Outcome measures for BEST1 gene augmentation therapy: restoration of RPE-photoreceptor interface homeostasis

Abstract

Purpose : Mutations in BEST1 gene are causally associated with a group of juvenile maculopathies characterized by abnormal EOG and variable severity and progression. We have previously described a combination of structural and biochemical factors that, in a mutation-independent manner, predispose the macular area to primary detachment in canine and human bestrophinopathies. Using a naturally-occurring canine bestrophinopathy model (cBest), we have also shown that AAV-mediated BEST1 gene augmentation therapy results in reversal of macular and extramacular lesions with either canine- or human-specific transgene. Here, we investigated whether the AAV-mediated BEST1 gene therapy also restores homeostasis of RPE-photoreceptor interface, which will impact the long-term efficacy of this treatment.

Methods : Thirteen dogs of both sexes (age 4-17mos) harboring homozygous (R25X or P463fs) or compound heterozygous (R25X/P463fs) mutations in BEST1 were subretinally injected with AAV2-VMD2-BEST1 (1.5x10¹⁰-6.5x10¹¹vg/ml) and imaged serially using cSLO/SD-OCT. Retinal structure was examined at 9-24mos post injection by H&E and immunohistochemistry (IHC) using RPE- and photoreceptor-specific markers. Treated retinas were evaluated by light microscopy or epifluorescence and confocal microscopy in comparison to untreated and WT control eyes.

Results : Based on SD-OCT analyses, subretinal lesions within the treated regions resolved 8-12 wks after therapy and the photoreceptor layer thickness, measured from external limiting membrane to RPE apical surface, returned to normal within the entire treated area. Both the in vivo imaging and IHC revealed no apparent adverse effects in the RPE or retina secondary to the BEST1 gene augmentation. Immunolabeling with EZRIN and MCT1 demonstrated extension of RPE apical projections corresponding to the vector treated bleb areas with augmented BEST1. In sharp contrast, the untreated regions showed absent or only vestigial RPE apical microvilli, associated with formation of new lesions or subtle RPE-neuroretina separation, and accumulation of subretinal debris.

Conclusions : AAV-mediated BEST1 gene augmentation therapy in canine models of bestrophinopathy promotes sustained reversal of macular lesions through reestablishment of a normal physical interaction between RPE and neuroretina, and restoration of homeostasis at the RPE-photoreceptor interface.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.