Early Retina-Wide Pathology at the RPE–PR Interface.

To understand the pathophysiology behind the impaired RPE–PR interaction, we first evaluated cBest retinas with clinically obvious disease. The key features of RPE apical membrane responsible for direct interaction with PR OSs were examined by immunohistochemistry (IHC) against EZRIN, a membrane–cytoskeleton linker protein essential for formation of RPE apical MV, and combined with human cone arrestin (hCAR) and peanut agglutinin lectin (PNA) labeling to distinguish the cone PR matrix-specific interface (Fig. 1). Confocal microscopy and analysis of 3D reconstruction images from the wild-type (WT) retina exposed a complex sheet-like structure of both inherent constituents of RPE apical membrane: cone- and rod-associated MV. Cone-MV (also known as RPE apical cone sheath) (41) were more pronounced than rod-MV, and formed a highly organized wrapping that tethered individual cone outer segments (COSs) to the RPE apical surface (Fig. 1A and Movie S1). In the subretinal space, this intercellular complex was further encased with an equally intricate cone-specific insoluble extracellular matrix sheath (cone-IPM) detected by selective binding of PNA lectin (42) (Fig. 1A and Movie S1). In diseased cBest retinas, however, such complex extracellular compartmentalization of COSs was lost, and the dearth of microvillar ensheathment was accompanied by hypertrophied RPE cells overloaded with lipofuscin granules and compromised insoluble cone-IPM (Fig. 1B and Movie S2). These observations were confirmed in three distinct cBEST1 genotypes (R25*/R25*, P463fs/P463fs, and R25*/P463fs) examined across both the tapetal and nontapetal portions of the retina in 22 eyes after disease onset (age range 45 to 270 wk).

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Fig. 1.

Retina-wide pathology of RPE apical microvillar projections associated with BEST1 mutations. (A and B) Confocal images illustrating the molecular pathology of cBest (R25*/R25*; 89 wk) (B) compared with wild-type (A) (42 wk). Retinal cryosections immunolabeled with anti-EZRIN (green) and human cone arrestin (red) and combined with peanut agglutinin lectin (cyan) and DAPI (blue) detail the structural alterations underlying loss of the native extracellular compartmentalization of cone photoreceptor outer segments (B). The RPE apical marker, EZRIN, recognizes both the smaller hair-like projections capping rod OS tips (rod-MV) as well as the more complex and substantial cone-associated MV (cone-MV). The bilayered extracellular ensheathment associated with a single cone OS (COS) is shown in the magnified photomicrographs (Right), along with the corresponding schematic representation. LF, lipofuscin. (Scale bars, 10 μm.) (C) Representative photomicrographs of 6-wk-old canine wild-type and cBest-mutant (R25*/P463fs) retinas immunolabeled with anti-BEST1 (red) and anti-SLC16A1 (green); white arrowheads point to a subset of cone-MV. H&E, hematoxylin & eosin staining; PRL, photoreceptor IS/OS layer. (Scale bars, 20 μm.) (D) Quantification of cone-MV numbers across the retina between cBest-mutant and age-matched control eyes. The y axis represents the average number of cone-MV per square millimeter for each color-coded retinal region examined (i, inferior; N, nasal; S, superior; T, temporal) with error bars ± SD (****P < 0.0001; n = 80 regions of interest per eye).

To evaluate the possibility that the structural cone-MV abnormalities are secondary to cone dysfunction and disease, we examined the RPE–COS interaction in a different canine IRD model: a primary cone photoreceptor channelopathy, CNGB3-associated achromatopsia (43). We first examined the RPE–COS complex at 6 wk of age; CNGB3-mutant retinas, harboring either a missense or locus deletion mutation (43), showed no apparent irregularities at the RPE–PR interface, and the proper localization of RPE apical markers was associated with specific anti-BEST1 labeling (Fig. S1 A and B). Double immunostaining demonstrated specific distribution of EZRIN along cone-MV interdigitating with hCAR-positive yet CNGB3-negative COSs (Fig. S1 A and B). Then we studied the consequences of CNGB3 channel subunit dysfunction in older (ages 57 and 85 wk) mutant retinas, which undergo a gradual cone PR degeneration (44), and found that the microvillar ensheathment of the RPE apical domain still remained largely intact (Fig. S1 C and D).

The findings in CNGB3-mutant retinas suggested that the structural alterations associated with cone-MV ensheathment in cBest were not secondary to a cone defect but specific to the RPE channelopathy triggered by mutations in BEST1. We then focused on the 6-wk time point, which is well before clinical disease onset and near the end of postnatal retinal differentiation in dogs (45) (Fig. 1 C and D). In contrast to the age-matched WT control, the lack of specific basolateral BEST1 immunolabeling in the cBest RPE was associated with a rather smooth apical surface and clearly underdeveloped (vestigial) apical microvilli (Fig. 1C, arrowheads). Quantification of the spatial density of cone-MV and the length of cone- and rod-MV was performed on deconvolved 3D Z-stack projection images at four retinal locations (Fig. 1D). Significant differences (P < 0.0001) in the mean number of cone-MV were found between cBest and WT in each retinal region examined (Fig. 1D). Even though the cone photoreceptor numbers were comparable to controls, the cone-MV in cBest were much fewer in number, sparsely distributed, and consistently appeared shorter and much finer than those in controls regardless of the topographical location. In control (WT) eyes, the average length of cone-MV was 17.4 (±0.25) μm in the tapetal superotemporal quadrant and 12.3 (±0.23) μm in the inferior nontapetal retina, whereas the length of rod-MV was 6.7 (±0.11) μm and 5.3 (±0.27) μm in the tapetal and nontapetal portions of the retina, respectively. In cBest, however, the average length of rare cone-MV extensions identified was substantially reduced (6.0 ± 0.31 and 6.5 ± 0.74 μm in the central tapetal and nontapetal inferior parts, respectively). A quantitative assessment of the minute rod-MV in cBest was beyond the limits of optical resolution.

cBEST1-Mutant Eyes Have Retina-Wide Microdetachments That Expand with Light Exposure.

To determine in vivo correlates of the early RPE–PR interface abnormalities detected by IHC, we used noninvasive imaging with optical coherence tomography (OCT) to evaluate cBest eyes at young ages, well before ophthalmoscopic lesions are detectable. Qualitatively, central retinas of all evaluated eyes showed an additional hyposcattering layer in the outer retina located distal to the outer nuclear layer (ONL) that was not detectable in WT eyes (Fig. 2A, arrow and double arrow). Unexpectedly, the hyposcattering layer was variable with repeated recordings in the same eye within a single experimental session. Further analyses uncovered that the width of the hyposcattering layer was greater in scans obtained toward the end of an imaging session, when the retina would have been exposed to greater retinal irradiance due to intervening autofluorescence imaging performed with bright short-wavelength lights (Fig. 2A, double arrow, more LA). The width of the hyposcattering layer was less in scans obtained early in the imaging session before autofluorescence imaging was performed (Fig. 2A, arrow, less LA).

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Fig. 2.

Light-mediated changes in the outer retinal structure. (A) Cross-sectional imaging along the horizontal meridian through the central area centralis (fovea-like area) in a 15-wk-old normal (WT) dog (Left) and an 11-wk-old cBest (R25*/P463fs) with less (Middle) and more (Right) light adaptation (LA). (Insets) En face reflectance images; the thin white arrows show the superotemporal location of the OCT. The arrowheads and single and double arrows are described in B. (B) Longitudinal reflectivity profiles (LRPs) (average of 85 single LRPs) at 3° nasal from the fovea-like area (T, temporal retina) and nasal edge of the optic nerve head (N, nasal retina) in WT dogs (12 eyes, age 15 to 17 wk) compared with cBest (6 eyes, age 11 wk) with less and more LA. Arrowheads mark IS/OS and RPE/T peaks; single and double arrows mark the additional hyporeflective layer in cBest. (C) Distance between IS/OS and RPE/T peaks in WT and cBest eyes under two LA conditions. Symbols with error bars represent mean (±2 SD) distance for each group of eyes at both locations. (D, Upper) Schematic description of the dark and light adaptation protocol. Animals were dark-adapted (D/A) overnight and OCT imaging was performed. Then, five increasing light exposures (L1 through L5) were used. Following each light exposure, OCT imaging was repeated. (D, Lower) Magnified views of the OCT scans in cBest with overlapping LRPs after overnight dark adaptation (Left) and after the maximum light exposure (Right). Arrowheads and single and double arrows are described in B. (E, Left) Distance between IS/OS and RPE/T peaks after overnight dark adaptation and following increasing exposures to light (L1 to L5, 488 nm; vertical lines) in cBest (n = 3; colored traces represent individual eyes) and WT eyes (gray traces). (E, Right) In a different subset of cBest eyes (n = 3; colored traces), results of using an abbreviated protocol involving only L4 and L5 exposures. (F) Spatial topography of IS/OS-to-RPE/T distance in mean WT compared with two representative cBest eyes [pseudocolor panels; EM356-OS: 297-wk-old cmr1/cmr3 (R25*/P463fs); LH30-OD: 12-wk-old cmr3 (P463fs/P463fs)]. Purple indicates gross retinal detachment. Grayscale panels show the point-by-point difference between each cBest eye and the mean WT.

Quantitative studies were performed by obtaining longitudinal reflectivity profiles and making measurements both at nasal and temporal retinal locations. WT eyes (n = 12, ages 15 to 17 wk) showed outer retinal hyperscattering peaks at the outer plexiform layer (OPL) and the external limiting membrane (ELM) defining the intervening hyposcattering layer as the ONL (Fig. 2B). Distal to the ELM was a hyperscattering peak corresponding to the junction between inner and outer segments of photoreceptors (IS/OS), a major peak originating near the RPE–tapetum interface (RPE/T), and an intervening minor hyperscattering peak corresponding to photoreceptor OS tips, which was often difficult to resolve (Fig. 2B). An abnormal hyposcattering layer (Fig. 2B, arrows) was detectable in cBest eyes (n = 6, age 11 wk) with less light exposure. With greater light exposure, the hyposcattering layer became deeper and more distinct (Fig. 2B, double arrows); both nasal and temporal retinal locations showed the same effect. The distance between the IS/OS and RPE/T peaks (Fig. 2 A and B, arrowheads) was measured. In WT eyes, the distance was 41.3 (±4.5) µm, whereas in cBest eyes this distance was significantly greater (P < 0.001) at 46.8 (±6.7) µm and 45.2 (±6.8) µm (less light exposure) and 55.8 (±10.5) µm and 53.5 (±6.3) µm (more light exposure) for nasal and temporal retinal regions, respectively (Fig. 2C).

Two types of experiments were designed to better understand the thickness of the hyposcattering layer as a function of light exposure. In the main experiment (WT, n = 12, age 15 to 17 wk; cBest, n = 3, age 13 wk), eyes were dark-adapted overnight and then sequential imaging was performed in the dark over a 2-h period with five intervening brief 488-nm light exposures of incrementally greater intensity ranging from very dim lights to moderate lights produced by standard clinical ophthalmic equipment (Fig. 2D). In a shorter experimental protocol, only the highest two light exposures were used in different eyes (cBest, n = 3, age 13 wk). After overnight dark adaptation, IS/OS–RPE/T distance was 40.0 (±4.5) µm in WT eyes, whereas it was 47.1 (±4.8) µm in cBest (Fig. 2E); the difference was statistically significant (P < 0.001). Increasingly brighter light exposures resulted in monotonic expansion of the IS/OS–RPE/T distance in cBest eyes, reaching an apparent plateau of 59.4 (±8.7) µm (Fig. 2E). In WT eyes, the effect of the light exposure was either negligible or small, with the IS/OS–RPE/T distance reaching a plateau of 40.9 (±4.3) µm. Thus, exposure to light appeared to cause an acute retinal microdetachment of up to 18.4 (±8.7) µm in cBest eyes within minutes at an age preceding any detectable ophthalmoscopic findings. The light-mediated microdetachment disappeared over a time span of less than 24 h (Fig. S2).

In preparation for localized gene therapy, retinotopic distribution of light-driven microdetachments was evaluated in fully light-adapted cBest and WT eyes (Fig. 2F). The mean IS/OS–RPE/T distance across WT eyes (n = 4, age 104 wk) was relatively homogeneous across superior and inferior retinal areas, with a clear boundary corresponding to the transition between tapetal and pigmented (nontapetal) retina. Greater distance in the tapetal retina of WT eyes was likely due to differences in the dominant contributors to the hyperscattering peak (tapetum in the tapetal retina versus pigmented RPE). In a cBest eye (R25*/P463fs) at age 297 wk, there was a relatively diffuse retina-wide microdetachment in addition to grossly obvious retinal detachment at the fovea-like region (Fig. 2F, demarcated with purple). In a younger cBest eye (P463fs/P463fs) at age 12 wk, there was a distinct band of greater microdetachment along the visual streak and surrounding the optic nerve head even though no ophthalmoscopic abnormalities were evident. Difference maps between mutant eyes and mean WT demonstrated the spatial distribution of the extent of microdetachments (Fig. 2F, Right).

To assess the potential adverse consequences on photoreceptors, ONL thickness was topographically mapped across the retinal areas with microdetachments (Fig. S3). The microdetachments did not result in thinning of the ONL that would be expected from photoreceptor degeneration. Instead, there was a tendency for the ONL in cBest to be homogeneously thicker than WT; hyperthick regions typically included the central-superior tapetal retina, but could also extend into the inferior nontapetal retina (Fig. S3A). Of importance, the hyperthick regions of the ONL, when examined microscopically, had numbers of PR nuclei that were comparable to controls. This suggests an expansion of internuclear spacing as the likely cause of hyperthick ONL observed by imaging.

Natural History of Canine Bestrophinopathy.

As a prerequisite to assessing gene therapy outcomes, the natural history of cBest was determined from a group of 18 dogs [12 male (M) and 6 female (F); age range 6 to 297 wk] (Table S1). cBest dogs were serially monitored by ophthalmoscopy and noninvasive imaging to detect the onset of earliest disease and understand disease progression. Based on the systematic in vivo imaging, the first disease signs were detected as early as 11 wk of age (mean age of 15 wk) as a subtle focal retinal elevation of the canine fovea-like regions (Fig. S4A). This discrete RPE–PR detachment on cross-sectional imaging, yet unnoticeable on en face imaging, was found to be consistent among cBest eyes examined (n = 34), regardless of genotype. From the subclinical stage, the disease progressed to form a macrodetachment (vitelliform stage) limited to the canine fovea and surrounded by microdetachment (Fig. 3A, Left). The primary lesion gradually evolved to manifest as a characteristic bullous detachment within the area centralis that encompassed the fovea-like region (Fig. 3A, Middle and Right and Fig. S4 B–D). The presence of distinctive hyperautofluorescence was evident in the inferior part of the lesion (Fig. 3A, Middle Insets; pseudohypopyon stage). The advanced disease stages involved a partial resorption and dispersion of the hyperautofluorescent material within the central lesion, associated with significant thinning of ONL (Fig. S4 E and F).

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Fig. 3.

BEST1 gene augmentation therapy results in sustained reversal of foveomacular lesions and restoration of RPE–PR interface structure. (A) Natural history of the central subretinal detachment documented by in vivo imaging in the right eye of cBest dog (EM356-OD; R25*/P463fs) at three time points. A series of representative near-infrared reflectance (NIR) images demonstrate the evolution of the focal lesion (green arrowhead) in the canine macula from the vitelliform stage (Left) through early pseudohypopyon (Middle) to an advanced pseudohypopyon stage (Right). (A, Insets) Autofluorescence and OCT images. (B) Subretinal injection with AAV2-cBEST1 (1.5E10 vg/mL) was performed in the eye shown in A at 52 wk of age, entirely encompassing the lesion depicted in the fundus photograph before injection (b.i.) (Left, Upper Inset). The subretinal bleb area is denoted by the dashed circle. The treated eye showed ophthalmoscopic resolution of lesion 1 wk postinjection (p.i.) (Left, Lower Inset); however, confirmatory OCT scans were not available at this age. Images acquired at 43 (Middle) and 245 wk (Right) p.i. document sustained reversal of the central lesion and fully reattached retina within the treated area. (B, Middle and Right, Insets) Autofluorescence and OCT images. EM356-OD and similar labels designate the individual animal and eye. (Scale bars, 200 μm.) (C and D) Restoration of RPE–photoreceptor interface structure post AAV-hBEST1 treatment in the cBest (R25*/R25*) model in comparison with control. (C and D, Upper) NIR images from EMC3 eyes exhibiting early vitelliform lesions shown at ages 19 (C) and 25 wk (D). Both eyes were injected at 27 wk of age with either BSS (C; EMC3-OD control eye) or AAV2-hBEST1 (2E11 vg/mL) (D; EMC3-OS study eye). Bleb boundaries are marked by dashed circles; the locations of corresponding OCT scans cut through the subretinal lesions before injection or through the matching locations mapped p.i. are marked by green horizontal lines; retinotomy sites are indicated by blue arrowheads. (Scale bars, 200 μm.) (C and D, Lower) Confocal fluorescence images of IHC assessment of AAV-hBEST1– or BSS-injected eyes; in contrast to the BSS control eye (C), cytoskeleton rescue and restoration of RPE–PR interface structure are shown in AAV-treated retina 103 wk p.i. (D). (Scale bars, 10 μm.)

In each case followed by serial imaging (Table S1), cBest manifested bilaterally and nearly always presented a remarkable symmetry, albeit with a variable rate of progression (Fig. 3A and Fig. S4). The gross retinal detachments, ophthalmoscopically visible in both eyes, either remained limited to the central retina or became more extensive with extracentral lesions scattered throughout, still with a strong predilection to the central cone-rich areas and associated with hyperthick ONL.

Subretinal BEST1 Gene Augmentation Therapy Stably Corrects Disease.

To evaluate proof of concept for AAV2-mediated subretinal gene augmentation therapy, 22 cBest eyes were injected [vector titers in the 0.1 to 5E11 vector genomes (vg) per mL range or balanced salt solution (BSS) control] at 27 to 69 wk (Table S1) with a canine (cBEST1) or human (hBEST1) transgene driven by the human VMD2 promoter (46). cBest dogs exhibiting different stages of focal or multifocal retinal detachments were either injected unilaterally with AAV leaving the fellow eye uninjected, or with AAV in one eye and control (BSS) injection in the contralateral eye; three cases manifesting multifocal disease were injected bilaterally with AAV targeting the superotemporal quadrant, while the retinal areas outside of the surgical bleb served as an internal control (Table S1).

A representative result is shown from a compound heterozygous (R25*/P463fs) dog displaying advanced central retinal detachment in the right eye (EM356-OD) (Fig. 3A) that underwent subretinal injection with cBEST1 at the age of 52 wk (Fig. 3B, Left), while the fellow eye was not injected (EM356-OS) (Fig. S4). Both eyes were monitored clinically and by in vivo imaging. Disease reversal was first apparent in the injected eye at 4 wk postinjection (p.i.) and retained a sustained effect long-term, as illustrated at 43 and 245 wk p.i. (Fig. 3B). In this and other cases with advanced disease that presented extensive accumulation of autofluorescent material within the subretinal macrodetachments (n = 13 eyes), hyperautofluorescent signals were still detectable for several months post AAV injection but gradually faded over time (Fig. 3B, Insets). Based on noninvasive imaging, both the focal as well as extracentral lesions within the AAV-BEST1–treated regions resolved 4 to 12 wk p.i., and localized retinal reattachments remained stable thereafter (Table S1). There was no evidence of inflammatory responses in any of the AAV-treated eyes, and the longitudinal in vivo evaluations did not reveal adverse effects on the RPE or neural retina.

AAV-mediated treatment with the hBEST1 also resulted in lesion reversal and long-term disease correction (n = 13 eyes). Representative in vivo imaging results and IHC evaluations (Fig. 3 C and D) from a cBest dog (R25*/R25*) showed that the early bilateral lesions present before treatment disappeared after the study eye (EMC3-OS) was treated with AAV-hBEST1 (2E11 vg/mL) (Fig. 3D), while the lesion in the contralateral control eye (EMC3-OD) injected with BSS continued to enlarge (Fig. 3C). Based on funduscopic examination, in the illustrated example as well as in all other cases the transient retinal detachment associated with vector or BSS delivery resolved within 24 to 48 h p.i.; however, the retinal lesions injected with BSS reappeared as early as 1 wk p.i., and progressed along the natural disease course (Fig. 3C). This was in sharp contrast to the AAV-treated eyes, where both the early as well as more advanced lesions resolved within the first 6 wk after hBEST1 gene therapy, and the treated areas thereafter remained disease-free (Fig. 3D). Ophthalmological examinations and IHC assessments using RPE- and PR-specific markers showed no adverse effects on the retina up to 207 wk p.i. (Fig. 3 and Figs. S5 and S6). Of particular importance, the assessments of the retinal preservation p.i. revealed a remarkable restoration of retinal architecture at the RPE–PR interface, including extension of cone-MV and actin cytoskeleton rescue, corresponding to the vector-treated bleb area with either the canine or human BEST1 transgenes (Fig. 3 C and D, Lower and Figs. S5 and S6). No differences in the clinical picture or response to the AAV-BEST1 treatment were observed between genders.

Correction of Light-Modulated Microdetachments with Gene Therapy.

To understand the consequences of BEST1 gene augmentation therapy on retinal regions without ophthalmoscopically detectable retinal detachments, IS/OS–RPE/T distance was measured topographically within and outside subretinal blebs (Fig. 4). A representative result with a control subretinal BSS injection at age 69 wk in a cBest (R25*/P463fs) dog showed homogeneous microdetachment covering all the imaged retina at age 87 wk (Fig. 4A). The average microdetachment extent (IS/OS–RPE/T distance of the BSS-injected mutant dog subtracted from colocalized measurements performed in WT eyes) was 11.6 μm in the superior retina and 16.7 μm in the inferior retina (Fig. 4B), consistent with uninjected cBest eyes. Subretinal AAV gene therapy, on the other hand, resulted in substantial reduction of the IS/OS–RPE/T distance in treated regions. EMC3-OS, EML4-OS, and LH21-OS demonstrate results in three genotypes treated with gene therapy using the human BEST1 transgene with titers of ∼2E11 vg/mL (Fig. 4A and Table S1). In each case, there was significant reduction of the IS/OS–RPE/T distance in the treated bleb. Notably, gross retinal detachments (purple) were only detectable outside the treatment region (Fig. 4A). Quantitative measurements showed complete amelioration of the microdetachments, with the IS/OS–RPE/T distance returning to WT levels both in superior and inferior retinal regions treated with subretinal gene therapy (Fig. 4B, filled symbols) but not in retinal regions away from the treatment bleb (Fig. 4B, unfilled symbols).

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Fig. 4.

Reversal of microdetachments across retinal regions after subretinal gene therapy. (A) Pseudocolor maps of IS/OS–RPE/T distance topography in cBest-mutant dogs [cmr1 (R25*/R25*), cmr1/cmr3 (R25*/P463fs), or cmr3 (P463fs/P463fs)] subretinally injected with BSS or AAV-hBEST1. All imaging was performed in fully light-adapted eyes. Treatment boundaries are based on fundus photographs of the bleb taken at the time of the injection (dotted lines) and, if visible, demarcations apparent at the time of imaging (dashed lines). All eyes are shown as equivalent right eyes with optic nerve and major blood vessels (black), tapetum boundary (yellow), and fovea-like region (white ellipse) overlaid for ease of comparison. EML9-OS and similar labels designate the individual animal and eye. Purple color depicts gross retinal detachment with an IS/OS–RPE/T distance of >100 μm. (B) The IS/OS–RPE/T distance difference from WT at the superior (Left) and inferior (Right) retinal locations in cBest eyes within the treated bleb (Tx; filled symbols) and untreated outside bleb (Ctrl; open symbols) regions. Dashed lines delimit the 95th percentile of normal variability. All AAV-exposed Tx regions are significantly (P < 0.001) different from untreated Ctrl. (C and E) Examples of eyes where the treatment effect extended well beyond the initial treatment bleb. Pseudocolor topographies of the IS/OS–RPE/T distance are shown pre- (Left) and posttreatment (Right). Purple represents gross retinal detachments. (C and E, Upper) Grayscale maps of the difference between each cBest eye and mean WT control. White represents gross retinal detachments. (D and F) Measurements of the colocalized difference of IS/OS–RPE/T distance between WT and cBest pre- (PreTx) and posttreatment (Tx) for the eyes shown in C and E, respectively. (D and F, Insets) Locations sampled within (PreTx and Tx) and outside (Ctrl) the blebs in the superior and inferior retinas. Note that Ctrl regions in EML9-OD show a definite treatment effect likely due to the diffuse microdetachment allowing for widespread vector-driven transduction.

The region of efficacy with subretinal gene therapy is often shown to extend beyond the bleb formed at the time of the surgery to include a penumbral region (47, 48). In cBest dogs with successful gene therapy, there was also a penumbral region but it appeared to be qualitatively larger than typically encountered previously (Fig. 4A). In some of the most extreme examples, pretreatment maps of retina-wide microdetachment were found to be necessary to demonstrate the extent of penumbral expansion. EML9-OD, for example, at age 29 wk showed a retina-wide microdetachment that was most extreme along the visual streak and included several regions with gross retinal detachments (Fig. 4C). Gene therapy was performed at 69 wk. At 87 wk, microdetachments across the whole retina imaged as well as the majority of the gross retinal detachments had disappeared (Fig. 4C), and quantitative measures showed a normal or thinner IS/OS–RPE/T distance in superior and inferior retinal locations (Fig. 4D). Importantly, IS/OS–RPE/T distance showed substantial improvements at retinal locations corresponding to the bleb formed at the time of the injection as well as in nasal retinal control regions in the same eye. This extreme example of penumbral expansion is likely explained by greater diffusion of the vector via the microdetachment in cBest eyes that resulted in the transduction of the RPE at sites substantially more distant than the initial bleb. A more typical example with a delimited penumbral expansion is illustrated for comparison. EML13-OS at age 37 wk showed microdetachments retina-wide that were especially prominent in the temporal retina and along the visual streak; there were also several gross retinal detachments along the visual streak (Fig. 4E). Gene therapy was performed at 45 wk. At 81 wk of age, both superior and inferior retina temporal to the optic nerve was lacking micro- and macrodetachments, whereas the untreated nasal retina had retained the microdetachments as well as formed a large number of macrodetachments (Fig. 4E). Quantitative results confirmed the treatment effect (Fig. 4F), which did not reach the nasal retina, unlike EML9-OD.

To understand the potential consequences of gene therapy on retinal degeneration, ONL thickness was mapped across treated eyes (Fig. S3B). Treated retinal regions showing disappearance of microdetachments tended to also correspond to normal ONL thickness, whereas untreated regions retaining microdetachments tended to show hyperthick or normal, or in some regions, thinned ONL (Fig. S3B). In summary, AAV-mediated gene augmentation therapy in canine bestrophinopathies appears to promote a sustained reversal of gross retinal detachments, reestablishment of a close contact between RPE and PRs, and return of ONL thickness to normal values.

Human Autosomal Recessive Bestrophinopathies: Structure and Function.

To facilitate clinical translation of successful gene therapy in BEST1-mutant dogs, studies were performed to better understand the human pathophysiology of autosomal recessive bestrophinopathies (ARBs) and to gain insight into the distribution of retina-wide disease beyond the gross ophthalmoscopically detectable lesions previously described. Data are shown (Fig. 5) from two patients: P1 was a 39-y-old woman with a best corrected visual acuity of 20/100 carrying biallelic BEST1 mutations (c.341T>C/c.400C>G), whereas P2 was a 36-y-old man with 20/60 acuity also carrying biallelic mutations (c.95T>C/c.102C>T) in BEST1. In both patients, mutant alleles segregated with clinically unaffected parents. Ultrawide imaging of RPE health taking advantage of the natural autofluorescence of lipofuscin granules they contain showed widespread and extensive abnormalities consisting of regions of relative hyper- or hypoautofluorescence and local heterogeneity. Of note, there was a distinct transition zone (Fig. 5A, arrowheads) in the nasal midperipheral retina demarcating the healthier nasal peripheral retina.

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Fig. 5.

Retinotopic phenotype in patients with ARB. (A) RPE health across the retinas of two ARB patients, P1 and P2, imaged with short-wavelength reduced-illuminance autofluorescence imaging (SW-RAFI), taking advantage of the natural RPE fluorophore lipofuscin. White arrows depict the location of the perimetric profiles and OCT scans; red and green rectangles show the regions of interest shown in other panels; and black arrowheads demarcate disease-to-health transition in the nasal midperipheral retina. (A, Inset) Normal SW-RAFI for reference. (B) Perimetric light sensitivity of rods in dark-adapted (Upper) and cones in light-adapted (Lower) eyes measured across the horizontal meridian. Gray regions represent normal sensitivity except for the physiological blindspot corresponding to the optic nerve (ONH). (C) Retinal cross-section with OCT along the horizontal meridian crossing the fovea. (C, Inset) Normal OCT for reference. (D and E) Detail of outer retinal lamination in patients compared with normal at the two regions of interest at the parapapillary retina (D, red) and midperipheral nasal retina (E, green). Layers and peaks are highlighted for visibility. Orange indicates interface near COS tips, blue indicates interface near ROS tips and RPE apical processes, brick indicates interface near the RPE and Bruch’s membrane. (F and G) Dark-adaptation kinetics measured in P1 at the parapapillary locus (F) and in P2 at the nasal midperipheral locus (G). Time 0 refers to the end of adaptation light. Baseline thresholds obtained after extended dark adaptation are shown before time 0.

Rod and cone function was sampled at high density along the horizontal meridian to better understand the topography of vision loss and its correspondence to retinal structural abnormalities. Both patients demonstrated a deep (>3 log) loss of rod-mediated sensitivity centrally in long-term dark-adapted eyes; there was relative preservation of rod function in the temporal field (nasal retina) in both patients and the parapapillary area in one patient (Fig. 5B, Upper). Surprisingly, cone-mediated function in light-adapted eyes demonstrated only a moderate loss (<1 log) or normal or near-normal results (Fig. 5B, Lower). Rod and cone function sampled across the full extent of the visual field corroborated and extended these findings and showed strong interocular symmetry (Fig. S7).

Cross-sectional imaging with OCT was performed to evaluate the retinal lamination abnormalities along the horizontal meridian crossing the fovea (Fig. 5C). There was not a consistent light-exposure history at the time of OCT imaging. Both patients showed a significant loss of ONL and abnormalities at the level of photoreceptor IS/OS in the outer retina across most of the central retina. P2, in addition, showed intraretinal cystic spaces and the detachment of the central retina from the RPE, likely due to accumulation of subretinal fluid. The retinal lamination showed relative normalization in the parapapillary region (Fig. 5C, red rectangles) and beyond the nasal midperipheral transition (Fig. 5C, green rectangles). Analyses of the two regions of interest showed detectable but abnormally thinned ONL, and detectable IS/OS and cone outer segment tip (COST) with low peak signal in both patients (Fig. 5 D and E). In P1, the distances from the ELM to IS/OS and IS/OS to COST were comparable to normal. There appeared to be a hyposcattering layer distal to COST, and the RPE appeared hyperthick (Fig. 5D, Middle). In P2, ELM to IS/OS appeared shorter than normal, whereas IS/OS-to-COST distance was comparable to normal. COST-to-ROST/RPE distance appeared greater than normal with an intervening indistinct hyposcattering layer; the RPE appeared to be comparable in thickness to normal (Fig. 5D, Right). Analysis of the outer retina in the nasal midperipheral region in P1 showed ELM-to-IS/OS, IS/OS-to-COST, and COST-to-ROST/RPE distances greater than normal and RPE thickness which was comparable to normal (Fig. 5E, Middle). P2 features appeared intermediate between P1 and normal (Fig. 5E, Right).

To understand the implications of structural abnormalities at the level of the outer retina and RPE for the kinetics of retinoid transfer between these cellular layers, dark-adaptation testing was performed. At the parapapillary location shown in Fig. 5D, dark-adapted thresholds of P1 were rod-mediated but 1.3 log unit-elevated (Fig. 5F). By 22.5 min following a light exposure, the P1 results had remained cone-mediated on a plateau whereas normal was already within 1 log unit of the final dark-adapted threshold. By 50 min, P1 rod results were still 1 log-elevated whereas normal recovery was complete (Fig. 5F). At the midperipheral nasal retinal location shown in Fig. 5E, dark-adapted thresholds of P2 were rod-mediated and ∼0.5 log unit-elevated compared with normal (Fig. 5G). By 14.5 min following a light exposure, there was first evidence of rod function which was only incrementally slower than the 11-min cone–rod break in normal. The rate of rod recovery was similar to normal (Fig. 5G). In summary, rod dark-adaptation kinetics of P1 at the parapapillary locus showed an extremely slow time course, whereas dark-adaptation kinetics of rod function of P2 at the midperipheral locus was closer to normal (Fig. 5 F and G).