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Delivery of CRISPR-Cas9 to the Brain for the Treatment of CNS Disease.

O Mokrane(2) A Vaideanu(2) M Mazza(1) I Uchegbu(2) A Schatzlein(2)

1:AstraZeneca; 2:UCL

Introduction: The utility of CRISPR-Cas9 gene-editing technology for the correction of pathogenic mutations in CNS disorders is limited because delivery across the BBB is restricted for large biomolecules. The Nose-to-Brain (N2B) delivery route is a non-invasive pathway to the CNS, gaining access via perivascular and perineural pathways in the olfactory epithelium.  Here we demonstrate that synthetic vectors based on Glycol Chitosan (GC) administered via the N2B route deliver CRISPR-Cas9 plasmid DNA (pDNA) to the CNS and lead to Cas9 protein expression.

Methods: CRISPR-GC polyplexes were formed via electrostatic interaction with GC and the in-vitro cellular delivery was assessed in a U87-MG astrocyte cell line via flow cytometry. The regional expression of Cas9 protein in the brain was evaluated via immunoblotting following nasal dosing in a mouse model.

Results: The polyplexes demonstrated a selective cellular uptake at pH 6.7, a property which is favourable for transport into the olfactory epithelium which sits at pH 5.5-6.5. The transfection efficiency was superior when compared to a commercial transfection reagent and the polyplexes initiated expression of the Cas9 gene-editing protein in U87-MG cells. Pilot preclinical studies of low-dose polyplexes identified the presence of Cas9 protein in the olfactory bulbs following nasal dosing in a mouse model.

Conclusion: To our knowledge, this is the first report to demonstrate the use of synthetic vectors to initiate Cas9 expression in the brain following N2B delivery. Further studies are underway to determine if higher polyplex doses can initiate Cas9 expression in the cerebral cortex, where therapeutic targets reside for gene-editing.

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