Establishing Clinical Trial Endpoints in Selecting Patients for RPGR Retinal Gene Therapy

Abstract

Purpose: Clinical trials for X-linked retinitis pigmentosa (RP) often assess retinal structure using optical coherence tomography (OCT) and function using microperimetry to evaluate initial eligibility and endpoints. Therefore, we seek to determine which parameters might be most sensitive in screening new patients for enrollment.

Methods: Thirty-one patients (62 eyes) with confirmed retinitis pigmentosa GTPase regulator (RPGR) mutations attending Oxford Eye Hospital were included in this retrospective analysis. Outer retinal structure was investigated by measuring the remaining ellipsoid zone (EZ) and external limiting membrane (ELM) on OCT. Visual function was evaluated by using 10-2 microperimetry mean sensitivity.

Results: The median age of patients with RPGR was 31 years (interquartile range [IQR] = 22–39 years). For the right and left eyes, respectively, the median EZ length through the foveal section was 921 µm (IQR = 607–1570) and 865 µm (IQR = 508–1442) and median ELM length was 2056 µm (IQR = 1336–2764) and 1860 µm (IQR = 1152–2680). Similarly, the median microperimetry sensitivity (MS) was 2.0 decibel (dB; IQR = 0.4–5.4) and 1.1 dB (IQR = 0.1–5.4). Linear mixed model regression analysis showed that EZ was significantly positively correlated to ELM (P < 0.001, R² = 0.931). EZ and ELM were significantly correlated with the microperimetry sensitivity with exponential relationship (P < 0.001, R² = 0.71 and 0.72, respectively). Using the exponential equation of regression line, EZ below approximately 600 µm (RE = 637 µm, 95% confidence interval [CI] = 397–877, LE = 586 µm, 95% CI = 356–817) results in microperimetry sensitivity of approximately 0 dB. There was high degree of inter-eye symmetry for progression of EZ, ELM, and microperimetry sensitivity. Age was significantly correlated with the analyzed parameters (P < 0.001), although with low R² for each of them.

Discussion: EZ may comprise a surrogate biomarker for prediction of visual function in X-linked RP caused by mutations in RPGR and, in turn, identification of appropriate patients for enrollment in clinical trials. As expected, age plays a role in predicting potential biomarkers and visual function, however, it should not be used to preclude patients for gene therapy due to the poor correlation and heterogeneity of disease onset.

Translational Relevance: Biomarkers of visual function in RPGR-associated RP may lead to identification of appropriate patients for enrollment in clinical trials.

Introduction

Retinitis pigmentosa (RP) is a genetically heterogenous group of disorders leading to loss of photoreceptors.^1–3 X-linked RP is the most common recessive form of RP; it is characterized by degeneration of rod and cone photoreceptors and is found in 15% to 30% of patients with RP.^4,5 Mutations in the gene encoding RP GTPase regulator (RPGR), a protein localized to the photoreceptor connecting cilia, account for the majority of X-linked RP (70%), whereas RP2 variants are mainly identified in the remaining cases.^6–10 Most studies agree that photoreceptor degeneration in X-linked RP starts at a younger age than in many other types of RP, along with faster progression, thus contributing to its more severe phenotype.^11–13 

RPGR-associated RP typically, although not always, presents as a rod-cone dystrophy.^14,15 Detection of disease progression during the early stages is often challenging, thus, assessment may require a combination of structural and functional measures. Visual acuity is moderately preserved until the photoreceptor degeneration encroaches centrally toward the fovea and, therefore, may be considered an unreliable indicator of visual function. Indeed, numerous studies have used a variety of visual field testing. Recently, macular integrity assessment (MAIA) microperimetry has been utilized as outcome measure in RPGR-associated RP gene therapy clinical trials. Despite the increasing popularity of microperimetry in the clinical trials space, specific criteria concerning its use during the course of the disease are yet to be explored.^16–20 

Existing research has widely focused on the relationship between visual function and retinal structure in inherited retinal disorders.^21–24 The advent of high-resolution optical coherence tomography (OCT) has provided new insights in morphological changes within individual retinal layers; especially the inner segment/outer segment (IS/OS) junction, also known as ellipsoid zone (EZ), and outer nuclear layer (ONL) have been studied in detail in RP.^21–24 Several studies have shown that structural measurements of EZ as a metric of disease severity have been well correlated with retinal function, therefore the EZ band may comprise an efficacious biomarker of progression, apparently more sensitive than functional measures such as full-field electroretinogram (ERG), and kinetic or static perimetry.^25,26 

Currently, there is no treatment for RPGR-related RP; however, retinal gene therapy may provide a promising therapeutic approach.^27–29 The identification of target patient populations as well as the adoption of clinically meaningful endpoints that would best determine the efficacy of a trial is of utmost importance. Factors that possibly impact on clinical trial inclusion criteria and delineation of participant cohorts may include pathogenic PRGR genetic mutations, retinal clinical phenotypes, age, and functional and structural status at baseline. Hence, prior to commencing any clinical trial extensive screening visits identification of a suitable patient population is a requirement. Any insights into structure-function correlation which may assist in screening populations at the earliest stages would dramatically ease the burden on clinicians and patients. This would also help to avoid patient disappointment if the patients are later found ineligible for trial inclusion.^30–34 

In the present study, we sought to characterize baseline measurements for retinal structure using OCT and visual function using microperimetry in patients with RPGR-associated RP that could lead to optimal recruitment of target patients for inclusion into retinal gene therapy clinical trials. The aim of this investigation was to identify potential objective biomarkers that might be suitable for screening new patients that could benefit the most from genetic treatment and contribute to the designing of clinical studies to provide the best chance for successful outcomes. 

Methods

Study Design

Patients with genetically confirmed RPGR-associated RP who attended Oxford Eye Hospital, UK, were screened prior to inclusion on a phase I/II clinical trial of retinal gene therapy (ClinicalTrials.gov identifier NCT03116113) and included in this retrospective analysis. Oxford Eye Hospital is a tertiary center in the Southeastern United Kingdom for assessing patients with inherited retinal degenerations, including X-linked RP. The study adhered to the tenets of the Declaration of Helsinki. 

Data were screened and patient data excluded from the analysis if imaging was of low quality. 

Microperimetry

MAIA confocal microperimetry (ICARE MAIA; Mainline Instruments Limited, Birmingham, UK) was used to assess macular sensitivity. Testing was performed for all participants in a darkened room (light level <1.0 lux) after 20 minutes of dark adaptation without pupil dilation. Examination was performed in both eyes using a standard 68-stimulus (10-2) grid covering the central 10 degrees of the macula. Testing involves Goldmann size III stimuli of various intensities (0–318 cd/m²), presented for 200 ms, on a mesopic background (1.27 cd/m²). The overall dynamic testing range was 0 to 36 decibels (dB). A red circle with a diameter of 1 degree was used as a central fixation target. Examinations were considered reliable based on the response frequency to 10 dB stimuli presented to the physiological blind spot approximately once every minute, termed “fixation losses.” Owing to the eye-tracking capability of the MAIA, these responses are commonly considered to be false positive results arising from incorrect button presses in the absence of any seen stimuli. As per MAIA guidelines, any examinations with fixation losses of 30% or greater were considered to be unreliable and were repeated. An average of all resulting individual point threshold sensitivities is provided as the mean sensitivity index as part of the standard output and collected for later analysis. All the tests were completed by trained optometrists. 

OCT Imaging Protocols

OCT scans were captured using high-resolution spectral-domain OCT (SD-OCT) imaging obtained using the Spectralis HRA+OCT system (Heidelberg Engineering GmbH, Heidelberg, Germany). Scans were obtained using standardized settings; 30 degrees macula volume scan consisting of 121 b-scans, using automated real-time tracking (ART). For each study participant, a scan of the macular area centered over the foveola was performed. Precise measurements of the retinal structure in the OCT scans were subsequently performed in the Heidelberg Eye Explorer software (HEYEX; Heidelberg Engineering GmbH) using the caliber tools. To quantify the retinal structure, horizontal b-scans centered on the fovea were analyzed. All scans included in the study were reviewed to ensure their quality was sufficient. Eyes with a history of concomitant ocular conditions or co-existed macular pathology, such as cystoid macular edema, that could distort measurements of retinal layers and obscure visualization of the EZ band were excluded to avoid limiting accurate assessment of disease progression and potential confounding effects. All measurements were made using a manual segmentation procedure by the same experienced examiner at the highest magnification available, as described in a previous study.²⁴ For all scans, the boundaries between the following structures were identified; the retinal pigment epithelium (RPE), the EZ, the ELM, the ONL. The nasal and temporal edges of the EZ band were defined as the locations where the EZ band met the RPE; the width of the preserved EZ band was defined as the horizontal distance between these two locations. Similarly, the remaining ELM overlying the EZ was defined as the horizontal distance between the points that its boundary reached the RPE, as seen in Figure 1. 

Figure 1.

 

Representative optical coherence tomography scan indicating the remaining ellipsoid zone (EZ) and external limiting membrane (ELM). The edges of the EZ and ELM band were defined as the locations where each band met the retinal pigment epithelium, while the preserved EZ and ELM were defined as the horizontal distance between these two locations, respectively.

Statistical Analysis

All data analysis was performed by using R (version 4.2.1)³⁵ and the lme4 package.³⁶ A linear mixed model was used to model the relationship between either ELM or EZ as the independent predictor variables and EZ as the outcome dependent variable. In order to take the right and left eyes from each patient into account, the patient ID was set as random intercept variable. 

Results

A total of 37 patients with X-RPGR-associated RP were identified in this retrospective analysis. After applying exclusion criteria, a total of 31 patients were included in the analysis. The median age of patients with RPGR-associated RP was 31 years (interquartile range [IQR] = 22–39 years). For the right and left eyes, respectively, the median EZ width through the foveal section was 921 µm (IQR = 607–1570) and 865 µm (IQR = 508–1442) and ELM width was 2056 µm (IQR = 1336–2764) and 1860 µm (IQR = 1152–2680). The median microperimetry sensitivity (MS) was 2.0 dB (IQR = 0.4–5.4) for the right eye and 1.1 dB (IQR = 0.1–5.4) for the left eye. Linear mixed model regression analysis showed that EZ width was significantly positively correlated to ELM width (P < 0.001, R² = 0.931), as shown in Figure 2. EZ and ELM widths were significantly correlated with microperimetry sensitivity with exponential relationship (P < 0.001, R² = 0.84 and 0.72, respectively) with representative plots shown in Figure 2. Using the exponential equation of regression line, EZ widths below approximately 600 µm (RE = 637 µm, 95% confidence interval [CI] = 397–877, LE = 586 µm, 95% CI = 356–817) results in microperimetry sensitivity of approximately 0 dB. There was high degree of inter-eye symmetry for declining progression of EZ, ELM, and microperimetry sensitivity. Age was significantly correlated with these covariates (P < 0.001), although with low R² for each of them, as shown in Figure 3. 

Figure 2.

 

Linear mixed model regression analysis showed that EZ was significantly positively correlated to ELM (P < 0.001, R² = 0.931). EZ and ELM were significantly correlated with microperimetry sensitivity (MS) with exponential relationship (P < 0.001, R² = 0.84 and 0.72, respectively).

Figure 3.

 

Age was significantly correlated with the EZ, ELM, and MS (P < 0.001), although with low R² for each of them was likely due to a greater importance of age at disease onset rather than simply age.

Discussion

Given the promising aspects of RPGR-associated RP gene therapy, it is worthwhile to evaluate the possible parameters that could contribute to optimal development and outcomes of relevant clinical trials.³⁰ Our investigation focused on identifying robust biomarkers crucial for patient selection for gene therapy, whereas the goal was to contribute to the optimization of trial design and the recruitment of appropriate candidates for emerging therapeutic interventions. 

X-linked RPGR-related RP is a condition characterized by progressive degeneration of photoreceptors. Within this, there exists a broad spectrum whereby the phenotypic presentation varies depending on the location of the variant along the gene. These range from rod-cone dystrophy, to cone-rod or cone dystrophies with a greater cone involvement associated with distal regions of the gene due to impaired RPGR glutamylation. Because variant location/phenotype correlation is a pivotal aspect in determining disease prognosis, it is critical that the patients’ group in clinical trials share similar characteristics.^{14,15,27,28,37–41} Our study focused specifically on individuals with the rod-cone dystrophy phenotype associated with RPGR mutations. By excluding cases with cone or cone-rod dystrophies, we aimed to ensure consistency in our analyses and interpretations. 

Patients with rod-cone RPGR-associated RP tend to manifest symptoms of nyctalopia in childhood followed by progressive peripheral visual field constriction and severe visual impairment by the fourth decade, whereas those with cone or cone-rod dystrophies may have comparatively early central visual deterioration in younger age.^12,14,15 Notably, our analysis revealed a weak relationship between age and disease severity, suggesting that factors beyond chronological age, such as time since disease onset, may better predict disease progression. Furthermore, progression rates in individuals with X-linked RP may be affected by genetic parameters; for example, individuals with mutations in ORF15 exhibit faster disease progression than those with mutations in exons 1 to 14, which in turn may be associated with differences in outcomes of functional and structural tests not directly depending on patients’ age.^4,8,30 These insights underscore the complexity of disease trajectory and the need for comprehensive assessment strategies in patient selection for gene therapy trials. 

Objective biomarkers that could delineate the stage and progression of the disease are often used in enrollment of gene therapy clinical trials. The currently available evidence indicates that there is a close association between visual function and structure in inherited retinal disorders.^25,26 In our study, we utilized functional and morphological assessment of the retina in individuals with RPGR-associated RP in an attempt to characterize these parameters during the course of the disease. Several studies have demonstrated that the point of EZ diminishment corresponds to the area of visual field sensitivity reduction,^25,26 whereas EZ serves as a critical anatomic landmark delineating the boundary between healthy and affected retina. In agreement to this, the average EZ width decrease rate has been found similar to the Goldmann visual field deterioration.¹⁶ The above evidence indicates that the EZ band may be an appealing surrogate biomarker for disease progression. The findings of our study notably demonstrated structural biomarkers, particularly measurements of EZ on OCT, as promising indicators of disease severity. In addition, we identified macular sensitivity as assessed in the central 10-2 microperimetry correlated significantly with morphological characteristics on OCT, namely the ELM and EZ width. Specifically, in this cohort of patients, EZ width below a certain threshold (600 µm) was likely to yield an extinguished microperimetry sensitivity, suggesting its potential utility as a screening tool for identifying suitable candidates for enrollment into gene therapy trials. Thus, EZ could notably evolve into a useful biomarker for determining appropriate patients for participation in clinical trials. 

To date, there is limited evidence concerning efficacious treatment for RPGR-associated RP.^27,28 In the context of ongoing efforts to optimize gene therapy trials, our findings may have significant implications. The identification of reliable biomarkers, such as EZ width, could streamline patient selection processes and enhance the accuracy of treatment efficacy assessment. We explored correlations between macular sensitivity and structural changes in order to identify parameters that could contribute to optimal recruitment of patients for gene therapy clinical trials. Demonstrating relationships between visual function and photoreceptors’ alterations could strengthen the value of structural parameters and potentially allow for more accurate and brief testing. In any case, our study contributes to a little-studied issue which warrants further investigation. Inevitably, certain limitations of the current study need to be further elucidated. First, our selection of functional tests included macular sensitivity on microperimetry, whereas we did not evaluate visual acuity or ERG measurements. Because visual acuity is only representative of the central 0.5 degrees of foveal function and can be preserved until later in disease progression, this test is considered of limited value. Visual acuity is generally considered an insensitive marker of severity leading to the adoption of microperimetry and mobility testing as the preferred clinical trial outcome measures in most recent RPGR clinical trials.²⁹ In addition, we do not routinely perform ERGs for the same reason. Recruitment in clinical trials, particularly for earlier disease stages, is predominantly based on functional measures via microperimetry and OCT structural markers, hence the focus of this paper on these measures. Second, concerning morphological investigations on OCT, a horizontal b-scan centered on the foveola was used for each patient, whereas the vertical scans and enface OCT images that would potentially provide further information were not analyzed in this study. In addition, EZ length was defined by the locations where it met the RPE; expectedly, the EZ band was not completely intact in the whole length, as disruption is an element in these patients. Undoubtedly, quantification of the preserved EZ area on en face OCT would potentially provide further information and comprise an additional tool for monitoring the EZ changes in these patients. Another limitation is that the sample size was relatively small; patients with RPGR-associated RP consist of a small cohort of RP, and previous studies have included similar sample sizes. Nonetheless, despite that this small sample enabled the detection of significant correlations in the investigated parameters, further studies are needed to confirm our results. Notably, all measurements were performed by the same experienced examiner enabling remarkable outcomes, however, additional masked investigations are required to support our findings and add value to our results. Finally, we should definitely not ignore the inherent limitations of the retrospective nature of our study design. These limitations underscore the need for further longitudinal studies to validate our findings and establish robust clinical endpoints for trial efficacy assessment. 

In conclusion, our study provides valuable insights into the potential of EZ width as a biomarker for patient selection in RPGR-associated RP gene therapy trials. By elucidating the complex interplay between structural and functional retinal biomarkers, we contribute to the ongoing efforts to advance therapeutic interventions for individuals affected by inherited retinal disorders. 

Acknowledgments

Supported by the NIHR Oxford Biomedical Research Centre (R.E.M.), and the MRC Clinician Scientist Fellowship (J.C.K.). 

Disclosure: E.E. Christou, None; A.S. Josan, None; J. Cehajic-Kapetanovic, None; R.E. MacLaren is listed as an inventor on a patent for RPGR gene therapy owned and licensed by the University of Oxford (P); Novartis (C), Scribe Therapeutics (C), Biogen Inc. (C), AGTC (C), and is a director of Beacon Therapeutics