Walker, Amy; (2019) Delivery of CRISPR/Cas9 by receptor-targeted nanoparticles as a corrective therapy for Cystic Fibrosis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cystic Fibrosis (CF) is the most common inherited genetic disorder, affecting around 1 in 2,500 babies born in the UK. Clinical manifestations are caused by mutations in the gene encoding the cystic fibrosis transmembrane regulator (CFTR), a membrane channel protein which regulates anion transport and mucociliary clearance. CRISPR/Cas9 is an attractive therapeutic option for CF as it could target the underlying cause of the disease, rather than treating symptoms. However, a major hurdle to overcome if CRISPR/Cas9 is to be deployed as a treatment is how to deliver the technology to the lung. We hypothesise that receptor targeted nanocomplexes (RTNs), previously shown to deliver DNA and siRNA to the lung, can be used to package and deliver the gene editing components. Here, we have successfully optimised these nanocomplexes specifically for the delivery of Cas9 over its various platforms and extensively characterised the particles. Using an epithelial GFP reporter system, we were able to achieve higher transfection levels than commercially available reagents, for both Cas9 mRNA and Cas9 protein. The 10th most common CF causing mutation, 3849+10kb C>T, generates a cryptic splice site, resulting in the formation of a pseudoexon containing a PTC, producing a truncated version of the protein. Using pairs of gRNAs, we were able to successfully remove the cryptic splice site using an NHEJ strategy, correcting aberrant splicing and, importantly, CFTR channel function as measured by Ussing Chamber. A homology-independent targeted integration (HITI) strategy was used to investigate the potential of restoring CFTR function regardless of mutation type. As proof-of-concept, we delivered GFP into AAVS1 locus of HEK293 cells, achieving a targeted knock-in efficiency of 14%.Finally, the use of RTNs to deliver CRISPR/Cas9 in vivo was explored successfully in an Ai9 mouse reporter model to restore tdTomato expression by paired gRNAs excision of a stop cassette, confirming RTNs can be used as a safe delivery method for repeated dosing of CRISPR/Cas9, and highlighting their translational potential.

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