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Jennifer Barber Phillips, Willem T Griffiths, Jeremy Wegner, Monte Westerfield; Preclinical testing of a therapy for the R245X mutation of PCDH15 (USH1F). Invest. Ophthalmol. Vis. Sci. 2016;57(12):1161.
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© 2017 Association for Research in Vision and Ophthalmology.
The R245X mutation in PCDH15 causes a severe form of Usher Syndrome prevalent in the Ashkenazi Jewish population. It results from a point mutation in exon 8, a 171bp, in-frame exon. We use gene editing techniques in zebrafish to test the feasibility of exon skipping as a therapy to treat the retinal manifestations of this pathogenic PCHD15 allele.
CRISPRs with zebrafish-optimized Cas9 were injected into zygotes to generate germline mutations. Genotyping was carried out by restriction digest and DNA sequencing. Optokinetic and startle response assays and histological analyses of ear and eyes were performed. Images were captured using a Zeiss LSM5 Confocal microscope.
Sequence analysis of zebrafish pcdh15a and pcdh15b duplicate genes confirm that exon 8 structure is conserved with humans. We generated frameshift mutations in the exon 8 sequence of zebrafish pcdh15a and pcdh15b to obtain truncations functionally analogous to the human R245X mutation. Early truncation of pcdh15a resulted in severe auditory and vestibular dysfunction, disrupted stereocilia morphology, and grossly normal retinal architecture and function consistent with previous reports of zebrafish orbiter mutants. Pcdh15a protein was undetectable in mutant animals by fluorescent antibody labeling. Surprisingly, truncating mutations in exon 8 of pcdh15b also caused swimming and balance problems and disrupted stereocilia, in contrast to previous morpholino studies. pcdh15b mutants showed a diminished, but measurable, optokinetic response, but no overt defects in photoreceptor morphology. To evaluate the functional consequence of deleting exon 8 from pcdh15a and pcdh15b, we used CRISPR/Cas9 targeted to flanking introns. DNA repair by non-homologous end joining then excluded the exon 8 sequence. Sequence analysis of gDNA and cDNA confirmed the deletions and revealed production of transcripts lacking exon 8.
Our analysis of truncating mutations in pcdh15a and pcdh15b reveal more functional overlap in sensory cell development and function than previously described, and provide us with the opportunity to model the pathogenicity of the R245X mutation more precisely. Our successful use of CRISPR/Cas9 technology to generate in-frame deletions of exon 8 allows us to assess how its absence affects protein function, and to determine the feasibility of exon skipping as a potential treatment for R245X patients.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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