STUDY POPULATION, ADVERSE EVENTS AND COMPLICATIONS
There were 15 patients (8 female and 7 male subjects) in the trial (). Other than a sibling pair (P9, P12), the patients were unrelated. P10 and P13 had the same mutant alleles (homozygous for R91W) but were not known to be related. Ninety-day and 1-year post-injection data from the three patients in Cohort 1 have been published (17
). The first three cohorts received single subretinal injections (). Cohorts 1 and 2 were young adult patients (ages 20-30 years) and dose-escalation occurred by doubling the initial injection volume of 150 μl of vector (Cohort 1) to 300 μl (Cohort 2). Cohort 3 patients were <18 years of age and we were advised by a UP regulatory body to reduce dosage in this first cohort of children, hence the 225 μl volume for these 3 patients. Cohorts 4 and 5 had two injections of 225 μl each (total 450 μl; ), first in young adults (ages 24 and 27 years) and then in ≤18 year-old patients. P15 had two injections but less total volume was injected. Infrared views of all 15 study eyes are shown with superimposed locations of the injection site and estimated boundaries of retinal detachments caused by the subretinal injections. The treated eyes are all portrayed as left eyes for comparison (); the actual eye treated is tabulated (). The postoperative course was similar in 13 of 15 patients with absorption of the subretinal fluid within 48 hours and no evidence of intraocular inflammation. P7 and P11 were exceptions with a second retinal detachment and choroidal effusions, respectively (see below). By 30-60 days postoperatively, all eyes were quiet and have remained so. Systemic safety parameters including physical examinations and blood (hematology, serum chemistry, coagulation) and urine testing showed no clinically significant abnormalities after gene transfer in all patients.
Figure 1 Fundus images with near-infrared illumination and sites of retinal detachments from subretinal injections of vector-gene in the 15 patients (P1-P15) with RPE65-LCA. Dotted circles on the images of individual patients represent the estimated areas of retinal (more ...)
Among the postoperative adverse events were retinal detachment, choroidal effusions, ocular hypotension in the immediate postoperative period and ocular hypertension associated with the administration of topical steroids. P7, Cohort 3, had a retinal detachment in the region of the single subretinal injection, one day after it was deemed flat by ophthalmoscopy. This was surgically repaired and there has not been further complication. P11, Cohort 4, was detected to have choroidal effusions on the 3rd day after surgery. The choroidals were treated with topical cycloplegics and increased topical steroids. The lack of resolution of the choroidals at day 30 led to a 3-week course of systemic steroids. By 149 days postoperatively, the choroidals had resolved by clinical examination and ultrasound. There was no measured ocular hypotension from the first measurement (postoperative day 3) through day 238. This patient, however, developed increased intraocular pressure which was detected on day 149 and presumed to be secondary to the extended use of topical steroids. Cessation of steroids followed and topical beta blockers were added. After a period of quiescence, the choroidals re-appeared on day 190 and were treated with further cycloplegics and a short course of topical steroids. There was resolution by day 238. Ocular hypotension was documented in four patients (P4, P9, P12, P15) during the early postoperative period (days 2-5; intraocular pressures were ≤6 mm Hg with an interocular difference of 5-10 mm Hg). By postoperative day 7, pressure in the study eyes increased and interocular asymmetry decreased in all patients. Ocular hypertension was also documented after topical steroid use in the postoperative period in P8, P12 and P15. Cessation of steroids and treatment with topical beta blockers led to the eyes becoming normotensive.
IMMUNE RESPONSE ASSAYS
Humoral immune responses were monitored by measuring levels of circulating antibody to AAV2 capsid at baseline and at postoperative days 14, 90, 270 and years 1, 2 and 3, depending on the treatment cohort (). All patients exhibited titers at baseline and at all post-treatment timepoints well below the normal population mean of 2,148,715 mU/ml (n=99 random samples) except P12 with a titer of 2,789,606 mU/ml at day 90, which is ~60% above this patient’s baseline value. Ten of the 14 patients with at least day 14 data showed no increase in antibody titer greater than two-fold from baseline;, and most experienced a decline. Of the remaining four, P2 exhibited a four-fold increase at day 14, and returned to below baseline at day 90 and years 1 and 3. P5 experienced a 3-fold increase at day 14, returned to baseline at day 90 and year 1, but then spiked again at year 2. This pattern of episodic antibody spikes over multiple years with a return to baseline is not consistent with a humoral immune response to a one-time vector administration, and is more likely a result of periodic re-exposure to wild type AAV2 through natural viral infections. P6 experienced a 7-fold titer increase at day 14 that peaked at 11-fold at day 60, and has subsequently declined to just over 2-fold at year 2. P8 also showed an increase in titer at day 14 (3-fold) that peaked at day 90 (4-fold) and is diminishing at year 1 (2-fold over baseline). P6 and P8 are circumstantially the only patients with serum antibody titer behaviors potentially consistent with a response to the vector, but coincidental exposure to wild type AAV2 either directly or by reactivation of latent AAV2 through natural Adenoviral or Herpes viral infection cannot be ruled out. Overall, the patterns of patient serum antibody titers to AAV2 over time post-treatment suggest limited or no systemic immune response to subretinal AAV2 vector delivery.
Anti-AAV2 Serum Antibody Titers at Baseline and Post-Treatment Timepoints
AAV2 capsid antigen-specific reactivity of peripheral lymphocytes (ASR) was monitored at baseline and at post-treatment days 14 and 90 and years 1, 2 and 3 (eTable 1
). Only P1 and P2 of the patients with ASR data exhibited a significant increase in stimulation index (SI) at any timepoint (minimal level of significance for SI ranges from 2 to 3). For both patients, the change in SI was only marginally significant and only a small increase from baseline: P1 at year 1 had a SI of 2.02, a small increase from the baseline value of 1.62, and P2 at day 90 showed a SI of 2.10, also a small change from 1.89 at baseline. We conclude that the AAV2 capsid antigen-specific lymphocyte proliferation response to a single subretinal AAV2 treatment elicits neither a consistent nor pronounced AAV2 antigen-specific immune response.
The T cell immune response to AAV2 capsid was monitored by IFN-γ ELISPOT assays. PBMCs from subjects at baseline and at days 14 and 90 and years 1, 2 and 3 after treatment, depending on the patient, were stimulated with AAV2 peptide library pools and assayed for IFN-γ secretion. With one exception, there were no positive responses to AAV2 peptide pools at any of the timepoints tested in the subjects thus far (eTable 2
). P9 exhibited a response to peptide pool 2C at day 90, but neither before nor after. This is inconsistent with T cell response to AAV2 vector, and a clear reason for this modest and transient increase at day 90 is not apparent. The study of AAV2-specfic memory T cells measured by the cultured ELISpot showed that some patients had pre-existing AAV2-specific T cells at baseline (Patients 2, 6, 9, 11-14; eTable 3
). Most of the patients that were positive at baseline remained positive by the cultured ELISpot and most of the patients that were negative remained negative. Only P5 and P7, who were negative at baseline, became positive post-treatment by cultured ELISpot but not by ex-vivo ELISpot. Although pre-existing AAV2-specific memory T cells were found in some patients, AAV2 administration to the eye did not expand these resting cells as demonstrated by the negative data obtained by the ex vivo ELISpot. The data suggest that even in the presence of peripheral AAV2-specfic memory T cells, AAV2 administration to the eye is not sufficient to activate them.
Biodistribution of vector in the peripheral blood was monitored at baseline and at days 1, 3 and 14 post-treatment by quantitative PCR with spike-in assays to control for PCR inhibition in specific samples. For all patients at all timepoints, there were no vector genome copies detectable, thus confirming the lack of escape of subretinally administered AAV2 vector into the circulation.
FULL FIELD AND FOCAL PSYCHOPHYSICS, PUPILLOMETRY AND MOBILITY
Visual function was assessed before and after treatment using FST, TPLR, dark-adapted static visual field testing, mobility performance and ETDRS visual acuity. We did not assume that the vector used in each patient was bioactive but tested it. At the end of each surgery, unused residual vector was injected subretinally into rd12
(Rpe65-deficient) mice and an ERG bioassay performed to quantify vector activity (18
). In all patients, the residual vector was proven active by this method (eFigure 1
Full field sensitivity
Visual function was measured psychophysically using FST in dark-adapted eyes with blue and red flashes (, eFigure 2
, ). At baseline, the mean (SE) FST sensitivity with blue flashes in all RPE65
-LCA eyes (study and control) was 1.17(0.087) log10
, a value that was substantially reduced compared to 6.61 (0.10) log10
in normal eyes. At baseline, there were no significant differences (p=0.16) between control and study eyes of trial patients. Intervisit test-retest variability of the FST measure at baseline in trial patients was similar to that published previously (30
). FST sensitivity to red flashes in all RPE65
-LCA eyes at baseline was 0.72 log10
(0.11), which is greatly reduced compared to the normal value of 4.37 (0.08) log10
measured under dark-adapted conditions, or the normal value of 2.48 (0.09) log10
measured at the cone plateau (eFigure 2
, ). Chromatic differences defined the photoreceptor type mediating FST responses. In all patients, red FST flashes were detected by cones whereas blue FST flashes could be detected by rods (P1, P3, P5, P7, P8, P9, P10), or cones (P11, P12, P15), or rods and cones (P2, P4, P6, P13, P14) at baseline (eFigure 2
Figure 2 Visual function in all clinical trial participants analyzed with full-field stimulus testing (FST) and the transient pupillary light reflex (TPLR). A, FST sensitivity (mean±SD) to blue stimuli measured under dark-adapted conditions in each eye (more ...)
Measures of Ocular Function at Baseline and Post-operatively
In the postoperative period, FST sensitivities to blue flashes showed highly significant differences compared to baseline in study eyes but not in control eyes (, , p<0.001, repeated measures ANOVA test of interaction). The postoperative improvement in study eyes was 1.59 (0.25) log10
. Chromatic differences supported mediation of blue FST flashes by rods postoperatively in all study eyes except for P6 (eFigure 2
FST sensitivities to red flashes showed highly significant differences compared to baseline in study eyes but not in control eyes (p=0.008, repeated measures ANOVA test of interaction). The postoperative improvement in study eyes was 0.45 (0.10) log10
(). Chromatic differences supported mediation of red FST flashes by cones postoperatively in all study eyes (eFigure 2
). The ages of the clinical trial participants did not have a significant effect on blue FST (P=.25, overall; P=.53, magnitude) or on red FST (P=.10, overall; P=.63, magnitude).
The transmission of information from retina to the brainstem was quantified objectively with the TPLR (). Measurements were made under fully dark-adapted conditions and luminance-response functions were available in 27 of 30 eyes. For P10 at baseline, the control eye showed a sub-criterion contraction whereas the study eye showed no contraction to maximal stimulation; his sensitivities were assigned to the reciprocal of the maximum stimulus luminance for statistical purposes. For the control eye of P13, TPLR was not recorded due to time constraints. At baseline, RPE65-LCA eyes required on average 5.6 log10 unit higher luminance of a full-field green flash in order to produce a criterion pupillary contraction compared to normal eyes (RPE65-LCA= −0.88 (0.15) log10 versus normal= 4.74 (0.064) log10). The magnitude of this defect was similar to the 5.5 log10 difference observed with blue FST between RPE65-LCA and normal eyes. At baseline, there were no significant differences (p=0.81) between the control and study eyes of trial patients ().
In the postoperative period (1-6 months), TPLR sensitivities showed highly significant differences compared to baseline in study eyes but not in control eyes (, , p<0.001, repeated measures ANOVA test of interaction). The magnitude of the postoperative TPLR sensitivity improvement in study eyes was 1.17 (0.20) log10 (, ) which corresponded to an intermediate value between the blue and red FST improvements observed. The ages of the clinical trial participants did not have a significant effect on the TPLR (P=.67, overall; P=.56, magnitude of the treatment effect).
Static visual fields
The significantly increased light sensitivity postoperatively in treated eyes using FST and TPLR prompted us to ask whether we could localize the increases in the visual field and how any localization of function was related to the sites of subretinal injection (). Visual field maps of sensitivity change from baseline in the study eyes showed good correspondence between the loci where there were significantly increased responses to stimuli and the estimated region where the retinal detachment with subretinal injection of agent occurred. Patients in Cohorts 4 and 5, who had a second subretinal injection site in the nasal retina, showed loci with significant responding in the temporal visual field. In summary, 11 of the 12 patients with visual field maps showed correspondence between most detected loci and the area of injection; these include P1-P4, P7-P9, P11, P12, P14 and P15. It is of interest that some of the detected loci in the patients, however, were outside the estimated injection area. The basis for this effect remains uncertain. It is to be noted that most of these unexpectedly responsive loci were in the far periphery. The results of P6 indicated response at a single peripheral locus but no evidence of a response in the subretinal injection area. This temporal inferior peripheral field locus was consistently detected and it is likely to be the source of the FST and TPLR responses in this patient (), who described this location of perception and its appearance postoperatively.
Figure 3 Dark-adapted visual field maps in study eyes to localize regions of improved sensitivity after treatment. All maps are depicted as left eyes for comparability and with an overlaid schematic of retinal features (optic nerve and posterior pole vessels) (more ...) Mobility testing
We also asked whether these localized changes from baseline had any impact on the ability of the subjects to negotiate an obstacle course. In 5 patients, representing Cohorts 4 and 5, we performed the study both before and after treatment (). A comparison of mobility performance for study eyes relative to postoperative values (first row) indicates overall a better performance after treatment for ambient illuminations between 0.2 to 4 lux. For the 100 lux illumination, however, patients were able to navigate the course practically without errors both at baseline and postoperatively and regardless of which eye was used. For the control eyes (second row) there were less pronounced differences in performance for the lower illumination levels, which suggests a learning effect. We also determined if the difference in performance between eyes (interocular difference, IOD) changed after treatment (third row). The results in P11 and P13 indicate greater IOD after treatment, with better performance of the treated eye relative to the control eye, at the lower illumination levels. P12 performed better with the treated eye only at the lowest illumination level; P10, P14 and P15 did not show notable effects.
Figure 4 Mobility performance of clinical trial participants as measured by the number of navigation incidents experienced while traveling an indoor course of fixed length, for five ambient illumination levels. A, Change from baseline performance for the study (more ...)
All study participants were assessed by the difference in performance between eyes, averaged across all post-treatment visits (). Results indicate a consistently lower number of incidents while navigating with the treated eye, with varying degrees of performance gain across participants. The mobility performance IOD results for all participants are summarized (). Mean differences between study and control eyes were significantly different from zero for the four lower illumination levels indicating that patients as a group navigated more efficiently when using their treated eyes under such conditions.
VISUAL ACUITY, FIXATION AND FOVEAL OPTICAL COHERENCE TOMOGRAPHY
At baseline, VA in control eyes was 0.96 (SE, 0.13) logMAR (corresponding to a mean Snellen acuity of 20/182; range 20/39 to worse than 20/2000) as compared to study eyes of 1.09 (0.11) logMAR (=20/246; range 20/43 to 20/1824) (, ). Postoperatively, the mean VA increased to 0.91 (0.13) logMAR in control eyes and to 0.97 (0.11) logMAR in study eyes, and both changes were significant (). In contrast to other psychophysical and to pupillometric measurements, repeated measures ANOVA showed no indication (p=0.16) of a difference in the magnitude of the postoperative acuity change between study and control eyes of −0.12 (0.05) and −0.05 (0.02) logMAR, respectively. Furthermore, in the great majority (28 of 30) of the eyes, mean postoperative acuity change did not reach or surpass the 0.30 logMAR (3-line) halving of visual angle limit which is a commonly accepted criterion for clinical significance (44
). It was noted that the second baseline VA was better than the first baseline measurement in 10 study eyes (range: −0.18 logMAR better to 0.08 logMAR worse) and in 9 control eyes (range: −0.10 logMAR better to 0.10 logMAR worse). Regression to the mean and learning curve effects could have potentially contributed to this tendency. Thus, an alternative analysis was performed using only the second baseline VA. This led to a decrease in the logMAR improvement postoperatively in both the study eyes (mean (SE) = −0.09 (0.05), p=0.099) and the control eyes (mean (SE) = −0.04 (0.02), p=0.077).
In order to understand better any possible VA changes resulting from gene therapy, fixation properties were analyzed in each eye at each visit. At baseline, in 12 of 15 study eyes mean fixation location corresponded to the anatomical foveal depression () and not unexpectedly these eyes had the highest VA (). Instability of fixation (including high frequency nystagmus and lower frequency wandering eye movements) around the mean was 1.97 (0.21) degrees and correlated (r2
=0.42) inversely with VA as previously published in this patient population (29
). Control eyes showed less fixation instability with a mean of 1.54 (0.14) degrees (data not shown) consistent with the better acuities recorded; the difference was borderline significant (p=0.052). Postoperatively, fixation remained foveal in both study and control eyes in this subset of 12 patients (the study eye of P2 developed a secondary fixation locus which was detectable with dimmer stimuli, reference 24
), and there were no significant changes to fixation instability ().
Figure 5 Retinal location and instability of fixation in RPE65-LCA eyes at baseline and postoperatively, and its relation to changes in visual acuity. A, D, Fixation clouds of all study eyes during a 10 sec epoch recorded while gazing to a 1 deg diameter stationary (more ...)
Three study eyes (P4, P5, and P10) with the worst baseline acuities () fixated parafoveally at mean eccentricities of 2.1, 5.1 and 3.4 degrees, respectively, from the anatomical foveal depression (). Their fixation instability was 2.3, 3.7, and 2.2 degrees, respectively. Control eyes of these three patients also fixated eccentrically; mean locus of fixation was 2.0 and 2.3 degrees eccentric for P4 and P5, respectively, whereas it could not be quantified in P10. Postoperatively, fixation remained extrafoveal for all three patients ().
Next, VA changes were considered in the context of the type of fixation. There was a larger VA improvement in patients with extrafoveal fixation (−0.30 (0.14) logMAR, ) compared to those with foveal fixation (−0.09 (0.04) logMAR, ). A repeated measures ANOVA demonstrated a statistically significant difference (p=0.036, test of interaction) between the magnitude of postoperative VA improvement in the patients with the two types of fixation. In subgroup analyses, acuity change in foveal-fixating study eyes, or extrafoveal-fixating study and control eyes did not achieve statistical significance (p=0.07, 0.17, 0.56, respectively); counterintuitively, foveal-fixating control eyes showed a significant (p=0.02) improvement of VA. Of note were the large VA improvements (P4=−0.29 and P10=−0.55 logMAR) in two of the extrafoveal-fixating eyes where the central retina including the fixation was involved in the subretinal injection, as compared to the lack of any such large improvement in the extrafoveal-fixating eye without a central detachment (P5=−0.06 logMAR), or in two foveal-fixating eyes with a foveal detachment (P1=0.20, P13=−0.11 logMAR). Until there are physiologically-based hypotheses for improvements in untreated control eyes or in eyes without foveal detachment, parsimony would suggest that small VA improvements in foveal-fixating eyes may have been influenced by the high expectations and motivation in patients taking part in an open-label study as well as a possible learning effect (45
). Clinically significant unilateral VA improvements in extrafoveal-fixating eyes, on the other hand, appear consistent with independent data (18
) showing improvements in extrafoveal cone sensitivity following gene therapy.
Retinal laminar architecture across the fovea was quantified in all patients. Foveal thickness in both eyes of the 15 patients pre- and post-operatively are summarized (). For control eyes, changes from baseline are within published intervisit variability for a retinal degeneration population (17
). For study eyes, there are two notable examples of foveal thinning in the short-term: P1 and P13. Long-term follow-up in P1 showed that foveal thinning was still present. In both P1 and P13, the fovea was detached in the subretinal injection (). P4, another patient with foveal detachment showed less pronounced but still significant thinning long-term but not short-term. P4’s control eye also showed long-term thinning but not to the degree as in the study eye. As a counter example, P5 who did not have foveal detachment showed similar results as P4. The other patients with a foveal detachment, P6 and P10, showed no such effects. All other study eyes without foveal detachment did not have thinning within the time period studied.
Figure 6 Foveal structure and quantitation of thickness using OCT scans in control and study eyes. A, Foveal thickness measurements in control and study eyes at baseline, and at short-term and long-term postoperative timepoints. Changes from baseline are displayed (more ...)
Representative horizontal OCT cross-sections and longitudinal reflectivity profiles through the fovea (highlighted and labeled for outer retinal laminae and with histograms of layer thickness) are shown for four study eyes at baseline and at early and later postoperative times (). P7, a patient without foveal detachment, shows no remarkable changes at 30 days and 18 months postoperatively. P1, P13 and P6 had foveal detachments and all showed disturbance of the IS/OS laminar architecture at 30 days postoperatively but recovery at later visits. Unlike P6, however, P1 and P13 also showed loss of ONL at 30 days and later timepoints. How do these structural findings relate to visual acuity? The only patient with clinically significant loss of visual acuity was P1 at last visit () and this patient had the most prominent foveal abnormalities by OCT.