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Gene therapy is a treatment concept that uses, in most cases, viral vectors to deliver a therapeutic transgene to target cells. Although the idea of gene therapy dates back over 50 years ago, due to the complexity of the treatment concept, it took until the last decade for the responsible agencies like FDA and EMA to recommend the first gene therapy products for clinical use. The development of these therapies relies on molecular engineering of specifically designed vectors and models to test the effectiveness and safety of the treatment. Despite an increasing effort to find effective surrogates, animal models are still irreplaceable in gene therapy development. Rodents are important for exploring pathways and disease mechanisms and identifying potential treatment targets. However, only the primate eye resembles the human eye to a degree where most structures are nearly identical. Some research questions can therefore only be answered using a nonhuman primate (NHP) model. In this review, we want to summarize these key features and highlight the importance of the NHP model for gene therapy development in ophthalmology.Bei der Gentherapie wird mittels eines meist viralen Vektors ein spezifisch entwickeltes therapeutisches Transgen in bestimmte Zielzellen eingeschleust. Zur Erreichung dieses Ziels waren viele Jahrzehnte an Forschung notwendig und erst seit ungefähr 10 Jahren gibt es die ersten am Menschen zugelassenen Gentherapien. Unverzichtbar für die Entwicklung sind die Methoden der modernen Molekulargenetik und -technik sowie geeignete Tiermodelle. Die zunehmend besseren Alternativen können die gängigen Tiermodelle zum jetzigen Zeitpunkt allerdings noch nicht vollständig ersetzen. Mausmodelle nehmen weiterhin aufgrund ihrer vielen Vorteile eine tragende Rolle ein. Jedoch bestehen grundlegende Unterschiede zur Physiologie und Anatomie insbesondere der Netzhaut des Menschen, weshalb sie für diesbezügliche Fragestellungen nur bedingt geeignet sind. Die Augen der Primaten sind hingegen denen des Menschen in strukturellem Aufbau der Netzhaut und des Auges sehr ähnlich. Dadurch besteht eine deutlich bessere Vergleichbarkeit für bestimmte Fragestellungen in der Entwicklung von Gentherapien am Auge. In diesem Artikel möchten wir die Relevanz von Tiermodellen an sog. nicht menschlichen Primaten in der Entwicklung von Gentherapien am Auge darstellen.

BackgroundVoretigene neparvovec is a gene therapeutic agent for treatment of retinal dystrophies caused by bi-allelic RPE65 mutations. In this study, we report on a novel and objective evaluation of a retinotopic photoreceptor rescue.MethodsSeven eyes of five patients (14, 21, 23, 24, 36 years, 1 male, 4 females) with bi-allelic RPE65 mutations have been treated with voretigene neparvovec. The clinical examinations included visual acuity testing, dark-adapted full-field stimulus threshold (FST), dark-adapted chromatic perimeter (DAC) with a 30-degree grid, and a 30 degrees grid scotopic and photopic chromatic pupil campimetry (CPC). All evaluations and spectral domain optical coherence tomography were performed at baseline, 1 month and 3 months.ResultsAll except the oldest patient had a measurable improvement of the rod function assessed via FST, DAC or scotopic CPC at 1 month. The visual acuity improved slightly or remained stable in all eyes. A cone function improvement as measured by photopic CPC was observed in three eyes. The gain of the dark-adapted threshold with blue FST and the DAC stimuli (cyan) average correlated strongly with age (R2>0.7). The pupil response improvement in the scotopic CPC correlated with the baseline local retinal volume (R2=0.5).ConclusionsThe presented protocols allow evaluating the individual spatial and temporal effects of gene therapy effects. Additionally, we explored parameters that correlated with the success of the therapy. CPC and DAC present new and fast ways to assess functional changes in retinotopic maps of rod and cone function, measuring complementary aspects of retinal function.

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.

Background/aimsVoretigene 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) 65 mutation-associated inherited retinal degenerations. Herein, we evaluated the development of retinal atrophy within in a single-centre patient cohort treated with VN.Methods13 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.ResultsAtrophic 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).ConclusionSubretinal 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.

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.