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AimsTo determine long-term safety and efficacy outcomes of a subretinal gene therapy for CNGA3-associated achromatopsia. We present data from an open-label, nonrandomised controlled trial (NCT02610582).MethodsDetails of the study design have been previously described. Briefly, nine patients were treated in three escalating dose groups with subretinal AAV8.CNGA3 gene therapy between November 2015 and October 2016. After the first year, patients were seen on a yearly basis. Safety assessment constituted the primary endpoint. On a secondary level, multiple functional tests were carried out to determine efficacy of the therapy.ResultsNo adverse or serious adverse events deemed related to the study drug occurred after year 1. Safety of the therapy, as the primary endpoint of this trial, can, therefore, be confirmed. The functional benefits that were noted in the treated eye at year 1 were persistent throughout the following visits at years 2 and 3. While functional improvement in the treated eye reached statistical significance for some secondary endpoints, for most endpoints, this was not the case when the treated eye was compared with the untreated fellow eye.ConclusionThe results demonstrate a very good safety profile of the therapy even at the highest dose administered. The small sample size limits the statistical power of efficacy analyses. However, trial results inform on the most promising design and endpoints for future clinical trials. Such trials have to determine whether treatment of younger patients results in greater functional gains by avoiding amblyopia as a potential limiting factor.

Purpose Subretinal injections (SRis) are commonly used in retinal gene therapy procedures to deliver adeno-associated virus (AAV) to photoreceptors and retinal pigment epithelial cells. We present an optimized surgical protocol to minimize off-target application of AAV in the vitreous, which in turn reduces the risk of extensive biodistribution and inflammation, ultimately leading to enhanced safety of the therapy. Design Experimental animal research study. Participants Eight cynomolgus monkeys (Macaca fascicularis). Methods Subretinal injections with an AAV2/8 vector were performed. The animals were allocated to 2 different vector dose groups (1×10ˆ11 and 5×10ˆ11 viral genomes [vg]). Samples of intravitreal fluid were taken at the end of the SRi procedure and again after a 3-minute lavage (wash-out) with balanced salt solution (BSS). Main Outcome Measures Intravitreal vector genome copies were analyzed with quantitative polymerase chain reaction and compared between groups. Results Even uneventful SRi leads to dissemination of millions of AAV particles (0.1–0.7% of viral vector loading dose) into the vitreous cavity. Three minutes of lavage led to a substantial decrease (on average 96%) of intravitreal vector load. Conclusions Multiple studies have shown that the intravitreal space is not as immune privileged as the subretinal space. Intravitreal AAV particles disseminate into the bloodstream, lead to increased biodistribution into lymphatic tissue, and help to stage an immune response with implications for both safety and efficacy. Therefore, minimizing off-target vector application after reflux of vector from the subretinal space is of significant interest. We show that a simple lavage of intravitreal fluid efficiently decreases the intravitreal vector load. Such a step should be considered when performing subretinal gene therapy.

ZusammenfassungSeit ihrer Erstbeschreibung im Jahr 1872 herrscht um den natürlichen Verlauf der Choroideremie eine rege und in Teilen bis heute andauernde akademische Debatte. Aufgrund der Seltenheit der Erkrankung verlief diese traditionell weitgehend am Rande des klinisch geprägten Interessenhorizonts. Nun jedoch hat die Entwicklung grundlegend neuer, potenziell verlaufsmodifizierender Therapieansätze die Frage nach dem natürlichen Verlauf der Chorioideremie ins Zentrum der Aufmerksamkeit eines größeren Fachpublikums gerückt. Dieser Übersichtsartikel subsumiert den aktuellen Stand der Fachliteratur zum natürlichen Verlauf dieser seltenen Erkrankung und illustriert dessen Eigenheiten anhand eines übersichtlichen 2-Phasen-Modells. Neben einer ausführlichen Besprechung klinisch gängiger Modalitäten liegt der Fokus des Manuskripts auf der Ausarbeitung forschungsrelevanter Fragestellungen wie der intraindividuellen Symmetrie sowie der Etablierung neuer Endpunkte. Darüber hinaus werden die Limitationen bisheriger Studien diskutiert und Empfehlungen für zukünftige Beobachtungsstudien entwickelt.

Purpose: Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause 70% to 90% of X-linked retinitis pigmentosa (XLRP3) cases, making this gene a high-yield target for gene therapy. This study analyzed the utility of relevant clinical biomarkers to assess symmetry and rate of progression in XLRP3. Methods: A retrospective, cross-sectional analysis of 50 XLRP3 patients extracted clinical data including visual acuity (VA), visual fields (I4e and III4e targets), foveal thickness, and ERG data points alongside molecular genetic data. Symmetry was assessed by using linear regression analysis. Kaplan-Meier survival curves (KMCs) and generalized linear mixed model calculations were used to describe disease progression. Results: Ninety-six percent of patients exhibited a rod-cone phenotype, and 4% a cone-rod phenotype. Open reading frame 15 (ORF15) was confirmed as a mutational hotspot within RPGR harboring 73% of exonic mutations. Significant variability, but no clear genotype-phenotype relationship, could be shown between mutations located in exons 1-14 versus ORF15. All biomarkers suggested a high degree of symmetry between eyes but demonstrated different estimates of disease progression. VA and foveal thickness, followed by perimetry III4e, were the most useful endpoints to evaluate progression. KMC estimates predicted a loss of 6/6 vision at a mean of 34 years (±2.9; 95% confidence interval). Conclusions: XLRP3 affects retinal structure and function symmetrically, supporting the use of the fellow eye as an internal control in interventional trials. VA and kinetic visual fields (III4e) seem promising functional outcome measures to assess disease progression. KMC analysis predicted the most severe decline in vision between the third and fourth decade of life.

IMPORTANCE: Choroideremia (CHM) is a rare, degenerative, genetic retinal disorder resulting from mutation of the CHM gene, leading to an absence of functional ras-associated binding escort protein 1 (REP1). There is currently no approved treatment for CHM. OBJECTIVE: To assess the safety and efficacy of retinal gene therapy with an adeno-associated virus vector (AAV2) designed to deliver a functional version of the CHM gene (AAV2-REP1) for treatment of patients with choroideremia. DESIGN, SETTING, AND PARTICIPANTS: Tübingen Choroideremia Gene Therapy (THOR) was a single-center, phase 2, open-label randomized clinical trial. Data were collected from January 11, 2016, to February 26, 2018. Twenty-four–month data are reported for 6 men with a molecularly confirmed diagnosis of CHM. Intention-to-treat analysis was used. INTERVENTIONS: Patients received AAV2-REP1 by a single, 0.1-mL subretinal injection of 10(11) genome particles during vitrectomy into 1 eye randomly assigned to receive treatment. MAIN OUTCOMES AND MEASURES: Primary end point was change in best-corrected visual acuity (BCVA) on the Early Treatment Diabetic Retinopathy Study chart from baseline to month 24 in the treated eye vs the control eye. Secondary end points included microperimetry variables, change in fundus autofluorescence, and spectral-domain optical coherence tomographic evaluations from baseline to month 24 in the treated eye vs the control eye. RESULTS: On enrollment, the mean (SD) age of the 6 men included in the study was 54.9 (4.1) years. The mean (SD) BCVA score was 60.3 (13.4) (approximately 20/63 Snellen equivalent) in the study eyes and 69.3 (20.6) (approximately 20/40 Snellen equivalent) in the control eyes. At 24 months, the BCVA change was 3.7 (7.5) in the treated eyes and 0.0 (5.1) in the control eyes (difference, 3.7; 95% CI, −7.2 to 14.5; P = .43). Mean change in retinal sensitivity was 10.3 (5.5) dB in the treated eyes and 9.7 (4.9) dB in the control eyes (difference, 0.6; 95% CI, −10.2 to 11.4; P = .74). A total of 28 adverse events were reported; all were consistent with the surgical procedure (eg, conjunctival hyperemia, foreign body sensation), and none were regarded as severe. CONCLUSIONS AND RELEVANCE: Among 6 participants, gene therapy with AAV2-REP1 was associated with maintenance or improvement of visual acuity, although no significant difference was found from control eyes. All safety issues were associated with the surgical procedure and none were judged severe. Continued investigations could more precisely define the efficacy and safety of gene therapy with AAV2-REP1 in CHM. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02671539

Since its first description in 1872, there has been a lively academic debate about the natural history of choroideremia. Due to the low prevalence of choroideremia, interest in this discussion has been limited to subspecialists. However, the current development of novel, potentially disease-modifying therapies has sparked the attention of a larger professional audience. This review summarises the literature around the natural history of the disease and illustrates its key aspects using a simple two-stage model. Apart from a comprehensive discussion of ubiquitous clinical modalities, the manuscript reviews scientifically relevant questions, such as intra-individual symmetry and the utility of novel endpoints for use in clinical studies. Furthermore, it examines the limitations of past and current studies and develops recommendations for further observational trials.Seit ihrer Erstbeschreibung im Jahr 1872 herrscht um den natürlichen Verlauf der Choroideremie eine rege und in Teilen bis heute andauernde akademische Debatte. Aufgrund der Seltenheit der Erkrankung verlief diese traditionell weitgehend am Rande des klinisch geprägten Interessenhorizonts. Nun jedoch hat die Entwicklung grundlegend neuer, potenziell verlaufsmodifizierender Therapieansätze die Frage nach dem natürlichen Verlauf der Chorioideremie ins Zentrum der Aufmerksamkeit eines größeren Fachpublikums gerückt. Dieser Übersichtsartikel subsumiert den aktuellen Stand der Fachliteratur zum natürlichen Verlauf dieser seltenen Erkrankung und illustriert dessen Eigenheiten anhand eines übersichtlichen 2-Phasen-Modells. Neben einer ausführlichen Besprechung klinisch gängiger Modalitäten liegt der Fokus des Manuskripts auf der Ausarbeitung forschungsrelevanter Fragestellungen wie der intraindividuellen Symmetrie sowie der Etablierung neuer Endpunkte. Darüber hinaus werden die Limitationen bisheriger Studien diskutiert und Empfehlungen für zukünftige Beobachtungsstudien entwickelt.

Purpose The purpose of this study was to evaluate whether clinical grade recombinant adeno-associated virus serotype 8 (rAAV8) leads to increased appearance of hyper-reflective foci (HRF) in the retina of non-human primates (NHPs) following subretinal gene therapy injection. Methods Different doses of rAAV8 vector (rAAV8. human phosphodiesterase 6A subunit (hPDE6A) at low dose: 1 × 1011 vector genomes (vg), medium dose: 5 × 1011 vg, or high dose: 1 × 1012 vg) were injected subretinally into the left eyes of NHPs in a formal toxicology study in preparation of a clinical trial. Right eyes received sham-injection. After 3 months of in vivo, follow-up retinal sections were obtained and analyzed. The number of HRF on spectral domain-optical coherence tomography (SD-OCT) volume scans were counted from both eyes at 30 and 90 days. Results Animals from the high-dose group showed more HRF than in the low (P = 0.03) and medium (P = 0.01) dose groups at 90 days. There was a significant increase in the mean number of HRF in rAAV8-treated eyes compared with sham-treated eyes at 90 days (P = 0.02). Sham-treated eyes demonstrated a nonsignificant reduction of HRF numbers over time. In contrast, a significant increase over time was observed in the rAAV8-treated eyes of the high dose group (P = 0.001). The presence of infiltrating B- and T-cells and microglia activation were detected in rAAV8-treated eyes. Conclusions Some HRF in the retina appear to be related to the surgical trauma of subretinal injection. Although HRF in sham-treated retina tends to become less frequent over time, they accumulate in the high-dose rAAV8-treated eyes. This may suggest a sustained immunogenicity when subretinal injections of higher doses of rAAV8 vectors are applied, but it has lower impact when using more clinically relevant doses (low and medium groups). Translational relevance An increase or persistence of HRFs following retinal gene therapy may indicate the need for immunomodulatory treatment.

Achromatopsia is an autosomal recessively inherited congenital defect characterized by a lack of cone photoreceptor function, leading to severely impaired vision. In this clinical study, achromatopsia patients were treated with a single subretinal injection of rAAV.hCNGA3 to restore cone function. The focus of this trial was on the safety of the treatment. After surgery, patients were monitored in eight extensive visits during the first year, followed by a 4-year follow-up period with annual visits. For essential complementation of the standard ophthalmological and systemic examinations, disease-specific methods were developed to assess the safety, efficacy, and patient-reported outcomes in this trial.

Purpose The purpose of this study was to characterise alterations in colour discrimination in a cohort of patients with choroideremia prior to gene therapy, using a test previously validated for use in patients with retinal dystrophies. Methods We tested 20 eyes of 10 patients with a diagnosis of choroideremia and an age-matched cohort of 10 eyes of 10 normal controls using the "Cambridge Colour Test" (CCT), in which subjects are required to distinguish the gap in a C presented in one of 4 orientations in a Stilling-type array. Colour discrimination was probed along eight axes in the CIE L*u*v* colour space, and the resulting data were plotted in the CIE 1976 chromaticity diagram and fitted with least-squares ellipses. Subsequently, we estimated the achromatic area for each subject by calculating the area of the resultant discrimination ellipse and calculated sensitivity thresholds along relevant colour confusion axes. Results Colour discrimination—as quantified by log10 of the ellipse area expressed in square 1/1000th^2 units in CIE 1976 — was 2.26 (range 1.82 to 2.67) for normal subjects and 3.85 (range 2.35 to 5.41) for choroideremia patients. There was a statistically significant correlation between both achromatic area and red-green colour discrimination at the CCT and BCVA, and to a lesser degree between blue colour discrimination at the CCTand BCVA. The majority of ellipses in choroideremia were aligned close to the tritan axis, and loss of sensitivity was significantly larger in the tritan direction than in the red-green. Conclusions The majority of our patients demonstrated greater loss in tritan discrimination than in red-green colour discrimination using the CCT. There was a significant correlation between achromatic area and BCVA. In keeping with our current understanding of the machinery of colour vision, there was a significant correlation between BCVA and colour discrimination thresholds, which was stronger for red-green colour discrimination, than for tritan colour discrimination.We propose that this and similar tests of colour discrimination may prove to be suitable tools for assessing functional outcomes in gene therapy trials for choroideremia.

Purpose To investigate shedding and biodistribution characteristics of recombinant adeno-associated virus serotype 8 (rAAV8) after single-dose subretinal or intravitreal injection in nonhuman primates (NHP, Macaca fascicularis) as a surrogate for environmental hazard and patient safety. Methods In a study for regulatory submission, 22 NHP were divided into four cohorts receiving either single subretinal injections of vehicle or clinical grade rAAV8 (1 × 1011 or 1 × 1012 vector genomes [vg]) versus single intravitreal application of 1 × 1012 vg. Viral shedding and biodistribution were monitored in biofluids for up to 91 days, followed by necropsy and tissue harvesting of all major organs, the visual pathway, and lymphatic tissue. Quantification of vector genomes was done by quantitative (q)PCR. Results Shedding occurred in a dose-dependent manner in all biofluids and persisted for a maximum of 7 days. Intravitreal delivery led to increased and persistent (up to 13 weeks) distribution of vector genomes in blood and draining lymphatic tissue, increased off-target deposition, and inefficient gene transfer to the retina. No vector targeting of the germ line was observed in any cohort. Conclusions These data illustrate that subretinal application of rAAV8 leads to a more favorable biodistribution profile compared to intravitreal injections. Extraocular biodistribution is limited after subretinal delivery, while intravitreal injection leads to both greater and more persistent systemic exposure, evident in blood and lymphatic tissues. With the knowledge on the dynamics of shedding in a setting mimicking clinical application, guidelines can be developed to refine clinical trial protocols to reduce the risk for trial subjects and their environment.

Purpose This study aimed to quantify the impact of high altitude on choroidal thickness and relate changes of altered choroidal blood flow to clinical parameters and acute mountain sickness (AMS). This work is related to the Tubingen High Altitude Ophthalmology (THAO) study. Methods Enhanced depth imaging spectral-domain optical coherence tomography was used to quantify macular choroidal layer thickness. Peripheral oxygen saturation, heart rate, and AMS scores were assessed in eight healthy subjects at baseline (altitude, 341 m) and at altitude (4559 m) for respective correlations. Results Longitudinal analysis revealed a significant (P = 0.011, ANOVA) increase in central choroidal thickness (CCT) during altitude exposure (CCT baseline = 271 ± 9 μm; CCT altitude = 288 ± 9 μm) due to an increased choroidal blood flow. Incidence of AMS at altitude was 50%, peripheral oxygen saturation decreased by 25%, and heart rate increased by 39%. All changes were completely reversible after descent to low altitudes. Conclusions A small but significant increase in choroidal thickness was observed upon acute altitude exposure to 4559 m. This increase in choroidal blood flow was not related to AMS and was fully reversible after return to low altitude.