demonstrated promising brain tumor accumulation that led to extensive EGFR and PLK1 gene editing in both U87MG and patient‐derived GSC xenograft mouse models with negligible off‐target gene editing detected through NGS. Additionally, CRISPR/Cas12a nanocapsules that concurrently targeted the EGFR and PLK1 oncogenes showed superior tumor growth suppression and significantly improved the median survival time relative to nanocapsules containing single oncogene knockouts, signifying the potency of the multi‐oncogene targeting strategy. The findings indicate that utilization of the CRISPR/Cas12a combinatorial gene editing technique presents a practical option for gene therapy in GBM. A biodegradable brain‐targeted CRISPR/Cas12a nanocapsule (ANC@RNP) simultaneously targeted EGFR and PLK1 for effective GBM therapy. ANC@RNP achieved extended blood half‐life, efficient blood‐brain barrier (BBB) penetration, active tumor targeting, and selective release. ANC@RNP lead to robust combinatorial gene editing and showed superior tumor growth suppression in U87MG and patient‐derived GSC orthotopic xenograft mouse models with negligible off‐target gene editing.