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.

In the course of the project Gene correction by double RNA trans-splicing we applied double RNA trans-splicing technology to correct mutations in the collagen 7 gene (COL7A1) during transcription via specific replacement of internal mutated sequences with wild-type copies in the corresponding RNA. Mutations in COL7A1 lead to dystrophic epidermolysis bullosa (DEB) characterized by a severe phenotype due to functional impairment or complete loss of type VII collagen protein in the patients skin. This devastating rare skin disease is accompanied by intense pain, requires 24/7 care and is incurable. The skin of affected patients, who in Austria are also known as butterfly children, detaches after mild mechanical trauma, often resulting in chronic wounds, persisting for months or even years. These chronic wounds are a major risk for the development of associated aggressive skin cancers. In the project two functional repair molecules, called double RNA trans-splicing molecules (dRTM), were derived using a recently in our laboratory established fluorescence-based selection model system. The resulting dRTMs were capable of inducing the exchange of two internal COL7A1 regions and were therefore adapted for the treatment of patient skin cells. The dRTMs were delivered into the patient cells using a retrovirus and subsequently integrated into the genome of the cells to obtain long-lasting production of the molecules. Molecular biological studies confirmed the repair capacity of the dRTMs leading to the partial restoration of type VII collagen production. Due to the low repair efficiency of the dRTMs we generated antisense RNAs (asRNAs), which specifically bind the COL7A1 target region aiming toincrease the overall repair efficiency. Individual asRNAs, tested via our fluorescence-based model system, had a positive impact on the repair efficiency of our selected dRTMs. In summary, we confirmed the two main hypotheses of the project. We showed for the first time the feasibility of the double RNA trans-splicing technology to specifically correct disease-associated mutations in patient cells. We were able to replace two distinct COL7A1 regions using selected dRTMs, thereby partially restoring the function of the impaired protein in the treated cells. Further, we demonstrated the potential of asRNAs to increase the trans-splicing efficiency of these dRTMs via our fluorescence-based model system, which has to be confirmed in patient cells in future experiments. Basically, we have developed a methodology which is especially suitable for the repair of large genes for which no alternative repair options are available.