Abstract
Contract No.: 305134; EC contribution: € 5,976,298; Total costs: € 7,769,833.46; Starting date: 01/11/2012; Duration: 48 months
Background and Objectives
Age-related macular degeneration (AMD) is a chronic progressive disease that appears to result from age-associated alterations that include impaired phagocytosis by retinal pigment epithelial (RPE) cells; alteration in Bruch's membrane, RPE cell, and photoreceptor degeneration; and eventually neural retinal ganglion degradation. AMD presents two distinct forms, a slowly progressing nonvascular atrophic form (dry or avascular AMD) and a rapidly progressing blinding form (neovascular AMD), in which choroidal vessels grow through Bruch's membrane into the subretinal space.
No treatment is available for avascular AMD. However, VEGF inhibitors have been approved and used to treat neovascular AMD, since VEGF is the major promoter of blood vessel growth. Treatment with VEGF inhibitors is effective in about 30% of patients who regain 3 or more lines of visual acuity. However, the effect of VEGF inhibitors is limited in time, thus necessitating repetitive, often monthly injections to maintain the therapeutic effect.
Since in vivo subretinal space avascularity depends on the anti-angiogenic activity of PEDF, the natural antagonist of VEGF, administration of PEDF to the subretinal space should inhibit choroidal neovascularization (CNV) in neovascular AMD. However, its short half-life of a few hours limits its therapeutic use. Since AMD is accompanied by RPE cell degeneration, an ideal treatment would be the replacement of the degenerated RPE cells with cells that secrete PEDF constitutively. RPE and iris pigment epithelial (IPE) cells, as a substitute for RPE cells, have been transplanted to the subretinal space.1,2 However, no significant improvements were observed, suggesting that the endogenously expressed PEDF secreted by the transplanted cells is not sufficient to control CNV.
Approach and Methodology
The overall objectives of TargetAMD are to genetically modify RPE and IPE cells to produce elevated levels of PEDF.3,4 The genetically modified cells will be transplanted to the subretinal space of neovascular AMD patients to inhibit CNV by augmented PEDF secretion. Specifically, RPE and IPE cells will be transfected with the PEDF gene using the hyperactive Sleeping Beauty (SB100X) transposon system. 5 Transposons are discrete sequences of DNA that can move from one location to another in the genome via a “cut and paste” mechanism. Similar to retroviral vectors, the SB100X system will deliver and integrate genetic material into a target cell genome, resulting in robust and stable expression of the transfected gene. The SB100X-mediated gene delivery shows efficient transgene delivery, integration into the cell genome, lower immunogenicity, ability to incorporate large inserts, reduced genomic instability of the host genome, and safety of transgene integration, since Sleeping Beauty integration is random at the genomic level 6 and does not integrate into transcription start sites.
TargetAMD aims at establishing transposon-based gene therapy for the treatment of neovascular AMD. Specifically, our aims are (1) development of protocols and devices for efficient transfection by electric pulses delivery of the low number of cells freshly isolated from an iris biopsy and a peripheral retina biopsy; (2) testing the use of SB100X transposase as mRNA, 7 PEDF transposon vectors with insulator sequences, 8 and plasmids free of antibiotic resistance markers (pFAR4) 9 ; (3) transplantation of rat RPE and IPE cells, transfected with the PEDF transgene, subretinally in a rat model of CNV; (4) safety analyses including distribution of the transplanted cells in rabbits and distribution of transposon integration sites in the genome; (5) production of GMP-grade plasmids for clinical use; (6) preparation of dossier of preclinical studies and application to appropriate agencies to obtain approval for clinical trials; (7) development of protocols and performing a phase Ib/IIa clinical trial in which autologous, freshly isolated IPE cells will be transfected with the PEDF gene and transplanted subretinally in 10 neovascular AMD patients during a single surgical session lasting approximately 60 min; (8) performing a phase Ib/IIa clinical trial in which autologous, freshly isolated PEDF-transfected RPE cells will be transplanted subretinally in 10 neovascular AMD patients.
Main Findings
Optimization of biosafety profile
1. The use of Sleeping Beauty transposase mRNA was repeatedly tested in ARPE-19 and primary human RPE cells from distinct donor eyes and compared with the use of SB100X transposase DNA. Both strategies were successful and effective with transfection efficiency of around 100% in ARPE-19 cells. However, SB100X transposase DNA exhibited better reproducibility; SB100X transposase mRNA showed higher intraindividual variations.
2. PEDF transposon vectors with incorporated insulator sequences showed protection from transactivation; however, in primary human RPE cells transfected with the insulator-carrying transposon plasmids, expression of the PEDF transgene was clearly decreased.
3. pFAR4 transposon derivatives showed increased transfection efficiencies compared with the respective pT2 transposon vectors.10–12 In transfected primary rat IPE and RPE, 13 bovine IPE, and human RPE cells, secretion of PEDF was clearly evident and persisted at constant levels without transgene silencing. Based on these findings, the decision was taken to use both the transposase expression plasmid and the PEDF encoding transposon plasmid in the pFAR4 back bone for the clinical trials.
Transfection of low numbers of primary human RPE cells
Since the final objective of TargetAMD is the isolation of IPE or RPE cells from a patient followed by transfection and subretinal transplantation of the transfected cells during a 60 min surgical session, it is necessary that transfection is accomplished in only the few cells (5×103 to 1×104) that can be isolated from a biopsy. Using the final SB100X and PEDF constructs at a specific ratio, validated for best transfection efficiency, protocols were developed for the transfection of 5×103 and 1×104 cells. For these experiments, cultured primary human RPE cells (from 20 different donors) were used. In these experiments, PEDF secretion was increased at least 20-fold, consistent with the increased PEDF gene expression of at least 65-fold as well as with a low relative number of copies of the PEDF gene integrated for 1×104 and for 5×103cells. Transfection was mediated by electroporation using the Cliniporator device, which has been modified for TargetAMD purposes. 14
Transfection of low numbers of freshly isolated bovine IPE cells
Around 105–104 cells could be isolated from small biopsies of iris. These cells were immediately transfected with the SB100X transposase and the PEDF transposon plasmids. Efficient transfection was verified for the small biopsy-derived cells by image-based cytometry. Using ELISA it was determined that the transfected cells secreted up to 25-fold more PEDF than nontransfected cells. Western blot analyses showed that the level of recombinant PEDF secreted remained stable for the 191 days that the cells have been followed by now. 15
Expected Outcome
Based on the results to date, the final objective of the project, namely, “the successful completion of two phase Ib/IIa clinical trials for the treatment of AMD using transposon-based gene therapy technology” will become a reality. The major challenge to the completion of the project was the ability to successfully and efficiently transfect the very few cells (5×103 to 1×104) that can be obtained from an iris or peripheral retina biopsy. TargetAMD has achieved this objective, since low numbers of IPE cells freshly isolated from iris biopsies have been successfully transfected using the Sleeping Beauty transposon system in conjunction with free of antibiotic resistance marker (pFAR4) miniplasmids. We have consulted with Swissmedic as to the requirements for conducting the clinical trials and are preparing the appropriate protocols and instituting the necessary procedures for the clinical trials.
