The Leber Congenital Amaurosis CEP290 cat model : working towards a cure

Leber Congenital Amaurosis (LCA) is an early-onset and severe inherited retinaldystrophy. The most commonly implicated gene is the centrosomal 290kDa (CEP290)gene. There are currently no treatments for LCACEP290. Animal models are integral fortreatment safety and efficacy testing, with a feline model for CEP290 retinopathiesshowing promise for this purpose. This model has a spontaneous mutation in CEP290resulting in a progressive retinal degeneration and vision loss. This mutation alterssplicing and is predicted to result in a truncated protein. CEP290 is integrally involved inthe transport within photoreceptor cells, localizing to the interconnecting cilium.The CEP290 mutant cat ("rdAc") has a milder phenotype than the phenotypemost commonly associated with CEP290 mutations in people. Understanding thereason for this milder phenotype was an important aim of this dissertation. We analyzedthe wild-type and truncated mutant transcript levels and total CEP290 protein levels incat retinal tissue across genotypes (wild-type, heterozygous, homozygous mutant). Ourfindings show that the milder phenotype in mutant cats is likely the result of low-levelproduction of wild-type transcript and protein combined with production of truncatedprotein that we suspect retains some function. These measures can also serve asobjective markers of disease progression or treatment success in future studies.Further objective markers were investigated utilizing spectral-domain opticalcoherence tomography. By analyzing photoreceptor layer thickness (receptor plus/REC+) and the ellipsoid zone (EZ), a zone noted to change with disease in people, weshowed that REC+ thickness and EZ integrity can be used as markers of diseaseprogression. These findings support the cat as a CEP290 model, and show the affectedcats have a degeneration with central retinal sparing as described in human patients.We also developed an in vitro tool by developing primary fibroblast cell culturesfrom skin samples of CEP290 cats. We showed that cilia formation, which involvesCEP290, can be induced, opening the door for future cilia studies. We assessed cilialength as an objective marker of disease, however, no difference was found in cilialength across genotypes. Utilizing this new tool, we tested a CRISPR/Cas-9 genomeediting treatment in an attempt to replace the mutated region with a sequence thatwould direct splicing to the correct location. We achieved high efficiency transduction ofCRISPR components, however, we failed to detect insertion of our chosen sequence.As we work towards a treatment, it is imperative to develop an optimized methodto deliver the treatment to feline photoreceptors. To achieve this aim we tested hybridadeno-associated viral (AAV) vectors, the retinal viral vector of choice, in the wild-typefeline retina. We showed that all serotypes studied, AAV2/2, 2/5, 2/8, and 2/9, transducephotoreceptor cells with AAV2/8 and 2/9 showing higher transduction and faster onset.Interestingly, we found more efficient transduction of cones than rods, an unusualfinding that speaks to the need to use caution when extrapolating across species.It is our hope that these data will help the scientific community move closer totreatments for CEP290 retinopathies. We have added to the understanding of the rdAccat model and determined objective markers, supported the cat as a model, and havemade progress towards a treatment.