Keratinocyte cell lines derived from severe generalized recessive epidermolysis bullosa patients carrying a highly recurrent COL7A1 homozygous mutation: models to assess cell and gene therapies in vitro and in vivo.

[dystrophic epidermolysis bullosa]

Recessive dystrophic epidermolysis bullosa (RDEB ) is caused by deficiency of type VII collagen due to COL 7 A 1 mutations such as c .6527 insC , recurrently found in the Spanish RDEB population . Assessment of clonal correction-based therapeutic approaches for RDEB requires large expansions of cells , exceeding the replication capacity of human primary keratinocytes . Thus , immortalized RDEB cells with enhanced proliferative abilities would be valuable . Using either the SV 40 large T antigen or papillomavirus HPV 16 -derived E 6 -E 7 proteins , we immortalized and cloned RDEB keratinocytes carrying the c .6527 insC mutation . Clones exhibited high proliferative and colony-forming features . Cytogenetic analysis revealed important differences between T antigen-driven and E 6 -E 7 -driven immortalization . Immortalized cells responded to differentiation stimuli and were competent for epidermal regeneration and recapitulation of the blistering RDEB phenotype in vivo . These features make these cell lines useful to test novel therapeutic approaches including those aimed at editing mutant COL 7 A 1 .