Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa Patients With Nonsense Mutations

Aim 1. Evaluate the Safety and Efficacy of Short Term Intravenous Gentamicin for RDEB Patients.

Overview & Rationale: The rationale for short-term systemic intravenous gentamicin in these patients is outlined in the sections above, namely the drug could treat multiple skin sites simultaneously including the esophagus. This approach has proven safe and efficacious in other genetic diseases such as CF and DMD22-25 Our HYPOTHESIS is that intravenous gentamicin will, like topical and intradermal gentamicin, create new C7 and AFs at the patient's DEJ, improve multiple skin sites simultaneously, improve the patient's clinical disease and improve his or her quality of life. In accordance with the enclosed letters from two experienced pediatricians, we believe this therapy will be safe. Because C7 and AFs are incredibly stable, we further hypothesize that newly induced C7 and AFs will persist for many months in the patient's skin. Using dosing regimens of gentamicin that have proven to be safe, we will: 1) determine the percentage increase of C7 expression in the patients' skin before and after treatment, 2) determine how durable and sustainable the new C7 and AFs are in the patients' skin after treatment, 3) determine if this treatment is safe in this patient population, 4) determine if this treatment improves the patients clinically and improves their quality of life and 5) generate data for a multi-centered Phase III study using a larger number of patients.

Patients/Intervention: We have already lined up 3 well-characterized, adult RDEB patients with nonsense mutations who wish to participate in this study. These patients had positive responses to topical and intradermal gentamicin in our clinical trial (Table II). The patients will receive intravenous gentamicin (7.5 mgs per kilogram), in 100 ml of 5% dextrose in saline, for 14 consecutive days, a dose proven to not cause ototoxicity or neprhotoxicity28 and to be efficacious in inducing PTC read-through in DMD patients.22 The first few infusions will be done in the Infusion Center of the Keck Hospital of USC. We will measure gentamicin trough levels (24 hours after infusion) and gentamicin peak levels (30 minutes after the infusion) during the second day and, if needed, adjust the gentamicin doses such that the peak level is between 20 and 40 μg/ml and the trough level <2 μg/ml. If no untoward side effects occur, the patients will then have their infusions in their home via a commercial infusion service.

Safety Evaluation: Patients will be consented, enrolled and evaluated on Day 0 and then evaluated on post-treatment days 30, 90, and 180. On Day 0, the patients will have a complete medical history, review of systems (ROS), physical examination, vital signs, and weight. They will also have baseline urine analysis, complete blood count (CBC), electrolytes, glucose, comprehensive metabolic panel (CMP), and calculated creatinine clearance. Pure tone audiometry and a calculated creatinine clearance will be done on Days 0, 7, 14 and 30 post-treatment. Stop criteria will be a decline of >15 dB on pure tone audiometry at 2 consecutive frequencies and a creatinine clearance <60ml/min.28 Whenever there is new protein created in a patient, there is the possibility that the patient's immune system will raise autoantibodies against it. This did not occur in our clinical trial with topical and intradermal gentamicin, but it behooves us to evaluate this again with intravenous gentamicin and potentially more auto-antigenic protein created. Using indirect immunofluorescence and our anti-C7 ELISA, we will test the subject's serum for anti-C7 antibodies at each visit.

Efficacy: A. Clinical Evaluations: At baseline Day 0 and at each follow-up visit, (days 30, 90 and 180) the patients will complete a Quality of Life Assessment tool, a Pruritus Score tool, and a validated epidermolysis bullosa disease activity index (EBDASI)29,30. Patients will keep a daily diary using a self-ROS questionnaire. Patients will be telephoned weekly by a USC study member inquiring about any new signs, symptoms or ROS changes since the advent of the study. At baseline Day 0, we will identify four Test Sites at least 25 cm2 in size to follow sequentially. Two will be from areas that at baseline have blisters or erosions and the other two from intact skin areas. The two Test Sites with active blisters and erosions will be traced using a transparency sheet and the areas of the lesions calculated on a digitizing tablet. Likewise, all of the Test Sites will have standardized photographs and the lesional areas calculated by computer-assisted morphometry, as previously published.29-31 Within the context of their daily diary, patients will note the number of new blisters and erosions that occur in the Test Sites. A USC study member will telephone the patient each week and inquire about the Test Sites and ask the patient to estimate the percentage area of the Test Site consumed by erosions and blisters. Also, at each follow-up visit, three dermatologists will observe the four Test Sites and count the number of bullae and erosions in each. The intact Test Site areas will be scored at each follow-up visit using the following 3-point scale: 0 = no lesions; +1 = any active lesions; +2 = active lesions consuming over 50% of the Test Site area. Tracings to evaluate the areas of blisters or open erosions will be done, and standardized photographs paired with computer-assisted morphometry to calculate the areas of any lesions will be performed. B. Evaluations of C7 and AF Expression: At each post-treatment time point, 8 mm punch biopsies from the four Test Sites will be divided into three parts for (i) H&E histology and quantitation of epidermal-dermal adherence, (ii) the expression of C7 at the DEJ relative to that of normal human skin using anti-C7 antibodies to the NC1 and NC2 domains of C7 and NIH J Image software. One major "milestone" of the study will be the expression of NC2 since this would clearly indicate restoration of full-length C7 and (III) evaluation of newly created AFs by immuno-EM and AF enumeration by computer-assisted morphometry. Performing these evaluations out to 6 months will determine how durable the newly created C7 and AFs are. In our recent clinical trial outlined above, it was surprising that gentamicin-induced C7 remained at a similar level at 3 months post-treatment as that of 1 month for Patient #3 (Please see Figure 1 and Table II).