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.
Recessive dystrophic epidermolysis bullosa (RDEB) is caused by deficiency of type VII collagen due to COL7A1 mutations such as c.6527insC, 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 SV40 large T antigen or papillomavirus HPV16-derived E6-E7 proteins, we immortalized and cloned RDEB keratinocytes carrying the c.6527insC mutation. Clones exhibited high proliferative and colony-forming features. Cytogenetic analysis revealed important differences between T antigen-driven and E6-E7-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 COL7A1.
Epithelial Biomedicine Division, Cutaneous Disease Modelling Unit, CIEMAT, Madrid, Spain.
Cristina
C
Chamorro
David
D
Almarza
Blanca
B
Duarte
Sara G
SG
Llames
Rodolfo
R
Murillas
Marta
M
García
Juan C
JC
Cigudosa
Luis
L
Espinosa-Hevia
Maria José
MJ
Escámez
Angeles
A
Mencía
Alvaro
A
Meana
Ramón
R
García-Escudero
Rosa
R
Moro
Claudio J
CJ
Conti
Marcela
M
Del Río
Fernando
F
Larcher
D000818:Animals; D002460:Cell Line; D064987:Cell- and Tissue-Based Therapy; D024143:Collagen Type VII / Q000235:genetics*; D016108:Epidermolysis Bullosa Dystrophica / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D015316:Genetic Therapy; D064593:Heterografts; D006720:Homozygote; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism* / Q000637:transplantation; D051379:Mice; D008957:Models, Genetic; D009154:Mutation; D012038:Regeneration
C569053:COL7A1 protein, human; D024143:Collagen Type VII
cell lines; genodermatoses; keratinocytes; viral oncogenes