Development of CRISPR gene therapy for blindness caused by Usher Syndrome

Thesis

Inherited retinal degenerations are a leading cause of blindness in the UK. Significant advances have been made to solve this issue in recent years, with a pioneering FDA approved gene therapy treatment (Luxturna®), which targets a loss of function mutation in the RPE65 gene. However, there remain notable shortcomings to this form of gene replacement therapy. In particular, the lack of viability for gene sequences exceeding the 4.7 kb adeno-associated virus (AAV) packaging limit or for toxic gain of function mutations. The USH2A gene at ~15.7 kb for instance is too large for AAV delivery: an effective vehicle capable of transducing photoreceptor cells for gene replacement therapy. This thesis explores the potential of CRISPR technology, derived from bacterial defence mechanisms, to overcome these challenges, delivering tools to precisely edit and correct small insertions, deletions and base transitions in USH2A without the need to deliver the full-length gene.

In the first stage of the thesis, a mouse model of the common USH2A (c.11864G>A; p.W3955X) mutation was characterised. The natural history study followed the mice until the 24-month time point, however no deficits in visual function were observed. Nonetheless, auditory function was impacted from birth and molecular and structural abnormalities were detected, including at the ultrastructural level. Initially an attempt was made to optimise a compact prime editor for single AAV packaging. However, the efficiency of the prime editor (comprising SaCas9 tethered to a truncated MMLV reverse transcriptase) was not adequate for therapeutic editing in vivo.

Advancing with the most effective optimised prime editor, two pilot studies were run in parallel: electroporation of the prime editor encoded by a minicircle and subretinal injection of the prime editor split across dual AAV vectors. Both delivery methods demonstrated on-target DNA editing and protein restoration. Moreover, off-target edits were minimal and retinal thinning was not significant relative to controls. The non-viral approach generated higher levels of editing and appeared less toxic than the viral approach. Additionally, the minicircle driven by the viral CMV promoter was silenced within 1 month.

Finally, an attempt was made to degrade prime editor protein within the retina following sufficient therapeutic editing. A ligand-responsive degron motif was tethered to the N terminus of the CRISPR construct. Following intravitreal injection of the ligand, prime editor protein was undetectable, although the effect was temporary and more suitable for non-viral delivery.

Authors: Major, L

• DOI: 10.5287/ora-8jewej4ge
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Usher Syndrome; inherited retinal diseases; prime editing; CRISPR