Application of therapeutic alternative splicing to Usher syndrome Type 2A

Khine Zaw

Usher Syndrome type 2A (USH2A), the most common genetic cause of combined deafness and blindness disease, is caused by biallelic mutations in the USH2A gene. The USH2A gene encodes the usherin protein predominantly found in photoreceptors of retina and hair cells of cochlea. In this study, patients with a confirmed diagnosis of USH2A-related retinopathy were assessed for potential splice modulation study. Dermal fibroblasts were propagated from skin biopsies and USH2A mutations were characterised. We found that a splice site mutation in intron 46 (c.9258+1 G>A) resulted in cryptic splicing with the deletion of the last 153 nucleotides of exon 46 while a synonymous mutation (c.949C>A) in exon 6 led to the deletion of the last 193 nucleotides of exon 6. The fibroblasts from a patient carrying biallelic c.949C>A and c.1256G>T were reprogrammed to induced pluripotent stem cells (iPSCs) that were then differentiated into retinal organoids. The patient iPSC-derived retinal organoids showed lower expression of retinal markers, recoverin (photoreceptor marker), ARL13B (ciliary marker) and usherin protein when compared to retinal organoids derived from iPSCs of a healthy donor. With the emergence of personalised medicine, antisense oligonucleotide (AO) modulation of pre-mRNA splicing has been applied to some rare diseases. AOs are short, synthetic, single-stranded nucleic acid analogues that can be designed to anneal to a premRNA and modulate the splicing process. We explored two approaches of splice modulation in this study: exon skipping and splice correction to produce internally truncated semifunctional usherin protein and to rescue expression of full-length functional, respectively. The former approach assumed that the protein domains encoded by the in-frame USH2A exon 13 and 62 are likely redundant and removing them would not affect the function of usherin. Different AO sequences to skip exons 13 and 62 were synthesised as the 2ʹ-O-methyl modified phosphorothioate chemistry (2ʹOMe) for screening in human hepatoma cell lines (Huh-7), retinal pigment epithelial cell lines (ARPE-19) or patient fibroblasts. The lead candidate AOs that induced the complete skipping of each targeted exon were purchased as phosphorodiamidate morpholino oligomers (PMO). Treatment with PMOs in the same cell lines exhibited efficient skipping of the target exons 13 and 62. Secondly, the splice correction AOs were designed for c.949C>A mutation and assessed in patient-derived iPSCs and retinal organoids. Despite preliminary, one out of three tested AOs for splice correction exhibited the expected outcome. When developing AOs to treat USH2A and other genetic diseases in our laboratory, we explored various chemical modification options for AOs with ineffective exon skipping. We modified some previously reported ineffective 2ʹOMe AO sequences targeting DMD exons 16, 23 and 51 as locked nucleic acid (LNA)/ 2ʹOMe mixmers. All the mixmers exhibited some skipping of the targeted exons however the mixmer targeting exon 51 activated cryptic splice sites and induced aberrant splicing with retention of 95 or 188 bases from the beginning of exon 51. In conclusion, we identified two USH2A pathogenic variants associated with premRNA splicing defects among eleven tested in total. We successfully designed AOs for USH2A exon 13 and 62 skipping and a potential candidate AO for correction of cryptic splice site induced by c.949C>A mutation. Out of three chemical modifications tested, PMOs are the most reliable AO chemistry with less undesired effects. We anticipate potential AO therapeutics for USH2A patients after further refinement of the AO sequences and functional validation studies.

Application of therapeutic alternative splicing to Usher syndrome Type 2A

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