Development of a human/murine COL7A1 hybrid mouse model for epidermolysis bullosaOngoing
|Project lead||Dr Ulrich Koller|
|Organisation||EB House Austria, Salzburg, AUSTRIA|
|Partner organizations & collaborators||Dr. Josefina Piñón Hofbauer from EB House Austria|
|Project budget||EUR 245,000.00|
|Start date / Duration||01. Jan 2017 / 60 months|
|Funder(s) / Co-Funder(s)||DEBRA Austria|
|Research area||Molecular therapy, EB genetics, epigenetics & biology|
Short lay summary
Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating blistering skin disease caused by mutations within the COL7A1 gene. Currently, there is no cure available for RDEB. The development of potential therapeutic options is complicated by the lack of available animal models. Due to poor survival rates, associated with a COL7A1 knock out, we aim to develop an inducible human/murine COL7A1 hybrid model for RDEB. This should lead to a better survival prognosis. The model will facilitate and accelerate the development of potential therapeutic options, such as CRISPR/Cas9 gene editing strategies for RDEB. Additionally, an RDEB model with a longer lifespan will enable the accurate modeling of RDEB-associated secondary complications such as wound healing deficiencies and the development of SCC.
The ultimate goal is to generate a mouse, which will carry one “hybrid” COL7A1 allele in which a murine exon (and surrounding intronic sequences) is replaced with its human counterpart that additionally carries a known RDEB disease-causing mutation. The second Col7a1 allele will bear a floxed murine exon that can be deleted upon expression of Cre-recombinase. As gene editing (CRISPR/Cas9) strategies are largely context-dependent and therefore personalized, this proposed hybrid model is specifically being generated for the development and testing of human-specific gene therapeutic molecules for correcting hotspot mutations in the selected human COL7A1 exon. Currently, we are in the process of exchanging the murine exon with its human counterpart in murine keratinocytes using CRISPR/Cas9. Analyses of the endogenously expressed hybrid molecule will be carried out via sqRT-PCR, Western blot analysis, immunofluorescence analysis and by the generation of 3D skin equivalents to confirm functional or non-functional C7 deposition at the BMZ. Once the hybrid molecule is shown to be functional, templates to incorporate the disease-causing mutation are generated for microinjection, along with gRNAs, into fertilized oocytes. Separately, we will generate an inducible murine Col7a1 knock out allele by introducing LoxP sites up- and downstream of the target murine exon of Col7a1. Subsequent Cre-recombinase treatment will lead to the excision of the exon and consequently to the disruption of the gene, which will be confirmed at mRNA, protein and cellular level in the course of the project. Crossing of these two knock-in mice should result in a mouse carrying the mutated human COL7A1 exon on the one allele and the floxed murine Col7a1 exon on the other allele. In addition, crossing to Cre-ERT models is essential to achieve forced deletion of the floxed allele upon administration of tamoxifen to the animals, and activation of the Cre recombinase. Exon deletion will lead to a Col7a1 KO, resulting in a gradual reduction and finally absent expression of type VII collagen within the BMZ of the skin, facilitating the investigation of numerous pathological aspects of the disease.
The mice will be fully characterized after the skin-specific and/or systemic induction of the C7 knock out, including a comprehensive immunophenotypic analysis. They will be used to develop different in vivo gene editing strategies, investigate wound healing deficiencies as well as the process of malignant transformation to SCC.