Sandra Johnen

The in vitro and in vivo behaviour of retinal pigment epithelial cells cultured on ultrathin collagen membranes.

The transplantation of pigment epithelial cells as a therapeutic modality for retinal degeneration requires that the transplanted cells form a monolayer in the subretinal space that will establish communication with photoreceptors. Since previous studies have shown that transplanted cells in suspension do not form a monolayer, it will be necessary to transplant preformed pigment epithelial cell monolayers at the location of the exposed photoreceptors. To establish cell monolayers, retinal pigment epithelial (RPE) cells were cultured on ultrathin collagen membranes. Cells were examined for morphology, for characteristics of differentiation and viability. Membrane degradation and long-term biocompatibility in vivo were assessed following subconjunctival and subretinal implantation in rabbits. These studies have shown that RPE cells adhere, proliferate, form monolayers, and acquire differentiated properties on a collagen membrane that has features similar to Bruchs membrane. Membranes transplanted subconjunctivally and subretinally exhibit excellent biocompatibility without any evidence of inflammation or rejection. RPE cells cultured on collagen membranes acquire differentiated characteristics similar to those of RPE cells in vivo and form complete monolayers that are amenable to be transplanted to the subretinal space. The collagen membranes are non-toxic and do not elicit any rejection or inflammatory response when implanted subconjunctivally or subretinally in rabbits.

Effects of Bevacizumab (Avastin) on Retinal Cells in Organotypic Culture

PURPOSE Repetitive intravitreal injections of bevacizumab are a successful treatment option for exudative age-related macular degeneration (AMD). The aim of this study was to evaluate the toxicity of bevacizumab in the adult mammalian neurosensory retina in culture. METHODS Adult porcine neurosensory retinas were cultured adjoined to the retinal pigment epithelium-choroid layer (retina-RPE-choroid complex) in static culture for 3 days, whereas neural retinas alone were cultured in a perfusion chamber for 3 days. Bevacizumab was added to the culture and perfusion medium at three concentrations (0.25 mg/mL [n = 6], 0.5 mg/mL [n = 6], and 1.25 mg/mL [n = 6]). Retina-RPE-choroid complex and neural retinas alone cultured without bevacizumab were used as controls. After 3 days in culture, the neural retinas alone and the retina-RPE-choroid complexes were analyzed histologically and immunohistochemically for the expression of glial fibrillary acidic protein (GFAP), vimentin, glutamine synthetase, rhodopsin, smooth muscle actin (SMA), and apoptosis. RESULTS No toxic effects on ganglion or photoreceptor cells were observed at any concentration of bevacizumab. The expression of GFAP and vimentin was slightly increased in Müller cells, whereas glutamine synthetase and rhodopsin were unaffected by bevacizumab. However, significantly enhanced SMA expression in retina blood vessels was observed in retinas cultured in the presence of bevacizumab. CONCLUSIONS Bevacizumab was well tolerated by ganglion and photoreceptor cells even at concentrations fivefold higher than those used clinically. The increased expression of SMA is an indication of the loss of functional VEGF modulating smooth muscle cells in mature vessels.

Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells.

PURPOSE Subretinal transplantation of retinal (RPE) or iris (IPE) pigment epithelial cells has been advocated as a treatment for retinal degeneration. However, to our knowledge, in patients with age-related macular degeneration no significant beneficial effects on vision have been shown. Since the transplanted cells did not appear to maintain a healthy avascular and neuroprotective environment, we postulate that it will be necessary to transplant cells that express elevated levels of anti-angiogenic and neuroprotective activities. In our study, we provide a protocol for the efficient stable gene transfer and sustained gene expression of pigment epithelium-derived factor (PEDF), a potent anti-angiogenic and neuroprotective factor, using the nonviral Sleeping Beauty transposon system (SB100X). METHODS Pigment epithelial cells were electroporated with a Venus reporter or a PEDF encoding plasmid, controlled by either CMV or CAGGS promoters. Transfection efficiencies and protein expression stability were evaluated by flow cytometry and immunoblotting. Gene expression profiles were analyzed by RT-PCR. RESULTS SB100X-based delivery resulted in efficiencies of 100% with the Venus gene and 30% with the PEDF gene. Cell sorting enabled establishment of pure PEDF-transfected ARPE-19 populations. Transfected RPE and IPE cells have been shown to maintain stable PEDF secretion for more than 16 and 6 months, respectively. CONCLUSIONS Transfection using the nonviral SB100X vector system avoids complications associated with viral gene delivery. SB100X-mediated transfer allows for stable PEDF gene integration into the cells genome, ensuring continuous expression and secretion of PEDF. Stable expression of the therapeutic gene is critical for the development of cell-based gene addition therapies for retinal degenerative diseases.

Current Treatment Limitations in Age-Related Macular Degeneration and Future Approaches Based on Cell Therapy and Tissue Engineering

Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. With an ageing population, it is anticipated that the number of AMD cases will increase dramatically, making a solution to this debilitating disease an urgent requirement for the socioeconomic future of the European Union and worldwide. The present paper reviews the limitations of the current therapies as well as the socioeconomic impact of the AMD. There is currently no cure available for AMD, and even palliative treatments are rare. Treatment options show several side effects, are of high cost, and only treat the consequence, not the cause of the pathology. For that reason, many options involving cell therapy mainly based on retinal and iris pigment epithelium cells as well as stem cells are being tested. Moreover, tissue engineering strategies to design and manufacture scaffolds to mimic Bruchs membrane are very diverse and under investigation. Both alternative therapies are aimed to prevent and/or cure AMD and are reviewed herein.

Selective photoreceptor degeneration by intravitreal injection of N-methyl-N-nitrosourea.

PURPOSE To characterize the effects of intravitreal injections of N-methyl-N-nitrosourea (MNU) in comparison to its systemic application as a measure of inducing unilateral photoreceptor degeneration. METHODS Eight-week-old male C57BL/6J mice received either intraperitoneal injections (three animals) or intravitreal injections (24 animals) of MNU in different concentrations and were observed over a period of 2 weeks using full-field electroretinography (ERG), spectral-domain optical coherence tomography (SD-OCT), and immunohistochemistry. RESULTS The intraperitoneal application of MNU showed moderate systemic toxic effects, indicated by a loss of body weight of 12% within the first 2 days. In both eyes the ERG became extinguished, and SD-OCT scans showed a thinning of the retina, predominantly in the outer nuclear layer (ONL). Immunohistochemistry demonstrated the selective loss of rods and cones. Mice that received intravitreal MNU injections displayed nearly no weight loss, and no degeneration of their general welfare was observed. After 2 weeks, ERG, SD-OCT, and immunohistochemistry revealed changes identical to those seen after systemic application in the injected eye, but not in the control eye. CONCLUSIONS The intraperitoneal application of MNU led to moderate systemic side effects in mice and to selective photoreceptor degeneration. Intravitreal injections of MNU also induced photoreceptor degeneration; however, no systemic side effects were observed. This tool may be helpful in larger species, where genetic models of receptor degenerations are not applicable but where the size of the eye is more suitable to study surgical or other approaches to treat blindness caused by receptor degeneration.

High efficiency non-viral transfection of retinal and iris pigment epithelial cells with pigment epithelium-derived factor.

Transplantation of pigment epithelial cells in patients with age-related macular degeneration and Parkinsons disease has the potential to improve functional rehabilitation. Genetic modification of cells before transplantation may allow the delivery of neuroprotective factors to achieve functional improvement. As transplantation of cells modified using viral vectors is complicated by the possible dissemination of viral particles and severe immune reactions, we have explored non-viral methods to insert genetic material in pigment epithelial cells. Using lipofection or nucleofection ARPE-19 cells, freshly isolated and primary retinal and iris pigment epithelial (IPE) cells were transfected with plasmids encoding green fluorescent protein (GFP) and with three plasmids encoding recombinant pigment epithelium-derived factor (PEDF) and GFP. Transfection efficiency was evaluated by fluorescence microscopy and stability of protein expression by immunoblotting. Pigment epithelial cells were successfully transfected with plasmid encoding GFP. Expression of GFP in ARPE-19 was transient, but was observed for up to 1 year in IPE cells. Analysis of pigment epithelial cells transfected with PEDF plasmids revealed that PEDF fusion proteins were successfully expressed and functionally active. In conclusion, efficient transfer of genetic information in pigment epithelial cells can be achieved using non-viral transfection protocols.

Going non-viral: the Sleeping Beauty transposon system breaks on through to the clinical side

Abstract Molecular medicine has entered a high-tech age that provides curative treatments of complex genetic diseases through genetically engineered cellular medicinal products. Their clinical implementation requires the ability to stably integrate genetic information through gene transfer vectors in a safe, effective and economically viable manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are prevalent in ongoing pre-clinical and translational research. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use. Here we review several recent refinements of the system, including the development of optimized transposons and hyperactive SB variants, the vectorization of transposase and transposon as mRNA and DNA minicircles (MCs) to enhance performance and facilitate vector production, as well as a detailed understanding of SB’s genomic integration and biosafety features. This review also provides a perspective on the regulatory framework for clinical trials of gene delivery with SB, and illustrates the path to successful clinical implementation by using, as examples, gene therapy for age-related macular degeneration (AMD) and the engineering of chimeric antigen receptor (CAR)-modified T cells in cancer immunotherapy.

Correlations between ERG, OCT, and Anatomical Findings in the rd10 Mouse

Background. To evaluate the correlation between ERG, OCT, and microscopic findings in the rd10 mouse. Methods. C57BL/6J wild type mice and rd10 mice were compared at the age of 2, 3, 5, 7, 9, 12, 24, and 48 weeks (each age group n = 3) using full-field electroretinography (ERG), spectral domain Optical Coherence Tomography (sd-OCT), fluorescein angiography (FA), Hematoxylin & Eosin histology (HE), and immunohistology (IH). Results. While in wild type mice, the amplitude of a- and b-wave increased with light intensity and with the age of the animals, the rd10 mice showed extinction of the ERG beginning with the age of 5 weeks. In OCT recordings, the thickness of the retina decreased up to 9 weeks of age, mainly based on the degradation of the outer nuclear layer (ONL). Afterwards, the ONL was no longer visible in the OCT. HE staining and immunohistological findings confirmed the in vivo data. Conclusion. ERG and OCT are useful methods to evaluate the retinal function and structure in vivo. The retinal changes seen in the OCT closely match those observed in histological staining.

Antiangiogenic and Neurogenic Activities of Sleeping Beauty-Mediated PEDF-Transfected RPE Cells In Vitro and In Vivo

Pigment epithelium-derived factor (PEDF) is a potent multifunctional protein that inhibits angiogenesis and has neurogenic and neuroprotective properties. Since the wet form of age-related macular degeneration is characterized by choroidal neovascularization (CNV), PEDF would be an ideal candidate to inhibit CNV and support retinal pigment epithelial (RPE) cells. However, its short half-life has precluded its clinical use. To deliver PEDF to the subretinal space, we transfected RPE cells with the PEDF gene using the Sleeping Beauty transposon system. Transfected cells expressed and secreted biologically active recombinant PEDF (rPEDF). In cultures of human umbilical vein endothelial cells, rPEDF reduced VEGF-induced cumulative sprouting by ≥47%, decreased migration by 77%, and increased rate of apoptosis at least 3.4 times. rPEDF induced neurite outgrowth in neuroblastoma cells and protected ganglion and photoreceptor cells in organotypic retinal cultures. In a rat model of CNV, subretinal transplantation of PEDF-transfected cells led to a reduction of the CNV area by 48% 14 days after transplantation and decreased clinical significant lesions by 55% and 40% after 7 and 14 days, respectively. We showed that transplantation of pigment epithelial cells overexpressing PEDF can restore a permissive subretinal environment for RPE and photoreceptor maintenance, while inhibiting choroidal blood vessel growth.

Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal blood vessels growing into the subretinal space, leading to retinal pigment epithelial (RPE) cell degeneration and vision loss. Vessel growth results from an imbalance of pro-angiogenic (e.g., vascular endothelial growth factor [VEGF]) and anti-angiogenic factors (e.g., pigment epithelium-derived factor [PEDF]). Current treatment using intravitreal injections of anti-VEGF antibodies improves vision in about 30% of patients but may be accompanied by side effects and non-compliance. To avoid the difficulties posed by frequent intravitreal injections, we have proposed the transplantation of pigment epithelial cells modified to overexpress human PEDF. Stable transgene integration and expression is ensured by the hyperactive Sleeping Beauty transposon system delivered by pFAR4 miniplasmids, which have a backbone free of antibiotic resistance markers. We demonstrated efficient expression of the PEDF gene and an optimized PEDF cDNA sequence in as few as 5 × 103 primary cells. At 3 weeks post-transfection, PEDF secretion was significantly elevated and long-term follow-up indicated a more stable secretion by cells transfected with the optimized PEDF transgene. Analysis of transgene insertion sites in human RPE cells showed an almost random genomic distribution. The results represent an important contribution toward a clinical trial aiming at a non-viral gene therapy of nvAMD.