Clinical trial NCT02698735
Conditions: Recessive Dystrophic Epidermolysis Bullosa
Sponsor: University of Southern California
Start date: 2016-02-25; Completion date: 2017-06-30
Enrollment: 5 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable, devastating, inherited skin disease for which there is only supportive care. RDEB is due to mutations in COL7A1 gene that encodes for type VII collagen (C7), the major component of anchoring fibrils (AFs) mediating epidermal-dermal adherence. Approximately 20% of COL7A1 mutations are nonsense mutations leading to premature stop codons and a truncated C7 with diminished function. The investigators demonstrated that aminoglycosides such as gentamicin readily induce premature termination codon (PTC) "read through" and produce biologically functional C7 in 22 reported COL7A1 nonsense mutations. Importantly, aminoglycoside-induced C7 reversed the abnormal RDEB cell phenotype and incorporated into the dermal-epidermal junction. Herein, the investigators propose the first clinical trial of gentamicin (topical and intradermal) in RDEB patients with nonsense mutations that the investigators have fully characterized. The milestones include increased C7 and AFs at the patients' dermal-epidermal junction and absence of significant gentamicin side effects.

Detailed description:
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.

Evaluation of RDEB intact skin treated by intradermal injections of gentamicin: Patients and Investigators will be blinded to the treatments of the Experimental Area and Control Area. Each week, the patient will evaluate the areas and grade them as follows: - 1 = new blister or erosion formation in the site, and +1 = no new blisters or erosions. At USC visits at 1 and 3 months, biopsies will be obtained from the sites and evaluated as above for the expression of C7 at the DEJ and enumeration of AFs.

Evaluation of Patients' Safety: Patients will have baseline histories, review of systems (ROS), vital signs (including weight) and physical examinations on Day 0 before treatment, and at Day 1 (one day after treatment) and then at 1 and 3 months during their visits to USC. At these same time points, blood tests will be performed and include a complete blood count, electrolytes, liver enzymes, erythrocyte sedimentation rate, creatinine, and BUN. Creatinine clearances will be also calculated, and the patient's treatment sites will be evaluated for erythema, edema, blistering and erosions. At Day 0, 1 month and 3 months after treatment, audiometry evaluations will be done. Patients will complete a ROS questionnaire daily at home, and be telephoned weekly by a USC study member inquiring about any new signs, symptoms, or ROS changes.

E. Characterization of Immune Responses to Gentamicin-Induced C7: Our study patients all express lower levels of C7 including the NC1 domain, which is the most antigenic domain of C7. Therefore, with the exception of patient B, we doubt that reading through the patient's PTC will induce a protein that is viewed as antigenic by the patient's immune system. Nevertheless, we hope that gentamicin will generate a functional, rather than non-functional, species of C7. To evaluate if this change triggers an immune response and generates anti-C7 antibodies, patient serum will be obtained at baseline, 1 month, and 3 months for evaluation of anti-C7 antibodies by salt-split IIF and ELISA. If a patient develops antibodies to C7, we will then examine their skin for C7 antibody deposits by DIF.

Arms: arm 1: Gentamicin antibiotic arm 2: Vehicle control
Interventions: intervention 1: Gentamicin intervention 2: Placebo
Intervention descriptions: intervention 1: Gentamicin was either formulated into a 0.1% ointment or solutions for injection were purchased directly from suppliers. intervention 2: There are two placebos used in this study. The ointment vehicle (same as used to formulate gentamicin) and vehicle solution for injection.

Protocol documents:
Prot_000.pdf: 
 
1 
 
 
 
 
 
 
 
Project Title: A Pilot Study of the Effect of Topical and  Intradermal Gentamicin on the Restoration of Full-
Length Type VII Collagen in RDEB Patients Carrying Nonsense Mutations  
 
 
 
 
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 
 


 
2 
 
 
 
Abstract: 
 
Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable, devastating, inherited skin disease for 
which there is only supportive care. RDEB is due to mutations in COL7A1 gene that encodes for type VII 
collagen (C7), the major component of anchoring fibrils (AFs) mediating epidermal-dermal adherence. 
Approximately 20% of COL7A1 mutations are nonsense mutations leading to premature stop codons and a 
truncated C7 with diminished function. We demonstrated that aminoglycosides such as gentamicin readily 
induce PTC “read through” and produce biologically functional C7 in 22 reported COL7A1 nonsense 
mutations. Importantly, aminoglycoside-induced C7 reversed the abnormal RDEB cell phenotype and 
incorporated into the dermal-epidermal junction. Herein, we propose the first clinical trial of gentamicin (topical 
and intradermal) in RDEB patients with nonsense mutations that we have fully characterized. The milestones 
include increased C7 and AFs at the patients’ dermal-epidermal junction, improved EB Disease Activity Score, 
and absence of significant gentamicin side effects. 


 
3 
 
Figure 1. (A&B) Gentamicin mediated dose-dependent induction of full-length C7 in RDEB Cells. RDEB 
keratinocytes (A) or fibroblasts (B), were treated with increasing concentrations of gentamicin and cell lysates 
were prepared and then subjected to 4-12% SDS-PAGE, followed by immunoblot analysis with an antibody to 
C7 or anti--tubulin (loading control) antibody. (C) Aminoglycoside-induced C7 reverses fibroblast 
hypermotility. RDEB fibroblasts were either untreated or treated with gentamicin (400 µg/ml) for 48 hours and 
then subjected to a colloidal gold salt migration assay. Note that hypermotility of both RDEB fibroblasts were 
reversed after gentamicin treatment. (D) Aminoglycoside-induced C7 incorporates into the DEJ of in vitro 
skin equivalents (SEs). The cryosections were subjected to IF using anti-C7 antibody. Left panels are SEs 
composed of untreated RDEB keratinocytes combined with untreated RDEB fibroblasts. Middle panels are SEs 
composed of RDEB keratinocytes treated with gentamicin (400 g/ml) combined with RDEB fibroblasts. Right 
panels are SEs composed of normal skin cells. 
Background and Significance: 
Recessive Dystrophic Epidermolysis Bullosa (RDEB) is an inherited skin disease characterized by skin fragility 
blisters, erosions, and scarring.1 RDEB is caused by mutations in the COL7A1 gene that encodes type VII 
collagen (C7), the major component of anchoring fibrils (AFs).2,3 Various therapeutic strategies have been 
envisioned for RDEB based on preclinical animal models: intradermal injection of allogeneic dermal fibroblasts or 
gene corrected RDEB fibroblasts,4,5 intradermal injection of lentiviral vectors expressing C7,6 intradermal injection 
or topical application of recombinant C7,7-9 intravenous (IV) injection of C7 protein or fibroblasts,10,11 and 
transplantation of gene-corrected keratinocyte autografts.12,13 Recently, proof-of-principle clinical trials have been 
initiated, including bone marrow/stem cell transplantation and intradermal injection of allogeneic fibroblasts.5,14 
None of these therapies are consistently effective, and some have associated morbidity and mortality.  
Over 700 distinct mutations have been identified in RDEB patients.15These mutations include missense, 
frame-shift, insertion, deletion, and nonsense changes. Between 10-25% of RDEB mutations are nonsense 
mutations that create a premature termination codon (PTC) resulting in a truncated, non-functional protein.  RDEB 
patients with nonsense mutations often have severe disease. Aminoglycoside antibiotics such as gentamicin have 
emerged as vanguard agents for the treatment of human genetic disorders due to their ability to suppress PTC 
mutations and restore synthesis of a full-length protein. They have been used successfully in a number of genetic 
disorders, such as cystic fibrosis (CF), Duchenne’s muscular dystrophy (DMD), and others.16-18  Gentamicin, has 
been used in humans for many years and is the most effective aminoglycoside for inducing PTC read-through. 
Topical gentamicin to the nasal epithelium of CF patients restores their mutated receptor, CFTR.19,20 Intravenous 
gentamicin has also improved disease in patients with CF and DMD.21,22  
 
Using RDEB keratinocytes and fibroblasts harboring nonsense mutations, we showed that aminoglycosides 
(including gentamicin) 
induced PTC read-
through and restored 
functional full length 
C7 that reversed the 
abnormal cell motility 
associated with 
RDEB (Figure 1A to 
1C).23 Using an in 
vitro, three-
dimensional, skin 
equivalent model, we 
showed that C7 
produced by 
aminoglycoside-
treated RDEB cells 
incorporated into the 
dermal-epidermal 
junction (DEJ) of the 
skin equivalents 
(Figure 1D). We also 
generated 22 known 
RDEB nonsense 
mutations via site-
directed mutagenesis 
and transfected these 
constructs into human 
293 cells. Aminoglycoside treatment of these cells induced PTC read-through and induction of full-length C7 with 
all 22 RDEB nonsense mutations. This is the first study demonstrating that aminoglycosides can induce PTC 
read-through and restore functional C7 in RDEB caused by nonsense mutations. Our results suggest that 
aminoglycoside-mediated nonsense mutation suppression may provide a novel, non-invasive treatment for a sub-
set of RDEB patients. The goal of this proposal is to determine if topical, intradermal, and intravenous gentamicin 
can suppress PTCs and induce full length, functional C7 in RDEB patients with nonsense mutations and to 
evaluate the safety, potential immune responses, efficacy, and duration of any beneficial effects. Our over-arching 
HYPOTHESIS is that the administration of gentamicin to RDEB patients will induce PTC read-through, create new 


 
4 
Patient
ID
Allele 1 / Allele 2
Clinical
Diagnosis
C7 Expression
at DEJ
Anchoring Fibrils by EM
Density
Morphology
A
G2517KfsX3 / G2517KfsX3
RDEB-sev,gen
Reduced
+
Very thin and wispy
B
R578X / R578X
RDEB-sev,gen
Absent
+
Short, rudimentary
C
R613X / R1683X
RDEB-sev,gen
Reduced
+
Thin, mild arching
D
IVS17-2delA/ R2814X
RDEB-sev,gen
Reduced
+
Thin and wispy
E
R236X / IVS85-1G>A
RDEB-sev,gen
Reduced
+
Thin and wispy
F
R578X / G1907D
RDEB-I
Normal
+++
Non-banded, arching
NHS
- / -
Normal
Normal
+++++
Thick, banded, arching, looping
C7 and AFs at the DEJ, and improve the clinical status of these patients. We also hypothesize that clinical 
improvement will correlate with the level of gentamicin-induced PTC readthrough and C7 production. Finally, we 
hypothesize that the gentamicin–induced C7 will persist for several months and will not cause any adverse effects. 
Based on these hypotheses we propose two specific aims:  
 
Aim: Evaluate the safety and efficacy of topical gentamicin for RDEB patients.   
A. Identification of 5 RDEB patients with nonsense mutations for a pilot clinical trial. 
We recently identified and characterized 22 bona fide RDEB patients clinically, genetically, pathologically, 
ultrastructurally and immunologically (Table 1).24 All 22 RDEB patients had abnormal AFs with reduced density 
and significant scarring of their skin. We placed these patients’ keratinocytes and fibroblasts into tissue culture 
allowing us to study their cellular phenotype and gene expression profiles.  Five of 22 RDEB patients contain one 
or two alleles of nonsense mutations (highlighted by bold in Table 1). We recently treated cultured fibroblasts of 
two patients (B and C) of these five patients with gentamicin.23 The gentamicin treatment resulted in read-through 
of the nonsense mutations and generation of full-length C7 (See Figure 1B RDEB3 and RDEB4). In CF patients, 
there is a good correlation between the in vitro cell culture read-through levels and in vivo efficacy suggesting that 
in vitro studies should be done first to predict which patients are most likely to respond and benefit from 
aminoglycosides.22 Therefore, we will treat the five gentamicin-responding RDEB patients with nonsense 
mutations identified in our recent study. We expect gentamicin treatment to be more effective in patients B and C 
who have two alleles with nonsense mutations than patients D to F who only have one nonsense mutated allele. 
In addition, one RDEB patient who has PTCs from frame shift mutations (A) will be treated to serve as a negative 
control patient since gentamicin is specific for nonsense mutation induced - PTCs. The Exclusion Criteria for these 
6 RDEB patients will include pregnant women, breast feeding women, pre-existing auditory impairment, renal 
impairment, allergies to aminoglycosides, and use of aminoglyosides within the past 6 months. The acceptable 
renal parameters with be a normal BUN, normal plasma creatinine and a normal estimated creatinine clearance. 
  
B. C7 and AF expression with topical and intradermal gentamicin treatment  
i. 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 g/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.  
 
ii. 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 l (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 gs/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.  


 
5 
 
iii. 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 icron 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.  
 
C. 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.  
 
Evaluation of RDEB intact skin treated by intradermal injections of gentamicin:  Patients and 
Investigators will be blinded to the treatments of the Experimental Area and Control Area. Each week, the 
patient will evaluate the areas and grade them as follows: - 1 = new blister or erosion formation in the site, and 
+1 = no new blisters or erosions. At USC visits at 1 and 3 months, biopsies will be obtained from the sites and 
evaluated as above for the expression of C7 at the DEJ and enumeration of AFs. 
 
D. Evaluation of Patients’ Safety: Patients will have baseline histories, review of systems (ROS), vital signs 
(including weight) and physical examinations on Day 0 before treatment, and at Day 1 (one day after treatment) 
and then at 1 and 3 months during their visits to USC. At these same time points, blood tests will be performed 
and include a complete blood count, electrolytes, liver enzymes, erythrocyte sedimentation rate, creatinine, and 
BUN. Creatinine clearances will be also calculated, and the patient's treatment sites will be evaluated for 
erythema, edema, blistering and erosions. At Day 0, 1 month and 3 months after treatment, audiometry 
evaluations will be done. Patients will complete a ROS questionnaire daily at home, and be telephoned weekly 
by a USC study member inquiring about any new signs, symptoms, or ROS changes. 
 
E. Characterization of Immune Responses to Gentamicin-Induced C7: Our study patients all express lower 
levels of C7 including the NC1 domain, which is the most antigenic domain of C7. Therefore, with the exception 
of patient B, we doubt that reading through the patient's PTC will induce a protein that is viewed as antigenic by 
the patient's immune system. Nevertheless, we hope that gentamicin will generate a functional, rather than non-
functional, species of C7. To evaluate if this change triggers an immune response and generates anti-C7 
antibodies, patient serum will be obtained at baseline, 1 month, and 3 months for evaluation of anti-C7 
antibodies by salt-split IIF and ELISA. If a patient develops antibodies to C7, we will then examine their skin for 
C7 antibody deposits by DIF. 
 
F. Discussion on Potential Toxicities Associated with Topical or Intradermal Gentamicin. 
In our in vitro skin cell cultures, read-through of non-sense mutations in the COL7A1 gene occurred at 
concentrations of gentamicin within the 0.2 - 0.4 mg/ml for cultured human keratinocytes and cultured human 
fibroblasts.  The commercially available FDA-approved topical gentamicin products (Gentamicin 0.1% Topical 
Cream) are 1 mg/ml, and they have been used for years in open skin wounds of patients with no untoward side 
effects such as ototoxicity or nephrotoxicity.  In fact, we have used these agents ourselves in a number of RDEB 
patients and have had no untoward side effects.  We believe that if there were a significant incidence of ototoxicity 
or nephrotoxicity from topical 0.1% gentamicin cream or ointment that these products would have been pulled 
from the market by the Federal Drug Administration (FDA) long ago. For infections in patients with normal renal 


 
6 
function, the recommended systemic gentamicin doses are between 3 mg - 5 mg per kilogram body weight per 
day with the duration of treatment being 10 days. In a 70 pound child (32 kilograms) such a dose would be 
between 96 mg and 160 mg per day or a total dose course between 960 mg and 1600 mg. 
 
One milliliter of ointment covers liberally a 4-cm2 area of skin or wound.  If we have one such wound or 4 cm2 
test sites and applied one milliliter of 0.1% gentamicin ointment to these sites the dose would one milligram 
per application.  If we apply this amount three times a day, the total daily dose to the skin would be 3 
milligrams per day.  If we apply the gentamicin for 14 days, the total dose will be 42 milligrams to the skin. 
Topical application of gentamicin to open wounds generates negligible amounts of gentamicin in the blood 
stream. Nevertheless, for arguments sake, lets say that 100% of the topically applied gentamicin went into the 
blood stream. Then, the worse case scenario would be that the subject would have a total systemic exposure 
of gentamicin of 42 milligrams vis a vis the minimal therapeutic dose of gentamicin of 960 mg.  Given that in 
reality only a very small amount of topical gentamicin enters the blood stream, we believe our proposed trial of 
topical 0.1% gentamicin ointment is very safe and will have minimal risk of nephrotoxicity.  
 
To confirm topical gentamicin safety with some objective data, a recent publication in the New England Journal of 
Medicine shows the safety of topically applied gentamicin. Please see the paper by Salah AB et al. entitled 
"Topical Paromycin With or Without Gentamicin for Cutaneous Leishmaniasis", New England Journal of Medicine 
volume 368, pages 524 - 532, 2013.  These investigators applied gentamicin 0.5% cream (five times stronger than 
the commercially available topical 0.1% gentamicin cream we plan to use in our study) in combination with 15% 
paromomycin to ulcerative cutaneous leishmaniasis lesions once daily for 20 days. There was no evidence of any 
renal toxicity or ototoxicity. 
 
For the intradermal gentamicin injection arm of the study, we will be injecting experimental areas totaling 4 cm2.  
These areas will be injected intradermally with 200 microliters of commercially available gentamicin solution, 0.04 
milligrams per microliter. Each injection will be 8 milligrams. If we inject once a day for a total of 2 days, the total 
dose to each subject will be 16 milligrams. In reality, none or a small fraction of the intradermally injected 
gentamicin will enter the subject's blood stream. Nevertheless, if we assumed that 100% of the intradermal 
injections went to the patient's blood stream, the total possible dose of gentamicin to which the patient is exposed 
would be 16 milligrams. Again this is markedly lower than the minimal recommended therapeutic dose of 
gentamicin of 960 milligrams. Therefore, like our proposed experiments with topical gentamicin ointment, we 
believe the proposed experiments with intradermally injected gentamicin are extremely safe. 
 
We believe the proposed experiments with topical and intradermally injected gentamicin are very safe, particularly 
since only a small fraction, if any, of the gentamicin administered in these ways will enter the patient's blood 
stream. Nevertheless, as outlined in our experimental protocol, will carefully monitor the subjects' creatinine, BUN 
and creatinine clearance for any early signs of renal toxicity from the topically applied gentamicin ointment. 
Moreover, we will have a pediatric nephrologist Dr. Lawrence Opas, the Chief of Pediatrics at LAC + USC Medical 
Center and a seasoned pediatric nephrologist advise us throughout this study and review the six subjects' 
laboratory data.   
 
References: 
1. Lin, AN and Carter, DM (1992). Epidermolysis bullosa: basic and clinical aspects. New York: Springer-Verlag NY, Inc. 
2. Uitto, J and Christiano, AM (1994). Arch Dermatol Res 287: 16–22. 
3. Sakai, LY, Keene, DR, Morris, NP, Burgeson, RE (1986). J Cell Biol 103: 1577–1586. 
4. Woodley, DT et al. (2003). J Invest Dermatol 121: 1021–1028. 
5. Wong, T et al. (2008). J Invest Dermatol 128: 2179–2189. 
6. Woodley, DT et al. (2004). Mol Ther 10: 318–326. 
7. Woodley, DT et al. (2004). Nat Med 10: 693-5. 
8. Remington, J et al. (2009). Mol Ther 17: 26–33. 
9. Wang XY et al. (2013) Mol Ther, 21:1335-1344.  
10. Woodley, DT et al. (2013). J Invest Dermatol 133: 1910–1913. 
11. Woodley, DT et al. (2007). Mol Ther 15: 628–635. 
12. Chen, M et al. (2002). Nat Genet 32: 670–675. 
13. Ortiz-Urda, S et al. (2002). Nat Med 8: 1166–70. 
14. Wagner, JE et al. (2010). N Engl J Med 363: 629–639. 
15. Wertheim-Tysarowska, K et al. (2012). Hum Mutat 33: 327–331. 
16. Barton-Davis, ER et al. (1999). J Clin Invest 104: 375–81. 


 
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17. Bedwell, DM et al. (1997). Nat Med 3: 1280–4. 
18. Linde, L and Kerem, B (2008). Trends Genet 24: 552–563. 
19. Wilschanski, M  et al. (2003).. N Engl J Med 349: 1433-1441. 
20. Wilschanski, M et al. (2000). Am J Respir Crit Care Med 181: 860-865. 
21. Malik, V et al. (2010). Annals of Neurology 67: 771-780. 
22. Sermet-Gaudelus, et al. (2007). BMC Medicine 5: 5. 
23. Cogan J et al. (2014). Mol Ther, 22: 1741-1752 
24. Woodley, DT et al. (2014). J Invest Dermatol 134:1138-40  
25. Loh CC et al. (2014) J Am Acad Dermatol 70:89-97 
 
 
Timeline, Milestones, Expected Measurable Outcomes and Deliverables: 
Timeline:  This is an 12-month grant. We will enroll 6 RDEB patients (5 with nonsense mutations and one 
without a nonsense mutation) that we have characterized and published before as shown in Table I. We will re-
evaluate their clinical disease, re-assess their RDEB disease classification, assess their basal levels of C7 and 
AFs, and ensure that they do not have any of our exclusion criteria. We will commence the study with Aim 1 to 
evaluate the safety and efficacy of topical and intradermal gentamicin for RDEB patients. Aim 1 will be 
completed in 12 months.  
 
11/1/15 – 12/26/15: Write and submit an IRB for USC institutional approval and prepare RDEB patients for the 
trial.  
1/1/16 – 3/31/16: Invite 6 RDEB patients for initial screening and begin Aim 1. We will also analyze the patients' 
baseline biopsies for C7 and AF expression.  
4/1/16 – 6/30/16: Patients will return to USC for a one-month follow-up visit, evaluation, photography paired with 
computer-assisted planimetry, and skin biopsies to evaluate C7 and AF expression.  
7/1/16 – 9/30/16: Patients will return to USC for a three-month follow-up visit, evaluation, photography paired 
with computer-assisted planimetry, and skin biopsies to evaluate C7 and AF expression. A data analysis of Aim 
1 will be completed.  
10/1/16 – 10/30/16: We will write a final report and a manuscript for publication. 
 
Milestones:  By 12 months into the grant we will have identified and completed the evaluation of 6 RDEB 
patients who have received topical and intradermal injections of gentamicin.  
 
Expected Measurable Outcomes and Deliverables: Our Outcome-Safety Parameters are un-ambiguous.  The 
patients’ vital signs, physical examinations, review of systems, diaries, weekly telephone interviews, and face-to-
face evaluations are all readily measurable and easy to document. Possible renal toxicity and ototoxicity from 
gentamicin exposure will be ascertained by means of serial creatine clearances, audiometry results, and serial 
signs and symptoms as reported by the patients. These parameters are deliverable with no technical hurdles.  
 
Our Outcome Laboratory Parameters are objective and without technical hurdles. The patients either will or will 
not have increased C7 and AFs at the DEJ of gentamicin Treated Sites compared with vehicle Control Sites 
when evaluated by straight forward, objective measures of fluorescence intensity according to Wong and co-
workers5 and computer-assisted electron micrographs. These parameters are objective and without technical 
hurdles. We will define “a significant restoration” of both functional C7 and AFs as an increase of 35% or more 
above baseline. This will be the threshold for our Minimal Pharmacological Dose (MPD).  We chose this criterion 
based on the following: 1) A morphometric analysis of AF numbers in RDEB skin revealed a decrease of at least 
77% below that of normal skin. 2) Studies with RDEB-like C7 knockout mice showed that restoration of AF 
numbers to 35% of normal was sufficient for reasonable epidermal-dermal adherence, and 3) Family members 
of RDEB patients who are heterozygous carriers of a COL7A1 null mutations and have 50% of the normal 
complement of C7 and AFs are phenotypically normal with no skin fragility or mechanobullous disease.  Taken 
together, these data indicate that about 35% of the normal levels of C7 and AFs are necessary for good 
epidermal-dermal adherence, and this should be the goal of our gentamicin therapy.  
 
Our Outcome Clinical Parameters are readily deliverable and without technical hurdles, but do rely on some 
blinded patient and blinded physician evaluator’s subjective measurements. Because the patients will not be 
aware of which sites received gentamicin or vehicle, they will be blinded. Therefore, the patient diaries of new 


 
8 
lesions and comparisons between sites should be valid.  Weekly telephone contact by us should reinforce the 
patients’ compliance in documenting these parameters. 

SAP_001.pdf: 
Statistical Analysis Plan 
 
This pilot, proof-of-concept, study is an double blinded placebo controlled study of 5 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 intravenous 
gentamicin. Quantitative C7 expression by IF will be performed by computer-assisted image analysis using 
NIH Image J software as previously described.16  The patients' results will compared against 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).50  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 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. To obtain statistical significance concerning the type VII collagen induction in 
response to gentamicin treatment, we will compile mean average fluorescence from 6 independent 
evaluations for C7 expression from each patient biopsy at 1 month after receiving either topical or 
intradermal gentamicin or placebo and determined statistical significance using 2-tailed Welch’s (unequal 
variance) t test. These tests will be performed for both intravenous and topical treatments, independently. A 
P value of less than 0.05 will be considered significant. 
 
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.