Polyethylenimine based magnetic nanoparticles mediated non-viral CRISPR/Cas9 system for genome editing.

Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9) system has become a revolutionary tool for gene editing. Since viral delivery systems have significant side effects, and naked DNA delivery is not an option, the nontoxic, non-viral delivery of CRISPR/Cas9 components would significantly improve future therapeutic delivery. In this study, we aim at characterizing nanoparticles to deliver plasmid DNA encoding for the CRISPR-Cas system in eukaryotic cells in vitro. CRISPR/Cas9 complexed polyethylenimine (PEI) magnetic nanoparticles (MNPs) were generated. We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to evaluate efficient homology- directed repair (HDR) and non-homologous end joining (NHEJ) events following transfection with NPs. MNPs have been synthesized by co-precipitation with the average particle size around 20 nm in diameter. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. Genome editing events were as efficient as compared to standard lipofectamine transfection. Our approach tested non-viral delivery of CRISPR/Cas9 and DNA template to perform HDR and NHEJ in the same assay. We demonstrated that PEI-MNPs is a promising delivery system for plasmids encoding CRISPR/Cas9 and template DNA and thus can improve safety and utility of gene editing.

Toward genome editing in X-linked RP-development of a mouse model with specific treatment relevant features.

Genome editing represents a powerful tool to treat inherited disorders. Highly specific endonucleases induce a DNA double strand break near the mutant site, which is subsequently repaired by cellular DNA repair mechanisms that involve the presence of a wild type template DNA. In vivo applications of this strategy are still rare, in part due to the absence of appropriate animal models carrying human disease mutations and knowledge of the efficient targeting of endonucleases. Here we report the generation and characterization of a new mouse model for X-linked retinitis pigmentosa (XLRP) carrying a point mutation in the mutational hotspot exon ORF15 of the RPGR gene as well as a recognition site for the homing endonuclease I-SceI. Presence of the genomic modifications was verified at the RNA and protein levels. The mutant protein was observed at low levels. Optical coherence tomography studies revealed a slowly progressive retinal degeneration with photoreceptor loss starting at 9 months of age, paralleling the onset of functional deficits as seen in the electroretinogram. Early changes to the outer retinal bands can be used as biomarker during treatment applications. We further show for the first time efficient targeting using the I-SceI enzyme at the genomic locus in a proof of concept in photoreceptors following adeno-associated virus mediated gene transfer in vivo. Taken together, our studies not only provide a human-XLRP disease model but also act as a platform to design genome editing technology for retinal degenerative diseases using the currently available endonucleases.

[Genome Editing Tools and their Application in Experimental Ophthalmology]. / Genome Editing Tools und ihr Einsatz in der experimentellen Augenheilkunde.

New genome editing tools in molecular biology are revolutionising precise genome surgery and have greatly influenced experimental ophthalmology too. Aside from the commonly used nuclease-based platforms, such as the zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), CRISPR/Cas systems, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, perform very efficiently in site-specific DNA cleavage within living cells. DNA double strand breaks (DSB) are repaired through two different conserved repair pathways: NHEJ (non-homologous end joining) and HDR (homology directed repair). By using the correct DNA templates, these repair pathways can be used to knock out defective genes or to repair mutations. Genome editing technology lays the ground for new strategies in basic science, biotechnology, and biomedical science, as well as clinical studies with genome editing. Therapeutic gene editing strategies are now concentrating on diseases in the retina, due to the comparatively easy accessibility of the eye and with local application in vivo.

Quantification of the vascular endothelial growth factor with a bioluminescence resonance energy transfer (BRET) based single molecule biosensor.

Neovascular pathologies in the eye like age-related macular degeneration (AMD), the diabetic retinopathie (DR), retinopathie of prematurity (ROP) or the retinal vein occlusion (RVO) are caused through a hypoxia induced upregulation of the vascular endothelial growth factor (VEGF). So far a correlation of intraocular VEGF concentrations to the impact of the pathologies is limited because of invasive sampling. Therefore, a minimally invasive, repeatable quantification of VEGF levels in the eye is needed to correlate the stage of VEGF induced pathologies as well as the efficacy of anti-VEGF treatment. Here we describe the development of three variants of enhanced BRET2 (eBRET2) based, single molecule biosensors by fusing a Renilla luciferase mutant with enhanced light output (RLuc8) to the N-terminus and a suitable eBRET2 acceptor fluorophore (GFP2) to the C-terminus of a VEGF binding domain, directly fused or separated with two different peptide linkers for the quantification of VEGF in vitro. The VEGF binding domain consists of a single chain variable fragment (scFv) based on ranibizumab in which the light- and the heavy- F(ab) chains were connected with a peptide linker to generate one open reading frame (orf). All three variants generate measureable eBRET2 ratios by transferring energy from the luciferase donor to the GFP2 acceptor, whereas only the directly fused and the proline variant permit VEGF quantification. The directly fused biosensor variant allows the quantification of VEGF with higher sensitivity, compared to the widely used ELISA systems and a wide dynamic quantification range in vitro. Our system demonstrates not only an additional in vitro application on VEGF quantification but also a promising step towards an applicable biosensor in an implantable device able to quantify VEGF reliably after implantation in vivo.

Correlation of central visual function and ROP risk factors in prematures with and without acute ROP at the age of 6-13†years: the Giessen long-term ROP study.

AIM: To correlate light increment sensitivity (LIS) and visual acuity (VA) with birth weight (BW), gestational age (GA) and stage of acute retinopathy of prematurity (ROP) (STG) in premature children at school age. METHODS: 180 children (150 former prematures and 30 age-matched term-born children) were enrolled at age 6-13 years. Former prematures were categorised by the results of the initial ROP screening based on digital wide-field fundus imaging: absence of ROP (n=100) and spontaneously resolved ROP (n=50). The latter group was further subdivided according to their STG (Stg 1; Stg 2; Stg 3). Both groups were categorised into sectors by BW (<1000 g; 1000-1500 g; >1500 g), and GA (≤28 weeks; >28<32 weeks; ≥32 weeks). VA was assessed with Early Treatment of Diabetic Retinopathy Study letters, LIS was measured at 0°, 2.8° and 8° in the visual field (Microperimeter MP1, Nidek Technologies), and spherical equivalent refraction assessed with a Nidek autorefractor (Nidek, Italy). RESULTS: Central and pericentral LIS (0° and 2.8°) and VA were significantly lower in all groups and sectors compared with term-born controls except for BW >1500 g for LIS and GA >28 to <32 W for VA. No significant differences were found for LIS at 8° in all groups. No correlation was found between LIS and VA on an individual basis. CONCLUSIONS: Low BW, GA and increasing severity of spontaneously resolving ROP were associated with significantly decreased central visual function. In addition to VA, LIS measurement further describes foveal function and is a unique parameter to assess parafoveal function.

[Specific gene therapy for hereditary retinal dystrophies - an update]. / Spezifische Gentherapie bei erblichen Netzhauterkrankungen - ein Update.

Treatment possibilities based on specific gene therapy strategies have become reality for a small number of patients with hereditary retinal dystrophies and are currently under investigation in several clinical trials worldwide. The most advanced studies are for patients suffering from mutations in the RPE65 gene. In addition, studies are ongoing for patients with disease causing mutations in the MERTK, REP1, ABCA4, or Myosin7A gene. Depending on the size of the gene copy to be transferred, two vectors are currently used in clinical trials: vectors based on adeno-associated virus (AAV) or on lentivirus (equine infectious anaemia virus, EIAV). An important aspect of current research includes the capacity to objectively measure the treatment effect in patients, since this is currently limited. This article gives an overview of the current state of specific gene therapy for hereditary retinal dystrophies.

[The treatment of inherited dystrophies and neovascular disorders of the retina by rAAV-mediated gene therapy]. / Die Behandlung hereditärer Dystrophien und neovaskulärer Erkrankungen der Retina durch AAV-vermittelten Gentransfer.

Over the last decade, significant progress has been made in the development of gene therapy strategies for the treatment of neovascular disorders and inherited dystrophies of the retina. Of all tested viral vectors, recombinant adeno-associated virus (rAAV) vectors, have been shown to be optimal vectors for gene transfer to the retina. Broadly speaking, two gene therapy strategies are used to treat retinal diseases; the first being corrective expression in the retina of the mutated gene (i. e., specific gene therapy) and the second being therapeutic expression of, for example, neurotrophic or antiangiogenic factors, in cases of neurodegenerative or neovascular, respectively, disorders (non-specific gene therapy). The naturally occurring RPE65 (-/-) Briard dog model has been successfully treated by specific gene transfer protocols and, based on these studies, the first clinical phase I trials are in preparation or have already begun. To avoid potential negative side effects due to the expression of neurotrophic and/or antiangiogenic factors in the retina, the expression of these transgenes needs to be regulated into a therapeutic window. Several regulatory systems have been tested in the retina of large animal models and may soon be used in clinical applications.

Oral administration of doxycycline allows tight control of transgene expression: a key step towards gene therapy of retinal diseases.

Gene transfer of neurotrophic or antiangiogenic factors has been shown to improve photoreceptor survival in retinal degenerative disorders (that is retinitis pigmentosa) and to prevent neovascularization in retinal vascular diseases (that is age-related macular degeneration, diabetic retinopathy). Expression of such neurotrophic or antiangiogenic factors after gene transfer requires the use of a regulatory system to control transgene expression to avoid unwanted side effects in cases of overexpression. In a previous study, we demonstrated that rAAV-mediated gene transfer of the tetracycline-regulatable (tetR) system allows transgene regulation in the retina of nonhuman primates after intravenous administration of doxycycline (Dox). The purpose of this study was to evaluate oral administration of Dox to control transgene expression in the retina, since the pharmacokinetics after oral administration of the inducer drug represent a key factor when considering advancing to clinical trials. We report on the outcome of this evaluation and demonstrate that oral administration of Dox at a dose that is clinically used in humans (5 mg kg(-1) per day) is capable to continuously induce transgene expression in all macaques tested for 6 months. Moreover, control of transgene expression persists up to 4 years post-subretinal injection, with maximal induced levels of transgene product remaining stable over time.

Restoration of vision in RPE65-deficient Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.

Previous studies have tested gene replacement therapy in RPE65-deficient dogs using recombinant adeno-associated virus 2/2 (rAAV2/2), -2/1 or -2/5 mediated delivery of the RPE65 gene. They all documented restoration of dark- and light-adapted electroretinography responses and improved psychophysical outcomes. Use of a specific RPE65 promoter and a rAAV vector that targets transgene expression specifically to the RPE may, however, provide a safer setting for the long-term therapeutic expression of RPE65. Subretinal injection of rAAV2 pseudotyped with serotype 4 (rAAV2/4) specifically targets the RPE. The purpose of our study was to evaluate a rAAV2/4 vector carrying a human RPE65cDNA driven by a human RPE65 promoter, for the ability to restore vision in RPE65-/- purebred Briard dogs and to assess the safety of gene transfer with respect to retinal morphology and function. rAAV2/4 and rAAV2/2 vectors containing similar human RPE65 promoter and cDNA cassettes were generated and administered subretinally in eight affected dogs, ages 8-30 months (n = 6 with rAAV2/4, n = 2 with rAAV2/2). Although fluorescein angiography and optical coherence tomography examinations displayed retinal abnormalities in treated retinas, electrophysiological analysis demonstrated that restoration of rod and cone photoreceptor function started as soon as 15 days post-injection, reaching maximal function at 3 months post-injection, and remaining stable thereafter in all animals treated at 8-11 months of age. As assessed by the ability of these animals to avoid obstacles in both dim and normal light, functional vision was restored in the treated eye, whereas the untreated contralateral eye served as an internal control. The dog treated at a later age (30 months) did not recover retinal function or vision, suggesting that there might be a therapeutic window for the successful treatment of RPE65-/- dogs by gene replacement therapy.