Improved Rod Sensitivity as Assessed by Two-Color Dark-Adapted Perimetry in Patients With RPE65-Related Retinopathy Treated With Voretigene Neparvovec-rzyl

Abstract

Purpose: The purpose of this study was to evaluate rod-mediated function with two-color dark-adapted perimetry (2cDAP) in patients with RPE65-related retinopathy treated with voretigene neparvovec-rzyl.

Methods: Following dilation and dark adaptation, 2cDAP and FST were performed. The 2cDAP was measured on an Octopus 900 perimeter (Haag-Streit) with cyan (500 nm wavelength) and red (650 nm wavelength) stimuli. Hill of vision (HOV) analysis was performed on 2cDAP perimetry with Visual Field Modeling and Analysis (VFMA). Full field threshold stimulus testing (FST) was also measured as a secondary measure of rod-mediated function, and assessed on a Diagnosys Espion with the ColorDome stimulator (Diagnosys LLC).

Results: Eight eyes from 4 patients who were treated with voretigene bilaterally had rod function assessed by 2cDAP testing at least 1 year after treatment. There was statistically significant improvement in 2cDAP following gene augmentation therapy. HOV VFMA analysis showed widespread improvements that extended beyond the treatment bleb and statistically significant improvement in HOV analysis volumetric measurements post-treatment to cyan and red stimuli. FST testing performed in six eyes from three patients demonstrated statistically significant improvement to all chromatic stimuli following treatment.

Conclusions: These findings demonstrated statistically significant improvement in 2cDAP and FST following treatment with voretigene.

Translational Relevance: These findings provide a sensitive method of assessing rod-mediated function in a topographic manner that may be useful in future clinical trials for inherited retinal dystrophies.

Introduction

The approval of voretigene neparvovec-rzyl as the first gene augmentation therapy for RPE65-related retinopathy heralded a new age for patients with inherited retinal dystrophies. As a wider spectrum of patients are treated, it has become apparent that novel outcome measures beyond those deployed in the original clinical trial are needed to better understand the efficacy of this treatment. We were specifically interested in measurements that could isolate improvement of rod function from cone function in a topographic manner. Many traditional measurements of visual function do not adequately separate rod and cone function. For example, kinetic fields, such as Goldmann, are performed under photopic conditions with 10 cd/m² background and microperimetry is performed under mesopic conditions with a 1.27 cd/m² background. Although dark-adapted, the multi-luminance mobility test (MLMT) is performed over a wide range of luminances such that the boundary between rod and cone mediated function cannot be determined. Full field electroretinograms (ERG) can separate rod from cone function, but it is often extinguished in patients with RPE65 mutations and does not provide topographic measurements. Similarly, full field stimulus thresholds (FST) are able to delineate rod function in this patient population but do not provide topographical information. 

Assessment of rod function with chromatic dark-adapted perimetry has been historically performed using a variety of methods since the 1980s. Due to the inherent rod dysfunction associated with retinitis pigmentosa (RP), chromatic dark-adapted perimetry was a useful method to isolate rod function in this disease. Early methodologies utilized perimeters modified with chromatic red (650 nm) and blue-green filters (500 nm), such as the Tubinger perimeter that evaluated threshold sensitivity along the vertical and horizontal meridians,^1–4 or the Lister perimeter.^5,6 More recent use of automated static perimeters with chromatic stimuli on the modified Humphrey perimeter,^7–9 the Medmont dark-adapted chromatic perimeter (Medmont Pty. Ltd. International, Victoria, Australia), and the Octopus perimeter (Haag Streit, Köniz, Switzerland) have provided a means to more readily evaluate rod function as an end point for clinical trials for inherited retinal dystrophies, such as in Stargardt disease^10–12 and achromatopsia.¹³ 

Chromatic dark-adapted perimetry on the Octopus perimeter allows the full-field to be tested without the patient having to eccentrically fixate. We further utilized visual field modeling analysis (VFMA) of chromatic dark-adapted perimetry, which provides a three-dimensional hill of vision (HOV) analysis model of perimetry and quantitative visual function indices for both the entire visual field and specified regions of interest.¹⁴ 

In this study, patients with RPE65-LCA were treated with voretigene neparvovec-rzyl, and rod-mediated function was evaluated using two-color dark-adapted perimetry (2cDAP) with VFMA. With VFMA three-dimensional topographical modeling, we observed widespread improvement of rod-mediated function that extended beyond the treatment bleb in six out of eight eyes. 

Methods

This study was approved by the Institutional Review Board of Oregon Health & Science University and met the tenets of the Declaration of Helsinki. All subjects provided written informed consent prior to completing any study procedures. 

Testing Strategy

Two-color dark-adapted perimetry (2cDAP) was performed using a modified Octopus 900 Perimeter (Haag-Streit, Köniz Switzerland) using 500 nm (cyan) and 650 nm (red) filters. Light baffling was used on the perimeter and in the testing room to prevent ambient light escape. Stray light was measured with an IL-1700 that measured ambient light at 1.54⁻⁴ cd/m². We used a 78-point grid that was evenly spaced with 11 points horizontal and 8 points vertical at the meridians with size V Goldmann targets and a 4-2-1 testing strategy. The maximum luminance (i.e. the 0 dB luminance) values were 174 cd/m² and 63.5 cd/m² for the cyan and red stimuli, respectively. Patients underwent a 60-minute dark adaptation, and testing was performed with the cyan followed by the red stimulus. The mean sensitivity represents an average of the 78 points and recorded in decibels of attenuation (dB). 

Hill of Vision Analysis

In addition to mean sensitivity, the HOV analysis was performed using VFMA software developed by Weleber et al.¹⁴ Three-dimensional HOV analysis models were generated, with HOV volumetric analyses determined for the total perimetry (V_total), the central 30 degrees (V₃₀), and for the area of the treatment bleb (V_bleb), and were reported in units of decibel-steradians (dB-sr). Treatment bleb areas were determined through surgical videos with intra-operative optical coherence tomography. 

Field Stimulus Thresholds

Full-field stimulus threshold (FST) was performed using a Diagnosys Epsion system with the ColorDome stimulator (Diagnosys LLC, Lowell, MA). Patients were dilated by topical tropicamide 1% and phenylephrine hydrochloride 2.5% and dark-adapted for 45 minutes. Eyes were tested monocularly with patching of the other eye during testing. The starting 0 dB illuminance was 0.1 cd·s/m² (25 cd/m² presented for 4 ms) with 2500 ms response time. The range of decibels available for the test ranged from −75 dB to +15 dB. The tester defined a starting value to initiate testing, and the proprietary program utilized a forced-choice testing strategy to test within a range of 10 dB around the starting value. Testing was performed with blue (448 nm), red (627 nm), and white stimuli. A meaningful change in FST has been reported as 10 dB or 1 log cd/m², with test-retest variability as 0.3 log cd/m².^15,16 

Statistical Analysis

We used linear mixed-effect model analysis with restricted maximum likelihood estimation allowing for random effects of each eye nested within individual subjects. The P values for the fixed effect of treatment were estimated using a t-distribution and are presented without adjustment. Statistical analysis was implemented using the lmer package in R software. 

Results

Demographics

Eight eyes from 4 patients (3 males and 1 female) with biallelic RPE65-associated retinopathy were treated with voretigene neparvovec-rzyl. The mean age at treatment with voretigene neparvovec-rzyl was 23.0 years old (range = 12–37 years). Two 2cDAP was performed bilaterally in 4 patients with a mean follow-up time of 19.75 months at last visit (Table 1). The FST was performed in a subset of 6 eyes from 3 patients, with a mean follow-up time of 22.3 months at the last visit. 

Table 1.

 

Patient Demographics

 

Two-Color Dark-Adapted Perimetry: Mean Sensitivity

Average mean sensitivities are summarized in Table 2. Briefly, the average of the mean sensitivity pre-operatively for the cyan stimulus was 13.65 dB ± 13.74 (mean ± SD, 95% confidence interval [CI] = 2.16–25.14). There was a statistically significant improvement in the mean sensitivity for the cyan stimulus postoperatively at the last follow-up, with an average mean sensitivity of 24.83 ± 16.31 (95% CI = 11.19–38.46, P < 0.01). There was also a statistically significant improvement in mean sensitivity for the red stimulus, with a pre-operative average of 5.86 ± 7.04 (9% CI = −0.02 to –11.75), and a postoperative average of 10.89 ± 8.56 (95% CI = 3.73–18.05) at last follow-up (P < 0.01; see Table 2, Fig. 1). Three out of four patients showed an improvement in both cyan and red perimetry following treatment. Furthermore, improvements were observed as early as 1 to 4 months post-treatment (Supplementary Fig. S1). Patients 1 to 3, who had notable improvements in perimetry, maintained their improved response at the last follow-up visit (Supplementary Table S1, Figs. 2–4). Patient 4, who was treated at age 37 years old, showed minimal changes in dark-adapted perimetry with a decline back to baseline responses at last follow-up (see Supplementary Table S1, Fig. 5). 

Table 2.

 

Two-Color Dark-Adapted Perimetry (2cDAP) Responses Pre- and Post-Treatment

 

Figure 1.

 

Two-color dark-adapted perimetry pre- and postoperatively at the last visit. Bars show average values for (A) mean sensitivity or (B) average hill of vision (HOV) volumetric analyses for the total perimetry (V_total), the central 30 degrees (V₃₀), or the treatment bleb (V_bleb) with error bars representing the standard deviation. Individual mean sensitivity and HOV analysis values for each patient and eye are shown. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

 

The 2cDAP mean sensitivity map (A, B) and VFMA with en face view (C, D) and at an angle (E, F) at baseline and last postoperative follow-up to cyan A to F and red stimuli (G-L) of patient 1, in visual field view.

Figure 3.

 

The 2cDAP mean sensitivity map (A, B) and VFMA with en face view (C, D) and at an angle (E, F) at baseline and last postoperative follow-up to cyan A to F and red stimuli (G-L) of patient 2, in visual field view.

Figure 4.

 

The 2cDAP mean sensitivity map (A, B) and VFMA with en face view (C, D) and at an angle (E, F) at baseline and last postoperative follow-up to cyan A to F and red stimuli (G-L) of patient 3, in visual field view.

Figure 5.

 

The 2cDAP mean sensitivity map (A, B) and VFMA with en face view (C, D) and at an angle (E, F) at baseline and last postoperative follow-up to cyan A to F and red stimuli (G-L) of patient 4, in visual field view.

Two-Color Dark-Adapted Perimetry: Hill of Vision Analysis

Average HOV volumetric analyses for V_total, V₃₀, and V_bleb for cyan and red stimuli, pre- and postoperatively are summarized in Table 2. There was a statistically significant improvement in all volumetric analyses to both cyan and red stimuli postoperatively (see Table 2, Fig. 1). Similar to mean sensitivity, changes were notable in patients 1 to 3 but minimal in patient 4 (see Supplementary Table S1, Figs. 2–5). Through HOV analysis, we observed improvements in patients 1 to 3 to the cyan stimuli that extended beyond the treatment area (V_bleb), which is best visualized on 3D topographic analyses (see Figs. 2–5, Supplementary Videos S1–S4). 

Field Stimulus Thresholds

Average threshold sensitivities for blue, white, and red FST at the last follow-up are summarized in Table 3. Briefly, the mean threshold sensitivity (dB) postoperatively was statistically lower than pre-operatively for blue, white, and red stimuli. Improvements in FST were above the 1 log change that is considered clinically significant was observed in patients 1 and 3. Improvements in FST were noted as early as 1 to 4 months postoperatively and generally remained stable or continued to improve (see Supplementary Table S1). 

Table 3.

 

FST Responses (log cd/m²) Pre- and Post-Treatment

 

Patient 1 demonstrated a shift from cone-mediated to rod-mediated thresholds following treatment, given the pattern among blue, white, and red thresholds described in Birch et al.¹⁷ Rod-mediated thresholds are represented by white threshold sensitivity below −30 dB or with blue-red differences ≥20 dB. Patients 2 and 3 demonstrated rod-mediated thresholds at baseline, which correlated with their chromatic perimetry. Following treatment, there were improved rod-mediated FST thresholds in both patients, however, patient 2 had good baseline FST responses which improved slightly but were likely limited by a ceiling effect. Patient 3 had a greater than 1 log improvement. Unfortunately, patient 4 did not have baseline FST testing performed, but would likely show a pattern of cone-mediated thresholds without significant improvement after treatment. 

Discussion

Novel visual function outcome measures are needed to assess the efficacy of the growing number of treatments being evaluated for inherited retinal degenerations. Measures of visual function in patients with these diseases are limited by severely low or negligible responses or are not sensitive at detecting progression. Previously, FST and MLMT have been used as outcome measures in clinical trials using gene therapy for RPE65-related retinopathy.^16,18,19 Our cohort showed comparable improvements in the threshold sensitivity with FST compared with previous clinical trials where improvements to a blue stimulus average 1.6 log units²⁰ or >2 log units to a white stimulus.¹⁶ 

Achromatic dark-adapted perimetry has been measured in previous AAV-RPE65 clinical trials^8,21–24; however, chromatic perimetry has not been explored in this population. The 2cDAP may assess rod-mediated function more specifically without interfering with responses from dark-adapted cones.^25,26 VFMA obtained from static perimetry provides further detailed descriptive and quantitative HOV analyses and was previously validated in retinitis pigmentosa patients.¹⁴ Our 4 patients showed a range of 2cDAP responses at baseline, with improvement in 3 out of 4 patients. Patient 1 showed low baseline dark-adapted perimetry that significantly improved on all measures in mean sensitivity and HOV analysis, and a shift from cone-mediated to rod-mediated thresholds on FST. Patient 2 showed relatively normal baseline dark-adapted perimetry that mildly improved with treatment. Patient 3 showed moderate baseline dark-adapted perimetry that improved with treatment. Patient 4, who had low baseline 2cDAP similar to patient 1, demonstrated minimal improvement after treatment. This difference in treatment response is likely attributed to age of treatment, with patient 1 receiving treatment at 12 years old as compared to patient 4 who was treated at 37 years old. 

Three out of four patients (patients 1–3) showed improvements to the cyan stimulus extending beyond the treatment area. Changes in 2cDAP mirrored with those measured by FST (see Supplementary Fig. S1), however, 2cDAP provides topographical information of improvements in rod-mediated function. Furthermore, 2cDAP with VFMA analysis provides the ability to evaluate changes in total perimetry, and in defined regions, such as the central macula or in the treatment bleb. HOV analysis in treatment blebs may be particularly useful for detecting localized improvements, such as that in choroideremia or following stem cell therapy, as compared to the more widespread effect of RPE65 gene augmentation. 

Patients with RPE65-RP are characterized as having biochemical insufficiency of the retinoid cycle with disproportionately preserved photoreceptors compared to functional vision loss.²⁷ Therefore, diffusion of retinoids to neighboring residual photoreceptors may be contributing to our finding of improvement in rod-mediated sensitivity beyond the treatment area. This has been supported by studies that show administration of oral synthetic retinoids may improve perimetry responses.²⁸ Alternatively, the location of the bleb may have shifted or extended postoperatively and the results may reflect treatment of a broader area than that visualized intraoperatively. In summary, 2cDAP with HOV analysis provides a useful outcome measure of rod-mediated function with topographic depiction of retinal sensitivity that is well-suited for evaluating treatment responses in clinical trials for inherited retinal conditions. 

Acknowledgments

The authors thank Sam Mershon, BS, at OHSU for statistical analysis consultation. 

Funded by the National Institutes of Health (P30EY010572, K08EY026650, and P30EY2162) and the Research to Prevent Blindness (unrestricted grant for Casey Eye Institute). 

Disclosure: C.A. Ku, None; A.D. Igelman, None; S.J. Huang, None; H. Vasconcelos, None; M.M. da Palma, None; S.T. Bailey, None; A.K. Lauer, None; R.G. Weleber, None; P. Yang, None; M.E. Pennesi, None 

Supplementary Material

Supplementary Video S1A and Supplementary Video S1B. 3-dimensional animation of VFMA of the left eye of patient 1 to the (A) cyan, and (B) red stimulus, before (left) and after (right) voretigene neparvovec-rzyl treatment. 

Supplementary Video S2A and Supplementary Video S2B. 3-dimensional animation of VFMA of the right eye of patient 2 to the (A) cyan, and (B) red stimulus, before (left) and after (right) voretigene neparvovec-rzyl treatment. 

Supplementary Video S3A and Supplementary Video S3B. 3-dimensional animation of VFMA of the left eye of patient 3 to the (A) cyan, and (B) red stimulus, before (left) and after (right) voretigene neparvovec-rzyl treatment. 

Supplementary Video S4A and Supplementary Video S4B. 3-dimensional animation of VFMA of the right eye of patient 4 to the (A) cyan, and (B) red stimulus, before (left) and after (right) voretigene neparvovec-rzyl treatment.