*4.5. BEST1*

Best vitelliform macular dystrophy (Best disease, BVMD) has autosomal dominant inheritance and is the most common disease associated with mutations in the gene *BEST1*. Four other disease phenotypes have been described in association with *BEST1* mutations: adult onset vitelliform macular dystrophy, autosomal recessive bestrophinopathy, autosomal dominant vitreoretinochoroidopathy and retinitis pigmentosa. BVMD is characterized by at least one vitelliform lesion in the macula but can present with multiple lesions. The disease slowly progresses to degenerate the RPE and retina in the a ffected regions, resulting in vision impairment [75]. In dogs, a similar disease has been described, termed canine multifocal retinopathy (CMR) and is caused by mutations in the *Best1* gene. In contrast to the BVMD in humans, CMR due to *Best1* mutations is an autosomal recessive disease and has a consistent and predictable disease phenotype. This consistency and the detailed natural history of the disease lends well to measuring the outcomes of translatable therapies.

Initially, two *Best1* mutations were identified in dogs: p.Arg25Ter (Great Pyrenees and masti ff-related breeds, *cmr1*) and p.Gly161Asp (Coton de Tulear, *cmr2*), with the former resulting in a premature stop codon and, presumably, a lack of Best1 protein [91]. Analysis of additional breeds

with CMR identified another breed (Lapponian herder, *cmr3*) with two deleterious mutations in exon 10, a 1 bp deletion leading to a frameshift and premature stop codon (p.Pro463fs) and a missense mutation (p.Gly489Val) [92]. Interestingly, the phenotype resulting from the three mutations in dogs is indistinguishable. The dogs present with multifocal regions of retinal separation with a pink or tan-colored subretinal fluid, which eventually leads to retinal degeneration. Using optical coherence tomography to image eyes from *cmr1*/*cmr1*, *cmr3*/*cmr3* and *cmr1*/*cmr3* dogs in vivo, the earliest detectable sign of the disease is at ~11 weeks of age in which there is a retinal elevation in the fovea-like region of the area centralis. As the disease progresses, this retinal elevation becomes a macrodetachment, surrounded by microdetachments. Rates of progression, detachment location and number varied, but the disease was typically localized to the more cone-rich regions of the retina [93].

Immunohistochemistry of CMR canine tissues showed a lack of RPE apical microvilli at the cone photoreceptor/RPE interface along with accumulated lipofuscin within the RPE. The loss of the RPE apical processes results in a loss of all direct contact of the cones to the RPE, severely impacting the physiological role the RPE has on retinal maintenance. It is hypothesized that the lack of RPE apical processes and subsequent weakened interphotoreceptor matrix is instrumental in the characteristic detachments and lesions observed in diseases caused by *BEST1* mutations [93,94]. Interestingly, microdetachments were identified in response to light exposure in pre-clinical CMR dogs. The detachments occurred between the photoreceptor inner/outer segments and the RPE/tapetum interface. These light-induced detachments occurred within minutes and increased in response to time of light exposure and would resolve within 24 h.

Using a rAAV2 vector delivered subretinally, CMR dogs were treated by gene augmentation with wildtype canine or human *BEST1*. Macro and microdetachments were resolved and RPE microvilli ensheathment of cone photoreceptors returned within the treatment area. This positive outcome was retained in the dogs for as long as 207 weeks post injection. The same outcome was present regardless of age of treatment (within 27–69 weeks of age), the stage of detachments or mutation (*cmr1*/*cmr1*, *cmr3*/*cmr3* or *cmr1*/*cmr3*). These results show promise for the treatment of human patients with *BEST1* mutations [93].
