Gene correction by "double RNA trans-splicing"

Spliceosome Mediated RNA Trans-splicing is a useful tool to correct genes on mRNA level. Currently, three modes of RNA trans-splicing (5`, 3` and double RNA trans-splicing) are available and were applied for different genetic diseases including epidermolysis bullosa, haemophilia and cystic fibrosis. Using RNA trans-splicing, the endogenous splicing machinery can be exploited to reprogram two pre-mRNAs to a new gene product of choice. An engineered RNA trans-splicing molecule (RTM), harbouring an exonic region of a gene of interest, facilitates the trans-splicing process by binding to the target pre-mRNA, thereby replacing the mutated gene region. The combination of both trans-splicing events (5` and 3`), called double RNA trans-splicing or internal exon replacement, is used to replace a central portion of a given transcript. Theoretically, it is an elegant approach, but its practical application is hindered by the low efficiency of the method. Data from our laboratory (Koller et al. in 2011) showed accurate double trans-splicing in a novel GFP-based screening system. Using this screening system we want find out factors that improve the efficiency of double RNA trans-splicing. We want to optimize the binding properties of designed RTMs and investigate the influence of antisense oligonucleotides by blocking the competitive cis-splice sites on the target pre-mRNA. Both, RTM binding and blocking of cis-splicing within the target pre-mRNA, will be crucial to increase the trans-splicing efficiency significantly. For the first time we want to use this approach to correct different COL7A1 mutations in EB patient cells, carrying mutations in three different exons of COL7A1. We assume that RNA trans-splicing is a promising tool to correct a broad range of disease-associated mutations, avoiding many side effects present in conventional gene therapeutic applications. The development of a gene therapy for type VII collagen deficiency would increase the chance to find a cure for dystrophic EB. Additionally, the implementation of the methodology of double RNA trans-splicing will help us to move closer to the treatment of other genetic diseases caused by mutations in especially large genes. COL7A1 with a size of over 9kb is therefore well applicable for double RNA trans-splicing, in which only a short RTM has to be designed, harbouring only a couple of short exons to introduce. Using the screening system, established in our laboratory, it should be possible to increase the trans-splicing efficiency of designed RTMs to higher levels to revert the phenotype of EB patient cells into wildtype.

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