Optimising CRISPR sgRNA delivery strategies for the treatment of retinal diseases

Thesis

Gene therapy for inherited retinal diseases (IRDs) has traditionally focussed on gene augmentation. However, the advent of CRISPR/Cas9 has changed the research landscape dramatically. Theoretically able to correct almost any mutation, CRISPR/Cas9 is an attractive tool for IRD therapeutics, many untreatable by gene augmentation. However, numerous challenges exist for Cas9 delivery to retina. This thesis explores non-viral, separate delivery of Cas9 sgRNA as a way to overcome these limitations.

The ability of unpackaged RNA oligonucleotides to enter retina from intravitreal injection has been shown by chemically modified antisense oligonucleotides (ASOs). Similar modifications applied to Cas9 sgRNA have been used effectively in vitro, and in vivo in liver, but not in the eye. In this thesis I investigate the use of chemically modified Cas9 sgRNA as a proof-of-concept for retinal therapeutics. First, I examined sgRNA modification pattern and number, and showed that variations can enhance or abrogate EGFP and Cas9 knockdown for SpCas9, SaCas9, and a SpCas9 prime editor in vitro in HEKdEGFP cells without toxicity. Next, I examined retinal distribution and survival of unpackaged sgRNA in vivo using qPCR and direct visualisation of fluorescently-labelled chemically modified sgRNA. This revealed that intravitreally-injected sgRNA enters the retinal ganglion cell (RGC) layer and survives for at least 1 month without toxicity, but is rapidly lost from vitreous, while subretinally-injected sgRNA survives in the retina for at least 8 weeks without toxicity.

Finally, I examined indel formation in vivo, targeting photoreceptor-level EGFP in the Nrl.EGFP mouse with subretinal unpackaged sgRNA and AAV8-GRK1-Cas9, and RGC-level EGFP using intravitreal unpackaged sgRNA in the Cas9EGFP mouse. This revealed that heavily modified SpCas9 sgRNA successfully effects indel formation in RGCs, but not in photoreceptors, perhaps due to sgRNA trapping in outer segments or within other retinal cells. As part of this thesis, I also characterised retinal Cas9 expression of several transgenic Cas9-expressing mice using Western blot, in vivo imaging, and IHC.

Authors: Han, RC

• DOI: 10.5287/ora-o17qygany
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: CRISPR; sgRNA; Inherited retinal dystrophies; Retina; Photoreceptors
• Subjects: Drug delivery systems; CRISPR-associated protein 9; Retinal degeneration; Ophthalmology; RNA targeting