Deliverables Collection of biological material from OPMD patients Over the course of the project we have identified a total of 14 families (31 patients) with OPMD in the Netherlands. OPMD is a very rare disorder affecting approximately 1:200.000 individuals in Western Europe. With 16 million inhabitants, we estimate a maximum of 80 patients in The Netherlands. A new process to form filaments of expanded PABPN1 in vitro An in vitro assay was developed to induce formation of fibrillar aggregates with the mutant protein. The filaments had the anticipated characteristics of amyloid structures and could be induced by seeding. Although conditions to induce filament formation could not be established for the full length PABPN1 protein, an N-terminal PABPN1 fragment was produced with the wild-type sequence, the +7 ala sequence and with the ala sequence. Upon incubation at high concentrations and for prolonged time periods, the formation of fibrillar aggregates was observed with the +7 ala mutant and, with a longer lag time and a more stringent concentration-dependence, also for the wild-type protein, but never for the ala mutant. This result was made public through a publication in a scientific journal. Generation of a Drosophila Model of OPMD We have generated transgenic Drosophila lines able to produce wildtype or alanine-expanded bovine PABPN1. We have characterised the transgenic Drosophila lines produced and addressed the role in OPMD of the different domains of PABPN1. This result will be made public through publications in scientific journals. New mammalian cellular models of OPMD We generated mouse muscle cell lines that express either normal or OPMD-associated PABPN1 transgenes. Contrasting to previous reports in the literature, we demonstrated that equivalent numbers of nuclear inclusions are formed in our PABPN1 wild-type and +7ala transfected cell lines, suggesting that it is merely the presence of the transgene that is the key to the formation of these structures. This result was made public through a publication in a scientific journal. Generation of a Camelid antibody against PABPN1 We have generated Camelid phage libraries, derived from an immunised and a non-immunised Llama against muscle proteins. Using this library we were able to select Camelid antibodies against the following targets: -actin-1, -tropomyosin, -sarcoglycan, -sarcoglycan, -sarcoglycan, telethonin, ZNF9, calpain-3, caveolin-3, dysferlin, dytrophin (spectrin repeat); dystrophin-related protein 2, troponin T, myosin, emerin, FRG1P and PABPN1. Characterization of the HCAb fragments selected against PABPN1 indicate that these can immunomodulate PABPN1 aggregates in vivo, thus offering a new therapeutic potential. This result will be made public through publication in scientific journals. Generation of vectors that drive efficient expression of wild-type and mutant PABPN1 cDNA We produced novel genetic vectors for PABPN1 expression based on ubiquitous chromatin opening elements. The new vectors for PABPN1 expression contain promotors that are able to overcome variegated expression and silencing that is commonly induced after transgene integration into the host cell genome. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
