Regulierbare somatische ex vivo Gentherapie zur Behandlung neurodegenerativer Erkrankungen mittels PEDF

Age-related macular degeneration (AMD) is one of the main causes for the blindness of the over 60-year-olds in Germany. One differentiates between the dry and the wet, neovascular form. The neovascular form is characterized by the formation of choroidal neovascularization due to an increased concentration of the pro-angiogenic Vascular Endothelial Growth Factor (VEGF). This form of AMD is currently being treated by means of anti-VEGF antibodies, which should stop neovascularization and thus the progress of the disease. However, since the various drugs have a short half-life, regular intravitreal injections are necessary to achieve a therapeutic effect. This form of therapy is on the one hand associated with high costs, on the other hand, the regular doctor's visit is not possible for the elderly with limited vision alone. There is also the risk of side effects, such as eye infections, for each injection. The establishment of an additive gene therapy is a promising alternative to current treatments. In this approach, autologous retinal (RPE) or iris (IPE) pigment epithelial cells will be transfected with the anti-angiogenic and neuroprotective Pigment Epithelium-Derived Factor (PEDF), the natural counterpart of the VEGF, and transplanted subretinally, where they secrete recombinant PEDF, thus counteracting the effect of VEGF to suppress neovascularization. For the transfection of the pigment epithelial cells, linearized plasmids and the non-viral Sleeping Beauty (SB) transposon system were used to ensure stable and long-term expression of the introduced PEDF gene, thereby avoiding the possible complications of viral gene transfer, such as immune responses and mutagenesis. This work focused on the analysis of the SB-mediated PEDF gene transfer, which was extended by the function of a doxycycline-dependent regulatory mechanism. The aim was to regulate the expression and thus the amount of secreted PEDF, which would allow a personalized therapy to be ensured. Furthermore, the use of the SB100X transposase as mRNA was investigated to improve system safety. The results of this work showed that the expression and thus the PEDF secretion can be regulated by means of the Tet-On system. By adding or omitting doxycycline in the cell culture medium, the expression of the respective transgene could be switched on and off again. This is an important therapeutic aspect if unforeseen side effects occur due to an increased PEDF secretion rate. In this case it would be conceivable that the expression of PEDF is regulated by administration of doxycycline, for example in the form of eye drops. Furthermore, it was shown that dose-dependent regulation of gene expression is possible: the higher the doxycycline concentration, the higher the expression of the respective transgene. This allows an individual adjustment of the secreted PEDF concentration, which could increase the success potential of a possible therapy. Experiments using SB100X transposase as mRNA showed stable and long-term transfection of the cells. These results bring the field of gene therapy a decisive step forward in the direction of individual gene therapy in which the dose of the secreted therapeutic agent, in this case PEDF, can be individually controlled and the expression of the gene can be switched off again if necessary. Gene transfer using the non-viral Sleeping Beauty Transposon System is a promising method that leads to long-term and stable gene expression. The use of the transposase as mRNA increases the safety of the system

SNF2016 project (31003A_160195/1)

Abstract"Transposon Based Gene Cell Therapy in The Treatment Of Dry Macular Degeneration

Transplantation of PEDF-transfected pigment epithelial cells inhibits corneal neovascularization in a rabbit model

Background: The purpose of this study was to investigate the effect of recombinant pigment epithelium-derived factor (rPEDF), secreted by ARPE-19 cells transfected with the human PEDF gene and transplanted subconjunctivally in normal and in rabbits in which corneal neovascularization was elicited by a chemical burn. Methods: Twenty grey Chinchilla Bastard rabbits were randomly assigned to four groups; neovascularization was induced in groups A, B, and C by alkali cauterization. Seven days later, group A received no cell implantation, non-transfected ARPE-19 cells were implanted subconjunctivally in group B, and PEDF-transfected ARPE-19 cells were implanted subconjunctivally in groups C and D (non-cauterized). In-vivo rPEDF secretion was analyzed by immunoblotting, and ELISA of extracts of conjunctival tissue samples taken at different time points. Digital photographs acquired on days 7, 14, and 21 after cauterization were evaluated for lead vessel length, vascular invasion area, and overall neovascularization rate. Results: At days 14 and 21 after cauterization, significant differences were observed between groups A, B, and C in lead vessel length (day 21: 5.91 ± 0.45, 5.11 ± 1.22, 3.79 ± 0.59mm, repectively), vascular invasion area (day 21: 35.5 ± 8.65, 34.86 ± 4.92, 19.2 ± 5.03mm2 respectively), and rate of corneal neovascularization. Compared to controls, neovascularization was reduced by 37.5% on day 14 and 47% on day 21. Analysis of conjunctival tissue extracts showed that rPEDF was secreted by the transplanted PEDF-transfected cells. Conclusion: Subconjunctivally transplanted, PEDF-transfected ARPE-19 cells secrete rPEDF, which inhibits the corneal neovascularization elicited by alkali cauterization

Transplantation of PEDF-transfected pigment epithelial cells inhibits corneal neovascularization in a rabbit model

The purpose of this study was to investigate the effect of recombinant pigment epithelium-derived factor (rPEDF), secreted by ARPE-19 cells transfected with the human PEDF gene and transplanted subconjunctivally in normal and in rabbits in which corneal neovascularization was elicited by a chemical burn

SNF2019 project (310030_182788/6)

Abstract"Integrative, transposon-based vectors in ocular gene therapy - safety improvement and in-process quality control

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, 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. Here, 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 non-viral 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 months and 6 months, respectively. Conclusions: Transfection using the non-viral SB100X vector system avoids complications associated with viral gene delivery. SB100X-mediated transfer allows for stable PEDF gene integration into the cell's 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