subretinal-implantation-rpe-on-carrier-minipigs-guidelines-for

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial ...

University Hospital Giessen and Marburg GmbH

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.

[The treatment of inherited dystrophies and neovascular disorders of the retina by rAAV-mediated gene therapy].

The gene therapy vector tgAAG76 (rAAV 2/2.hRPE65p.hRPE65) is in joint development by Targeted Genetics Corp, Moorfields Eye Hospital and the University of London. The vector is a recombinant adeno-associated virus vector that contains the human RPE65 gene under the control of the human RPE65 promoter region and the bovine growth hormone polyadenylation signal. The vector was designed for administration into the subretinal space of patients affected by a hereditary blinding disorder, Leber congenital amaurosis type 2, which is caused by mutations in the RPE65 gene. Interim results from an ongoing phase I/II clinical trial assessing tgAAG76 in three patients with Leber congenital amaurosis type 2 were considered to accomplish the primary outcome of the trial, which was the safety of the procedure, with no severe side effects observed to date. One of the three patients had a significant increase in sensitivity to light and the better capacity to ambulate an obstacle course under dim light conditions compared with baseline. Completion of the clinical trial was anticipated in the second half of 2010.

tgAAG76, an adeno-associated virus delivered gene therapy for the potential treatment of vision loss caused by RPE65 gene abnormalities.

Retinal gene therapy mediated by adeno-associated virus (AAV) based gene transfer was recently proven to improve photoreceptor function in one form of inherited retinal blinding disorder associated with mutations in the RPE65 gene. Several clinical trials are currently ongoing, and more than 30 patients have been treated to date. Even though only a very limited number of patients will greatly benefit from this still experimental treatment protocol, the technique itself has been shown to be safe and will likely be used in other retinal disorders in the near future. A canine model for achromatopsia has been treated successfully as well as mouse models for different forms of Leber congenital amaurosis (LCA). For patients with autosomal dominant retinitis pigmentosa (adRP), a combined gene knockdown and gene addition therapy is being developed using RNA interference to block mRNA of the mutant allele. For those patients suffering from RP with unknown mutations, an AAV based transfer of bacterial forms of rhodopsin in the central retina might be an option to reactivate residual cones in the future.

Gene therapy for vision loss -- recent developments.

Over the last decade, huge progress has been made in the understanding of the molecular mechanisms underlying inherited retinal dystrophy (IRD), as well as in the development and implementation of novel therapies, especially in the field of gene therapy. The use of mutant animal models, either naturally occurring or generated by genetic modification, have contributed greatly to our knowledge on IRD. Yet, these mutant animal models do not always mimic the retinal phenotype that is observed in humans with mutations in the orthologous gene, often due to species-specific characteristics of the retina, and/or diverse functions of the gene products in different species. In this manuscript, we compare general and ocular characteristics of a series of widely used vertebrate animal models, i.e. zebrafish, chicken, rodents, cats, dogs, sheep, pigs and monkeys, in terms of genetic architecture and sequence homology, methods to modify genomes, anatomy of the eye, and structural details of the retina. Furthermore, we present an overview of mutant vertebrate animal models that have been used to study or develop treatments for the various genetic subtypes of IRD, and correlate the suitability of these models to the specific characteristics of each animal. Herewith, we provide tools that will help to select the most suitable animal model for specific research questions on IRDs in the future, and thereby assist in an optimal use of animals and resources to further increase our understanding of inherited retinal dystrophies, and develop novel treatments for these disorders. 

The pros and cons of vertebrate animal models for functional and therapeutic research on inherited retinal dystrophies.

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. 

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

In vivo genome editing represents an emerging field in the treatment of monogenic disorders, as it may constitute a solution to the current hurdles in classic gene addition therapy, which are the low levels and limited duration of transgene expression. Following the introduction of a double strand break (DSB) at the mutational site by highly specific endonucleases, such as TALENs (transcription activator like effector nucleases) or RNA based nucleases (clustered regulatory interspaced short palindromic repeats - CRISPR-Cas), the cell's own DNA repair machinery restores integrity to the DNA strand and corrects the mutant sequence, thus allowing the cell to produce protein levels as needed. The DNA repair happens either through the error prone non-homologous end-joining (NHEJ) pathway or with high fidelity through homology directed repair (HDR) in the presence of a DNA donor template. A third pathway called microhomology mediated endjoining (MMEJ) has been recently discovered. In this review, the authors focus on the different DNA repair mechanisms, the current state of the art tools for genome editing and the particularities of the retina and photoreceptors with regard to in vivo therapeutic approaches. Finally, current attempts in the field of retinal in vivo genome editing are discussed and future directions of research identified.

In vivo genome editing as a potential treatment strategy for inherited retinal dystrophies.

In this paper we describe a two-component BRET (bioluminescence resonance energy transfer)-based method to detect vascular endothelial growth factor (VEGF) molecules in unknown samples as the basis for subsequent in vivo use. A luminescent VEGF binding molecule, which binds in the receptor binding motif of VEGF, is used as the energy donor, transferred to a fluorophore-coupled VEGF binding molecule (acceptor), which binds to the neuropilin binding motif of VEGF, thus enabling energy transfer from the donor to the acceptor molecule. This leads to the emission of light at a longer wavelength and thus the generation of an increased BRET signal only when VEGF is bound to both the donor and acceptor molecules. We further describe a novel BRET pair that uses the  mutant luciferase RLuc8 and the chemically engineered fluorophore PerCP-Cy5.5, which exhibits superior peak separation of approximately 300 nm. The implantation of capsules consisting of the two BRET components in solution, permeable for VEGF for its in vivo detection, would provide a new and improved method for monitoring VEGF-induced pathologies and thus an adjustment of therapy to patient needs.

Detection of the Vascular Endothelial Growth Factor with a Novel Bioluminescence Resonance Energy Transfer Pair Using a Two-Component System.

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated proteins) technology enables rapid and precise genome editing at any desired genomic position in almost all cells and organisms. In this study, we analyzed the impact of different repair templates on the frequency of homology-directed repair (HDR) and non-homologous end joining (NHEJ). We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to quantify HDR and NHEJ events following transfection with Cas9, eight different guide RNAs, and a 1,000 bp donor template generated either as circular plasmid, as linearized plasmid with long 3' or 5' backbone overhang, or as PCR product. The sequence to be corrected was either centrally located (RS55), with a shorter 5' homologous region (RS37), or with a shorter 3' homologous region (RS73). Guide RNAs targeting the transcriptionally active strand (T5, T7) showed significantly higher NHEJ frequencies compared with guide RNAs targeting the transcriptionally inactive strand. HDR activity was highest when using the linearized plasmid with the short 5' backbone overhang and the RS37 design. The results demonstrate the importance of the design of the guide RNA and template DNA on the frequency of DNA repair events and, ultimately, on the outcome of treatment approaches using HDR.

Optimizing the DNA Donor Template for Homology-Directed Repair of Double-Strand Breaks.

Common genome-editing strategies are either based on non-homologous end joining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology-directed repair [HDR]) or short (microhomology-mediated end joining [MMEJ]) homologous sequences. In the current study, we aim to develop a model system to test the activity of MMEJ after CRISPR/Cas9-mediated cleavage in cell culture. Following successful proof of concept in an episomally based reporter system, we tested template plasmids containing a promoter-less luciferase gene flanked by microhomologous sequences (mhs) of different length (5, 10, 15, 20, 30, and 50 bp) that are complementary to the mouse retinitis pigmentosa GTPase regulator (RPGR)-ORF15, which is under the control of a CMV promoter stably integrated into a HEK293 cell line. Luciferase signal appearance represented successful recombination events and was highest when the mhs were 5 bp long, while longer mhs revealed lower luciferase signal. In addition, presence of Csy4 RNase was shown to increase luciferase signaling. The luciferase reporter system is a valuable tool to study the input of the different DNA repair mechanisms in the replacement of large DNA sequences by mhs.

Development of a Reporter System to Explore MMEJ in the Context of Replacing Large Genomic Fragments.

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.

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

To study the efficacy of a novel needle for intravitreal injection (IVI) in comparison to the conventional needle under experimental conditions.

Novel Needle for Intravitreal Drug Delivery: Comparative Study of Needle Tip Aspirates, Injection Stream and Penetration Forces.

The choice of surgical treatment for chronic, persistent and large full-thickness macular holes (FTMH) continues to be undefined and challenging, as some of these cases remain refractory to the treatment. We report the efficacy of combination of inverted internal limiting membrane flap technique (IILMFT) and subretinal application of the fluid (SR fluid application) technique for treatment of refractory FTMHs.

Combination of Inverted ILM Flap Technique and Subretinal Fluid Application Technique for Treatment of Chronic, Persistent and Large Macular Holes.

Cigarette smoke has been identified as a major risk factor for the development of age-related macular degeneration (AMD). As an alternative to conventional cigarettes (C-cigarette), electronic cigarettes (E-cigarette) have been globally promoted and are currently widely used. The increasing usage of E-cigarettes raises concerns with regard to short- (2 weeks), medium- (3 months), and long- (8 months) term consequences related to retinal tissue. In this report, a controlled study in mouse models was conducted to probe the comprehensive effects of E-cigarette vapor on retina, retinal pigmented epithelium (RPE), and choroidal tissues by (1) comparing the effects of C-cigarette smoke and E-cigarette vapor on retina separately and (2) determining the effects of E-cigarette vapor on the RPE and analyzing the changes with regard to inflammatory (IL-1β, TNFα, iNOS) and angiogenic (VEGF, PEDF) mediators in retina/RPE/choroid by ELISA assays. The data showed that C-cigarette smoke exposure promoted an inflammatory reaction in the retina in vivo. Mice exposed to E-cigarette (nicotine-free) vapor developed inflammatory and angiogenic reactions more pronounced in RPE and choroid as compared to retinal tissue, while nicotine-containing E-cigarette vapor caused even a more serious reaction. Both inflammatory and pro-angiogenic reactions increased with the extension of exposure time. These results demonstrate that exposure to C-cigarette smoke is harmful to the retina. Likewise, the exposure to E-cigarette vapor (with or without nicotine) increases the occurrence and progression of inflammatory and angiogenic stimuli in the retina, which might also be related to the onset of wet AMD in humans. KEY MESSAGES: C-cigarette smoke exposure promotes an inflammatory reaction in the retina in vivo. Mice exposed to E-cigarette (nicotine-free) vapor develop inflammatory and angiogenic reactions more pronounced in RPE and choroid compared to retinal tissue, while nicotine-containing E-cigarette vapor causes even a more serious reaction. Both inflammatory and pro-angiogenic reactions increase with the extension of E-cigarette vapor exposure time.

Retinal tissue develops an inflammatory reaction to tobacco smoke and electronic cigarette vapor in mice.

The development of primary human retinal pigmented epithelium (hRPE) for clinical transplantation purposes on biodegradable scaffolds is indispensable. We hereby report the results of the subretinal implantation of hRPE cells on nanofibrous membranes in minipigs.

Article	Year
[The treatment of inherited dystrophies and neovascular disorders of the retina by rAAV-mediated gene therapy]. Klinische Monatsblatter fur Augenheilkunde\| PubMed ID: 19085778	2008
tgAAG76, an adeno-associated virus delivered gene therapy for the potential treatment of vision loss caused by RPE65 gene abnormalities. Current opinion in molecular therapeutics\| PubMed ID: 20677098	2010
Gene therapy for vision loss -- recent developments. Discovery medicine\| PubMed ID: 21122474	2010
The pros and cons of vertebrate animal models for functional and therapeutic research on inherited retinal dystrophies. Progress in retinal and eye research\| PubMed ID: 25936606	2015
Quantification of the vascular endothelial growth factor with a bioluminescence resonance energy transfer (BRET) based single molecule biosensor. Biosensors & bioelectronics\| PubMed ID: 27459244	2016
In vivo genome editing as a potential treatment strategy for inherited retinal dystrophies. Progress in retinal and eye research\| PubMed ID: 27623223	2017
Detection of the Vascular Endothelial Growth Factor with a Novel Bioluminescence Resonance Energy Transfer Pair Using a Two-Component System. Sensors (Basel, Switzerland)\| PubMed ID: 28098756	2017
Optimizing the DNA Donor Template for Homology-Directed Repair of Double-Strand Breaks. Molecular therapy. Nucleic acids\| PubMed ID: 28624224	2017
Development of a Reporter System to Explore MMEJ in the Context of Replacing Large Genomic Fragments. Molecular therapy. Nucleic acids\| PubMed ID: 29858075	2018
Toward genome editing in X-linked RP-development of a mouse model with specific treatment relevant features. Translational research : the journal of laboratory and clinical medicine\| PubMed ID: 30213530	2019
Novel Needle for Intravitreal Drug Delivery: Comparative Study of Needle Tip Aspirates, Injection Stream and Penetration Forces. Clinical ophthalmology (Auckland, N.Z.)\| PubMed ID: 33642853	2021
Combination of Inverted ILM Flap Technique and Subretinal Fluid Application Technique for Treatment of Chronic, Persistent and Large Macular Holes. Ophthalmology and therapy\| PubMed ID: 34231188	2021
Retinal tissue develops an inflammatory reaction to tobacco smoke and electronic cigarette vapor in mice. Journal of molecular medicine (Berlin, Germany)\| PubMed ID: 34264377	2021
Subretinal Implantation of Human Primary RPE Cells Cultured on Nanofibrous Membranes in Minipigs. Biomedicines\| PubMed ID: 35327471	2022

Knut Stieger

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