Supplementary Appendix

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1 This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Edwards TL, Jolly JK, MacLaren RE, et al.. N Engl J Med 206;374: DOI: 0.056/NEJMc50950

2 Supplement to:. TL Edwards, JK Jolly, M Groppe, AR Barnard, CL Cottriall, T Tolmachova, GCM Black, AR Webster, AJ Lotery, GE Holder, K Xue, SM Downes, MP Simunovic, MC Seabra, RE MacLaren TABLE OF CONTENTS PAGE Supplementary Methods 2. Clinical assessments 2 2. Visual field analyses 2 3. Electrophysiology 3 Supplementary Results 4 Figure S: Overall visual acuity changes over time 4 Figure S2: Difference between treated and control eyes over time 5 Figure S3: Electrophysiology 6 Figure S4: Goldmann visual fields 7 Figure S5: Change in static automated perimetry threshold sensitivity 8 Table S: Summary of Adverse Events 9 References 0 Page of 0

3 Supplementary Methods. Clinical assessments All patients had best corrected visual acuity measured in each eye separately after refraction in accordance with the established protocols of the Early Treatment for Diabetic Retinopathy Study (ETDRS) with a masked examiner. Details of patient demographics and baseline measurements of visual function are described elsewhere Visual field analyses Loss of peripheral vision is a characteristic of choroideremia, though peripheral islands of vision often remain later in the disease process. Peripheral fields were assessed using both Goldmann perimetry and the Humphrey Visual Field Analyser (HVFA); whilst no treatment benefit was anticipated in terms of peripheral vision (as the areas of residual retina corresponding to peripheral islands of vision were not treated directly), we sought to exclude accelerated deterioration following treatment. Each patient underwent three HVFA tests prior to surgery to control for any learning effect and the final test was taken as baseline. Tests were repeated at one and two years. Standard automated perimetry (SAP) data were obtained using the 30-2 program of the HVFA. The 30-2 program assesses threshold sensitivity for 76 points spaced 6 apart horizontally and vertically from their nearest neighboring points throughout the central 60 of the visual field. Sensitivity was assessed for Goldman size III (0.4 ) 2 white stimuli presented in a Ganzfeld bowl under photopic conditions (neutral adapting background at 0 cd/m 2 ). Each field test was conducted using a full thresholding strategy using a rapid interleaved staircase procedure previously described in detail 3. Fixation was monitored by the examiner using an in-built camera based fixation monitor and by the instrument itself using gaze-tracking software and the so-called Hiejl-Krakau psychophysical method 4. Data were scrutinized for reliability by assessing false positive (acceptable if <5%), false negative (acceptable if <33%) and fixation loss (acceptable if <20%) rates: if these fell outside the accepted limits, the data were discarded and the field test attempted again. Change in decibel sensitivity was determined at each field location and the results were averaged: results stated as <0 db in the visual field plot were attributed a value of 0 db in our analysis. In one patient (P4), follow-up 24-2 field results were obtained, however none of the additional peripheral points included in the 30-2 test were seen at baseline in this patient and hence mean change in sensitivity was calculated assuming that the nonsampled points remained at 0 db at 2 years. Average change in SAP sensitivity (2 years versus baseline) was ± 0.30 db (mean ± SEM) in treated eyes and ± 0.8 db (mean ± SEM) in control eyes. These results need to be interpreted in light of the effects of the observed media opacity following treatment, which would be predicted to cause a decline in sensitivity. Although Page 2 of 0

4 such changes would also be anticipated to affect microperimetric sensitivity estimates, the latter may be more robust to such changes for two reasons. First, the deleterious effects of media opacity may be masked by improvements due to treatment (because a smaller, more central, area is sampled in microperimetry). Second, the examiner selects the de facto optimum optical pathway for the Maxwellian viewing system when optimizing the image of the ocular fundus obtained by the microperimeter SLO (analogous techniques have been employed previously as a means of determining retinal function in the context of media opacity) 5. Goldmann perimetry was conducted by an experienced perimetrist using size V (.7 ) stimuli at an intensity of 000 asb (38 cd.m -2 ) presented on a standard 3.5 (0 cd.m -2 ) asb background similar to that used in the HVFA. Field plots were converted into field areas (deg 2 ) and change in field area was calculated (2 year vs. baseline). Change in total field area for a V-4e stimulus was -6 ± 880 deg 2 (mean ± SEM) in control eyes and +9 ± 208 deg 2 (mean ± SEM) in treated eyes. Taken together, the perimetric data do not suggest any deleterious effects as a result of the gene therapy procedure in our group of subjects. 3. Electrophysiology The protocol of electrophysiological testing included full field tests conducted under scotopic and photopic conditions 6. Additionally, multifocal ERG (mferg) and PERGs were obtained 7-8. All tests conformed to ISCEV standards. Page 3 of 0

5 Supplementary Results Figure S: Overall visual acuity changes over time Visual acuity change from baseline in the treated (green) and untreated (red) eyes of each participant (P 6). The ETDRS vision chart presents 5 letters on each line e.g. a gain of 5 letters is equivalent to a one line improvement in vision. At the most recent follow up date, the treated eye had become the dominant eye in 5 out of 6 patients. Cataract surgery was performed at the following time points in 4 out of 6 patients due to development of postvitrectomy cataract: P2 (35 months), P4 (40 months), P5 (33 months) and P6 (28 months). The black vertical arrows denote the last best corrected visual acuity reading prior to cataract surgery on the treated eyes (P and P3 have not yet undergone cataract surgery). P6 was the only patient in whom the treated eye got worse, however, the decline was late after recovery back to normal 6 months after surgery. Page 4 of 0

6 Figure S2: Difference between treated and control eyes over time The difference between treated and untreated eyes normalized to baseline is shown over time for each of the six participants over the 3.5 year period. The greatest relative improvements were seen in P (+39 letters) and P4 (+24 letters). The relative gains seen in P2 (+8 letters) and P5 (+5 letters) were mainly due to losses in their unoperated control eyes during this period. P3 remained stable and an letter decline was observed in the treated eye of P6 compared to his unoperated eye over this period, although this did not occur until after the 6 month follow up visit, which is more in keeping with slow progressive degeneration rather than any negative effects of the retinal detachment surgery. P6 also received the lowest vector dose of the cohort. All treated eyes underwent retinal detachment to deliver the vector. Page 5 of 0

7 Figure S3: Electrophysiology Pattern electroretinogram (PERG) in the 6 patients at baseline and two years post gene replacement therapy in the treated (red box) and untreated eyes. The only change identified by a masked specialist electrophysiologist (GEH) was in P3, showing a deterioration in the RE (control) but improvement in the LE (treated). No changes were seen in any of the other patients, although there was insufficient signal to identify a PERG waveform at baseline in all but P6. The baseline recordings in the treated eye of P were contaminated by muscle and movement artefact as this patient was unable to fixate with this eye before gene therapy surgery. A normal control is shown for comparison. The protocol of electrophysiological testing included full field tests conducted under scotopic and photopic conditions. Additionally, multifocal ERG (mferg) and PERGs were obtained. All tests conformed to ISCEV standards 6-8. No further testing was planned beyond the two year time point, which formally defined the end of the trial for each patient. Page 6 of 0

8 Figure S4: Goldmann visual fields Kinetic perimetry was performed using a V4e stimulus size at baseline, at one and at two years. No deleterious effect of gene therapy on the peripheral field was observed. The outer edge on each diagram represents the extent of the peripheral field measured by the Goldmann perimeter (90 degrees either side of fixation along the horizontal meridian and 70 degrees either side of fixation along the vertical meridian). The mean (± SEM) change in total field area for a V-4e stimulus was +9 ± 208 deg 2 in treated eyes and -6 ± 880 deg 2 in control eyes. Page 7 of 0

9 Figure S5: Change in static automated perimetry threshold sensitivity The 30-2 Humphrey visual field program assessed 76 points within the central 60 of the visual field using Goldman size III white stimuli presented on a photopic background (0cd/m 2 ). Baseline and two year data were compared in the treated (icosahedron) and untreated eyes for each patient. Fields for right and left eyes are presented on the right and left respectively. Horizontal and vertical eccentricity in degrees are plotted on the x and y axes respectively, whilst difference in threshold is plotted on the z axis. Mean (± SEM) change in sensitivity (2 years versus baseline) was db ± 0.30dB in treated eyes and +0.05dB ± 0.8dB in control eyes. Page 8 of 0

10 Table S: Summary of Adverse Events Adverse Event Number of patients Number of events Any adverse event 9 Ocular. Transient blurred vision 2. Transient visual distortion 3. Raised intraocular pressure 4. Transient photopsia 5. Transient "violet colored vision 3 3 Respiratory/Cardiovascular 0 0 Neurological 0 0 Gastrointestinal 0 0 Genitourinary 0 0 Skin 0 0 Pain. Eye pain Musculoskeletal. Fracture after fall Serious adverse event 0 0 Page 9 of 0

11 References. Klein R, Klein BE, Moss SE, DeMets D. Inter-observer variation in refraction and visual acuity measurement using a standardized protocol. Ophthalmology 983;90(): MacLaren RE, Groppe M, Barnard AR, et al. Retinal gene therapy in patients with choroideremia: initial findings from a phase /2 clinical trial. The Lancet 204;383(9923): Simunovic MP, Cullerne A, Colley A, Wilson TD. How well does color perimetry isolate responses from individual cone mechanisms? J Glaucoma 2004;3(): Heijl A, Krakau CE. An automatic static perimeter, design and pilot study. Acta Ophthalmol 975;53(3): Minkowski JS, Palese M, Guyton DL. Potential acuity meter using a minute aerial pinhole aperture. Ophthalmology 983;90(): Marmor MF, Fulton AB, Holder GE, et al. ISCEV Standard for full-field clinical electroretinography (2008 update). Doc Ophthalmol. 2009;8(): Hood DC, Bach M, Brigell M, et al. ISCEV standard for clinical multifocal electroretinography (mferg) (20 edition). Doc Ophthalmol 202;24(): Bach M, Brigell MG, Hawlina M, et al. ISCEV standard for clinical pattern electroretinography (PERG): 202 update. Doc Ophthalmol 203;26(): 7. Page 0 of 0