T.P.25 Development of the exon exchange method for repair of mutant nebulin transcripts

Abstract

Abstract The nebulin gene (NEB) has 183 exons encoding transcripts up to 26 kb in length. Mutations found in NEB are dispersed throughout the gene, i.e. no mutational hot spots are evident. Mutations cause autosomal recessive nemaline myopathy, distal nebulin myopathy and core-rod myopathy, for which no therapy is available. The size of NEB limits the options of gene therapy development. Thus, our research has focused on methods correcting the mutation carrying transcripts. Targeted exon exchange requires the spliceosome provided by the cell in addition to the target pre-mRNA and pre-trans-splicing molecules (PTMs) both produced from expression vectors transfected into C2C12 mouse myoblasts. We have developed wild type and mutant NEB minigenes for production of target pre-mRNA and PTMs for internal exon exchange of one or several NEB exons at a time. We have previously obtained successful results from our 3′ exon exchange and first internal exon exchange experiments and continue to develop the internal exon exchange technique further. NEB introns are usually short and binding of PTM can interfere with exon recognition. We have designed PTMs targeted to larger introns on both sides of an area of two exons close to each other to overcome this problem. Exon exchange is assessed by RT-PCR and sequencing and at the protein level by Western blotting and immunofluorescence staining. Our preliminary results from co-transfection of mutant minigenes and PTMs indicate successful, but weak trans-splicing at the RNA level. Immunofluorescence staining of co-transfected C2C12 cells show protein production in some cells, which may indicate successful trans-splicing, but the results need to be confirmed by western blotting. Being able to exchange several exons at a time from the 183-exon-containing NEB at the RNA level would be an advantage in therapy as the same therapy molecule would be useful for patients with mutations in any of the exons in the exchanged area.