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Jennifer Barber Phillips, Jeremy Wegner, Monte Westerfield; In-frame exon skipping fails to rescue retinal or hair cell defects in zebrafish models of PCDH15 (USH1F). Invest. Ophthalmol. Vis. Sci. 2017;58(8):4501.
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The R245X mutation in PCDH15 R245X mutation causes a severe form of Usher Syndrome. The affected codon lies in an in-frame exon that encodes part of two consecutive E-cadherin domains in the extracellular region of PCDH15. This region is proximal to a protein-protein interaction domain required in stereocilia, but of unknown function in retinal tissue. To evaluate in-frame exon-skipping as a therapy for PCHD15(R245X) patients, we deleted Exon 8 from zebrafish pcdh15 genes and assayed visual and auditory function.
CRISPRs with zebrafish-optimized Cas9 were injected into zygotes to generate germline mutations. Sequence analysis of gDNA and cDNA confirmed the deletions and revealed production of transcripts lacking exon 8. Behavioral and histological analyses were performed to assay hearing, balance, cellular morphology and cell death. Images were captured with an iOS camera or Zeiss LSM5 Confocal microscope.
We analyzed behavior, cell morphology and photoreceptor cell death in zebrafish pcdh15aE8△and pcdh15bE8△ lines, each containing in-frame deletions of Exon 8. We also examined truncating mutations in exon 8 of both zebrafish PCDH15 orthologs (pcdh15aE8X and pcdh15bE8X, respectively) showing partially overlapping functions with a greater requirement for pcdh15a in inner ear and for pcdh15b in retina. We used these lines as positive controls for experiments with the in-frame exon deletions. pcdh15aE8△ and pcdh15bE8△ animals recapitulated the swimming and balance phenotypes seen in the E8 truncating mutants for each gene, with no improvements or differences noted in mechanosensory hair cell structure or function. Mild photoreceptor cell morphology defects and low levels of photoreceptor death that was not exacerbated by exposure to increased illumination observed in pcdh15aE8X larvae were recapitulated in pcdh15aE8△ larvae. Severe PRC morphology defects and marked PRC death that increased with elevated light intensity was the same in pcdh15bE8X and pcdh15bE8△ larvae.
We conclude that disruption of EC1 and EC2 domains is incompatible with normal PCDH15 protein function in auditory and visual sensory cells, even when more C-terminal portions of the protein remain intact. Our rapid and inexpensive evaluation of this therapeutic approach in zebrafish disease models informs future approaches for alternative allele-specific treatments of the R245X mutation
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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