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Transposon-based, targeted ex vivo gene therapy to treat age-related macular degeneration (AMD)

Periodic Report Summary 3 - TARGETAMD (Transposon-based, targeted ex vivo gene therapy to treat age-related macular degeneration (AMD))

Project Context and Objectives:
Age-related macular degeneration (AMD) presents two forms: an avascular form in which retinal pigment epithelial (RPE) cells degenerate over decades and blindness occurs when the RPE degenerates in the macula. The second form, neovascular AMD (nvAMD), manifests and progresses rapidly, such that blindness can ensue within a few months if left untreated or unrecognized by the patient. nvAMD presents a different pathology than the avascular form; in nvAMD choroidal blood vessels invade Bruch’s membrane, enter the subretinal space, leak fluid, disrupt the retinal architecture and impair the function of the RPE cells, which degenerate. Choroidal blood vessel growth (CNV) appears to be stimulated by overexpression of vascular endothelial growth factor (VEGF), while in the healthy retina, VEGF activity is balanced by the activity of the pigment epithelium-derived factor (PEDF). In nvAMD an increase in the level of PEDF should rebalance VEGF activity and prevent CNV. However, since PEDF has a half-life of only six hours, administration of PEDF is not a practical option. The standard treatment is focused on controlling CNV and consists of frequent, lifelong intravitreal injections of anti-VEGF biopharmaceuticals. The treatment stabilises or retards the progress of the disease in 90% of patients; however, only 30% experience significant improvement of visual acuity. The ideal treatment would be the transplantation of cells that would secrete high levels of PEDF to counteract the action of VEGF. However, transplantation of autologous RPE or iris pigment epithelial (IPE) cells did not show a significant benefit, suggesting that the transplanted cells did not provide sufficient PEDF to suppress CNV.
The purpose of TargetAMD is to transfect with the PEDF gene IPE cells and transplant the cells to the subretinal space of nVAMD patients where they will overexpress and secrete PEDF. In a phase Ib/IIa clinical trial, within a single surgical session lasting about 60 minutes, IPE cells will be isolated from a patient’s iris biopsy, transfected with the PEDF gene and transplanted into the subretinal space of the same patient. The PEDF gene is delivered to the cells using the non-viral integrative Sleeping Beauty transposon system (SB100X). The PEDF gene and the SB100X transposase will be encoded in free of antibiotic resistance marker (pFAR4) miniplasmids and delivered into the cell by electroporation. The combination of the SB100X and the pFAR4 miniplasmid to deliver PEDF to IPE cells, or any gene to any cell, has a number of advantages, including higher transfection efficiency, higher transgene expression, random integration of the transgene and therefore increased safety. The safety of the SB100X transposon has been demonstrated in a number of preclinical disease models such as Huntington’s disease and sickle cell anaemia. The TargetAMD consortium will accomplish the first European human clinical trial using the SB100X combined with the pFAR4plasmids. The project objectives can be summarised briefly as:
(a) Assessment of the safety of the SB100X-pFAR4 vector system to deliver the PEDF gene to cells; (b) optimisation of electroporation necessary for PEDF gene delivery to cells; (c) determination of efficacy of PEDF-transfected rat RPE and IPE cells transplanted in a rat model of CNV; (d) exclusion of systemic biodistribution of transfected cells or naked DNA in rabbit model; (e) GMP manufacture of pFAR4 plasmids; (f) preparation of application dossier containing pre-clinical data to regulatory authorities for approval of the gene therapy clinical trial; (g) completion of a phase Ib/IIa clinical trial in which IPE cells from 10 nvAMD patients will be isolated, transfected with the PEDF gene and transplanted subretinally to the same patients during a single surgical session; (h) dissemination of results to the scientific community and general public; (i) exploitation of newly developed devices and reagents.
Project Results:
The objectives as stated in the grant application have been mostly completed and the clinical trial (CT) will proceed as planned.
Even though the safety of the SB100X transposon system is inherently superior to viral systems, the TargetAMD consortium tested additional safety strategies. Based on the results, the consortium defined the final constructs to be used for the CT, would be pFAR4-ITRs CMV PEDF BGH and pFAR4-ITRs CMV SB100X. Preclinical and clinical batches of the pFAR4 plasmids have been manufactured by partner AmBTU.
Efficient transfection of low numbers (5,000-10,000) of human RPE/IPE cells immediately after isolation has been accomplished successfully. Reliability of transfection of human PE cells with the PEDF gene (Gene Therapy Medicinal Product, GTMP) has been verified in transfections of PE cells isolated from ~300 human donor eyes. Efficacy of transfection and quality of the GTMP were analysed by determination of PEDF expression, copies of PEDF gene integrated, PEDF protein secretion, and cell viability. The GMP-grade GTMP production has been established; SOP preparation and personnel training will be completed by a validation run with human donor eyes during the next 4 weeks. A GMP-grade electroporation buffer was developed and produced for the specific use in TargetAMD. Similarly, the electroporation device, the Cliniporator®, was adapted to the transfection of low cell numbers and microcuvettes were developed that support efficient electroporation. The modified Cliniporator® has obtained the necessary approvals for use in proposed CT and has been installed and validated in the clean room. Protection of Intellectual Property (IP) rights are being explored. The IP transfer offices of the partners are currently negotiating interests for the product developed, i.e. plasmids, buffer, and microcuvette.
Efficacy of transplanting PEDF-transfected IPE or RPE cells has been demonstrated by the significant reduction of laser-induced CNV in a rat model. Safety of the proposed treatment has been shown by the lack of tumorigenic potential of PEDF-transfected cells in a soft agar assay and in a biodistribution study in rabbits by the absence of local or systemic toxicity and of transfected cells or plasmid DNA/RNA in any organ except for the retina, the transplantation site. The lack of tumorigenicity is supported by analysis of the close-to-random integration profile of the PEDF gene into the PE genome, without preference for transcription start sites or oncogenes.
Appropriate SOPs have been developed to define the whole process from iridectomy to transplantation to achieve quality and uniformity of performance, thus reducing possible failure to comply with GMP regulations. Required documents for the CT have been prepared, i.e. a Patient Information brochure and Informed Consent form, a Case Report Form, the Investigator’s Brochure and the Investigational Medicinal Product Dossier. The preparation of the documents for the CT was facilitated by a close collaboration with the Swiss Regulatory Authority, Swissmedic. In addition to phone calls and email communications, the consortium met with Swissmedic twice for Scientific Advice and once for a Presubmission Meeting to insure that the application dossier for the clinical trial met all requirements. To be able to start the clinical trial as soon as approval is obtained, Partner UNIGE has screened their patient database for eligible patients with the goal of having approval by Swissmedic and begin patient recruitment by September 2017.
The consortium is actively promoting the visibility of the project by regular updates of the TargetAMD website ( the TargetAMD logo, symposiums, presentations at international conferences, information events for medicinal personnel, patients and relatives, and articles. The two first peer-reviewed paper were published in 2017 and four more are in preparation.
Potential Impact:
The results shown in the present report indicate that the final aim of TargetAMD “the successful completion of a phase Ib/IIa clinical trial for the treatment of AMD using transposon-based gene therapy technology” will be completed successfully. The major challenges to be overcome for the successful completion of the clinical trial were the efficient transfection of the low cell numbers of freshly isolated human RPE or IPE cells from retina or iris biopsies and the manufacture of the GTMP under GMP guidelines to be immediately transplanted to the subretinal space, which required a GMP-compliant facility adjacent or close to the operating room. Both of these challenges have been successfully overcome. The consortium has developed microcuvettes, a buffer and electroporation procedure that reproducibly result in efficient transfection. A GMP-compliant facility close to the operating room has been made available and the logistics of transporting the biopsy to the facility and return the GTMP to the operating room have been established and codified in SOPs. The three new products developed and associated IP, i.e. the SB100X-pFAR4 plasmids, the electroporation buffer, and the microcuvette, are in discussion as to be best exploited.
The first-in-man phase Ib/IIb clinical trial using the SB100X-pFAR4 system for gene delivery will begin in September 2017 and should be completed by January of 2017. Based on results from animal models of CNV, the consortium expects that the clinical trial will show that the proposed treatment of nvAMD, i.e. the transplantation of PEDF-transfected IPE cells, will be safe. The innovation of the treatment lies not only in the methods developed, e.g. transfection of few cells with the non-viral SB100X-pFAR plasmids and the transplantation of the cells without culturing, but also in the fact that the treatment and surgical procedure is personalised in so far as the cells will be isolated from a patient iris biopsy, immediately genetically modified and transplanted into the same patient during a single surgical procedure lasting 60 minutes. Such innovation results from having the foresight to collaborate with scientists from different fields of science.
For nvAMD patients the novel treatment will replace the current costly therapy that requires often monthly, life-long intraocular injections of anti-VEGF biopharmaceuticals; the treatment developed under the aigis of TargetAMD will be a one-in-a-lifetime treatment. The socioeconomic burden will decrease significantly, since the minimum costs of current treatment averages over 30,000 Euro per year and since the TargetAMD treatment does not require special methods, it can become a routine procedure that can be performed by most ophthalmology hospital clinics.
Beyond the benefit to nvAMD patients and to health care, the ability to transfect few somatic cells, the safety and efficiency of the transfection using non-viral means and personalisation of treatment developed by the TargetAMD consortium will open new possibilities for the treatment of other intractable diseases.
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