OCT Biomarkers in Ocular CLN2 Disease in Patients Treated With Intraventricular Enzyme Replacement Therapy

Abstract

Purpose: Bilateral progressive, symmetrical loss of central retinal thickness (CRT) has been described in neuronal ceroid lipofuscinosis type 2 (CLN2) disease. This study details the pattern of morphological changes underlying CRT loss and disease progression in patients receiving intracerebroventricular (ICV) enzyme replacement therapy (ERT) with cerliponase alfa.

Methods: Spectral-domain optical coherence tomography macular cube scans were collected from 16 patients with classic CLN2 disease receiving ICV ERT. Detailed retinal structure analyses were performed on manually segmented horizontal B-scans through the fovea to determine the thickness of six retinal parameters and the extent of ellipsoid zone (EZ) loss.

Results: Anatomical changes primarily occurred in photoreceptor (PR)-related retinal parameters and correlated with ocular disease severity. Retinal degeneration began with initial focal parafoveal EZ discontinuities signaling the onset of rapid PR degeneration in a predictable pattern: parafoveal PR involvement with foveal sparing followed by profound parafoveal and foveal PR loss with additional thinning beyond the central retina. PR degeneration began with outer segment loss and progressed to outer nuclear layer (ONL) involvement. Longitudinal analyses confirmed these observations. The rate of PR loss was fastest at the fovea at ∼58 mm per year and became slower at locations farther away from the fovea.

Conclusions: Retinal degeneration in CLN2 disease is primarily associated with PR loss in a predictable pattern, with EZ disruption signaling early PR stress. CRT, ONL thickness, and PR layer thickness are useful anatomical biomarkers for understanding disease progression and treatment efficacy in CLN2. Studies using en face images will further clarify CLN2-related retinal degeneration.

Neuronal ceroid lipofuscinosis (NCL) is a heterogeneous spectrum of progressive neurodegenerative lysosomal storage diseases caused by mutations in one of at least 13 genes.¹ Type 2 NCL, or CLN2 disease, is caused by autosomal recessive mutation in the CLN2 gene. Classic CLN2 disease typically presents in children between the ages of 2 and 4 years with language developmental delay, seizures, and psychomotor decline, followed by profound, progressive neurological and cognitive degeneration, loss of vision, and, finally, death in early adolescence.¹ A less common, attenuated phenotype has also been described in which vision loss is delayed, presenting as late as adolescence.^1,2 

Tripeptidyl peptidase 1 (TPP1) is a soluble lysosomal protein that removes tripeptides from small proteins within lysosomes.^3,4 Mutations in the CLN2/TPP1 gene lead to severe reductions of TPP1 enzymatic activity, which in turn results in the accumulation of subunit c of mitochondrial adenosine triphosphate synthase and intracellular accumulation of curvilinear autofluorescent lipopigment storage material.⁵ The accumulation of lysosomal storage material associated with CLN2 mutations is particularly toxic to the central nervous system (CNS) and retina, leading to neurodegeneration seen in the classic and atypical phenotypes as described above.⁵ 

Retinal degeneration associated with classic CLN2 disease manifests as a bilateral, progressive, symmetrical decline beginning at the central macula and expanding in late-stage disease beyond the central retina.⁶ The retinal degeneration is characterized by rapid loss of central retina thickness (CRT) measured by optical coherence tomography (OCT) over a critical 2-year period as disease severity rapidly progresses.^7–9 

The natural history of retinal degeneration-associated CRT loss in classic CLN2 disease follows a predictable pattern along a sigmoidal curve.⁷ This can be described in four distinct phases: The pre-degenerative phase is characterized by a relatively normal plateau of CRT, followed by the early degenerative phase, in which the gradual onset of CRT loss and accompanying visual symptoms begin. Subsequently, the phase of accelerated decline in CRT occurs over a period of approximately 2 years. Finally, another plateau is reached in late-stage disease due to a floor effect when most of the photoreceptors within the central fovea have been lost.^7,8,10 With the magnitude of retinal degeneration, visual function is presumed to be severely compromised at the central fovea. However, until recently, this has not been quantified. A pilot study of an automated visual acuity (VA) test that employs optokinetic nystagmus detection (Threshold Visual Acuity Test; University of Auckland, Auckland, NZ) demonstrated a high correlation between CRT loss and loss of VA in children with CLN2 disease (Ohnsman CM, et al. IOVS 2021;62:ARVO E-Abstract 3141). 

Current treatment for CLN2 disease consists of intracerebroventricular enzyme replacement therapy (ERT) with cerliponase alfa, a recombinant human proenzyme of TPP1.^1,11 Intracerebroventricular infusion of cerliponase alfa via a Rickham or Ommaya reservoir device every 2 weeks has been shown to reduce the accumulation of lysosomal storage material and attenuate motor and language decline.¹¹ However, CNS-directed infusions of cerliponase alfa most likely do not reach the retina in humans and sufficiently treat the ophthalmologic manifestations of CLN2.^11,12 This study sought to further characterize the progression of CLN2-associated retinopathy using OCT anatomical biomarkers, particularly those associated with vision loss despite receiving intraventricular cerliponase alfa therapy. 

Methods

Ethics Approval and Consent

This observational study was approved by the local ethics committees (Ethikkommission Ärztekammer Hamburg; approval number for the international DEM-CHILD NCL patient database, PV7199) and adhered to the tenets of the Declaration of Helsinki. Consent for study participation was obtained from parents and/or legal guardians of all study participants after they received an explanation of the nature of the observational study and its potential consequences. 

OCT Imaging and Patient Staging

Macular cube scans were collected by spectral-domain OCT (SD-OCT) using the EnVisu C2300 OCT system (Leica Microsystems, Wetzlar, Germany), DRI OCT Triton 1.2.8 (Topcon Healthcare, Tokyo, Japan), and SPECTRALIS OCT2 Flex version 1.10.2.0 (Heidelberg Engineering, Heidelberg, Germany) from patients with CLN2 disease who were undergoing routine cerebral magnetic resonance imaging at the University Medical Center Hamburg-Eppendorf (Huang WC, et al. IOVS 2022;62:ARVO E-Abstract 2448). One eye per patient was analyzed given that disease progression is highly symmetrical between eyes (Huang WC, et al. IOVS 2022;62:ARVO E-Abstract 2448).⁷ All observations were considered to be characteristic of classic CLN2 disease, as ocular manifestations of CLN2 disease are unaffected by ERT with cerliponase alfa.^8,11,12 Ocular CLN2 disease severity for each patient was staged based on the OCT parameters of the Weill Cornell Late Infantile Neuronal Ceroid Lipofuscinosis Ophthalmic Severity Scale and designated as stages 1 to 5, where stage 5 is the most severe stage (Fig. 1A).⁶ 

Figure 1.

 

Representative horizontal B-scans of ocular CLN2 and representative retinal thickness parameters in a normal eye. (A) Select horizontal B-scans depicting OCT characteristics of ocular CLN2 disease according to the Weill Cornell Late Infantile Neuronal Ceroid Lipofuscinosis Ophthalmic Severity Scale. The insets in stages 4 and 5 present OCT scans from the same macula cubes, 2 mm inferior to the horizontal meridian, where there is ONL+HFL retention in stage 4 (white arrow), but not in stage 5. Yellow arrows indicate the following: ELM thickening in stage 1; focal EZ band disruption in stage 2; complete EZ loss within the retinal regions marked by yellow arrows in stage 3; and RPE atrophy in stage 5. (B) Retinal thickness parameters are denoted by line segmentation: RNFL, INL, ONL, PR+RPE, and OS+RPE of a stage 1 patients with intact retinal structure.

Sectorial and Detailed Cross-Sectional OCT Analyses

Sectorial full retinal thickness (FRT) analyses were conducted automatically with manual corrections (performed by WCH) in all Early Treatment Diabetic Retinopathy Study (ETDRS) sectors using OCT Explorer 3.8.0 and Heidelberg Eye Explorer 3.1.0.55 (Huang WC, et al. IOVS 2022;62:ARVO E-Abstract 2448). Because parafoveal FRT did not differ significantly from sector to sector, the average values of parafoveal sectors (1–3 mm from foveal center) were reported. Likewise, average values of perifoveal (3–6 mm from foveal center) sectorial FRT were reported. 

The thickness of six retinal parameters was measured by manual segmentation (performed by WCH) of horizontal B-scans through the fovea using Engauge Digitizer 12.1 software (Huang WC, et al. IOVS 2022;62:ARVO E-Abstract 2448). Retinal thickness parameters included FRT, retinal nerve fiber layer (RNFL), inner nuclear layer (INL), outer nuclear layer plus Henle fiber layer (ONL+HFL), photoreceptor (PR) plus retinal pigment epithelium (RPE), and outer segment (OS) plus RPE (Fig. 1B). Due to limitations of image quality, ganglion cell layer segmentation could not be performed reliably. The mean ± SD of each retinal thickness parameter was plotted against retinal distance from the fovea in either direction (i.e., temporal and nasal). Normative data along the horizontal meridian in adult populations were plotted for comparison purposes.^13,14 Comparable measurements in children have been reported up to 2 mm along the horizontal meridian.¹⁵ 

Macular cube B-scans were captured with spacing ranging from 120 to 250 µm, which did not provide sufficient resolution for measurements using en face images. The extent of ellipsoid zone (EZ) loss was therefore approximated based on the retinal distance in two directions (i.e., horizontal and vertical) where the EZ band signal was not detectable and averaged. Horizontal EZ loss was measured on B-scans through the fovea, and vertical EZ loss was estimated by inspecting the three-dimensional macular cube where EZ signal was absent on frames superior and inferior to the fovea. For purposes of approximating the areas of EZ loss, a circular pattern was estimated at each disease stage based on the patient with the largest EZ loss within each OCT stage. The extent of the estimated EZ loss at each disease stage was then overlaid on an ETDRS grid. 

Longitudinal OCT Measurements

To confirm cross-sectional observations, longitudinal data for individual patients were analyzed when available (Huang WC, et al. IOVS 2022;62:ARVO E-Abstract 2448). The rate of change for PR-related thickness parameters at three retinal locations (fovea and temporal 1.5 mm and 3.0 mm) along the horizontal meridian was estimated between the first available time point and the last available time point if the patient was in early stages (stages 1 and 2). Because retinal thickness reaches a floor, defined as a difference in thickness between previous and subsequent visits within intrarater variability for that parameter, during stage 3, the PR-related thickness at these locations was determined between the first available time point and the earliest follow-up time point at which the floor was reached.¹⁶ 

In a subset of horizontal OCT scans (n = 8), intrarater variability was estimated by repeating manual segmentations by the same grader (WCH) after a 2-day washout period. Differences in ONL+HFL, OS+RPE, and PR+RPE thickness between the repeats were estimated. The variation cutoffs for the three parameters were determined based on the 95% confidence interval of the differences between repeated thickness measurements. 

Results

A total of 16 eyes from 16 patients (seven males and nine females), ages 33 to 153 months (mean, 72.56 ± 27.39 months) were evaluated. Longitudinal data were available for nine patients. Therefore, some eyes were evaluated more than once. Visual assessment of retinal layers showed that OCT anatomical changes primarily occurred in PR-related retinal parameters and correlated with ocular disease severity (Fig. 1A). In stage 1, B-scans appeared normal except for discrete thickening of the external limiting membrane (ELM) in four of six patients. Early stage 2 was characterized by central ONL+HFL thinning with focal areas of parafoveal EZ disruption that were early signs of retinal stress. By late stage 2, complete parafoveal EZ loss was observed, followed by parafoveal ONL+HFL reduction with relative foveal sparing. Stage 3 was characterized by profound ONL+HFL degradation and extensive EZ loss throughout the fovea; ONL+HFL thickness and the EZ were relatively maintained in areas beyond the central retina. In stage 4, ONL+HFL thinning extended beyond the central retina with some ONL preservation in regions greater than 2 mm superior and inferior to the fovea, associated with increased EZ loss. By stage 5, marked ONL+HFL thinning and complete EZ loss were present throughout the entire macular cube. 

The total number of eyes evaluated at stage 1 was six; stage 2, 10; stage 3, four; stage 4, four; and stage 5, two. Longitudinal data were available beginning at stage 1 in three patients and stage 2 in six patients. Progressive retinal degeneration was observed in these patients in the follow-up visits. 

Cross-Sectional Data

Reduced Macula Sectorial FRT With Progressive CLN2 Disease

The relationship between ocular CLN2 disease severity and ETDRS sectors was assessed within and outside of the central subfield by plotting CLN2 OCT stages against sectorial FRT measurements. Progressive FRT thinning in the central, parafoveal, and perifoveal regions of the ETDRS grid correlated with CLN2 disease progression (Figs. 2A–2C). CRT loss occurred rapidly in stage 2 and stage 3. Progressive reduction of sectorial FRT in parafoveal sectors occurred in stages 2 through 4, whereas perifoveal sectors had a gradual decline across all stages. A small increase in FRT was observed in stage 5. 

Figure 2.

 

Relationships among sectorial ETDRS full retinal thickness, ONL+HFL, and CLN2 OCT staging in ERT-treated classic CLN2 disease. (A–C) CLN2 OCT staging compared with central, parafoveal, and perifoveal FRT thickness in ETDRS sectors. (D) CLN2 OCT staging compared with foveal ONL+HFL thickness. (E) Decreasing CRT within ETDRS sectors plotted against foveal ONL thickness. Black dots represent subjects with cross-sectional data; colored dots present subjects with longitudinal data.

Retinal Degeneration in CLN2 Was Primarily Associated With PR Loss

Given that PRs are the predominant cell type within the central subfield region of the ETDRS grid, ONL+HFL thickness was examined to determine the contribution of PR loss to CRT decline. Foveal ONL+HFL thicknesses showed a pattern of decline as OCT CLN2 disease severity increased, similar to that of CRT loss (Fig. 2D). A linear decline in foveal ONL+HFL thickness with an increase in CRT loss was observed, as shown in Figure 2E. The linear correlation between the two parameters was statistically significant (r = –0.9; P < 0.01). 

Assessment of horizontal B-scans showed that the greatest loss of FRT occurred at the central fovea, reaching its lowest point at stage 4 (Fig. 3). OCT anatomical changes occurred in PR-related retinal parameters and correlated with disease severity. The pattern of PR+RPE loss with respect to disease stages was similar to FRT decline. PR+RPE loss was observed around the fovea in stage 2 with some degree of foveal PR+RPE retention. Significant loss of PR+RPE within ∼1.5 mm of the fovea was observed in stage 3. PR+RPE continued to degenerate beyond the central region in later disease stages. 

Figure 3.

 

B-scan analyses of six retinal thickness parameters in subjects with enzyme replacement therapy-treated classic CLN2 disease. Plots of retinal thickness layers are presented from inner- to outermost retinal location. The retinal thickness parameters are represented as mean ± SD with respect to retinal distance. Gray bands represent normative data along the horizontal meridian in adult populations. Comparable measurements have been reported in children up to 2 mm around the fovea along the horizontal meridian. ND, not detected.

The pattern of PR loss was further analyzed by segmenting the PR+RPE into ONL+HFL and OS+RPE layers. ONL+HFL and OS+RPE thicknesses showed patterns of reduction similar to those of FRT and PR+RPE in each disease stage. Parafoveal reduction of OS+RPE thickness was detected in stage 2. This was accompanied by parafoveal thinning of the ONL+HFL. The OS+RPE layer was not detected ∼1 mm from the fovea in stage 3 where ONL+HFL thinning was also observed. By stage 4, OS+RPE was not detected in most patients (five of seven), but remnants of the ONL outside of the foveal region remained (Fig. 1A). OS+RPE was not detected throughout the macular cube scans in all patients in stage 5 (n = 3). Additionally, islands of ONL were detected outside of the fovea in stage 5, and ONL+HFL thickness was further reduced compared with stage 4. 

Evaluation of inner retinal structures showed a thin INL across the horizontal meridian in a subset of patients (12 out of 16). Change in INL thickness was variable with disease progression, including thickening in two patients with late-stage disease. RNFL thicknesses exhibited normal variations across all five stages. 

Pattern of EZ Loss

Initial signs of EZ loss began in early stage 2, with small parafoveal focal EZ disruptions (Fig. 4). The pattern of progressive EZ loss, assumed to be circular, was predicted to progress to a continuous parafoveal ring of undetectable EZ signal in late stage 2, followed by complete EZ loss in the foveal region, as observed in stage 3. Eventually, the area of EZ loss expanded throughout the entire macular cube by stage 5. The extent of EZ abnormality appeared to be inversely related to CRT. 

Figure 4.

 

(A) EZ loss in patients with ERT-treated classic CLN2 disease increased with ocular disease severity according to the Weill Cornell Late Infantile Neuronal Ceroid Lipofuscinosis Ophthalmic Severity Score. The magnitude of EZ loss seemed to be generally associated with CRT reduction. (B) The representative pattern of EZ loss was derived from measurements of patients with the greatest extent of EZ loss at each stage. Focal EZ disruption was observed in early stage 2 patients and complete parafoveal EZ loss in late stage 2.

Longitudinal Data Confirmed Cross-Sectional Observations

Longitudinal data were available for nine patients between 39 months and 111 months (approximately 3–9 years) of age, ranging from disease stages 1 through 5. The number of follow-up visits ranged from one to six, with a median of two follow-up visits. Mean follow-up time was approximately 8 months between visits. Visual inspection of OCT scans across the horizontal meridian of subjects with progressive disease showed a pattern of retinal degeneration similar to that observed among cross-sectional subjects. Representative OCT scans from a single patient are shown in Figure 5A. In this subject, CLN2 disease progressed from stage 2 to stage 5 over a period of approximately 30 months. Foveal ONL+HFL was detectable at stage 2 and markedly reduced by stage 3, approximately 10 months later. In stage 4, profound ONL+HFL thinning at the fovea with preservation beyond the central macula was observed. 

Figure 5.

 

Longitudinal analyses of ERT-treated classic CLN2 disease progression. (A) Representative B-scans of ocular CLN2 disease progression from a single ERT-treated patient. Note the loss of ONL (orange) with disease progression. (B) Foveal PR+RPE, ONL+HFL, and OS+RPE thicknesses were plotted against age. Patients with longitudinal data are connected by lines. ND, not detected.

When subjects with longitudinal data entered the degenerative phase beginning at stage 2, foveal PR+RPE, ONL+HFL, and OS+RPE followed a similar pattern of rapid decline (Fig. 5B), although the magnitude of PR loss appeared to be location dependent (see Supplementary Fig. S1 for graphical analysis), with OS+RPE loss preceding complete ONL+HFL reduction. Based on intrarater variability, cutoffs of 6.44, 3.84, and 5.69 µm for ONL+HFL, OS+RPE, and PR+RPE, respectively, were used to determine the follow-up visit at which the patient entered the floor phase. The rates of decline in ONL+HFL, OS+RPE, and PR+RPE were estimated, and the fastest PR loss occurred at the fovea (Table). The pattern of EZ loss was similar to cross-sectional data observations with complete EZ loss within the macular cube at stage 5. Eight of nine patients had reduced INL thickness at the earliest time point studied, without progressive thinning over time. 

Table.

 

Annualized Rate of Retinal Layer Loss

 

Discussion

Assessment of OCT findings showed that retinal degeneration in classic CLN2 disease is primarily associated with PR loss. Changes in PR-related retinal thickness parameters, including the entire PR (represented by PR+RPE), its component layers of OS+RPE and ONL+HFL, and EZ integrity, corresponded with CLN2 ocular disease severity. Additionally, declines in foveal ONL+HFL thickness correlated with CRT loss and CLN2 ocular disease severity. 

The data from this study provide insight into the sequence of retinal degeneration in CLN2 disease. Our results suggest that PR damage begins as focal EZ disruption, progresses through the inner and outer segments, and then to the ONL+HFL. Furthermore, PR damage appears to begin parafoveally with initial foveal sparing, followed by progression toward the fovea, as well as perifoveally, until ultimately it is present throughout the entire macula. We speculate that the high ratio of support cells, such as Müller and RPE cells, to cones at the fovea and high foveal macular pigment distribution help to maintain foveal cone PRs during retinal stress in the earlier disease stages, with relative preservation of foveal PRs compared with parafoveal PRs in stages 1 and 2.^17–19 

The rate of PR+RPE loss was measured to understand the relationship between CRT loss and PR loss, as cones are the predominant cell type at the fovea. Knowing that the rate of change is not linear except in the phase of accelerated decline, care was taken to limit this calculation to appropriate time points, using intrarater variability as a cutoff to aid in identifying the time point at which the floor was reached. Retinal–neuronal remodeling in late stages (i.e., stage 5) further complicated the rate estimation (Fig. 2A).²⁰ With these observations in mind, the mean annualized rate of PR+RPE loss at the fovea during the rapid decline phase was 57.34 ± 29.04 µm, occurring between 39 and 111 months (approximately 3–9 years) of age, although more longitudinal data are required to verify these estimates. This rate was similar to the rate of CRT loss reported by Kovacs et al.⁷ of 67 to 74 µm between 48 months and 72 months of age and the maximum annual decrease in CRT of 23 µm observed by Dulz et al.⁸ These observations are consistent despite the differences in estimation methodologies. 

The findings of this study also demonstrate that the rate of PR+RPE loss changed in a location-dependent manner, with the highest rate of PR+RPE loss at the fovea. These data support a pattern of cone–rod degeneration where rapid PR loss occurs within regions of high metabolic activity.²¹ Given that patients with CLN2 disease experience a reduction of enzymatic TPP1 activity, resulting in lysosomal accumulation of metabolic waste, as shown in an in vitro CLN2-derived ocular model, this process is likely the primary cause of PR death.²² 

The close association between EZ integrity and visual function has been demonstrated in other types of retinal degeneration and is therefore a recognized anatomical surrogate for vision loss among inherited retinal disorders.^23–27 ONL+HFL integrity is also indicative of PR health and, similar to the EZ, has been shown to correlate with visual function.²⁸ In this cohort of patients, EZ loss preceded ONL+HFL loss and progressed at a greater rate compared with ONL+HFL. Taken together, these data demonstrate a rapid loss of PRs that would directly lead to reduced visual function. Loss of VA is highly correlated with loss of CRT thickness in this population, as demonstrated by the functional assessment of VA through the use of a novel optokinetic detection technology (Ohnsman CM, et al. IOVS 2021;62:ARVO E-Abstract 3141; Ohnsman CM, et al. IOVS 2022;63:ARVO E-Abstract 4266). 

In contrast to the outer retina, TPP1 deficiency resulted in a modest effect on inner retinal structures in this ERT-treated cohort. RNFL thickness in CLN2 disease was within normal variations in all disease stages. Mild INL thinning was observed across all disease stages in most patients; however, longitudinal data did not show progressive loss of INL thickness over time. Given the limited number of longitudinal patients, the significance of nonprogressive INL thinning remains unclear. Nevertheless, the totality of outer and inner retinal layer observations indicate that PR loss is the major contributor of FRT reduction in CLN2 disease. 

Identifying OCT biomarkers that signal the imminent onset of retinal degeneration in CLN2 disease could provide clues about optimal timing of therapeutic interventions. Based on data presented here, the earliest signal of retinal degeneration was a discrete area of foveal ELM thickening in stage 1. ELM thickening and granular deposits of residual debris attached to the ELM have been observed among a subset of patients with early-stage Stargardt disease.^29,30 Additionally, similar areas of ELM thickening were identified on OCT scans from a non-human primate CLN7 disease model at ages 3 to 5 years.³¹ These ELM-specific changes may be the result of cellular repair processes mediated by Müller cells in response to pre-degenerative retinal stress.³² Unfortunately, ELM thickening was not consistently observed in all patients and would therefore be an unreliable biomarker. 

The presence of focal areas of parafoveal EZ loss with relative foveal sparing in stage 2, which occurs later than foveal ELM thickening, is another potential biomarker. Further evaluation of en face scans will be useful for verifying this finding and characterizing it in more detail. However, by the time that this phenomenon has occurred, progressive retinal degeneration has begun. 

In summary, retinal degeneration in CLN2 disease is primarily associated with PR loss. Changes in PR-related thickness parameters, primarily ONL+HFL thickness, correlate with CRT loss and CLN2 ocular disease severity. CRT is a useful anatomical biomarker in CLN2 disease until maximal foveal thinning occurs late in the disease, when parafoveal and perifoveal sector changes become more significant for understanding disease progression and treatment efficacy. Future studies including age-matched controls, fundus or fundus autofluorescence images, and en face images will further clarify progression of retinal degeneration in CLN2 disease. Additional longitudinal data are needed to characterize the pattern and progression of EZ loss in CLN2 disease. 

Acknowledgments

Supported by REGENXBIO Inc., Rockville, MD, which provided support for the study and participated in the study design; conducting the study; data collection, management, and interpretation; and review of the manuscript. Third-party writing assistance was provided by Stephanie S. Wenick and funded by REGENXBIO Inc. 

Disclosure: W.C. Huang, REGENXBIO Inc. (E); C.M. Ohnsman, REGENXBIO Inc. (E); Y. Atiskova, Neurogene (C); P. Falabella, REGENXBIO Inc. (E); M.S. Spitzer, AbbVie (C), Apellis Pharmaceuticals (C), Bayer AG (C), Biogen (C), Hoffmann-La Roche (C), GenSight Biologics (C), Neurogene (C), Novartis Pharmaceuticals (C); A. Schulz, Amicus Therapeutics (C), BioMarin Pharmaceutical (C), Neurogene (C), REGENXBIO Inc. (C), Taysha GTx (C); S. Dulz, Amicus Therapeutics (C), BioMarin Pharmaceutical (C), Exicure (C), Neurogene (C), REGENXBIO Inc. (C)