
==== Front
Brain
Brain
brainj
Brain
0006-8950
1460-2156
Oxford University Press UK

38538211
10.1093/brain/awae096
awae096
Original Article
AcademicSubjects/MED00310
AcademicSubjects/SCI01870
Central visual pathways affected by degenerative retinal disease before and after gene therapy
Ashtari Manzar Center for Advanced Retinal and Ocular Therapeutics (CAROT), University of Pennsylvania, Philadelphia, PA 19104, USA
Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA 19104, USA
Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA

Bennett Jean Center for Advanced Retinal and Ocular Therapeutics (CAROT), University of Pennsylvania, Philadelphia, PA 19104, USA
Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA 19104, USA

Leopold David A Laboratory of Neuropsychology, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA

Correspondence to: Manzar Ashtari, PhD University of Pennsylvania Room 201 Anatomy/Chemistry Building Philadelphia PA 19102, USA E-mail: ashtari@pennmedicine.upenn.edu
9 2024
28 3 2024
28 3 2024
147 9 32343246
23 8 2023
09 2 2024
20 2 2024
21 8 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.
2024
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Abstract

Genetic diseases affecting the retina can result in partial or complete loss of visual function. Leber’s congenital amaurosis (LCA) is a rare blinding disease, usually inherited in an autosomally recessive manner, with no cure. Retinal gene therapy has been shown to improve vision in LCA patients caused by mutations in the RPE65 gene (LCA2). However, little is known about how activity in central visual pathways is affected by the disease or by subsequent gene therapy.

Functional MRI (fMRI) was used to assess retinal signal transmission in cortical and subcortical visual structures before and 1 year after retinal intervention. The fMRI paradigm consisted of 15-s blocks of flickering (8 Hz) black and white checkerboards interleaved with 15 s of blank (black) screen. Visual activation in the brain was assessed using the general linear model, with multiple comparisons corrected using the false discovery rate method.

Response to visual stimulation through untreated eyes of LCA2 patients showed heightened fMRI responses in the superior colliculus and diminished activities in the lateral geniculate nucleus (LGN) compared to controls, indicating a shift in the patients’ visual processing towards the retinotectal pathway. Following gene therapy, stimuli presented to the treated eye elicited significantly stronger fMRI responses in the LGN and primary visual cortex, indicating some re-engagement of the geniculostriate pathway (GS) pathway. Across patients, the post-treatment LGN fMRI responses correlated significantly with performance on a clinical test measuring light sensitivity.

Our results demonstrate that the low vision observed in LCA2 patients involves a shift in visual processing toward the retinotectal pathway, and that gene therapy partially reinstates visual transmission through the GS pathway. This selective boosting of retinal output through the GS pathway and its correlation to improved visual performance, following several years of degenerative retinal disease, is striking. However, while retinal gene therapy and other ocular interventions have given hope to RPE65 patients, it may take years before development of therapies tailored to treat the diseases in other low vision patients are available.

Our demonstration of a shift toward the retinotectal pathway in these patients may spur the development of new tools and rehabilitation strategies to help maximize the use of residual visual abilities and augment experience-dependent plasticity.

Gene therapy can improve vision in patients with the retinal disease Leber’s congenital amaurosis. Ashtari et al. reveal a shift in visual processing towards the retinotectal pathway in affected individuals, and show that gene therapy partly reinstates visual transmission through the geniculostriate pathway.

visual system
retinotectal pathway
geniculostriate pathway
brain plasticity
retinal gene therapy
NEI 10.13039/100000053 R01EY025287-01A1 Center for Advanced Retinal and Ocular Therapeutics 10.13039/100019107 University of Pennsylvania Perelman School of Medicine F.M. Kirby Foundation 10.13039/100001205 Research to Prevent Blindness 10.13039/100001818 National Institute of Mental Health Intramural Research Program 10.13039/100000025 ZIAMH002838
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pmcIntroduction

Our visual experiences and interactions with the environment are supported by multiple parallel pathways into the brain, the anatomical organization and physiological specializations of which are well studied.1,2 Little is known, however, about how pathologies affecting vision, such as degenerative diseases of the outer retina, affect the balance of visual processing in these pathways. Nor is it known how emerging therapeutic interventions aimed at sight restoration might impact this balance. The present study uses functional MRI (fMRI) to investigate these topics.

In humans and other primates, the main pathways supporting visual perception transmit signals from the retina by way of relay in the lateral geniculate nucleus (LGN) to the striate cortex (primary visual cortex, or V1). In normally sighted individuals, ∼90% of retinal ganglion cells of different sorts project to the LGN,3 with visual image information then passed along to V1. The retinogeniculate projections are differentiated into two main populations targeting the magnocellular and parvocellular portions of the LGN, along with more heterogeneous ganglion cell populations targeting the koniocellular LGN layers.1 These afferents are matched in the LGN by corticothalamic inputs from V1 and elsewhere, which target functionally similar layers of the LGN.4 Visual stimulation of the geniculostriate (GS) pathway leads to particularly strong fMRI responses in the primary visual cortex and weaker responses in extrastriate areas such as MT/V5. Following damage to the GS pathway, some extrastriate areas continue to respond to retinal stimulation, albeit with a greatly reduced magnitude.5,6 Perceptually, the GS pathway is sometimes conceived as a bottleneck for conscious visual processing, as its damage virtually abolishes subjective visual experience.7

Following damage to the GS pathway, residual visual responses can be explained by the ∼10% of retinal ganglion cells in primates that innervate other central structures, most prominently the superior colliculus (SC).3 A major role of these retinotectal (RT) pathways is to relay visual information to extrastriate cortical areas via thalamic nuclei, including the pulvinar8 and LGN9-12 (for a review, see Baldwin and Bourne13). Following V1 damage, these RT pathways are believed to support residual visual guided behaviours and corresponding responses in the cerebral cortex.6,14-17 The most prominent cortical target site of the RT pathways is MT/V5, which also receives strong GS pathway input directly from V1. In addition to the GS and RT pathways, small populations of specialized ganglion cells project to other targets,2,18 including directly to retinotopically organized subdivisions of the pulvinar19,20 and pregeniculate nucleus.21

How is the engagement of central visual pathways affected by degenerative retinal disease, and to what extent do any changes reverse their course following genetic therapy leading to partial visual recovery? Here we investigate these questions by studying fMRI responses in patients with Leber’s congenital amaurosis (LCA), a rare blinding disease usually inherited in an autosomally recessive manner. LCA is typically symptomatic at birth or during the first few months of life and affects around 1 in 81 000 people.22 LCA is associated with mutations in any of more than 20 different genes, with one of the most common forms, LCA2, due to bi-allelic RPE65 mutations (LCA2). LCA2 leads to slow degeneration of the retina, affecting both cone and rod function,23 and progressive vision loss or blindness as the patient ages.22,24 Vision loss is most commonly manifest clinically as low spatial resolution and low contrast and light sensitivity.25 The mechanistic bases of these visual deficits are difficult to pinpoint, in part because the affected retinal cells exhibit pathway convergence, with rods and cones projecting to the same ganglion cells, and divergence, with ganglion cell subpopulations projecting to multiple central targets.11,26,27

We used fMRI to measure the magnitude of visual responses in GS and RT pathways in patients with LCA2 disease, first following several years of retinal degeneration and second, following therapeutic retinal gene therapy with subretinal injection of AAV2.hRPE65, known to improve vision and to increase responses in the visual cortex.28-30 In the first experiment, we show an altered pattern of visual fMRI responses in the central visual pathways of LCA2 patients relative to matched controls. Namely, whereas the disease diminished visual responses in the LGN and visual cortex, it led to significantly elevated visual responses in the SC, suggesting an unexpected strengthening of the RT pathway. In the second set of experiments, we report that ocular gene therapy led to a partial restoration of activity in the LGN and V1 but did not affect the magnitude of responses in the already strengthened SC. Together, these results indicate that the retinal degeneration caused by LCA2 leads to a permanent shift in the balance of processing in the RT and GS pathway. We submit that this new understanding of central visual processing in retinal disease can facilitate strategies to augment vision in the large proportion of patients who currently do not have access to ocular gene therapy.

Materials and method

Study participants

Seven affected individuals and seven normally sighted control subjects participated in this study. The affected patients were participants in a gene therapy clinical drug trial (phase 1, Clinical Trial #NCT00516477, www.clinicaltrials.gov), which was initiated as part of the proof of safety and efficacy of gene augmentation therapy for LCA2 patients. This study was carried out in two stages. In the first stage (phase 1/1), patients underwent treatment of one eye, namely that with the poorer vision. This was done in the context of a dose-escalation trial, with subretinal adeno-associated virus (AAV) injection at low, medium and high doses of the recombinant AAV, voretigene neparvovec-rzyl (LuxturnaTM).28,31 A battery of exploratory measures revealed improvements in these individuals,28 functional enhancement of cortical visual responses30 and associated structural changes to visual pathways,32 with no clear dose-related effect. The success of the initial dose escalation trial led to a follow-on study (phase 1/2) in the same group of patients (Clinical Trial #NCT01208389), in which only the high-dose retinal gene therapy was delivered, in this case to the previously untreated eye.33-35 The present fMRI investigations were carried out just before and 1 year after treatment of the second eye in the follow-on study.

For the fMRI testing, initial scanning was carried out in nine patients following phase 1/1 and prior to phase 1/2. However, two of the original patients were subsequently excluded from further fMRI testing, one due to glaucoma (Patient CH13) and one due to the frequent appearance of phosphenes (Patient CH11). Thus, seven patients participated in the fMRI studies.36 Four of these participants were children and three adults. In the present study, these individuals were considered as a single cohort, in part because previous studies did not identify age as a significant variable for disease presentation and severity of visual functions.31,37

One of the inclusion criteria was confirmation of bi-allelic RPE65 mutations. In addition, participants had clinical diagnoses of LCA, were age 8 years or older at the time of intervention and had visual acuity worse than Snellen 20/160 or a visual field less than 20° in the eye to be injected. The latter are legal definitions of blindness. Additional details of inclusion/exclusion criteria are presented in earlier reports.28,38 Detailed demographics of the LCA2 patients including the mutation type and their originally treated eye are presented in Table 1. All patients received subretinal injection of AAV2.hRPE65v2 gene directed primarily to the superior temporal retina location.28,31

Table 1 LCA2 patient demographics

Subject ID	Age (years) at follow-on study	Sex	Newly treated eye	Years/original treated eye	RPE65 mutation	
CH08	12	Male	Left	3.39/Right	F350fs/F530fs	
CH09	8	Male	Right	3.33/Left	R124X/Lys297del1ggA	
CH10	10	Male	Left	3.20/Right	IVS1+5 g>a/Phe530del1ttc	
CH12	44	Female	Left	2.69/Right	K303X/W431C	
NP01	30	Female	Left	3.41/Right	E102K/E102K	
NP02	30	Male	Left	4.06/Right	E102K/E102K	
NP15	11	Male	Left	2.07/Right	D167W/H313R	

Seven normally sighted control subjects, who were demographically matched to the LCA patients, were recruited by flyers and word of mouth. Controls were excluded if they had any current or past psychiatric diagnosis or a history of drug or alcohol abuse. Additional exclusion criteria for all subjects included mental retardation, known neurological disorder, a history of head injury or any focal findings revealed by MRI, or current use of psychotropic medications. All healthy control participants were initially phone screened and, upon their qualification, were invited to participate in the study. Subjects were also excluded from the neuroimaging study if they had a positive pregnancy test; expressed claustrophobia; had a metallic implanted prosthetic or device (e.g. cardiac pacemaker) or other contraindications for MRI; had excessive metallic dental work (including braces and non-removable retainers); or were non-compliant. All of the affected LCA2 patients met the same inclusion characteristics28,31 and were participants in the follow-on study (NCT01208389), where the same dose of AAV2.hRPE65v2 was administered to each of the contralateral eyes.31 While the patients received various strength of the therapeutic does (low, medium, high) for their original treated eye, all subjects in the follow-on study (NCT01208389) received the high dose.

Detailed statistical comparison for matching of patients and controls are presented in Table 2. In addition to their consent process to enter the clinical trial study, all patients and controls were separately consented for the current neuroimaging study. All subjects provided written informed consent (≥18 years old) or written assent and parental permission (<18 years old). Consent was obtained according to the Declaration of Helsink, and it was approved by the ethical committees of both the University of Pennsylvania and the Children’s Hospital of Philadelphia.

Table 2 Statistical comparison of participants

Demographics	Controls	Patients	P-value	
n	7	7		
Age at baseline, years	
 Mean (SD)	21.5 (13.5)	20.71 (13.91)	0.91a	
 Range	(9.0–44.0)	(8.0–44.0)		
Sex	1.00b	
 Female	0 (0.0%)	2 (28.5%)		
 Male	7 (100.0%)	5 (71.5%)		
Race	1.00b	
 White	7 (100%)	7 (100%)		
Handedness	1.00b	
 Left	0 (0.0%)	1 (14.3%)		
 Right	7 (100.0%)	6 (85.7%)		
SD = standard deviation.

aEqual variance t-test.

bFisher's exact.

Behavioural tests

Full-field sensitivity threshold

The full field sensitivity threshold (FST) is one of the exploratory clinical tests for the LCA2 patients used in the initial gene therapy studies39,40 and has been used as an outcome measure to assess the efficacy in the registrational trial.33,34,37,41,42 FST measures sensitivity of the entire visual field (full-field stimulus) by recording the patient’s behavioural detection of monocularly presented stimuli. This establishes the lowest detectable luminance flash of red, blue and white light. The subjects are asked to indicate, by pressing a button, when they can perceive the stimulus. In the FST testing, the two colours of red and blue are used to estimate the level of rod and cone dysfunction and the white colour is used for mixed photoreceptors.40 The FST responses are used to assess photoreceptor function, such as whether patients have viable rod or cone vision and which photoreceptor types are affected most.40 Results of the FST clinical tests for the LCA2 patients are presented in Supplementary Table 1. FST test was not used at the beginning of the clinical trial and as a result, two subjects did not undergo FST testing (Supplementary Table 1).

Visual acuity

Visual acuity (VA) is a primary outcome safety or efficacy measure in many studies involving ocular diseases. Visual acuity was measured monocularly for the LCA2 patients by trained vision examiners using a standard protocol involving Early Treatment Diabetic Retinopathy Study (ETDRS) charts and letter counts.43 Letter scores were converted to the log of the minimum angle of resolution (logMAR), on a scale ranging from 0.00 to 2.00, with higher values indicating poorer vision. Eyes that could detect hand motions were assigned a score that was one line worse than the largest printed line on the chart tested at a standardized distance of 4 m to provide the most conservative evaluation in terms of underestimating the actual extent of visual impairment.28 The visual acuity test results for the LCA2 patients are presented in Supplementary Table 1. The visual acuity result for one subject was not included due to a macular hole.28,44

MRI

Data were acquired using the research-dedicated 3 T Siemens Verio MRI scanner at the Children’s Hospital of Philadelphia (CHOP) using a 12-channel head coil. All subjects participated in two imaging sessions associated with different trial phases. Session 1 (phase 1/1, NCT00516477) occurred just prior to the injection of the contralateral, previously untreated, eye. Session 1 was used to assess the effect of LCA2 disease relative to controls, as well as for the within-session evaluation of the effects of gene therapy in each individual subject. Session 2 (phase 1/2, NCT01208389) occurred approximately 1 year after gene therapy of the contralateral eye. Session 2 was used for the longitudinal evaluation of gene therapy in individual patients, comparing the responses to the initially untreated eye before or after treatment. All fMRI tests were conducted monocularly. Scanning was carried out by a single expert operator and monitored to be free of motion and other artefacts at the time of acquisition.

Anatomical imaging

A 3D isotropic structural high resolution T1 sequence was acquired with inversion preparation pulse (IR-Prep: repetition time = 2080 ms, echo time = 2.54 ms, bandwidth = 180 Hz/Px, matrix size = 320 × 320, field of view = 256 × 256 mm2, 192 axial slices, slice thickness = 0.8 mm, inversion time = 1200 ms with flip angle = 8°, NEX = 1, echo spacing = 7.8, iPAT = 2 and scan time = 7:04 min). This sequence was obtained for visual activation localization and the generation of inflated hemispheres (www.brainvoyager.com).

Functional MRI

Functional MRI sequence

Functional data were acquired using blood oxygenation level-dependent (BOLD) imaging, acquiring 3 mm isotropic resolution (matrix, 64 × 64; repetition time/echo time, 3000/30 ms) with a total acquisition time of 4 and 23 s. Detailed information for data acquisitions is presented elsewhere.30,32,35 All subjects were monitored using the real time feature of the MRI system.36 Real time monitoring of the subjects allows for motion detection at the time of data acquisition as well as ensuring an active participation of the individual being scanned. Using the real time feature, subject movement exceeding ≥0.6 mm translation or ≥0.6° for rotation led to the termination of the scan, at which the subject was informed to stay still and the experiment was restarted.

Functional MRI paradigm

The fMRI paradigm used for evaluation of visual functions for phase 1 (NCT00516477) and phase 1/2 clinical trial (NCT01208389) participants was identical and consisted of 15-s blocks of flickering (8 Hz) black and white checkerboards, which consisted of three contrasts of high, medium and low, interleaved with 15 s of blank (black) screens.30,34,35 Subjects were asked to fixate on a yellow cross in the centre of the checkerboard patterns or, if they could not see the cross, were asked to look straight ahead. Visual stimuli were presented using Resonance Technology VisuaStim (www.mrivideo.com) goggles featuring a digital display and a 30° horizontal field of view. The visual paradigm was programmed in E-Prime (http://www.pstnet.com/eprime.cfm). All fMRI for both patients and controls were restricted to monocular stimulus presentation, with the VisuaStim goggles providing independent digital stimulation to a given eye while the other eye was cut off completely.

Functional MRI image preprocessing

Details for analysis of functional data are presented in earlier reports.30,32,35,36 In summary all functional MRI data were processed using Brain Voyager version 21.4 (BV21.4) (www.brainvoyager.com, Maastricht, The Netherlands). Preprocessing of data included slice scan time correction, 3D motion correction, high-pass temporal filtering and spatial smoothing. Sinc interpolation was used for scan time correction to ensure that all voxels in the volume represented the signal simultaneously. A high-pass temporal filter with a cut-off frequency of two cycles per run was applied to remove low frequency drift noise. Spatial smoothing was performed using a conservative 3 mm full-width at half-maximum Gaussian filter. Statistical analyses were performed using the general linear model (GLM) as implicated in BV21.4.45 All three of the high, medium and low contrast conditions were analysed together by specifying a design matrix, defined as blocks with checkerboard presentation versus blank black screens as control blocks, followed by application of a haemodynamic response function and correction for multiple comparisons using the false discovery rate (FDR).

Functional MRI statistical analyses

Using BV21.4 for each functional run of an individual subject, a protocol file (PRT) was created representing the duration and timing of each condition for the visual tasks. A design matrix was then constructed using the defined PRT and data dispersion due to haemodynamic response with a double-gamma function.46 Task-based visual paradigm for each subject and separate runs were then analysed using the GLM, which is mathematically similar to a multiple regression analysis and is based on a univariate statistic for each voxel of the acquired brain volumes. The visual task was analysed by contrasting the sum of high, medium and low contrast checkerboard blocks with the rest blocks. To correct for the multiple comparison the FDR approach47 was used. While we were able to correct the larger cortical regions of interest (ROIs) [Brodmann area (BA)17, BA18, BA19 and MT/V5] for multiple comparison (FDR) at q = 0.05 level, the fMRI results for the small subcortical regions, such as LGN, SC and pulvinar, were not corrected for multiple comparison but were evaluated at a statistically significant uncorrected P = 0.05. All fMRI results for individual participants were transferred to the Talairach (TAL) atlas space to accurately measure the activation levels within all selected ROIs in the same TAL space.

Region of interest selection and extraction

In general, the ROIs were categorized into cortical and subcortical groups. The subcortical group of ROIs consisted of the LGN, SC and pulvinar. A sample of the extracted subcortical ROIs along with fMRI activations resulted from unilateral ocular stimulations and the superimposition of the two are depicted in the Supplementary Fig. 1. The cortical ROIs were comprised of the striate cortex (V1, primary visual cortex BA17), extrastriate cortex (BA18 and BA19) and the motion sensitive medial temporal brain area (MT/V5). A sample of the extracted regions of interests for cortical vision centres along with the superimposition of the fMRI activations on these centres are depicted in the Supplementary Fig. 2.

To extract the subcortical ROIs, 3D anatomical data of each subject were preprocessed using BV21.4 with the processing steps of intensity inhomogeneity correction, resampling from 0.8 to 1 mm isovoxel and spatial transformation to AC-PC and TAL space. The TAL transferred images for each subject and all their MRI imaging from two time points were registered to create an average image per subject. An atlas was then created by co-registration of all average T1-weighted images. The subcortical ROIs were delineated on the atlas images and propagated to the baseline and 1 year time points for the LCA2 patients and baseline images of the sighted controls. All subcortical ROIs were carefully examined for individual subjects and fine tuned to ensure the delineated ROIs encompassed the structures of interest consistent with the subject’s anatomy in TAL space. A typical extraction of the LGN, SC and pulvinar are shown in the top row of Supplementary Fig. 1 along with subcortical distribution of fMRI results (Supplementary Fig. 1) and superimposed fMRI activations onto the subcortical ROIs (Supplementary Fig. 1).

The extraction of the cortical ROIs was facilitated through cortex-based alignment (CBA) algorithm implemented in BV21.4. CBA is used to improve the spatial correspondence mapping between subjects’ cortical curvatures beyond the TAL volume space matching (the registration used for subcortical ROIs). This method matches gyri and sulci locations across the brains of each individual subject to bring major cortical landmarks into alignment beyond standard normalization. To perform CBA, the final TAL transferred brain images were extracted from surrounding head tissue and segmented to define the grey/white matter boundary to reconstruct a folded surface representation for the left and right hemisphere.48 CBA registration is then performed to align all the reconstructed folded meshes49 for patients and controls. Furthermore, the use of CBA transformation and registration matrix allows for a more precise alignment of subject-specific ROIs, or Brodmann areas, such as the primary visual cortex (BA17), extrastriate visual areas (BA18 and BA19) and MT/V5 cortical labels. Supplementary Fig. 2 shows an example of the cortical ROIs for B17, BA18 and BA19, and MT/V5 (Supplementary Fig. 2, first row) and the superimposed cortical activations onto cortical ROIs (Supplementary Fig. 2, second row).

Results

A schematic diagram of the study design is depicted in Fig. 1. We used fMRI to investigate visual processing in subcortical and cortical structures of LCA2 patients and matched normal sighted controls to characterize the visual responses in GS and RT pathways. In all cases, fMRI responses were assessed using a standard checkerboard stimulus and a boxcar fMRI design. In the first part of the study, the focus was to study the effects of retinal disease on visual pathways, comparing fMRI responses originating in patients’ untreated eyes to those of matched controls (Fig. 1A). In the second part of the study, we investigated the relative impact of therapeutic retinal gene therapy with (subretinal injection of AAV2.hRPE65; the drug that was later named LuxturnaTM and approved by the US FDA) (Fig. 1B) on the GS and RT pathways. The fMRI responses from the patients’ untreated eyes were compared to the responses of their treated eyes. Patients were studied in either the same session, where the eye treatment preceded the study by an average of 3 years (Fig. 1C) and in a longitudinal experimental design, in which fMRI assessment of the untreated eye was followed by its treatment and subsequent assessment 1 year later (Fig. 1D).

Figure 1 Schematic diagram of study design. (A) The first fMRI study focused on examining the effects of LCA2 disease on the visual system by comparing the fMRI responses of the untreated eye to those of controls using the same visual stimuli. (B) The wild-type copy of the RPE65 cDNA was delivered through the AAV2 vector to the superior temporal location of the retina. (C) The second fMRI study was to assess the effects of retinal gene therapy on the visual system by comparing the visual responses from the previously treated eye to the responses from the untreated eye within the same fMRI scanning session. Here the untreated eye is shown in black and the eye with intervention is shown in red colour. (D) The third fMRI experiment was carried out 1 year after the untreated eye received subretinal viral injection. The study was carried out in two separate fMRI scanning sessions 1 year apart. In the first session the fMRI was performed on the untreated eye and in the second session (1 year later) fMRI was performed on the same eye 1 year after its retinal intervention using the same visual stimuli. LCA = Leber’s congenital amaurosis.

Shift in LCA2 patients’ visual processing to the retinotectal pathway

The profile of subcortical and cortical responses to monocular visual stimulation differed markedly in LCA2 patients compared to control subjects (Fig. 2A and Supplementary Fig. 3). Consistent with previous findings,30 the patients showed greatly reduced responses in the visual cortex, namely cortical area V1 (P = 1.2 × 10−5) and extrastriate areas BA18/19 (P = 0.01). In addition, they showed weakened visual responses in the LGN (P = 0.004) (Fig. 2A). This weakening of responses in the two main components of the GS pathway presumably reflected decreased transmission of visual signals through retinogeniculate projections stemming from damage to the retinal circuity. More surprisingly, the fMRI responses in the SC were strongly and significantly elevated (P = 4 × 10−4) in LCA2 patients compared to controls. These contrasting subcortical effects are visible in individual subjects (Fig. 2B), comparing LGN (blue boxes) and SC (yellow arrows) responses between control subjects and LCA2 patients. No such elevation was observed in the pulvinar (Supplementary Fig. 3). This alteration in the subcortical activation pattern suggests that the retinal degeneration accompanying LCA2 disease shifts the transmission of visual signals more strongly to the RT pathway and away from the GS pathway.

Figure 2 Comparison of the LGN and superior colliculus fMRI activations. (A) The average lateral geniculate nucleus (LGN) activation in response to vision stimulation in LCA2 patients was significantly (P = 0.003) lower than in normal sighted controls. In contrast, the average superior colliculus (SC) activation was significantly (P = 0.0004) higher in the LCA2 patients compared to controls. (B) Patterns of fMRI activations for LGN (blue boxes) and SC (yellow arrows) in controls (left column) and LCA2 patients (right column). (C) In controls, the activation in V1 (shown in green) was significantly (P = 0.005) higher than in MT/V5 (shown in purple) area. No such difference was observed in the LCA2 patients. In fact, while sighted controls presented with significantly lower activation levels within MT/V5 versus V1, LCA2 patients showed slightly higher levels of activations in MT/V5 versus V1, although not significant (NS). This may show the resilience of the large receptive field cell populations within the MT/V5 area to the RPE65 disease compared to midget cell population in the primary visual cortex (V1). LCA = Leber’s congenital amaurosis.

As area MT/V5 is a critical cortical target of RT pathways, we additionally investigated whether its visual activation was also affected in LCA2 patients. Comparing its absolute response to controls did not, like the SC, show increased visual responses in LCA2 patients (Supplementary Fig. 3). Most subjects showed decreased responses in MT/V5, presumably because of the diminished activity in V1, whose input to MT/V5 is substantial.50 To explore further, we asked whether the relative activation of V1 and MT/V5 was affected by the rebalancing of pathways in LCA2 patients. This was indeed the case: whereas V1 responses were stronger than MT/V5 responses in control subjects (P = 0.005), they were slightly lower or of equivalent magnitude in LCA2 patients (Fig. 2C). Together, these findings are consistent with the hypothesis that, following a period of gradual retinal degeneration in LCA2 patients, visual processing shifts to the RT pathway and away from the GS pathway.

Partial restoration of the geniculostriate pathway following gene therapy

Retinal degeneration in LCA2 patients has been successfully treated using gene therapy, resulting in a strengthening of cortical responses.30,35 With access to these patients, it is possible to investigate how the disease-related shift toward the RT pathway is affected by such treatment. The following sections employ same-session and longitudinal fMRI designs to study the recovery of visual responses in the brain, and a link to visual performance, for the treated patients.

Same-session functional MRI assessment of gene therapy

The ‘same session’ design compared visual fMRI responses following stimulation through the treated versus untreated eye in the phase 1 clinical trial (NCT00516477, Fig. 1C, refer to the ‘Materials and methods’ section). Briefly, fMRI during monocular visual stimulation of the left and right eyes was performed separately, with the eye order randomized across subjects. All seven LCA2 patients had received their initial, unilateral retinal gene therapy treatment an average of 3 years earlier (Table 1).

Figure 3A shows the fMRI activation maps, highlighting responses on the LGN and SC (cyan boxes) in the left columns and on V1 (green boxes) of all LCA2 patients in the right columns. Retinal gene therapy treatment led to overall higher magnitude visual fMRI responses in the cortex, consistent with previous findings.30,34,35

Figure 3 Same session study design fMRI results. Within-subject comparison of visual activation of treated versus untreated eye of LCA2 patients, with each eye’s data collected in the same fMRI scanning session. fMRI experiments were performed monocularly for both patients and controls to evaluate the treated and untreated eye separately. (A) The subcortical (blue boxes) and cortical visual activation patterns (green boxes) are shown for each patient for the same session design, conventions as above. Stimulation of the treated eye led to stronger activation in both the lateral geniculate nucleus (LGN) and V1 regions of interest (ROIs). (B) ROI (Supplementary Figs 1and 2) quantification revealed that stimulation of the treated eye led to significantly stronger fMRI activation in the LGN (P = 0.003) and V1 (P = 0.015) than stimulation of the untreated eye. Treatment did not affect activation of the superior colliculus (SC), which remained elevated relative to controls following stimulation of both treated (P = 0.004) and untreated (P = 0.03) eyes. There were no significant differences in pulvinar activations between patients and controls. Sighted controls showed significantly enhanced fMRI responses to the same visual stimulation of the treated or untreated eye of the LCA2 patients in LGN and V1 areas. Conversely, control responses to the visual stimuli in the SC area was significantly lower compared to the SC responses in the treated or untreated eyes. No significant changes were observed in pulvinar activations when comparing sighted controls and patients.

Important for the current study, analysis of responses in both subcortical and cortical visual structures revealed that the treatment affected the GS and RT pathways differently (Fig. 3B). Namely, significant response enhancements were observed in the treated eye relative to the untreated eye in the LGN (P = 0.003) and V1 (P = 0.015), the two main components of the GS pathway. These increases were significant but also remained significantly below the corresponding response magnitudes in control subjects. In contrast to the GS structures, no such enhancement was observed in either the SC (P = 0.89) or the pulvinar (P = 0.72), two main components of the RT pathway (Fig. 3B). Activation levels in extrastriate visual areas BA18/19 and MT/V5 were marginally, but not significantly, enhanced following gene therapy (Supplementary Fig. 5). Together, these data suggest that the AAV treatment applied to the retina preferentially affected cell types and visual projections used by the main, GS pathway and had relatively little impact on the RT pathway.

Functional MRI correlation with improvements in visual performance

To what extent might the increased transmission along the GS pathway lead to improvements in visual performance? Two important clinical outcomes described previously in LCA2 patients were (i) visual acuity for a subset of patients under photopic conditions; and (ii) heightened sensitivity to full field stimuli under scotopic conditions.28,29,39,40 To determine whether the augmented brain responses may be directly related to visual performance, we correlated these measures with fMRI responses in the GS system of the same patients. We focused on the responses of the LGN as the subcortical retinorecipient structure of the GS pathway.

For each subject, the magnitude of LGN fMRI activations to monocular visual stimulation in the treated and untreated eyes were compared to two clinical measures assessed monocularly in the same eyes, the FST and the VA test (refer to the ‘Materials and methods’ section). The FST and VA data for individual LCA2 patients are presented in Supplementary Table 1. The activation of the LGN was tightly correlated (r = −0.90, P = 0.04) across subjects with performance on the FST clinical assessment of the treated eyes (Fig. 4A and Supplementary Fig. 4). The correlation trend was similar for the VA, though the results did not reach statistical significance (Fig. 4B) nor did the overall VA performance differ significantly between the treated and untreated eyes (Supplementary Table 1). Nonetheless, significant correlation in the case of FST is consistent with the hypothesis that the partial restoration of the GS pathway underlies the demonstrated improvements in visual performance.

Figure 4 Lateral geniculate nucleus activation correlations with clinical measures. Pearson correlations were performed between averaged lateral geniculate nucleus (LGN) fMRI responses and two important LCA2 patient’s outcome measures: full field sensitivity threshold (FST) responses to blue light and visual acuity (VA). (A) FST responses to the blue colour stimuli from the treated eye also correlated negatively (r = −0.90, P = 0.04) with the magnitude of LGN fMRI responses, where lower FST values indicate higher visual sensitivity. (B) Across subjects, visual acuity measures of the treated eye correlated negatively with the magnitude of LGN fMRI responses of the same eye (r = −0.67, P = 0.07), where lower LogMAR values indicate higher visual acuity. Two patients did not undergo the FST tests as this clinical test was not available at the time of their retinal intervention and one subject’s visual acuity test was excluded due to a macular hole (Supplementary Table 1).

Longitudinal functional MRI assessment of gene therapy

The second, ‘longitudinal’ study design tested the same seven patients that participated in the phase 1/2 follow-on study (NCT01208389, Fig. 1D). In this case, initial visual fMRI scans were carried out in the untreated eye and the results were compared with fMRI scans to the same eye ∼1 year following gene therapy treatment.

The results of the longitudinal study (Fig. 5) were qualitatively and quantitatively similar to those of the same-session design described above (Fig. 3). One year following gene therapy, visual fMRI responses in the LGN (P = 0.006) and V1 (P = 0.004) were strongly elevated after retinal intervention. Likewise, this enhancement was restricted to the GS pathway and was not observed in either the SC (P = 0.33), the pulvinar (P = 0.68) (Fig. 5B) or the extrastriate visual areas BA18/19 or MT/V5 (Supplementary Fig. 5). It is important to point out that convergent findings from the same-session and longitudinal experiments is an internal replication, as it involves different eye cases, albeit in the same patients. The overall similarity of the results in the two different designs thus strengthens the finding that gene therapy treatment protocol selectively strengthens GS pathway.

Figure 5 Longitudinal study design fMRI results. Longitudinal, within-subject comparison of visual activation before and 1 year after RPE65 treatment. Functional MRI experiments were performed monocularly for both patients and controls to evaluate the treated and untreated eye separately. (A) The cortical and subcortical visual activation patterns are shown for each patient for the longitudinal same-subject design, same conventions as before. Following treatment, stimulation of the treated eye led to stronger activations in lateral geniculate nucleus (LGN) and V1 in most patients. (B) Region of interest (ROI) quantification of visual responses in the untreated eye at baseline versus responses from the same eye 1 year after retinal gene therapy and compared to responses from sighted controls. One year after treatment, monocular stimulation of the treated eye led to significant higher visual activation of the LGN (P = 0.006) and V1 (P = 0.004). The treatment did not affect the superior colliculus (SC) activation before and after gene therapy, but the SC activation was elevated both before (P = 0.00043) and after (P = 0.00047) gene therapy compared to sighted controls. There were no significant (NS) differences in pulvinar (Pul) activations between patients compared to controls. Thus, the findings from the longitudinal design matched those of the same-session study design. Similar to the same session fMRI results, in the longitudinal study, controls showed significantly enhanced fMRI responses to the treated or untreated eye of the LCA2 patients in LGN and V1 ROIs. On the contrary, SC responses of the control group was significantly lower compared to the SC responses of the treated or untreated eyes.

Discussion

Moderate-to-severe vision impairments affect millions of individuals worldwide.51 Beyond limiting visual abilities, low vision affects a person’s sense of independence, ability to carry out daily life activities, and safety. Although retinal gene therapy and other retinal interventions have given hope to the low vision patient population,52,53 it may take years before all genetic forms of retinal degenerative disease can benefit from such advances. Unfortunately, retinal disorders are progressive in nature, where not only the retina, but also downstream visual pathways will deteriorate over time,54-57 leaving little hope for a successful vision restoration by gene augmentation therapy outside of the window of opportunity. Maximal use of residual visual capabilities may be the key to maintain experience-dependent plasticity of the visual circuits until retinal therapeutic interventions become available. Thus, understanding the distinct information channels affected by retinal degeneration and the alternate pathways used to compensate for patients’ visual disabilities is of great importance.

Prior to the present study, little was known about the central pathways contributing to residual vision in chronic retinal diseases such as LCA. Having access to a unique population of LCA2 patients in the present study allowed us to study this question using standard fMRI paradigms before and after treatment, compared to matched control subjects and using two different within-subject treatment designs.

A shift to the retinotectal pathway in congenital retinal disease

One significant and unexpected finding from the present study was a significant enhancement of visual responses in the SC in LCA2 patients. This change, together with higher relative response in MT/V5 compared to V1 in these patients, seem to indicate that the RT pathway has become more dominant following a period of retinal degeneration. The diminished visual signals through the GS pathway may have its origin in the selective loss of or dysfunction of certain photoreceptor populations. As detailed in a recent report,42 the gene implicated in this patient population encodes retinal pigment epithelium 65 kDa protein (RPE65), which plays a key role in the retinoid cycle and phototransduction in photoreceptor cells.58 When RPE cells lack this protein, they cannot provide sufficient 11-cis-retinal to photoreceptors for them to respond efficiently to light. Over time, as the disease progresses, both the sensitivity and density of the photoreceptor mosaic are reduced. This combined effect may particularly degrade visual information entering the GS pathway via midget ganglion cells, as this cell class draws upon input from densely packed photoreceptors.1 Anatomical evidence suggests that chronic under-utilization of the GS pathway in LCA2 patients has long-term consequences, resulting in axonal changes within the GS track32 as well as retraction and/or elimination of the dendritic population of the primary visual cortex in animals.59,60 With greatly reduced GS input, the primary visual cortex may cease to function normally, diminishing, or in some severe cases eliminating, conscious visual experience.

What mechanism underlies the strengthening of SC responses in the LCA2 patients? One general explanation is an effective rebalancing or unmasking of SC visual responsiveness stemming from altered excitatory and inhibitory balance of circuit elements involved in early visual processing. For example, if the primary effect of the retinal disease is to diminish GS activation, this might have the consequence of gradually upregulating or strengthening corticotectal input from V1 and elsewhere known to innervate the superficial visual layers of the SC.61 Alternatively, the increased responses could stem from disinhibition, where excitatory cortical input to inhibitory interneurons is weakened. Finally, the prolonged absence of excitatory corticotectal projections could lead to an upregulation of the synaptic efficacy of direct retinal projections to SC. At present, which of these or any other mechanisms underlie the observed rebalancing of pathways remains a matter of speculation.

What are the potential ramifications of strengthening the RT pathway as a means of compensating for diminished GS input? The RT pathways are often deemed secondary, in some cases thought to support unconscious or minimally conscious processes related to orienting, dynamic actions, navigation, emotion or other cognitive operations.62,63 In primates, the SC has been studied in the context of saccadic eye movements, detecting salient or threatening features, or even analysing facial information.64,65 Recent work in the macaque suggests that the SC plays a pivotal role in directing visual attention in the temporal cortex,66 suggesting that its role in human cognition may be greater than previously believed. Evolutionarily, the RT pathway has long been the principal pathway carrying signals to the vertebrate telencephalon, with an expansion of the GS pathway in mammals and the extreme dominance being unique to primates.67

The congenital nature of LCA2 raises the possibility of a gradual recalibration of pathways during development, rather than an abrupt transition. The early life maturation of visual pathways itself involves a changing balance of retinofugal pathways. Recent evidence suggests that, in addition to projecting to the SC and LGN, the primate retina initially projects directly to the inferior pulvinar,68 constituting a shortcut for delivering visual information to extrastriate cortical area MT/V5 during infancy, and then disappearing early in life as a part of normal development.20 Damage early in life to the GS pathway can result in a decreased dependence on V1 and a persistence of the retinopulvinar pathway.69 While we did not observe changes in the fMRI response magnitude of the pulvinar, a developmental retention of this pathway in LCA2 patients is an important factor that might impact the overall balance of activity and, for example, explain the preserved visual responses in MT/V5.

At the time of testing, the strengthened responses in SC, together with relatively strong responses in MT/V5 despite diminished V1 input, suggest that the RT pathways may play an important role in the patients’ residual vision as information carried by the GS pathway is degraded. The RT pathway draws upon both parasol and diverse bistratified ganglion cells in the retina. The larger receptive fields and higher contrast gain of these populations compared to midget cells likely make them more resilient to photoreceptor damage of the type that is eventually experienced in RPE65 patients. In fact, it is likely that patients rely increasingly on RT pathways as the disease progresses. In contrast to the GS pathway, the RT pathway appears resistant to degeneration, as it is known to be partially spared in blindsight and amblyopia patients as well.70,71 Studies of blindsight patients have demonstrated that, with severe damage to the primary visual cortex (i.e. the GS pathway), cortical area MT/V5 exhibits heightened visual responses (for a review, see Werth72) presumably through fibres bypassing V1 and conveying the visual information to extrastriate cortex directly (i.e. a RT pathway).73-75 Interesting, an analogous resilience of the MT/V5 responses was recently demonstrated by our group in an fMRI study through population receptive fields (pRF) technique involving a group of low vision patients with choroideremia.76 Thus, the reliance on the parallel visual channels of the RT pathway may not be unique to LCA2 but may be a more common consequence of retinal degeneration.

Retinal gene therapy partially restores use of the geniculostriate pathway

Many ocular diseases leading to low vision are not yet candidates for gene therapy. Fortunately, LCA2 patients benefit from such treatment, as demonstrated by the positive data leading to FDA approval of Luxturna.41,42 One contributing factor to this success is the relatively long survival time of photoreceptors in this disease, even as they are severely dysfunctional, thus providing a wide time window for intervention. The gene therapy approach involves delivery of a normal copy of the native human RPE65 cDNA to the diseased retinal pigment epithelium (RPE) cells through a subretinal injection of a recombinant AAV.77-79 Despite the empirical successes of this revolutionary treatment, much remains to be learned about the mechanism by which the retinal intervention affects function in the brain’s pathways mediating different aspects of vision.

Our experiments using both fMRI and visual performance to test the effects of gene therapy on subcortical visual pathways converged on similar conclusions. Namely, the LCA2 disease, followed by the revitalization of the retina through RPE gene therapy, primarily affected the retinal cells projecting to the LGN, feeding the GS pathway and had relatively little effect on the RT pathway. It is interesting to note that the retained elevation of visual responses in the SC following gene therapy may indicate that, despite the improved capacities of the GS pathway, the patients’ RT pathway remained strengthened even after the retinal intervention. Hence, the upregulation of the GS pathway did not automatically lead to a downregulation of the RT pathway.

According to Jacobson and colleagues,37 LCA2-directed gene therapy has not been proven to increase foveal cone vision but there is clear evidence for increase in targeting efficiency of extrafoveal rod photoreceptors. In a separate report, Cideciyan and colleagues80 determined that the effects of gene therapy are much higher for the rods than cones. For cones, there were increases of up to 1.7 log units (i.e. 50-fold); and for rods, there were gains of up to 4.8 log units (i.e. 63 000-fold).80 This dramatic difference in the treatment effects on rods and cones could also be because the gene vector is normally injected in the retinal areas that are mainly populated by the rod photoreceptors with fewer extra foveal cones.28,31 However, it is known that the GS pathway carries both rod and cone inputs,26,27 resulting in potential enhanced visual performance due to cone or rod improvements. Interestingly, the selective enhancement of visual fMRI responses in the GS pathway exhibited a significant link to the rod related visual performance (FST) and at a trend level for the VA, even across the limited number of patients tested in the present study. Notably, the phase 3 clinical trial leading to drug approval showed similar improvements in those parameters.41

This relatively abrupt recovery of vision through the selective boosting of retinal function that, in turn, reactivated the GS pathway, following several years of retinal dysfunction and reliance on alternative, RT pathways, is striking. These findings not only provide useful information regarding the manner in which human vision draws upon these two evolved visual pathways, but also indicates that a single genetic manipulation can lead to positive clinical outcomes over a wide range of conditions, in this case improving vision, as reflected by performance in light sensitivity and to some extent visual acuity.

Recent studies have now established the amazing capacity of the human’s visual system for neuroplasticity throughout lifespan.32,81-84 A growing body of research also shows the potential for visual restoration training to enhance residual vision in patients with cortical blindness82 or retinal degeneration.85 Identifying the visual pathways that enable residual vision in low vision individuals could also play a critical role in devising targeted rehabilitation programmes engaging both the RT and GS pathway to further augment patients’ visual functions.

Limitations of the present study

The capacity to use fMRI to test visual responses in LCA2 patients in treated and untreated eyes, and before and after gene therapy, allowed us to discover the shift toward the RT pathway and the partial restoration of the GS pathway following gene therapy. At the same time, a number of variables were beyond our control, most notably the number of subjects in the study, their ages, the specific presentations of the disease, or subject-specific details regarding the retinal treatment. Due to the relatively small number of patients, it was not possible for us to break down the fMRI findings by age or severity of the visual impairments. We were, however, in a sense able to exploit the variability in disease severity by demonstrating a strong correlation with an fMRI measure of visual responses and the visual performance on a clinical test, indicating that despite the other factors, a significant proportion of the disease treatment variation was common to the fMRI and behavioural measures.

A puzzling finding was the lack of activity change observed in the pulvinar. This negative finding is somewhat difficult to interpret. This is in part because the pulvinar is a large and heterogeneous structure, with the portions most relevant for the RT pathways quite restricted and relatively small relative to resolution of the fMRI acquisition. Functional MRI activity in the pulvinar is also notoriously difficult to measure, compared to other structures, such as the visual cortex and SC. Thus, at present we do not place much emphasis on the lack of observed changes in of pulvinar activity following gene therapy, but simply report the findings.

Supplementary Material

awae096_Supplementary_Data

Acknowledgements

We would like to express our special gratitude to Mr Mohammad Ashtari for providing technical support on advanced excel programming to quantify fMRI results.

Data availability

The data for this study are currently on the University of Pennsylvania secure share drive where the access is password protected and not publicly available. Those who are interested can directly contact the corresponding author to gain access to all the images acquired on patient and control subjects. All image analyses packages used are free and accessible for public use.

Funding

This study was supported by a grant from the National Eye Institute, NEI (grant number: R01EY025287-01A1) and the Center for Advanced Retinal and Ocular Therapeutics (CAROT) at the University of Pennsylvania Perelman School of Medicine and the F.M. Kirby Foundation. Supported in part by an unrestricted grant from the Research to Prevent Blindness. D.A.L. was supported by the National Institute of Mental Health Intramural Research Program (grant number: ZIAMH002838).

Competing interests

J.B. reports that she and her husband were co-authors on a patent licensed by Spark Therapeutics that described retinal gene therapy in LCA2 patients but that they waived any potential financial gain in 2002. J.B. consulted for Spark Therapeutics on preclinical data and how it could relate to delivery of voretigene neparvovec-rzyl to patients in Canada and Japan. J.B. reports serving on the boards and scientific advisory boards of several different gene therapy companies. She also has some intellectual property that is managed by the University of Pennsylvania.

Supplementary material

Supplementary material is available at Brain online.
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