Genome editing for genodermatoses therapy: correction of a Netherton syndrome patient's cells carrying a deep intronic pathogenic variation

M GARCIA(1,3,5) D de Prado(4) V Galvez(3,4,5) B Duarte(3,4,5) A Vicente(2) F Larcher(1,3,4,5) R Murillas(3,4,5) A Mencia(4,5)

1:Universidad Carlos III de Madrid; 2:Hospital Sant Joan de Déu; 3:Instituto de Investigación Sanitaria Fundación Jiménez Díaz; 4:CIEMAT; 5:CIBERER

The establishment of effective preclinical protocols for gene editing in skin is necessary for translation to the clinic.Precise CRISPR-mediated deletions can be generated very efficiently in the genome of patients' skin cells to correct the effect of different types of deleterious variations underlying genodermatoses. Gene correction by gene editing allows endogenous regulation of expression to be maintained while respecting the endogenous spatiotemporal expression patterns of the corrected genes. Deep intronic variants that disrupt transcription causing altered gene splicing can be addressed by making small deletions.

Netherton syndrome, an inherited form of ichtyosis that causes atopic skin manifestations, growth retardation and hair defects is associated with genetic variations in SPINK5, most of them resulting in absence of LEKTI, a protease inhibitor precursor protein encoded by this gene. We have characterized a novel pathogenic allele in a Netherton patient that consists of a rare deep intronic SPINK5 variation that, in the particular intronic genetic background of this allele, results in an out of frame pseudoexon retention. Ex vivo editing to remove the pseudoexon sequence was performed to restore proper SPINK5 splicing ,and thus LEKTI expression, in skin cells from this patient. Human skin tissue regenerated from the gene-edited cells showed LEKTI expression in suprabasal layers of epidermis and normal epidermal differentiation.