The therapeutic effect of retinal gene therapy using CRISPR/ Cas9-mediated genome editing and knockout applications is dependent on efficient and safe delivery of gene-modifying tool kits. Recently, transient administration of single guide RNAs (sgRNAs) and SpCas9 proteins delivered as ribonucleoproteins (RNPs) has provided potent gene knockout in vitro.To improve efficacy of CRISPR-based gene therapy, we delivered RNPs containing SpCas9 protein complexed to chemically modified sgRNAs (msgRNAs). In K562 cells, msgRNAs significantly increased the insertion/deletion (indel) frequency (25%) compared with unmodified counterparts leading to robust knockout of the VEGFA gene encoding vascular endothelial growth factor A (96% indels). Likewise, in HEK293 cells, lipoplexes containing varying amounts of RNP and EGFP mRNA showed efficient VEGFA knockout (43% indels) and strong EGFP expression, indicative of efficacious functional knockout using small amounts of RNP. In mice, subretinal injections of equivalent lipoplexes yielded 6% indels in Vegfa of isolated EGFP-positive RPE cells. However, signs of toxicity following delivery of lipoplexes containing high amounts of RNP were observed. Although the mechanism resulting in the varying efficacy remains to be elucidated, our data suggest that a single subretinal injection of RNPs carrying msgRNAs and SpCas9 induces targeted retinal indel formation, thus providing a clinically relevant strategy relying on nonviral delivery of shortlived nuclease activity.

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Purpose Animal models of choroidal neovascularization (CNV) are extensively used to characterize the pathophysiology of chorioretinal diseases with CNV formation and to evaluate novel treatment strategies. This systematic review aims to give a detailed overview of contemporary animal models of CNV. Methods A systematic search was performed in PubMed and EMBASE from November 20, 2015, to November 20, 2020, for mammalian animal models of CNV. Following inclusion by two investigators, data from the articles were extracted according to a predefined protocol. Results A total of 380 full articles, representing 409 independent animal models, were included. Mice were by far the most utilized animal (76%) followed by rats and non-human primates. The median age of rodents was 8 weeks but with a wide range. Male animals were used in 44% of the studies, but 32% did not report the sex. CNV was laser induced in 89% of the studies, but only 44% of these reported sufficiently on standard laser parameters. Surprisingly, 28% of the studies did not report a sample size for quantitative CNV evaluation. Less than half of the studies performed quantitative in vivo evaluation, and 73% evaluated CNV quantitatively ex vivo. Both in vivo and ex vivo evaluations were conducted primarily at day 7 and/or day 14. Conclusions The laser-induced mouse model is the predominant model for experimental CNV. The widespread use of young, healthy male animals may complicate clinical translation, and inadequate reporting challenges reproducibility. Definition and implementation of standardized methodologic and reporting guidelines are attractive.

Age-related macular degeneration (AMD) is the leading cause of blindness affecting the elderly in the Western world. The most severe form of AMD, wet AMD (wAMD), is characterized by choroidal neovascularization (CNV) and acute vision loss. The current treatment for these patients comprises monthly intravitreal injections of anti-vascular endothelial growth factor (VEGF) antibodies, but this treatment is expensive, uncomfortable for the patient, and only effective in some individuals. AMD is a complex disease that has strong associations with the complement system. All three initiating complement pathways may be relevant in CNV formation, but most evidence indicates a major role for the alternative pathway (AP) and for the terminal complement complex, as well as certain complement peptides generated upon complement activation. Since the complement system is associated with AMD and CNV, a complement inhibitor may be a therapeutic option for patients with wAMD. The aim of this review is to (i) reflect on the possible complement targets in the context of wAMD pathology, (ii) investigate the results of prior clinical trials with complement inhibitors for wAMD patients, and (iii) outline important considerations when developing a future strategy for the treatment of wAMD.

Gene therapy is a promising therapeutic modality for ocular diseases arising in and affecting the retina and choroid. In this context, delivering gene therapy to the multifunctional retinal pigment epithelium (RPE) cells situated between the retina and choroid is desired. Efficacy assessment of any gene therapy strategy, whether it is gene augmentation, inhibition, or editing is initially tested in vitro in cell models, where delivery is simple and efficient. However, efficacy assessment in vivo in animal models is far more complex and several factors can influence the result significantly. Here we report a simple fluorescence activated cell sorting (FACS)-based enrichment method for direct assessment of efficacy and potential off-target effects of gene therapy co-delivered with an eGFP reporter to murine RPE cells using subretinal administration. Isolation of true eGFP+ RPE cells by FACS is notoriously difficult due to their intrinsic autofluorescence resulting in decreased sensitivity and false positives. Combining retinal dissection and harvest of RPE cells with a FACS-gating strategy utilizing the GFP filter and a neighboring filter, to separate the eGFP signal from autofluorescence, allows a significant enrichment of gene therapy-targeted eGFP+ RPE cells. In our hands the method may provide quantitative and qualitative advances in terms of up to 7-fold enrichment of true eGFP+ RPE cells compared to a standard protocol. The isolated cells can subsequently be utilized for reliable assessment of changes in DNA, RNA, or protein. This method allows proof-of-principle analysis of early gene therapy development and investigation of new delivery strategies or therapeutic approaches targeting RPE cells in vivo.