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Purpose: This review explores the role of pigment epithelium-derived factor (PEDF) in retinal degenerative and vascular disorders and assesses its potential both as an adjunct to established vascular endothelial growth factor inhibiting treatments for retinal vascular diseases and as a neuroprotective therapeutic agent., Methods: A comprehensive literature review was conducted, focusing on the neuroprotective and anti-angiogenic properties of PEDF. The review evaluated its effects on retinal health, its dysregulation in ocular disorders, and its therapeutic application in preclinical models. Advances in drug delivery, including gene therapy, were also examined., Results: PEDF, initially identified for promoting neuronal differentiation, is also a potent endogenous angiogenesis inhibitor. Strong anti-angiogenic and neuroprotective effects are observed in preclinical studies. It has pro-apoptotic and antiproliferative effects on endothelial cells thereby reducing neovascularization. Although promising, clinical development is limited with only a single conducted phase I clinical trial for macular neovascularization. Development of PEDF-derived peptides enhances potency and specificity, and emerging gene therapy approaches offer sustained PEDF expression for long-term treatment. However, questions regarding dosage, durability, and efficacy remain, particularly in large animal models., Conclusions: PEDF shows significant therapeutic potential in preclinical models of retinal degeneration and vascular disorders. Despite inconclusive evidence on PEDF downregulation as a primary disease driver, many studies highlight its therapeutic benefits and favorable safety profile. Advances in gene therapy could enable long-acting PEDF-based treatments, but further research is needed to optimize dosage and durability, potentially leading to clinical trials and expanding treatment options for retinal disorders.

Neovascular age-related macular degeneration (nAMD) is a frequent cause of vision loss among the elderly in the Western world. Current disease management with repeated injections of anti-VEGF agents accumulates the risk for adverse events and constitutes a burden for society and the individual patient. Sustained suppression of VEGF using gene therapy is an attractive alternative, which we explored using adeno-associated virus (AAV)-based delivery of novel RNA interference (RNAi) effectors in a porcine model of choroidal neovascularization (CNV). The potency of VEGFA -targeting, Ago2-dependent short hairpin RNAs placed in pri-microRNA scaffolds (miR-agshRNA) was established in vitro and in vivo in mice. Subsequently, AAV serotype 8 (AAV2.8) vectors encoding VEGFA -targeting or irrelevant miR-agshRNAs under the control of a tissue-specific promotor were delivered to the porcine retina via subretinal injection before CNV induction by laser. Notably, VEGFA -targeting miR-agshRNAs resulted in a significant and sizable reduction of CNV compared with the non-targeting control. We also demonstrated that single-stranded and self-complementary AAV2.8 vectors efficiently transduce porcine retinal pigment epithelium cells but differ in their transduction characteristics and retinal safety. Collectively, our data demonstrated a robust anti-angiogenic effect of VEGFA -targeting miR-aghsRNAs in a large translational animal model, thereby suggesting AAV-based delivery of anti-VEGFA RNAi therapeutics as a valuable tool for the management of nAMD., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Animal models of choroidal neovascularization (CNV) are extensively used in translational studies of CNV formation and to evaluate angiostatic treatment strategies. However, the current paucity of large animal models compared with rodent models constitutes a knowledge gap regarding the clinical translation of findings. Ocular anatomical and physiological similarities to humans suggest the pig as a relevant model animal. Thus, a systematic survey of porcine CNV models was performed to identify pertinent model parameters and suggest avenues for model standardization and optimization. A systematic search was performed in PubMed and EMBASE on November 28, 2022 for porcine models of CNV. Following inclusion by two investigators, data from the articles were extracted according to a predefined protocol. A total of 14 articles, representing 19 independent porcine CNV models were included. The included models were almost equally divided between laser-induced (53%) and surgically-induced (47%) models. Different specified breeds of domestic pigs (71%) were most commonly used in the studies. All studies used normal animals. Female pigs were reported used in 43% of the studies, while 43% did not report on sex of the animals. Younger pigs were typically used. The surgical models reported consistent CNV induction following mechanical Bruch's membrane rupture. The laser models used variants of the infrared diode laser (40%) or the frequency-doubled Nd:YAG laser (50%). Both lasers enabled successful CNV induction with reported induction rates ranging from 60 to 100%. Collateral damage to the neuroretina was reported for the infrared diode laser. CNV evaluation varied across studies with fluorescein angiography (50%) as the most used in vivo method and retinal sections (71%) as the most used ex vivo method. In interventional studies, quantification of lesions was in general performed between 7 and 14 days. The field of porcine CNV models is relatively small and heterogeneous and almost equally divided between surgically-induced and laser-induced models. Both methods have allowed successful modeling of CNV formation with induction rates comparable to those of non-human primates. However, the field would benefit from standardization of model parameters and reporting. This includes laser parameters and validation of CNV formation as well as methods of CNV evaluation and statistical analysis., Competing Interests: Declarations of competing interest None., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The year 2023 marks the 25th anniversary of the discovery of RNAi. RNAi-based therapeutics enable sequence-specific gene knockdown by eliminating target RNA molecules through complementary base-pairing. A systematic review of published and ongoing clinical trials was performed. Web of Science, PubMed, and Embase were searched from January 1, 1998, to December 30, 2022 for clinical trials using RNAi. Following inclusion, data from the articles were extracted according to a predefined protocol. A total of 90 trials published in 81 articles were included. In addition, ongoing clinical trials were retrieved from ClinicalTrials.gov, resulting in the inclusion of 48 trials. We investigated how maturation of RNAi-based therapeutics and developments in delivery platforms, administration routes, and potential targets shape the current landscape of clinically applied RNAi. Notably, most contemporary clinical trials used either N -acetylgalactosamine delivery and subcutaneous administration or lipid nanoparticle delivery and intravenous administration. In conclusion, RNAi therapeutics have gained great momentum during the past decade, resulting in five approved therapeutics targeting the liver for treatment of severe diseases, and the trajectory depicted by the ongoing trials emphasizes that even more RNAi-based medicines also targeting extra-hepatic tissues are likely to be available in the years to come., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Purpose: To investigate the level and localization of the multifunctional receptor sortilin in the diabetic retina, as well as the effect of sortilin inhibition on retinal neurodegeneration in experimental diabetes., Methods: The localization of sortilin and colocalization with the p75 neurotrophin receptor (p75NTR) and Müller cell (MC) markers were determined using immunofluorescence on retinal sections from human patients with diabetes and streptozotocin-induced diabetic C57BL/6J male mice. In the diabetic mice, levels were further quantified using Western blot and quantitative PCR. Therapeutic studies were performed on diabetic mice using intravitreally injected anti-sortilin antibodies. Neuroprotection was evaluated in vivo by optical coherence tomography and by quantification of retinal ganglion cells (RGCs) in flat mounts., Results: Increased levels of sortilin were observed in human and murine diabetic retinas compared with nondiabetic control retinas. Sortilin was highly localized to retinal MCs, and, notably, colocalization with p75NTR was only seen in diabetic retinas. A remarkable protective effect of sortilin inhibition on inner retinal cells was observed in diabetic mice. At eight weeks after diabetes induction, inner retinal thickness was reduced by 9.7% (-12.7%, -6.6%; P < 0.0001; n = 11-12) in the PBS-injected control group compared with the anti-sortilin injected group. Similarly, the count of RGCs was reduced by 20.5% (-30.8%, -10.2%; P = 0.0009) in the PBS-injected control group compared with the anti-sortilin-injected group., Conclusions: Sortilin is upregulated in the diabetic retina, and sortilin inhibition effectively protects against neuronal loss. Thus sortilin emerges as a novel pharmacological target in diabetic retinal neurodegeneration-an important early event in the pathogenesis of diabetic retinopathy.

Introduction: The purpose of this study was to determine if conjunctival lymphangiogenesis can be induced using adenoviral delivery of vascular endothelial growth factor C (VEGF-C)., Methods: Seventeen New Zealand white rabbits received a subconjunctival injection containing 3.5 × 107 plaque-forming units of an adenoviral vector containing the gene-encoding VEGF-C (Ad-VEGF-C). The contralateral eye was used for control experiment (the same volume of either saline or an empty vector). After 2 weeks, the animals were examined with trypan blue conjunctival lymphangiography, and the eyes were harvested for histology and immunohistochemistry (podoplanin and CD31)., Results: Trypan blue conjunctival lymphangiography revealed significantly more extensive conjunctival vessel network in the Ad-VEGF-C group compared with control: 1.35 ± 0.67 versus 0.28 ± 0.17 vessel length/analysed area (p = &lt;0.0001). This finding was confirmed with immunohistochemistry, where a significant increase in the number of lymphatic vessels was found compared to control; 34 ± 9 per mm2 versus 13 ± 8 per mm2 (p = 0.0019). Furthermore, there was a significant increase in lymphatic cross-sectional area; 32,500 ± 7,900 µm2 per mm2 versus 17,600 ± 9,700 µm2 per mm2 (p = 0.0149). Quantification of blood vessels revealed no significant difference in blood vessel density between Ad-VEGF-C and control; 19 ± 9 per mm2 versus 14 ± 8 per mm2 (p = 0.1971). There was no significant difference in total blood vessel area; 13,200 ± 7,600 µm2 per mm2 versus 7,100 ± 3,000 µm2 per mm2 (p = 0.0715). Eyes treated with an adenoviral vector (VEGF-C or empty vector) responded with a reactive cellular response, predominantly lymphocytes, towards the vector., Conclusion: The study demonstrates the feasibility of inducing conjunctival lymphangiogenesis with a single subconjunctival injection of Ad-VEGF-C. Future studies will explore how this can be used with a therapeutic purpose., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

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.

Retinal gene therapy using RNA interference (RNAi) to silence targeted genes requires both efficacy and safety. Short hairpin RNAs (shRNAs) are useful for RNAi, but high expression levels and activity from the co-delivered passenger strand may cause undesirable cellular responses. Ago2-dependent shRNAs (agshRNAs) produce no passenger strand activity. To enhance efficacy and to investigate improvements in safety, we have generated VEGFA -targeting agshRNAs and microRNA (miRNA)-embedded agshRNAs (miR-agshRNAs) and inserted these RNAi effectors in Pol II/III-driven expression cassettes and lentiviral vectors (LVs). Compared with corresponding shRNAs, agshRNAs and miR-agshRNAs increased specificity and safety, while retaining a high knockdown efficacy and abolishing passenger strand activity. The agshRNAs also caused significantly smaller reductions in cell viability and reduced competition with the processing of endogenous miR21 compared with their shRNA counterparts. RNA sequencing (RNA-seq) analysis of LV-transduced ARPE19 cells revealed that expression of shRNAs in general leads to more changes in gene expression levels compared with their agshRNA counterparts and activation of immune-related pathways. In mice, subretinal delivery of LVs encoding tissue-specific miR-agshRNAs resulted in retinal pigment epithelium (RPE)-restricted expression and significant knockdown of Vegfa in transduced RPE cells. Collectively, our data suggest that agshRNAs and miR-agshRNA possess important advantages over shRNAs, thereby posing a clinically relevant approach with respect to efficacy, specificity, and safety., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Purpose: The purpose of this study was to develop a porcine model for photocoagulation induced choroidal neovascularization (CNV) with high success rate and minimal thermic damage to the neuroretina., Methods: Experimental CNV was induced by laser photocoagulation in both eyes of 16 domestic pigs. In the left eyes, photocoagulation was preceded by subretinal injection of saline to protect the neuroretina from thermic damage, whereas the right eyes were treated with photocoagulation only. The development of the CNV after 3, 7, 14, 28, and 42 days was evaluated by optical coherence tomography (OCT) scanning, fluorescein angiography, and OCT angiography, and by histology after enucleation., Results: From day 7 after the photocoagulation, OCT showed subretinal density in all lesions of 14 alive animals, and either fluorescein or OCT angiography confirmed CNV formation in 11 of 14 of the eyes that had received photocoagulation alone and those in which photocoagulation had been preceded by subretinal injection of saline. In all cases pretreated with subretinal saline, the neuroretina was protected from immediate thermic damage. The formation of CNVs were confirmed by histology. For both groups, the largest lesions were observed within 14 days after photocoagulation., Conclusions: Injection of subretinal saline can protect the retina from thermic damage induced by retinal photocoagulation without reducing the success rate in producing experimental CNV. The effect of interventional studies aimed at reducing photocoagulation induced experimental CNV in pigs can be evaluated within 2 weeks after photocoagulation., Translational Relevance: This model provides a fundament to develop and evaluate novel treatment methods for neovascular retinal diseases.

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 short-lived nuclease activity., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

Vascular endothelial growth factor A (VEGFA) is involved in the pathogenesis of vasoproliferative retinal diseases, such as exudative age-related macular degeneration (AMD). The objective of this study was to investigate whether dual-acting therapy based on the simultaneous expression of anti-VEGFA microRNAs (miRNAs) and the secreted, antiangiogenic protein pigment endothelial-derived factor (PEDF) delivered by adeno-associated virus (AAV) vectors provides improved protection against choroidal neovascularization (CNV). To investigate this, a multigenic AAV vector allowing retina pigment epithelium (RPE)-specific expression of anti-VEGFA miRNAs and PEDF was engineered. Robust expression of PEDF, driven by the RPE-specific vitelliform macular dystrophy 2 promoter, was observed in human cells and in mouse retina. A significant reduction in CNV was observed in a laser-induced CNV mouse model 57 days post-injection of the AAV5 particles conveying either anti-VEGFA miRNA and PEDF dual therapy or anti-VEGFA miRNA monotherapy. Overall, CNV reduction was most prominent in animals receiving dual-acting therapy. In both cases, the reduction in CNV was accompanied by a significant attenuation of VEGFA. In conclusion, the presented data reveal that gene therapy targeting VEGFA via multigenic AAV vectors displays combined efficacy, suggesting that dual-acting therapy is an important tool in future eye gene therapy for the treatment of neovascular ocular diseases, including AMD., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genome editing and knockout by virus-based delivery of CRISPR/Cas9 may provide a new option to cure inherited and acquired ocular diseases. Here we describe development and application of lentivirus-based delivery vectors enabling knockout of the Vegfa gene. We show that Streptococcus pyogenes (Sp) Cas9 and single-guide RNAs (sgRNAs) delivered by such vectors selectively can ablate the vascular endothelial growth factor A (Vegfa) gene in mouse retina following a single administration. These findings may contribute to the development of a new therapeutic path in the treatment of ocular diseases including exudative age-related macular degeneration (AMD).

Delivery of genes to mouse liver is routinely accomplished by tail-vein injections of viral vectors or naked plasmid DNA. While viral vectors are typically injected in a low-pressure and -volume fashion, uptake of naked plasmid DNA to hepatocytes is facilitated by high pressure and volumes, also known as hydrodynamic delivery. In this study, we compare the efficacy and specificity of delivery of vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentiviral vectors to mouse liver by a number of injection schemes. Exploiting in vivo bioluminescence imaging as a readout after lentiviral gene transfer, we compare delivery by (1) "conventional" tail-vein injections, (2) "primed" injections, (3) "hydrodynamic" injections, or (4) direct "intrahepatic" injections into exposed livers. Reporter gene activity demonstrate potent and targeted delivery to liver by hydrodynamic injections. Enhanced efficacy is confirmed by analysis of liver sections from mice treated with GFP-encoding vectors, demonstrating 10-fold higher transduction rates and gene delivery to ∼80% of hepatocytes after hydrodynamic vector delivery. In summary, lentiviral vector transfer to mouse liver can be strongly augmented by hydrodynamic tail-vein injections, resulting in both reduced off-target delivery and transduction of the majority of hepatocytes. Our findings pave the way for more effective use of lentiviral gene delivery in the mouse., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are key regulators of gene expression in humans. Overexpression or depletion of individual miRNAs is associated with human disease. Current knowledge suggests that the retina is influenced by miRNAs and that dysregulation of miRNAs as well as alterations in components of the miRNA biogenesis machinery are involved in retinal diseases, including age-related macular degeneration (AMD). Furthermore, recent studies have indicated that the vitreous has a specific panel of circulating miRNAs and that this panel varies according to the specific pathological stress experienced by the retinal cells. MicroRNA (miRNA) profiling indicates subtype-specific miRNA profiles for late-stage AMD highlighting the importance of proper miRNA regulation in AMD. This review will describe the function of important miRNAs involved in inflammation, oxidative stress and pathological neovascularization, the key molecular mechanisms leading to AMD, and focus on dysregulated miRNAs as potential therapeutic targets in AMD., (© 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Generation of lentivirus (LV)-based vectors holding multiple gene cassettes for coexpression of several therapeutic factors provides potent tools in both gene delivery studies as well as in gene therapy. Here we describe the development of such multigenic LV gene delivery vectors enabling cell-specific coexpression of antiangiogenic microRNA (miRNA) and protein factors and, if preferred, a fluorescent reporter, from RNApol(II)-driven expression cassettes orientated in a back-to-back fashion. This configuration may contribute to the development of new combination therapies for amelioration of diseases involving intraocular neovascularization including exudative age-related macular degeneration (AMD).

Virus-based gene therapy by CRISPR/Cas9-mediated genome editing and knockout may provide a new option for treatment of inherited and acquired ocular diseases of the retina. In support of this notion, we show that Streptococcus pyogenes (Sp) Cas9, delivered by lentiviral vectors (LVs), can be used in vivo to selectively ablate the vascular endothelial growth factor A (Vegfa) gene in mice. By generating LVs encoding SpCas9 targeted to Vegfa, and in parallel the fluorescent eGFP marker protein, we demonstrate robust knockout of Vegfa that leads to a significant reduction of VEGFA protein in transduced cells. Three of the designed single-guide RNAs (sgRNAs) induce in vitro indel formation at high frequencies (44%-93%). A single unilateral subretinal injection facilitates RPE-specific localization of the vector and disruption of Vegfa in isolated eGFP + RPE cells obtained from mice five weeks after LV administration. Notably, sgRNA delivery results in the disruption of Vegfa with an in vivo indel formation efficacy of up to 84%. Sequencing of Vegfa-specific amplicons reveals formation of indels, including 4-bp deletions and 2-bp insertions. Taken together, our data demonstrate the capacity of lentivirus-delivered SpCas9 and sgRNAs as a developing therapeutic path in the treatment of ocular diseases, including age-related macular degeneration., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lentivirus-based vectors have been used for the development of potent gene therapies. Here, application of a multigenic lentiviral vector (LV) producing multiple anti-angiogenic microRNAs following subretinal delivery in a laser-induced choroidal neovascularization (CNV) mouse model is presented. This versatile LV, carrying back-to-back RNApolII-driven expression cassettes, enables combined expression of microRNAs targeting vascular endothelial growth factor A (Vegfa) mRNA and fluorescent reporters. In addition, by including a vitelliform macular dystrophy 2 (VMD2) promoter, expression of microRNAs is restricted to the retinal pigment epithelial (RPE) cells. Six days post injection (PI), robust and widespread fluorescent signals of eGFP are already observed in the retina by funduscopy. The eGFP expression peaks at day 21 PI and persists with stable expression for at least 9 months. In parallel, prominent AsRED co-expression, encoded from the VMD2-driven microRNA expression cassette, is evident in retinal sections and flat-mounts, revealing RPE-specific expression of microRNAs. Furthermore, LV-delivered microRNAs targeting the Vegfa gene in RPE cells reduced the size of laser-induced CNV in mice 28 days PI, as a consequence of diminished VEGF levels, suggesting that LVs delivered locally are powerful tools in the development of gene therapy-based strategies for treatment of age-related macular degeneration.

Despite the identification of numerous schizophrenia-associated genetic variants, few have been examined functionally to identify and characterize the causal variants. To mitigate this, we aimed at identifying functional variants affecting miRNA function. Using data from a large-scale genome-wide association study of schizophrenia, we looked for schizophrenia risk variants altering either miRNA binding sites, miRNA genes, promoters for miRNA genes, or variants that were expression quantitative trait loci (eQTLs) for miRNA genes. We hereby identified several potentially functional variants relating to miRNA function with our top finding being a schizophrenia protective allele that disrupts miR-206׳s binding to NT5C2 thus leading to increased expression of this gene. A subsequent experimental follow-up of the variant using a luciferase-based reporter assay confirmed that the allele disrupts the binding. Our study therefore suggests that miR-206 may contribute to schizophrenia risk through allele-dependent regulation of the genome-wide significant gene NT5C2., (Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.)

Background/aims: Microgravity (µg) has adverse effects on the eye of humans in space. The risk of visual impairment is therefore one of the leading health concerns for NASA. The impact of µg on human adult retinal epithelium (ARPE-19) cells is unknown., Methods: In this study we investigated the influence of simulated µg (s-µg; 5 and 10 days (d)), using a Random Positioning Machine (RPM), on ARPE-19 cells. We performed phase-contrast/fluorescent microscopy, qRT-PCR, Western blotting and pathway analysis., Results: Following RPM-exposure a subset of ARPE-19 cells formed multicellular spheroids (MCS), whereas the majority of the cells remained adherent (AD). After 5d, alterations of F-actin and fibronectin were observed which reverted after 10d-exposure, suggesting a time-dependent adaptation to s-µg. Gene expression analysis of 12 genes involved in cell structure, shape, adhesion, migration, and angiogenesis suggested significant changes after a 10d-RPM-exposure. 11 genes were down-regulated in AD and MCS 10d-RPM-samples compared to 1g, whereas FLK1 was up-regulated in 5d- and 10d-RPM-MCS-samples. Similarly, TIMP1 was up-regulated in 5d-RPM-samples, whereas the remaining genes were down-regulated in 5d-RPM-samples. Western blotting revealed similar changes in VEGF, β-actin, laminin and fibronectin of 5d-RPM-samples compared to 10d, whereas different alterations of β-tubulin and vimentin were observed. The pathway analysis showed complementing effects of VEGF and integrin β-1., Conclusions: These findings clearly show that s-µg induces significant alterations in the F-actin-cytoskeleton and cytoskeleton-related proteins of ARPE-19, in addition to changes in cell growth behavior and gene expression patterns involved in cell structure, growth, shape, migration, adhesion and angiogenesis., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo.

Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF) expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF). Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration.

Intraocular neovascular diseases are the leading cause of blindness in the Western world in individuals over the age of 50. Age-related macular degeneration (AMD) is one of these diseases. Exudative AMD, the late-stage form, is characterized by abnormal neovessel development, sprouting from the choroid into the avascular subretinal space, where it can suddenly cause irreversible damage to the vulnerable photoreceptor (PR) cells essential for our high-resolution, central vision. The molecular basis of AMD is not well understood, but several growth factors have been implicated including vascular endothelial growth factor (VEGF), and the advent of anti-VEGF therapy has markedly changed the outcome of treatment. However, common to all current therapies for exudative AMD are the complications of repeated monthly intravitreal injections, which must be continued throughout one's lifetime to maintain visual benefits. Additionally, some patients do not benefit from established treatments. Strategies providing long-term suppression of inappropriate ocular angiogenesis are therefore needed, and gene therapy offers a potential powerful technique. This study aimed to develop a strategy based on RNA interference (RNAi) for the sustained attenuation of VEGF. We designed a panel of anti-VEGF short hairpin RNAs (shRNA), and based on the most potent shRNAs, microRNA (miRNA)-mimicked hairpins were developed. We demonstrated an additive VEGF silencing effect when we combined the miRNAs in a tricistronic miRNA cluster. To meet the requirements for development of medical treatments for AMD with long-term effects, the shRNA/miRNA is expressed from vectors based on adeno-associated virus (AAV) or lentivirus (LV). Both vector systems have been found superior in terms of transduction efficiency and persistence in gene expression in retinal cells. The capacity of AAV-encoded RNAi effector molecules to silence endogenous VEGF gene expression was evaluated in mouse models, including the model of laser-induced choroidal neovascularization (CNV), and we found that subretinal administration of self-complementary (sc)-AAV2/8 encoding anti-VEGF shRNAs can impair vessel formation. In parallel, a significant reduction of endogenous VEGF was demonstrated following injection of scAAV2/8 vectors expressing multiple anti-VEGF miRNAs into murine hind limb muscles. Furthermore, in an ongoing project we have designed versatile, multigenic LV vectors with combined expression of multiple miRNAs and proteins, including pigment epithelium-derived factor (PEDF), a multifunctional, secreted protein that has anti-angiogenic and neurotrophic functions. Co-expression of miRNAs and proteins from a single viral vector increases safety by minimizing the viral load necessary to obtain a therapeutic effect and thereby reduces the risk of insertional mutagenesis as well as the immune response against viral proteins. Our results show co-expression of functional anti-VEGF-miRNAs and PEDF in cell studies, and in vivo studies reveal an efficient retinal pigment epithelium (RPE)-specific gene expression following the incorporation of the vitelliform macular dystrophy 2 (VMD2) promoter, demonstrating the potential applicability of our multigenic LV vectors in ocular anti-VEGF gene therapy, including combination therapy for treatment of exudative AMD. In conclusion, these highly promising data clearly demonstrate that viral-encoded RNAi effector molecules can be used for the inhibition of neovascularization and will, in combination with the growing interest of applying DNA- or RNA-based technologies in the clinic, undoubtedly contribute to the development of efficacious long-term gene therapy treatment of intraocular neovascular diseases., (© 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Background: Strategies leading to the long-term suppression of inappropriate ocular angiogenesis are required to avoid the need for repetitive monthly injections for treatment of diseases of the eye, such as age-related macular degeneration (AMD). The present study aimed to develop a strategy for the sustained repression of vascular endothelial growth factor (VEGF), which is identified as the key player in exudative AMD., Methods: We have employed short hairpin (sh)RNAs combined with adeno-associated virus (AAV) delivery to obtain the targeted expression of potent gene-regulatory molecules. Anti-VEGF shRNAs were analyzed in human retinal pigment epithelial (RPE) cells using Renilla luciferase screening. For in vivo delivery of the most potent shRNA, self-complementary AAV vectors were packaged in serotype 8 capsids (scAAV2/8-hU6-sh9). In vivo efficacy was evaluated either by injection of scAAV2/8-hU6-sh9 into murine hind limb muscles or in a laser-induced murine model of choroidal neovascularization (CNV) following scAAV2/8-hU6-sh9 subretinal delivery., Results: Plasmids encoding anti-VEGF shRNAs showed efficient knockdown of human VEGF in RPEs. Intramuscular administration led to localized expression and 91% knockdown of endogenous murine (m)VEGF. Subsequently, the ability of AAV2/8-encoded shRNAs to impair vessel formation was evaluated in the murine model of CNV. In this model, the sizes of the CNV were significantly reduced (up to 48%) following scAAV2/8-hU6-sh9 subretinal delivery., Conclusions: Using anti-VEGF vectors, we have demonstrated efficient silencing of endogenous mVEGF and showed that subretinal administration of scAAV2/8-hU6-sh9 has the ability to impair vessel formation in an AMD animal model. Thus, AAV-encoded shRNA can be used for the inhibition of neovascularization, leading to the development of sustained anti-VEGF therapy., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background: Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that plays a critical role in several diseases, including cancer, rheumatoid arthritis and diseases of the eye. Persistent regulation of VEGF by expression of small interfering RNAs targeting VEGF represents a potential future strategy for treatment of such diseases. As a step toward this goal, the present study combines the potency of VEGF-targeted miRNA mimics, produced from a miRNA cluster, with delivery by adeno-associated virus (AAV)-based vectors., Methods: Nine different engineered tri-cistronic miRNA clusters encoding anti-VEGF effectors were generated and tested in adult human retinal pigment epithelial (ARPE-19) cells using Renilla luciferase screening, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting and immunostaining analysis. In vivo efficacy was tested by the injection of scAAV2/8 vectors expressing the most effective miRNA cluster into murine hindlimb muscles, followed by quantitative RT-PCR., Results: Plasmids containing anti-VEGF miRNA clusters showed efficient silencing of VEGF and demonstrated a combined gene silencing effect for miRNA clusters composed of multiple miRNA-mimicked RNA interference effectors. The most potent molecule, miR-5,10,7, resulted in a knockdown of VEGF by approximately 75%. Injection of scAAV2/8 vectors expressing miR-5,10,7 into murine hindlimb muscles, resulted in a 44% reduction of endogenous VEGF., Conclusions: We have developed miRNA clusters encoding anti-VEGF effectors and shown, in a mouse model, that VEGF is efficiently down-regulated by scAAV2/8-delivered miRNA clusters, allowing potent attenuation of VEGF. These findings may contribute to the development of gene therapy based on AAV-mediated delivery of miRNA clusters., (Copyright © 2012 John Wiley & Sons, Ltd.)