CRISPR/Cas9 gene editing of brain cancer stem cells using lipid-based nano-delivery

Abstract Despite advances in cancer therapy glioblastoma (GBM) remains one of the deadliest brain tumours. Effective therapy is restricted by the presence of multiple resistance mechanisms. Physical barriers such as the blood-brain barrier limit the brain delivery of therapeutic compounds. In addition, the presence of a subset of GBM-stem-like cells (GSCs), characterized by radio/chemoresistance, and the intratumor heterogeneity impede standard therapies from being effective. New therapeutic approaches are urgently needed. Given its high specificity, CRISPR/Cas9-mediated genome editing provides new prospects for novel therapeutic targets. While promising, in vivo application of CRISPR/Cas9 is currently hampered by poor pharmacokinetics and limited ability to cross biological membranes. The present research is designed to utilise stable nucleic acid lipid nanoparticles (SNALPs) for in vivo delivery of CRISPR/Cas9 to GSC by disrupting the epidermal growth factor receptor variant III (EGFRvIII), a GBM associated mutation, responsible for tumour cell proliferation, angiogenesis and invasion. Near Infrared fluorescence labelling and live optical imaging confirmed SNALPs uptake in GSC tumours implanted intracranially in mice after intravenous injection. Higher uptake of SNALPs in tumourous tissues compared to healthy brain tissues was further confirmed by ex vivo imaging and flow cytometry. EGFRvIII-specific sgRNA has been designed and validated in GSCs using commercial transfection regents. Studies are underway to load CRISPR/Cas9 mRNA/gRNA into SNALPs to test their in vitro gene editing efficacy. Successful in vivo delivery of CRISPR/Cas9 will represent a promising approach for identifying GBM therapeutic targets in vivo which in the long-run can be applied for GBM treatment.