Phase I/IIa Gene Transfer Clinical Trial for Duchenne Muscular Dystrophy Using rAAVrh74.MCK.GALGT2
Phase: PHASE1
Conditions: Duchenne Muscular Dystrophy
Sponsor: Kevin Flanigan

Phase I/II gene transfer clinical trial for Duchenne Muscular Dystrophy using rAAVrh74.MCK.GALGT2 
Kevin M. Flanigan, M.D. 
 
Protocol version 8.0 (27March2019) 
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Phase I/IIa gene transfer clinical trial for Duchenne Muscular Dystrophy using 
rAAVrh74.MCK.GALGT2  
CLINICAL PROTOCOL 
 
Version: 8.0 (27March2019) 
Principal Investigator: Kevin Flanigan, M.D. 
 
Co-Investigators:  
 
Paul T. Martin, Ph.D. 
Louis G. Chicoine, M.D. 
Megan Waldrop, M.D. 
John Cheatham, M.D. 
Brian Boe, MD 
Rachel Schrader, CPNP-CP 
The Research Institute at Nationwide Children’s Hospital 
Center for Gene Therapy 

Phase I/II gene transfer clinical trial for Duchenne Muscular Dystrophy using rAAVrh74.MCK.GALGT2 
Kevin M. Flanigan, M.D. 
 
Protocol version 8.0 (27March2019) 
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Table of Contents 
1.0 Protocol Synopsis ..................................................................................................... 1 
2.0 Introduction .............................................................................................................. 4 
2.1 
Abstract ..................................................................................................................................................................... 4 
2.2 
Clinical Trial and Principal Investigator .................................................................................................................. 5 
2.3 
Background and Significance .................................................................................................................................... 6 
2.3.1 
Disease Characteristics .................................................................................................................................... 6 
2.3.2 
Disease Pathogenesis ....................................................................................................................................... 6 
2.3.3 
Current Treatment for DMD ............................................................................................................................ 6 
2.3.4 
Rationale for Gene Therapy using GALTGT2 .................................................................................................... 7 
3.0 Clinical Research Plan ............................................................................................. 7 
3.1 
Study Population ....................................................................................................................................................... 7 
3.2 
Inclusion/Exclusion Criteria ..................................................................................................................................... 7 
3.3 
Informed Consent ...................................................................................................................................................... 8 
3.4 
Establish Subject Identification Number ................................................................................................................... 9 
3.5 
Dosing Plan ............................................................................................................................................................... 9 
3.6 
Gene Transfer Protocol ........................................................................................................................................... 10 
3.6.1 
Baseline Measures Prior to Vector Injection  (Day -45 to Day -1) ................................................................. 10 
3.6.2 
Pre Infusion Visit (Day -1) .............................................................................................................................. 11 
3.6.3 
Day of Gene Transfer (Day 0) ......................................................................................................................... 11 
3.6.4 
Vascular delivery via intravascular limb infusion for gene therapy (ILI-GT) .................................................. 12 
3.7 
Post Gene Transfer Monitoring Plan ...................................................................................................................... 14 
3.7.1 
Up to 48-Hour Inpatient Monitoring (Days 1 and 2) ...................................................................................... 14 
3.7.2 
Post-Gene Transfer Monitoring (Days 7, 14, 30) ........................................................................................... 15 
3.7.3 
Post-Gene Transfer Monitoring after Day 30 (Days 45, 60, 75, 90, 180; Months 12, 18, and 24) ................. 16 
3.8 
Muscle biopsy .......................................................................................................................................................... 17 
3.9 
Study Timeline ......................................................................................................................................................... 18 
3.10 
Long-Term Monitoring ....................................................................................................................................... 19 
3.11 
Outcome Measures ............................................................................................................................................. 19 
3.12 
Statistical Analysis .............................................................................................................................................. 20 
4.0 Adverse Event Reporting ...................................................................................... 21 
4.1 
Classification of Adverse Events: ............................................................................................................................ 21 
4.2 
Dose limiting toxicity (DLT) .................................................................................................................................... 21 
4.3 
Stopping/Discontinuation Rules .............................................................................................................................. 22 
4.4 
Expected Adverse Events Related to Disease Progression ...................................................................................... 22 
4.5 
Dose Escalation....................................................................................................................................................... 22 
5.0 Adverse event monitoring ..................................................................................... 23 
5.1 
Definition of an Adverse Event ................................................................................................................................ 23 
5.2 
Serious adverse event (SAE) .................................................................................................................................... 24 

Phase I/II gene transfer clinical trial for Duchenne Muscular Dystrophy using rAAVrh74.MCK.GALGT2 
Kevin M. Flanigan, M.D. 
 
Protocol version 8.0 (27March2019) 
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5.3 
Life-threatening (21 CFR 312.32(a)) ...................................................................................................................... 24 
5.4 
Obligations of the Investigator ................................................................................................................................ 24 
5.5 
Safety Reporting ...................................................................................................................................................... 25 
5.5.1 
Serious Adverse Event Reporting: Content and Format ................................................................................ 26 
5.6 
Unexpected Adverse Events ..................................................................................................................................... 26 
5.7 
Follow-up of Adverse Events ................................................................................................................................... 26 
5.8 
Adverse Event Reporting from Primary Cary Physician ......................................................................................... 26 
6.0 Study Reports ......................................................................................................... 27 
6.1 
Final Study Report .................................................................................................................................................. 27 
6.2 
Annual Study Reports .............................................................................................................................................. 27 
6.3 
Data and Safety Monitoring Plan ............................................................................................................................ 27 
6.3.1 
The Data Safety Monitoring Board ................................................................................................................ 27 
6.3.2 
DSMB Reporting and Meetings ...................................................................................................................... 28 
6.3.3 
Membership................................................................................................................................................... 28 
6.4 
Clinical Monitoring of the Study ............................................................................................................................. 29 
6.4.1 
Data Management and Study Forms ............................................................................................................. 29 
7.0 References ............................................................................................................... 31 
 

Phase I/II gene transfer clinical trial for Duchenne Muscular Dystrophy using rAAVrh74.MCK.GALGT2 
Kevin M. Flanigan, M.D. 
 
Protocol version 8.0 (27March2019) 
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1.0 PROTOCOL SYNOPSIS 
Title 
Phase I/IIa gene transfer clinical trial for Duchenne Muscular Dystrophy 
using rAAVrh74.MCK.GALGT2 
Study Number 
 
Cohort 1: N = 1 [Low dose: 2.5 x 1013 vg/kg per leg, delivered bilaterally 
(total 5.0 x 1013 vg/kg)] 
 
Cohort 2: N=1 [High dose: 5x1013 vg/kg per leg, delivered bilaterally (total 
1.0 x 1014 vg/kg)] 
 
Clinical Study Phase Phase I/IIa trial 
Number of Centers 
Single site (Nationwide Children’s Hospital)  
Study Objectives 
The primary objective is assessment of safety.   
Study Design 
Dose escalation study rAAVrh74.MCK.GALGT2: 
- 
 Intravascular limb infusion for gene transfer delivered bilaterally 
(two cohorts) via a major lower limb artery to the whole lower limb 
of DMD subjects. 
Patient Population 
Inclusion Criteria 
• Ambulant patients age 4 years or older  
• Confirmed mutations in the DMD gene using a clinical accepted 
technique that completely defines the mutation   
• Measurably impaired muscle function (defined as less than 80% of 
the predicted value for age 100 MWT), but with sufficient muscle 
preservation to ensure assessment of muscle transfection based on 
clinical evaluation by the PI and expert colleagues.  This degree of 
preservation will include: 
o Ability to extend the knee fully against gravity  
o Preserved ambulation with ability to walk ≥ 350 meters 
during the 6MWT  
o A magnetic resonance image of the quadriceps showing 
preservation of sufficient muscle mass to permit 
transfection 
• Males of any ethnic group will be eligible 
• Ability to cooperate with muscle testing 
• Stable dose of corticosteroid therapy (including either prednisone, 
prednisolone, deflazacort or their generic forms) for at least 12 
weeks prior to gene transfer  
Exclusion Criteria 
• Active viral infection based on clinical observations 
• The presence of a DMD mutation without weakness or loss of 
function  
• Subject is amenable to or is currently being treated with eteplirsen 
• 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 

 
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2.3 
Background and Significance 
 
2.3.1 Disease Characteristics 
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 
births5.  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 occurring, in the pre-
corticosteroid treatment era, by age 126.  Historically patients died from respiratory 
complications, but improved pulmonary and ventilatory measures (including nocturnal 
ventilatory support) have further improved survival7.  Prolonged survival from in turn  unmasks 
a decline in cardiac function,  marked by dilated cardiomyopathy, raising additional treatment 
challenges in the late-stage patient.    
 
2.3.2 Disease Pathogenesis 
Nearly 30 years ago, the DMD gene was cloned, defining the molecular basis of the disease8 
and leading to the identification of dystrophin as the deficient protein in muscle from DMD 
patients9.  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 
regions10.  Originally multiplex PCR kits were developed that were able to detect 95%-98% of 
all deletions11, 12, but improved molecular methods of diagnosis – including multiplex ligation-
dependent probe amplification (MLPA)13,  multiplex amplifiable probe hybridization 
(MAPH)14, and comparative genomic hybridization (CGH) allow screening of all exons for 
copy number variation, facilitating detection of deletions and duplications.  Methods of direct 
sequence analysis of the entire coding region are now in routine use15, allowing detection of 
nearly all clinically relevant disease mutations15.  
 
2.3.3 Current Treatment for DMD 
The only therapeutic approach that has clearly demonstrated unequivocal efficacy for treatment 
of DMD is treatment with the glucocorticoids prednisone or deflazacort.  Treatment with one or 
another of these agents has been repeatedly shown to result in increased strength and delay loss 
of ambulation16-18.  Deflazacort has recently gained FDA approval for treatment of boys with 
DMD, based largely on a slightly more favorable side effect profile19, and although it is not yet 
commercially available in the US many patients order it from overseas. However, treatment 
with these drugs is associated with significant side effects, including weight gain, Cushingoid 
features, hypertension, cataract formation, loss of bone density, vertebral compression fractures, 
long bone fractures, and behavioral problems. Molecular therapies provide a promising 
alternative to glucocorticoids.  One approach is direct gene replacement.  Other approaches 
include exon skipping and nonsense mutation suppression (or “readthrough”).  Exon skipping 
utilizes antisense oligomers to induce altered pre-mRNA splicing to restore an open reading 
frame, and a single such agent (eteplirsen) has received FDA approval, based largely on a small 
but statistically significant increase in dystrophin expression20, 21.  However, both exon skipping 
and the as-yet unapproved nonsense suppression approach22-24 would only be beneficial to small 
subsets of patients with appropriate mutations.  For example, eteplirsen would be beneficial to 
only 13% of patients, and the nonsense suppression agent ataluren to only to 15%.  

 
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3.6 
Gene Transfer Protocol 
 
3.6.1 Baseline Measures Prior to Vector Injection  (Day -45 to Day -1) 
Informed consent will be obtained prior to the collection of any data and any study related 
procedures. Baseline measures from subjects will be obtained prior to gene transfer. A complete 
physical examination, medical history and concomitant medication history will be obtained. 
Vitals will be obtained, including temperature, respiratory rate, heart rate, blood pressure, 
height, and weight. Pre-treatment studies will also include 
 
 echocardiogram, ECG, the North Star Ambulatory Assessment 
(NSAA), a video recorded six minute walk test (6MWT), the 100 meter walk/run test, the 
maximum voluntary isometric contraction testing (MVICT) of knee extension and knee flexors 
bilaterally, pulmonary function testing (PFTs) including spirometry, and an open muscle biopsy.  
 
Lab work will include the following: 
• Complete blood cell count (CBC) and differential, platelets 
• Aspartate aminotransferase (AST)/alanine transaminase (ALT) 
• Gamma-glutamyl transpeptidase (GGT)* 
• A Comprehensive Metabolic Panel (CMP) that includes: 
o Albumin/Serum total protein 
o Serum total bilirubin 
o Electrolytes 
o Calcium 
o Creatinine/Blood Urea Nitrogen (BUN) 
o Serum glucose 
o Alkaline phosphatase  
• Prothrombin time (PT)/activated partial thromboplastin time (PTT) 
• Amylase 
• Creatine kinase (CK) 
• Cystatin C 
• Antibody testing for Hepatitis B,C and HIV1 and 2. 
• Urinalysis 
• Whole blood for biobanking 
• Urine for biobanking  
* For subjects taking drugs that potentially induce GGT synthesis through the cytochrome P450 system, the 
clinically significant range will be estimated at two times the levels obtained from the baseline screening test. 
 
Additionally, ELISA will be performed for detection of total antibodies to AAVrh74 and anti-
Sda, and ELISpots for T-cell responses to AAVrh74 and GALGT2. 
 
A baseline open muscle biopsy will be performed in one quadriceps muscle.  (Note that the 
rationale and methods of muscle biopsy site selection and performance for this and subsequent 
biopsies are discussed in Section 3.8) 
 
The baseline evaluation will take place over two visits (one to two weeks apart).  Because a pre-
existing antibody response would be exclusionary, ELISA and ELISpot testing will be 
performed at the first visit, allowing the results to be reviewed prior to the muscle biopsy with 
its attendant risks (including sedation).    
 

 
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A photograph will be taken of each injection site. 
On the second day after gene transfer (Day 2) the subject will be discharged from the hospital 
after a physical examination by the principal investigator or designee, as well as review and 
documentation of concomitant medications, medical history and all adverse events/serious 
adverse events following injection. 
 
 
 
3.7.1.1 Post-injection immune suppression 
All subjects will be discharged from the hospital on oral prednisone or prednisolone at 1 
mg/kg/day, which will be adjusted according to IFN-gamma T-cell studies in the following 
weeks.   
 
If the 30 day post-infusion IFN-gamma T-cell response shows ≤50 SFC per 1 x 106 PBMCs, the 
subject’s prednisone or prednisolone dose will be decreased back to the standard clinical dose of 
0.75 mg/kg/day.  Those subjects who had been on deflazacort may be switched to the standard 
clinical dose of 0.9 mg/kg day.  For either regimen, approximations of these target doses may be 
allowed to account for pill formulations (for prednisone and deflazacort), as in clinical practice.  
If either the AST and ALT exceeds >2.5X the subject’s baseline values, and results are 
confirmed on a follow-up blood test, the prednisone or prednisolone regimen will be maintained 
at 1 mg/kg/day until the enzyme levels fall  within a range , 2.5X the baseline value.   
 
If the IFN-gamma T-cell response shows ≥50 SFC per 1 x 106 PBMCs, we may increase the 
dose to approximately 2 mg/kg/day depending on T-cell response measured by ELISpot assay, 
and prolong a subsequent tapering protocol based on the individual subject’s immune response 
profile as assessed by subsequent ELISpot assays (as on the schedule discussed below). Based 
upon other gene transfer trials performed at NCH, we anticipate that oral prednisone or 
prednisolone may be administered for up to 120 days post gene transfer prior to return to the 
standard clinical corticosteroid regimen of each subject. 
 
 
3.7.2 Post-Gene Transfer Monitoring (Days 7, 14, 30)  
Subjects will return for follow up visits on Days 7, 14, and 30 after treatment, with repeat 
physical examination, review of concomitant medications, updates to the medical history, 
adverse events, vital signs (heart rate, respiratory rate, temperature, weight, and blood pressure) 
and blood and urine studies at each visit.  
 
Lab work will include the following: 
• Complete blood cell count (CBC) and differential, platelets 
• Aspartate aminotransferase (AST)/alanine transaminase (ALT) 
• Gamma-glutamyl transpeptidase (GGT)* 
• A Comprehensive Metabolic Panel (CMP) that includes: 
o Albumin/Serum total protein 
o Serum total bilirubin 
o Electrolytes 
o Calcium 
o Creatinine/Blood Urea Nitrogen (BUN) 
o Serum glucose 
o Alkaline phosphatase  

 
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• Prothrombin time (PT)/activated partial thromboplastin time (PTT) 
• Amylase 
• Urinalysis 
• 
 
• ELISpots for T-cell responses to AAVrh74 and GALGT2 
• Creatine kinase (CK)# 
• 
 
* For subjects taking drugs that potentially induce GGT synthesis through the cytochrome P450 system, the 
clinically significant range will be estimated at two times the levels obtained from the baseline screening test. 
# CK will not occur on Day 14 
 
On Day 30, additional tests will include an ELISA for detection of total antibodies to AAVrh74, 
Cystatin C, and whole blood banking to investigate vector shedding.  
 
To facilitate these scheduled visits, all subjects will remain in the Columbus metropolitan area 
for a minimum of two weeks following gene transfer.  If there is a systemic immune response 
that leads to acute respiratory distress syndrome, which implies a severe inflammation of the 
lung parenchyma, the patient would be hospitalized and treated aggressively with fluid 
replacement for hypotension if necessary and receive pulse methylprednisolone treatment (1 
gram per day) over 3-5 days.   
 
 
3.7.3 Post-Gene Transfer Monitoring after Day 30 (Days 45, 60, 75, 90, 180; Months 12, 18, 
and 24) 
 
After the follow-up visit at Day 30 post treatment, subjects will continue outpatient active 
monitoring through 2 years post-gene transfer. Subjects will return for follow-up visits at Days 
60, 90, and 180 and at Months 12, 18 and 24 after treatment. Please refer to the graphical Study 
Timeline of Events below for a summary of studies and procedures at each visit. 
At Days 45 and 75, targeted safety labs will be drawn, assessing liver function (AST, ALT, and 
GGT).  For convenience, for subjects outside of the Columbus area, these may be locally drawn 
and shipped to the NCH clinical laboratory.   
The Day 60 visit safety assessments include updates to the medical history, physical 
examination, concomitant medications, vital signs (heart rate, respiratory rate, temperature, 
weight, and blood pressure) and blood work, including: AST, ALT, GGT, ELISA for detection 
of serum total antibodies to AAVrh74 and ELISpots for T-cell responses to AAVrh74 and 
GALGT2.  Urine and whole blood will be banked for possible vector shedding studies. 
The Day 90, Day 180, 12 month, 18 month, and 24 month visits include updates to the 
medical history, physical examination, concomitant medications, vital signs (heart rate, 
respiratory rate, temperature, weight, and blood pressure, height), ECG, echocardiogram, and 
the following blood and urine studies:  
• Complete blood cell count (CBC) and differential, platelets. 
• Aspartate aminotransferase (AST)/alanine transaminase (ALT) 
• Gamma-glutamyl transpeptidase (GGT)* 
• A Comprehensive Metabolic Panel (CMP) that includes: 
o Albumin/Serum total protein 
o Serum total bilirubin 
o Electrolytes 

 
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3.9 
Study Timeline 
Table 2. Schedule of Events 
 
 
Note: There will be a flexibility of working days for each of the planned study visits including screening to adjust the schedule to any 
unanticipated event.  The windows for each appointment are as follows: 
• 
Days 7 and 14 ± 2 days 
• 
Day 30 ± 3 days 
• 
Days 60 and 90 ± 7 days 
• 
Day 180, and Months 12, 18 and 24 ± 14 days 
    
Study Interval 
Pre Infusion 
Visit
Hospital Admission 
Vector Injection
Visit #
Visit 12
Visit 2
Visit 4
Visit 5
Visit 6
Labs8
Visit 7
Labs8
Visit 82
Labs8
Visit 92
Visit 102
Visit 112
Visit 122
Study Procedures 
Day -1
Day 0
Day 1
Day 2
Day 7
Day 14
Day 30
Day 45
Day 60
Day 75
Day 90
2 weeks 
post 
steroids
Day 180
Month 12
Month 18
Month 24
Informed consent1 
X
Medical History
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Physical exam 
X
X
X6
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
Concomitant medications 
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Vital signs
X
X
X7
X
X
X
X
X
X
X
X
X
X
X
Height
X
X
X
X
X
X
Weight
X
X
X
X
X
X
X
X
X
ECHO
X
X
X
X
X
X
EKG
X
X
X
X
X
X
North Star Ambulatory 
Assessment
X
X
X
X
X
X
Video recorded 6MWT
X
X
X
X
X
X
100 m walk/run
X
X
X
X
X
X
MVICT
X
X
X
X
X
X
PFTs/Spirometry 
X
X
X
X
X
Anaesthesia
X
X
X
X
X
CBC/Diff
X
X
X
X
X
X
X
X
X
X
X
AST/ALT
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Serum GGT 
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Serum total protein/albumin
X
X
X
X
X
X
X
X
X
X
Serum Total bilirubin 
X
X
X
X
X
X
X
X
X
X
Electrolytes
X
X
X
X
X
X
X
X
X
X
Calcium
X
X
X
X
X
X
X
X
X
X
Creatinine/BUN
X
X
X
X
X
X
X
X
X
X
Random Glucose
X
X
X
X
X
X
X
X
X
X
Alkaline Phosphatase
X
X
X
X
X
X
X
X
X
X
PT/PTT/INR
X
X
X
X
X
X
X
X
X
X
X
Amylase
X
X
X
X
CK
X
X
X
X
X
X
X
X
X
X
Cystatin C
X
X
X
X
X
X
X
Antibody testing Hep. B, C 
and HIV
X
Urinalysis 
X
X
X
X
X
X
X
X
X
X
X
ELISA
X
X
X
X
X
X
X
X
ELISpot
X
X
X
X
X
X
X
X
X
X
X
Steroid Taper5
X
X
X
X
X
X
X
X
X
X
X
Admit to Hospital
X
Photograph injection site
X
X
Ultrasound (Injection needle 
guide)
X
Study agent administration
X
Whole Blood Banking
X
X
X
Urine Banking
X
X
X
X
X
X
X
7. Day 0 Vital Signs (Heart rate, respiratory rate, pulse oximetry, temperature, and blood pressure) will be measured before and immediately after the infusion, and at least every five minutes during 
the infusion, and repeated at 15 minutes post-infusion. VS will be obtained hourly for 4 hours following the injection and then every 4 hours until discharge.
1. If there are changes to the study, parents will be re-consented at their next visit.
8. The immunology studies will continue at post-treatment day 7, 14, 30, 45, 75, and 9, 12, 18, and 24 months post-gene transfer. Samples scheduled in between f/u visits will be collected at locally 
and ship to us. Immunology studies include ALT, AST and GGT.
(ILI-GT PROTOCOL) STUDY TIMELINE
Inpatient
Follow-Up (Outpatient) 
Visit 32
Days -45
to -1
Screening
2. Visit will occur over multiple days. 
3. Muscle strength testing at baseline (days -45 to -1) and visits 8-10 will be performed at least 1 day preceding the biopsy. 
4. An open muscle biopsy will be peformed on one leg at the baseline, and on the other leg at 3 months post gene transfer.
 
.
5. Prophylactic prednisolone/prednisone taper begins on Day -1 and is tapered according to AST, ALT, and ELISpot results.  Anticipate that most subjects will be on prednisolone for up to 120 days.
6. Day 0 physical exam will consist of the anesthesia evaluation prior to sedation.

 
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qualifies for reporting. The investigator will confer with the DSMB and FDA before continuing 
to enroll.  
 
4.3 
Stopping/Discontinuation Rules 
An independent Data Safety Monitoring Board (DSMB) will be established by the funding 
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 two 
or more Grade 3 or higher adverse events that are unanticipated and possibly, probably, or 
definitely related to the study drug. This will include any subject death, 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 the decision is made to continue, the study will 
proceed according to Section 4.5 (Dose Escalation) of this protocol. 
 
4.4 
Expected Adverse Events Related to Disease Progression 
Subjects enrolled in under this clinical protocol are expected to present clinically with adverse 
events related to natural progression of the disease. The draft guidance entitled “Duchenne 
Muscular Dystrophy Developing Drugs for Treatment over the Spectrum of Disease” 
(http://www.parentprojectmd.org/site/DocServer/Guidance_Document_Submission_-
_Duchenne_Muscular_Dystrop.pdf?docID=15283) provides the basis of expected disease-
related adverse events. 
 
Adverse events determined to be due to the underlying disease progression will be recorded but 
will not be subject to the expedited reporting requirements outlined in Section 5.0. All AEs 
related to the disease and unrelated to the gene therapy administration will be reported annually 
to the FDA, NIH/OBA and IRB. 
 
4.5 
Anticipated Adverse Event Lab Findings related to Intervention: 
 
• Asymptomatic elevations in transaminases are a feature of Duchenne muscular dystrophy, as 
they are related to release of AST and ALT from muscle tissue and correlate with levels of 
creatine kinase.  These are a feature of the disease itself.  Levels of AST up to 12.3X and ALT 
up to 22.6X the upper limit of normal are expected to be seen at baseline, and in the setting 
of normal GGT function are not indicative of muscle injury.  They are thus not exclusionary 
for enrollment, and will not be recorded as adverse events prior to gene transfer.   
Following gene transfer, transient transaminases (up to levels of 2.5x the patient’s baseline 
value) with preserved liver synthetic function and no significant elevation in GGT are 
anticipated, and will be recorded as Grade 1 adverse events, but not considered as adverse 
reaction in the presence of a normal GGT value. Mild decreases in leukocytes and 
lymphocyte counts within the first 30 days after gene transfer have been observed in other 
trials of AAV mediated gene transfer.  Such transient decreases in lymphocytes or 
leukocytes will be recorded as adverse events but considered anticipated.   
• Leukocytosis and neutrophilia during prednisone or prednisolone treatment: 
An elevation of up to 5000 cells/mm3 above the subject’s baseline value is expected in the 
neutrophil count (the primary granulocyte in circulation) and, consequently, in the leukocyte 
count within 5 hours of initiating prednisone or prednisolone therapy, based upon studies in 
healthy adult volunteers35. This occurs secondary to release of granulocytes from the 

 
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marginated pool into the circulation accompanied by an increase in the size of the marginated 
granulocyte pool due to steroids.36 This steroid effect may be seen with increased steroid 
dosing.   
 
4.6 
Dose Escalation 
There will be at least 4-6 weeks between dosing of subjects within a cohort.  The allowance of 
four weeks between dosing of subjects provides time for an internal review of the safety 
analysis from five time points (days 1, 2, 7, 14 and 30) prior to dosing of the next subject and 
time for review of the safety analysis of all the subjects within a cohort by the investigators and 
the DSMB. The 30-day post-injection data from one subject in the first cohort will be reviewed 
and discussed with the DSMB prior to dose escalation to the higher dose cohort. Dose 
escalation will be based on dose-limiting toxicity (DLT). 
 
The investigators will confer with the IRB, and DSMB on all Grade 3 or higher adverse events 
that are possibly, probably, or definitely related to the study agent before continuing enrollment.  
Based on the outcome of the safety and efficacy analysis at the end of each cohort decisions will 
be made to proceed with dose escalation for the following cohort. 
 
5.0 ADVERSE EVENT MONITORING 
5.1 
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/
UCM227351.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: 1 = mild, 2 = moderate, 3 = 
severe, 4 = life threatening or debilitating, and 5 = fatal as indicated above in section 7.5.  
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. 

 
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5.2 
Serious adverse event (SAE)  
An adverse event or suspected adverse reaction is considered “serious” if, in the view of either 
the investigator or funding agency, 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 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 3, 4, and 5.  
 
Category 3:  Severe adverse event; inability to carry on normal activities; required professional 
medical attention 
Category 4:   Life-threatening or permanently disabling adverse event 
Category 5:   Fatal adverse event 
 
5.3 
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 funding agency, 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).  
5.4 
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 adverse event reporting requirements in 
accordance with federal regulations, state laws, and the local institutional policies and 
procedures, as applicable. 
  

 
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The Principal Investigator will be responsible for ensuring that the reporting requirements are 
fulfilled and will be held accountable for any reporting lapses. 
5.5 
Safety Reporting  
 
The investigator or his designee will report all serious and unexpected adverse events to the 
IRB, NIH OBA, and DSMB according to regulatory requirements described as follows: 
 
DSMB and OBA/IRB: All Serious Adverse Events (SAEs) and Dose Limiting Toxicities 
(DLTs) will be reported to the DSMB and NIAMS, through KAI, and the IRB within 
48 hours of notification, regardless of expectedness, relatedness, or if they meet the 
definition for unanticipated problems to the clinical trial  
FDA/NIH OBA/IRB: 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 by 
the study investigator concurrently to the FDA/NIH and the OBA/IRB as soon as 
possible, but not later than 7 calendar days after the funding agency’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 concurrently to the FDA/NIH 
OBA/IRB as soon as possible, but not later than 15 calendar days after the funding agency’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. 
 
If, after further evaluation, an adverse event initially considered not to be associated with the 
use of the gene transfer product is subsequently determined to be associated, then the event will 
be reported concurrently to the FDA/NIH OBA/IRB as soon as possible, but in no case later 
than 15 calendar days after the determination is made. 
 
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 and the 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 and the DSMB within 15 calendar days of the determination. 
 
Any finding from tests in laboratory animals that suggests a significant risk for human research 
participants including reports of mutagenicity, teratogenicity, or carcinogenicity will be reported 
to FDA/NIH OBA/IRB and the DSMB as soon as possible to the , but not later than 15 
calendar days after initial receipt of the information. 
 
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. 
 
 
 

 
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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. 
6.0 STUDY REPORTS  
6.1 
Final Study Report 
The final study report will include data through the final study visit but will not include long-
term follow-up information.   
6.2 
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 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. 
6.3 
Data and Safety Monitoring Plan 
 
6.3.1 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 rAAVrh74.MCK.GALGT2 trial.”  
 
Responsibilities of the DSMB are to:  
• Review the research protocol, informed consent documents and plans for data and safety 
monitoring; 

 
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• Evaluate the progress of the trial, including periodic assessments of data quality and 
timelines, 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 trials; 
• 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  
• Assist by commenting on any problems with study conduct, enrollment, and sample size 
and/or data collection. 
 
6.3.2 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 last subject in each cohort, which will be 
approximately every 6 months, 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 gender, age, race and ethnicity and other 
relevant demographic characteristics. The narrative for each participant should briefly 
describe his/her 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 
 
6.3.3 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. Flanigan 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  

 
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• Gene Therapy  
• Muscular Dystrophy Clinical Care  
• Clinical Research and Clinical Trials   
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 annually.  
6.4 
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. 
 
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. 
 
The Safety Monitor will be a contracted Clinical Monitor through Novella Clinical every two 
months. The Monitor will be responsible for source verification against the Electronic Database 
Capture System, ensure adverse events are reported and review the regulatory binder.  
6.4.1 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, etc) and transferred to 

 
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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. 
  

 
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