Phase I/IIA Gene Transfer Clinical Trial for LGMD2D (Alpha-Sarcoglycan Deficiency) Using scAAVrh74.tMCK.hSGCA
Phase: PHASE1
Conditions: Limb-Girdle Muscular Dystrophy, Type 2D
Sponsor: Sarepta Therapeutics, Inc.

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Center for Gene Th era PV 
Phase I/Ila gene transfer clinical trial for LGMD2D using scAAVrh74.tMCK.hSGCA 
 
Phase I/Ila gene transfer clinical trial for LGMD2D (alpha-sarcoglycan deficiency) using 
scAAVrh74.tMCK.hSGCA 
CLINICAL PROTOCOL 
IND#: 15800 
Version: 11.0 {10 Feb 2017) 
Sponsor Investigator:
 
Funded by: 
NIH/NIAMS 
The Research Institute at Nationwide Children's Hospital 
Center for Gene Therapy 
Protocol Version11.0: 10 Feb 2017 
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Phase 1/tla gene transfer clinical trial for LGM020 using scAAVrh74.tMCK.hSGCA 
 
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TABLE OF CONTENTS 
TABLE Of CONTENTS ................................................................................................... ERROR! BOOKMARK NOT 0£FINEO.~ 
3.0 CLINICAL TRJAL ANO PRINCIPAL INVESTIGATOR .................................................................................................. 18 
4.0SPECIFICAIMS ....................................................................................................................................................... 9 
4. l Primary ObjectWe ............................................................................................................................................. 9 
4.2 Seeondory Objective ..................................................................................................................................... !fl..W 
4.3 
 ..................................................................................................................................... 10 
S.O BACKGROUND AND SIGNIFICANCE .....•................•..................•........................................................................... 10 
6.0CLINICAL RESEARCH PLAN ................................................................................................................................... 13 
6.1. Study Population ............................................................................................................................................ 13 
6.2. Pre-Treatment Assessment: ........................................................................................................................... 14 
6.3 Protocol for Gene Transfer ............................................................................................................................. 19 
6.4 Post- Gene Transfer Monitoring ..................................................................................................................... 2 7 
6.5 Long·Term Monitoring .................................................................................................................................... 31 
6.6 Outcome Measures ......................................................................................................................................... 31 
6.8. Study Timeline ........................................................................................................................................... 343-5 
7 .0 TeST MATERIAL AND ADMINISTRATION .......................................................................................................... 36¼7 
7.1 Description of Biological Product ................................................................................................................ 
7.2 Study De sign ............................................................................................................................................... JJJ.JB 
7.4 Dcse Limiting ToxJclry ................................................................................................................................. 41~ 
7.5 Stopplng/DisconUnuation Rules ................................................................................................................. 444$ 
7.,6 Dose Escalation ........................................................................................................................................... 4546 
7. 7 Concomitant Medications/Therapies ......................................................................................................... 
8.0 ADVERSE EVENT MONITORING ANO REPORTING ........................................................................................... 464+ 
8.1 Oefinition of an Mverse Event ................................................................................................................... 464+ 
8.2 Obligations of the lnve:Stigator ................................................................................................................... WJ, 
8.3 Safety Reporting ......................................................................................................................................... :2J.~ 
8.4 Unexpected Adverse Events ........................................................................................................................ 54~ 
8.S Follow-up of Adverse Events ....................................................................................................................... 54!;.5 
9.0 lONG--TERM FOLLOW-UP ................................................................................................................................ S4s.6 
9.1 Adverse Event Reporting from Primary Core Physician .............................................................................. 55.S+ 
10.0 STUDY REPORTS ............................................................................................................................................ SJ:iS8 
10.l Final Study Report ................................................................................. , ............................................................. ......... .... 2.§.5,S 
10.2 Annual Study Reports ............................................................................................................................... 56!-8 
11.0 HUMAN SUBJECTS ......................................................................................................................................... SSW 
11.1 .Human Subjects Involvement ond Choroct~ristics .................................................................................... 
11.2 Sources of Materials ................................................................................................................................. ~6{) 
11.3 Potential Risks .......................................................................................................................................... 2.2_6{) 
11.4 Mequacy of Protection against Risks ....................................................................................................... 6466 
11.5 Potentfol Benef,ts of the Proposed Research to the Subjects and Others ................................................. 687(} 
11.6 Inclusion of Minorities in Clinical Research ............................................................................................... li9+¾ 
12.0 DATA AND SAFETY MONITORING Pl.AN ......................................................................................................... 70+;! 
12.1 The Doto Safety Monitoring Beard ........................................................................................................... 7~ 0 
13.0 CLINICAL MONITORJNG OF THE STUDY ......................................................................................................... 7476 
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Phase I/Ila gene transfer clinical trial for LGMD2D using scAAVrh74.tMCK.hSGCA 
 
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13.1 Data Management and Study Forms ........................................................................................................ 7577-
14.0 AGENT ACCOUNTABJLITY ............................................................................................................................. , 7678 
14.l Handling of 1nvestigotional Agent ............................................................................................................ 76+8 
14.2 Disposltlan of Study Agent. ....................................................................................................................... 788(} 
15.0 BRIEF DESCRIPTIONS OF RESEARCH AREAS ................................................................................................... 7889 
16.o R~FERtNcrs ...................................................................................................................................................... 8284 
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1.0 PROTOCOL SYNOPSIS 
Title 
Phase I/Ila gene t ransfer clinical trial for LGMD2D (alpha-sarcoglycan deficiency) using 
scAAVrh74.tMCK.hSGCA. 
Study Number 
N = 7: [Low dose single leg (n=l) and bilareral (n =3) and High dose bilateral (n=3)] 
Clinical Study Phase 
Phase I/Ila t rial 
Number of Centers 
Single site (Nationwide Children's Hospital) 
Study Objectives 
Primary objective is safety 
Study Design 
Dose escalation study of self-complementary (sc) AAVrh74.tMCK.hSGCA delivered to 
single leg (low dose n=l) and bilaterally (n=6) via a major lower limb artery to the whole 
lower limb of LGMD2D (alpha-SG deficient) subjects. 
Patient Population 
'Inclusion Criteria 
• 
Subjects age 7 or older; cohort lA must be adult and wheelchair-dependent 
• 
Proven alpha-sarcoglycan deficiency by muscle biopsy or ONA testing . 
• 
Subject enrolled in Cohort lA must be adult and wheelchair dependent 
• 
Subjects enrolled in Cohorts 1B or 2 must be able to walk independently, but 
must exhibit signs of lower extremity weakness (i.e. a Gowers' sign, use a 
handrail for climbing stairs) and walk~ 80% of predicted distance on the 6MWT 
based on normative data. 
• 
Males and females of any ethnic group will be eligible 
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• 
Ability to cooperate with muscle testing . 
• 
Willingness of sexually active subjects with reproductive capacity to practice 
reliable method of contraception {If appropriate), during the first six months 
after gene therapy (females) or until two negative sperm samples are obtained 
post gene transfer (males). 
Exclusion Criteria 
• 
Active viral infection based on clinical observations . 
• 
The presence of SGCA mutations without weakness or loss of function . 
• 
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% 
• 
Serological evidence of HIV infection, or Hepatitis B or C infection . 
• 
Diagnosis of (or ongoing treatment for) an autoimmune disease . 
• 
Abnormal laboratory values considered clinically significant (GGT > 3XULN, 
bilirubin 3.0 mg/dl, creatinine 
1.8 mg/dl, Hgb < 8 or> 18 g/DI; WBC > 
15,000 per cmm). 
• 
Concomitant lllness or requirement for chronic drug treatment that in the 
opinion of the Pt creates unnecessary risks for gene transfer. 
• 
Pregnancy . 
• 
Subjects with AAVrh74 or AAVB binding antibody titers~ 1:50 as determined by 
ELISA immunoassay. 
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Study Procedures 
Primary Outcome 
Secondary Outcomes 
Study Duration 
Sample Size 
Statistical Analysis 
Long-term follow-up 
The vector will be delivered via direct intravascular delivery to a major lower limb artery 
to single leg (cohort lA) or to both legs sequentially (cohort 18-2). Study subjects will 
receive general anesthesia during the procedure following Children's Hospital protocol. 
Detailed description of the vector administration procedure can be found in section 6.3 
of this clinical protocol. 
Safety is a primary outcome for this clinical gene transfer trial. 
We propose the following efficacy outcome measures. 
1. 
The 6 minute walk test (GMWT) (primary variable to measure efficacy). 
2. 
 
 
3. 
 
4. 
 
 
A statistically significant change in distance walked on the GMWT will be considered 
evidence of a positive result. 
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, 60, 90, and 180 and at months 
9 (physical therapy only), 12, 18, and 24. Immune studies will continue with blood 
samples collected locally and shipped to us at different time points up to two years. 
Cohort lA: One (n=l) adult LGMD2D wheelchair-dependent subject will receive single-
limb perfusion at the low dose. 
Cohort 18: Three (n=3) LGMD2D subjects will receive bilateral whole limb perfusion at 
the low dose. 
Cohort 2: Three (n=3) LGMD2D subjects will receive bilateral whole limb perfusion at the 
high dose. 
• 
Cohort lA and 18 receive low Dose: lxl012 vg/kg per lower extremity (lA 
single lower limb; 1B both lower limbs) 
• 
Cohort 2 will receive 3xl0
12 vg/kg per lower extremity in both lower limbs 
If safety data is satisfactory at low dose in single-limb (Cohort 1A}, we will use the same 
dose for bilateral lower limb infusion (Cohort 1B). With satisfactory safety data we will 
increase to high dose for bilateral lower limb infustion (Cohort 2). 
This is a Phase I/Ila trial, with safety as the primary measure. The GMWT will be the 
leading or first order variable for efficacy measures (secondary outcome).
 
 
 
 
 
 
 
 
 The efficacy measures will 
be tested pre- and post-gene transfer at 60, 90 and 180 days and at months 9, 12, 18 and 
24. 
 
 
 
Subjects will be seen at the end of 1st and 2nd year for a physical exam, strength testing, 
and immune studies. 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. 
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Phase I/Ila gene transfer clinical trial for LGMD2D using stAAVrh74.tMCK.hSGCA 
 
2.0 A BSTRACT 
The proposed clinical trial is a dose escalation study of self-complementary 
scAAVrh74.tMCK.hSGCA vector and transgene to LGMD2D {alpha-SG deficient) subjects 
delivered via a major lower limb artery of each leg sequentially by isolated limb infusion {IU)1. 
Three cohorts (Cohorts lA, 1B, and 2) will undergo gene transfer in a standard three-six dose 
escalation scheme to establish maximum tolerated dose (MTD} using toxicity. One (n=l) adult 
wheelchair-dependent patient will be enrolled in Cohort lA, three (n =3} subjects will be 
enrolled in Cohort 1B, and three (n =3} subjects will be enrolled in Cohort 2. The first cohort 
(lA) will receive a vector dose of 1 x 1012vg/kg (optimized weight to height} in a single limb with 
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delivery to the whole limb. This same dose will be delivered to both limbs in Cohort 1B. Cohort 
2 will receive a total dose of 3 x 10
12vg/kg per limb delivered to both lower extremities. The 
vector will be infused into an indwelling vascular sheath placed in the femoral artery. This will 
be a one-time vector infusion to an isolated limb with an approximately 10-minute dwell time. 
Cohort 2 will receive 6 x 1012 vg/kg total vector dose - split between the 2 lower extremities (3 
x 1012 vg/kg per limb) delivered to the whole limb according to the same protocol. The total 
vector genome dose for each subject will be adjusted by rounding to the closest 5 kg (see 
section 6.2.7.3 Dose selection). 
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The primary objective of this study is the assessment of the safety of intravascular 
administration of scAAVrh.74.tMCK.hSGCA delivered via the femoral artery to lower extremities 
of LGMD2D (alpha-SG deficient) subjects. Safety monitoring during infusion will include: 
activated clotting times. Safety endpoints will be assessed by changes in hematology, serum 
chemistry, urinalysis, immunologic response to rAAVrh74 and hSGCA, and reported history and 
observations of symptoms. 
The 6 minute walk test (6MWT) will be used as the secondary outcome for this study. 
 
 
 These quantitative physical therapy measures will be done at baseline, 
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day 60, 90, 180, month 9, 18 and at the end of l
5t and 2nd years. 
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Subjects will be evaluated at baseline, infusion visit (days 0-1), and return for follow up 
visits on days 2, 7, 14, 30, 60, 90, and 180 and at months 9, 12, 18, and 24. 
 
 
 This is the first vascular delivery clinical trial 
for muscular dystrophy, and should be considered a major milestone for the disease. 
3.0 CLINICAL TRIAL AND PRINCIPAL INVESTIGATOR 
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Phase I/Ila gene transfer clfnical trial for LGMD2D using scMVrh74.tMCK.hSGCA 
 
The study will be carried out at Nationwide Children's Hospital. 
 
 
 
 will serve as Principal Investigator. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
This gene therapy trial is a follow up to the intramuscular trial where sustained gene expression 
was documented at 6 months in LGMD2D subjects without significant adverse events. Since 
that time, intravascular studies through lower limb arteries of non-human primates (rhesus 
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macaque) have demonstrated gene delivery using the same vector proposed in the clinical trial 
with the addition of a FLAG tag to differentiate endogenous a-SG from the protein expressed by 
scAAVrh.74.tMCK.SGCA. This vascular delivery clinical trial for LGMD2D (alpha-sarcoglycan 
deficiency) will be a dose escalation, Phase I/Ila study. The study is designed to assess safety, as 
well as clinical response to the scAAVrh.74.tMCK.hSGCA transgene. Seven subjects with alpha-
sarcoglycan deficiency by muscle biopsy or DNA testing will be enrolled for this trial. The FDA 
has requested that the first subject be non-ambulatory and receive vector infusion to only one 
limb to assess safety. 
4.0 SPECIFIC AIMS 
4.1 PRIMARY OBJECTIVE 
Determine the safety of intravascular administration of self-complementary 
scAAVrh74.tMCK.hSGCA delivered via a major lower limb artery targeting all of the lower limb 
muscles of LGMD2D (alpha-SG deficient) subjects. 
4.2 SECONDARY OBJECTIVE 
Assessment of the efficacy of the same vector measured by: 
• 
The distance walked in 6 minutes (GMWT, leading or first order variable). 
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4.3 
 
 
 
 
 
 
 
 
5.0 BACKG ROUND AND SIGNIFICANCE 
The LGMDs include 21 disorders with known chromosomal linkage. Seven, designated 
LGMDlA-G, have autosomal dominant inheritance (3 of which have known gene defects). 
Fourteen disorders are inherited as recessive autosomal conditions, referred to as LGMD2A-N 
(all with known gene defects). 
LGMD2D is one of a family of disorders known as the limb-girdle dystrophies (Table 6.1 A-B). 
AUTDSOMAL RECESSM-l.GMD 
DISEASE 
LGM02A 
LGM02B 
LGM02C 
LGM020 
LGM02F 
t--
LGM02G 
LGM02H 
LGMD21 
l GM02J 
LGMD2K 
PROTEIN 
Calpain3 
Dysferlin 
v - Sarcoglycan 
q - Sarcoglycan 
P -Sa rcoglycan 
6 • Sarcoglycan 
Telethon in 
TR1M32 
Fukutin-related protein 
Titin 
Q,M annosyl transferase-1 
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GENE 
CAPN3 
DYSF 
SGCG 
SGCA 
SGCB 7 
SGCO 
TCAP 
TRIM 32 
FKRP 
TTN 
POMTl 
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LGMD2L• 
Fukutln 
FKTN 
LGMD2M 
0-Mannose 13 -1, 2•N• 
POMGnl 
acetyigiucosaminyi 
transferase 
LGMD2N 
0 -Mannosyl transferases-2 
POMT2 
*No International agreement has been reached for 
the nomenclature of this LGMD. Recent 
publications assigned the LGMD type 2L to the 
ANOS gene mutations. 
AtnOSOMAL DOM•NT•LGMD 
DISEASE 
PROTEIN 
GENE 
LGMDlA 
Myotllln 
Myotilin 
LGMDlB 
LaminA/C 
LMNA 
LGMDlC 
Caveolin-3 
CAV3 
LGMDlD 
Oesmin 
Desmin 
LGMOlE 
OnaJ homolog 
DNAJB6 
subfamily B member 6 
LGMDlF 
Transportin -3 
TNP03 
I LGMDlG 
Heterogeneous nuclear 
HNRPDL 
ribonucleoprotein 0-like 
The clinical trial pertains directly to LGMD2D subjects with a-SG deficiency, and may have 
implications for all sarcoglycan deficiencies (see Table 6.lA-8 classification of LGMDs). 
Supportive care is the only treatment option available for LGMD2D subjects. 
The clinical features of LGMD2D overlap with the dystrophinopathies.5·8 The phenotypes range 
from severe to mild, similar to Duchenne (DMD) and Becker muscular dystrophies (BMD). The 
disease occurs worldwide. The usual age at onset ranges from 3 to 15 years, although some 
cases have delayed walking as in DMD. Subjects most commonly present with difficulty 
running, jumping and climbing stairs about age 3-5. Serum CK is markedly elevated (SO to 100 X 
normal is not unusual). Muscle weakness is generalized; biceps brachii are weaker compared to 
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DMD. Calf hypertrophy is common. Subjects become wheelchair dependent by age 13-15 years 
on average. Respiratory impairment occurs as the disease progresses leading to a need for 
ventilatory assistance. Scoliosis and joint contractures develop over time. Of particular 
importance regarding gene therapy as a potential treatment modality for LGMD2D is that 
cardiac muscle is spared in most cases (in contrast to the dystrophinopathies). In addition, no 
cognitive deficiencies are seen, again setting the st age for potential gene replacement of 
muscle without need to go to other body systems. 
The other sarcoglycanopathies, LGMD2C (v·SG), 2E {~-SG), and 2F (6-SG), are similar in 
spectrum of presentation to LGMD2D. LGMD2C and 2F, tend to be more severe. Confusion 
with DMD is common except that both genders are equally affected in LGMD2D. In our 
experience, there is significant overlap in phenotype between the sarcoglycanopathies and 
other forms of autosomal recessive LGMDs, especially deficiencies of dysferlin (LGMD2B), 
cailpain-3 (LGMD2A), and fukutin-related protein (FKRP, LGM D21). In addition, many of the 
LGMDs have residual protein expression in muscle since most are t he result of missense 
mutations. 
 
2 
The sarcoglycanopathies have an estimated prevalence of 1 per 178,000 affected (MDA 
Website and ORD). If successful in the LGMD2D population, studies could be expanded to 
include other LGMDs that could potentially benefit from vascular gene delivery increasing the 
impact with benefit to 1 per 123,000. The findings in this study also have direct implications for 
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the DMD population with an estimated annual incidence of newborn males of 1 in 5000 
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births.9•10 In the USA, it is estimated that there are approximately 45,333 boys affected with 
DMD in anyone year (ORD). This may be an underestimate since we are doing so much better 
at prolonging the lives of these boys and young men. Furthermore, it has been estimated that 
the cost per year per subject with DMD is $126,000 (MDA website). Using this as a yardstick, 
the cost for the sarcoglycanopathies per subject based on the severity of the condition is 
approximately the same. For the DMD population, the cost estimates for the disease exceed $5 
billion and $2SOM for the sarcoglycanopathies. The goal of this project is to promote 
ambulatory independence for subjects with LGMD2D. 
 
 The cascading effect to 
other neuromuscular disorders would also be very significant and would easily justify this initial 
investment. Apart from the overall cost for each subject, improving the quality of life for the 
muscular dystrophy subject could be significant by prolonged ambulation, enhancing self-
esteem and overall feelings of well-being11• 
6.0 CLINICAL RESEARCH PLAN 
6.1. STUDY POPULATION 
-- -·-· ---------
Seven LGMD2D subjects with alpha-sarcoglycan deficiency by muscle biopsy or DNA testing, will 
be enrolled at Nationwide Children's Hospital for the gene transfer study. Subjects will 
encompass any ethnic, racial, or gender background. Patients must have proven muscle 
weakness to be enrolled. 
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6.2. PRE-TREATMENT ASSESSMENT: 
A. Establish Subject Eligibility 
I. 
Inclusion Criteria 
1. Cohort lA must be adult and wheelchair-dependent; Cohorts 1B and 2 will be subjects 
age 7 or older. 
2. Confirmed alpha-sarcoglycan deficiency or identified SGCA DNA mutation. 
3. Subjects enrolled in Cohorts 1B or 2 must be able to walk independently, but must 
exhibit signs of lower extremity weakness (i.e. a Gowers' sign, use a handrail for 
climbing stairs) and walk~ 80% of predicted distance on the 6MWT based on normative 
data. 
4. Males and females of any ethnic group will be eligible. 
5. Ability to cooperate with muscle testing. 
6. Willingness of sexually active subjects with reproductive capacity to practice reliable 
method of contraception (if appropriate), during the first six months after gene therapy 
(females) or until two negative sperm samples are obtained post gene transfer (males). 
II. 
Exclusion Criteria 
1. 
Active viral infection based on clinical observations. 
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2. 
The presence of SGCA mutations without weakness or loss of function. 
3. 
Symptoms or signs of cardiomyopathy, including: 
4. 
5. 
6. 
7. 
• 
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%. 
Serological evidence of HIV infection, or Hepatitis B or C infection. 
Diagnosis of (or ongoing treatment for) an autoimmune disease. 
Abnormal laboratory values considered clinically significant (GGT > 3XULN, bilirubin 3.0 
mg/dL, creatinine 
1.8 mg/dl, Hgb < 8 or> 18 g/01; WBC > 15,000 per cmm). 
Concomitant illness or requirement for chronic drug treatment that in the opinion of the 
Pl creates unnecessary risks for gene transfer. 
8. 
Pregnancy. 
9. 
Subjects with AAVrh74 or AAV8 binding antibody titers~ 1:50 as determined by ELISA 
immunoassay. 
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 
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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 IRS-approved document as well as any subsequent approved modified 
consent document(s) must be provided to NIH/OBA 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 Measures Prior to Injection (day -45 to day -1) 
After obtaining informed consent and completing the registration procedures, a baseline 
subject history will be collected, including records of all medications and supplements that the 
subject is currently taking. The following assessments will be performed to confirm subject 
eligibility for this study. Baseline tests which must be completed prior to gene transfer include 
the following: 
Day -45 to day -1 before gene transfer 
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• 
Physical exam 
• 
EKG 
• 
Echocardiogram 
• 
Antibody (lgG and lgM) testing for hepatitis B, and C, and for HIV 
• 
Complete blood count (CBC) with differential and platelets, with smear 
• 
Serum total protein 
• 
Serum gamma-glutamyl transferase (GGT*) 
• 
Serum total bilirubin 
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• 
Glucose 
• 
Creatine kinase (CK) 
• 
Creatinine/BUN 
• 
Cystatin C 
• 
Alkaline phosphatase 
• 
ALT 
• 
AST 
• 
Amylase 
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• 
Electrolytes 
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Fibrinogen 
• 
Prothrombin time (PT), partial thromboplastin time (PTT), JNR 
• 
Urinalysis 
• 
Serum binding antibody to rAAVrh74 and AAV8 
• 
ELISpot assay to capsid proteins and alpha-sarcoglycan 
• 
Pregnancy test (if appropriate) 
• 
 
 
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• 
 
• 
 
• 
Six minute walk test (6MWT). 
• 
 
• 
 
 
• 
 
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* Because GGT is very sensitive to alcohol consumption, subjects will be required to avoid 
alcohol the week before testing. 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. 
6.3 PROTOCOL FOR GENE TRANSFER 
The clinical trial is a dose escalation study of scAAVrh74.tMCK.hSGCA delivered by isolated limb 
infusion via a sidearm sheath (REF CP-08403; Manufacturer: Arrow) placed percutaneously in 
the femoral artery and allowed to circulate through the limb vasculature. Prior to vector 
delivery the limb circulation is isolated from the central circulation by the careful placement of 
occlusive balloon catheters (Tyshak 11 Balloon Dilatation Catheter; Manufacturer: B. Braun 
Intervention Systems Inc., and NuMED, Inc.) one placed through the arterial sheath in a 
cephalad direction positioned above the profunda artery and a second occlusive balloon 
positioned via a similarly placed sidearm sheath placed in the femoral vein. Following 
placement of the arterial and venous sidearm sheaths and before the insertion of the occlusive 
balloons the subject is anticoagulated with unfractionated heparin. The balloons are then 
positioned under flouroscopy guidance and inflated to create stasis in the limb during the 
infusion procedure. 
 
 
 
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For the subjects in cohort lA the arterial and venous sheaths will be placed and procedure will 
continue as described above. for the subjects in cohorts 1B and 2-both limbs-the vascular 
sheaths will be placed on both sides (left and right) and the the subject will be anticoagulated 
and the occlusive balloon catheters positioned one side and the subject dosed. Upon 
completion of dosing of the first extremity, the other will be done. Three cohorts will undergo 
gene transfer in a dose escalation scheme to establish maximum tolerated dose (MTD) using 
0 
toxicity. Cohort lA (n=l) will receive a vector dose of 1 x 1012 vg/kg in a single lower extremity. 
Q 
In Cohort 1B (n=3), three subjects will receive the same vector dose per limb delivered 
bilateral!ly to both lower extremities for a total dose of 2 x 1012vg/kg. This will be a one-time 
vector infusion as described above. Subjects in Cohort 2 (n=3) will receive 3 x 1012 vg/kg vector 
dose per extremity (6 x 1012 vg/kg total dose) delivered according to the identical protocol. 
 
 
 
 
 
 
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In an attempt to dampen the host immune response to our AAV-based therapy, subjects will be 
started on a prophylactic daily dose of prednisone (or alternative glucocorticoid in dose 
equivalency) at approximately lmg/kg/day with a maximum dose of 60 mg/day. Post-
treatment, we may increase this dose to approximately 2 mg/kg/day with a maximum dose of 
60 mg/day, depending on T cell responses measured by ELISpot assay. Treatment will be 
initiated one day prior to the gene transfer and the subjects will remain on prednisone 
treatment for approximately one month. A tapering protocol for prednisone will be 
implemented based on individual subjects' immune response to the gene transfer, again 
Q 
assessed by the ELISpot assay. If GGT elevations are seen after 30 days; the prednisone 
treatment will be continued or restarted at an appropriate dose for duration necessary to quell 
the immune reaction with a maximum of 60 mg per day. The duration of treatment will be 
determined based on weekly levels of GGT and immune responses. 
0 
Subjects will be admitted to the Nationwide Children's Hospital the day of the gene transfer. 
Procedures will be performed under sterile conditions in the Cardiac Catheterization Suite. An 
intravenous catheter with heparin lock will be placed in the arm for delivery of sedation. 
I. Injection Preparation 
Preparation of the scAAVrh74.tMCK.hSGCA gene vector will be done by the Nationwide 
Children's Hospital research pharmacist according to the Manual of Operating Procedures 
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(MOOP). Immediately prior to transportation to the clinical setting, appropriate dilutions of the 
test article will be completed by the pharmacy. 
 
 
Documentation of the dilution will be completed by the pharmacy following standard pharmacy 
protocol. 
The vector-containing syringes will be delivered to the designated Cardiac Catheterization Suite 
at Nationwide Children's Hospital. It will be delivered under ambient temperature (not frozen) 
and administered to the subject within 8 hours of preparation. Handling of 
scAAVrh74.tMCK.hSGCA gene will follow compliance standards for Biosafety Level 1 vectors. 
(http:ljwww4.od.nih.gov/oba/RAC/guidelines 02/ APPENDIX G.htm# Toc7246561) 
II. Vector Administration Protocol 
The overall procedure is such that the vector will be delivered via direct intravascular delivery 
into the femoral artery of the leg followed by an 8-10 minute dwell time.1 Subjects will 
undergo appropriate anesthesia (general or conscious sedation as appropriate) for their age 
and muscle disease per Children's Hospital Department of Anesthesia protocol. Under sterile 
conditions, the following protocol will be performed: 
a. The subject is transferred from a hospital bed to the Cardiac Catheterization Lab and 
positioned in the usual supine manner, attached to appropriate monitoring and placed 
under general anesthesia or conscious sedation as determined by the anesthesiologist 
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utilizing NCH anesthesia protocols that minimize the risk of anesthetic reaction in 
muscular dystrophy. The subject will be prepped and draped in the usual sterile fashion. 
A baseline activated clotting time {ACT) level will be obtained from a peripheral IV. 
b. The femoral artery and femoral vein will be entered percutaneously using the Seldinger 
technique and guide wire advanced and positioned under fluoroscopy. A sidearm sheath 
will be advanced over the guide wire-one into the femoral artery and one into the 
femoral vein and both flushed with heparinized normal saline. Before the occlusion 
balloons are inserted through the sidearm sheaths, 200 IU/kg of heparin will be 
administered via a peripheral arm IV and allowed to circulate for 3-5 minutes, at which 
time a blood sample will be drawn from the peripheral IV and an ACT will be measured 
with the goal of> 200 seconds from an anticipated base line of 90-120 seconds drawn 
systemically {arm-PIV). If the post heparin ACT is less< 200 seconds, a second dose of 
heparin, 50-100 IU /Kg will be administered. An immediate follow up ACT will be 
obtained with the targeted ACT goal of> 200 seconds. This process will be repeated 
until ACT's are within the designated range and repeated every 30-60 minutes to assess 
ongoing anticoagulation levels. 
c. The procedure for vascular delivery of scAAVrh.74tMCK.hSGCA to the isolated limb can 
be summarized as follows: 
• 
The area near the inguinal ligament will be infused with several mls of Marcaine or 
Lidocaine for local anesthetic effect. Percutaneous access of the femoral artery will 
be obtained using the Seldinger technique and advancing an introducer and dilator 
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over a guide wire. The dilator and wire are removed and sidearm sheath (REF CP-
08403; Manufacturer: Arrow) placed and flushed appropriately with heparinized 
normal saline. 
• 
Percutaneous access of the femoral vein is obtained in a similar fashion. 
• 
Both sets of femoral vessels (right and left, artery and vein) will be cannulated before 
heparinization of the subject. 
• 
Before the occlusion balloons are inserted through the sidearm sheaths, 200 IU/kg of 
heparin will be administered via a peripheral arm IV and allowed to circulate for 3-5 
minutes, at which time a blood sample will be drawn from the peripheral IV and an 
ACT will be measured with the goal of> 200 seconds from an anticipated base line of 
90-120 seconds drawn systemically (arm-PIV). If the post heparin ACT is less< 200 
seconds, a second dose of heparin, 50-100 IU /Kg will be administered. An immediate 
follow up ACT will be obtained with the targeted ACT goal of> 200 seconds. This 
process will be repeated until ACT's are within the designated range. 
• 
Using fluoroscopic guidance a Tyshak II Balloon Dilation catheter is advanced over a 
guide wire through the sheath into the femoral artery above the take-off of the 
femoral profunda artery and positioned to achieve arterial stasis when the occlusive 
balloon is inflated. Positioning the arterial balloon catheter above the femoral 
prnfunda artery take off enables perfusion and targeting of proximal muscles of the 
hip via the anterior circumflex artery. 
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• 
Again with fluoroscopic guidance a Tyshak II Balloon Dilation catheter is advanced 
over a guide wire through the sheath into the femoral vein to approximately the 
same level as the arterial balloon catheter and positioned to achieve venous stasis 
when the balloon is inflated. 
• 
If needed a tourniquet may be placed over the region of the balloons to augment 
vascular stasis and isolation of the limb circulation. 
• 
 
 
• 
 
 
 
 
 
 
 
 
 
 
 
 
 
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• 
 
 
 
 
 
 
 
 
• 
At the conclusion of the post flush the balloon catheters and tourniquet are taken 
down. 
• 
For subjects being dosed in both limbs the balloon catheters will be placed and 
positioned via fluoroscopic guidance and the treatment of the second limb 
undertaken as described above. 
• 
At the end of the procedure all occlusive balloon catheters, and sheat hs, will be 
removed. 
• 
Direct pressure is used to control bleeding at the site and obt ain hemostasis. 
• 
Following gene transfer, subjects will return to a designated monitored bed for a 
minimum of 6 hours within Nationwide Children's Hospital. Vital signs will be 
obtained per Hospital post-op policy and hourly for four hours following the injection 
and then every 4 hours prior to discharge. 
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Subjects will be monitored for bleeding, perfusion to the extremity, compartment syndrome 
and neuromuscular integrity. Compartment syndrome will be assessed by direct limb palpation 
and Doppler ultrasound (as needed}. Neuromuscular integrity will be determined by routine 
assessment of clinical pain and strength. 
6.4 POST- GENE TRANSFER MONITORING 
• 
Subjects will return to a designated bed following gene transfer with close monitoring 
of vital signs. CK levels will be checked after the procedure. Concomitant medications 
and all adverse events/serious adverse events will also be monitored and documented 
following injection. Subjects will be discharged the day after gene transfer (if no 
adverse side effects are observed). 
Subjects will return for follow up visits on days 2, 7, 14, 30, 60, 90 and 180 and at months 9 
(physical therapy and immunology only), 12, 18, and 24. Toxicity monitoring on each of these 
dates (except visit 9) (including day 1-2 post-gene transfer) will include: 
• 
Physical Exam 
• 
GGT* will be drawn as above and at 45 and 75 days post gene transfer to assess 
delayed sub-clinical hepatitis. GGT is assessed in preference to ALT/AST which are 
elevated in muscular dystrophy. 
• 
Total bilirubin 
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• 
Glucose 
• 
PT/PTT/INR 
• 
CBC/Diff/Platelet with smear 
• 
Serum Creatine kinase (CK) 
• 
Creatinine/BUN 
• 
Cystatin C 
• 
Alkaline phosphatase 
• 
AST 
• 
Amylase 
• 
ALT 
• 
Electrolytes 
• 
Total Protein 
• 
Urinalysis 
• 
Immune studies (day 7 visit and after) 
• 
 
 
• 
 
• 
 
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• 
Six minute walk test (GMWT). (60 day visit and after) 
• 
 
• 
 
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• 
Photograph of incision site and surrounding area to be completed until day 14 
* Subjects will be required to avoid alcohol the week before testing. Far 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. 
Because of the possible concerns with viral shedding, all sexually active participants will be 
asked to refrain from sexual intercourse or practice barrier contraception for six months 
(females) or until two consecutive semen samples (adult males) are tested negative for the 
presence of vector. As part of this study we must ask for a semen sample from all sexually 
active males 18 years and older. 
Semen samples will be requested on day 90 from all sexually active males age 18 years or older, 
for PCR analysis for vector genomes. This interval is based on selecting a date post-gene 
transfer that is longer than the 70 days required from the beginning of the first meiotic division 
to the completion of spermatogenesis. As mature sperm spend an additional 5-15 days in the 
epididymis, a date three months after gene transfer is therefore optimal to look for any 
possible germ line incorporation. Testing will continue until 2 consecutive negative samples are 
obtained. 
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Blood samples will be obtained for immune studies at days 7, 14, 30, 60, 90, 180 post gene 
transfer and 9, 12, 18, and 24 months; this will include testing for binding antibody to 
rAAVrh74, as well as ELISpot to detect T cell response to capsid antigens. GGT will be measured 
at 45 and 75 days post gene transfer to assess delayed sub-clinical hepatitis. 
6.5 LONG-TERM MONITORING 
Subjects will be seen at the end of 1st and 2nd years for a physical exam, strength testing, and 
immune studies. We will follow the most recent FDA guidelines with regard to long-term 
subject follow up following gene transfer. As discussed and based on prior experience with 
rAAV or transgene, there is 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 
0 
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. 
0 
6.6 OUTCOME MEASURES 
The study is a Phase I/Ila clinical trial inclusive of safety and efficacy outcome measures 
A. Safety is the primary outcome for this clinical gene transfer trial. Stopping criteria are 
based on development of unacceptable toxicity defined as the occurrence of any one 
Grade Ill or higher treatment-related toxicities, or two or more Grade II treatment-
related toxicities. 
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B. Efficacy outcome measures include: 
i. Strength and functional testing will be performed at baseline and at days 60, 90, 180, 
and at 9, 12, 18, and 24 months. This will include the 6MWT distance as the primary 
functional variable. 
 
 
 
 
 Pre- and post-treatment strength, distance, and time will be 
evaluated by using paired t-tests. 
 
 
 
 
 
 
 
 
 
 
 
 
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iii. Immune response will be assessed by IFN-y ELISpots to alpha-sarcoglycan and AAV 
capsid. A rise in IFN-y of 50 spot forming colonies per one million PBMCs towards virus 
or transgene will be considered significant. An additional measure of immune response 
will be the binding antibody assay to AAV. 
6.7 STATISTICAL ANALYSIS 
• 
 
 
 
  
 
 
 
  
 
Data will be shared with the DSMB as gathered for each study subject. Related serious adverse 
events will be discussed with the 05MB and reported to the FDA. 
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6.8. STUDY TIMELINE 
0 
Table 2 
I 
STUDY TIMELINE 
I Baseline 
-
idy Interval 
I 
Vector Infusion 
Follow Up 
I 
(Inpatient) 
I 
Screening 
I 
Visit# 
. 
1 
2 
3 
" 
I 
e 
7 
8 
.. 
10 
11 
12 
13 
'14 ' 
Study 
-45 to-1 
Oday 
1 
2 
1 
14 
30 
45 
60 
75 
90 
180 
9mo 
1 yr 
18 
2yr 
rocedures 
day 
day 
day 
day 
day 
day 
day 
day 
day 
day 
mo 
sit Window 
+/. 7 
+l-1 
+/-1 
+/-7 
+/- 7 
+/-7 
+/- 21 
+/-21 
+/- 21 
+/-21 
+/- 21 
+/- 21 
+/. 21 
+/- 21 
+/-21 
+/- 21 
days 
days 
days 
days 
days 
days 
days 
days 
days 
days 
days 
days 
days 
days 
days 
days 
Informed 
X 
Consent 
:Heal History 
X 
yslcal Exam 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
'.CHO/EKG 
X 
0-
hestX-Ray 
X 
patitls , B & 
X 
C, HIV 
afety labs* 
XE 
X 
X 
X 
X 
X 
x•• 
X 
x•• 
X 
X 
X 
X 
X 
gnancyTest 
X 
Jmen Test
0 
X 
Physical I 
XA 
XA 
Therapy 
x-" 
X 
X 
X 
X 
X 
sessmentsc 
ne Transfer 
X 
1munology 
studles8 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
otograph of 
X 
X 
X 
X 
X 
jectlon site 
:,ncomitant 
To be collected from time of consent until final study visit 
edicatlons 
1fety labs (CBC/Dlff/Platelet with smear, PT/PTT/INR, Alkallne Phosphatase, ALT, Amylase, AST, BUN, CK, Creatlnlne, GGT, Glucose, Electrolytes, 
11 Protein, Total Blrli ubln, Cystln C, Urinalysis) 
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--
.. GG I only (day 45 and day 75) 
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Table 2. Legend to annotations 
A. 
 
 
B. Immunology studies will be done pre-gene transfer (-45 to -1) and continue at post-
treatment day 7, 14, 30, 60, 90, 180, and at months 9, 12, 18 and 24 post-gene transfer. 
Immunology studies consist of measuring binding antibodies to rAAVrh74, (ELISA), and 
detecting T cell responses to both rAAVrh74 and alpha-sarcoglycan peptides (ELISpot). 
C. Physical therapy assessments (
, 6MWT, 
}will be 
performed as appropriate based on clinical judgement, age and/or ambulatory status. 
D. Vector identification by DNA-PCR in semen on male subjects, testing will continue every 
7 days until obtaining 2 consecutive negative samples. 
E. 
 
 
 
 
 
F. Fibrinogen level will be drawn 
G. 
 
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Note: There will be a flexibility of+/- 7 or+/- 21 working days as outlined 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. 
7.0 TEST MAT ERIAL AND ADMINI ST RATION 
7.1 DESCRIPTION OF BIOLOGICAL PRODUCT 
The biological product is a non-replicating self complementary recombinant adeno-associated 
virus termed scAAVrh74.tMCK.hSGCA. The vector contains the human alpha-sarcoglycan gene 
under the control of a triple E-box muscle creatine kinase (tMCK) muscle specific promoter. 
This cGMP product was produced at the Clinical Manufacturing Facility (CMF) at Nationwide 
Children's Hospital using practices consistent with the US Food and Drug Administration's 
"Guidance for Industry- cGMP for Phase 1 lnvestigational Drugs", July 2008. The material 
produced will be used to support a vascular delivery phase 1/lla clinical trial. The manufacturing 
process includes procedures and practices to ensure the safety, identity, quality, purity and 
strength of the manufactured biologic. The HEK293 cell bank used in the production of the 
product meets specifications for product release as detailed in FDA and ICH guidelines. The 
production process is governed by over 100 standard operating procedures (SOP) and 
production documents. 
 
 The vector is supplied as a frozen liquid that is 
thawed before dilution and clinical administration. The vector DNA structure is illustrated 
below. 
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Self-Complementary scAA Vrh. 74. tMCK.hSGCA 
7.1.1 PRODUCT DEFINITION 
Product Name: 
scAAVrh74.tMCK.hSGCA 
Gene Insert: 
Human alpha-sarcoglycan cDNA 
Control Elements: 
triple E box mouse MCK promoter 
AAV Serotype: 
rh74 
 
 
 
_.... 
 
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7.1.2. NAME AND ADDRESSES OF MANUFACTURER 
The HEK293 Master Cell Bank and scAAVrh74.tMCK.hSGCA clinical grade vector was 
manufactured at: 
Nationwide Children's Hospital 
Clinical Manufacturing Facility 
Center for Gene Therapy 
700 Children's Dr. 
Columbus, Oh 43205 
7.2 STUDY DESIGN 
Eligibility for subjects 7 years and older will be established by SGCA mutations, ability to 
cooperate for testing, willingness to practice contraception during the study (if appropriate), 
negative pregnancy test (for females, if appropriate), and no evidence of cardiomyopathy. HIV 
infection, hepatitis B, or C, or known autoimmune diseases are exclusion criteria. 
IRB approved consent {18yrs. of age and older) and assent (ages 12 years up to 18 years old) 
forms will be obtained by the Principal Investigator
 or Co-investigators and signed by 
parents and subjects. Pre-gene transfer immune studies include serum binding antibodies to 
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AAV and ELISpot assays for both AAV capsid and a-SG proteins. 
 
 
 
This will be a vascular delivery trial through the femoral artery of a single leg 
(cohort lA) and both lower extremities (cohorts 1B and 2) using scAAVrh74.tMCK.hSGCA. This 
will be a dose escalation study of scAAVrh74.tMCK.hSGCA delivered through the femoral artery 
to an isolated limb to maximize limb vector exposure. Three cohorts of LGMD2D subjects will 
be included. The lower limb muscles will be targeted to demonstrate increased muscle 
strength with the intent of promoting extended independent ambulation. Subjects in Cohort 
lA will receive a dose of lx1012 vg/kg per limb (Ideal weight to height). This first subject will 
receive a perfusion to a single lower extremity with a total dose of 1 x 1012vg/kg. If safety is 
achieved, three subjects in Cohort 1B will receive the same dose per limb delivered to both 
lower extremities for a total dose of 2 x 1012vg/kg. Subjects in Cohort 2 (n=3) will receive 
6x1012 vg/kg total dose- split between the 2 extremities (3 x 1012 vg/kg per limb) delivered to 
the whole limb muscles according to the same protocol. Subjects will be appropriately sedated 
for the procedure with the assistance of an anesthesiologist. A detailed description of the 
infusion procedure is described in section 6.3 of this protocol. The goal for this study is to have 
efficient gene transfer, muscle fiber transduction, and gene expression levels sufficient to 
produce clinically meaningful results (improved ambulation= distance walked on GMWT). 
7.3 DOSE SELECTION 
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There will be three cohorts of LGMD2D subjects [Cohorts lA (n=l), 1B (n=3), and 2 (n: 3)] with 
dosing permitted by toxicology- biodistribution study and predicted by pre-clinical studies in 
non-human primates. The trial will start with low dose gene transfer (1 X 1012 vg/kg per limb); 
the first patient (Cohort 1A) enrolled will receive a single unilateral infusion to one lower 
extremity. The same dose will be delivered to three subjects (Cohort 1B) infused to both lower 
extremities. The total vector genomes dosed for each subject will be adjust ed by rounding to 
the closest 5 kg per the discretion of the Pl or designated co-investigator. The subjects in Cohort 
2 (n=3) will receive a total dose of 3 x 1012vg/kg per limb delivered to both extremities. Table 
6.3 below shows dosing for the clinical trial based on weights from clinic subjects eligible for the 
clinical gene transfer trial. 
20-29 
30-39 
40.49 
so- 59 
60 - 69 
70 • 79 
80- 89 
90 - 99 
100 . 
110-
Patient wt {kg): 
-··-
109 
119 
Cohort 
Dose/quad 
---- . To1al Cose / Patient jtotal vector genomes) 
-
(1) 
LO 
lel2vg/kg/limb 
4el.3 
6el3 
Sell 
1.0e14 
1.2e14 
1.4e14 
l ,6e14 
1.8e14 
2.0e14 
2..2e14 
12) 
HD 
3e12vg/kg/limb 
l .2el4 
1..8el4 
2.4e14 
l.Oe14 
3.6el4 
4.2el4 
4-llel4 
S.4el4 
6e14 
6.6e14 
Reference Table: Total Dose (vg) per subject based on bodyweight (kg) 
* LD = low dose; HD= high dose 
If safety data is obtained at low dose, the third cohort of three additional subjects will receive 
the next escalating dose (6x1012vg/kg total dose) split between the two limbs. 
Subjects within a cohort will be entered at intervals of approximately 4-6 weeks. Additional 
time will be allowed to review the biopsy results of the first subject in the first cohort prior to 
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120 
129 
2.4e14 
7.2e14 
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dose escalation to the next cohort. Intervals within subjects in the second cohort can be 
reduced base on safety observed in first cohort. 
7.4 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". 
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatorylnformation/Guidances/ 
UCM 227351.pdf 
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 
s 
Fatal adverse event 
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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", and described in section 7.4 
of this protocol. 
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 event 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. 
0 
Laboratory tests with values within the clinically significant range will be repeated during the 
Q 
same visit whenever possible (refer to Table 6.4 for a complete list of clinically significant range 
values). 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 as clinically indicated. 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 t he 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 for addition to the subject's research chart. 
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Only those adverse events requiring treatment will qualify as DLT. The Pl 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 investigator 
receives the safety information and determines that the information qualifies for reporting. The 
investigator will confer with the DSMB and FDA before continuing enrollment. 
Table 6.4 Clinically Significant Laboratory Range 
Test 
Units 
Reference Range 
CUnh:aUy Slgnlflcant Range 
Hemoglobin 
g/dl 
2Y-5Y 
11.5- 13.5 
< 8.0or > 18 
6Y-11Y 
11.5-15.5 
12Y-Adult 
12.0-16.0 
wee 
K/cu mm 
4Y-5Y 
5.5-15.S 
S3.5 or :i: 20 
6Y - 9Y 
5.0 - 14.5 
10Y- 20Y 
4.5 - 13.5 
21Y-Adult 
4.5-11.0 
BUN 
mg/dl 
5-18 
~5 
PT 
sec 
12.4-14.7 
s 5.0 or.: 35.0 
PTT 
sec 
24 - 36 
.: 12S 
Platelet 
K/cu mm 
140-440 
s 90 ore: 800 
Amylase 
U/L 
0-17Y 
<110 
:!:200 
18Y-Adult 
30 -110 
Total Protein 
g/dl 
3Y-15Y 
5.8 - 8.7 
s4.4 or .t 9.5 
16Y-Adult 
6.4-8.4 
Total Blllrubln 
mg/dL 
0.1-1.0 mg/dL 
2:3.0 
Creatlnine 
mg/dl 
4Y-7Y 
0.3-0.8 
U.8 
SY-l0Y 
0.3-0.9 
11Y-12Y 
0.4-1.0 
13Y-17Y 
0.5-1.2 
18Y-Adult 
0.6-1.3 
GGT" 
U/L 
8-78 
3XULN 
ULN= upper limit normal 
* Subjects with history of alcohol abuse will be excluded from the study. Since GGT is very 
sensitive to alcohol consumption, subjects will be required to avoid alcohol the week before 
testing. 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. 
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7.5 STOPPING/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 
o Severe or medically significant but not immediately life-threatening; 
o 
Hospitalization or prolongation of hospitalization indicated; 
o 
Disabling; limiting self care ADL **. 
• 
Grade 4 
o life-threatening consequences; 
o Urgent intervention indicated. 
• 
Grade S 
o 
Death related to AE. 
Activities of Daily Living {ADL) 
*Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the 
telephone, managing money, etc. 
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**Self care AOL refer to bathing, dressing and undressing, feeding self, using the toilet, taking 
medications, and not bedridden. 
If after review by the DSMB the decision is made to continue, the study will proceed according 
to Section 6.2.7.6 (Dose escalation) of this protocol. 
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7.6 DOSE ESCALATION 
There will be 4 weeks between dosing of subjects within a cohort. For the first cohort, the 
allowance of four weeks between dosing of subjects provides time for safety review from five 
time points (days 1, 2, 7, 14 and 30) prior to dosing of the next subject and time for review of 
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the safety analysis of all the subjects within a cohort by the investigators and the 05MB. 
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This timeline will also provide time for review of any clinically significant improvement based on 
the 6MWT data. 
 
 
The investigators will confer with the IRB, DSMB, and FDA/CBER 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. 
7.7 CONCOMITANT MEDICATIONS/THERAPIES 
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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 
medical history. The Pl 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 postoperat ive bleeding and should not 
lead to modification or cessation of such therapy.13•15 
8.0 ADVERSE EVENT MONITORING AND REPORTING 
8.1 DEFINITION OF AN ADVERSE EVENT 
As stated above (6.2.7.4) 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 bioavailabillty and bioequivalence studies 
found in 21 CFR part 320. "Safety Reporting Requirements for INDs and BA/BE Studies". 
http:ljwww.fda.gov/downloads/ Drugs/GuidanceComplianceRegulatorylnformation/ 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. 
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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 *. 
Grade 3 Severe or medically significant but not immediately life-threatening; hospitalization or 
prolongation of hospitalization indicated; disabling; limiting self care AOL**. 
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 (AOL) 
*Instrumental AOL refer to preparing meals, shopping for groceries or clothes, using the 
telephone, managing money, etc. 
**Self care AOL refer to bathing, dressing and undressing, feeding self, using the toilet, taking 
medications, and not bedridden. 
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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 312.3Z(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 
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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 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 AOL**. 
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 Pl will fulfill the reporting responsibilities to FDA/ OBA on behalf of Nationwide Children's 
Hospital using the web-based Adverse Event reporting system {GeMCRIS). 
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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. 
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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 
follows: 
NIAMS and DSMB: 
All Serious Adverse Events (SAEs) and Dose Limiting Toxicities (DLTs) will be reported to the 
NIAMS and the DSMB through KAI within 48 hours of notification, regardless of relatedness to 
the clinical trial. KAI Research, Inc. serves as the NIAMS Executive Secretary. 
FDA/NIH OBA/IRB 
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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 OBA/I RB as soon as possible, but not later than 7 calendar days 
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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 concurrently to the FDA/NIH 
OBA/I RB 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. 
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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/I RB as soon as possible, but in no case later 
than 15 calendar days after the determination is made. 
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/IRS/ NIAMS and the DSMB through KAI 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/IRS/ NIAMS and the DSMB through KAI within 15 calendar days ofthe 
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/ NIAMS and the DSMB through KAI 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 
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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; (S) NIH OBA protocol number; (6) FDA's 
Investigation al 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: 
http://osp.od.ni h.~ov /sites/default/tiles/resources/Adverse Event Template .docx 
A copy of this report will also be sent to the IRS, IBCSC, FDA, and DSMB according to regulatory 
requirements described in section Safety Reporting. 
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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 
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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 FROM 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 
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physician prov,ide information regarding every routine visit and any intercurrent event taking 
place during the two years following gene transfer. Laboratory reports, hospitalizations, 
clinical notes and any other relevant medical records will be requested at the time of their 
occurrence. If non-routine visits are reported to us by the primary care physician, the study 
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 
pirocedures, as applicable. 
During the consent process, the study investigator will emphasize the importance of subject 
communication with our study team. Any routine or non-routine doctor's visits or medical care 
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received during the two years following gene transfer should be reported to the study team. 
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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 
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Within 60 days after the one-year anniversary of the date on which the investigation al 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 NlH 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 
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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, 
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the number of participants who died during participation in the investigation and causes of 
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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. 
11.0 HUMAN SUBJECTS 
11.1 HUMAN SUBJECTS INVOLVEMENT AND CHARACTERISTICS 
The proposed study will involve seven subjects with alpha-sarcoglycan deficiency (LGMD2D] 
subjects. All subjects with this condition that are enrolled must have muscle weakness by 
clinical exam. LGMD2D is established on the basis of an alpha-sarcoglycan gene mutation with 
reduced or abnormally expressed alpha-sarcoglycan in the muscle. Subject selection will not 
exclude anyone on the basis of race, ethnic, or gender background. For complete details see 
the Inclusion and Exclusion Criteria Section. 
11.2 SOURCES OF MATERIALS 
Research material obtained from identifiable living human subjects includes reports of: history 
and physical assessments, blood, urine, chest X-ray, ECHO, EKG, 
 
, 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 
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file by the Principal Investigator. All material or data collected as part of the study will be 
obtained specifically for research purposes. 
11.3 POTENTIAL RISKS 
Potential risks to study subjects include risks associated with administration of the study agent 
and with study procedures. 
11.3.1 RISKS ASSOCIATED WITH SCAAVRH74.TMCK.HSGCA GENE TRANSFER 
This is the first time that this viral serotype, scAAVrh74.tMCK.hSGCA, will be given to human 
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subjects. Thus, the risks of participating in this study are not known. Studies on animals that 
received larger doses injected into muscle showed no side effects. 
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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.16-18 In summary, our data suggest that rAAV genomes persist in mouse muscle as 
transcriptionally active large and small concatameric episomes and when considering potential 
and theoretical integration events, rAAV vectors appear to possess a similar risk profile as 
plasmid DNA injected into muscle.17 The majority of vector DNA appears to persist as episomal 
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rather than integrated DNA, which makes it very unlikely that mutagenesis will occur. 16 
Therefore, we think the chance that cancer would develop is very small. 
l iver 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 
vector-induced DNA damage based on the number of AAV genomes per cell in tumor samples. 
19•20 Also, other larger scale studies in newborn and adult mice have not shown this effect. 21 It 
remains difficult to ascertain the true risk of neoplasia, although it seems very small. 
Subjects may also develop immune response to rAAVrh74. 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. 
AAV injected by vascular delivery could also spread to other organs. The consequences are not 
known. 
11.3.2 RISKS ASSOCIATED WITH PROCEDURES 
11.3 .2.1 RISK RELATED TO GENERAL ANEST HESIA 
Although rare, unexpected severe complications with anesthesia can occur and include the 
remote possibility of infection, bleeding, drug reactions, blood clots, loss of sensation, loss of 
limb function, paralysis, stroke, brain damage, heart attack or death. 
11.3.2.2 CANNULATION OF THE FEMORAL ARTERY AND FEMORAL VEIN 
The risks of limb artery canulation include rarely permanent ischemic damage, sepsis, local 
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infection, pseudoaneurysm, hematoma, and bleeding. large artery canulation Is used routinely 
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in the following clinical situations: extracorporal membrane oxygenation (in >10 kg patients) 
and isolated limb perfusion for the treatment of limb soft tissue sarcomas. Small artery 
canulation is used in acute care settings for invasive blood pressure monitoring and blood 
sampling. At the puncture site: bruising at the site where the catheter is inserted is common 
(usually in the groin). Occasionally, a larger bruise/swelling (hematoma) which may require 
transfusion, surgery or delayed discharge may occur. The risk of this is less than 4% (or 1 in 25). 
The risk of the vessel becoming blocked or being significantly damaged at the insertion site is 
less than 0.5% (or 1 in 200). To minimize risk to the limb and subject we are targeting the 
femoral vessels in the current protocol. In addition the subject will be heparinized (monitored 
by Activated Clotting Time measurements) to minimize the risk of thrombosis/emboli. In ILP/lll 
procedures there is a small risk of compartment syndrome. This outcome is minimized in our 
protocol by utilizing pressure servo-regulating pump flows. 
11.3.2.3 ARTERIOGRAM 
Fluoroscopy is used to assess vascular architecture, patency, and stasis prior to vector 
administration. Exposure to radiation and radio-opaque contrast material carry a small risk to 
the subject as the dose of radiation is limited to 8-16 minutes .. Therefore, a 8-16-minute study 
would give an effective dose of about 6,400-12,800 mrem. Per NIH Guidelines a dose range of 
300 mrem to 5000 mrem is termed "minimal". Although contrast exposure can on extremely 
rare occurrences cause an allergic reaction that may be life threatening most cases of contrast 
related reactions are limited to localized erythema and sensation of warmth. 
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11.3.2.4 PREDNISONE 
The possible side effects of prednisolone (steroid) 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. 
11.3.2.S LYM PHEDEMA 
A possible long-term side effect of gene transfer procedure is a permanent swelling of the 
treated ltmbs, known as lymph edema that develops because of a build-up of fluid in the tissues 
surrounding the injected area. This can happen if the vessels are damaged or blocked during 
the gene transfer procedure. 
11.3.2.6 PERIPHERAL NEUROPATHY 
You may get numbness or tingling after the procedure. This will be in your foot if you have ILP 
on your leg. It can be due to the inadvertent manipulation on your nerves around the area of 
procedure and is called peripheral neuropathy. Tell your doctor if this happens. It usually 
improves slowly over a few months but is sometimes permanent. 
11.3.2.7 ISCHEMIC INJURY 
Due to manipulation of the blood vessels on the limb area during the procedure there is a 
chance that restriction of blood supply can damage tissues in the area. This occlusion if 
prolonged can cause permanent pain, ulcers, or infection of tissue area. 
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11.3.2.8 REPERFUSION INJURY 
Your limb may become red and swollen. This usually starts about 48 hours after your treatment 
and is most noticeable after a week. The swelling gradually reduces over 2-3 months. The 
redness will gradually fade to brown and become lighter over the next few months. Your skin 
color should go back to normal after about six months, but some people are permanently left 
with a slight darkening of the skin. 
11.3.2.9 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. 
11.3.2.10 
 
 
 
 
 
 
 
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11.3.2.11 MYOGLOBINURIA/RHABDOMYOLYSIS 
There have been cases where myoglobinuria as a result of rhabdomyolysis has occurred after 
ILP infusion of scAAVrh74.tMCK.hSGCA. There is an increased risk ofthis due to the disease 
state of LGMD2D, but there have been cases seen in the subjects within this study. This is 
thought to possibly be caused by the increase in muscle strength after gene transfer that leads 
subjects to exercise more rigorously. Subjects will be asked to decrease exercise to a low to 
moderate level following the transfer and to visually monitor their urine for any brown 
discoloration. Subjects will also be warned to avoid over-exhaustion and to stay well hydrated. 
If brown urine discoloration or muscle pain were to occur, they should alert the Pl as soon as 
possible or go to the nearest Emergency Room. 
11.3.3 RISKS RELATE D TO SUBJECT EVALUATIONS BEFORE AND AFTER GENE TRANSFER 
Multiple blood draws could lead to pain at the site of venipuncture, as well as bruising that 
could persist for several days, and rarely, infection. Subjects may also feel light-headed or faint 
from the blood draw. 
Muscle testing as a secondary outcome measure could result in soreness for 1 to 2 days after 
muscle testing. 
11.4 ADEQUACY OF PROTECTION AGAINST RISKS 
11.4.1 INFORMED OF RISKS 
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 
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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 scAAVrh74.tMCK.hSGCA 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. 
11.4.2 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 scAAVrh74.tMCK.hSGCA gene. 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 {during the first six months after gene therapy (females) or until two 
negative sperm samples are obtained post gene transfer (males). As part of this study we must 
ask for a semen sample from all sexually active males 18 years and older. 
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Some of vector can be excreted from the body for at 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. 
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11.4. 3 CONFIDE NTI ALI TY 
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, Ohio Reproductive Medicine, and an outside contracted monitor of 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. 
11.4.4 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
. 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 
 
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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. 
11.5 POTENTIAL BENEFITS OF THE PROPOSED RESEARCM TO THE SUBJECTS AND 
OTHERS 
This is primarily a safety study of transfer of scAAVrh74.tMCK.hSGCA with the potential to 
increase lower limbs muscle strength. If muscle strength is increased in knee extension/flexor it 
has the potential to make a difference for subjects in prolonging ambulation and preventing 
falls. 
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11.6 INCLUSION OF MINORITIES IN CLINICAL RESEARCH 
11.6.1 SELECTION CRITERIA 
Seven LGM D2D subjects will be enrolled in this trial. Eligibility criteria will include subjects with 
proven alpha-sarcoglycan deficiency by muscle biopsy or DNA testing. Participants will 
encompass any ethnic or racial background. 
11.6.2 TARGETED/PLANNED ENROLLMENT 
The number of subjects enrolled from each ethnic group will mirror that seen in the clinic 
population. 
Table 6.5. Local distribution of the disease (percent) 
Gender 
American 
Asian or 
Hispanic 
Black, 
White, 
Total 
Indian 
Pacific 
Non-
Non-
Alaskan 
Islander 
Hispanic 
Hispanic 
Native 
Female 
0 
0 
10 
0 
30 
40 
Male 
0 
0 
40 
0 
20 
60 
Total 
0 
0 
so 
0 
50 
100 
11.6.3 PROPOSED DATES OF ENROLLMENT AND RECRUITMENT 
I 
I 
We anticipate beginning enrollment in January 2014, with completion of enrollment December 
2017. This protocol is listed in the Clinical Trials Data Bank of the NIH. 
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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 
partidpant safety and study progress for the "Phase I/Ila gene transfer clinical trial for LGMD2D 
(alpha-sarcoglycan deficiency) using scAAVrh74.tMCK.hSGCA" trial. 
Responsibilities of the 05MB 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; 
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• 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. 
DSMB Reporting and Meetings 
Reports describing the status of the study will be prepared by the Principal Investigator's staff 
and sent to the 05MB through KAI at the end of each cohort, or at the DSM B's request. 
An initial meeting {either by teleconference, webcast,or paper report) with the DSMB is 
scheduled prior to study initiation and at the DSM B's request. Reports will be submitted at 
least 2 weeks 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); 
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• 
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) has been approved by the study's 
sponsor (National Institute of Health). 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 and discussed with the DSMB 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, 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, lBCSC, NIH-OBA and FDA, 
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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
 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 
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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 annually. 
13.0 CLINICAL MON ITORING 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 
Pl 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 appllicable, subject's consent to continue participation will again be obtained. 
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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 
Q 
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 Pl 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 Pl 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 
Q 
will be extracted from source documents (lab reports, echo reports ... ) and transferred to the 
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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 INYESTIGATIONAL AGENT 
0 
t,lnvestigator shall take adequate precautions, including storage of the investigation al drug in a 
Q 
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). 
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. 
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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 AAV to individuals will be reported to 
 
 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
, 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. All study staff must have documentation of taking the 
laboratory training safety module provided by the IBCSC. The link to the on line training is the 
following: 
http:// rex/Trai n ing/Pages/1 BCSC%20Tra in ing. aspx 
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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. 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).
11 (Code 
of Federal Regulations, Title 21, Part 312 .62, Section a). 
15.0 BRIEF DESCRIPTIONS OF RESEARCH AREAS 
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: 
 
Co-Investigator: 
 
Co-Investigator: 
 
Co-Investigator: 
 
Co•lnvestigator: 
 
Co-Investigator: 
 
Co-Investigator:
 
Co-Investigator: 
 
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Co-Investigator: 
 
Safety Monitor:
 
Clinical Evaluators: 
 
 
Program Manager: 
 
Research Coordinators: 
 
 
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: 
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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; 
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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 
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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. 
16.0 REFERENCES 
1 
Seinen, J. M. & Hoekstra, H. J. Isolated limb perfusion of soft tissue sarcomas: A 
comprehensive review of literature. Cancer treatment reviews, 
doi: 10.1016/j.ctrv .2012 .10.005 (2012}. 
2 
Mendell, J. R. et al. Dystrophin immunity in Duchenne's muscular dystrophy. N Engl J 
Med 363, 1429-1437, doi:10.1056/NEJMoa1000228 (2010}. 
3 
Mendell, J. R. et al. Limb-girdle muscular dystrophy type 2D gene therapy restores 
alpha-sarcoglycan and associated proteins. Ann Neurol 66, 290-297, 
doi:10.1002/ana.21732 (2009}. 
4 
Mendell, J. R. et al. Sustained alpha-sarcoglycan gene expression after gene transfer in 
limb-girdle muscular dystrophy, type 2D. Ann Neural 68, 629-638, 
doi:10.1002/ana.22251 (2010). 
5 
Laval, S. H. & Bushby, K. M. limb-girdle muscular dystrophies--from genetics to 
molecular pathology. Neuropathol Appl Neurobiol 30, 91-105, doi:10.llll/j.1365-
2990.2004.00555.x (2004). 
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6 
Klinge, L. et al. Sarcoglycanopathies: can muscle immunoanalysis predict the genotype? 
Neuromuscul Disord 18, 934-941, doi:S0960-8966(08}00615-9 
[piiJl0.1016/j.nmd.2008.08.003 (2008). 
7 
Bueno, M. R. et ol. A common missense mutation in the adhalin gene in three unrelated 
Brazilian families with a relatively mild form of autosomal recessive limb-girdle muscular 
dystrophy. Hum Mo/ Genet 4, 1163-1167 (1995). 
8 
Carrie, A. et al. Mutational diversity and hot spots in the alpha-sarcoglycan gene in 
autosomal recessive muscular dystrophy {LGMD2D). J Med Genet 34, 470-475 (1997). 
9 
Emery, A. E. Population frequencies of inherited neuromuscular diseases--a world 
survey. Neuromuscul Disord 1, 19-29 (1991). 
10 
Emery, A. E. The muscular dystrophies. Lancet 359, 687-695, doi:10.1016/S0140-
6736(02)07815-7 (2002). 
11 
Grootenhuis, M.A., de Boone, J. & van der Kaai, A. J. Living with muscular dystrophy: 
health related quality of life consequences for children and adults. Health and quality of 
life outcomes 5, 31, doi:10.1186/1477-7525-5-31 (2007). 
12 
Gao, G. et al. Clad es of Adeno-associated viruses are widely disseminated in human 
tissues. J Virol 78, 6381-6388, doi:10.1128/JVl.78.12.6381-6388.200478/12/6381 [pii] 
(2004). 
13 
Liu, G. et al. The Effect of Preoperative Aspirin and/or Heparin Therapy on Coagulation 
and Postoperative Blood Loss after Coronary Artery Bypass Surgery. Crit Care Resusc 1, 
139, doi:AACCM-JJune99.5 [pii) {1999). 
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14 
Veikutiene, A. et al. [Influence of preoperative treatment with aspirin or heparin on 
platelet function and intensity of post operative bleeding in early period after coronary 
artery bypass surgery]. Medicina (Kaunas) 41, 577-583 (2005). 
15 
Sun, J. C., Crowther, M.A., Warkentin, T. E., Lamy, A. & Teoh, K. H. Should aspirin be 
discontinued before coronary artery bypass surgery? Circulation 112, e85-90 (2005). 
16 
Song, S., Lai pis, P. J., Berns, K. I. & Flotte, T. R. Effect of DNA-dependent protein kinase 
on the molecular fate of t he rAAV2 genome in skeletal muscle. Proc Natl Acad Sci US A 
98, 4084-4088, doi:10.1073/pnas.061014598061014598 [pii] (2001). 
17 
Schnepp, B. C., Clark, K. R., Klemanski, D. L., Pacak, C. A. & Johnson, P. R. Genetic fate of 
recombinant adeno-associated virus vector genomes in muscle. J Virol 11, 3495-3504 
(2003). 
18 
Duan, D. et al. Circular intermediates of recombinant adeno-associated virus have 
defined structural characteristics responsible for long-term episomal persistence in 
muscle tissue. J Virol 72, 8568-8577 (1998). 
19 
Donsante, A. et al. Observed incidence of tumorigenesis in long-term rodent studies of 
rAAV vectors. Gene Ther 81 1343-1346, doi:10.1038/sj.gt.3301541 (2001). 
20 
Monahan, P. E., Jooss, K. & Sands, M. S. Safety of adeno-associated virus gene therapy 
vectors: a current evaluation. Expert Opin Drug Saf 1, 79-91 (2002). 
21 
Song, S. et al. Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after 
portal vein injection of recombinant adeno-associated virus (rAAV) vectors. Gene Ther 8, 
1299-1306, doi:10.1038/sj.gt.3301422 (2001). 
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Phase I/Ila gene transfer clinical trial for LGM020 using scAAVrh74.tMCK.hSGCA 
 
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