P36-A151†Primary human RPE cells as individual starting material for non-viral gene transfer of therapeutically active factors.

PURPOSE: Our aging society leads to an increasing incidence of neurodegenerative diseases. To date, the development of defined therapies has been hampered because the pathological mechanisms are poorly understood. Cell-based additive gene therapies to enhance the expression of protective factors are considered a promising modality for the treatment of neurodegenerative diseases, such as agerelated macular degeneration (AMD). We have developed a method to stably overexpress the genes encoding pigment epithelium-derived factor (PEDF) and brain-derived neurotrophic factor (BDNF) into the genome of primary human retinal pigment epithelial (RPE) cells by electroporation using the Sleeping Beauty (SB) transposon system. BDNF is the most abundant neurotrophin in the central nervous system. PEDF is a multifunctional protein with anti-angiogenic and neurotrophic properties. METHODS: Primary RPE cells were isolated from various human donor eyes and maintained individually in culture. After reaching confluence, RPE cells were trypsinized and co-transfected in suspension with two plasmids encoding SB100X transposase and the transposon carrying a PEDF and BDNF transcription cassette, respectively. The results of transfection were evaluated by different methods including microscopy, immunoblotting, ELISA, and quantitative PCR (qPCR). RESULTS: Seeding of sufficient numbers of primary human RPE cells allows cultivation and growth into an integrated monolayer of pigmented, hexagonally shaped cells, independent of the donor age (65.3 ± 9.94 a, min: 49 a, max: 83 a, n = 12), post-mortem time of isolation (37.3 ± 17.0 h, min: 16 h, max: 68 h), and cultivation time (27.6 ± 14.1 d, min: 13 d, max: 61 d). Successful transfection was demonstrated in experiments performed independently. Applied electrical pulses had no negative effects on cell morphology. Gene expression of PEDF and BDNF was significantly increased compared with non-transfected control cells. Secretion of recombinant PEDF and BDNF proteins was also significantly elevated and remained stable over time. CONCLUSION: The studies using primary human RPE cells are an important step in the development of a cell-based PEDF or BDNF gene therapy that could be applied as an advanced therapy medicinal product to treat AMD or other degenerative retinal diseases.

A workflow to visualize vertebrate eyes in 3D.

PURPOSE: To establish a workflow to visualize the surgical anatomy in 3D based on histological data of eyes of experimental animals for improving the planning of complex surgical procedures. METHODS: Four C57BL/6J wild-type(wt) mouse eyes, three Brown Norway rat eyes and four Chinchilla Bastard rabbit eyes were enucleated and processed for standard histology with serial sections and hematoxylin and eosin staining. Image stacks were processed to obtain a representation of the eye anatomy in 3D. In addition, virtual image stacks and 3D point clouds were generated by processing sagittal sections of eyes with stepwise 180° rotation and projection around the eye axis to construct a rotationally symmetric 3D model from one single sagittal section. RESULTS: Serial sections of whole eyes of mice, rats and rabbits showed significant artifacts interfering with a practical image stack generation and straightforward 3D reconstruction despite the application of image registration techniques. A workflow was established to obtain a 3D image of the eye based on virtual image stacks and point cloud generation by rotation of a single sagittal section of the eye around the symmetry axis. By analyzing the tissue shrinkage during histological processing true biometric reconstructions of the eyes were feasible making the resulting model usable for 3D modeling and simulation, e.g. for planning of complex surgical procedures in different species. CONCLUSION: Because serial sections of the eye with standard histological protocols yielded too many artifacts for a straightforward 3D visualization we reconstructed a pseudorealistic 3D model based on virtual image stacks and point cloud generation calculated from a single sagittal section of the eye. Such a model detailing microscopic structures of the whole eye will allow for a specific planning of surgical procedures in small animal eyes in order to prevent surgical complications in a very early stage of an experiment and it will support the design and development of complex intraocular implants. It will therefore be helpful in surgical teaching and improve laboratory animal welfare by an expected reduction of experimental animal numbers. Further processing including integration of mechanical tissue properties is needed to convert these 3D models into a practical virtual reality teaching and simulation platform for eyes of several species.

Mammalian Animal and Human Retinal Organ Culture as Pre-Clinical Model to Evaluate Oxidative Stress and Antioxidant Intraocular Therapeutics.

Oxidative stress (OS) is involved in the pathogenesis of retinal neurodegenerative diseases such as age-related macular degeneration (AMD) and diabetic retinopathy (DR) and an important target of therapeutic treatments. New therapeutics are tested in vivo despite limits in terms of transferability and ethical concerns. Retina cultures using human tissue can deliver critical information and significantly reduce the number of animal experiments along with increased transferability. We cultured up to 32 retina samples derived from one eye, analyzed the model's quality, induced OS, and tested the efficiency of antioxidative therapeutics. Bovine, porcine, rat, and human retinae were cultured in different experimental settings for 3-14 d. OS was induced by a high amount of glucose or hydrogen peroxide (H2O2) and treated with scutellarin, pigment epithelium-derived factor (PEDF), and/or granulocyte macrophage colony-stimulating factor (GM-CSF). The tissue morphology, cell viability, inflammation, and glutathione level were determined. The retina samples showed only moderate necrosis (23.83 ± 5.05 increased to 27.00 ± 1.66 AU PI-staining over 14 d) after 14 days in culture. OS was successfully induced (reduced ATP content of 288.3 ± 59.9 vs. 435.7 ± 166.8 nM ATP in the controls) and the antioxidants reduced OS-induced apoptosis (from 124.20 ± 51.09 to 60.80 ± 319.66 cells/image after the scutellarin treatment). Enhanced mammalian animal and human retina cultures enable reliable, highly transferable research on OS-triggered age-related diseases and pre-clinical testing during drug development.

Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells.

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal neovascularization (CNV), which leads to retinal pigment epithelial (RPE) cell and photoreceptor degeneration and blindness if untreated. Since blood vessel growth is mediated by endothelial cell growth factors, including vascular endothelial growth factor (VEGF), treatment consists of repeated, often monthly, intravitreal injections of anti-angiogenic biopharmaceuticals. Frequent injections are costly and present logistic difficulties; therefore, our laboratories are developing a cell-based gene therapy based on autologous RPE cells transfected ex vivo with the pigment epithelium derived factor (PEDF), which is the most potent natural antagonist of VEGF. Gene delivery and long-term expression of the transgene are enabled by the use of the non-viral Sleeping Beauty (SB100X) transposon system that is introduced into the cells by electroporation. The transposase may have a cytotoxic effect and a low risk of remobilization of the transposon if supplied in the form of DNA. Here, we investigated the use of the SB100X transposase delivered as mRNA and showed that ARPE-19 cells as well as primary human RPE cells were successfully transfected with the Venus or the PEDF gene, followed by stable transgene expression. In human RPE cells, secretion of recombinant PEDF could be detected in cell culture up to one year. Non-viral ex vivo transfection using SB100X-mRNA in combination with electroporation increases the biosafety of our gene therapeutic approach to treat nvAMD while ensuring high transfection efficiency and long-term transgene expression in RPE cells.

Early Alterations of RNA Binding Protein (RBP) Homeostasis and ER Stress-Mediated Autophagy Contributes to Progressive Retinal Degeneration in the rd10 Mouse Model of Retinitis Pigmentosa (RP).

The retinal degeneration 10 (rd10) mouse model is widely used to study retinitis pigmentosa (RP) pathomechanisms. It offers a rather unique opportunity to study trans-neuronal degeneration because the cell populations in question are separated anatomically and the mutated Pde6b gene is selectively expressed in rod photoreceptors. We hypothesized that RNA binding protein (RBP) aggregation and abnormal autophagy might serve as early pathogenic events, damaging non-photoreceptor retinal cell types that are not primarily targeted by the Pde6b gene defect. We used a combination of immunohistochemistry (DAB, immunofluorescence), electron microscopy (EM), subcellular fractionation, and Western blot analysis on the retinal preparations obtained from both rd10 and wild-type mice. We found early, robust increases in levels of the protective endoplasmic reticulum (ER) calcium (Ca2+) buffering chaperone Sigma receptor 1 (SigR1) together with other ER-Ca2+ buffering proteins in both photoreceptors and non-photoreceptor neuronal cells before any noticeable photoreceptor degeneration. In line with this, we found markedly altered expression of the autophagy proteins p62 and LC3, together with abnormal ER widening and large autophagic vacuoles as detected by EM. Interestingly, these changes were accompanied by early, prominent cytoplasmic and nuclear aggregation of the key RBPs including pTDP-43 and FET family RBPs and stress granule formation. We conclude that progressive neurodegeneration in the rd10 mouse retina is associated with early disturbances of proteostasis and autophagy, along with abnormal cytoplasmic RBP aggregation.

GMP-Grade Manufacturing and Quality Control of a Non-Virally Engineered Advanced Therapy Medicinal Product for Personalized Treatment of Age-Related Macular Degeneration.

The introduction of new therapeutics requires validation of Good Manufacturing Practice (GMP)-grade manufacturing including suitable quality controls. This is challenging for Advanced Therapy Medicinal Products (ATMP) with personalized batches. We have developed a person-alized, cell-based gene therapy to treat age-related macular degeneration and established a vali-dation strategy of the GMP-grade manufacture for the ATMP; manufacturing and quality control were challenging due to a low cell number, batch-to-batch variability and short production duration. Instead of patient iris pigment epithelial cells, human donor tissue was used to produce the transfected cell product ("tIPE"). We implemented an extended validation of 104 tIPE productions. Procedure, operators and devices have been validated and qualified by determining cell number, viability, extracellular DNA, sterility, duration, temperature and volume. Transfected autologous cells were transplanted to rabbits verifying feasibility of the treatment. A container has been engineered to ensure a safe transport from the production to the surgery site. Criteria for successful validation and qualification were based on tIPE's Critical Quality Attributes and Process Parameters, its manufacture and release criteria. The validated process and qualified operators are essential to bring the ATMP into clinic and offer a general strategy for the transfer to other manufacture centers and personalized ATMPs.

Molecular and Functional Characterization of BDNF-Overexpressing Human Retinal Pigment Epithelial Cells Established by Sleeping Beauty Transposon-Mediated Gene Transfer.

More and more patients suffer from multifactorial neurodegenerative diseases, such as age-related macular degeneration (AMD). However, their pathological mechanisms are still poorly understood, which complicates the development of effective therapies. To improve treatment of multifactorial diseases, cell-based gene therapy can be used to increase the expression of therapeutic factors. To date, there is no approved therapy for dry AMD, including late-stage geographic atrophy. We present a treatment option for dry AMD that transfers the brain-derived neurotrophic factor (BDNF) gene into retinal pigment epithelial (RPE) cells by electroporation using the plasmid-based Sleeping Beauty (SB) transposon system. ARPE-19 cells and primary human RPE cells were co-transfected with two plasmids encoding the SB100X transposase and the transposon carrying a BDNF transcription cassette. We demonstrated efficient expression and secretion of BDNF in both RPE cell types, which were further increased in ARPE-19 cell cultures exposed to hydrogen peroxide. BDNF-transfected cells exhibited lower apoptosis rates and stimulated neurite outgrowth in human SH-SY5Y cells. This study is an important step in the development of a cell-based BDNF gene therapy that could be applied as an advanced therapy medicinal product to treat dry AMD or other degenerative retinal diseases.

BioAdhere: tailor-made bioadhesives for epiretinal visual prostheses.

Introduction: Visual prostheses, i.e. epiretinal stimulating arrays, are a promising therapy in treating retinal dystrophies and degenerations. In the wake of a new generation of devices, an innovative method for epiretinal fixation of stimulator arrays is required. We present the development of tailor-made bioadhesive peptides (peptesives) for fixating epiretinal stimulating arrays omitting the use of traumatic retinal tacks. Materials and methods: Binding motifs on the stimulating array (poly[chloro-p-xylylene] (Parylene C)) and in the extracellular matrix of the retinal surface (collagens I and IV, laminin, fibronectin) were identified. The anchor peptides cecropin A (CecA), KH1, KH2 (author's initials) and osteopontin (OPN) were genetically fused to reporter proteins to assess their binding behavior to coated microtiter plates via fluorescence-based assays. Domain Z (DZ) of staphylococcal protein A was used as a separator to generate a bioadhesive peptide. Following ISO 10993 "biological evaluation of medical materials", direct and non-direct cytotoxicity testing (L-929 and R28 retinal progenitor cells) was performed. Lastly, the fixating capabilities of the peptesives were tested in proof-of-principle experiments. Results: The generation of the bioadhesive peptide required evaluation of the N- and C-anchoring of investigated APs. The YmPh-CecA construct showed the highest activity on Parylene C in comparison with the wildtype phytase without the anchor peptide. eGFP-OPN was binding to all four investigated ECM proteins (collagen I, laminin > collagen IV, fibronectin). The strongest binding to collagen I was observed for eGFP-KH1, while the strongest binding to fibronectin was observed for eGFP-KH2. The selectivity of binding was checked by incubating eGFP-CecA and eGFP-OPN on ECM proteins and on Parylene C, respectively. Direct and non-direct cytotoxicity testing of the peptide cecropin-A-DZ-OPN using L-929 and R28 cells showed good biocompatibility properties. Proof-of-concept experiments in post-mortem rabbit eyes suggested an increased adhesion of CecA-DZ-OPN-coated stimulating arrays. Conclusion: This is the first study to prove the applicability and biocompatibility of peptesives for the fixation of macroscopic objects.

Effects of Hydrostatic Pressure on Electrical Retinal Activity in a Multielectrode Array-Based ex vivo Glaucoma Acute Model.

Glaucoma is a heterogeneous eye disease causing atrophy of the optic nerve head (ONH). The optic nerve is formed by the axons of the retinal ganglion cells (RGCs) that transmit visual input to the brain. The progressive RGC loss during glaucoma leads to irreversible vision loss. An elevated intraocular pressure (IOP) is described as main risk factor in glaucoma. In this study, a multielectrode array (MEA)-based ex vivo glaucoma acute model was established and the effects of hydrostatic pressure (10, 30, 60, and 90 mmHg) on the functionality and survival of adult male and female wild-type mouse (C57BL/6) retinae were investigated. Spontaneous activity, response rate to electrical and light stimulation, and bursting behavior of RGCs was analyzed prior, during, and after pressure stress. No pressure related effects on spontaneous firing and on the response rate of the RGCs were observed. Even a high pressure level (90 mmHg for 2 h) did not disturb the RGC functionality. However, the cells' bursting behavior significantly changed under 90 mmHg. The number of spikes in bursts doubled during pressure application and stayed on a high level after pressure stress. Addition of the amino sulfonic acid taurine (1 mM) showed a counteracting effect. OFF ganglion cells did not reveal an increase in bursts under pressure stress. Live/dead staining after pressure application showed no significant changes in RGC survival. The findings of our ex vivo model suggest that RGCs are tolerant toward high, short-time pressure stress.

New epiretinal implant with integrated sensor chips for optical capturing shows a good biocompatibility profile in vitro and in vivo.

BACKGROUND: Retinal degenerative diseases, e.g., retinitis pigmentosa, cause a severe decline of the visual function up to blindness. Treatment still remains difficult; however, implantation of retinal prostheses can help restoring vision. In this study, the biocompatibility and surgical feasibility of a newly developed epiretinal stimulator (OPTO-EPIRET) was investigated. The previously developed implant was extended by an integrated circuit-based optical capturing, which will enable the immediate conversion of the visual field into stimulation patterns to stimulate retinal ganglion cells. RESULTS: The biocompatibility of the OPTO-EPIRET was investigated in vitro using the two different cell lines L-929 and R28. Direct and indirect contact were analyzed in terms of cell proliferation, cell viability, and gene expression. The surgical feasibility was initially tested by implanting the OPTO-EPIRET in cadaveric rabbit eyes. Afterwards, inactive devices were implanted in six rabbits for feasibility and biocompatibility testings in vivo. In follow-up controls (1-12 weeks post-surgery), the eyes were examined using fundoscopy and optical coherence tomography. After finalization, histological examination was performed to analyze the retinal structure. Regarding the in vitro biocompatibility, no significant influence on cell viability was detected (L929: < 1.3% dead cells; R-28: < 0.8% dead cells). The surgery, which comprised phacoemulsification, vitrectomy, and implantation of the OPTO-EPIRET through a 9-10 mm corneal incision, was successfully established. The implant was fixated with a retinal tack. Vitreal hemorrhage or retinal tearing occurred as main adverse effects. Transitional corneal edema caused difficulties in post-surgical imaging. CONCLUSIONS: The OPTO-EPIRET stimulator showed a good biocompatibility profile in vitro. Furthermore, the implantation surgery was shown to be feasible. However, further design optimization steps are necessary to avoid intra- and postoperative complications. Overall, the OPTO-EPIRET will allow for a wide visual field and good visual acuity due to a high density of electrodes in the central retina.

Human platelet lysate as a replacement for fetal bovine serum in human corneal stromal keratocyte and fibroblast culture.

The isolation and propagation of primary human corneal stromal keratocytes (CSK) are crucial for cellular research and corneal tissue engineering. However, this delicate cell type easily transforms into stromal fibroblasts (SF) and scar inducing myofibroblasts (Myo-SF). Current protocols mainly rely on xenogeneic fetal bovine serum (FBS). Human platelet lysate (hPL) could be a viable, potentially autologous, alternative. We found high cell survival with both supplements in CSK and SF. Cell numbers and Ki67+ ratios increased with higher fractions of hPL and FBS in CSK and SF. We detected a loss in CSK marker expression (Col8A2, ALDH3A1 and LUM) with increasing fractions of FBS and hPL in CSK and SF. The expression of the Myo-SF marker SMA increased with higher amounts of FBS but decreased with incremental hPL substitution in both cell types, implying an antifibrotic effect of hPL. Immunohistochemistry confirmed the RT-PCR findings. bFGF and HGF were only found in hPL and could be responsible for suppressing the Myo-SF conversion. Considering all findings, we propose 0.5% hPL as a suitable substitution in CSK culture, as this xeno-free component efficiently preserved CSK characteristics, with non-inferiority in terms of cell viability, cell number and proliferation in comparison to the established 0.5% FBS protocol.

A multielectrode array-based hypoxia model for the analysis of electrical activity in murine retinae.

Several eye diseases, for example, retinal artery occlusion, diabetic retinopathy, and glaucoma, are associated with retinal hypoxia. The lack of oxygen in the retina, especially in retinal ganglion cells (RGCs), causes cell damage up to cell degeneration and leads to blindness. Using multielectrode array recordings, an ex vivo hypoxia acute model was established to analyze the electrical activity of murine wild-type retinae under hypoxic stress conditions. Hypoxia was induced by exchanging the perfusion with oxygen-saturated medium by nitrogen-saturated medium. Hypoxic periods of 0 min (control) up to 60 min were tested on the retinae of adult female C57BL/6J mice. The electrical RGC activity vanished during hypoxia, but conditionally returned after the reestablishment of conventional test conditions. With increasing duration of hypoxia, the returning RGC activity decreased. After a hypoxic period of 30 min and a subsequent recovery time of 30 min, 59.43 ± 11.35% of the initially active channels showed a restored RGC activity. The survival rate of retinal cells after hypoxic stress was analyzed by a live/dead staining assay using two-photon laser scanning microscopy. For detailed information about molecular changes caused by hypoxia, a microarray gene expression analysis was performed. Furthermore, the effect of 2-aminoethanesulfonic acid (taurine, 1 mM) on retinae under hypoxic stress was tested. Treatment with taurine resulted in an increase in the RGC response rate after hypoxia and also increased the survival rate of retinal cells under hypoxic stress, confirming its potential as promising candidate for neuroprotective therapies of eye diseases.

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy.

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients >60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells using the Sleeping Beauty Transposon System.

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 µL of resuspension buffer and combined with 2 µL of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

Blockade of Retinal Oscillations by Benzodiazepines Improves Efficiency of Electrical Stimulation in the Mouse Model of RP, rd10.

Purpose: In RP, photoreceptors degenerate. Retinal prostheses are considered a suitable strategy to restore vision. In animal models of RP, a pathologic rhythmic activity seems to compromise the efficiency of retinal ganglion cell stimulation by an electrical prosthesis. We, therefore, strove to eliminate this pathologic activity. Methods: Electrophysiologic recordings of local field potentials and spike activity of retinal ganglion cells were obtained in vitro from retinae of wild-type and rd10 mice using multielectrode arrays. Retinae were stimulated electrically. Results: The efficiency of electrical stimulation was lower in rd10 retina than in wild-type retina and this was highly correlated with the presence of oscillations in retinal activity. Glycine and GABA, as well as the benzodiazepines diazepam, lorazepam, and flunitrazepam, abolished retinal oscillations and, most important, increased the efficiency of electrical stimulation to values similar to those in wild-type retina. Conclusions: Treatment of patients with these benzodiazepines may offer a way to improve the performance of retinal implants in cases with poor implant proficiency. This study may open the way to a therapy that supports electrical stimulation by prostheses with pharmacologic treatment.

Establishment and Characterization of a Unilateral UV-Induced Photoreceptor Degeneration Model in the C57Bl/6J Mouse.

Purpose: To investigate whether UV irradiation of the mouse eye can induce photoreceptor degeneration, producing a phenotype reminiscent of the rd10 mouse, left eyes of female C57Bl/6J mice were irradiated with a UV LED array (370 nm). A lens was placed between the cornea and LED, allowing illumination of about one-third of the retina. The short-term and long-term effects on the retina were evaluated. Methods: First, a dose escalation study, in which corneal dosages between 2.8 and 9.3 J/cm2 were tested, was performed. A dosage of 7.5 J/cm2 was chosen for the following characterization study. Before and after irradiation slit-lamp examinations, full-field electroretinography, spectral domain optical coherence tomography and macroscopy were performed. After different time spans (5 days to 12 weeks) the animals were sacrificed and the retinae used for immunohistochemistry or multielectrode array testing. Right eyes served as untreated controls. Results: In treated eyes, spectral domain optical coherence tomography revealed a decrease in retinal thickness to 53%. Full-field electroretinography responses decreased significantly from day 5 on in treated eyes. Multielectrode array recordings revealed oscillatory potentials with a mean frequency of 5.2 ± 0.6 Hz in the illuminated area. Structural changes in the retina were observed in immunohistochemical staining. Conclusions: UV irradiation proved to be efficient in inducing photoreceptor degeneration in the mouse retina, while leaving the other retinal layers largely intact. The irradiated area of treated eyes can be identified easily in spectral domain optical coherence tomography and in explanted retinae. Translational Relevance: This study provides information on anatomic and functional changes in UV-treated retina, enabling the use of this model for retinitis pigmentosa-like diseases in animals suited for experimental retinal surgery.

The very large electrode array for retinal stimulation (VLARS)-A concept study.

OBJECTIVE: The restoration of vision in blind patients suffering from degenerative retinal diseases like retinitis pigmentosa may be obtained by local electrical stimulation with retinal implants. In this study, a very large electrode array for retinal stimulation (VLARS) was introduced and tested regarding its safety in implantation and biocompatibility. Further, the array's stimulation capabilities were tested in an acute setting. APPROACH: The polyimide-based implants have a diameter of 12 mm, cover approximately 110 mm2 of the retinal surface and carrying 250 iridium oxide coated gold electrodes. The implantation surgery was established in cadaveric porcine eyes. To analyze biocompatibility, ten rabbits were implanted with the VLARS device, and observed for 12 weeks using slit lamp examination, fundus photography, optical coherence tomography (OCT) as well as ultrasound imaging. After enucleation, histological examinations were performed. In acute stimulation experiments, electrodes recorded cortical field potentials upon retinal stimulation in the visual cortex in rabbits. MAIN RESULTS: Implantation studies in rabbits showed that the implantation surgery is safe but difficult. Retinal detachment induced by retinal tears was observed in five animals in varying severity. In five cases, corneal edema reduced the quality of the follow-up examinations. Findings in OCT-imaging and funduscopy suggested that peripheral fixation was insufficient in various animals. Results of the acute stimulation demonstrated the array's ability to elicit cortical responses. SIGNIFICANCE: Overall, it was possible to implant very large epiretinal arrays. On retinal stimulation with the VLARS responses in the visual cortex were recorded. The VLARS device offers the opportunity to restore a much larger field of visual perception when compared to current available retinal implants.

[Dry AMD - Cellular and Genetic Therapies]. / Trockene AMD ­ zelluläre und gentherapeutische Behandlungsansätze.

The growing incidence of neurodegenerative diseases is based on our increasingly aging society as well as the difficulties in establishing defined therapy regimens. For dry age-related macular degeneration (AMD) and the later stage of geographic atrophy (GA), various treatment options exist that only decelerate the progression of the disease. However, no therapy is currently available that can restore the degenerated retinal pigment epithelium (RPE) and/or photoreceptor cells. Cellular and gene-based approaches aim for the regeneration of the degenerated cells and/or the continuous secretion of cell-protecting agents. The article describes the approaches that are currently being investigated in different clinical trials. These trials are based on the use of cell-based drug delivery systems, stem cells of different origins as well as virus-mediated gene therapy approaches. Finally, we give an overview of ongoing therapeutic developments and present our own research activities, which consist of a combination of pigment epithelial cell transplantation and additive non-viral gene therapy to treat retinal degenerative diseases.

Preclinical Evaluation of a Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System in Choroidal Neovascularization.

Age-related macular degeneration (AMD) is a progressive retinal disorder characterized by imbalanced pro- and antiangiogenic signals. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells were co-transfected with pFAR4-inverted terminal repeats (ITRs) CMV-PEDF-BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats, and transfected primary cells (transfected RPE [tRPE] and transfected IPE [tIPE] cells) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinases 2 and 9 (MMP-2/9), and microglial/macrophage markers were measured. Injection with tRPE/IPE cells significantly reduced the leakage area at 7 and 14 days and the CNV area at 7 days. There was a significant increase in PEDF and the PEDF/VEGF ratio with tRPE cells and a reduction in the MMP-2 activity. Our data demonstrated that ex vivo non-viral gene therapy reduces CNV and could be an effective and safe therapeutic option for angiogenic retinal diseases.

The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells.

The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells.