Abstract
Purpose :
Usher syndrome (USH) is a leading cause of deafness-blindness and variants in PCDH15 are responsible for USH type 1F (USH1F). Previous studies showeded three alternative spliced cytoplasmic domains (CD1, CD2 and CD3) of PCDH15., however expression and functional data in the murine retina were lacking. Herein, we investigated the spatiotemporal expression pattern of Pcdh15 isoforms in the developing and mature mouse retina. We evaluated the visual function of three mouse lines, lacking one out of three Pcdh15 cytodomains (Pcdh15-CD1, -CD2, -CD3). Finally, we developed and evaluated the impact of dual-AAV based PCDH15 gene delivery in the mouse retina.
Methods :
Immunoblotting and immunofluorescent imaging showed using CD1, CD2 and CD3 domain specific antibodies showed the expression and subcellular distribution of protocadherin-15 isoforms. Mice lacking Pcdh15-CD1, -CD2 or -CD3 isoforms were assessed for structure and function was assessed by optical coherence tomography (OCT) and electroretinography (ERG). Two halves of human PCDH15 were packaged in adeno-associated virus (AAV) and subretinal injection were performed in Pcdh15 mutant and control mice followed by serial ERG analysis.
Results :
Both protocadherin-15-CD1 and -CD3 isoforms were detected but no expression of CD2 isoform was found. Consistent with immunoblotting, we also observed expression of protocadherin-15-CD1 and -CD3 isoforms in multiple retinal layers, including photoreceptors, at specific ages from P1 to P42. Consistent with expression data we found intact vision in Pcdh15-CD2 knockout mice at 2-months of age. Currently, we are performing ERG analysis and retinal structural analysis of older mice from each of the mutant lines. In parallel, we also packaged two halves of human PCDH15 in AAV-Anc80L65 capsid and performed subretinal injections in. Finally, Pcdh15 null mice injected with dual AAV at P20-P21 or P30, revealed significant improvement of vision.
Conclusions :
Our current findings indicate that although retina expresses multiple isoforms of protocadherin-15, but either not all of them require for normal vision or have functional redundancy. Furthermore, dual AAV based PCDH15 vectors mediated gene replacement therapy revealed promising results and significantly restored vision in Pcdh15 mutant mice.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.