Amlexanox Enhances Premature Termination Codon Read-Through in COL7A1 and Expression of Full Length Type VII Collagen: Potential Therapy for Recessive Dystrophic Epidermolysis Bullosa

Abstract

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare monogenic blistering disorder caused by the lack of functional type VII collagen, leading to skin fragility and subsequent trauma-induced separation of the epidermis from the underlying dermis. A total of 46% of patients with RDEB harbor at least one premature termination codon (PTC) mutation in COL7A1, and previous studies have shown that aminoglycosides are able to overcome RDEB PTC mutations by inducing "read-through" and incorporation of an amino acid at the PTC site. However, aminoglycoside toxicity will likely prevent widespread clinical application. Here the FDA-approved drug amlexanox was tested for its ability to read-through PTC mutations in cells derived from patients with RDEB. Eight of 12 different PTC alleles responded to treatment and produced full length protein, in some cases more than 50% relative to normal controls. Read-through type VII collagen was readily detectable in cell culture media and also localized to the dermal-epidermal junction in organotypic skin culture. Amlexanox increased COL7A1 transcript and the phosphorylation of UPF-1, an RNA helicase associated with nonsense-mediated mRNA decay, suggesting that amlexanox inhibits nonsense-mediated mRNA decay in cells from patients with RDEB that respond to read-through treatment. This preclinical study demonstrates the potential of repurposing amlexanox for the treatment of patients with RDEB harboring PTC mutation in COL7A1.

Figure 1

Amlexanox induces full length type VII collagen synthesis in RDEB patient derived cells. Keratinocytes (a and b) and fibroblasts (c) isolated from different RDEB patients were treated with 250 μM amlexanox (A) or gentamicin 1 mM (G). Expression of type VII collagen after treatment was compared to normal human keratinocytes or fibroblasts (NHK or NHF) using Western blotting. For optimization of detection, the amount of protein loaded from RDEB keratinocytes (not fibroblasts) was twice the amount of protein loaded from control cells. Levels of type VII collagen expression were quantified in image J relative to β-actin and non-treated controls (NT). Graph shows protein levels as a percentage of normal cells.

Figure 2

Read-through type VII collagen is stable and locates to the dermal-epidermal junction. RDEB cells harboring G2073D/R578X mutations and R2610X/R2610X mutations were assessed for levels of intracellular type VII collagen (a) and levels of secreted type VII collagen (b) using Western blotting. Treatment with amlexanox and gentamicin stabilizes secreted levels of the protein. For optimization of detection, the amount of protein loaded from RDEB cells was twice the amount of protein loaded from control cells. (c) Treatment of organotypic skin cultures with amlexanox for two weeks increases type VII collagen deposition at the dermal-epidermal junction (DEJ). Organotypic skin cultures were prepared using RDEB cells or wild type cells, and synthesis of type VII collagen at the DEJ was evaluated using immunofluorescence staining. The magnification bar = 400 μM.

Figure 3

Amlexanox increases transcript levels of COL7A1 in 3 out of 4 RDEB patient cells. (a) Evaluation of baseline levels of COL7A1 transcript compared to normal keratinocytes and fibroblasts shows relatively similar levels of expression. (b) Treatment with amlexanox (250 μM) increases COL7A1 transcript in all but one population of RDEB cells. Gentamicin (1 mM).

Figure 4

Read-through synthesis of type VII collagen correlates with increase in UPF1 phosphorylation state. RDEB or wild type cells were cultured in 100 mm dishes and treated with either amlexanox or gentamicin for 48 hours. Levels of UPF1-P were quantified in image J relative to total levels of UPF1 and non-treated controls.