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Purpose: Subretinal (SR) injection in porcine models is a promising avenue for preclinical evaluation of cell and gene therapies. Targeting of the subretinal fluid compartment (bleb) is critical to the procedure, especially if treatment of the cone-rich area centralis is required (i.e., visual streak [VS] in pigs). To our knowledge, this study is the first to investigate the influence of injection site placement on VS involvement in the pig eye., Methods: We performed 23-gauge pars plana vitrectomy followed by SR injection in 41 eyes of 21 animals (Sus scrofa domesticus). In 27 eyes (65.9%), the injection site was placed superior to the VS, and in 14 eyes (34.1%) it was placed inferior to it. Using intraoperative imaging, blebs were classified based on their propagation behavior relative to the VS., Results: In 79% of cases, blebs from inferior injection sites developed away from the VS, exhibiting a mean ± SEM vertical anisotropy (AP) of 0.67 ± 0.11. In contrast, blebs from superior injection sites tended to develop toward the VS with an AP of 1.27 ± 0.18 (P = 0.0070). Blebs developed away from the VS in only 41% of injections (P = 0.0212). Inferior blebs were orientated close to 0° (horizontal), whereas superior blebs displayed varied orientations with a mean angle of 56° (P = 0.0008)., Conclusions: Bleb propagation was anisotropic (i.e., directionally biased) and dependent on injection site placement. Superior injection sites led to superior VS detachment. Morphological analysis suggested increased adhesion forces at the VS and superior vascular arcades. This study will aid the planning of surgeries for targeted subretinal delivery in pig models.

Purpose: Autosomal recessive retinitis pigmentosa (arRP) can be caused by mutations in the phosphodiesterase 6A (PDE6A) gene. Here, we describe the natural course of disease progression with respect to central retinal function (i.e., visual acuity, contrast sensitivity, and color vision) and establish a detailed genotype--phenotype correlation., Methods: Forty-four patients (26 females; mean age ± SD, 43 ± 13 years) with a confirmed genetic diagnosis of PDE6A-associated arRP underwent comprehensive ophthalmological examinations including best-corrected visual acuity (BCVA) with Early Treatment Diabetic Retinopathy Study charts, contrast sensitivity (CS) with Pelli-Robson charts at distances of 3 m and 1 m, and color vision testing using Roth 28-Hue and Panel D-15 saturated color cups., Results: The most frequently observed variants were c.998+1G>A/p.?, c.304C>A/p.R102S, and c.2053G>A/p.V685M. Central retinal function in patients homozygous for variant c.304C>A/p.R102S was better when compared to patients homozygous for variant c.998+1G>A/p.?, although the former were older at baseline. Central retinal function was similar in patients homozygous for variant c.304C>A/p.R102S and patients heterozygous for variants c.304C>A/p.R102S and c.2053G>A/p.V685M, although the latter were younger at baseline. Annual decline rates in central retinal function were small., Conclusions: We conclude that the severity of the different disease-causing PDE6A mutations in humans with respect to central visual function may be ranked as follows: c.2053G>A/p.V685M in homozygous state (most severe) > c.998+1G>A/p.? in homozygous state > c.304C>A/p.R102S and c.2053G>A/p.V685M in compound-heterozygous state > c.304C>A/p.R102S in homozygous state (mildest). The assessment of treatment efficacy in interventional trials will remain challenging due to small annual decline rates in central retinal function.

Purpose: To study longitudinal changes of anti-drug antibody (ADA) titers to recombinant adeno-associated virus serotype 8 (rAAV8) capsid epitopes in nonhuman primates (NHP) and patients., Methods: Three groups of six NHP each received subretinal injections (high dose: 1 × 1012 vector genomes [vg], low dose: 1 × 1011 vg, or vehicle only). Four additional animals received intravitreal injections of the high dose (1 × 1012 vg). Three patients received 1 × 1010 vg as subretinal injections. ELISA quantified ADA levels at baseline and 1, 2, 3, 7, 28, and 90 days after surgery in NHP and at baseline and 1, 3, and 6 months after surgery in patients., Results: Two out of 22 animals lacked ADA titers at baseline and developed low ADA titers toward the end of the study. Titers in the low-dose group stayed constant, while two of six animals from the high-dose group developed titers that rose beyond the range of the assay. All animals from the intravitreal control group showed a rise in ADA titer by day 7 that peaked at day 28. Preliminary data from the clinical trial (NCT02610582) show no humoral immune response in patients following subretinal delivery of 1 × 1010 vg., Conclusions: No significant induction of ADA occurred in NHP when mimicking the clinical scenario of subretinal delivery with a clinical-grade rAAV8 and concomitant immunosuppression. Likewise, clinical data showed no humoral immune response in patients. In contrast, intravitreal delivery was associated with a substantial humoral immune response. Subretinal delivery might be superior to an intravitreal application regarding immunologic aspects.

Purpose: To investigate shedding and biodistribution characteristics of recombinant adeno-associated virus serotype 8 (rAAV8) after single-dose subretinal or intravitreal injection in nonhuman primates (NHP, Macaca fascicularis) as a surrogate for environmental hazard and patient safety., Methods: In a study for regulatory submission, 22 NHP were divided into four cohorts receiving either single subretinal injections of vehicle or clinical grade rAAV8 (1 × 1011 or 1 × 1012 vector genomes [vg]) versus single intravitreal application of 1 × 1012 vg. Viral shedding and biodistribution were monitored in biofluids for up to 91 days, followed by necropsy and tissue harvesting of all major organs, the visual pathway, and lymphatic tissue. Quantification of vector genomes was done by quantitative (q)PCR., Results: Shedding occurred in a dose-dependent manner in all biofluids and persisted for a maximum of 7 days. Intravitreal delivery led to increased and persistent (up to 13 weeks) distribution of vector genomes in blood and draining lymphatic tissue, increased off-target deposition, and inefficient gene transfer to the retina. No vector targeting of the germ line was observed in any cohort., Conclusions: These data illustrate that subretinal application of rAAV8 leads to a more favorable biodistribution profile compared to intravitreal injections. Extraocular biodistribution is limited after subretinal delivery, while intravitreal injection leads to both greater and more persistent systemic exposure, evident in blood and lymphatic tissues. With the knowledge on the dynamics of shedding in a setting mimicking clinical application, guidelines can be developed to refine clinical trial protocols to reduce the risk for trial subjects and their environment.

Purpose: Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause 70% to 90% of X-linked retinitis pigmentosa (XLRP3) cases, making this gene a high-yield target for gene therapy. This study analyzed the utility of relevant clinical biomarkers to assess symmetry and rate of progression in XLRP3., Methods: A retrospective, cross-sectional analysis of 50 XLRP3 patients extracted clinical data including visual acuity (VA), visual fields (I4e and III4e targets), foveal thickness, and ERG data points alongside molecular genetic data. Symmetry was assessed by using linear regression analysis. Kaplan-Meier survival curves (KMCs) and generalized linear mixed model calculations were used to describe disease progression., Results: Ninety-six percent of patients exhibited a rod-cone phenotype, and 4% a cone-rod phenotype. Open reading frame 15 (ORF15) was confirmed as a mutational hotspot within RPGR harboring 73% of exonic mutations. Significant variability, but no clear genotype-phenotype relationship, could be shown between mutations located in exons 1-14 versus ORF15. All biomarkers suggested a high degree of symmetry between eyes but demonstrated different estimates of disease progression. VA and foveal thickness, followed by perimetry III4e, were the most useful endpoints to evaluate progression. KMC estimates predicted a loss of 6/6 vision at a mean of 34 years (±2.9; 95% confidence interval)., Conclusions: XLRP3 affects retinal structure and function symmetrically, supporting the use of the fellow eye as an internal control in interventional trials. VA and kinetic visual fields (III4e) seem promising functional outcome measures to assess disease progression. KMC analysis predicted the most severe decline in vision between the third and fourth decade of life.

Purpose: Despite ever-growing adoption of subretinal (SRi) and intravitreal injections (IVTi) in ocular gene therapy trials, concerns regarding possible deleterious effects of the SRi on the outer retina are yet to be addressed. SRi offers several advantages over IVTi, such as a better photoreceptor transduction efficiency and a limited off-target exposure. We assessed structural changes in the outer retina in nonhuman primates following either SRi or IVTi of a gene therapeutic or control solution and compared both techniques in a noninferiority analysis., Methods: In a toxicology study, 22 cynomolgus monkeys underwent single intraocular injections with rAAV2/8 or vehicle; 18 animals received SRi, 4 animals received IVTi. Outer nuclear layer (ONL) thickness change on optical coherence tomography was used for a noninferiority analysis. Preservation of the physiological foveal bulge was used as a secondary outcome measure., Results: The average ONL change from baseline after 2 weeks was -6.54 ± 5.16 (mean ± SD μm) and +1.50 ± 4.36 for SRi and IVTi groups accordingly. At 13 weeks, the SRi group maintained a difference of -6.54 ± 9.66 while IVTi group gained +1.00 ± 4.24. The ellipsoid zone line was transiently lost after SRi and completely recovered by 13 weeks in 77% of eyes. One SRi case resulted in subfoveal pigment accumulation and 39% ONL thinning., Conclusions: Despite limited ONL thinning following SRi, the observed effect was under the predefined clinical significance threshold. The SRi has proven not to be inferior to the IVTi in terms of ONL thickness loss and estimated loss of visual acuity.

Purpose: To establish a feasible and sensitive pupillographic protocol to assess outer and inner retinal function for the first gene therapy trial in achromatopsia patients (ACHM) with mutations in CNGA3., Methods: Twenty-seven CNGA3-ACHM patients and 22 age-matched control subjects were tested using chromatic pupillography. Three different protocols were established to assess the pupillary light reflex parameters and to create the final protocol. In the individual protocols, various stimulus parameters (i.e., intensity, duration, wavelength, adaptation states) were applied to evaluate the impact of these stimuli on the pupillary response in untreated ACHM patients., Results: In the light-adapted conditions, CNGA3-ACHM patients showed significantly reduced maximal amplitudes compared with the control group when using a 1-second high intensity (28-lux corneal illumination) blue or red stimulus (P < 0.005). In the dark-adapted conditions, CNGA3-ACHM patients unexpectedly revealed significantly increased maximal amplitudes when stimulating with red (1 second) or blue (4 ms and 1 second) stimuli of low intensity (0.01-lux corneal illumination; P < 0.05). Pupil responses of CNGA3-ACHM patients after high intensity (28 lux) red and blue 1-second stimuli were within the normal range., Conclusions: Chromatic pupillography demonstrated significant reduced pupil responses to stimuli addressing primarily cone function, an increased sensitivity to rod-favoring stimuli and evidence for disinhibition of intrinsically photosensitive retinal ganglion cells in CNGA3-ACHM patients. A final protocol was established based on these findings. These conclusions may be useful for the objective assessment of efficacy gained by gene therapy or other innovative interventions in this hereditary retinal disorder.

Purpose: This study aimed to quantify the impact of high altitude on choroidal thickness and relate changes of altered choroidal blood flow to clinical parameters and acute mountain sickness (AMS). This work is related to the Tübingen High Altitude Ophthalmology (THAO) study., Methods: Enhanced depth imaging spectral-domain optical coherence tomography was used to quantify macular choroidal layer thickness. Peripheral oxygen saturation, heart rate, and AMS scores were assessed in eight healthy subjects at baseline (altitude, 341 m) and at altitude (4559 m) for respective correlations., Results: Longitudinal analysis revealed a significant (P = 0.011, ANOVA) increase in central choroidal thickness (CCT) during altitude exposure (CCT baseline = 271 ± 9 μm; CCT altitude = 288 ± 9 μm) due to an increased choroidal blood flow. Incidence of AMS at altitude was 50%, peripheral oxygen saturation decreased by 25%, and heart rate increased by 39%. All changes were completely reversible after descent to low altitudes., Conclusions: A small but significant increase in choroidal thickness was observed upon acute altitude exposure to 4559 m. This increase in choroidal blood flow was not related to AMS and was fully reversible after return to low altitude.

Purpose: This study aimed to quantify the impact of exposure to high altitude on individual layers of the cornea in regard to central corneal thickness (CCT) and the geometry of the anterior chamber angle (ACA). This work is related to the Tübingen High Altitude Ophthalmology study., Methods: Anterior segment spectral domain optical coherence tomography was used to quantify changes in individual corneal layers and to study ACA and angle opening distance (AOD). Peripheral oxygen saturation, heart rate, and scores of acute mountain sickness (AMS) were assessed in 14 healthy subjects at baseline (341 m) and altitude (4559 m) for respective correlations., Results: Longitudinal analysis revealed a significant (P < 0.05) increase of CCT during altitude exposure (CCT(baseline) = 539.27 ± 32.30 μm; CCT(day1) = 558.87 ± 29.39 μm; CCT(day3) = 567.17 ± 33.40 μm; mean ± SD) due to stromal edema. This change was completely reversible upon descent. Geometric measures of aqueous outflow structures remained consistent with no significant changes in AOD or ACA. Incidence of AMS on day 1 was 64% followed by a decrease in AMS scores over time spent at high altitude; while AMS correlated significantly with stromal edema formation just after arrival (r = 0.71; P = 0.01), no correlation was found on day 3 (r = 0.05; P = 0.87); no correlations were found for vital parameters., Conclusions: Significant stromal edema was found during exposure to high altitude in healthy subjects. This seems to occur due to decreased atmospheric pressure under hypoxia but independent of systemic acclimatization. Other measures of anterior chamber geometry remained stable during the challenge to hypoxic conditions at high altitude.

Purpose: Transcorneal electrical stimulation (TES) has been beneficial in several neurodegenerative ocular diseases, but the exact mechanisms remain to be elucidated. This study was conducted to investigate the effects of TES on the retinas of wild-type Brown Norway (BN) rats by gene expression profiling and to assess its effects on retinal function and morphology., Methods: TES was applied to BN wild-type rat retinas in vivo for 1 hour (1-ms biphasic pulses at 20 Hz; 200 μA). RNA was isolated and processed for microarray-based profiling 4 hours after TES; differentially expressed genes from TES compared with those from sham-treated animals were validated by quantitative real-time polymerase chain reaction. Furthermore, the effect of TES was assessed at the structural and functional levels using electroretinography, confocal scanning laser ophthalmoscopy, optical coherence tomography, and immunohistochemistry., Results: Transcriptome changes associated with TES versus sham-stimulated BN wild-type retina were identified. Four hundred ninety genes were differentially expressed in TES and included potentially neuroprotective genes such as Bax or members of the tumor necrosis factor family (Tnfrsf11b, Tnrsf12a, Tnfsf13b, Tnfsf13). ERG recordings showed physiological retinal function after TES, and structural in vivo and ex vivo studies revealed intact retinal anatomy., Conclusions: These results demonstrate that TES applied to the retina of the wild-type BN rats induces distinct transcriptome level changes and may help in the understanding of the mechanisms underlying TES. In addition, TES treatment indicates no negative effect on structure and function of the wild-type BN retina up to 35 hours after application.

Purpose: Retinal blood vessel diameter and arteriovenous ratio (AVR) are commonly used diagnostic parameters. Because vascular walls are typically not visible in funduscopy, clinical AVR estimation is based on the lumen rather than the entire vessel diameter. Here the authors used a transgenic mouse model to quantify AVR in vivo based on total vessel dimensions (wall and lumen)., Methods: Confocal scanning laser ophthalmoscopy (cSLO) and indocyanine green angiography of the retinal vasculature were performed in wild-type and transgenic mice expressing green fluorescent protein (GFP) under the transcriptional control of the smooth muscle type α-actin (αSMA) promoter. Spectral-domain-OCT and ERG were performed to control for integrity of retinal structure and function in vivo and histology to demonstrate the location of GFP expression., Results: Native cSLO imaging and angiography yielded only inner vessel diameters similar to those observed through clinical funduscopy. In αSMA-GFP mice, autofluorescence imaging of the GFP-marked vascular walls also allowed the determination of outer vessel diameters. The mean AVR based on either inner diameter (AVR(id) = 0.72 ± 0.08) or outer diameter (AVR(od) = 0.97 ± 0.09) measurements were significantly different (P < 0.01)., Conclusions: Transgenic αSMA-GFP expression in murine vessel wall components allowed quantification of retinal vessel outer diameters in vivo. Although arterioles and venules differ in lumen and vessel wall width, they share a common outer diameter, leading to an AVR(od) close to unity. Because vessel walls are primary targets in common hypertensive and metabolic diseases, αSMA-GFP transgenic mice may prove valuable in the detailed assessment of such disorders in vivo.

Purpose: To identify individual cone photoreceptors in a transgenic mouse line in vivo based on selective expression of green fluorescent protein (GFP) using cSLO (confocal scanning laser ophthalmoscopy) and to use this approach to monitor cone cell fate in mouse models of retinal degeneration., Methods: Transgenic mice expressing GFP under the control of a red-green opsin promoter (RG-GFP mice) were analyzed in vivo with respect to GFP expression in cone cells using cSLO and functional integrity using electroretinography (ERG). Histology was performed to correlate the pattern of GFP expression with light microscopic data. Longitudinal monitoring of cone survival was evaluated in crossbreds of RG-GFP mice with cpfl1 and Rpe65(-/-) mutant mice, respectively., Results: The authors found that RG-GFP transgenic mice had a stable GFP expression that did not interfere with retinal function up to at least 3 months of age. Thus, a longitudinal analysis of cone degeneration in individual RG cpfl1 and RG Rpe65(-/-) cross-bred mice in vivo was successfully performed and demonstrated distinct time frames of cone survival in the particular mouse model., Conclusions: Monitoring GFP expression in cone photoreceptor cells, such as in the RG-GFP mouse, is a promising in vivo approach for the analysis of cone survival in mice.

Purpose: Spectral domain optical coherence tomography (SD-OCT) allows cross-sectional visualization of retinal structures in vivo. Here, the authors report the efficacy of a commercially available SD-OCT device to study mouse models of retinal degeneration., Methods: C57BL/6 and BALB/c wild-type mice and three different mouse models of hereditary retinal degeneration (Rho(-/-), rd1, RPE65(-/-)) were investigated using confocal scanning laser ophthalmoscopy (cSLO) for en face visualization and SD-OCT for cross-sectional imaging of retinal structures. Histology was performed to correlate structural findings in SD-OCT with light microscopic data., Results: In C57BL/6 and BALB/c mice, cSLO and SD-OCT imaging provided structural details of frequently used control animals (central retinal thickness, CRT(C57BL/6) = 237 +/- 2 microm and CRT(BALB/c) = 211 +/- 10 microm). RPE65(-/-) mice at 11 months of age showed a significant reduction of retinal thickness (CRT(RPE65) = 193 +/- 2 microm) with thinning of the outer nuclear layer. Rho(-/-) mice at P28 demonstrated degenerative changes mainly in the outer retinal layers (CRT(Rho) = 193 +/- 2 microm). Examining rd1 animals before and after the onset of retinal degeneration allowed monitoring of disease progression (CRT(rd1 P11) = 246 +/- 4 microm, CRT(rd1 P28) = 143 +/- 4 microm). Correlation of CRT assessed by histology and SD-OCT was high (r(2) = 0.897)., Conclusions: The authors demonstrated cross-sectional visualization of retinal structures in wild-type mice and mouse models for retinal degeneration in vivo using a commercially available SD-OCT device. This method will help to reduce numbers of animals needed per study by allowing longitudinal study designs and will facilitate characterization of disease dynamics and evaluation of putative therapeutic effects after experimental interventions.

Purpose: To correlate the dimension of the visual field (VF) tested by Goldman kinetic perimetry with the extent of visibility of the highly reflective layer between inner and outer segments of photoreceptors (IOS) seen in optical coherence tomography (OCT) images in patients with retinitis pigmentosa (RP)., Methods: In a retrospectively designed cross-sectional study, 18 eyes of 18 patients with RP were examined with OCT and Goldmann perimetry using test target I4e and compared with 18 eyes of 18 control subjects. A-scans of raw scan data of Stratus OCT images (Carl Zeiss Meditec, AG, Oberkochen, Germany) were quantitatively analyzed for the presence of the signal generated by the highly reflective layer between the IOS in OCT images. Starting in the fovea, the distance to which this signal was detectable was measured. Visual fields were analyzed by measuring the distance from the center point to isopter I4e. OCT and visual field data were analyzed in a clockwise fashion every 30 degrees , and corresponding measures were correlated., Results: In corresponding alignments, the distance from the center point to isopter I4e and the distance to which the highly reflective signal from the IOS can be detected correlate significantly (r = 0.75, P < 0.0001). The greater the distance in VF, the greater the distance measured in OCT., Conclusions: The authors hypothesize that the retinal structure from which the highly reflective layer between the IOS emanates is of critical importance for visual and photoreceptor function. Further research is warranted to determine whether this may be useful as an objective marker of progression of retinal degeneration in patients with RP.