Proof of concept of CRISPR/Cas9 to treat corneal dystrophies

Abstract

Introduction The corneal dystrophies are a group of inherited disorders that affect the shape or transparency of the cornea. The unique qualities of the cornea coupled with their mendelian traits make the them an ideal target for gene therapy. The main aim of this thesis is to investigate the potential to use CRISPR/Cas9 to treat the autosomal dominant TGFBI corneal dystrophies.

Methods Paper II: Plasmid expressing CRISPR/Cas9 targeted to Luc2 was delivered to the cornea via an intrastromal pressure injection. Knockdown of luciferase was measured using an IVIS in vivo imager. The allele-specificity of CRISPR/Cas9 was assessed in vitro using a dualluciferase assay and an in vitro digestion. Paper III: A morpholino targeted to tgfbi was electroporated into the regenerating adult zebrafish tail fin, and the effect of Tgfbi knockdown was measured by the quantification of regenerated tissue. 10X phased sequencing and EBV-transformation of isolated lymphocytes was performed on a patient harbouring a R124H granular corneal dystrophy type II (GCD2) mutation. The phasing information was used to design allele-specific sgRNAs which were then tested using an in vitro digest and targeted resequencing across the target locus in TGFBI. qPCR was used to infer the efficiency of a dual-cut using a combination of sgRNAs. Paper IV: The transduction efficiency of 3 AAV-GFP serotypes in immortalised human corneal cells was assessed using flow cytometry. In vivo transduction of AAV-GFP following intracameral injection was assessed using the IVIS in vivo imager and fluorescent microscopy. AAV-Cas9 and AAV-sgRNA-GFP were co-injected, DNA was extracted from the whole cornea and TIDE analysis was performed to determine efficiency of indels.

Results Paper II: The failure of a mutation-dependent approach to target 20% of TGFBI missense mutations was demonstrated. Comparison of two widely used allele-specific strategies revealed a PAM-specific approach conferred superior specificity than that of a guide-specific approach. The inability of S.pyogenes Cas9 to distinguish between single base pair changes in the guide sequence was confirmed. Paper III: Knockdown of Tgfbi in the regenerating zebrafish tail fin was shown to impair wound healing. The ability to selectively target the mutant allele by means of non-diseasecausing SNPs, which are associated with a PAM on the same allele as the disease-causing mutation, was demonstrated. The addition of a 50:50 ssODN with the ribonucleoprotein complex was shown to significantly increases the frequency of a dual-cut event. Paper IV: AAV-2/9 was shown to transduce all corneal layers in vivo following a single intracameral injection. A dual-AAV-2/9 CRISPR/Cas9 system was shown to generate 25.7% within the whole cornea. Conclusion Cas9 lacks the specificity to discriminate between single base pair mismatches within the 20bp guide sequence, however mutations within the 2bp PAM are much less tolerated. As such, using currently available nucleases a PAM-specific approach is necessary to discriminate between alleles. AAV was demonstrated as a robust vehicle to deliver geneediting reagents to the corneal layers.

Date of Award	Oct 2018
Original language	English
Sponsors	Avellino Lab USA
Supervisor	Andrew Nesbit (Supervisor), Tara Moore (Supervisor) & David Courtney (Supervisor)