Volume 10, Pages 258-268 (August 2016) Long-term outcomes of gene therapy for the treatment of Leber's hereditary optic neuropathy  Shuo Yang, Si-qi Ma, Xing Wan, Heng He, Han Pei, Min-jian Zhao, Chen Chen, Dao-wen Wang, Xiao-yan Dong, Jia-jia Yuan, Bin Li  EBioMedicine  Volume 10, Pages 258-268 (August 2016) DOI: 10.1016/j.ebiom.2016.07.002 Copyright © 2016 The Ohio State University Wexner Medical Center Terms and Conditions

Fig. 1 Fundus photography obtained before and after the intravitreal injection of rAAV2-ND4 (n=8 patients). A representative 30° fundus photograph showing the disc and macula of injected and uninjected eyes before (A) and at 3months (B), 6months (C), 12months (D), 24months (E), and 36months (F) after intravitreal injection. The retinal structure appears normal in all photographs with no apparent abnormalities. EBioMedicine 2016 10, 258-268DOI: (10.1016/j.ebiom.2016.07.002) Copyright © 2016 The Ohio State University Wexner Medical Center Terms and Conditions

Fig. 2 Improvement in logMAR visual acuity from baseline after the intravitreal injection of rAAV2-ND4 (n=8 patients). Visual acuity improvements from baseline in injected and uninjected eyes at 3, 6, 12, 24, and 36months after the intravitreal injection of rAAV2-ND4. Visual acuity improved in the injected eyes of Patients 2, 4, 6, 7, 8, and 9 and in the uninjected eyes of Patients 2, 3, 4, 6, and 9. However, visual acuity decreased in some patients until 36months after gene therapy, but the mean visual acuity of the injected and uninjected eyes improved in all 8 patients. Mean logMAR BCVA in the injected eyes of all 8 patients who received unilateral gene therapy improved by 0.26±0.29, 0.40±0.29, 0.33±0.41, 0.35±0.39, and 0.39±0.43 at 3, 6, 12, 24, and 36months, respectively. Overall, patients had the best BCVA 6months after treatment. Mean logMAR BCVA of the uninjected eyes in these same 8 patients improved by 0.10±0.16, 0.16±0.18, 0.23±0.28, 0.15±0.21, and 0.26±0.21 at 3, 6, 12, 24, and 36months, respectively. EBioMedicine 2016 10, 258-268DOI: (10.1016/j.ebiom.2016.07.002) Copyright © 2016 The Ohio State University Wexner Medical Center Terms and Conditions

Fig. 3 The visual field index (VFI), mean defect (MD), and pattern-reversal visual evoked potential P100 waveform in the injected and uninjected eyes of 8 patients who received unilateral gene therapy. The mean VFI (A) and MD (B) values of the injected and uninjected eyes before and 3, 6, 12, 24, and 36months after intravitreal injection. Five patients had VFI and MD improvements after treatment, but Patient 5 had no improvement. Error bars represent one standard deviation. The latency (C) and amplitude (D) of the P100 waveform in the injected and uninjected eyes of the 8 patients who received unilateral gene therapy are shown. After gene therapy, P100 latency decreased and P100 amplitude increased in both the injected and uninjected eyes within 6months. This indicated an improvement in optic nerve function after gene therapy. Error bars represent one standard deviation. Mean VFI in the 8 patients who received unilateral gene therapy was 11±0.15%, 28±0.23%, 23±0.22%, 18±0.23%, 28±0.18%, and 27±0.22% before and 3, 6, 12, 24, and 36months after treatment, respectively. The mean VFIs of the uninjected eyes were 12±0.07%, 29±0.25%, 27±0.29%, 20±0.21%, 34±0.28%, and 30±0.25% before and 3, 6, 12, 24, and 36months after treatment, respectively (Tables 3 and 4, Fig. 3A, B). EBioMedicine 2016 10, 258-268DOI: (10.1016/j.ebiom.2016.07.002) Copyright © 2016 The Ohio State University Wexner Medical Center Terms and Conditions

Fig. 4 Optical coherence tomography (OCT) measurements of retinal nerve fiber layer (RNFL) thicknesses in 8 patients who received unilateral gene therapy. A. Temporal RNFL thickness. B. Inferior RNFL thickness. C. Superior RNFL thickness. D. Nasal RNFL thickness. E. Mean RNFL thickness. Average RNFL thicknesses before and 3, 6, 12, 24, and 36months after treatment were 47.31±7.87, 46.78±7.54, 48.47±7.79, 46.46±7.79, 43.13±6.07, and 46.19±8.67μm, respectively. RNFL thicknesses of the uninjected eyes before and 3, 6, 12, 24, and 36months after treatment were 50.19±13.40, 47.97±9.70, 48.19±8.78, 48.08±8.11, 45.25±7.69, and 45.69±6.01μm, respectively. Specific measurements revealed a decreasing trend in RNFL thickness over time. There were no significant changes in RNFL thickness in the injected eyes over time (baseline: 47.31±7.87μm; at 36months: 46.19±8.67μm, p=0.691). However, RNFL thickness in the uninjected eyes began to decline 24months after treatment (baseline: 50.19±13.40μm; at 36months: 45.69±6.01μm, p=0.245). EBioMedicine 2016 10, 258-268DOI: (10.1016/j.ebiom.2016.07.002) Copyright © 2016 The Ohio State University Wexner Medical Center Terms and Conditions

Fig. 5 Effects of gene therapy on the uninjected eyes of mice. A–B. The retinal ND4 protein level, measured using Western blot analysis, after injection of the second eye. There was no significant difference between Groups 1 and 2 (p=0.22) or between Groups 3 and 4 (p=0.78). C. Serum AAV2 concentration as measured using ELISA. There was no significant difference between Groups 1 and 2 (p=0.86) or between Groups 3 and 4 (p=0.51). D. Serum AAV2 antibody concentration as measured using ELISA. There was no significant difference between Groups 1 and 2 (p=0.20) or between Groups 3 and 4 (p=0.20). Error bars represent one standard deviation. EBioMedicine 2016 10, 258-268DOI: (10.1016/j.ebiom.2016.07.002) Copyright © 2016 The Ohio State University Wexner Medical Center Terms and Conditions

Fig. 6 Clinical anti-AAV2-neutralizing antibody assay results following gene therapy administration in the fellow eye and fundus photography of patient 1 in both eyes 12months apart. A. The percentage of green fluorescent protein (GFP) expression inhibition (1:20). B. The percentage of GFP expression inhibition (1:60, 1:180, 1:540, and 1:1620). Three months after receiving gene therapy, GFP expression inhibition increased when a 1:20 serum concentration was compared with serum-free medium. The inhibition of GFP expression returned to pre-treatment levels 6months after gene therapy. Photographs of the right (C) and left (D) fundi are shown. The photographs were taken 3, 6, and 12months after the administration of an intravitreal rAAV2-ND4 injection in the left eye. At 12months, an intravitreal rAAV2-ND4 injection was also administered in the right eye. Photographs were also taken at 3, 6, 12, 24, and 36months after treatment of the right eye. No apparent retinal abnormalities were identified. EBioMedicine 2016 10, 258-268DOI: (10.1016/j.ebiom.2016.07.002) Copyright © 2016 The Ohio State University Wexner Medical Center Terms and Conditions

Fig. 7 Improvement in logMAR visual acuity and changes in visual field parameters in a patient who received gene therapy in both eyes 12months apart (Patient 1). A. Visual acuity improved from baseline in the first eye following gene therapy in that eye, but decreased 6months after gene therapy in the second eye. B. Serial visual field index (VFI) measurements showed that bilateral VFI decreased after the second eye received gene therapy. C. Mean defect (MD) changes showed that bilateral MD worsened after gene therapy administration in the second eye, indicating greater visual field damage. EBioMedicine 2016 10, 258-268DOI: (10.1016/j.ebiom.2016.07.002) Copyright © 2016 The Ohio State University Wexner Medical Center Terms and Conditions

Fig. 8 Retinal nerve fiber layer thickness (RNFL) and visual evoked potentials (VEPs) after bilateral administration of gene therapy (Patient 1). Injections were administered 12months apart. A. The RNFL thickness did not change in either eye during the 48-month observation period. B. The amplitude of the VEP P100 component decreased in both eyes following the administration of gene therapy in the second eye. C. The VEP latency increased after the administration of gene therapy in the second eye. Both VEP changes are indicative of reduced optic nerve function. EBioMedicine 2016 10, 258-268DOI: (10.1016/j.ebiom.2016.07.002) Copyright © 2016 The Ohio State University Wexner Medical Center Terms and Conditions

Fig. S1 Study flow diagram showing the article selection process for the meta-analysis performed on the literature. EBioMedicine 2016 10, 258-268DOI: (10.1016/j.ebiom.2016.07.002) Copyright © 2016 The Ohio State University Wexner Medical Center Terms and Conditions

Fig. S2 Visual function recovery time in injected eyes, uninjected eyes, and spontaneous visual recovery eyes. Visual acuity improvements in injected eyes mainly occurred between 3 and 6months after gene therapy, while improvements in uninjected eyes mainly occurred between 3 and 12months. In contrast, visual acuity improvements in spontaneousvisual recovery eyes was highly variable and occurred between 1h and 72months. The timing between the injected and uninjected eyes was not significantly different (Mood's median test, Z=−0.913, p=0.361), but the timing between injected and spontaneous visual recovery eyes (Z=−2.79, p=0.009) and between uninjected and spontaneous visual recovery eyes (Z=−2.21, p=0.026) was significantly different. EBioMedicine 2016 10, 258-268DOI: (10.1016/j.ebiom.2016.07.002) Copyright © 2016 The Ohio State University Wexner Medical Center Terms and Conditions

Fig. S3 Direct material exchange between optic nerve axons. A. Sprague-Dawley rats administered a unilateral intravitreal injection of fluorogold (FG). B. A small proportion of FG did diffuse to the contralateral optic nerve anterior to the optic chiasm (2A, 2B). C. The FG reached the optic chiasm via optic nerve axons of the injected eye. Most of the FG that diffused to the contralateral optic nerve remained posterior to the optic chiasm. Only a small proportion of FG diffused to the ipsilateral optic nerve posterior to the optic chiasm. Therefore, direct material exchange occurred between the optic nerves (A, C). This mechanism may play a role in visual function improvements in the uninjected eye in LHON patients who receive gene therapy. Scale bar=100μm. EBioMedicine 2016 10, 258-268DOI: (10.1016/j.ebiom.2016.07.002) Copyright © 2016 The Ohio State University Wexner Medical Center Terms and Conditions