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

Periodic Report Summary 1 - TARGETAMD (Transposon-based, targeted ex vivo gene therapy to treat age-related macular degeneration (AMD))

Project Context and Objectives:
Age Related Macular Degeneration (AMD) is a chronic progressive disease that appears to result from age-associated alterations including impaired phagocytosis by retinal pigment epithelial (RPE) cells, alteration in Bruch’s membrane, inflammations leading to RPE cell and photoreceptor degradation and eventual neural retinal ganglion degradation. AMD presents two distinct forms, a slow progressing non-vascular atrophic form (avascular AMD) and a rapidly progressing, blinding form (neovascular AMD) in which choroidal vessels grow through Bruch’s membrane into the subretinal space.

Given that vascular endothelial growth factor (VEGF) is the major promoter of blood vessel growth, VEGF inhibitors have been approved and used to treat neovascular AMD. The most common and efficacious inhibitors are antibodies against VEGF, specifically ranibizumab (Lucentis®), a Fab fragment of the humanised monoclonal VEGF-A antibody and its parent antibody bevacizumab (Avastin®). While most patients treated with anti-VEGF do not regain significant vision, choroidal neovascularisation (CNV) is arrested. Even so about 30% of patients regain 3 or more lines of visual acuity. However, the effect of anti-VEGFs is limited by their short half-life in vivo, thus necessitating repetitive, often monthly injections to maintain the therapeutic effect. In addition to the logistics of bringing elderly, visually impaired patients to the clinic and the cost of such frequent treatments, the repetitive injections carry substantial risks to the patient, e. g. retinal detachment, cataract formation, endophthalmitis or submacular haemorrhage.

Since in vivo, subretinal space avascularity depends on the anti-angiogenic activity of pigment epithelium-derived factor (PEDF), a natural inhibitor of VEGF, subretinal administration of PEDF should inhibit CNV in vascular AMD. However, its short half-life of a few hours limits its therapeutic use. Since in avascular AMD RPE cells degenerate, an ideal treatment would be to replace the degenerated RPE cells, which constitutively secrete PEDF. RPE and iris pigment epithelial (IPE) cells, as a substitute for RPE cells, have been transplanted to the subretinal space; however, no significant improvements were observed, suggesting that the endogenous PEDF secreted is not sufficient to control the CNV.

The objectives of TargetAMD are to increase the PEDF levels by introducing the human PEDF gene into autologous RPE and IPE cells ex vivo followed by the transplantation to the subretinal space of AMD patients. RPE and IPE cells will be transfected with the PEDF gene using the Sleeping Beauty Transposon System. Transposons are discrete DNA sequences that can move from one location and become integrated at another location of one cell’s genome via a “cut and paste” mechanism. Sleeping Beauty (SB), and notably its hyperactive form SB100X, have been used in animal models to correct genetic defects, e. g. Huntington’s disease and sickle cell anemia. The advantages of SB100X to deliver genetic information are efficiency of transgene delivery into cell genome, ability to incorporate large inserts, and safety of transgene integration since SB transposon does not preferentially integrate into transcriptional sequences.

TargetAMD aims at establishing transposon-based technology as a clinically acceptable gene delivery modality. Specifically: (a) for the transfection of RPE and IPE cells, the SB100X transposase and the PEDF genes will be cloned into Free of Antibiotic Resistance marker (pFAR4) plasmids, since regulatory agencies recommend avoiding the use of antibiotic resistance genes; (b) electroporation conditions for the delivery of the pFAR4 plasmids will be optimised; (c) attempts will be made to target gene integration into predetermined regions of the RPE / IPE cell genomes; (d) rat RPE / IPE cells will be transfected with the PEDF gene and transplanted subretinally in a rat model of CNV; (e) distribution of the injected cells will be analysed in rabbits; (f) plasmids will be produced as GMP-grade for clinical use; (g) dossier of preclinical studies and application to appropriate agencies to obtain approval for clinical trial will be prepared; (h) a phase Ib/IIa clinical trial in which autologous, freshly isolated PEDF-transfected IPE cells will be transplanted subretinally in 10 patients will be carried out; (i) a phase Ib/IIa clinical trial in which autologous, freshly isolated PEDF-transfected RPE cells will be transplanted subretinally in 10 patients will be carried out. During the duration of the grant all partners will be engaged in disseminating information obtained to both the scientific and public community.

Project Results:
Since the start of TargetAMD in November of 2012, the Consortium has been actively pursuing the goals set to accomplish its final objective, a phase Ib/IIa clinical trial in which autologous IPE cells transfected with the PEDF gene will be transplanted subretinally in neovascular AMD patients and a phase Ib/IIa clinical trial in which autologous RPE cells transfected with the PEDF gene will be transplanted subretinally in neovascular AMD patients. To such end, Free of Antibiotic Resistance Marker (pFAR4) plasmids were constructed by inserting genes encoding either PEDF or the SB100X transposase. Afterwards, the plasmids were used to transfect primary IPE and RPE cells and the expression and secretion of PEDF were analysed.

Even though SB100X has been shown to be safe in a number of animal studies, additional safety features were tested, specifically (1) the use of SB100X mRNA as transient recombinase source; (2) the addition of insulator sequence(s) to prevent possible transactivation of endogenous genes at the integration site; (3) the addition of sequences to target transposon integration into specific “safe harbours” of the genome.

The results of these studies have shown that insulator sequences depressed PEDF expression and thus were not considered to be added to the final plasmid construct. Cells transfected with the SB100X transposase encoded by either DNA or mRNA expressed PEDF efficiently. However, large intra-individual variations in the expression of PEDF were detected with mRNA (and not DNA) as source of the transposase coding sequence. Based on the results of these experiments, the final constructs, pFAR4-CMV-PEDF and pFAR4-CMV-SB100X DNA, were chosen for further preclinical studies and for use in the clinical trials. Since the final constructs have been chosen, a manufacturer for the production of GMP-grade plasmids has been identified.

Since the final objective of TargetAMD is the isolation of IPE or RPE cells from a patient followed by transfection and transplantation of the transfected cells subretinally during a 60 minutes surgical session, it is necessary that transfection is accomplished in only the few cells (5,000 to 10,000) that can be isolated from a biopsy. Using the final constructs, protocols were developed for the transfection of 5x103 and 10x103 cells. For these experiments both primary bovine IPE (n=14) and human RPE (n=14) cells and freshly isolated bovine IPE (n=17) cells were used. In all these experiments transfection efficiency and PEDF secretion were high with PEDF copies integrated of 9.2 ± 12.7 for 1x104 cells and 3.3 ± 2.3 for 5x103cells.

Since no animal-derived products are allowed to be part of product to be used in the clinical trials, buffers have been developed that will support transfection efficiency and cell integrity. Twenty-one buffers were formulated and analysed, of which 4 supported transfection efficiency and cell integrity, when cells were transfected with pFAR4-ITRs-CAGGS-Venus. Notably, one buffer showed a higher level of transgene expression than culture media containing fetal bovine serum.

As for documentation efforts that are necessary for approval of the clinical trials, the Investigational Brochure design, the Clinical Trial Protocol, as well as the Case Report Form have been completed. The Patient Information and Consent Form have been prepared and the Investigational Medicinal Product Dossier is available including all data obtained to this point. In addition, the Coordinator has met with the Swiss Regulatory Authorities (Swissmedic) to discuss the clinical trial.

The TargetAMD website ( and has been completed and has been published. To increase the recognition value for visitors of the website the TargetAMD logo is a pivotal instrument, augmenting corporate identity and visual appearance of the project.

Figure 1 The TargetAMD logo

Potential Impact:
Based on the results to date, the final objective of the proposal, namely “the successful completion of a phase Ib/IIa clinical trial for the treatment of AMD using transposon-based gene therapy technology” will become a reality. The major challenge to the successful completion of the project was the ability to successfully and efficiently transfect very few cells (5,000 to 10,000) that can be obtained from an iris or peripheral retina biopsy. TargetAMD has achieved this objective, since as few as 5,000 RPE cells from human donors have been successfully transfected using the Sleeping Beauty transposon system, leading to efficient integration of the transgene into the host cell’s genome and efficient and stable PEDF expression and secretion. In addition, the use of PEDF and SB100X transposase-encoding plasmids that are Free of Antibiotic Resistance Markers (pFAR4) constitutes an important step toward translating laboratory research to the clinic.

Two phase Ib/IIa clinical trials are proposed in TargetAMD; in one clinical trial, RPE cells are isolated from the peripheral retina of neovascular AMD patients. The cells will immediately be transfected with the PEDF gene and will be transplanted in the subretinal space of the patient during a 60-minutes surgical session. In the second clinical trial IPE cells will be isolated from an iris biopsy, transfected with the PEDF gene and transplanted in the subretinal space of the patient during a 60 minute surgical session. The trial with IPE cells is essential since the isolation of RPE cells from the peripheral retina is a traumatic intervention and the cells may carry the defect that allowed the development of CNV. IPE cells, which have the same embryonic origin as RPE cells, are simple to isolate and have been shown to have the same phenotypic characteristics as RPE cells. Thus, IPE cells could be appropriate substitutes for RPE cells.

Current treatment for neovascular AMD requires frequent, often monthly intravitreal injections of anti-VEGFs. Considering that the cost of one injection of Lucentis®, the approved anti-VEGF for intraocular use, is $1,000.00 per injection, the cost per patient per year is $12,000.00. Such costs are a burden for industrialised countries and unaffordable for poor countries. In addition, the repetitive intraocular injections carry a substantial risk of side effects. TargetAMD would solve these difficulties since one single subretinal transplantation of PEDF-transfected cells would prevent CNV for the life of the patient, since the transplanted cells would express PEDF permanently. The protocols being developed by TargetAMD are such that any ophthalmic surgeon should be able to carry out the procedure, thus allowing treatment to be widely available.

Currently, about 30% of patients regain visual acuity from the treatment with anti-VEGFs, suggesting that in the other 70% the retinal pigment epithelial cells are irreversibly damaged or degenerated. Since the TargetAMD protocol comprises the transplantation of RPE or IPE cells, a larger number of patients should benefit, specifically those patients with irreversibly damaged or degenerated RPE cells. The only patients that would not benefit from the treatment developed by TargetAMD would be patients in whom both RPE and photoreceptors are degenerated.

Beyond ophthalmology, the work we have done is important to bring visibility to the value of transposons for clinical use. Since transposons can safely integrate the transgene into the host cell’s genome, their wider use would prevent the side effects that accompany virally mediated gene transfer. Finally the ability to deliver a transgene efficiently into as few as 5,000 freshly isolated cells, should advocate for the use of ex vivo genetic cell modifications for use in the treatment of other diseases.

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