Background/aims: Voretigene neparvovec (VN) is the first and only subretinal gene therapy approved by the Food and Drug Administration and European Medicines Agency. Real-world application has started in 2018 in patients with vision impairment due to biallelic retinal pigment epithelium (RPE)65mutation-associated inherited retinal degenerations. Herein, we evaluated the development of retinal atrophy within in a single-centre patient cohort treated with VN.Methods: 13 eyes of eight patients treated with VN were retrospectively analysed for areas of retinal atrophy over a period of 6–24 months following surgery. Ultrawide field images were used to measure the area of atrophy. Fundus autofluorescence imaging is presented as an instrument for early detection of signs of retinal atrophy in these patients.Results: Atrophic changes beyond the retinotomy site were observed in all eyes. Areas of atrophy developed within the area of detachment (bleb) in all eight patients and outside the bleb in three patients. Changes in autofluorescence preceded the development of retinal atrophy and were already evident 2 weeks after surgery in the majority of patients. The areas of atrophy increase with time and progression continued over year 1. Functional outcomes remained stable (VA, FST, visual field).Conclusion: Subretinal injection of VN can lead to RPE atrophy with consequent photoreceptor loss in and outside of the bleb area. Fundus autofluorescence is an important tool to monitor atrophic changes in patients after gene therapy. Interestingly, while areas of atrophy also included central areas, the functional benefits of the treatment did not appear to be affected and remained stable.

Aims To 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). Methods Details 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. Results No 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. Conclusion The 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.

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. Experimental animal research study. Eight cynomolgus monkeys (Macaca fascicularis). Subretinal injections with an AAV2/8 vector were performed. The animals were allocated to 2 different vector dose groups (1×10^ˆ11 and 5×10^ˆ11 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). Intravitreal vector genome copies were analyzed with quantitative polymerase chain reaction and compared between groups. 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. 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.

Progressive retinal atrophy has been described after subretinal gene therapy utilizing the adeno-associated virus (AAV) vector platform. To elucidate whether this atrophy is a consequence of inherent properties of AAV, or if it is related to the surgical trauma of subretinal delivery, we analyzed data from an Investigational New Drug-enabling study for PDE6A gene therapy in nonhuman primates. Animal study (nonhuman primates), retrospective data analysis. Forty eyes of 30 healthy nonhuman primates (macaca fascicularis) were included in the analysis. Two AAV dose levels (low: 1x10E11, high: 1x10E12) were compared with sham injection (balanced saline solution; BSS). Twenty untreated eyes were not analyzed. Animals were treated with a sutureless 23G vitrectomy and single subretinal injections of AAV.PDE6A and/or BSS. The follow-up period was 12 weeks. Atrophy development was followed using fundus autofluorescence (AF), OCT, fluorescence angiography, and indocyanine green angiography. Area [mm²] of retinal pigment epithelium atrophy on AF. Presence of outer retinal atrophy on optical coherence tomography. Area [mm²] of hyperfluorescence in fluorescence angiography and hypofluorescence in indocyanine green angiography. Progressive atrophy at the injection site developed in 54% of high-dose-treated, 27% of low-dose-treated, and 0% of sham-treated eyes. At the end of observation, the mean ± SD area of atrophy in AF was 1.19 ± 1.75 mm², 0.25 ± 0.50 mm², and 0.0 ± 0.0 mm², respectively (sham × high dose: P = 0.01). Atrophic lesions in AF (P = 0.01) and fluorescence angiography (P = 0.02) were significantly larger in high-dose-treated eyes, compared with sham-treated eyes. Rate of progression in high-dose-treated eyes was 4.1× higher compared with low-dose-treated eyes. Subretinal injection of AAV.PDE6A induced dose-dependent, progressive retinal atrophy at the site of injection. Findings from multimodal imaging were in line with focal, transient inflammation within the retina and choroid and secondary atrophy. Atrophic changes after gene therapy with AAV-based vector systems are not primarily due to surgical trauma and increase with the dose given. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Purpose: PDE6A -associated retinitis pigmentosa (RP) is a rare inherited retinal disease leading to severe vision loss and blindness, with no available treatment. This study assessed the safety and vision outcomes of a gene therapy using an adeno-associated virus (AAV) vector encoding PDE6A (AAV8.hPDE6A). Methods: In an open-label, non-randomised controlled phase I/IIa trial, nine patients with biallelic PDE6A variants received a single subretinal injection of AAV8.hPDE6A. Doses were either 1.0×10¹⁰ (n=6) or 5.0×10¹⁰ (n=3) total vector genomes. Safety was the primary endpoint, assessed via clinical examinations, laboratory analyses and optical coherence tomography imaging. Secondary outcomes included changes in visual function, such as best corrected visual acuity (BCVA), contrast sensitivity, colour perception, dark adaptation thresholds, visual fields, patient-reported outcomes and chromatic pupil campimetry over 1 year. Results: The mean patient age was 40.1 years, with baseline BCVA ranging from 40 to 82 letters (0.9–0.1 logMAR). No systemic adverse events occurred, and most ocular events resolved without treatment. Persistent adverse events included small peripheral atrophic areas (n=2), disturbed colour discrimination (n=3), cataract (n=1), slight central retinal thinning (n=5) and moderate visual acuity loss (n=2, 1 in each dose group). BCVA, full-field stimulus thresholds and other visual function measures showed statistically non-significant changes, with a trend towards worsening of retinal sensitivity in the treated eyes. Conclusion: Subretinal gene therapy with AAV8.hPDE6A did not improve visual function over 1 year and posed risks, including central retinal thinning and visual acuity decline. This is in contrast to the safety and efficacy profile established in preclinical models.

Objectives: To analyse the incidence of persistence and recurrence after the peeling of idiopathic epiretinal membrane (ERM) and to describe its clinical features. Methods: This retrospective study included 666 eyes (645 patients) that underwent macular surgery for ERM removal. Optical coherence tomographic (OCT) images taken within three months after surgery and at the following visits, clinical parameters and surgery related factors were analysed to investigate the incidence and associated factors of ERM persistence and recurrence. Postoperative ERM types were categorised depending on the size ( < 100 µm, ≥100 µm) and the location (foveal, parafoveal, outside the parafovea) Results: The mean follow-up time was 29.4 months. ERM persistence (examination within 3 months) was found in 29.6% of all eyes. Only 1.9% of the eyes presented foveal ERM persistence. Foveal recurrence, defined as reappearance or growth of persistent ERM covering the fovea, was found in 8.2%. In 84.4% of eyes with foveal ERM recurrence, postoperative persistence of ERM of varying severity were identified. None of the pre-operative or surgery related factors were found significantly associated with ERM recurrence. Persistent ERM within the parafovea was the most significant risk factor for foveal ERM recurrence. Conclusion: Recurrence of ERM is generally preceded by the persistence of ERM fragments found in the early postoperative period. Growth of ERM persistence from the parafoveal region was often the origin of foveal ERM recurrence. Insufficient peeling seems to be the most significant predisposing factor for foveal ERM recurrence.