Towards Personalized Gene Targeting-Based Therapeutics for Recessive Dystrophic Epidermolysis Bullosa Patients of Spanish Origin

Dystrophic Epidermolysis Bullosa (DEB) is a genoderma-toses characterized by fragility of the skin. Tissue separation occurs below the basement membrane at the level of the anchoring fibrils. Mutation analysis has demonstrated that COL7A1 is the defective gene in all forms of DEB. The phe-notypical variability depends on the different type of mutations in DEB alleles and their position within the gene. From the identification of an increasing number of mutations, however, some general genotype-phenotype corrdation has been drawn. The most severe form of DEB, the recessive DEB severe generalized (RDEBsg), is characterized by mutilating scarring of the hands and feet, joint contractures, structures of the upper gastrointestinal tract, and the development of aggressive squamous cell carcinomas. These features frequently shorten the life span of the affected individuals to three or four decades. RDEBsg, is frequently due to the presence of mutations leading to premature termination codon of translation (PTC) on both alleles. Although in DEB the pathogenic mutations are generally family specific, a high recurrent mutation in COL7A1 (NM_000094.3:c.6527insC) (42.5% allelic frequency) was disdosed in the Spanish population. Our cohort comprises 19 carriers of the 6527insC mutation (10 in 9 in heterozygous and homozygous) and 22 patients with other mutations. We have described new SNPs in COL7A1 (NM-000094.3:c.11639C> T, 19020G> T, 23353G> C, 23354C> G, 24558C> T, 25215C> T, 8141C> G, 29056C> T, 31427C> A), as well as, determined seven haplotypes in our population. We found a unique haplotype that segregates with the 6527insC mutation supporting the hypothesis that 6527insC mutation originated from a single mutational event. These genetic findings prompted us to search for new therapeutic approaches based on targeted homologous recombination. Strategies involving nucleofection and AAV-mediated gene delivery of knock-in constructs suitable to correct the 6527insC mutation present in Spanish RDEBsg patients are currently under development in our laboratory. Gene tar-geting-based ex vivo gene therapy followed by bioengineered skin equivalent transplantation may be a rational and clinically relevant approach for permanent correction of Spanish RDEBsg patients.