Conditions: Recessive Dystrophic Epidermolysis Bullosa
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 6
Healthy volunteers: no

Brief summary:
Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable, devastating, inherited skin disease caused by mutations in the COL7A1 gene that encodes for type VII collagen (C7), the major component of anchoring fibrils (AFs), structures that mediate epidermal-dermal adherence. Thirty percent of RDEB patients have nonsense mutations. The investigators recently demonstrated in 5 such patients that intradermal and topical gentamicin induced "read-through" of their nonsense mutations and created robust and sustained new C7 and AFs at the dermal-epidermal junction (DEJ) of their skin and also stimulated wound closure and reduced new blister formation. No untoward side effects occurred. Herein, the investigators propose evaluating the safety and efficacy of intravenous gentamicin in these patients. In theory, this intravenous administration has the possibility of treating simultaneously all of the patients' skin wounds. The investigators also propose optimizing the concentration and manner of delivery of topical gentamicin. The unambiguous milestones will be increased C7 and AFs in the patients' DEJ, improved EB Disease Activity Scores, and absence of significant gentamicin side effects.

Detailed description:
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).

Aim 2: Evaluate the Safety and Efficacy of Higher Dose Topical Gentamicin for RDEB Patients.

Overview and Rationale: In our gentamicin clinical trial outlined earlier, there was significant variation in the production of new C7 and AFs, as shown in Table II. Patients #2 and #5 only restored C7 to 20% of normal skin in response to gentamicin. We only used one concentration of gentamicin, 0.1% ointment, and our in vitro studies with RDEB fibroblasts from these same two patients showed that the C7 response to gentamicin was dose-dependent.26 In these patients, there could have been sub-optimal dosing or sub-optimal access of the gentamicin to the patient's dermal fibroblasts and basal keratinocytes. In this aim, we will use 0.5% gentamicin ointment rather than 0.1% and determine if the C7 response is more robust in these two patients. Based on our encouraging data shown in Figure 2, we will also treat intact skin and determine if higher dose gentamicin can prophylactically prevent frank skin blisters and erosions from forming.

Patients/Intervention: We will study two RDEB children tested in our previous trial for this study. In addition, we also have 2 other children and 2 other adult patients who have contacted us and wish to participate in this study (Table III). Two of these patients share the same mutations (R578X and R2814) as our current patients. We will select a total of four Test Sites (two with open wounds of at least 3 cm by 3 cm and two areas of intact skin nearby the Test Sites with blisters or erosions) and apply 0.5% gentamicin ointment two times a day under an occlusive dressing of the patients' choice for two weeks. This dose was used in combination with 15% paromomycin to ulcerative cutaneous leishmaniasis lesions once daily for 20 days in two publications without renal or ototoxicity detected.32,33 Safety and Efficacy: We will essentially assess in these patients the safety and efficacy parameters outlined in Aim #1 at months 1, 3 and 6 after topical application. Potential renal toxicity and ototoxicity will be monitored by creatinine clearance and pure tone audiometry at day 0 and at each follow up visit.

Aim 3: Evaluate the Safety and Efficacy of Intradermal Administration of Gentamicin with a MR2 Micro-needle Roller Device.

Overview and Rationale: In our current clinical trial outlined above, there were excellent C7 responses to intradermal injections of gentamicin solution in several patients (Table II). Nevertheless, these injections were very localized with diffusion of the drug probably less than an inch from the single injection site. Recent evidence has shown that a MR2 microneedle roller device can readily deliver lidocaine into the lower epidermis and upper papillary dermis over a wide area of skin with good efficacy, safety and excellent patient tolerance.34 This may be an opportunity to deliver intradermal gentamicin solution (40 mg/ml) to wide areas of skin in a simple manner that can be done in an outpatient office with minimal logistical issues and avoidance of systemic exposure to the drug. Advantages include the possibility of delivering in a "pulse " fashion significant levels of gentamicin to the papillary dermis over wide areas of skin with minimal discomfort and minimal systemic exposure of gentamicin. Avoiding systemic exposure would be valuable for RDEB patients who have ototoxicity or renal compromise, poor venous access or cannot tolerate intravenous infusions.

Patients \& Interventions: We will enroll two new RDEB patients (either adults or children) shown in Table III in this "proof-of-principle" study. Skin Test Sites will be areas measuring at least 40 cm2, and we will test one site of intact unwounded skin and one site of skin with lesions (blisters or erosions). The areas will be cleansed with 4% Hibiclens and then the MR2 Microneedle Roller Device passed over the area in two directions at 90 degrees. This device induces tiny micro-wounds in the skin to the depth of the papillary dermis. Gentamicin sterile solution (40 mg/ml) will then be liberally applied over the area and the area immediately occluded with a polyrurethane membrane (Opsite). Another equal sized Test Site of intact skin will be tested without prior intervention with the Microneedle Roller. The same gentamicin solution will be applied to the site and immediately occluded with Opsite. This will determine if the gentamicin solution can penetrate intact skin.

Safety and Efficacy: We will measure gentamicin in the serum of these patient 30 minutes after the microneedle delivery to determine if there is any systemic exposure when gentamicin is administered in this manner. Efficacy of the gentamicin administration will be assessed identically to those in Aim#1 by IF and immuno-EM analysis in conjunction with the patients keeping a daily diary of the treatment sites, weekly photographs of Test Sites, weekly staff interviews of the patients, and physician assessments at patient visits at months 1, 3 and 6 after administration of gentamicin. Potential renal toxicity and ototoxicity will be monitored by creatinine clearance and pure tone audiometry at day 0 and each follow up visit.

Arms:
arm 1: Topical gentamicin; Topical gentamicin with microneedle roller assistance; IV gentamicin. While the intervention is the same drug, the topical gentamicin is compounded into a 0.5% ointment and the IV gentamicin is prepared to 7.5 mg/kg body weight and administered over a 30 minutes.

Interventions: intervention 1: Gentamicin Sulfate
Intervention descriptions: intervention 1: Participants will be divided into three cohorts: topical gentamicin; Topical gentamicin with microneedle roller assistance; IV gentamicin. While the intervention is the same drug, the topical gentamicin is compounded into a 0.5% ointment and the IV gentamicin is prepared to 7.5 mg/kg body weight and administered over a 30 minutes.

Protocol document:
Prot_000.pdf: 
Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa Patients With Nonsense Mutations 
Gentamicin for RDEB  
NCT03012191             March 02, 2017 
1
 
 
 
 
 
 
 
 
 
 
Project Title: A Pilot Study of the Restoration of Full-Length Type VII Collagen in RDEB Patients  
with Nonsense Mutations After Topical, Intradermal and Intravenous Gentamicin Treatment 
 
HSIRB Title: Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa Patients With Nonsense 
Mutations 
 
HSIRB # HS-16-00788 
 
Brief Title: Gentamicin for RDEB 
 
NCT# NCT03012191 
 
March 02, 2017 
 
Principle Investigators: 
 
Mei Chen, Ph.D. 
Professor and Director of Research 
USC Laboratories for Investigative Dermatology 
USC /Norris Comprehensive Cancer Center 
University of Southern California 
1441 Eastlake Avenue, NOR Room 6322 
Los Angeles CA 90033 
Tel: 323-865-0621 
E-mail: chenm@usc.edu 
 
David T. Woodley, M.D. 
Professor and Emeritus Founding Chair 
Department of Dermatology 
USC /Norris Comprehensive Cancer Center 
University of Southern California 
1441 Eastlake Avenue, Topping Tower #3405 
Los Angeles CA 90033 
Tel: 323-865-0956 
E-mail: david.woodley@med.usc.edu 
 
Host Institution: 
University of Southern California  
Keck School Medicine of USC 
 
 


Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa Patients With Nonsense Mutations 
Gentamicin for RDEB  
NCT03012191             March 02, 2017 
2
 
 
 
Abstract: 
 
Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable, devastating, inherited skin disease caused 
by mutations in the COL7A1 gene that encodes for type VII collagen (C7), the major component of anchoring 
fibrils (AFs), structures that mediate epidermal-dermal adherence. Thirty percent of RDEB patients have 
nonsense mutations. We recently demonstrated in 5 such patients that intradermal and topical gentamicin 
induced “read-through” of their nonsense mutations and created robust and sustained new C7 and AFs at the 
dermal-epidermal junction (DEJ) of their skin and also improved wound closure and the quality of their skin. No 
untoward side effects occurred. Herein, we propose evaluating the safety and efficacy of intravenous gentamicin 
in these patients. We also propose optimizing the concentration and manner of delivery of topical gentamicin. 
The unambiguous milestones will be increased C7 and AFs in the patients’ DEJ, improved EB Disease Activity 
Scores, and absence of significant gentamicin side effects. 


Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa Patients With Nonsense Mutations 
Gentamicin for RDEB  
NCT03012191             March 02, 2017 
3
A. Background, Significance and Preliminary Data: 
Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited, incurable, mechano-bullous disease 
characterized by blisters, erosions and scarring of the skin and mucosa.1 RDEB is caused by mutations in the 
COL7A1 gene that encodes type VII collagen (C7), a major component of anchoring fibrils (AFs), structures that 
hold the two main layers of skin together - the epidermis and the dermis.2,3 Based largely on "pre-clinical" animal 
models, we and others envisioned several therapeutic strategies for RDEB. These include intradermal injections 
of allogeneic and RDEB gene-corrected dermal fibroblasts4,5, topical and intradermal administration of C7 itself6-
8, intradermal injection of viral vectors expressing full-length C7 and intravenous C7 or fibroblasts that home to 
RDEB skin wounds9-11, and transplantation of gene-corrected keratinocyte autografts.12,13 Recently, proof-of-
principle clinical trials have been conducted, including bone marrow/stem cell transplantation, intradermal 
injection of allogeneic fibroblasts and transplantation of gene-corrected keratinocyte autograft sheets onto RDEB 
skin wounds.14,15 These strategies are logistically arduous and not consistently effective or safe. The mainstays 
of RDEB treatment currently are only palliative and supportive measures.  
 
Over 800 distinct mutations (missense, frame-shift, insertion, deletion, and nonsense changes) have 
been identified in RDEB patients.16,17  Our recent search of an RDEB patient registry data base shows that the 
prevalence of nonsense mutations in RDEB approaches 30% (161 out of 506 patients),16,17 and these patients 
often have a more severe form of the  disease.37 Aminoglycoside antibiotics can read-through nonsense 
mutations and generate full-length, functional proteins in cystic fibrosis (CF), Duchennes' muscular dystrophy 
(DMD) and other genetic diseases with nonsense mutations18,19 Gentamicin is an aminoglycoside antibiotic that 
has been used for infectious diseases for decades. It also is the most effective aminoglycoside for reading 
through nonsense mutations in CF and 
DMD and can be delivered topically or 
intravenously.20-25 Using cultured skin 
cells from 4 RDEB patients with 
nonsense mutations, we recently 
published that aminoglycosides were 
able to induce PTC read-through in the 
COL7A1 gene and produce full-length 
C7.26 Importantly, aminoglycoside-
induced C7 reversed the abnormal 
RDEB cell phenotype and incorporated into the DEJ of skin equivalents. In addition, we showed that 
aminoglycosides were capable of promoting PTC read-through and inducing full-length C7 in human 293 cells 
transfected with C7 expression constructs harboring 22 different known RDEB nonsense mutations.26 With these 
exciting data in hand, we were fortunate to secure funding from Epidermolysis Bullous Research Partnership 
(EBRP) and translated these in vitro findings into patients. For this clinical trial, we recruited five patients with 
nonsense mutations from our 22 RDEB patient cohort27and treated them with topical and intradermal gentamicin 
(please see details of NCT02698735 on ClinicalTrials.gov). Table I summarizes the participants. The study was 
double-blinded and placebo-controlled at its onset. For the topical arm, gentamicin 0.1% ointment or the 
ointment vehicle were applied three times a day to two open erosion Test Sites of at least 2 x 2 cm in size for 
two weeks. For the intradermal arm, in the same patients, a sterile gentamicin solution (40 mgs/ml) or sterile 
saline placebo were injected intradermally into two skin Test Sites, remote from the topical Test Sites, on days 1 
and 2. On each day 200 μl (8 mgs) were injected into each site for a total dose of 16 mgs. Follow-up visits were 
at weeks 4 and 12. The patients completed a weekly telephone questionnaire. They also kept a daily diary and 
photographed their skin test sites once a week. A number of safety parameters were also assessed including a 
complete blood count, blood urea nitrogen, creatinine, electrolytes, liver function tests, pure-tone audiometry, 
and serum ELISAs and indirect immunofluorescence for the possible development of anti-C7 autoantibodies. 
Briefly, the results of the safety parameters showed no untoward, gentamicin-induced altered audiometry or 
laboratory abnormalities. In addition, we did not detect any gentamicin-induced anti-C7 antibodies in the 
patients.  
All 4 patients trended towards clinical improvement in the gentamicin Test Sites with less new blister 
formation, rapid closure of existing erosions and improved skin durability compared to the placebo Test Sites. 
Figure 1A shows wound sites that received topical gentamicin or placebo from Patient #3. Topical gentamicin 
rapidly closed the Test Site wound and kept it closed for 12 weeks. Patients reported improved wound closure 
and skin quality. Figure 1B and 1C shows the patient's expression of C7 and AFs at her DEJ before and after 
topical or intradermal gentamicin. At Day 0, there was minimal or no C7 at the patient's DEJ. At the 4-week visit, 
there was minimal C7 at skin sites that received the topical and intradermal placebos. In contrast, there was 
abundant C7 expression at the DEJ of the patient's skin after topical or intradermal gentamicin at week 4. 
 
Table I. Summary of COL7A1 mutations, basal C7 levels and AFs in 5 RDEB 
patients.* indicate children; pAb, polyclonal antibody; mAb, monoclonal antibody.  
	
Patient 
ID	
	
Allele 1 / Allele 2	
	
C7 Level at 
DEJ by pAb	
	
C7 Level at 
DEJ by mAb 	
Anchoring Fibrils by EM	
Density	
Morphology	
1	
R578X / V168GfsX12	
2%	
Absent	
+	
Very thin and wispy	
2*	
R578X / R578X	
Absent	
Absent	
+	
Short, rudimentary	
3	
IVS17-2delA/ R2814X	
4.5%	
Absent	
+	
Thin and wispy	
4	
R236X / IVS85-1G>A	
11.6%	
2%	
+	
Thin and wispy	
5*	
R613X / R1683X	
<1%	
Absent	
+	
Thin, mild arching	
NHS	
- / -	
100%	
100%	
+++++	 Thick, banded, arching, looping	


Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa Patients With Nonsense Mutations 
Gentamicin for RDEB  
NCT03012191             March 02, 2017 
4
Surprisingly, gentamicin-induced C7 was stably retained at the DEJ at 12 weeks after initial application. 
Immuno-EM with a 
monoclonal antibody to C7 
revealed marked labeling 
of new AFs (arrows) in the 
patient's skin 4 weeks after 
topical gentamicin. At 12 
weeks, there were 
plentiful, well-labeled AFs 
in both the topical and 
intradermal gentamicin 
sites. In contrast, there 
were neither labeling nor 
visible AFs in the placebo 
treated sites. Table II 
summarizes the 
immunofluorescence (IF) 
results of the study. The 
numbers are the 
percentage of C7 
expression compared with 
normal skin. All 5 patients 
responded to topical and 
intradermal gentamicin 
with maximal C7 levels 
restored for each patient 
ranging from 19% to 166% 
of that of normal human 
skin. The study of our 5th patient is not yet completed.  In a preliminary pilot study with one patient, we noted 
that the topical application of gentamicin to unwounded skin resulted in new C7 in Patient #4 to 65% of that of 
normal skin (E2 in Figure 2). This raises the possibility that 
perhaps topical gentamicin can also penetrate intact skin.  
 
B: Innovation: Our clinical trial above showed that topical and 
intradermal gentamicin induced new C7 and new AFs. This has 
never been shown before. We now wish to determine if systemic 
intravenous gentamicin can improve multiple skin wounds 
simultaneously (including the esophagus) in the patients and 
perhaps prevent new blister formation. With this systemic 
administration and improvement in multiple wound sites, 
there may be improved patient clinical scores and 
improved patient quality of life. This type of systemic 
treatment was completed in CF and DMD patients,22-25 but 
never before in RDEB patients. Secondly, in this proposal, 
we wish to determine if we can optimize topical gentamicin 
delivery into the patient’s papillary dermis using a five-fold 
increase in the concentration of the drug or delivering the 
gentamicin with a micro-needle device.  
 
C: Approach: 
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 
 
Figure 1. Gentamicin improved wound closure and restored C7 and AFs in RDEB patient. (A) 
Shown are patient #3's wound sites treated with topical gentamicin (TOP-GENT) or placebo (TOP-
PLAC) at day 0, and weeks 4 and 12. (B) Cryosections from day 0, weeks 4 and 12 were subjected 
to immunofluorescence labeling with a monoclonal antibody against C7, after treatment with topical 
gentamicin (TOP-GENT), topical placebo vehicle (TOP-PLAC), intradermally injected gentamicin (ID-
GENT) or intradermally injected saline placebo (ID-PLAC). Note that topical or intradermal 
gentamicin induced strong C7 at the DEJ. (C) Skin sections were subjected to immuno-EM following 
incubation with a monoclonal antibody, NP185 to C7. Please note that in the placebo treated wounds 
(TOP-PLAC, top left panel), there was no labeling of the DEJ and no visible AFs. In contrast, skin 
biopsy samples from both topical (TOP-GENT) and intradermally injected gentamicin sites (ID-
GENT) exhibited heavy gold labeling of the lamina densa and anchoring plaques. In addition, many 
well-labeled, wheat-stack shaped AFs are readily observed (arrows). Scale bars: 250 nm. 
TOP - GENT 
ID - GENT 
C) 
Week 4 
Week 12 
TOP - PLAC 
TOP - GENT 
TOP - GENT 
TOP - PLAC 
ID - GENT 
ID - PLAC 
NORMAL 
Week 4 
Week 4 
Week 4 
Week 4 
Week 12 
Week 12 
Day 0 
e 
d 
Day 0 
Week 4 
Week 12 
A) 
PT 3 
B) 
TOP - GENT 
TOP - PLAC 
TOP - GENT 
ID - GENT 
TOP - GENT 
ID - GENT 
 
Table II: Image J analysis of IF results of C7 levels at 
DEJ in comparison with normal human skin.  
Patient 
ID 
Topical 1M 
Topical 3M 
ID 1M 
ID 3M 
Gent. 
Plac. 
Gent. 
Plac. 
Gent. 
Plac. 
Gent. 
Plac. 
1 
N/A 
0 
50 
0 
42 
0 
40 
0 
2 
19 
0 
18 
0 
19 
0 
1 
0 
3 
153 
0 
165 
0 
121 
0 
120 
0 
4 
166 
7 
50	
6 
108 
5 
51 
3 
5 
20 
0 
NYD 
NYD 
NYD 
NYD 
NYD 
NYD 
 
Figure 2. Gentamicin restored C7 in RDEB patient. (A) Shown 
are Patient #4's open (E1) and closed (E2) wound sites at day 0, 
and weeks 2 and 8 after topical gentamicin. (B) Cryosections 
from topical placebo and gentamicin at week 8 were subjected to 
IF with a polyclonal antibody against C7. (C) Image J analysis of 
IF results of C7 levels at DEJ compared to normal human skin.  
Week 2 
Day 0 
Week 8 
E1 
E2 
A) 
B) NORMAL 
E1 
E2 
TOP-PLAC 
C) 


Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa Patients With Nonsense Mutations 
Gentamicin for RDEB  
NCT03012191             March 02, 2017 
5
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).  


Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa Patients With Nonsense Mutations 
Gentamicin for RDEB  
NCT03012191             March 02, 2017 
6
 
Aim 2: Evaluate the Safety and Efficacy of Higher Dose Topical Gentamicin for RDEB Patients. 
Overview and Rationale: In our gentamicin clinical trial outlined earlier, there was significant variation in the 
production of new C7 and AFs, as shown in Table II. Patients #2 and #5 only restored C7 to 20% of normal skin 
in response to gentamicin. We only used one concentration of gentamicin, 0.1% ointment, and our in vitro 
studies with RDEB fibroblasts from these same two patients showed that the C7 response to gentamicin was 
dose-dependent.26 In these patients, there could have been sub-optimal dosing or sub-optimal access of the 
gentamicin to the patient’s dermal fibroblasts and basal keratinocytes. In this aim, we will use 0.5% gentamicin 
ointment rather than 0.1% and determine if the C7 response is more robust in these two patients. Based on our 
encouraging data shown in Figure 2, we will also treat intact skin and determine if higher dose gentamicin can 
prophylactically prevent frank skin blisters and erosions from forming. 
 
Patients/Intervention: We will study two RDEB children tested in 
our previous trial for this study. In addition, we also have 2 other children 
and 2 other adult patients who have contacted us and wish to participate 
in this study (Table III). Two of these patients share the same mutations 
(R578X and R2814) as our current patients. We will select a total of four 
Test Sites (two with open wounds of at least 3 cm by 3 cm and two areas 
of intact skin nearby the Test Sites with blisters or erosions) and apply 
0.5% gentamicin ointment two times a day under an occlusive dressing of 
the patients' choice for two weeks. This dose was used in combination 
with 15% paromomycin to ulcerative cutaneous leishmaniasis lesions 
once daily for 20 days in two publications without renal or ototoxicity detected.32,33  
 
Safety and Efficacy: We will essentially assess in these patients the safety and efficacy parameters 
outlined in Aim #1 at months 1, 3 and 6 after topical application. Potential renal toxicity and ototoxicity will be 
monitored by creatinine clearance and pure tone audiometry at day 0 and at each follow up visit. 
 
Aim 3: Evaluate the Safety and Efficacy of Intradermal Administration of Gentamicin with a MR2 Micro-
needle Roller Device. 
Overview and Rationale: In our current clinical trial outlined above, there were excellent C7 responses to 
intradermal injections of gentamicin solution in several patients (Table II). Nevertheless, these injections were 
very localized with diffusion of the drug probably less than an inch from the single injection site. Recent evidence 
has shown that a MR2 microneedle roller device can readily deliver lidocaine into the lower epidermis and upper 
papillary dermis over a wide area of skin with good efficacy, safety and excellent patient tolerance.34 This may 
be an opportunity to deliver intradermal gentamicin solution (40 mg/ml) to wide areas of skin in a simple manner 
that can be done in an outpatient office with minimal logistical issues and avoidance of systemic exposure to the 
drug. Advantages include the possibility of delivering in a "pulse " fashion significant levels of gentamicin to the 
papillary dermis over wide areas of skin with minimal discomfort and minimal systemic exposure of gentamicin. 
Avoiding systemic exposure would be valuable for RDEB patients who have ototoxicity or renal compromise, 
poor venous access or cannot tolerate intravenous infusions. 
 
Patients & Interventions: We will enroll two new RDEB patients (either adults or children) shown in 
Table III in this “proof-of-principle” study. Skin Test Sites will be areas measuring at least 40 cm2, and we will 
test one site of intact unwounded skin and one site of skin with lesions (blisters or erosions). The areas will be 
cleansed with 4% Hibiclens and then the MR2 Microneedle Roller Device passed over the area in two directions 
at 90 degrees. This device induces tiny micro-wounds in the skin to the depth of the papillary dermis. 
Gentamicin sterile solution (40 mg/ml) will then be liberally applied over the area and the area immediately 
occluded with a polyrurethane membrane (Opsite). Another equal sized Test Site of intact skin will be tested 
without prior intervention with the Microneedle Roller. The same gentamicin solution will be applied to the site 
and immediately occluded with Opsite. This will determine if the gentamicin solution can penetrate intact skin.  
 
Safety and Efficacy:  We will measure gentamicin in the serum of these patient 30 minutes after the 
microneedle delivery to determine if there is any systemic exposure when gentamicin is administered in this 
manner. Efficacy of the gentamicin administration will be assessed identically to those in Aim#1 by IF and 
immuno-EM analysis in conjunction with the patients keeping a daily diary of the treatment sites, weekly 
photographs of Test Sites, weekly staff interviews of the patients, and physician assessments at patient visits at 
months 1, 3 and 6 after administration of gentamicin. Potential renal toxicity and ototoxicity will be monitored by 
creatinine clearance and pure tone audiometry at day 0 and each follow up visit.  
D: References: 
(1) Lin AN and Carter DM (1992) Epidermolysis bullosa: basic and clinical aspects. NY: Springer-Verlag NY, Inc. 
 
Table III.  Four potential RDEB patients 
with nonsense mutations. *Children 
	
Patient 
ID	
	
Allele 1 / Allele 2	
6	
R578X/G1907D	
7	
R1933X/G1907D	
8*	
Y311X/IVS106+11+26del16	
9*	
R2814X/G2132D	


Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa Patients With Nonsense Mutations 
Gentamicin for RDEB  
NCT03012191             March 02, 2017 
7
(2) Uitto J and Christiano AM (1994) Arch Dermatol Res 287:16-22. (3) Sakai LY et al. (1986) J Cell Biol 103:1577-86. 
(4) Woodley DT et al. (2003) J Invest Dermatol 121:1021-8. (5) Ortiz-Urda S et al. (2003) J Clin Invest 111:251-5. 
(6) Woodley DT et al. (2004) Nat Med 10:693-5. (7) Remington J et al. (2009) Mol Ther 17:26-33. 
(8) Wang XY et al. (2013) Mol Ther 21:1335-44. (9) Woodley DT et al. (2013) J Invest Dermatol 133:1910-3.  
(10) Woodley DT et al. (2007) Mol Ther 15:628-35. (11) Hou Y et al. (2015) J Invest Dermatol 135:3060-72. 
(12) Chen M et al. (2002) Nat Genet 32:670-5. (13) Ortiz-Urda S et al. (2002) Nat Med 8:1166-70. 
(14) Wong T et al. (2008) J Invest Dermatol 128:2179-89. (15) Wagner JE et al. (2010) N Engl J Med 363:629-39. 
(16) Wertheim-Tysarowska K et al. (2012) Hum Mutat 33:327-31. (17) Van den Akker PC et al. (2011) Hum Mutat 32:1100-7. 
(18) Linde L and Kerem B (2008) Trends Genet 24:552–563. (19) Bidou L et al. (2012) Trends Mol Med 18:679-88.  
(20) Wilschanski M et al. (2003) N Engl J Med 349:1433-41.  
(21) Wilschanski M et al. (2000) Am J Respir Crit Care Med 181:860-5. (22) Malik V et al. (2010) Ann Neurol 67:771-80. 
(23) Sermet-Gaudelus I et al. (2007) BMC Med 5:5. (24) Clancy JP et al. (2001) Am J Respir Crit Care Med 163:1683-92. 
(25) Politano L et al. (2003) Acta Myol 22:15-21. (26) Cogan J et al. (2014). Mol Ther, 22: 1741-1752 
(27) Woodley DT et al. (2014) J Invest Dermatol 134:1138-40. (28) Bass KD et al. (1998) J Pediatr Surg 33:1104-7. 
(29) Loh CC et al. (2014) J Am Acad Dermatol 70:89-97. (30) Murrell DF (2015) Br J Dermatol 173:1357-8. 
(31) Thawer HA et al. (2002) Ostomy Wound Manage 10:46-53 
(32,33) Ben Salah, A et al. (2009) PLoS Negl Trop Dis 3:e432. and (2013) N Engl J Med 368:524-32. 
(34) Ornelas J et al. (2016) JAMA Dermatol 152:476-7. (35) Woodley DT et al. (1990) JAMA 263:3057-9.  
 
 

SAP_001.pdf: 
Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa Patients With Nonsense Mutations 
Gentamicin for RDEB  
NCT03012191 
March 02, 2017 
 
 
 
Project Title: A Pilot Study of the Restoration of Full-Length Type VII Collagen in RDEB Patients  
with Nonsense Mutations After Topical, Intradermal and Intravenous Gentamicin Treatment 
 
HSIRB Title: Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa Patients With Nonsense 
Mutations 
 
HSIRB # HS-16-00788 
 
Brief Title: Gentamicin for RDEB 
 
NCT# NCT03012191 
 
March 02, 2017 
 
Principle Investigators: 
 
Mei Chen, Ph.D. 
Professor and Director of Research 
USC Laboratories for Investigative Dermatology 
USC /Norris Comprehensive Cancer Center 
University of Southern California 
1441 Eastlake Avenue, NOR Room 6322 
Los Angeles CA 90033 
Tel: 323-865-0621 
E-mail: chenm@usc.edu 
 
David T. Woodley, M.D. 
Professor and Emeritus Founding Chair 
Department of Dermatology 
USC /Norris Comprehensive Cancer Center 
University of Southern California 
1441 Eastlake Avenue, Topping Tower #3405 
Los Angeles CA 90033 
Tel: 323-865-0956 
E-mail: david.woodley@med.usc.edu 
 
Host Institution: 
University of Southern California  
Keck School Medicine of USC 
 
 
 
 
 
 
 
 
 
 
 
 


Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa Patients With Nonsense Mutations 
Gentamicin for RDEB  
NCT03012191 
March 02, 2017 
 
 
 
Statistical Analysis Plan 
 
This pilot, proof-of-concept, study is an un-blinded, single cohort, open study of 7 RDEB patients with 
proven nonsense mutations in their COL7A1 gene.  All of the patients have had their keratinocytes and 
fibroblasts placed into culture, and the presence of gentamicin in the cultures induced read-through of their 
nonsense mutation and the production of full-length type VII collagen (C7) in vitro. They all have proven 
diminished C7 and anchoring fibrils (AFs) in the dermal-epidermal junction of their skin. This is a unique and 
ideal cohort for the questions that are posed in this grant proposal. The expression of C7 and AFs will be 
measured in biopsy specimens taken before and at 30, and 90 days after start of treatment with topical, 
intradermal, and intravenous gentamicin. Quantitative C7 expression by IF will be performed by computer-
assisted image analysis using NIH Image J software as previously described.  The patients' results will 
compared against their baseline expression and the expression of C7 in normal human skin, a positive 
control run with each experiment.  AFs will be assessed by immuno-electron microscopy and enumeration 
of AFs as performed in our previous study (Woodley DT et al., JAMA).  The results of C7 and AF expression 
levels and changes (as well as other biological measures reflecting the effects of gentamicin [e.g. changes 
in the sizes of lesions] and clinical (EBDASI scores) and patient reports will be summarized with plots to 
display trends over time and standard descriptive statistics (point estimates and associated 95% confidence 
intervals).  The co-primary efficacy endpoints will be  (1) “C7 success” defined as developing a C7 
expression level that is at least 35% of the normal control and (2) “AF success” defined as developing an 
AF expression level that is at least 35% of the normal control.  Historical data indicate that the likelihood of 
an increase of 35% or greater is extremely, extremely unlikely; hence such an increase in C7 or AF 
expression will be attributed to gentamicin activity. At the 30- and 90-day assessments, the number of 
patients with a successful increase in C7 expression and in AF expression will be reported, as well as the 
mean/median changes and associated 95% confidence intervals.  These analyses will be descriptive; the 
purpose is to document large, biologically/clinically important changes and to use this information to guide 
the design of future studies. 
 
Safety parameters including audiometry and standard routine laboratory tests for blood, liver, and kidney 
function will be performed and the normal limits are well established.   Adverse events and toxicities will be 
recorded for all patients and scored using the MedDRA 19.0 criteria; in particular, based on known toxicities 
associated with gentamicin, calculated creatinine clearance and pure tone audiometry will be measured as 
described above, and based on these, “stop criteria” will be used to stop treatment.   All patients who begin 
treatment with gentamicin will be accounted for in all analyses.