Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa (RDEB) Nonsense Mutation Patients

Evaluate C7 and AF expression with topical and intradermal gentamicin treatment Topical application of gentamicin to skin lesion: We will apply commercially available gentamicin 0.1% ointment three times a day under Tegaderm occlusion for two weeks to a target RDEB skin erosion. Likewise, we will apply the ointment control vehicle three times a day to a similarly sized RDEB erosion. This dosage schedule is based on the successful clinical trial in CF patients that resulted in clinical efficacy without side effects.19 In our in vitro cell culture studies, we showed that a single dose of gentamicin (400 ug/ml) induced C7 expression at a level of 20-40% of that seen in normal cells (Figure 1A and 1B).23 In our clinical trial, the experimental ointment will contain 1 milligram of gentamicin per milliliter of ointment vehicle.

Injection of gentamicin into the high dermis of RDEB patient skin: In addition, we will identify target 2.0 cm x 2.0 cm areas of unwounded intact skin within areas prone to blister formation and intradermally inject commercially available sterile gentamicin solution (40 mg/ml in saline). A similar control area will be injected with equal volumes of saline. We will inject 200 ul (8 milligrams) into each site once on day 0 and once again on day 1. A single injection will be administered to the site on each of the two days. The total dose will be 16 milligrams injected into the upper dermis where it will contact the patient's dermal fibroblasts and basal keratinocytes. In our published in vitro read-through study, each 1 cm2 of cultured cells was exposed to 400 ugs/ml of gentamicin that showed read-through activity with no cytoxocity. For the proposed intradermal study, we will be using a total dose approximately 5-fold greater than the in vitro dose.

Initial and follow-up Parameters: Prior to any treatment, the RDEB patients will be subjected to a 9 mm shave biopsy of intact skin that will be divided into 3 parts and evaluated for H&E histology, transmission electron microscopy and direct immunofluorescence for C7 expression. At 1 and 3 months after gentamicin treatment of RDEB erosions or intact skin in blister prone areas, we will biopsy the treated sites and repeat the histological, ultrastructural and C7 expression evaluations. For the assessment of C7 expression at the DEJ by immunofluorescence (IF), 5 micron cryosections will be probed with anti-C7 polyclonal antibodies to the NC1 and NC2 domains of C7. The increased expression of the NC2 domain of C7 at the DEJ of gentamicin-treated skin or erosions will serve as one major "milestone" in this study since it would indicate PTC read-through and restoration of a full-length C7. The third part of the biopsy will be evaluated ultrastructurally, and AFs will be enumerated by computer-assisted morphometry. These studies will assess if there is restoration of normal AFs. Skin sections from normal human subjects will serve as positive controls, while skin sections from the vehicle control site will serve as negative controls.

Patient clinical assessment: Skin Erosion Sites: Patients will be blinded to the gentamicin and vehicle treatments of the Experimental Site and Control Site. Each week, the patients will assess the sites and grade their healing as follows: - 1 = enlargement of the erosion compared to its initial size; 0 = no change in the size of the erosion; +1 = partial healing and a smaller erosion than its initial size, and +2 = complete closure of the wound.

Secondly, baseline photographs of the erosions will be generated and the area of the erosions calculated by computer-assisted planimetry. Identical assessments will be made at 1 and 3 months post treatment. Therefore, a second "milestone" for this study will be decreased surface areas of gentamicin-treated erosions compared with vehicle control-treated erosions.