Efficient CRISPR/Cas9n-mediated repair of a COL17A1 mutation

In the past genetic diseases were counted as incurable. The discovery of bacterial nucleases, which are able to bind and cleave distinct genomic sequences, is the basis for the development of powerful technologies, dealing with the repair of genetic mutations ex vivo. The list of genetic disorders for which nuclease-based gene editing tools have been applied is rapidly growing. Skin diseases are optimal targets for these gene therapeutic interventions in particular due to a number of reasons: the stem cells of the skin are well accessible, are easy to cultivate in vitro and can be re-transplanted onto the patient after their expansion to skin sheets. The junctional form of epidermolysis bullosa (JEB), characterized by a severe phenotype, is caused by mutations within the COL17A1 gene, leading to functional impairment or complete loss of type XVII collagen in the patient. This devastating rare skin disease is accompanied by intense pain, requires care 24/7 and is incurable. The skin of affected patients, who in Austria are also known as butterfly children, detaches after mild mechanical trauma. Our project deals with the development and improvement of a nuclease-based gene therapy for JEB via repair of a distinct mutation within the COL17A1 gene. Our main focus is largely on the efficienc y and specificity of this applied technology. Despite the high specificity of programmable nucleases, the high potential risk of off-target events is recognized. These may lead to sequence modifications within semi-homologous genomic regions. In this project we are aiming at the potential traceless gene correction using the Cas9 mutant D10A (Cas9n) associated with high safety. The mutant Cas9n demonstrates a reduced off-target activity in comparison to the wild-type Cas9. The efficiency of gene editing via the mutant can be increased significantly by simultaneous binding and DNA cleaving of two Cas9n in near proximity to each other. For COL17A1 repair the respective designer nucleases are introduced together with DNA oligonucleotides, comprising the homologous wildtype COL17A1 sequence, into JEB patient keratinocytes. The wildtype COL17A1 sequences are inserted at the COL17A1 targeting locus upon activated DNA repair mechanisms in treated cells leading to the correction of the genetic mutation. The expression of COL7A1 and the restoration of type XVII collagen in corrected cells will be analysed by sqRT-PCR, immunofluorescence and Western blot analysis. The accurate deposition of restored type XVII collagen will be analysed in generated skin equivalents transplanted onto an appropriate mouse model. Besides the repair analysis of the respective COL17A1 mutation, safety profiles for the gene editing application will be established. Possible off-target regions will be analysed via sequencing to detect possible unwanted modifications within the genome to evaluate the applicability of this approach for an ex vivo treatment of patients with JEB.

Genetic skin diseases are particularly interesting for the development of designer nuclease-based therapies: stem cells within the skin are readly accessible, can be cultivated in the laboratory, and can be transplanted back onto the skin. The junctional form of the rare skin disease epidermolysis bullosa (JEB) is caused by, among other things, mutations in the COL17A1 gene, which can lead to a complete loss of the corresponding collagen XVII protein (C17). Our project involved the development of a designer-nuclease-based gene therapy for JEB to correct disease-associated mutations in COL17A1 in an efficient and specific manner. During the course of the project, we developed several promising gene editing strategies for COL17A1 repair. Treatment with CRISPR molecules resulted in restoration of normal C17 levels in JEB cells. Furthermore, the protein was correctly deposited between the skin layers of artificially-generated skin. Molecular biological analyses showed that the gene treatment worked accurately, with no undesirable changes in the JEB cells. Continuous progress in CRISPR gene therapy research offers hope for a future clinical application.