Phase I/II Clinical Intramuscular Gene Transfer of rAAV1.CMV.huFollistatin344 Trial to Patients With Duchenne Muscular Dystrophy
Phase: PHASE1
Conditions: Duchenne Muscular Dystrophy
Sponsor: Jerry R. Mendell

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
1 
Protocol Version 5.0 – 16January2015 
 
 
 
 
 
 
 
 
 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 
trial to patients with Duchenne muscular dystrophy  
C L I N I C A L  P R O T O C O L  
IND#: 14845 
Follistatin gene therapy trial to include boys with Duchenne muscular dystrophy 
(DMD). 
Version: 5.0 (16 January 2015) 
Sponsor Investigator: Jerry R. Mendell, M.D. 
 
The Research Institute at Nationwide Children’s Hospital                            
Center for Gene Therapy 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
2 
Protocol Version 5.0 – 16January2015 
TABLE OF CONTENTS 
1.0 PROTOCOL SYNOPSIS .................................................................................................................... 4 
2.0 ABSTRACT ........................................................................................................................................ 6 
3.0 CLINICAL TRIAL AND PRINCIPAL INVESTIGATOR ................................................................ 6 
4.0 SPECIFIC AIMS ................................................................................................................................ 6 
4.1 Primary Objective ........................................................................................................................... 6 
4.2 Secondary Objective ....................................................................................................................... 7 
5.0 BACKGROUND AND SIGNIFICANCE .......................................................................................... 7 
6.0 CLINICAL RESEARCH PLAN ....................................................................................................... 12 
6.1. Study Population .......................................................................................................................... 12 
6.2. Pre-Treatment Assessment ........................................................................................................... 12 
6.3 Protocol for Gene Transfer ........................................................................................................... 15 
6.4 Post Gene Transfer Monitoring ..................................................................................................... 16 
6.5 Assessment of Endpoints .............................................................................................................. 17 
6.6 Statistical Analysis ........................................................................................................................ 19 
6.7 Study Timeline .............................................................................................................................. 20 
7.0 TEST MATERIAL AND ADMINISTRATION .............................................................................. 21 
7.1 Description of Biological Product ................................................................................................. 21 
7.2 Dose Selection .............................................................................................................................. 21 
7.3 Dose Limiting Toxicity ................................................................................................................. 22 
7.4 Stopping and Discontinuation Rules ............................................................................................. 23 
7.5 Concomitant Medications and Therapies ...................................................................................... 24 
8.0 ADVERSE EVENT MONITORING AND REPORTING .............................................................. 24 
8.1 Definition of an Adverse Event .................................................................................................... 24 
8.2 Obligations of the Investigator ...................................................................................................... 26 
8.3 Safety Reporting ........................................................................................................................... 26 
8.4 Unexpected Adverse Events ......................................................................................................... 28 
8.5 Follow Up of Adverse Events ....................................................................................................... 28 
9.0 Long-Term Follow Up ...................................................................................................................... 28 
9.1 Adverse Event Reporting with Primary Care Physician ............................................................... 28 
10.0 Study Reports .................................................................................................................................. 29 
10.1 Final Study Report ...................................................................................................................... 29 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
3 
Protocol Version 5.0 – 16January2015 
10.2 Annual Study Reports ................................................................................................................. 29 
11.0 Human Subjects .............................................................................................................................. 30 
11.1 Sources of Material ..................................................................................................................... 30 
11.2 Potential Risks ............................................................................................................................ 30 
11.3 Adequacy of Protection against Risks......................................................................................... 31 
11.4 Potential Benefits of Research to Subjects and Others ............................................................... 33 
11.5 Inclusion of Minorities ................................................................................................................ 33 
12.0 DATA AND SAFETY MONITORING PLAN .................................................................... 34 
12.1 THE DATA SAFETY MONITORING BOARD ....................................................................... 34 
13.0 CLINICAL MONITORING OF THE STUDY .............................................................................. 36 
13.1 Data Management and Study Forms ........................................................................................... 37 
14.0 AGENT ACCOUNTABILITY ....................................................................................................... 37 
14.1 Handling of Investigational Agent .............................................................................................. 37 
14.2 Disposition of Study Agent ......................................................................................................... 38 
15.0 BRIEF DESCRIPTIONS OF RESEARCH AREAS ...................................................................... 38 
REFERENCES ....................................................................................................................................... 41 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
4 
Protocol Version 5.0 – 16January2015 
 
1.0 PROTOCOL SYNOPSIS 
 
Title 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to 
patients with Duchenne muscular dystrophy.   
Study Number 
N = 6 DMD boys 
Clinical Study Phase 
Phase I/IIa trial 
Number of Centers 
Single site (Nationwide Children’s Hospital)  
Study Objectives 
Safety and increase distance in 6 MWT 
Study Design 
rAAV1.CMV.huFollistatin344 injections to gluteus muscles, quadriceps, tibialis anterior  
Patient Population 
Inclusion Criteria 
1. Age 7 or older 
2. Confirmed DMD gene mutations  
3. Impaired muscle function based on clinical evidence including difficulty 
climbing stairs, getting from the floor (Gowers’ sign), and weakness of 
individual muscles of extremities 
4. Males of any ethnic group will be eligible 
5. Ability to cooperate with study procedures including muscle testing. 
6. Willingness of sexually active subjects with reproductive capacity to practice 
reliable method of contraception  
7. Subjects must be on stable dose of prednisone or deflazacort for three months at 
time of enrollment.  Subjects will continue the steroid dose  post gene transfer as 
part of the standard of care for DMD patients. Dose adjustment could be 
considered if there are adverse effects.   
Exclusion Criteria 
8. Active viral infection based on clinical observations. 
9. The presence of a DMD gene mutation without weakness or loss of function  
10. Symptoms or signs of cardiomyopathy, including:  
o 
Dyspnea on exertion, pedal edema, shortness of breath upon lying flat, 
or rales at the base of the lungs 
o 
Echocardiogram with ejection fraction below 40% 
11. Serological evidence of HIV infection, or Hepatitis A, B or C infection 
12. Diagnosis of (or ongoing treatment for) an autoimmune disease  
13. Concomitant illness or requirement for chronic drug treatment that in the opinion 
of the PI creates unnecessary risks for gene transfer 
14. Subjects with   rAAV1 binding antibody titers > 1:50 as determined by ELISA 
immunoassay 
15. Abnormal laboratory values for liver, kidney, CBC, in the clinically significant 
range, based upon normal values in the Nationwide Children’s Hospital 
Laboratory 
Study Procedures 
The vector will be delivered to both limbs via multiple, direct intramuscular injections of 
rAAV1.CMV.huFollistin344; the number of injections per muscle will depend on the size 
of the patient.  A total dose of 2.4 X 1012 vg/kg (1.2 X 1012 vg/kg/limb) will be delivered 
to the lower limbs of 6 DMD subjects.  
Patients will also receive conscious sedation by an anesthesiologist at Nationwide 
Children’s Hospital for the injections.  
Primary Outcome 
Safety is a primary outcome for this clinical gene transfer trial. 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
5 
Protocol Version 5.0 – 16January2015 
Secondary Outcomes 
• 
The distance walked on the 6MWT is the major functional outcome to be evaluated. 
Other tests include time to walk and run, strength test, functional test, and reaching 
ability.   
• 
MRI studies of leg muscles will be done pre and post treatment (6, 12 and 24 
months) to study muscle size, fibrosis and fat content.   
• 
EIM measurement will quantify the pathological status of muscles.  
• 
Muscle biopsies on quadriceps muscles (a muscle biopsy on one leg at baseline 
screening visit and the post gene transfer biopsy on the opposite leg at day 180), will 
include general histological evaluation. Assessment will consist of morphometric 
analyses for  quantification of fiber size, fibrosis, inflammatory process, satellite 
cells and regeneration. Muscle tissue obtained at biopsy will also be assessed for 
viral DNA (qPCR), and follistatin transgene expression.  . 
Study Duration 
We will evaluate short-term safety over a two year period.  Subjects will be tested at 
baseline and return for follow up visits on days 7, 14, 30, 45, 60, 90, 180, and 9, 12, 18 
and 24 months post-gene transfer.  
Sample Size 
Six DMD patients will receive rAAV1.CMV.huFollistatin344 to both limbs by multiple 
injections to gluteal muscles, quadriceps and tibialis anterior muscles. 
Statistical Analysis 
This is a phase I/IIA trial, with safety as the primary measure. For each measure based on 
differences between pre- and post-gene transfer examinations (clinical) evaluated by 
paired t test, with a p value of < 0.05 indicating significance.   
Long-term follow-up 
We will follow the most recent FDA guidance with regard to long-term subject follow up 
post gene transfer.  As indicated by the guidelines, our proposed vector has a very low 
probability of gene transfer-related delayed adverse events.  We will, however, evaluate 
short-term safety over a two-year period that incorporates the active phase of the protocol. 
If newly identified risks are associated with our product, or if the subjects suffer any 
adverse events during this period, we will initiate a long-term follow-up according to the 
FDA guidelines.   
 
 
 
 
 
 
 
 
 
 
 
 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
6 
Protocol Version 5.0 – 16January2015 
 
2.0 ABSTRACT 
The proposed clinical trial is an outgrowth of the safety record and functional improvement seen 
in the BMD follistatin gene therapy trial.  In this study we propose to inject 
AAV1.CMV.huFS344 at a total dose of 2.4 X 1012 vg/kg to six DMD patients.  This dose will be 
divided between gluteal muscles, quadriceps and tibialis anterior.  This is a wider distribution of 
vector than given to BMD patients, who overall improved the distance walked on the 6MWT 
without adverse events related to viral transduction into a single muscle.  
The primary objective of this study is safety and endpoints will include hematology, serum 
chemistry, urinalysis, immunologic response to rAAV1 and follistatin, and reported history and 
observations of symptoms.  Efficacy measures will be used as secondary outcomes and include 
the distance walked on the 6MWT, functional tests by PT, life quality questionnaire, MRI, EIM, 
and muscle biopsy.  Subject will have follow up visits on days 7, 14, 30, 45, 60, 90, 180 and 9, 
12, 18 and 24 months post-gene transfer.      
3.0 CLINICAL TRIAL AND PRINCIPAL INVESTIGATOR 
The study will be carried out at the Research Institute at Nationwide Children’s Hospital.  Dr 
Mendell, Professor of Pediatrics and Neurology, Director of the Center for Gene Therapy at the 
Research Institute at Nationwide Children’s Hospital, Columbus, OH and Director of the 
Neuromuscular Disease Center, will serve as Principal Investigator.  Dr Mendell brings to this 
trial more than forty years of clinical trial experience in neuromuscular disorders.  With regard to 
gene therapy, he served as principal investigator for the first viral mediated gene transfer for 
muscular dystrophy (Duchenne muscular dystrophy) (DMD, IND BB-IND 12936 Serial # 004 
using a hybrid AAV2.5 consisting of AAV2 with 5 amino acid substitutions from AAV1, CMV 
promoter, and mini-dystrophin).1  In DMD, rAAV.2.5.CMV.mini-dystrophin was delivered by 
intramuscular injection to the biceps muscle.  In a subsequent gene transfer study for limb girdle 
muscular dystrophy, type 2D subjects with α-SG deficiency received intramuscular injections to 
the extensor digitorum brevis muscle using rAAV1.tMCK.α-SG  (IND BB-IND 13434 Serial # 
002)2, 3.  He also holds an approved IND (#14845) for AAV1.CMV.follistatin gene therapy in 
Becker muscular dystrophy (BMD) and sporadic inclusion body myositis (sIBM), a study that is 
currently underway with intramuscular injections to the quadriceps muscles bilaterally.  No 
adverse events have been encountered in any of these trials.   
4.0 SPECIFIC AIMS 
4.1 PRIMARY OBJECTIVE 
 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
7 
Protocol Version 5.0 – 16January2015 
Determine the safety of intramuscular administration of rAAV1.CMV.huFS344 for DMD 
patients via direct intramuscular injection (IM protocol) to the gluteal muscles, quadriceps and 
tibialis anterior. 
4.2 SECONDARY OBJECTIVE 
Efficacy measures will be used as secondary outcomes and include the distance walked on the 
6MWT, functional tests by PT, life quality questionnaire, MRI, EIM, and muscle biopsy.  
Subject will have follow up visits on days 7, 14, 30, 45, 60, 90, 180 and 9, 12, 18 and 24 months 
post-gene transfer.         
5.0 BACKGROUND AND SIGNIFICANCE 
DMD is the most common, severe childhood form of muscular dystrophy.  Inheritance follows 
an X-linked recessive pattern.  Birth prevalence has been estimated at 1 in 5000 live male births.4  
Approximately one-third of cases represent new mutations of the DMD gene with the remaining 
inherited on the X chromosome from a carrier mother.  Questions usually begin to surface 
between ages 3 to 5 regarding reduced motor skills that alert a need for diagnostic evaluation.  
DMD is relentlessly progressive with loss of ambulation by age 12.5  Historically patients died 
from respiratory complications.  Now, a variety of factors protect the respiratory system related 
to improved supportive equipment, antibiotics, vaccines, and other ancillary methods.6  
Prolonging life unmasks decline in cardiac function with complications of dilated 
cardiomyopathy.  This poses further clinical challenges and a need for recognition and medical 
intervention that did not previously exist.  Non-progressive cognitive dysfunction might be 
present in DMD and BMD.  
 
Disease Pathogenesis 
More than 20 years ago the DMD gene was cloned defining the molecular basis of the disease.7  
The identification of the dystrophin as the deficient protein followed closely on the heels of this 
discovery.8  Dystrophin is a 427kDa cytoskeletal protein required for muscle fiber stability.  Loss 
of this protein results in susceptibility to repeated cycles of necrosis and regeneration with 
satellite cell depletion, diminished regenerative capacity of the muscle, ending in fat and 
connective tissue replacement (fibrosis). The mutation spectrum within the DMD gene reveals 
that deletions of one or more exons are found in ~65% of cases clustered in two hotspot regions.9  
Originally multiplex PCR kits were developed that were able to detect 95%-98% of all 
deletions10, 11.  Detection of duplications, representing about 6% of the DMD mutations, initially 
required Southern blots to detect.  Overtime the demand for more rapid, less expensive detection 
methods have encouraged the introduction of additional tools to identify the full spectrum of 
mutations (deletions, duplications, splice-site and point mutations).  Multiplex ligation-
dependent probe amplification (MLPA)12 or multiplex amplifiable probe hybridization 
(MAPH)13 will screen all exons providing detection of most deletions and duplications.  If 
MLPA or MAPH are negative, the gene should be scanned for subexonic rearrangements or 
point mutations using DNA sequence analysis.14  This has become more than an academic 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
8 
Protocol Version 5.0 – 16January2015 
exercise because of treatment paradigms that depend on the full characterization of the mutation 
endpoints that help establish if patients are candidates for molecular therapies. 
 
Treatment for DMD 
Despite virtually hundreds of clinical trials in DMD, only one treatment has consistently 
demonstrated efficacy.  Unequivocal evidence for glucocorticoid-induced improvement was 
established through a double-blind, randomized controlled trial in a large cohort of subjects 
(n=103).15 Patients received 0.75 mg /kg/day or 1.5 mg/kg/day versus placebo.  At six months 
there was approximately equal efficacy for the high and low dose prednisone groups compared to 
placebo in muscle strength (manual muscle testing score), the time needed to rise from supine to 
a standing (prednisone 3.4 vs. placebo 6.2 seconds), to walk 9 m (prednisone 7.0 vs. placebo 9.7 
seconds), to climb four stairs (prednisone 4.0 vs. placebo 7.1 seconds), and in forced vital 
capacity (prednisone 1.7 vs. placebo 1.5 liters) (P<0.001 for all comparisons).  Similar results 
were later reported with deflazacort (0.9 mg/kg/day), an alternative, sodium-sparing 
corticosteroid that was also shown to prolong ambulation (untreated DMD patients stopped 
walking at 9.8 + 1.8 years compared to deflazacort-treated at 12.3 + 2.7 years, P<.005).16  A 
dose-related effect was observed in two follow up studies that demonstrated lesser but similar 
benefits using prednisone 0.30 and 0.35 mg/kg/day17, 18  A weekend dosing regimen (10 
mg/kg/wk: half on Saturday and half on Sunday) showed equal efficacy to daily steroids.19  
Additional follow up studies reported that the number of boys having scoliosis surgery in treated 
groups was significantly less than untreated boys (P < 0.05);20, 21 and treatment extended 
independent ambulation by 3.3 years compared to untreated (9.2 +1.48 years vs. 12.5+ 3.02 (p < 
0.0001).21  
The side-effect profile of glucocorticoids consistently demonstrates weight gain with a 
cushingoid appearance.  DMD boys on steroids are also at risk for hypertension, cataract 
formation, loss of bone density, vertebral compression fractures, and long bone fractures.  Long-
term administration may be limited in some cases by steroid-induced behavioral problems 
frequently observed with this class of drugs.  
Molecular therapies provide a promising alternative to glucocorticoids.  In addition to direct gene 
replacement, three molecular pharmacologic approaches have demonstrated promising findings 
representing current research aspirations.  These include exon skipping, readthrough of stop 
codon mutations, and replacement of dystrophin using utrophin, a dystrophin-like cytoskeletal 
protein.  The presence of revertants fibers encouraged scientists to develop a pharmacologic path 
to reproduce and expand this spontaneous natural achievement in order to generate dystrophin 
expression levels that would result in clinically meaningful outcomes.  
Exon skipping is well underway in clinical trials but is still early in demonstration of efficacy. 
Limitations include development of products related to specific exons.  The target of exon 
skipping is at the pre-mRNA level allowing one or more exons to be omitted to restore the 
dystrophin reading frame.  This is accomplished with splice-switching oligomers, typically 20-30 
nucleotides in length and complementary in sequence to regions of the pre-mRNA transcript.  
Pre-clinical efficacy has been demonstrated in the mdx mouse, dystrophin/utrophin knock-out 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
9 
Protocol Version 5.0 – 16January2015 
mouse, and CXMD dog22-24 using both 2’O-methyl-ribooligonucleoside-phoshophorothioate 
(2OMe) and phosphorodiamidate morpholino oligomers (PMOs).  Two proof-of-principle 
clinical trials in DMD used a 2OMe oligomer (PRO051, ProsensaTherapeutics) or a PMO 
(Sarepta Therapeutics) delivered directly to muscle targeting exon 51.25, 26  Evidence favoring 
exon skipping was validated by RT-PCR and a newly synthesized dystrophin protein correctly 
localized to the sarcolemma.  Phase I/II extension studies were performed with both oligomers 
following systemic delivery.  In the PRO051 trial, four doses were tested in the initial 5 week 
phase (0.5, 2.0, 4.0, and 6.0 mg/kg) prior to an open-label 12-week extension phase.27  Dose-
related efficacy was achieved with evidence of new dystrophin expression in approximately 60-
100% of muscle fibers in 10 of 12 patients and modest improvement in the 6-minute walk test.  
In the AVI phase II open-label study with AVI-4658 (Eteplirsen®) conducted in the UK, 7 of 19 
patients saw a modest response with a mean increase of sarcolemmal dystrophin from 8.9% to 
16.4%.28  The results were variable with one patient responding following treatment with 2 
mg/kg, while all other responders received the higher dosing levels of 10 and 20 mg/kg.  The 
PMO has now been tested in a phase I/II randomized, double-blind, placebo-controlled, multiple 
dose efficacy trial at Nationwide Children’s Hospital, Columbus, OH, USA to assess 30 and 50 
mg/kg dosing over 24 weeks (http://www.clinicaltrials.gov).  At the 24-week time point, 
dystrophin was increased and there was stability in the distance walked on the 6MWT.  This was 
followed by an open label extension study with all data reported at the 1 year time point showing 
increase dystrophin and stability in the 6MWT (Mendell et al Ann Neurol 2013;74:637-47).  
A second molecular approach involves suppression of stop codon mutations of the DMD gene 
that comprise approximately 15% of DMD cases.  Two pharmacologic tactics have shown pre-
clinical efficacy.  In mdx mice, mutation suppression was shown with the aminoglycoside 
antibiotic, gentamicin.29  In a follow up clinical study, treatment of four DMD/BMD subjects 
failed to show a benefit.30  A subsequent clinical trial, also in a small cohort, challenged the 
findings suggesting that readthrough had occurred, demonstrating full-length dystrophin as the 
product of gentamicin treatment.31  In a more definitive trial, done by Dr. Mendell at Nationwide 
Children’s Hospital, DMD patients (n = 16) with stop codons, treated weekly or twice weekly for 
six months (7.5 mg kg IV), showed a significant increase in dystrophin levels with the highest 
levels reaching 13 and 15% of normal.32  Muscle strength was stabilized and a modest increase 
in forced vital capacity was achieved.  Although this study demonstrates the therapeutic potential 
of gentamicin, higher doses might be necessary to improve functional outcomes.  The known 
renal toxicity of aminoglycoside antibiotics and the inconvenience of intravenous administration 
pushed the field in the direction of identifying an orally administered agent.  
Ataluren, formerly referred to as PTC124 (PTC therapeutics), fulfilled the requirement of an 
orally administered pharmacologic readthrough agent for stop codon mutations.33  Pre-clinical 
studies in the mdx mouse demonstrated dystrophin expression in skeletal, cardiac, and diaphragm 
muscle and protected skeletal muscle from eccentric contraction-induced injury.  A phase I study 
in healthy volunteers established safety and tolerability at doses exceeding what was required for 
pre-clinical efficacy.34  In a phase IIa proof-of-concept 28-day study in DMD/BMD patients, 
dystrophin appeared to increase post treatment.  A randomized, double-blind, placebo-controlled 
phase IIb trial followed, evaluating safety and efficacy over a 48 week treatment period.  PTC, 
Inc. released preliminary results indicating a very strong safety profile; however, the primary 
endpoint of the 6 minute walk test did not reach statistical significance 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
10 
Protocol Version 5.0 – 16January2015 
[http://clinicaltrials.gov/ct2/show/NCT00592553].  Continued subgroup analysis to look at 
efficacy is underway.  Influence for further pursuit of treatment in DMD may come from on-
going clinical trials in cystic fibrosis35 and 
hemophilia[[http://clinicaltrials.gov/ct2/show/results/NCT00947193], presuming results are 
favorable. Upregulation of utrophin is another therapeutic strategy for DMD that has shown 
promise in the mdx mouse.  Utrophin shares 80% sequence homology with dystrophin and has 
been shown to partially restore function as a dystrophin surrogate in pre-clinical transgenic mice 
36 and gene replacement studies.37  In normal muscle utrophin expression is limited to the 
neuromuscular and myotendinous junctions but in mdx mice and DMD patients it is 
overexpressed throughout the sarcolemma of all muscle fibers, putatively compensating for the 
absence of dystrophin.  Upregulation of utrophin holds a particular advantage because of the 
unlikely occurrence of an immune response as seen following mini-dystrophin gene 
replacement.1  
Several small molecules demonstrate upregulation of the utrophin gene through the utrophin-A 
promoter.  SMT C1100 is a novel small molecule that was identified through an exhaustive high-
throughput small molecule screen.  When used in the mdx mouse, utrophin staining is increased 
at the sarcolemma and dystrophic muscle pathology is reversed.38  Summit PLC, a UK 
biotechnology company, has advocated for its use in DMD clinical trials. A similar small 
molecule transcriptionally upregulating utrophin, BMN195 (Biomarin Pharmaceuticals), was 
tried in a phase I safety trial in healthy volunteers but disappointingly failed to achieve adequate 
concentrations in the blood.  The non-steroidal anti-inflammatory drug nabumetone39 (also 
known as Relafen®) was shown to upregulate endogenous utrophin mRNA and protein in 
C2C12 cells.39  It remains a candidate agent considering its current use in the treatment of 
juvenile rheumatoid arthritis, although its side effect profile as an NSAID with warnings 
regarding life-threatening heart or circulation problems, including heart attack or stroke may be a 
limiting factor as a life-long therapy.  
Heregulin represents an additional molecule capable of transactivating the utrophin-A promoter.  
When delivered to the mdx mouse for 3 months by intraperitoneal injections of a small peptide 
encoding the epidermal growth factor-like region of heregulin ectodomain, utrophin was 
upregulated, accompanied by increased resistance to eccentric contraction, and a reduction of 
muscle pathology.40  The amelioration of dystrophic phenotype by heregulin-mediated utrophin 
up-regulation offers a pharmacological therapeutic modality that deserves consideration in future 
planning to alleviate DMD.  
Another approach to upregulate utrophin employs a recombinant biglycan (rhBGN) that can be 
delivered systemically in the mdx mouse and ameliorate pathology and improve function at the 
diaphragm.41  There is clear evidence that biglycan can recruit utrophin to the sarcolemma.  
rhBGN has shown promise when used in cell culture and in vivo without changing mRNA levels 
of utrophin indicating that functional benefits arise from posttranscriptional effects.  rhBGN is 
being evaluated for clinical trials in DMD.  

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
11 
Protocol Version 5.0 – 16January2015 
 
Rationale for Follistatin (FS344) Gene Therapy 
The planned approach for gene therapy for DMD is an extension of our current follistatin (FS) 
gene therapy trial in BMD where we have found that gene transfer to the quadriceps muscles 
bilaterally improved the distance walked on the 6 minute walk test in 4 of 6 patients.  The results 
were robust in 2 patients with improvement over 100 meters at 1 year and 58 m and 30 m in two 
other patients. We believe that these results are also favorable for treating DMD patients and we 
presented these findings to the FDA along with our safety data as an amendment to our IND 
14845.  The FDA permitted us to treat this younger population without additional experiments. 
The paper describing the treatment of Becker patients has just been published in Molecular 
Therapy:  Mendell, JR, et al. A Phase I/IIa Follistatin Gene Therapy Trial for Becker Muscular 
Dystrophy, available on-line October 2014. 
Myostatin is a transforming growth factor-beta family member that normally acts to limit 
skeletal muscle growth.  Mice genetically engineered to lack myostatin have about twice the 
amount of muscle mass, and similar effects are seen in cattle, sheep, dogs, and human. 42-45  FS is 
a potent myostatin antagonist with the added advantage of controlling muscle mass through 
pathways independent of the myostatin signaling cascade. 42  Myostatin null mice carrying a 
follistatin transgene demonstrate four times the muscle mass of wild-type.  There are two 
isoforms of follistatin generated by alternative splicing and initially translated to isoforms FS317 
and FS344. 46  Post translational modification of each cleaves a 29 amino acid signal peptide 
giving rise to FS288 and FS315.  FS288 functions collaboratively in reproductive physiology 
with activin and inhibins of the hypothalamic pituitary-gonadal axis. 47  FS315 more reliably 
targets skeletal muscle, has no known cardiotoxicity or other adverse effects and is ideal for gene 
delivery to muscle. AAV1.CMV.FS344 delivered by direct intramuscular injection to quadriceps 
and tibialis anterior muscles of the mdx mouse increased muscle mass and strength throughout 
the lower extremities with a demonstrable remote effect on these same parameters in the upper 
limbs and increased muscle mass in the paraspinal muscles. 48  This we attributed to the muscle 
acting as a secretory site for follistatin with the circulating isoform reaching remote sites 49  
AAV1.CMV.FS344 was further tested in the non-human primate to explore a paradigm 
applicable to clinical trial.  In the cynomolgus macaque we injected AAV1.FS344 directly into 
the quadriceps muscle resulting in an increase in size and strength of this muscle. 50  It should be 
noted that when we injected the non-human primate in one muscle (as opposed to multiple 
muscles in the mouse) we did not see remote effects.  These pre-clinical studies in the absence of 
toxicity enabled a Phase I/IIa clinical trial in patients with BMD (IND 14845).  The BMD 
clinical results are the first ever gene therapy to muscle showing a functional benefit (increase 
distance walked on the 6MWT).  We are now ready to move ahead to treat boys with DMD.  
 
 
 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
12 
Protocol Version 5.0 – 16January2015 
6.0 CLINICAL RESEARCH PLAN  
6.1. STUDY POPULATION  
Six DMD subjects age 7 years and older with confirmed DMD gene mutations based on 
mutation will be enrolled at Nationwide Children’s Hospital for the gene transfer study. Subjects 
will encompass any ethnic or racial background. 
6.2. PRE-TREATMENT ASSESSMENT 
A. Establish Subject Eligibility 
I. 
Inclusion Criteria 
1. Age 7 or older 
2. Confirmed DMD gene mutations  
3. Impaired muscle function based on clinical evidence including difficulty climbing 
stairs, getting from the floor (Gowers’ sign), and weakness of individual muscles 
of extremities 
4. Males of any ethnic group will be eligible 
5. Ability to cooperate with study procedures including muscle testing. 
6. Willingness of sexually active subjects with reproductive capacity to practice 
reliable method of contraception  
7. Subjects must be on stable dose of prednisone or deflazacort for three months at 
time of enrollment.  Subjects will continue the steroid dose  post gene transfer as 
part of the standard of care for DMD patients. Dose adjustment could be 
considered if there are adverse effects.   
II. 
Exclusion Criteria 
1. Active viral infection based on clinical observations. 
2. The presence of a DMD gene mutation without weakness or loss of function  
3. Symptoms or signs of cardiomyopathy, including:  
- 
Dyspnea on exertion, pedal edema, shortness of breath upon lying flat, or rales at 
the base of the lungs 
- 
Echocardiogram with ejection fraction below 40% 
4. Serological evidence of HIV infection, or Hepatitis A, B or C infection 
5. Diagnosis of (or ongoing treatment for) an autoimmune disease  

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
13 
Protocol Version 5.0 – 16January2015 
6. Concomitant illness or requirement for chronic drug treatment that in the opinion of the 
PI creates unnecessary risks for gene transfer 
7. Subjects with   rAAV1 binding antibody titers > 1:50 as determined by ELISA 
immunoassay 
8. Abnormal laboratory values for liver, kidney, CBC, in the clinically significant range, 
based upon normal values in the Nationwide Children’s Hospital Laboratory 
 
B. Informed Consent  
Legally effective and properly executed written informed consent, in compliance with 21 CFR 
50 and the International Conference on Harmonization (ICH) guidelines, will be obtained from 
each subject before the subject is entered into the trial or before any unusual or non-routine 
procedure is performed that involves risk to the subject. 
Attention will be directed to the basic elements that are required for incorporation into the 
informed consent under US Federal Regulations for Protection of Human Subjects [21CFR 
50.25(a)].  The final IRB-approved document as well as any subsequent approved modified 
consent document(s) must be provided to correspondent agencies for regulatory purposes.  If 
new information related to the study arises, subjects will be asked to sign a revised document.    
Signed consent forms will remain in each subject's research chart and be available for the 
verification by study monitors at any time.  Subjects will be given a signed, dated copy of their 
consent form documents. 
 
C. Establish Subject Identification Number 
All subjects will be given a unique sequentially assigned subject number. Subjects will be 
identified by number only to protect identity. 
D. Baseline/Screening Assessment (day-45 to day -1) 
 
System 
Assay 
Normal Range 
Abnormal 
(Clinically Significant) 
Liver Function 
GGT 
8-80 U/L 
>3 times 
upper limit of normal 
 
Total Bilirubin 
0.1-1 mg/dL 
≥3 mg/dL 
Renal Function 
Creatinine 
0.3-1.3 mg/dL 
>1.8 mg/dL 
Hematologic 
Hemoglobin 
Age dependent 
For all ages: 
≤ 8 or ≥ 18 g/dL 
Hematologic 
White Blood 
Cells 
Age dependent 
For all ages: 
≤ 3.5 or ≥ 20 x 103 cells/mL 
Or 
Absolute neutrophil count of ≤ 1.5 x 103 
cells/mL 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
14 
Protocol Version 5.0 – 16January2015 
After obtaining informed consent and completing the registration procedures a baseline patient 
history will be collected, including records of all medications and supplements that the patient is 
taking.  The following assessments will be performed to confirm subject eligibility for this study.  
Baseline tests which must be completed prior to treatment administration include the following:  
 
Day -45 to day -1 before gene transfer  
 
• Medical history 
• Physical exam 
• EKG  
• ECHO 
• Chest X-ray 
• MRI of the gluteal muscles, quadriceps and TA (should not be repeated if the patient had 
the MRI test done within the previous year) 
• Hepatitis A,B, &C Screen 
 
• HIV screening  
• CBC/Diff/Platelet with smear 
• Total Protein 
• Alanine transaminase (ALT) 
• Aspartate Aminotransferase (AST) 
• Serum gamma-glutamyl transferase (GGT) 
• Total bilirubin 
• Glucose 
• Creatine kinase (CK) 
• Creatinine 
• BUN 
• Cystatin C 
• Alkaline phosphatase  
• Amylase 
• Prothrombin time (PT), partial thromboplastin time (PTT) 
• Urinalysis 
 
• Neutralizing antibody and/or binding antibody to AAV1 
• ELISA assay to detect antibody to follistatin 
• ELISPOT assay to detect T cell response to AAV1 capsid proteins and follistatin  
• Quantification of blood circulating follistatin 
• Six minute walk test (6MWT), functional and strength measures by PT 
• Life quality questionnaire 
• Pulmonary Function Test (PFT) 
• Electrical Impedance Myography (EIM) to quantify pathological status of muscles: 
Skulpt, Inc. has developed a handheld device specifically for performing EIM 
measurements.  This device will be used to measure EIM in this study.  The device is not 
yet approved by FDA, but has been tested in over 30 other individuals with no adverse 
events (AEs) (serious or otherwise) reported to date and currently being tested in SMA 
infants as part of the Network for Excellence in Neuroscience Clinical Trials 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
15 
Protocol Version 5.0 – 16January2015 
(NeuroNEXT) trials, and SMA gene therapy trial.  The device has undergone 
independent electrical safety testing by Intertek (www.intertek.com) to the requirements 
contained in the following standards:  
 
IEC 60601-1 Issue 1998/12/01 Ed: 2 Medical Electrical Equipment Part 1: General 
Requirements and Safety; (Amd. 1-1991(CENELEC EN 60601-1: 1990) (Amd. 2-1995) 
(Corrigendum-1995)) 
 
• Baseline muscle biopsy on quadriceps muscles of one leg  
• Subjects will be instructed to perform strengthening/endurance exercises at home 3 times 
per week, and asked to report the frequency of exercise. They will be incentivized 
through a video game and it will be emphasized that this should be relatively mild effort. 
The subjects will be incentivized to complete the exercise program by offering them $10 
per week for completion of log showing that exercise has been done.   
* Study participants will continue prednisone post gene transfer as part of the standard 
management of DMD boys.  Glucocorticoids are considered standard of care for DMD and most 
boys will be on a steroid regimen at the time they are recruited.  
6.3 PROTOCOL FOR GENE TRANSFER  
I. 
Injection preparation 
Preparation of the rAAV1.CMV.huFS344 will be in the investigational drug service based on the 
drug order from and following pharmacy standard protocol.  The vector shall be diluted and 
manipulated in polypropylene syringes only.  Polystyrene and polycarbonate materials 
should never come in contact with the vector. 
Prior to transportation to the clinical setting appropriate dilutions of the test article will be 
completed by the pharmacy.  The dose will be diluted in normal saline, divided in 9 ml per leg 
with 0.5 ml delivered per site on each leg.  Documentation of the dilution will be completed by 
the pharmacy following standard pharmacy protocol. The formulated syringes will be maintained 
refrigerated at 2-8C until transport to the patient procedure room where it will be delivered on 
wet ice in the appropriately labeled cooler.  
 
The final volume delivered to the PI will be 18 ml total volume distributed in 18 polypropylene 
syringes of 1 ml each, nine syringes being delivered to the right leg and nine to the left leg.  The 
vector-containing syringe will be delivered to the designated procedure room at Nationwide 
Children’s Hospital.  It will be delivered on wet ice (not frozen) and administered to the subject 
within 8 hours of of thaw. Handling of rAAV1.CMV.huFS344 will follow compliance standards 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
16 
Protocol Version 5.0 – 16January2015 
for Biosafety Level 1 vectors. 
(http://www4.od.nih.gov/oba/RAC/guidelines_02/APPENDIX_G.htm#_Toc7246561)  
 
II. 
Vector Administration Protocol 
Conscious sedation will be given to all DMD participants by an anesthesiologist who will be 
with patient at the time of injection.  In addition, the skin over the gene transfer site will be pre-
treated with a lidocaine/prilocaine eutectic mixture incorporated in a cream base (EMLA cream) 
or a cellulose disk (EMLA patch).  Comparable cream-based anesthesia such as xylocaine cream 
might be used.  Procedures will be performed under sterile conditions.  The injection site will be 
cleansed with three successive applications of non-iodine containing surgical prep swabs and 
draped with disposable sterile drapes.  A standard clinical Doppler ultrasound will be used with a 
sterile sheath around the transducer to maintain asepsis of the injection field.  This will increase 
the precision of gene transfer to be sure that we inject muscle and not connective tissue or fat.   
The total dose of vector per extremity, 1.2 X 1012 vg/kg will be distributed between the targeted 
muscles that include gluteals, quadriceps, and tibialis anterior.  The initial sites of injection, at 
least three injections per muscle, will be targeted based on MRI showing preserved muscle.  The 
sites will be confirmed at the time of injection by ultrasound guidance for the needle injections.  
The procedure should take approximately 30 minutes to inject muscles of both extremities.  
 
6.4 POST GENE TRANSFER MONITORING 
 
6.4.1 IMMEDIATELY FOLLOWING GENE TRANSFER  
Vital signs including blood pressure, heart rate, respiratory rate and temperature will be 
performed post-administration.  Concomitant medications and all adverse events/serious adverse 
events will also be monitored and documented following injection.  Patients will be discharged 
the day after gene therapy (if no side effects are observed). 
 
6.4.2 FOLLOW UP  
Subjects will return for follow up visits on days 7, 14, 30, 45, 60, 90,180, and 9, 12, 18, and 24 
months post-gene transfer.  Toxicity monitoring on each of these dates (including day 1 post-
gene transfer) are described in the following sections of the protocol. 
We will follow the most recent FDA guidelines with regard to long-term patient follow up 
following gene transfer.  Our proposed vector has a very low probability of gene transfer-related 
delayed adverse events.  We will, however, evaluate short-term safety over a two-year period 
that incorporates the active phase of the protocol (see assessment of endpoints below). If newly 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
17 
Protocol Version 5.0 – 16January2015 
identified risks are associated with our product, or if the patients suffer any adverse events during 
this period, we will initiate a long-term follow-up according to the FDA guidelines.   
 
6.5 ASSESSMENT OF ENDPOINTS 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
18 
Protocol Version 5.0 – 16January2015 
A. Safety monitoring post-gene transfer 
Monitoring will occur on Study Days 1, 7, 14, 30, 45, 60, 90,180, and continue at 9, 12, 18, and 
24 months post gene transfer.  The following parameters will be included at different time points 
during the monitoring evaluations (see study timeline section 6.7):  
 
• 
Physical Exam  
• 
ALT 
• 
AST 
• 
GGT 
• 
Total bilirubin 
• 
Glucose 
• 
PT/PTT 
• 
CBC/Diff/Platelet with smear 
• 
Serum Creatine kinase (CK) 
• 
Creatinine/BUN 
• 
Cystatin C 
• 
Alkaline phosphatase  
• 
Amylase 
• 
Total Protein 
• 
Urinalysis 
• 
Blood antibody to AAV1 and follistatin 
• 
ELISPOT assay to detect T cell response to AAV1 capsid proteins and follistatin  
• 
Blood circulating follistatin 
• 
Photograph of injection site/ surrounding area 
• 
Adverse events 
• 
Pulmonary Function Test (PFT) 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
19 
Protocol Version 5.0 – 16January2015 
B. Efficacy Monitoring 
 
I. 
Primary Outcome 
Safety is a primary outcome for this clinical gene transfer trial. 
II. 
Secondary Outcomes:  
• 
The distance walked on the 6MWT is the primary functional outcome to be evaluated. 
Time to walk and run, strength test, functional test, and reaching ability will be included 
in PT testing.  Functional testing will be performed at baseline and at days 30, 60, 
90,180, and 9, 12, 18 and 24 months post-gene transfer. 
• 
Life quality questionnaire will be completed at baseline and 6, 12, 18 and 24 months 
post-gene transfer. 
• 
In order to maximize potential benefit of follistatin gene therapy, subject will be 
instructed to continue strengthening exercise at home at least 3 times per week as 
instructed by physical therapists (PT), and asked to report the frequency of exercise.  
The subjects who follow the exercise instruction will be incentivized by video display 
and offered $10 per week if they fill out their log showing bike riding complete.   
• 
MRI studies of leg muscles will be done pre and post gene transfer (6, 12 and 24 
months) to study muscle size, fibrosis and fat content.   
• 
EIM measurement will quantify the pathological status of muscles.  
• 
Muscle biopsies on quadriceps muscles (a muscle biopsy on one leg at baseline 
screening visit and the post gene transfer biopsy on the opposite leg at day 180), will 
include general histological evaluation. Assessment will consist of morphometric 
analyses for  quantification of fiber size, fibrosis, inflammatory process, satellite cells 
and regeneration. Muscle tissue obtained at biopsy will also be assessed for viral DNA 
(qPCR), and follistatin transgene expression 
6.6 STATISTICAL ANALYSIS 
This is a phase I/IIA trial, with safety as the primary measure. For each measure based on 
differences between pre- and post-gene transfer examinations (clinical) analyses will be based on 
a paired t test, with a p value of < 0.05 indicating significance.  
 
 
 
 
 
 
 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
20 
Protocol Version 5.0 – 16January2015 
6.7 STUDY TIMELINE  
 
STUDY TIMELINE 
Study Interval 
Baseline 
Screening 
Vector 
Injection 
(Inpatient) 
Follow Up 
(Outpatient) 
Visit 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
Days in Study 
-45 to -1 
-1 
0 
1 
7 
14 
30 
45 
60 
90 
180 
194* 
9 
mo 
1 yr 
18 
mo 
2 yr 
Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam 
& vitals 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
ECHO/EKG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Chest X-Ray 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MRI 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
 
X 
Hepatitis A, B & 
C HIV 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Safety labs 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Urinalysis 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Pulmonary 
function test 
X 
 
 
 
 
 
X 
 
 
X 
X 
 
X 
X 
 
 
PT evaluation 
X 
 
 
 
 
 
X 
 
X 
X 
X 
 
X 
X 
X 
X 
Life quality 
questionnaire 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
X 
X 
EIM 
X 
 
 
 
 
 
 
 
 
X 
X 
 
 
X 
 
 
Immunology 
studies 
X 
 
 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
Circulating 
follistatin 
X 
 
 
 
 
 
X 
 
 
X 
X 
 
X 
X 
X 
X 
Gene Transfer 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Muscle Biopsy 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
Photograph of 
injection site 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse Events 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
Medications 
To be collected from time of consent until final study visit, recorded on separate CRF 
 
Note: There will be a flexibility of a +/- 7 working days for each of the planned study visits including 
screening to adjust the schedule to any unanticipated event.   There will also be potential additions to care 
based on best medical management if the need arises including additional fluids to restore blood volume.   
  
*: The follow up post-biopsy visit scheduled 2 weeks post-biopsy is made optional to give subjects the 
choice to have the stiches removed at their local doctor’s office. 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
21 
Protocol Version 5.0 – 16January2015 
7.0 TEST MATERIAL AND ADMINISTRATION 
7.1 DESCRIPTION OF BIOLOGICAL PRODUCT 
The biological drug product is a non-replicating, adeno-associated virus termed 
rAAV1.CMV.huFS344. The product will follow the IND 14845 CMC section, identical to what 
has been produced for the BMD study that is now completed. The product will be produced at 
the Clinical Manufacturing Facility (CMF) at Nationwide Children’s Hospital according to 
current Good Manufacturing Practices (cGMP).  The manufacturing process includes procedures 
to ensure the safety, identity, quality, purity and strength of the manufactured biologic.  The 
HEK 293 cell bank used in the production of the product will meet specifications for product 
release as detailed in FDA and ICH guidelines.  The production process is governed by over 90 
standard operating procedures (SOP), Master Batch Records, Methods and registered Forms.  
The vector will be formulated in 20 mM Tris (pH 8.0), 1 mM MgCl2 and 200 mM NaCl 
containing 0.001% pluronic F68.  The vector will be supplied as a frozen solution that is thawed 
before clinical administration.   
 
7.1.1 PRODUCT DEFINITION 
 
Product Name:  
rAAV1.CMV.huFS344 
Gene Insert: 
Human cDNA for FS344 (alternatively spliced follistatin) 
Control Elements:  
CMV promoter 
AAV Serotype:  
Serotype AAV1 
 
7.2 DOSE SELECTION 
The dose is based on our BMD gene therapy trial using the same vector.  In that study we used 
two doses without safety concerns.  No dose limiting toxicity was found.  For the low dose 
rAAV1.CMV.huFS344 trial (n=3) we injected 12 sites in each extremity (quadriceps only).  The 
total dose per patient was 6 X 1011 vg/kg (3e11 vg/kg/quadriceps) and the high dose was 1.2 X 
1012 vg/kg per patient (6e11vg/kg/quadriceps).  There did not seem to be a significant dose 
response effect. In the low dose cohort patients improved in the distance walked in the 6MWT as 
follows: 58m, 125m, and 9 m.  In the high dose cohort, patients improved in the 6MWT by 108m 
and 29m.  One patient in the high dose group did not improve.  Based on our toxicology studies 
prepared for IND 14845 that included a high dose cohort in C57Bl mice at 2.0 x 1013 vg/kg 
without any organ system toxicity and the clinical trial done at 1.2 X 1012 vg/kg, we are 
proposing that all six DMD boys receive 2.4 X 1012 vg/kg per patient.   

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
22 
Protocol Version 5.0 – 16January2015 
 
7.3 DOSE LIMITING TOXICITY  
In reporting adverse events we will follow the final regulations issued by the Food and Drug 
Administration addressing the safety reporting requirements for investigational new drug 
applications (INDs) found in 21 CFR part 312 and for bioavailability and bioequivalence studies 
found in 21 CFR part 320. “Safety Reporting Requirements for INDs and BA/BE Studies”. 
The classification for adverse events to be used is the following:  
1  
Mild adverse event; did not require treatment 
2  
Moderate adverse event; resolved with treatment 
3  
Severe adverse event; inability to carry on normal activities; required professional 
 
medical attention 
4  
Life-threatening or permanently disabling adverse event 
5  
Fatal adverse event In this grading system, “severe” is not equivalent to seriousness. 
 
The definitions to be employed will follow the final regulations issued by the FDA in September 
2010 “Safety Reporting Requirements for INDs and BA/BE Studies. 
 
Dose limiting toxicity (DLT) is defined as any adverse event that is possibly, probably, or 
definitely related to the study agent.  This would include any grade 3 according to the 
classification given above.  Study enrollment will be halted by the investigators when any 
subject experiences a Grade 3, or higher adverse event toxicity that is possibly, probably, or 
definitely related to the study drug. 
Laboratory tests with values within the clinically significant range will be repeated during the 
same visit whenever possible.  If the test result returns after the subject leaves the clinic, they 
will be immediately contacted.  For local residents they will be asked to return to the outpatient 
clinic for a repeat test.  For non-local residents, arrangements will be made to have the blood test 
redrawn in a laboratory close to home or by their primary care physician.  To avoid any 
confusion for the primary care physician, they will be informed (with permission from the 
subject) of their participation in the study at the time of gene transfer.  If the AE requires 
treatment, this will be carried out by the primary care physician or a doctor of choice selected by 
the subject.  We will obtain copies of repeat laboratory tests and any relevant medical records 
that will be added to the subject’s research chart.  
Only those adverse events requiring treatment will qualify as DLT.  The PI will fulfill the 
reporting responsibilities under 21 CFR 312.32(c), to notify FDA in an IND safety report of 
potentially serious risks, as soon as possible, but no later than 15 calendar days after the 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
23 
Protocol Version 5.0 – 16January2015 
investigator receives the safety information and determines that the information qualifies for 
reporting.  The investigator will confer with the DSMB, IRB, IBCSC, CBER, FDA, OBA, and 
NIH before continuing enrollment. 
Table of Clinically Significant Laboratory Ranges 
 
7.4 STOPPING AND DISCONTINUATION RULES 
An independent Data Safety Monitoring Board (DSMB) will be established by the sponsoring 
agency including a safety monitor for the study.  Safety data will be monitored on a continual 
basis throughout the trial.  The DSMB can recommend early termination of the trial for reasons 
of safety.  Study enrollment will be halted by the investigators when any subject experiences a 
Grade 3, or higher adverse event toxicity that is possibly, probably, or definitely related to the 
study drug.  This will include:  
Grade 3 Severe or medically significant but not immediately life-threatening; hospitalization or 
prolongation of hospitalization indicated; disabling; limiting self care ADL**. 
Grade 4 Life-threatening consequences; urgent intervention indicated. 
Grade 5 Death related to AE. 
Activities of Daily Living (ADL) 
*Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the 
Test 
Units 
Reference Range 
Clinically Significant 
Range 
Hemoglobin 
g/dL 
2Y – 5Y                          11.5 – 13.5 
6Y – 11Y                        11.5 – 15.5 
12Y – Adult                    12.0 – 16.0 
< 8.0 or > 18  
WBC 
K/cu mm 
4Y – 5Y                           5.5 – 15.5 
6Y – 9Y                           5.0 – 14.5 
10Y – 20Y                       4.5 – 13.5 
21Y – Adult                     4.5 – 11.0 
≤3.5 or ≥ 20  
BUN 
mg/dL 
5 - 18 
≥35 
PT 
sec 
12.4 – 14.7 
≤ 5.0 or ≥ 35.0 
PTT 
sec 
24 - 36 
≥ 125 
Platelet 
K/cu mm 
140 - 440 
≤ 90 or ≥ 800 
Amylase 
U/L 
0 – 17Y                                 <110 
18Y – Adult                     30  - 110 
≥ 200 
Total Protein 
g/dL 
3Y – 15Y                         5.8 – 8.7 
16Y – Adult                     6.4 – 8.4 
≤4.4 or ≥ 9.5  
Total Bilirubin 
mg/dL 
0.1 – 1.0 mg/dL 
≥ 3.0  
Creatinine 
mg/dL 
4Y – 7Y                           0.3 – 0.8 
8Y – 10Y                         0.3 – 0.9 
11Y – 12Y                       0.4 – 1.0 
13Y – 17Y                       0.5 – 1.2 
18Y – Adult                     0.6 – 1.3  
≥ 1.8  
GGT* 
U/L 
8 – 78 
3XULN 
 ULN= upper limit normal 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
24 
Protocol Version 5.0 – 16January2015 
telephone, managing money, etc. 
**Self care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, taking 
medications, and not bedridden. 
If after review by the DSMB, IRB, IBCSC, NIH-OBA and FDA, the decision is made to 
continue according to this study protocol. 
 
7.5 CONCOMITANT MEDICATIONS AND THERAPIES 
Prescribed and over the counter medications used in the prior two weeks will be recorded at the 
baseline visit and changes in these medications will be recorded during each subsequent follow 
up visits.  The PI will encourage participants to maintain the medication and supplements they 
are on at enrollment through the course of the study.  Subjects on aspirin or drugs that could 
affect coagulation will continue their medication as indicated.  Several investigations show that 
preoperative aspirin ingestion and intravenous heparin therapy can be administered safely 
without concerns about the risk of postoperative bleeding and should not lead to modification or 
cessation of such therapy.51-53 Subjects must already be on prednisone for three months at time of 
enrollment as part of the inclusion criteria. 
8.0 ADVERSE EVENT MONITORING AND REPORTING 
8.1 DEFINITION OF AN ADVERSE EVENT 
As stated above  this protocol will follow the final regulations issued by the Food and Drug 
Administration addressing the safety reporting requirements for investigational new drug 
applications (INDs) found in 21 CFR part 312 and for bioavailability and bioequivalence studies 
found in 21 CFR part 320. “Safety Reporting Requirements for INDs and BA/BE Studies”. 
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/U
CM227351.pdf 
Adverse Events will be collected throughout the study from enrollment to last follow up visit. 
Adverse Event (AE):  Adverse event means any untoward medical occurrence associated with 
the use of a drug in humans, whether or not considered drug related. 
Adverse events will be graded by the investigator accordingly:  
Grade 1 Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated. 
Grade 2 Moderate; minimal, local or noninvasive intervention indicated; limiting age-appropriate 
instrumental ADL*. 

Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
25 
Protocol Version 5.0 – 16January2015 
Grade 3 Severe or medically significant but not immediately life-threatening; hospitalization or 
prolongation of hospitalization indicated; disabling; limiting self care ADL**. 
Grade 4 Life-threatening consequences; urgent intervention indicated. 
Grade 5 Death related to AE. 
A Semi-colon indicates ‘or’ within the description of the grade. 
A single dash (-) indicates a grade is not available. 
 
Activities of Daily Living (ADL) 
*Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the 
telephone, managing money, etc. 
**Self care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, taking 
medications, and not bedridden. 
Association or relatedness to the study agent, study procedures and the subject's pre-existing 
disease will be graded as follows: 5 = unrelated, 4 = unlikely, 3 = possibly, 2 = probably, and 1 = 
definitely related.  
Adverse reaction: An adverse reaction means any adverse event caused by a drug. Adverse 
reactions are a subset of all suspected adverse reactions for which there is reason to conclude that 
the drug caused the event. 
Suspected adverse reaction (21 CFR 312.32(a)) Suspected adverse reaction means any adverse 
event for which there is a reasonable possibility that the drug caused the adverse event. For the 
purposes of IND safety reporting, ‘reasonable possibility’ means there is evidence to suggest a 
causal relationship between the drug and the adverse event. A suspected adverse reaction implies 
a lesser degree of certainty about causality than adverse reaction, which means any adverse event 
caused by a drug. 
Serious adverse event (SAE)  
An adverse event or suspected adverse reaction is considered “serious” if, in the view of either 
the investigator or sponsor, it results in any of the following outcomes: Death, a life-threatening 
adverse event, inpatient hospitalization or prolongation of existing hospitalization, a persistent or 
significant incapacity or substantial disruption of the ability to conduct normal life functions, or a 
congenital anomaly/birth defect. Important medical events that may not result in death, be life-
threatening, or require hospitalization may be considered serious when, based upon appropriate 
medical judgment, they may jeopardize the subject and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition. Examples of such medical 
events include allergic bronchospasm requiring intensive treatment in an emergency room or at 

Center for Gene Therapy 
 
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home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the 
development of drug dependency or drug abuse.  
To reiterate, an SAE is an event in categories graded 3, 4, and 5.   
Grade 3 Severe or medically significant but not immediately life-threatening; hospitalization or 
prolongation of hospitalization indicated; disabling; limiting self care ADL**. 
Grade 4 Life-threatening consequences; urgent intervention indicated. 
Grade 5 Death related to AE. 
Life-threatening (21 CFR 312.32(a)) An adverse event or suspected adverse reaction is 
considered “life-threatening” if, in the view of either the investigator or sponsor, its occurrence 
places the subject at immediate risk of death. It does not include an adverse event or suspected 
adverse reaction that, had it occurred in a more severe form, might have caused death. 
The PI will fulfill the reporting responsibilities to FDA/ OBA on behalf of Nationwide 
Children’s Hospital using the web-based Adverse Event reporting system (GeMCRIS).  
  
8.2 OBLIGATIONS OF THE INVESTIGATOR 
The Principal Investigator will submit an electronic report to NIH Office of Biotechnology 
Activities (NIH OBA) through the GeMCRIS web-based reporting system on any serious 
adverse event that is both unexpected and associated with the use of the gene transfer product 
(i.e., there is reasonable possibility that the event may have been caused by the use of the 
product; the investigator will not await definitive proof of association before reporting such 
events); as well as a written report on any finding from tests in laboratory animals that suggests a 
significant risk for human research participants including reports of mutagenicity, teratogenicity, 
or carcinogenicity.  The report will be clearly labeled as a “Safety Report” and will be submitted 
to the FDA, NIH Office of Biotechnology Activities (NIH OBA) and to the local Institutional 
Biosafety Committee within the timeframes set forth in section Safety Reporting. 
 
The Principal Investigators will adhere to any other serious adverse event reporting requirements 
in accordance with federal regulations, state laws, and the local institutional policies and 
procedures, as applicable.  The Principal Investigator will be responsible for ensuring that the 
reporting requirements are fulfilled and will be held accountable for any reporting lapses. 
 
8.3 SAFETY REPORTING 
The investigator or his designee will report all serious and unexpected adverse events to the IRB, 
IBCSC, CBER, FDA, OBA, NIH, and DSMB according to regulatory requirements described as 

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follows: All Serious Adverse Events (SAEs) and Dose Limiting Toxicities (DLTs) will be 
reported to the DSMB within 48 hours of notification, regardless of relatedness to the clinical 
trial.   
Any serious adverse event that is fatal or life-threatening, that is unexpected, and associated with 
the use of the gene transfer product will be reported to the FDA as soon as possible, but not later 
than 7 calendar days after the sponsor’s initial receipt of the information.  Serious adverse events 
that are unexpected and associated with the use of the gene transfer product, but are not fatal or 
life-threatening, will be reported to the FDA as soon as possible, but not later than 15 calendar 
days after the sponsor’s initial receipt of the information.   Changes in this schedule will be 
permitted only where, under the FDA IND regulations [21 CFR 312(c) (3)], changes in this 
reporting schedule have been approved by the FDA and are reflected in the protocol. 
Follow up  
Relevant additional clinical and laboratory data will become available following the initial 
serious adverse event report.  Relevant follow-up information to an IND safety report will be 
submitted concurrently to the FDA/NIH OBA/IRB/ DSMB as soon as the information is 
available and will be identified as such, i.e., "Follow-up IND Safety Report.”   If a serious 
adverse event occurs after the end of a clinical trial and is determined to be associated with the 
use of the gene transfer product, that event will be reported concurrently to the FDA/NIH 
OBA/IRB/ DSMB within 15 calendar days of the determination. 
Should a serious adverse event deemed possibly, probably or definitely related to the study agent 
occur during administration, the study agent will be discontinued, appropriate treatment will be 
given under medical supervision and the subject will be examined as frequently as necessary 
thereafter until symptoms cease or stabilize. 
 
8.3.1 SAFETY REPORTING: CONTENT AND FORMAT 
 
The serious adverse event report will include, but need not be limited to: (1) the date of the 
event; (2) designation of the report as an initial report or a follow-up report, identification of all 
safety reports previously filed for the clinical protocol concerning a similar adverse event, and an 
analysis of the significance of the adverse event in light of previous similar reports; (3) clinical 
site; (4) the Principal Investigator; (5) NIH OBA protocol number; (6) FDA’s Investigational 
New Drug (IND) application number; (7) vector type , e.g., adeno-associated virus; (8) vector 
subtype, if relevant; (9) gene delivery method, e.g., in vivo transduction; (10) route of 
administration, e.g., intramuscular; (11) dosing schedule; (12) a complete description of the 
event; (13) relevant clinical observations; (14) relevant clinical history; (15) relevant tests that 
were or are planned to be conducted; (16) date of any treatment of the event; and (17) the 
suspected cause of the event.  These items will be reported electronically through the GeMCRIS 
reporting system (E-mail address for Reporting Adverse Events: GeMCRIS@od.nih.gov ) by 
using the recommended Adverse Event Reporting Template available on NIH OBA’s web site 
at:  

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Mendell, Jerry R. 
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http://www.google.com/url?sa=t&rct=j&q=adverse%20event%20reporting%20template&source
=web&cd=1&cad=rja&ved=0CD0QFjAA&url=http%3A%2F%2Foba.od.nih.gov%2Foba%2Fra
c%2FAdverse_Event_Template.pdf&ei=cEoJUvjaC-amygHxuYGgDA&usg=AFQjCNEkxJ-
bsheco4BAh62i2kJ1VJh3Yw&bvm=bv.50500085,d.aWc 
8.4 UNEXPECTED ADVERSE EVENTS 
Unexpected adverse events are those which are not previously reported with recombinant AAV 
vectors, commonly not seen in association with the subject's underlying disease or with the 
procedures to be used in this study, or are related to a known toxicity but differ because of 
greater severity or specificity. 
8.5 FOLLOW UP OF ADVERSE EVENTS 
All adverse events will be followed until resolution or stabilization. 
9.0 LONG-TERM FOLLOW UP 
We will follow the most recent FDA guidance with regard to long-term patient follow up 
following gene transfer.  As indicated by the guidelines, our proposed vector has a very low 
probability of gene transfer-related delayed adverse events.  If newly identified risks are 
associated with our product, or if the subjects suffer any adverse events during this period, we 
will initiate a long-term follow-up according to the FDA guidelines.  We will, of course, notify 
CBER if there is any indication of need to extend follow-up period.  All subjects will be 
provided with written instructions on how to contact the Principal Investigator or study 
coordinator if they experience any serious adverse event that they consider possibly related to 
study treatment or study participation.  This information will also be included in the Informed 
Consent document.  All subjects will be instructed to notify the Principal Investigator of a 
change of address or contact information. 
The final results of the clinical trial will be shared with the participants at the completion of the 
study when all data has been collected and analyzed. However, if significant findings become 
available that might increase the risk of the subjects or might affect their decision to remain in 
the study, then information will be made available as soon as it is available.  
At the time of death, no matter what the cause, permission for an autopsy will be requested of 
their families.  Subjects will be asked to advise their families of this request and of its scientific 
and medical importance. 
9.1 ADVERSE EVENT REPORTING WITH PRIMARY CARE PHYSICIAN 
Close communication will be established with the primary care physician of all study 
participants and will be maintained throughout the study. The important hallmarks of the study 
along with the proposed reporting plan will be explained.  We will request that the primary care 
physician provide information regarding non-routine visits during the two years following gene 
transfer.  If non-routine visits are reported to us by the primary care physician, the study 

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investigator will initiate an investigation to determine the possibility of an adverse event related 
to the gene transfer and will adhere to the adverse event reporting requirements in accordance 
with federal regulations, state laws, and the local institutional policies and procedures, as 
applicable. 
During the consent process, the study investigator will emphasize the importance of subject 
communication with our study team.  Any non-routine doctor’s visits or medical care received 
during the two years following gene transfer should be reported to the study team.  The study 
doctor will explain to the participant that copies of any relevant medical records of those visits 
will be requested from their medical care provider. 
10.0 STUDY REPORTS 
10.1 FINAL STUDY REPORT 
The final study report will include data through the final study visit but will not include long-
term follow-up information.   
10.2 ANNUAL STUDY REPORTS 
 
Within 60 days after the one-year anniversary of the date on which the investigational new drug 
(IND) application went into effect, and after each subsequent anniversary until the trial is 
completed, the Principal Investigator will submit information set forth as follows:  
(a) Clinical Trial Information.  This will be a brief summary of the status of the trial in progress 
or completed during the previous year.  The summary will include the following information for 
the trial: (1) the title and purpose of the trial; (2) clinical site; (3) the Principal Investigator; (4) 
clinical protocol identifiers, including the NIH OBA protocol number, CCH IRB and IBCSC 
protocol numbers, and the FDA IND application number; (5) participant population (such as 
disease indication and general age group); (6) the total number of participants planned for 
inclusion in the trial; the number entered into the trial to date; the number whose participation in 
the trial was completed; and the number who dropped out of the trial with a brief description of 
the reasons; (7) the status of the trial, e.g., open to accrual of subjects, closed but data collection 
ongoing, or fully completed, and (8) if the trial has been completed, a brief description of any 
study results. 
(b) Progress Report and Data Analysis.  Information obtained during the previous year's clinical 
and non-clinical investigations, including:  (1) a narrative or tabular summary showing the most 
frequent and most serious adverse experiences by body system; (2) a summary of all serious 
adverse events submitted during the past year; (3) a summary of serious adverse events that were 
expected or considered to have causes not associated with the use of the gene transfer product 
such as disease progression or concurrent medications; (4) if any deaths have occurred, the 
number of participants who died during participation in the investigation and causes of death; 
and (5) a brief description of any information obtained that is pertinent to an understanding of the 

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gene transfer product’s action, including, for example, information about dose-response, 
information from controlled trials, and information about bioavailability.  
(c) A copy of the updated clinical protocol including a technical and non-technical abstract. 
11.0 HUMAN SUBJECTS 
The proposed study will involve a total of 6 Duchenne Muscular Dystrophy (DMD) subjects.  
All subjects with this condition that are enrolled must have muscle weakness by clinical exam.  
DMD is established on the basis of confirmed DMD gene mutations.  Subject selection will not 
exclude anyone on the basis of race or ethnic background.  For complete details see the Inclusion 
and Exclusion Criteria Section. 
11.1 SOURCES OF MATERIAL  
Research material obtained from identifiable living human subjects includes reports of: history 
and physical assessments, blood, urine, chest X-ray, ECHO, EKG, EIM, pulmonary function 
tests, and MRI, clinical assessments and admission/ discharge summaries for hospitalization 
occurring while the subject is on study, adverse events, and autopsies.  Copies of case report 
forms, original test results, subject medical records, signed subject informed consent, 
correspondence, and any other documents of the subjects, relevant to the conduct of the study 
will be kept on file by the Principal Investigator.  All material or data collected as part of the 
study will be obtained specifically for research purposes. 
11.2 POTENTIAL RISKS 
Potential risks to study subjects include risks associated with administration of the study agent 
and with study procedures. 
 
11.2.1 RISKS ASSOCIATED WITH rAAV.CMV.HUFS344 GENE TRANSFER 
There is a very small chance that the study agent could damage the DNA in the cells of the 
subject's muscle.  In the unlikely event that this occurred, it could put the subject at risk for 
developing cancer in the future.  In animal studies to date, we have not seen the development of 
cancer.  In addition, we and others have shown that the majority of vector DNA appears to 
persist as episomal rather than integrated DNA, which makes it very unlikely that mutagenesis 
will occur.54-56  In summary, our data suggest that AAV genomes persist in mouse muscle as 
transcriptionally active large and small concatameric episomes and when considering potential 
and theoretical integration events, AAV vectors appear to possess a similar risk profile as 
plasmid DNA injected into muscle.55  The majority of vector DNA appears to persist as episomal 
rather than integrated DNA, which makes it very unlikely that mutagenesis will occur. 54 
Therefore, we think the chance that cancer would develop is very small. 
Liver tumors were reported in one study performed in newborn mice at another center with a 
different rAAV vector.  There is no evidence to indicate that these tumors were caused by 

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vector-induced DNA damage based on the number of AAV genomes per cell in tumor samples. 
57, 58  Also, other larger scale studies in newborn and adult mice have not shown this effect. 59  It 
remains difficult to ascertain the true risk of neoplasia, although it seems very small.  
Subjects may also develop immune response to AAV1.  This could lead to immune rejection of 
virus precluding gene expression.  We will measure both binding antibodies and T cell responses 
to the AAV capsid following gene transfer.  
 
11.2.2 RISKS ASSOCIATED WITH PROCEDURES 
11.2.2.1 BLOOD DRAWS 
The risks of venous blood draws include discomfort at the site of puncture; possible bruising and 
swelling around the puncture site; rarely an infection; and, uncommonly, fainting, nausea, 
vomiting, and light-headedness from the procedure. 
 
11.2.2.2 GENE TRANSFER TO MUSCLES 
In this trial, patients will receive at least 18 injections per extremity.  This will be done under 
sterile conditions by the PI accompanied by the ultrasonographer.  Both will be gowned and 
gloved.  Patients will also receive conscious sedation that will be directed by the anesthesiologist 
at Nationwide Children’s Hospital. There is a low risk of complications that could include 
hypotension, respiratory compromise, and complications from low blood pressure such as stroke 
or myocardial infarction.  These would be rare and unlikely complications of the procedure.  
11.2.2.3 MUSCLE BIOPSY 
The muscle biopsy has potential complications from procedure that include infection at the biopsy site, 
bleeding and/or pain at the site of the biopsy, and psychological trauma from the scar at the site of the 
incision; in some cases because of keloid formation this can be quite large.   
11.2.2.4 STEROIDS  
The possible side effects of prednisolone or Deflazacort (steroids) include acne, increased hair growth, 
thinning of the skin, glaucoma, roundness of the face, changes in behavior, disturbance of sleep, and 
increase of blood glucose level.  
 
11.3 ADEQUACY OF PROTECTION AGAINST RISKS 
11.3.1 INFORMED OF RISKS  
 

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Volunteer subjects will be recruited from the Nationwide Children’s Hospital, Columbus-MDA 
Clinic or self-referred in response to advertisement.  This protocol will also be listed in the 
Clinical Trials Data Bank of the NIH.  Potential study subjects who demonstrate an interest in 
participating in the study will receive a copy of the informed consent form to review and an 
explanation of the nature, duration, and purpose of the study and possible consequences of the 
study from one of the investigators in a language they can understand.  They will be informed 
that they may withdraw from the study at any time and for any reason without jeopardizing their 
future treatment.  They will be asked to follow-up with necessary safety evaluations if they have 
received study agent prior to their desire to withdraw.  They will be given full information 
regarding potential side effects of rAAV1 and follistatin gene.  Subsequently, an investigator will 
request the subject's permission to participate in the study.  Volunteer participants who sign the 
study specific subject informed consent form approved by the Institutional Review Board of 
Nationwide Children’s Hospital will be screened for eligibility to participate in the gene transfer 
study.  
11.3.2 MINIMIZATION OF POTENTIAL RISK 
Participants will be provided with contact numbers for any questions or concerns arising 
regarding the possible effects of the administration of rAAV1.CMV.huFS344.  Results of all 
laboratory and safety exams regarding subject enrollment and following gene transfer will be 
reviewed with the subject.  Since the effects on the reproductive system are not fully established 
and the possible concerns with viral shedding, subjects with reproductive capacity must be 
willing to refrain from sexual intercourse  or use effective contraception for the active phase of 
the study until two negative sperm samples are obtained post gene transfer (males).  
Some of the vector can be excreted from the body for a least a week after injection.  This 
shedding of vector can be found in the blood, urine, saliva, and stool for up to a week following 
injection.  For a period of two weeks after gene injection, people who may come into contact 
with participants’ bodily fluids and waste must regularly wash their hands with soap and those 
who will have direct contact with their bodily fluids and waste must were protective gloves. 
 
11.3.3 CONFIDENTIALITY  
Access to the database will be limited to the key research personnel to ensure confidentiality.  
Case report forms, informed consent forms, laboratory study reports, and demographic profiles 
will be kept locked in a secure record storage and archiving under the responsibility of research 
staff.  The subjects will be informed of what information is stored and of the secured access to 
their information by the following regulatory bodies: FDA-CBER, IRB, IBCSC, NCH internal 
monitors, and an outside contracted monitor designated for the study called a “CRO”.  Subjects 
will be alerted that others may have an interest in the innovative character of the protocol and in 
the status of the treated subjects.  They will be informed that the institution and investigators will 
make efforts to provide protection from the media in an effort to protect the participants' privacy.  
They will be consulted as to whether, when, or how their identity is publicly disclosed. 
 

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11.3.4 PROVISIONS FOR INJURY 
If a subject experiences an injury that is directly related to this study, the event will be followed 
up until its resolution.  Nationwide Children’s Hospital will provide medical treatment 
reasonably necessary for such injury or illness at no cost to the subject or his insurance company 
for medical expenses up to $10,000. In order for The Research Institute at Nationwide Children’s 
Hospital to pay for these medical expenses the illness or injury must not be related to subject’s 
underlying medical condition and subject must have followed the study directions.  
If research subject seeks care for a research related injury or illness from a medical provider 
other than Nationwide Children’s Hospital, then The Research Institute at Nationwide Children’s 
Hospital will pay up to $10,000 to reimburse the research subject for treatment of such injury or 
illness directly resulting from the study.  Any claim for reimbursement must be supported by 
documentation such as a bill or invoice from a medical provider.  In order for The Research 
Institute at Nationwide Children’s Hospital to pay for these medical expenses the research 
subject’s illness or injury must not be related to his/her underlying medical condition and 
research subject must have followed the study directions. 
The Research Institute at Nationwide Children’s Hospital’s reimbursement for injury or illness as 
a result of research subject’s participation in the study does not include any payment for lost 
wages, lost time, emotional distress or pain.  This does not mean that the research subject gives 
up any of his legal rights to seek compensation for his illness or injury. 
 
11.4 POTENTIAL BENEFITS OF RESEARCH TO SUBJECTS AND OTHERS 
This is primarily a safety study of transfer of rAAV1.CMV.huFS344 .  Based on the results of 
the BMD clinical trial using the same vector, subjects participating in this trial could improve 
their walking ability. There is also anticipated benefit to determine the safety of this vector 
system that may provide efficient and sustained efficacy.   
11.5 INCLUSION OF MINORITIES  
11.5.1 SELECTION CRITERIA 
Six DMD subjects will be enrolled in this trial.  Eligibility criteria will include subjects (age 7 
and older) with proven DMD mutations.  Participants will encompass any ethnic or racial 
background.   
11.5.2 TARGETED / PLANNED ENROLLMENT 
The number of subjects enrolled from each ethnic group will mirror that seen in the clinic 
population. 
Local Distribution of Disease (Percent) 

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Gender 
American 
Indian Alaskan 
Native 
Asian or 
Pacific 
Islander 
Hispanic 
Black, 
Non-
Hispanic 
White, 
Non-
Hispanic 
Total 
Female 
0 
0 
0 
0 
0 
0 
Male 
0 
0.7 
1.4 
0.7 
97.2 
100 
Total 
0 
0.7 
1.4 
0.7 
97.2 
100 
 
 
11.5.3 PROPOSED DATES OF ENROLLMENT AND RECRUITMENT 
We anticipate beginning enrollment in Winter 2014, with completion of enrollment December 
2015.  This protocol will be listed in the Clinical Trials Data Bank of the NIH.  
12.0 DATA AND SAFETY MONITORING PLAN 
 
12.1 THE DATA SAFETY MONITORING BOARD 
The Data and Safety Monitoring Board (DSMB) will act in an advisory capacity to review 
participant safety and study progress for the “Phase I/II gene transfer clinical trial for Duchenne 
Muscular Dystrophy using rAAV1.CMV.huFS344.”  
 
Responsibilities of the DSMB are to:  
• Review the research protocol, informed consent documents and plans for data and safety 
 monitoring;  
• Evaluate the progress of the trial, including periodic assessments of data quality 
  and timeliness, participant recruitment, accrual and retention, participant risk 
  versus benefit, trial site performance, and other factors that can affect study outcome;  
• Consider factors external to the study when relevant information becomes available, such as 
 scientific or therapeutic developments that may have an impact on participant safety or the 
 ethics of the trial;  
• Review study performance, make recommendations and assist in the resolution of problems 
 reported by the Principal Investigator;  
• Protect the safety of the study participants;  
• Review safety data to determine whether to recommend dose escalation;  
 • Ensure the confidentiality of the trial data and the results of monitoring; and, 

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 • Assist by commenting on any problems with study conduct, enrollment, and sample size 
 and/or data collection.  
 
DSMB Reporting and Meetings 
Reports describing the status of the study will be prepared by the Principal Investigator’s staff 
and sent to the DSMB at the end of each cohort, or at the DSMB’s request.   
An initial meeting (either by teleconference or webcast) with the DSMB will be scheduled prior 
to study initiation and after Day 30 visit of the first subject, and approximately every 6 months 
thereafter, or at the DSMB’s request.  Reports will be submitted prior to a scheduled meeting for 
review by the DSMB.  
Reports will include the following: 
A brief narrative of the study status, including the target enrollment, current and  projected time 
to completing enrollment.  Any significant events and/or difficulties should be briefly described 
in this narrative. 
• A brief narrative for each participant describing age, race and ethnicity and other relevant 
 demographic characteristics.  The narrative for each participant should briefly describe his 
 study status (i.e., dose level, visit number, adverse event information); 
• A timeline outlining the study progress relative to visit number for each participant, as well as
 time points for each SAE/Dose Limiting Toxicity. A total for Adverse Events (AEs) for each 
 participant should be included. 
• A summary of AEs by severity levels; 
• A listing of AE details grouped by participant; 
• A listing of SAE details grouped by participant; 
• A listing of deaths 
• A summary of clinically significant laboratory test results 
• A listing of protocol deviations 
 
Stopping/Discontinuation Rules 
An independent Data Safety Monitoring Board (DSMB) will be designated by the study’s 
sponsor.  Safety data will be monitored on a continual basis throughout the trial.  The DSMB can 
recommend early termination of the trial for reasons of safety.  Study enrollment will be halted 
by the investigators when any subject experiences a Grade 3 or higher adverse event toxicity that 
is possibly, probably, or definitely related to the study drug.  This will include any subject death, 

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Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
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important clinical laboratory finding, or any severe local complication in the injected area related 
to administration of the study agent.  If after review by the DSMB, IRB, 
IBCSC, NIH-OBA and FDA, the decision is made to continue, the study will proceed according 
to Dose Escalation plan. 
 
MEMBERSHIP  
The DSMB membership consists of persons completely independent of the investigator who 
have no financial, scientific, or other conflicts of interest with the trial.  Current or past 
collaborators or associates of Dr. Mendell must note any conflict of interest before their 
eligibility to serve on the DSMB is approved.  
The DSMB will include experts in or representatives of the fields of:  
• Neurology and Neuromuscular Diseases  
• Immunology  
• Gene Therapy  
• Muscular Dystrophy Clinical Care  
• Clinical Research and Clinical Trials   
 
Conflict of Interest  
Individuals invited to serve on the DSMB as either voting or non-voting members must disclose 
any potential conflicts of interest, whether real or perceived.  Conflicts of interest can include 
professional, proprietary, and miscellaneous interests as described in the NIH Grant Policy 
Statement and 45 CFR Part 94.  Potential conflicts that develop during a member’s tenure on a 
DSMB must also be disclosed.  Written documentation attesting to an absence of conflict of 
interest is required.  
 
13.0 CLINICAL MONITORING OF THE STUDY 
The study will be monitored in compliance with the relevant parts of 21 CFR and according to 
the ICH GCP Guidelines. 
The procedures outlined in the protocol and case report forms will be carefully reviewed by the 
PI and staff prior to study initiation to ensure appropriate interpretation and implementation.  No 
deviations from the protocol shall be made except in emergency situations where alternative 
treatment is necessary for the protection, proper care and well-being of subjects. 

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Amendments will be submitted to the Nationwide Children’s Hospital IRB for their review and 
approval prior to implementation.  When an amendment to a protocol substantially alters the 
study design or increases potential risk to the study subject, the Informed Consent form will be 
revised and if applicable, subject's consent to continue participation will again be obtained.   
 
13.1 DATA MANAGEMENT AND STUDY FORMS 
All data and observations will be documented on electronic Case Report Forms (CRF) by source 
documentation using the Open Clinica Electronic Data Capture designed for the study.  A Safety 
Monitor will have access to the data to monitor adherence to the protocol and to applicable FDA 
regulations, and the maintenance of adequate and accurate clinical records.  An electronic Case 
Report Form will be completed for every subject that was registered for participation in the 
study.  The Case Report Form will be reviewed in detail.  Case Report Forms will be completed 
as information becomes available. 
 
Case Report Forms will be reviewed in detail by the Safety Monitor in a regular basis for which 
the Safety Monitor will have access to subject medical records, laboratory data, and other source 
documentation. Safety monitor will make a decision as to the data acceptability.  If errors or 
omissions are found in the course of a data audit, or if clarification of data is required, the 
electronic Case Report Form(s) in question will be corrected by the PI or his designee.  Data 
Resolution may be generated on omissions or clarifications, to be completed, electronically 
signed and dated, and maintained as a part of the eCRF.  The PI will sign and accept the 
indicated electronic Case Report Form. This signature will indicate that thorough inspection of 
the data therein has been made and will thereby certify the contents of the form.  
In collaboration with the study team, the Research Informatics Core designed a data collection 
system (Open Clinica) for managing the clinical trial. A web-based database was created and it 
will be managed by authorized users. CRFs will be transcribed to this web-based database. Data 
will be extracted from source documents (lab reports, echo reports…) and transferred to the 
database as well.  All source documents will be kept in the Subject Research Chart.  The secured 
portal will feature view and edit capability with field validations for quality controls, change 
history attribute and reporting.  
 
An outside contracted monitor of the study called a “CRO” will also monitor the study on a 
regular basis to make sure the study is conducted in compliance with all regulatory aspects of the 
protocol. 
14.0 AGENT ACCOUNTABILITY 
14.1 HANDLING OF INVESTIGATIONAL AGENT 

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"Investigator shall take adequate precautions, including storage of the investigational drug in a 
securely locked cabinet or other securely locked, substantially constructed enclosure, access to 
which is limited, to prevent theft or diversion of the substance into illegal channels of 
distribution." (Code of Federal Regulations, Title 21, Part 312 .69). 
The pharmacy will maintain records of all products received, compounded, dispensed, returned 
and destroyed.   
All materials used for injection, including sterile drapes, needles and syringes in contact with the 
vector will be sealed in leak proof primary and secondary containers.  To ensure the highest 
safety precautions, all waste will be double bagged in autoclave bag bearing the biohazard 
symbol and sealed with autoclave tape bearing name and room number.  The bag will then be 
autoclaved and disposed of in a biohazard waste container with name and room number. 
All surfaces will be wiped with 10% bleach with a 70% ethanol rinse.  Immediate 
decontamination procedures are performed whenever overt spills, splashes or other 
contaminating events occur.  All absorbent material is placed onto a blue absorbent pad and 
transferred into a properly labeled biohazard bag. 
Spills will be covered with absorbent paper towels and freshly prepared 10% bleach will be 
applied starting at the perimeter and working towards the center.  At least 30 minutes will be 
allowed after contact time before clean up.  In case of large spills, IBCSC and Environmental 
Services will be notified immediately.   
If an accidental exposure occurs (such as accidental needle stick), the exposure site will be 
washed with soap and water. 
Spills and accidents resulting in potential exposure of rAAV to individuals will be reported to 
Dr. Mendell, IBCSC, and the Employee Health Services within 24 hours.  Medical evaluations, 
surveillance, and treatment will be provided by the EHS.  All information concerning the 
incident is to be documented and submitted to Dr. Mendell, IBCSC, and the EHS. 
Only authorized personnel will be permitted in the surgery room during injection process.  
During the injection all personnel will be required to wear lab coat, gloves, respiratory mask, 
glasses, and toe covered shoes.   
14.2 DISPOSITION OF STUDY AGENT 
 
Investigator is required to maintain adequate records of the disposition of the drug, 
including dates, quantity, and use by subjects.  The NCH Pharmacy Disposal Policy 3-F-6 will 
be followed.  If the investigation is terminated, suspended, discontinued, or completed, the 
investigator shall return the unused supplies to the sponsor, or otherwise provide for disposition 
of the unused supplies (as authorized by the sponsor)."  (Code of Federal Regulations, Title 21, 
Part 312 .62, Section a). 
15.0 BRIEF DESCRIPTIONS OF RESEARCH AREAS 

Center for Gene Therapy 
 
Mendell, Jerry R. 
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Coordinating Center 
The Nationwide Children's Neuromuscular Research Institute is the Coordinating Center for the 
entire study.  Personnel consist of the following individuals: 
Study Principal Investigator: Jerry R. Mendell, M.D. 
Program Manager: Kathleen Church, MSW 
Clinical Evaluators: Linda Lowes. PT, PhD, Katherine Berry, Lindsay Alfano, PT.  
Safety Monitor: Gloria Galloway, M.D. 
Co-Investigator: Kevin Flanigan, M.D. 
Co-Investigator: Zarife Sahenk, M.D., PhD 
Co-Investigator: Louise Rodino-Klapac, PhD 
Co-Investigator: Mark Hogan, M.D. 
Co-Investigator: David Arnold, M.D. 
Co-Investigator: Samiah Al-Zaidy, M.D. 
Co-Investigator: Glenn A. Walter, PhD 
Research Coordinators: Brent Yetter, Ana Maria Gomez, MD; Sohyun McElroy, PhD. 
 
The Coordinating Center is responsible for the following: 
• Development and maintenance of the Manual of Operating Procedures 
• Enrollment of participants 
• Obtaining informed consent (by Principal Investigator only) 
• Adverse event monitoring and reporting 
 
Overview of Investigator Responsibilities 
 
 In conducting the clinical study, the Principal investigator is responsible: 
 

Center for Gene Therapy 
 
Mendell, Jerry R. 
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Protocol Version 5.0 – 16January2015 
1. For ensuring that a clinical investigation is conducted according to applicable regulations; 
2. For protecting the rights, safety, and welfare of subjects under the investigator's care; and 
3. For the control of the study drug under investigation  
4. To conduct the study in accordance with the relevant, current protocol(s) and to only 
make changes in the protocol after notifying the sponsor, except when necessary to 
protect the safety, rights, or welfare of subjects; 
5. To personally conduct or supervise the research study; 
6. To inform the subjects, that the drugs are being used for investigational purposes and to 
ensure that the requirements relating to obtaining informed consent in 21 CFR Part 50 
and institutional review board (IRB) review and approval in 21 CFR Part 56 are met; 
7. To report to the sponsor adverse experiences that occur in the course of the 
investigations(s) in accordance with 21 CFR 3 12.64; 
8. To ensure that all associates, colleagues, and employees assisting in the conduct of the 
study are informed about their obligations in meeting the above commitments; 
9. To maintain adequate and accurate records and to make those records available to FDA 
for inspection in accordance with 21 CFR 3 12.68; 
10. That IRB will be responsible for the initial and continuing review and approval of the 
clinical investigation; 
11. To promptly report to the IRB all changes in the research activity and all unanticipated 
problems involving risks to human subjects or others; 
12. To not make any changes in the research without IRB approval, except where necessary 
to eliminate apparent immediate hazards to human subjects; 
13. To comply with all other requirements regarding the obligations of clinical investigators 
and all other pertinent requirements in 2 1 CFR 3 12. 
 
Nationwide Children’s Hospital 
Nationwide Children’s Hospital is one of the nation’s most progressive and sophisticated health 
care institutions.  A multitude of comprehensive programs, integrating medical and surgical sub-
specialties, is the core for tertiary care provided at Children's Hospital.  Services are integrated 
between the inpatient and outpatient areas to enable a full continuum of subject care. 
The Nationwide Children’s Hospital’s services for this study are 
• Providing research beds and hospitals facilities for the inpatient phase of the research 
study 

Center for Gene Therapy 
 
Mendell, Jerry R. 
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• Subject care services and nursing support during the inpatient phase of the research study 
 
Clinical Study Center/ Outpatient Neuromuscular Clinic 
• Providing facilities for the follow up visits after gene transfer. 
 
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Center for Gene Therapy 
 
Mendell, Jerry R. 
Phase I/IIa Clinical intramuscular gene transfer of rAAV1.CMV.huFollistatin344 trial to patients with DMD 
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Protocol Version 5.0 – 16January2015