Trans-splicing gene therapy in the K14 gene (Bauer 3 + Ext Wally)

What did this project achieve?

Specific alterations in the keratin 14 (KRT14) gene underly generalized-severe epidermolysis bullosa simplex (gen-sev EBS). In contrast to other subtypes of EB, gen-sev EBS is mostly inherited in an autosomal dominant way. This means, that also a low percentage of the altered gene is sufficient to trigger the disease. This makes it a challenge to gene therapy, as a high rate of gene correction is necessary. Our project aimed to develop a safe and efficient repair system to restore changes in the keratin 14 gene. In a previous project, we were able to develop therapeutic molecules, which can correct alterations in the KRT14 gene. The functionality of these molecules was tested in several assays, showing that patient cells behave similarly to skin cells of healthy donors upon treatment. In this project, patient skin cells were treated with the therapeutic molecules and an artificial piece of skin (i.e. skin equivalent) was generated from those cells. The skin equivalents were then transplanted onto the back of mice, to show the stability of this corrected skin and also its development over a certain time period. After that, biopsies were taken and characterized by different staining methods, which aim to show characteristic features of human skin and also the mechanic stability. To sum up, we could show that we can successfully repair the keratin 14 gene using a pre-trans-splicing molecule. Experiments on mice showed, that a stable skin piece can be developed from treated patient skin cells, that morphologically look like healthy skin and render stability to the skin. We assume that this is a potential tool, especially for the treatment of dominant forms of EB, where conventional gene therapeutic approaches come to their limits.