Gentamicin induces functional type VII collagen in recessive dystrophic epidermolysis bullosa patients

Abstract

Background: Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable disease caused by mutations in the gene encoding type VII collagen, the major component of anchoring fibrils (AF). We previously demonstrated that gentamicin produced functional type VII collagen in RDEB cells harboring nonsense mutations. Herein, we determined whether topical or intradermal gentamicin administration induces type VII collagen and AFs in RDEB patients.

Methods: A double-blind, placebo-controlled pilot trial assessed safety and efficacy of topical and intradermal gentamicin in 5 RDEB patients with nonsense mutations. The topical arm tested 0.1% gentamicin ointment or placebo application 3 times daily at 2 open erosion sites for 2 weeks. The intradermal arm tested daily intradermal injection of gentamicin solution (8 mg) or placebo into 2 intact skin sites for 2 days in 4 of 5 patients. Primary outcomes were induction of type VII collagen and AFs at the test sites and safety assessment. A secondary outcome assessed wound closure of topically treated erosions.

Results: Both topical and intradermal gentamicin administration induced type VII collagen and AFs at the dermal-epidermal junction of treatment sites. Newly created type VII collagen varied from 20% to 165% of that expressed in normal human skin and persisted for 3 months. Topical gentamicin corrected dermal-epidermal separation, improved wound closure, and reduced blister formation. There were no untoward side effects from gentamicin treatments. Type VII collagen induction did not generate anti-type VII collagen autoantibodies in patients' blood or skin.

Conclusion: Topical and intradermal gentamicin suppresses nonsense mutations and induces type VII collagen and AFs in RDEB patients. Gentamicin therapy may provide a readily available treatment for RDEB patients with nonsense mutations.

Trial registration: ClinicalTrials.gov NCT02698735.

Funding: Epidermolysis Bullosa Research Partnership, Epidermolysis Bullosa Medical Research Foundation, NIH, and VA Merit Award.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. CONSORT flow diagram.

Flowchart summarizing RDEB patient enrollment and completion of the trial.

Figure 2. Topical and intradermal gentamicin treatment generated new type VII collagen in RDEB patients.

IF staining with a polyclonal antibody to type VII collagen on skin biopsy specimens taken at day 0, 1 month (1M), and 3 months (3M) after treatment with topical gentamicin (T-gent) or topical placebo (T-plac) (A) and intradermally injected gentamicin (I-gent) or placebo (I-plac) (B). Note that gentamin induced type VII collagen at the DEJ of the skin of all 5 patients who received topical gentamicin applied to open erosive test sites (A) and that intradermally injected gentamicin induced type VII collagen at the DEJ of all 4 patients who received the injections (B). All images were obtained using the same camera at identical exposure times. (C) Mean average fluorescence intensity for type VII collagen immunoreactivity was obtained from the images shown in panels A and B. Frozen sections were probed with a polyclonal antibody to type VII collagen, and the intensity of type VII collagen at the DEJ of each specimen was measured by computer-assisted ImageJ software and compared with the intensity of type VII collagen in NHS. The y axis shows the intensity of type VII collagen in the DEJ of the test sites expressed as a percentage of the average obtained from NHS (100%). Data represent the mean ± SD. (D) Mean average fluorescence for type VII collagen expression from each patient was determined and compiled for each treatment. Mean ± SEM are shown to the right of individual sample data. P = 0.037 (n = 5) for topical and P = 0.043 (n = 4) for intradermal. Statistical significance was determined by 2-tailed Welch’s t test. PT1, patient 1; e, epidermis; d, dermis. Scale bars: 50 μm.

Figure 3. Topical or intradermal gentamicin treatment generated new AFs in RDEB patients.

Skin sections taken from patients 3 and 4 at 1 month and 3 months after treatment, as indicated, were subjected to IEM by labeling en bloc with a murine monoclonal antibody, mAb 185, to type VII collagen, followed by anti-mouse IgG–conjugated immunogold particles (black dots). The photomicrographs consist of low magnification images shown in A with black boxes highlighting the regions shown in the high magnification images (B). Note that in the placebo-treated test sites, there was no labeling of the DEJ and there were no visible AFs in either patient. In contrast, skin biopsy samples from both topical and intradermally injected gentamicin test sites exhibited intense gold labeling of the lamina densa and many well-labeled AFs (arrows). Scale bars: 250 nm (A); 100 nm (B).

Figure 4. Topical gentamicin improved wound closure.

Representative photographs of the open erosion test sites prior to treatment and at day 0, 1 month, and 3 months after treatment with either topical gentamicin or topical placebo in patient 1 (A), patient 2 (B), patient 3 (C), and patient 5 (D). Yellow dotted lines indicate the treated areas. Scale bars: 1 cm.

Figure 5. Topical and intradermal gentamicin do not induce anti–type VII collagen antibodies.

(A) Sera were obtained from RDEB patients before and 1 month after receiving gentamicin treatments and subjected to an ELISA using a commercially available ELISA kit against type VII collagen. Note that low-level anti–type VII collagen IgG antibodies were detected in 2 of the RDEB patients at day 0. However, none of the patients’ sera exhibited any increases in anti–type VII collagen IgG antibodies at 1 month after gentamicin treatment. Serum from a patient with EBA was used as a positive control, while serum from a normal human subject (NHS) was used as a negative control. Data represent the mean ± SD. (B) Skin biopsies from the gentamicin-treated test sites were obtained 1 month and 3 months after treatment and subjected to direct IF using an FITC-conjugated goat anti-human IgG antibody. Note that no IgG deposits of human anti–type VII collagen IgG were detected in any of the test sites. Scale bar: 50 μm.