Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 115; no. 28; pp. E6536 - E6545
• Main Authors: Lincoln, Vadim, Cogan, Jon, Hou, Yingping, Hirsch, Michaela, Hao, Michelle, Alexeev, Vitali, De Luca, Michele, De Rosa, Laura, Bauer, Johann W., Woodley, David T., Chen, Mei
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 10.07.2018
• Series: PNAS Plus
• Subjects: Anchoring; Biological Sciences; Cell Adhesion Molecules - genetics; Cell Adhesion Molecules - metabolism; Cell lines; Cell morphology; Codon, Nonsense; Codons; Cytology; Epidermolysis bullosa; Epidermolysis Bullosa, Junctional - genetics; Epidermolysis Bullosa, Junctional - metabolism; Epidermolysis Bullosa, Junctional - pathology; Expression vectors; Filaments; Genotype & phenotype; Gentamicin; Gentamicins - pharmacology; HEK293 Cells; Humans; Integrin alpha6beta4 - genetics; Integrin alpha6beta4 - metabolism; Junctional epidermolysis bullosa; Kalinin; Keratinocytes; Keratinocytes - metabolism; Keratinocytes - pathology; Laminin; Morphology; Mutagenesis - drug effects; Mutation; Nonsense mutation; Phenotypes; PNAS Plus; Proteins; Restoration; Skin diseases; Three dimensional models; gentamicin; epidermolysis bullosa; readthrough
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1803154115

Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin β3) mutations account for 80% of patients with H-JEB, and ∼95% of H-JEB–associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin β3-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin β3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin β3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6β4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.