Skin gene therapy for acquired and inherited disorders

The rapid advances associated with the Human Genome Project combined with the development of proteomics technology set the bases to face the challenge of human gene therapy. Different strategies must be evaluated based on the genetic defect to be corrected. Therefore, the re-expression of the normal counterpart should be sufficient to reverse phenotype in single-gene inherited disorders. A growing number of candidate diseases are being evaluated since the ADA deficiency was selected for the first approved human gene therapy trial (Blaese et al., 1995). To cite some of them: sickle cell anemia, hemophilia, inherited immune deficiencies, hyper-cholesterolemia and cystic fibrosis. The approach does not seem to be so straightforward when a polygenic disorder is going to be treated. Many human traits like diabetes, hypertension, inflammatory diseases and cancer, appear to be due to the combined action of several genes and environment. For instance, several wizard gene therapy strategies have recently been proposed for cancer treatment, including the stimulation of the immune system of the patient (Xue et al., 2005), the targeting of particular signalling pathways to selectively kill cancer cells (Westphal and Melchner, 2002) and the modulation of the interactions with the stroma and the vasculature (Liotta, 2001; Liotta and Kohn, 2001).Our work is supported by grants SAF-2004-07717 from Ministerio de Ciencia y Tecnología (Spain) and LSHG-512073 from UE to M. Del Rio, LSHG-503447 from UE to J.L. Jorcano and LSHG-512102 from UE to F. Larcher. We express our gratitude to Dr. Y. Gache, Dr. F. Spirito and Dr. G. Meneguzzi for providing EM pictures to illustrate this work

Deletion of a Pathogenic Mutation-Containing Exon of COL7A1 Allows Clonal Gene Editing Correction of RDEB Patient Epidermal Stem Cells

Recessive dystrophic epidermolysis bullosa is a severe skin fragility disease caused by loss of functional type VII collagen at the dermal-epidermal junction. A frameshift mutation in exon 80 of COL7A1 gene, c.6527insC, is highly prevalent in the Spanish patient population. We have implemented geneediting strategies for COL7A1 frame restoration by NHEJ-induced indels in epidermal stem cells from patients carrying this mutation. TALEN nucleases designed to cut within the COL7A1 exon 80 sequence were delivered to primary patient keratinocyte cultures by non-integrating viral vectors. After genotyping a large collection of vector-transduced patient keratinocyte clones with high proliferative potential, we identified a significant percentage of clones with COL7A1 reading frame recovery and Collagen VII protein expression. Skin equivalents generated with cells from a clone lacking exon 80 entirely were able to regenerate phenotypically normal human skin upon their grafting onto immunodeficient mice. These patientderived human skin grafts showed Collagen VII deposition at the basement membrane zone, formation of anchoring fibrils, and structural integrity when analyzed 12 weeks after grafting. Our data provide a proof-of-principle for recessive dystrophic epidermolysis bullosa treatment through ex vivo gene editing based on removal of pathogenic mutationcontaining, functionally expendable COL7A1 exons in patient epidermal stem cells.The study was mainly supported by DEBRA International—funded by DEBRA Austria (grant termed as Larcher 1). Additional funds come from Spanish grants SAF2013-43475-R and SAF2017-86810-R from the Ministry of Economy and Competitiveness and PI14/00931 and PI17/01747 from the Instituto de Salud Carlos III, all of them co-funded with European Regional Development Funds (ERDF)