The human visual cortex responds to gene therapy-mediated recovery of retinal function

Abstract

Leber congenital amaurosis (LCA) is a rare degenerative eye disease, linked to mutations in at least 14 genes. A recent gene therapy trial in patients with LCA2, who have mutations in RPE65, demonstrated that subretinal injection of an adeno-associated virus (AAV) carrying the normal cDNA of that gene (AAV2-hRPE65v2) could markedly improve vision. However, it remains unclear how the visual cortex responds to recovery of retinal function after prolonged sensory deprivation. Here, 3 of the gene therapy trial subjects, treated at ages 8, 9, and 35 years, underwent functional MRI within 2 years of unilateral injection of AAV2-hRPE65v2. All subjects showed increased cortical activation in response to high- and medium-contrast stimuli after exposure to the treated compared with the untreated eye. Furthermore, we observed a correlation between the visual field maps and the distribution of cortical activations for the treated eyes. These data suggest that despite severe and long-term visual impairment, treated LCA2 patients have intact and responsive visual pathways. In addition, these data suggest that gene therapy resulted in not only sustained and improved visual ability, but also enhanced contrast sensitivity.

Figures

Figure 1. Results for subject CH09.

(A) fMRI results for high-contrast stimulus presented to the treated left eye showed significant (fdr < 5%, corrected P < 0.005, cca ≥ 100 mm2) bilateral activations extending from medial to lateral aspects of the occipital poles. All fMRI results show left and right inflated (medial view) and flatmap cortical representations. (C) Medium-contrast stimulus presented to the left eye also showed significant (fdr < 5%, corrected P < 0.002, cca ≥ 20 mm2) bilateral activations. Activations for high- and medium-contrast stimuli were primarily distributed to the upper bank of the CF. (B) The high-contrast stimulus presented to the untreated right eye showed patches of activation outside the primary visual cortex at a lower (uncorrected) statistical threshold and a smaller extent threshold. (D) No activation, even at an uncorrected relaxed statistical threshold, was detected for the right eye presented with the medium-contrast stimulus. (E and F) Measured VFs at baseline (before surgery) and at follow-up. (G) The predicted VF area in E was based on the observed subretinal injection site at the time of treatment, indicated by white arrowheads within the composite retinal fundus image. CH09’s predicted VF was symmetric to the vertical meridian with a greater area below than above the horizontal meridian. Such VF distribution is predictive of cortical activation that is symmetrically distributed to both hemispheres, with a larger area of activation in the upper bank of the CF. CH09’s fMRI results partially correlated with both predicted and measured VFs.

Figure 2. Results for subject CH08.

(B) fMRI results showed significant (fdr < 5%, corrected P < 0.005, cca ≥ 100 mm2) bilateral activations in the primary visual cortex extending from medial to lateral and posterior to anterior aspects of the occipital cortex after presentation of high-contrast stimuli to the treated right eye. (A) In contrast, the untreated left eye was nearly unresponsive to the high-contrast stimuli, with activation detectable only at a less stringent statistical threshold and much lower extent threshold (uncorrected P < 0.005, cca ≥ 50mm2). (D) Activation for medium-contrast stimuli presented to the treated eye was also bilateral, but with slightly more activation in the right hemisphere (fdr < 5%, corrected P < 0.004, cca ≥ 50 mm2). (C) No cortical activation was observed with the untreated eye exposed to the medium-contrast stimuli. (E and F) Measured VFs at baseline (before surgery) and at follow-up. (G) The predicted VF area in E was based on the observed subretinal injection site at the time of treatment, indicated by white arrowheads within the composite retinal fundus image (taken just prior to surgery). Based on the injection site located superior to the macula, the portion of the VF predicted to improve was the central inferior field. Such a VF is predictive of activation distributed bilaterally with the activation primarily located in the superior portion of the CF. CH08’s fMRI results were equally distributed about the CF and correlated more with his measured VFs. However, as suggested by the predicted and measured VFs, activation was distributed roughly equally to both hemispheres.

Figure 3. Results for subject CH13.

(B) fMRI results 1 year after gene transfer for the treated right eye presented with the high-contrast stimuli showed significant (fdr < 5%, corrected P < 0.003, cca ≥ 100 mm2) unilateral cortical activation, primarily confined to the left lateral occipital lobe. (A) No activation was observed after exposure of the untreated left eye to the high-contrast stimuli. No activation was observed after medium-contrast stimulus was presented to the treated (D) or untreated (C) eye. (E and F) Measured and predicted VFs. The predicted VF for the treated eye showed a lateralized predicted VF in the lower right quadrant. This predicted VF was mostly confined to the right of the vertical meridian and located primarily below the horizontal meridian. Such distribution predicts cortical activation in the left upper area of the cortex, with no activation in the contralateral hemisphere. The island of activation for the treated eye occurred in the predicted hemisphere (see B), although it was located far lateral to primary visual cortex. (G) These results are consistent with the location of CH13’s subretinal injection. Composite of retinal fundus images demonstrating pigmentary deposits, vascular attenuation, and “window defects” caused by degeneration particularly in the central macula. As a result, CH13 had a subretinal injection located more peripheral (predominantly superotemporal to the macula).

Figure 4. Results for control subject NC01.

Because vision tests showed normal vision for both left and right eyes, results from the right eye only are shown. fMRI results showed significant (fdr P < 0.003, cca ≥ 100 mm2) bilateral activations in the primary visual cortex extending from posterior to anterior and medial to some lateral aspects of the occipital cortex after presentation of high-contrast (A) and medium-contrast (B) stimuli to his right eye. Medial cortex activation was distributed symmetrically to the upper and lower banks of the CF in both the right and left hemispheres. Similar to the LCA2 participants, and as predicted based on the scotopic light stimulus and/or the size of squares used in the checkerboard stimuli, NC01 did not show any activation in the occipital pole, where foveal activation would otherwise be represented. (C) VF map, which shows symmetrical distribution with respect to the horizontal meridian but covers a greater area to the right of the vertical meridian. Such VF is predictive of bilateral fMRI activations that are equally spread to the superior and inferior aspects of CF. In agreement with his VF, activation was distributed equally to superior and inferior aspects of CF for both hemispheres. Also, as predicted by his VF, activations were bilateral with slightly more activation in the left lateral visual cortex.

Figure 5. fMRI stimuli and design.

(A) Checkerboard stimuli with a constant light intensity of 5 lux, at 3 levels of contrast (high, 100%; medium, 34%; low, 10%), were presented in a boxcar block design. (B) The checkerboard paradigm consisted of 15-second active blocks of contrast-reversing (8 Hz) checkerboards interleaved with 15-second presentation of a blank (black) screen as control blocks (rest period). 3 blocks of each contrast were interspersed randomly and interleaved with 9 rest blocks. Subjects were asked to press a button when they detected a checkerboard pattern. L, low contrast; M, medium contrast; H, high contrast.