1. Correction of recessive dystrophic epidermolysis bullosa by homology-directed repair-mediated genome editing
- Author
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Matthew H. Porteus, Rosa Romano, Rodolfo Murillas, Wai Srifa, Jose Bonafont, Fernando Larcher, Marta García, Marcela Del Rio, Ángeles Mencía, Rosario Hervás-Salcedo, Sriram Vaidyanathan, Laura Ugalde, Blanca Duarte, E. Chacón-Solano, Comunidad de Madrid, Ministerio de Economía y Competitividad (España), and Ministerio de Ciencia e Innovación (España)
- Subjects
Keratinocytes ,Genetic enhancement ,Robótica e Informática Industrial ,Gene Expression ,medicine.disease_cause ,0302 clinical medicine ,Genome editing ,Epidermolysis ,Drug Discovery ,CRISPR ,Skin ,Gene Editing ,0303 health sciences ,Mutation ,Crispr ,Gene Transfer Techniques ,Aav ,Dependovirus ,Epidermolysis Bullosa Dystrophica ,030220 oncology & carcinogenesis ,Molecular Medicine ,Electrónica ,Epidermolysis bullosa ,Rdeb ,Collagen Type VII ,Genetic Vectors ,Hdr ,Genes, Recessive ,Biology ,Viral vector ,Cell Line ,Homology directed repair ,03 medical and health sciences ,Gene therapy ,Genetics ,medicine ,Humans ,Molecular Biology ,Gene ,Biología y Biomedicina ,030304 developmental biology ,Pharmacology ,Ingeniería Mecánica ,Recombinational DNA Repair ,Genetic Therapy ,medicine.disease ,Cancer research ,CRISPR-Cas Systems - Abstract
Genome-editing technologies that enable the introduction of precise changes in DNA sequences have the potential to lead to a new class of treatments for genetic diseases. Epidermolysis bullosa (EB) is a group of rare genetic disorders characterized by extreme skin fragility. The recessive dystrophic subtype of EB (RDEB), which has one of the most severe phenotypes, is caused by mutations in COL7A1. In this study, we report a gene-editing approach for ex vivo homology-directed repair (HDR)-based gene correction that uses the CRISPR-Cas9 system delivered as a ribonucleoprotein (RNP) complex in combination with donor DNA templates delivered by adeno-associated viral vectors (AAVs). We demonstrate sufficient mutation correction frequencies to achieve therapeutic benefit in primary RDEB keratinocytes containing different COL7A1 mutations as well as efficient HDR-mediated COL7A1 modification in healthy cord blood-derived CD34+ cells and mesenchymal stem cells (MSCs). These results are a proof of concept for HDR-mediated gene correction in different cell types with therapeutic potential for RDEB. This work was supported by Spanish grants PI17/01747, PI20/00615, AC17/00054 (MutaEB-E-rare), and CIBERER ER18TRL714 from the Instituto de Salud Carlos III and grant SAF2017-86810-R from the Ministry of Economy and Competitiveness , all co-funded with European Regional Development Funds , and Avancell-CM grant ( S2017/BMD-3692 ). Authors are indebted to Almudena Holguín and Nuria Illera for grafting experiments, and to Jesus Martínez and Edilia De Almeida for animal maintenance and care.
- Published
- 2020