Generation and phenotypic characterization of a transgenic pig for the Usher syndrome type 1C

Abstract

Purpose : The human Usher syndrome (USH) is the most common form of inherited deaf-blindness, often accompanied by vestibular dysfunction. There is no therapy for the ophthalmic component of USH to date. Rodent models with defects in USH genes do not show a strong ophthalmologic phenotype. The lack of USH models has not only hindered comprehensive functional analyses of USH1 proteins, but also the evaluation of therapeutic assessments for USH1 in the eye. Here, we aim to generate and phenotypically characterize a transgenic USH1C pig model.

Methods : We modified the porcine USH1C gene by cloning the human USH1C exon 2 bearing the R31X nonsense mutation into a porcine BAC clone via bacterial recombineering. The modified BAC vector was CRISPRed into porcine primary cells and subsequently used as donor cells for somatic nuclear transfer. Bi-allelic USH1C^R31X pigs were analyzed by brainstem auditory evoked response tests, electro-retinography (ERG) recordings and optical coherence tomography. In addition, we performed RNAseq/NGS, qRT-PCR, Western blots, immunohistochemistry, light- and electron microscopy, biomarkers and behavior tests.

Results : We successfully generated USH1C^R31X pigs which do not express USH1C/harmonin. USH1C^R31X piglets are born deaf and display vestibular dysfunctions. Inner ear hair cells have disordered hair bundles with disordered stereocilia bundles. ERGs under both scotopic and photopic conditions are altered. Behaviour tests reveal reduced visual function. Biomarkers for retina degeneration are upregulated. The outer nuclear layer is thinner and the disk stacks in the outer segment of photoreceptor cells are disordered.

Conclusions : Our data reveal inner ear defects and ocular alterations in USH1C^R31X pigs representing the phenotype in USH1 patients. Thus, we successfully generated the first mammalian and large animal model for USH1, recapitulating the human disease. The USH1C^R31X pigs will provide insights into the molecular mechanisms of the disease and will be used for preclinical evaluation of gene and cell based therapy strategies.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.