A Pilot Study for the Safety and Expression of Dystrophin in Skeletal Muscle After SPOT-mRNA03 Administration in Duchenne Muscular Dystrophy (DMD) Patients
Phase: EARLY_PHASE1
Conditions: Duchenne Muscular Dystrophy (DMD)
Sponsor: Shanghai Siponuoyin Biotechnology Co Ltd

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Clinical Study Protocol 
 
 
A pilot study for the safety and expression of dystrophin in skeletal 
muscle after SPOT-mRNA03 administration in Duchenne Muscular 
Dystrophy (DMD) patients 
 
NCT Number: 
Not yet assigned 
Project Number： 
SPOT-MG02 
Protocol Number： 
FM-T3-SH 
Institution： 
Shanghai Children's Medical Center 
Principal Investigator： 
Wang Jiwen 
Co-sponsor： 
Shanghai Siponuoyin Biotechnology Co., Ltd 
CRO： 
Beijing Leadingpharm Medicine Technology 
Development Co., Ltd 
Version： 
V2.1 
Date： 
March 20, 2025 
CONFIDENTIAL 
The information contained in this document is the property of Spot Biosystems Ltd., 
and may not be reproduced, published or disclosed without written consent from Spot 
Biosystems Ltd. 
 
 

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Investigator’s Statement 
I the undersigned, have reviewed this protocol (protocol no: FM-T3-SH, Version: v2.1, 
Date: March 20,2025) and I agree to conduct this protocol in accordance with International 
Conference on Harmonization (ICH) guidelines on Good Clinical Practice (GCP) and the 
Declaration of Helsinki. This protocol will be carried out upon the approval from the Ethics 
Committee (EC) or Institutional Review Board (IRB). 
I understand that the protocol may not be modified without written approval of the sponsor 
and that all changes to the protocol must be submitted to the applicable regulatory 
authorities and the EC/IRB prior to implementation.  
 
 
 
Principal Investigator:  
 
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Collaborator’s Statement 
 
I the undersigned, have reviewed this protocol (protocol no: FM-T3-SH, Version: v2.1, 
Date: March 20,2025) and I agree to conduct this protocol in accordance with International 
Conference on Harmonization (ICH) guidelines on Good Clinical Practice (GCP) and the 
Declaration of Helsinki. I will fulfill the responsibilities of the sponsor including initiation, 
monitoring, and funding of this trial. I agree to conduct this clinical study in accordance 
with the design and provisions in the protocol. 
 
 
 
 
Signature of Responsible Party:  
 
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Signature 
 
 
 
 Date (mm/dd/yyyy) 
 
Name:  
 
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Collaborator: 
  
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CONTENTS 
INVESTIGATOR’S STATEMENT ............................................................................................................... 2 
COLLABORATOR’S STATEMENT ............................................................................................................. 3 
ABBREVIATION LIST ................................................................................................................................. 7 
SYNOPSIS ................................................................................................................................................. 9 
1. 
BACKGROUND ............................................................................................................................... 18 
1.1. 
DYSTROPHIN GENE AND ITS MUTATION TYPES .............................................................................. 18 
1.2. 
CLINICAL THERAPY FOR DMD PATIENTS ...................................................................................... 18 
1.3. 
GENE THERAPY FOR DMD ............................................................................................................ 19 
1.4. 
APPROVED DRUGS FOR DMD AND THEIR SCOPE OF APPLICATION ................................................ 20 
1.4.1. 
Steroid drugs ....................................................................................................................... 20 
1.4.2. 
Gene therapy for DMD ........................................................................................................ 21 
1.5. 
GLOBAL ONGOING STUDY ON DMD TREATMENT DRUGS .............................................................. 23 
2. 
SPOT-MRNA03 INTRODUCTION ..................................................................................................... 23 
2.1. 
SPOT-MRNA03 DESIGN ............................................................................................................... 23 
2.2. 
MECHANISM OF ACTION OF SPOT-MRNA03 ................................................................................ 24 
3. 
RISK AND BENEFIT ASSESSMENT .................................................................................................... 24 
3.1 
RISK ASSESSMENT ........................................................................................................................ 24 
3.2 
BENEFIT ASSESSMENT .................................................................................................................. 26 
4. 
STUDY OBJECTIVES AND ENDPOINTS ............................................................................................. 27 
5. 
STUDY DESIGN ............................................................................................................................... 27 
5.1. 
OVERALL DESIGN ......................................................................................................................... 27 
5.2. 
DURATION OF STUDY ................................................................................................................... 28 
5.3. 
TERMINATION CRITERIA ............................................................................................................... 28 
5.4. 
SCHEDULE OF EVENTS .................................................................................................................. 29 
6. 
STUDY POPULATION ...................................................................................................................... 34 
6.1 
INCLUSION CRITERIA .................................................................................................................... 34 
6.2 
EXCLUSION CRITERIA ................................................................................................................... 34 
7. 
SUBJECT CODING AND GROUPING ................................................................................................. 35 
8. 
STUDY PROCEDURES ...................................................................................................................... 36 
8.1. 
TREATMENT AND FOLLOW-UP ...................................................................................................... 36 
8.1.1. 
Screening（D-30 ~ -8）...................................................................................................... 36 
8.1.2. 
Baseline（D-7~ -1） ........................................................................................................... 37 
8.1.3. 
Treatment（D1 ~ 29） ........................................................................................................ 37 
8.1.4. 
Follow-up ............................................................................................................................ 39 
8.2. 
SUPPLY OF INVESTIGATIONAL DRUGS/TREATMENTS ..................................................................... 39 

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8.2.1. 
Name, Source and Strength ................................................................................................. 39 
8.2.2. 
Preparation and Storage ..................................................................................................... 40 
8.2.3. 
Route of Administration and Dosage Adjustment ................................................................ 40 
8.3. 
CONCOMITANT MEDICATION ........................................................................................................ 40 
8.4. 
PATIENT COMPLIANCE AND WITHDRAWAL .................................................................................. 41 
8.4.1. 
Screening Failure ................................................................................................................ 41 
8.4.2. 
Withdrawal Criteria ............................................................................................................ 42 
8.4.3. 
Management of Subjects During the Study .......................................................................... 42 
9. 
STUDY ASSESSMENT ...................................................................................................................... 43 
9.1 
PK ASSESSMENT........................................................................................................................... 43 
9.1.1 
Assessment Indicators .............................................................................................................. 43 
9.1.2 
Timepoints of Sampling ........................................................................................................... 43 
9.2 
PHARMACODYNAMIC ASSESSMENT .............................................................................................. 43 
9.2.1 
Dystrophin Expression ............................................................................................................. 43 
9.2.2 
Electromyography.................................................................................................................... 44 
9.2.3 
MRI .......................................................................................................................................... 44 
9.3 
IMMUNOGENICITY ASSESSMENT ................................................................................................... 44 
9.3.1 
Assessment Indicators .............................................................................................................. 44 
9.3.2 
Timepoints of Sampling ........................................................................................................... 45 
9.3.3 
Detection Method..................................................................................................................... 45 
9.4 
SAFETY ASSESSMENT ................................................................................................................... 45 
9.4.1 
Safety Parameters .................................................................................................................... 45 
9.4.2 
Baseline Signs and Symptoms .................................................................................................. 45 
9.4.3 
Laboratory Safety Assessment ................................................................................................. 46 
9.4.4 
Vital Signs and Physical Examination ..................................................................................... 47 
9.4.5 
12-Lead ECG ........................................................................................................................... 47 
9.4.6. 
NSAA Scale ........................................................................................................................... 47 
10. 
ADVERSE EVENT REPORTING ..................................................................................................... 49 
10.1. 
DEFINITION OF ADVERSE EVENT .................................................................................................. 49 
10.1.1. 
Definition ............................................................................................................................. 49 
10.1.2. 
Categorization of AEs .......................................................................................................... 49 
10.1.3. 
Causes of AEs ...................................................................................................................... 50 
10.2. 
ADVERSE EVENT RECORDING AND REPORTING ............................................................................ 51 
10.3. 
RISK PREVENTION AND HANDLING............................................................................................... 51 
11. 
STATISTICAL ANALYSIS ............................................................................................................... 52 
11.1. 
SAMPLE SIZE ................................................................................................................................ 52 
11.2. 
POPULATIONS FOR ANALYSIS ....................................................................................................... 52 
11.2.1 
Full Analysis Set .................................................................................................................. 52 
11.2.2 
Per-Protocol Set .................................................................................................................. 52 
11.2.3 
Safety Analysis Set ............................................................................................................... 52 
11.2.4 
Pharmacokinetic Dataset .................................................................................................... 52 
11.3. 
EFFICACY ANALYSIS AND STATISTICAL METHODS ...................................................................... 53 

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11.3.1 
Analysis of Primary Endpoints ............................................................................................ 53 
11.3.2 
Analysis of Secondary Endpoints ......................................................................................... 53 
11.4. 
SAFETY ANALYSIS AND STATISTICAL METHODS .......................................................................... 54 
11.5. 
MID-TERM ANALYSIS ................................................................................................................... 54 
11.6. 
DATA MONITORING COMMITTEE .................................................................................................. 54 
12. 
DATA COLLECTION AND MANAGEMENT .................................................................................... 54 
12.1. 
CRF/EDC ..................................................................................................................................... 54 
12.2. 
DATA MANAGEMENT ................................................................................................................... 55 
13. 
ETHICAL CONSIDERATIONS ........................................................................................................ 55 
13.1. 
ETHICS COMMITTEE/INSTITUTIONAL REVIEW BOARD .................................................................. 55 
13.2. 
INFORMED CONSENT .................................................................................................................... 56 
13.3. 
PROTOCOL AMENDMENT .............................................................................................................. 56 
14. 
CONFIDENTIALITY ...................................................................................................................... 56 
LITERATURE ........................................................................................................................................... 58 
 
Tables 
Table 1 Summary of gene therapy strategies for DMD [15] ................................................................. 19 
Table 2 Approved drugs for the treatment of DMD [16][17] ............................................................... 20 
Table 3 Global information of the ongoing new drugs for DMD ......................................................... 23 
Table 4 The objectives and endpoints of this study .............................................................................. 27 
Table 5 Schedule of Events ................................................................................................................... 30 
Table 6 The CTCAE displays grade 1 through 5 criteria of adverse event ........................................... 50 
Table 7 Causality or relatedness assessment criteria in AEs and investigational drugs ....................... 50 
 
 
 

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Abbreviation List 
Abbreviations 
Full Name 
AE 
Adverse Event 
AR 
Adverse Reaction 
BMI 
Body Mass Index 
CBC 
Complete Blood Count 
CN 
Copy Number 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
CRU 
Clinical Research Unit 
CTCAE 
Common Terminology Criteria For Adverse Events 
D/d 
Day 
DMD 
Duchenne Muscular Dystrophy 
EC 
Ethics Committee 
ECG 
12-Lead Electrocardiogram 
ELISA 
Enzyme-Linked Immunosorbent Assay 
EMG 
Electromyography 
EOS 
End Of Study 
EVs 
Extracellular Vesicles 
FAS 
Full Analysis Set 
FIH 
First In Human  
h 
Hour 
IC 
Informed Consent 
IIT 
Investigator-Initiated Clinical Trial 
IRB 
Institutional Review Board 
ISRs 
Injection Site Reactions 
IV 
Intravenous 
GCP 
Good Clinical Practice 
MRI 
Magnetic Resonance Imaging 
mRNA 
Messenger RNA 
NMPA 
National Medical Products Administration 
NSAA 
North Star Ambulatory Assessment 
PET 
Pulmonary Function Test 
PK 
Pharmacokinetics 
PD 
Pharmacodynamics 

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Abbreviations 
Full Name 
PP 
Per-Protocol Set 
qPCR 
Quantitative Polymerase Chain Reaction 
SAE 
Serious Adverse Event 
SAS 
Safety Analysis Set 
SAP 
Statistical Analysis Plan 
TRAEs 
Treatment Related Adverse Reactions 
W/w 
Week 

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Synopsis 
Title: 
A pilot study for the safety and expression of dystrophin in skeletal 
muscle after SPOT-mRNA03 administration in Duchenne Muscular 
Dystrophy (DMD) patients 
Project Number: SPOT-MG02 
Protocol 
Number: 
FM-T3-SH 
Collaborator: 
Shanghai Siponuoyin Biotechnology Co., Ltd 
Clinical 
Trial 
Phase: 
Investigator-initiated clinical trial (IIT) 
Investigational 
Drug： 
SPOT-mRNA03 injection 
Indication: 
Duchenne Muscular Dystrophy（DMD） 
Study 
Population 
Ambulatory boys with DMD, aged 2~6 years 
Sample Size 
6 subjects 
Duration 
of 
Study 
The screening period is 0-30 days, with 4 weeks of treatment period 
and a follow-up period of 6 months.  
Objectives 
Primary objective: 
To evaluate the safety and tolerability of SPOT-mRNA03 administered 
by intravenous infusion (IV) to DMD patients. 
Secondary objectives: 
⚫ To evaluate the changes of dystrophin mRNA in serum and 
muscles of DMD patients after IV infusion of SPOT-mRNA03. 
⚫ To evaluate the changes in dystrophin expression and engraftment 
in muscles of DMD patients after IV infusion of SPOT-mRNA03. 
⚫ To evaluate the changes in serum anti-dystrophin antibodies and 
cytokines in DMD patients after IV infusion of SPOT-mRNA03. 

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Endpoints 
Primary endpoints: 
⚫ 
Collection and quantification of adverse events (description of 
event, time of onset and resolution, assessment of severity and 
causal relationship to the investigational drug), as well as the data 
of physical examination, vital signs, 12-lead ECG, clinical 
laboratory test results (hematology, biochemistry, urinalysis) in 
this study. 
Secondary endpoints: 
⚫ 
Changes of dystrophin mRNA concentration in serum before and 
different timepoints after the initial administration (see Table 5 for 
schedule) as well as before and after the 2nd, 4th, 6th, and 8th 
administration and during follow-up. And the changes in 
Dystrophin mRNA concentration in muscles at baseline and D29 
after the completion of the last dose (if the biopsy time is between 
12-24 hours after the last dose) (by qPCR). 
⚫ 
Changes in the expression and engraftment level of dystrophin in 
muscle biopsies at baseline and D29 (after the completion of the 
last dose) (by Western Blot and IHC methods). 
⚫ 
Changes in anti-dystrophin antibodies and cytokines in the serum 
at baseline and before the 2nd, 4th, 6th, and 8th administration and 
during follow-up (see Table 5 for schedule). 
Study 
Description 
This is a FIH, open-label, single-arm, and single-center exploratory 
clinical study of SPOT-mRNA03 administered via IV infusion for 
DMD patients. The primary objective of this study is to evaluate the 
safety and tolerability of SPOT-mRNA03 for DMD patients. And the 
secondary objectives are to preliminarily investigate the concentration 
change of dystrophin mRNA, the expression and engraftment level 
changes of dystrophin protein, as well as changes in cytokines and 
immunogenicity. The study has two ascending dose cohorts: 
Ⅰ cohort：5.0 × 109 CN dystrophin mRNA / kg 
Ⅱ cohort：5.0 × 1010 CN dystrophin mRNA / kg 
The study will have a screening period of 30 days, during which 

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patients or their legal guardian written informed consent will be 
obtained before screening assessments and eligibility will be 
determined. A total of 6 DMD patients will participate in the study. 
there are 3 patients in each dose cohort [No previous treatment with 
corticosteroids]. All subjects started taking 0.05-0.1mg/kg/day 
(adjusted according to the actual clinical situation) tacrolimus or 
sirolimus orally at D-3 (3 days before initial dose of SPOT-mRNA03) 
for 4 weeks. All subjects are first administered via intravenous infusion 
on D1, and then administered twice a week (once every 4 days) for a 
total of 8 doses. Four weeks after the initial administration of the 
subjects in the previous dose cohort, if there are no serious adverse 
events related to the treatment, it will be determined that the subjects 
in next dose cohort could be administered after discussion between the 
investigators and the sponsor. 
Safety evaluations on subjects are conducted during each 
administration and follow-up. Muscle biopsies are performed at 
baseline and D29 after administration (see Table 5 for details). Samples 
for biopsy will be taken from gastrocnemius/biceps brachii muscle at 
baseline and from the gastrocnemius and biceps brachii muscles on 
D29. Western blot (WB) is used to detect the expression of dystrophin 
protein. Immunohistochemistry (IHC) is used to detect fiber intensity 
and percentage of dystrophin positive fibers, and qPCR is used to 
detect changes in dystrophin mRNA at baseline and D29 (if the biopsy 
time is between 12~24 hours after the last dose). 
The blood samples are collected for detection of dystrophin mRNA, 
cytokines and immunogenicity (see Table 5 for details) besides for 
laboratory testing. Perform MRI and EMG examinations at baseline 
and at week 16 after administration. 
Investigational 
Drug 
SPOT-mRNA03：Colorless and odorless clear liquid; 
Active Pharmaceutical Ingredients: Dystrophin mRNA-loaded EVs. 
Pharmaceutical 
excipients：CryoStorCS10 
(containing 
10% 
dimethyl sulfoxide (DMSO)), phospate-buffered saline (PBS), human 

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serum albumin (HSA), (pharmaecuetical grade) 
Strength：1.0 × 1010  CN dystrophin mRNA / mL  
Storage：-60℃ ~ -90℃ 
Route 
of 
Administration 
Intravenous infusion: twice a week (once every 4 days). Each subject 
will receive a total of 8 treatments. 
Each vial of SPOT-mRNA03 needs to slowly thaw at 4 ℃/ice before 
use. SPOT-mRNA03 after thawing should be used as soon as possible. 
The total volume of infusion is 40 mL. 
Choose an appropriate intravenous infusion site based on the subject's 
venous condition, with an infusion time of 60~120 minutes. If the 
subject experiences mild allergic reactions such as dizziness, chills, 
rash or extravasation during the infusion process, the infusion should 
be immediately suspended. After recovery, the investigator will 
determine whether to continue the administration. The infusion speed 
can be appropriately reduced (as determined by the investigator), and 
the infusion time can be correspondingly extended (as determined by 
the investigator). After completion of infusion, record the actual 
dosage and start and end time of infusion. 
Take photos for the infusion site before and after each infusion, and 
observe the infusion reaction. 
Concomitant 
Medication 
The newly generated dystrophin will be recognized by the immune 
system of the subjects as an exogenous protein, triggering an immune 
response. Any immune response to dystrophin can affect its 
engraftment and potentially induce further muscle damage, reversing 
any benefits that patients should have received. Therefore, the subjects 
should begin to orally take 0.05-0.1mg/kg (adjusted according to the 
actual clinical situation) tacrolimus or sirolimus daily at D-3 (before 
the initial administration of SPOT-mRNA03) for 4 weeks. 
Inclusion 
Criteria 
Only those who meet all the following criteria can be enrolled: 
1) According to the requirements of the region/country and/or 
IRB/IEC, the patient and/or legal guardian have signed a written 

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informed consent form and are aware of all relevant study content. 
2) Ambulatory boys aged 2 to 6 years, inclusive. 
3) Confirmed diagnosis of DMD through multiplex ligation-
dependent probe amplification (MLPA) and whole-exome 
sequencing. 
4) Tolerance for muscle biopsy under anesthesia with no absolute 
contraindications to the procedure. 
5) Heart, liver, lung, and kidney functions are sufficient: 
a) The left ventricular ejection fraction (LVEF) should be ≥ 
50%; 
b) Forced vital capacity (FVC) ＞ 50% of the expected value, 
and do not require nighttime ventilation; 
c) Patient's glomerular filtration rate (GFR) > 30 mL/min/1.73 
m2 
Exclusion 
Criteria 
Those who meet any one of the following criteria must be excluded: 
1) Complications other than DMD that may cause muscle weakness 
and/or motor dysfunction. 
2) There are severe intellectual disabilities (such as severe autism, 
severe cognitive impairment, and severe behavioral disorders) that, 
according to the investigator's judgment, can affect the study. 
3) Hospitalization for respiratory failure within 8 weeks prior to 
screening. 
4) Asthma or underlying lung diseases that are poorly controlled, such 
as bronchitis, bronchiectasis, emphysema, or recurrent infectious 
pneumonia that investigator believes may affect respiratory 
function. 
5) Severe uncontrolled heart failure (NYHA III-IV), including any of 
the following conditions： 
a) Intravenous administration of diuretics or positive inotropic 
drugs is required within 8 weeks prior to screening. 
b) Hospitalization due to worsening heart failure or arrhythmia 
within 8 weeks prior to screening. 

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6) Abnormal laboratory values considered clinically significant: 
a) GGT > 3 × upper limit of normal 
b) Bilirubin ≥ 3.0 mg/dL 
c) Creatinine ≥ 1.8 mg/dL 
d) Hemoglobin < 8 or > 18 g/dL 
e) White blood cell count > 18,500/μL 
7) There are arrhythmias that require antiarrhythmic treatment. 
8) Subjects who are undergoing immunosuppressive therapy. 
9) Has used other gene therapy, investigational drugs, or any 
treatment aimed at increasing dystrophin expression. 
10) Subjects with a history of major surgeries within 12 weeks prior to 
the initial infusion or planning to undergo major surgeries (such as 
scoliosis surgery) during this study. 
11) Subjects who are allergic to investigational products or local 
anesthetic drugs or have a history of severe allergies or genetic 
allergic reactions. 
12) Within 6 months prior to the initial infusion, the subjects are 
exposed to another investigational drug or are participating in an 
intervention clinical trial. 
13) Subjects with positive of hepatitis B core antibody or hepatitis C 
antibody or HIV antibody during screening. 
14) Investigator believes that the presence of any other serious 
diseases, medical conditions, or chronic drug treatment needs can 
pose unnecessary risks to gene transfer. 
Withdrawal 
Criteria 
1) Considering the safety, the investigator believes that withdrawing 
from the study is in the best interest of the subjects. 
2) Subjects with poor compliance cannot insist on completing the 
trial as planned or have other circumstances that may affect the 
judgment of the research results. 
3) The subject voluntarily requested to withdraw from the trial and 
withdraw the informed consent. 

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End of Study 
After the enrolled subjects complete all administration and follow-up 
at week 28, the study ends.  
Muscle 
Sample 
Collection 
Muscle biopsy will be performed at baseline and D29 (after the last 
dose). Samples of each subject for biopsy will be taken from 
gastrocnemius/biceps brachii muscle at baseline (one site) and from 
the gastrocnemius and biceps brachii muscles on D29 (two sites). 
Select a suitable location of the gastrocnemius muscle/biceps brachii 
muscle, confirm the puncture site, disinfect with iodine, and after local 
anesthesia, use puncture needle to penetrate the tissue and muscle, 
extract muscle tissue with a size of 0.5 cm × 1 cm × 0.5 cm and sent to 
a pathological laboratory for muscle biopsy. Camera the biopsy site 
before and after each muscle biopsy. 
The expression of dystrophin in muscle tissue will be detected using 
the following methods: 
• Evaluate expression level of dystrophin (measured by western 
blot) at baseline and D29 (after the completion of the last dose). 
• Evaluate 
engraftment 
of 
dystrophin 
(measured 
by 
immunofluorescence for fiber intensity and percentage of 
dystrophin positive fibers) at baseline and D29 (after the 
completion of the last dose). 
The concentration of dystrophin mRNA in muscle tissue will be 
detected: 
• Evaluate changes in dystrophin mRNA (detected by qPCR) at 
baseline and D29 (if the biopsy time is between 12~24 hours 
after the last dose). 
Safety 
Assessment 
Vital signs (pulse, respiratory rate, blood pressure, temperature) and 
physical examination will be performed at each visit.  
Safety assessments including laboratory tests (hematology, chemistry, 
urinalysis) and 12-lead ECG will be performed on some visits (see 
Table 5 for details). 
ISRs (redness, swelling, heat, pain, and itching) will be observed 
within 30 minutes each post-treatment. 

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According to the Common Terminology Criteria for Adverse Events 
(CTCAE, version 5.0), all adverse events that occur throughout the 
study period will be evaluated and graded. 
The investigator will collect a description of the events, time of onset 
and resolution, assessment of severity and causal relationship to SPOT-
mRNA03. 
Pharmacokinetic
s 
 Assessment 
Using qPCR to detect the changes of dystrophin mRNA in serum 
samples of subjects. Blood samples for PK test will be collected before 
and 0h+10min after the 1st, 2nd, 4th, 6th, and 8th infusion. Intensive 
blood collection will be carried out at 1 h ± 10 min, 2 h ± 20 min, 8 h 
± 1 h, 24 h ± 1 h, 48 h ± 2 h, and 72 h ± 3 h after the initial infusion. 
PK blood samples will also be collected during follow-up. If 
dystrophin mRNA cannot be detected at W8 follow-up, blood samples 
may not be collected for the test in subsequent follow-up. 
Pharmacodynam
ic Assessment 
Muscle biopsies will be performed at the baseline and D29 after the 
last dose to detect the expression of dystrophin in the muscle.  
Quantity of dystrophin is measured by western blot of biopsied muscle 
tissue. Levels are described as change from baseline, in order to 
remove any background signal in the assay. Levels are expressed as a 
percent of control (i.e., as a percent of the level of normal, wild-type 
dystrophin present in muscle tissue of healthy individuals without 
DMD or Becker muscular dystrophy). 
Perform MRI and electromyogram examinations at baseline and W16 
to assess the health of muscles and the nerve cells that control them. 
Immunogenicity 
• 
Cytokine test includes TNF-α, INF-γ, IL-2, IL-6, and IL-10. 
Collect blood samples before administration and during follow-up 
(see Table 5 for detailed schedule). If cytokine levels return to 
normal at W8 follow-up, blood samples may not be collected for 
the test in subsequent follow-up. 
• 
Anti-dystrophin antibody level can be measured by ELISA. 
Collect blood samples at baseline (if does not exceed 7 days from 
baseline to the first dose, no additional collection is required 

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before the firs dose) and before administration, and follow-up (see 
Table 5 for detailed schedule). If anti-dystrophin antibody cannot 
be detected at W8 follow-up, blood samples may not be collected 
for the test in subsequent follow-up. 
Statistical 
Analysis 
of 
Population 
1) Full analysis set (FAS): FAS will be used to report medication 
compliance and to summarize demographic characteristics (age, 
gender, race, height and weight) and background characteristics 
(medical history, concomitant medications, and physical examination, 
etc.) for all randomized subjects by treatment group. 
2) Safety analysis set (SAS): All subjects who have been randomized 
into cohorts, have used the investigational drug, and have safety 
evaluation data post-treatment constitute the safety population of this 
study. The safety population will be used for safety analysis. 
Statistical 
Method 
Due to the small sample size and open-label design of this study, only 
descriptive statistics will be conducted for all endpoints, including the 
number of subjects, mean and standard deviation, minimum and 
maximum values of continuous variables, and the number and 
percentage of categorical variables. 

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1. Background 
Duchenne muscular dystrophy (DMD) is the most common X-linked recessive male hereditary 
disease and the most common fatal monogenic disease. The incidence rate of DMD in male 
newborns is about 1/3500. Approximately 400-500 cases of DMD are born in China every year, 
with a total of approximately 70.000 confirmed cases, making it one of the countries with the 
highest number of DMD patients in the world. There is no obvious geographical distribution 
characteristic of DMD patients in China [1]. 
1.1. Dystrophin gene and its mutation types 
DMD is caused by mutations in the gene encoding the dystrophin protein. This gene is located in 
the Xp21.2 region of chromosome, with a total length of 2.3 Mb and 79 exons. The mRNA 
sequence encoded by this gene has a total length of 11 Kb. Dystrophin gene is the largest known 
human gene [2][3][4]. The normal dystrophin protein size generated by the translation of this gene 
in muscle cells is 427 kDa, and it is expressed in muscle cells throughout the body, including the 
heart, skeletal muscles, smooth muscles, retina, and brain [5].  
The mutation types in the dystrophin gene are mainly exon deletions/duplications, while mutations 
in large segments (≥ 1 exon) with deletions or duplications account for about 79% (of which 
deletions account for about 68% and duplications account for about 11%). The most common 
hotspots of deletions and duplications are located in exons 45-55 and 3-9 of the gene [2][4]. Other 
types of small mutations (<1 exon) account for approximately 21% (of which point mutations 
account for about 11%, small deletions account for about 5%, small insertions account for 
about 2%, and splice site mutations account for about 3%) [4][5]. These point mutations are 
distributed throughout the entire DMD gene and there are no obvious hotspots. DMD patients 
are unable to synthesize normal dystrophin in their bodies due to mutations in the dystrophin 
gene. 
The phenotype of DMD mainly depends on whether mutations in the dystrophin gene have 
disrupted the open reading frame. Out-frame mutations lead to transcription and translation 
disorders of the dystrophin gene, resulting in a more severe DMD phenotype. In-frame 
mutations, while retaining an open reading frame, can transcribe and translate into truncated, 
partially functional dystrophin proteins, resulting in milder phenotypes, such as Becker 
muscular dystrophy (BMD) [2]. 
1.2. Clinical therapy for DMD patients 
Based on the guidelines of the Neurology Branch of the Chinese Medical Association [7], 
the therapy of DMD patients mainly involves Multi-Disciplinary Treatment (MDT) 

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symptomatic treatment, usually starting in boys aged 4 or above. MDT mainly includes drug 
treatment, rehabilitation treatment, treatment of respiratory complications, treatment of heart 
disease, surgical orthodontic treatment, and other treatments etc [1]. Although advances in 
multidisciplinary nursing and disease management strategies for DMD have slowed down 
disease progression, the disease is still incurable [6][8][9][10].  
At present, the drug treatment for DMD patients mainly is glucocorticoids, such as 
prednisone (0.75mg/kg/d), which can improve the strength and lung function of DMD 
patients, reduce the need for spinal surgery, and delay the occurrence of cardiomyopathy. But 
hormone therapy may cause many side effects, including obesity, Cushing's face, hirsutism, 
osteoporosis, and so on. To reduce side effects, the dose can be reduced by one-third if 
necessary [7]. 
In recent years, in addition to hormone therapy, with the increasing maturity of core 
technologies such as gene delivery vector technology and gene editing technology, gene 
therapy has also been gradually developing and heating up. Due to its potential to cure rare 
diseases in one go, it has long received global attention and is known as the "dawn of rare 
disease treatment". 
1.3. Gene therapy for DMD 
The DMD gene therapy strategy is designed for different types of gene mutations aimed at 
restoring dystrophin expression in muscle tissue. It mainly includes readthrough therapy [11], 
exon skipping therapy [12], vector mediated gene replacement therapy [13], clustered 
regularly interspaced short palindromic repeat，CRISPR/ CRISPR-associated nuclease 9，
(Cas9）gene editing therapy [14]. 
The above four therapies are suitable for DMD patients with different mutation types, and 
each has its own advantages and disadvantages, see Table 1. 
Table 1 Summary of gene therapy strategies for DMD [15] 
Therapy 
strategy 
Mechanism of 
action 
Applicable 
to patients Advantage 
Disadvantage 
R&D progress 
Readthrough 
Prevent 
signal 
recognition 
of 
termination 
codon 
and 
induce 
read- 
through. 
Nonsense 
mutation 
Good safety 
and 
tolerability 
Applicable 
to 
few patients and 
limited clinical 
benefits 
Some 
drugs 
approved 
by 
EMA 
Exon 
skipping 
therapy 
Antisense 
oligonucleotides 
mask 
mRNA 
splicing signals 
and 
achieve 
exon skipping. 
Exon 
deletion 
Good safety 
and 
tolerability 
Limited clinical 
benefits
，
frequent 
administration 
requirement and 
low myocardial 
Some drugs have 
been 
approved 
by 
FDA, 
and 
some 
clinical 
trials 
are 
ongoing. 

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transduction 
efficiency 
Vector 
mediated 
dystrophin 
gene 
replacement 
therapy 
AAV 
loaded 
micro-
dystrophin 
AAV 
antibody 
negative 
Restore 
stable high 
expression 
of 
dystrophin 
High demand for 
AAV, 
time-
consuming 
manufacturing, 
high cost, and 
susceptibility to 
immune 
reactions 
Accompanied by 
questioning and 
controversy, the 
gene 
therapy 
drug-Elevidys, 
jointly 
developed 
by 
Sarepta 
& 
Roche, has been 
approved 
for 
market by FDA. 
CRISPR/Cas9 
gene editing 
AAV 
loaded 
CRISPR/Cas9 
precise 
repair 
gene mutation  
AAV 
antibody 
negative 
one-time 
treatment 
High demand for 
AAV, 
time-
consuming 
manufacturing, 
high 
cost, 
susceptibility to 
immune 
reactions, 
and 
off-target risk 
The most studies 
are in the animal 
experimental 
stage, and one 
clinical trial has 
been terminated 
EMA ：European Medicines Agency，FDA ：U.S. Food and Drug Administration，AAV ：Adeno-
associated virus，CRISPR ：Clustered regularly interspaced short palindromic repeat，Cas9 ：CRISPR-
associated nuclease 9  
1.4. Approved drugs for DMD and their scope of application 
In recent years, the approved drugs for DMD treatment are mainly steroid and gene therapy 
products. See Table 2. 
Table 2 Approved drugs for the treatment of DMD [16][17] 
Drug 
classification 
Drug name 
R&D 
company 
Approval 
time 
Indication 
Exon 
skipping 
therapy（ASO） 
Casimersen 
Sarepta 
2021 
DMD patients with exon 45 gene 
mutation 
Viltolarsen 
Nippon 
Shinyaku Co 
Ltd 
2020 
DMD patients with exon 53 gene 
mutation 
Golodirsen 
Sarepta 
2019 
DMD patients with exon 53 gene 
mutation 
Eteplirsen 
Sarepta 
2016 
DMD patients with exon 51 gene 
mutation 
Corticosteroid 
Deflazacort 
PTCT 
2017/2019 
DMD patients aged 2 years and above 
Vamorolone 
Catalyst 
Pharms 
2023 
DMD patients aged 2 years and above 
Readthrough 
Ataluren 
PTCT 
2014 
Ambulatory 
DMD 
patients 
with 
nonsense mutation 
AAV 
vector 
mediated 
dystrophin gene 
replacement 
therapy 
Elevidys 
Sarepta 
2023 
Used to treat DMD outpatient patients 
aged 4-5 with confirmed mutations 
1.4.1. Steroid drugs 
In 2017, the corticosteroid drug Deflazacort was approved by the US Food and Drug 

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Administration (FDA) for the treatment of DMD patients. On October 26, 2023, the FDA 
approved the marketing application of Vamorolone oral suspension 40 mg/mL for the 
treatment of DMD patients aged 2 years and above. Vamorolone is a first-in-class steroid 
drug, which differs from normal steroid drugs in that it can selectively activate certain 
signaling pathways of steroids, resulting in better safety [16]. 
1.4.2. Gene therapy for DMD 
In 2014, EMA approved the drug Ataluren (trade name: Translarna) to enter the European 
market. This drug can reduce the sensitivity of ribosomes to early termination codons, 
allowing mRNA to continue translation without stopping at the termination codon during 
translation (i.e. "readthrough"). It can be used to treat gene nonsense mutations, with 
approximately 10% of DMD patients [17]. 
The four exon skipping therapies approved by the FDA for the treatment of DMD are 
Eteplarirsen, Golodirsen, Viltolasen, and Casimersen. These drugs can only be used for DMD 
patients with specific gene mutations: Eteplarirsen (Exonys 51), approved for marketing on 
September 19, 2016, is used to treat approximately 13% of DMD patients with exon 51 
mutations [19]. Golodirsen (Vyondys 53), approved for sale on December 12, 2019, and 
Viltolarsen, approved for sale in Japan and the United States in March and August 2020, 
respectively, are used to treat approximately 8% of DMD patients with exon 53 mutations 
[20][21]. Casimersen (Amondys 45), approved for marketing on February 25, 2021, is used to 
treat approximately 8% of DMD patients with exon 45 mutations [22]. These four drugs are all 
ASO that modify the splicing of dystrophin mRNA to translate into truncated, partially 
functional dystrophin, transforming more severe DMD phenotype into lighter BMD phenotype 
[23][24]. These types of drugs have a low affinity for proteins, so they will be quickly cleared 
in the body after systemic injection. The biggest challenge for this type of drug currently is the 
difficulty of entering the cell, therefore, it is necessary to administer larger drug doses or 
increase the frequency of administration to improve bioavailability. All four drugs require 
regular intravenous injection [25]. More importantly, these products are conditionally marketed 
and have not yet completed the required confirmatory clinical studies, so their clinical benefits 
are not fully understood.  
On June 22, 2023, the FDA accelerated the approval of SRP-9001 (trade name: Elevidys) of 
Sarepta Therapeutics for the treatment of DMD patients aged 4-5. SRP-9001 (AAV loading 
micro-dystrophin) has also become the world's first disposable gene therapy for DMD. This 
drug uses the AAVrh74 vector. The gene loading capacity of this type of vector is limited, and 

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it cannot load entire dystrophin gene. Therefore, the main functional regions of dystrophin gene 
can only be selected for Elevidys design based on existing studies. After administration of 
Elevidys, the final product generated in the body, the micro-dystrophin (138 kDa), only 
contains the selected domain of the normal wild-type dystrophin (427 kDa). The micro-
dystrophin lacks the domain that can bind to neuronal nitric oxide synthase and α- syntrophic 
factors, which of them can protect muscle cells by synergistically regulating blood flow. So 
this gene therapy method can only alleviate the symptoms of DMD patients. Although in theory, 
the function of the micro-dystrophin may be similar to that of normal dystrophin, but it is a 
novel protein that does not exist in nature. It is different from both the normal wild-type and 
the endogenous shortened form of dystrophin in BMD patients, as well as the internally 
truncated dystrophin produced through exon skipping drug therapy expression. The 
relationship between micro-dystrophin and various shortened forms of dystrophin is still 
unclear, and whether its actual performance is close enough to that of natural dystrophin 
remains questionable. In addition, there is no clear answer to how high the expression level 
of micro-dystrophin can benefit patients clinically. Meanwhile, due to the lack of 
epidemiological data on this protein, it is not clear how it affects the pathophysiology of 
DMD patients [26]. 
The use of AAVrh74 vector also limits the clinically treatable population, requiring patients 
with anti-AAVrh74 antibody titers<1:400 to be selected. The existing clinical studies of 
Elevidys have shown that after large-scale administration, the titer of anti-AAVrh74 antibody 
in patients significantly increases. Therefore, even if the patient's initial dose is insufficient 
or there is no therapeutic efficacy after treatment, they cannot accept additional doses of the 
same drug for re-administration. And due to the possibility of immune cross reactivity with 
other AAV subtypes, DMD patients who have not benefited from Elevidys treatment may 
not be able to accept gene therapy based on other serotype AAV vectors in the future [26]. 
In addition, multiple experts have raised doubts about the structure, animal study, and clinical 
trial results of Sarepta's micro-dystrophin. They questioned whether there is a significant 
correlation between the micro-dystrophin level and muscle performance in DMD patients. 
Although the FDA has approved the product for market through accelerated approval, it 
requires further post market study to be conducted [26]. 
At present, there are no approved DMD gene therapy in China. From the above description, 
although gene therapy brings hope for the clinical treatment of DMD patients, the gene 
therapy products currently on the market have different shortcomings. Existing clinical 
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Therefore, R&D personnel still need to make efforts, as well as a large amount of funding to 
develop more promising and effective gene therapy products for the clinical treatment of 
DMD patients. 
1.5. Global ongoing study on DMD treatment drugs 
According to the Clinicaltrials.gov [18], there are a total of 381 clinical trials for the 
treatment of DMD (2023/8/29). The clinical trial information of new drugs that are currently 
ongoing or in recruitment status is detailed in Table 3. Whether from the approved DMD 
drugs or the current number of ongoing new drugs, the industry's enthusiasm for DMD drug 
development is gradually increasing. 
Table 3 Global information of the ongoing new drugs for DMD 
Drug name 
R&D company 
Treatment 
strategies 
Phase 
ID 
CAP-1002 
Capricor Inc 
Cell therapy 
Ⅲ 
NCT05126758 
CRD-TMH-001 
Cure Rare Disease 
Gene editing 
Ⅰ 
NCT05514249 
PF-06939926 
Pfizer 
Gene 
replacement 
therapy 
Ⅲ 
NCT04281485 
SGT-001 
Solid Biosciences 
Ⅰ/Ⅱ 
NCT03368742 
GNT 0004 
Genethon-Sarepta 
Ⅲ 
/ 
RGX-202 
REGENXBIO  
Ⅱ 
NCT05693142 
AOC 1044 
Avidity Biosciences 
Exon 
skipping 
therapy 
Ⅱ 
NCT05670730 
WVE-N531 
Wave Life Sciences 
Ⅱ 
NCT04906460 
DYNE-251 
Dyne Therapeutics 
Ⅱ 
NCT05524883 
SQY51 
Sqy Therapeutics 
Ⅱ 
NCT05753462 
Brogidirsen 
NS Pharma 
Ⅱ 
NCT05996003 
ENTR-601-44 
Entrada Therapeutics 
Ⅱ 
/ 
PGN-EDO51 
PepGen 
Ⅱ 
NCT06079736 
Renadirsen 
Daiichi Sankyo 
Ⅱ 
NCT04433234 
Vesleteplirsen 
Sarepta Therapeutics 
Ⅱ 
NCT04004065 
TAS-205 
Taiho 
Pharmaceutical 
Co., Ltd. 
Chemical 
drug 
Ⅲ 
NCT04587908 
EDG-5506 
Edgewise 
Ⅲ 
NCT05540860 
Givinostat 
Italfarmaco Group 
NDA  
NCT01761292 
Edasalonexent 
Catabasis 
Ⅲ 
NCT03703882 
Ezutromid 
Summit Therapeutics 
Ⅱ 
NCT02858362 
Pamrevlumab 
FibroGen 
Monoclonal 
antibody 
Ⅲ（Failed） 
NCT04632940 
2. SPOT-mRNA03 Introduction 
2.1. SPOT-mRNA03 design 
SPOT-mRNA03 is a gene therapy product for DMD, using the patented technology of Spot 
Biotech - Cellular nanoporation (CNP) (which can rearrange cell membrane molecules and 
improve membrane permeability through high-intensity electric field, thereby constructing 
a channel for introducing exogenous molecules into cells [27]) for transfecting human 

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dermal fibroblasts (HDFs), introducing target mRNA molecules and molecular targeted 
peptides (which can be co-expressed with transmembrane proteins of EVs). Compared with 
the traditional transfection technology, this technology can significantly improve the 
transfection efficiency, promote the cells to load about 14kb of full-length dystrophin 
mRNA molecules encoding dystrophin with high flux, and simultaneously generate a large 
number of EVs loaded with target dystrophin mRNA. After transfection, collect the 
supernatant containing the target product, purify and concentrate it, add phosphate buffer, 
cell cryopreservation solution CS10, and human serum albumin to prepare SPOT-mRNA03 
injection. 
2.2. Mechanism of Action of SPOT-mRNA03 
SPOT-mRNA03 is an EVs loaded full-length dystrophin mRNA molecule, and the EVs 
membrane surface expresses a molecular targeted peptide, which can be used as a gene 
therapy product for DMD. In order to improve the targeting ability of SPOT-mRNA03 into 
cells, the molecular targeting peptide was co-transfected with the transmembrane protein of 
EVs, thus introducing the molecular targeting peptide onto the EVs membrane. After 
intravenous injection of SPOT-mRNA03 into DMD patients, the targeting ability of SPOT-
mRNA03, endowed by molecular targeting peptides on the EVs membrane, can guide SPOT-
mRNA03 into the patient's muscle cells, allowing dystrophin deficient receptor cells to utilize 
intracellular ribosomes and amino acids to translate and secrete endogenous full-length intact 
dystrophin. The generated dystrophin can tightly bind with various proteins in the inner, 
transmembrane, and outer regions of the cell membrane, such as sarcoglycan and 
dystroglycan, and are interrelated to form a whole inside and outside the cell membrane. It 
maintains material exchange and connection inside and outside the cell membrane, protects 
the integrity and stability of the cell membrane structure, and thus protects muscles from 
damage and/or promotes muscle repair and regeneration. 
3. Risk and Benefit Assessment 
3.1 Risk Assessment 
The main components of SPOT-mRNA03 are EVs with molecular targeted peptides secreted 
by fibroblasts and full-length dystrophin mRNA molecules loaded on them. After 
administration, SPOT-mRNA03 can target muscle cells and generate dystrophin. The main 
component of SPOT-mRNA03, EVs, can be secreted by all cells in the human body, with low 
immunogenicity and good biocompatibility. It is hard to induce the production of antibodies 
and to cause immunogenicity in the body. EVs of SPOT-mRNA03, integrating corresponding 

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molecular targeted peptides on the lipid layer membrane, possess strong targeting ability, 
which can directly deliver loaded dystrophin mRNA to target cells. The dystrophin mRNA 
sequence loaded on SPOT-mRNA03 is the same as the mRNA molecular sequence encoding 
dystrophin in the human body. In addition, the mRNA molecule can only be delivered to the 
cytoplasm through EVs, without entering the nucleus, so there is no risk of foreign gene 
integration. Therefore, compared to AAV gene therapy products, SPOT-mRNA03 has higher 
safety. 
According to the mechanism of action of SPOT-mRNA03, endogenous full-length intact 
dystrophin can be generated and secreted in dystrophin deficient receptor. Because there are 
not the intact dystrophin proteins in DMD patients, it may be recognized by the immune 
system as an exogenous protein in the patient's body, leading to a more severe immune 
response and potentially inducing further muscle damage, reversing any benefits that the 
patient should have received. There have been reports in other clinical studies of gene therapy 
for DMD [27]. According to reports, independent clinical studies of three different AAV 
products have shown a total of approximately 200 patients, of which 5 experienced severe 
immune reactions. Symptom onset occurred 3 to 6 weeks after administration: all five 
patients had severe weakness of the proximal and distal limb muscles that led to loss of 
ambulation, as well as weakness of the bulbar and respiratory muscles, which led to receipt 
of transient ventilatory support in three of the patients (two with noninvasive ventilation and 
one with endotracheal intubation). In addition, all patients had symptoms of myositis, and 
three patients also experienced symptoms of myocarditis. Therefore, various 
immunomodulatory treatments were used among the trials, including pulse dose 
glucocorticoids, intravenous immunoglobulin, plasmapheresis, and tacrolimus. The above 
clinical symptoms were resolved within 3 months after treatment. To prevent the occurrence 
of immune response, this study orally administered tacrolimus or sirolimus daily for 4 weeks 
starting from D-3 before treatment of the study drug. Since these two drugs are 
immunosuppressants, it is necessary to closely monitor the adverse reactions of medication 
to prevent the occurrence of infections. The detailed adverse reactions of this drug can be 
found in Error! Reference source not found.. 
In addition, SPOT-mRNA03 belongs to a hypertonic solution with high osmotic pressure 
(>600 Osm/L) and may lead to extravasation and phlebitis if via intravenous infusion. 
Therefore, appropriate venous access should be selected for administration, and appropriate 
vascular access device should be used. During the infusion process, administer slowly, 

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carefully observe the patient's infusion site, evaluate the infusion site reaction, and strictly 
prevent adverse events such as extravasation and venous infusion complications. If 
extravasation is observed, stop administration immediately. 
The preclinical sin- and multi- dose toxicity study of SPOT-mRNA03 showed that SPOT-
mRNA03 had no significant systemic adverse reactions in the animal body. At present, there 
are no similar products to SPOT-mRNA03 on the market, and there are also no corresponding 
reports of ongoing clinical trials. There are over 400 registrations or related clinical trials 
based on EVs, most of which focus on the diagnosis, treatment, or prognostic strategies for 
various cancers, cardiovascular diseases, and neurodegenerative diseases. So far, these 
clinical trials have reported that EVs based therapies have no significant toxicity or adverse 
effects, indicating the safety and feasibility of the technology. 
In summary, from the perspective of product design, production, and mechanism of action, 
the most likely adverse reactions caused by SPOT-mRNA03 infusion in DMD patients are 
infusion reaction and immune reaction, which may pose safety risks in the clinical application 
of this product. However, the toxicity studies of SPOT-mRNA03 and other relevant reports 
of gene therapy indicate that the safety risks of SPOT-mRNA03 in clinical application are 
controllable. 
3.2 Benefit Assessment 
SPOT-mRNA03 is the latest gene therapy product for DMD. After administration to DMD 
patients, the molecular targeting peptide on the EVs membrane can guide SPOT-mRNA03 
to enter the patient's muscle cells, allowing dystrophin deficient receptor cells to utilize 
intracellular ribosomes and amino acids to translate and secrete endogenous full-length intact 
dystrophin. Dystrophin is a cytoskeletal protein that has an anti-mechanical stretching effect 
and can prevent damage to muscle cells during contraction. The generated dystrophin can 
tightly bind with various proteins in the inner, transmembrane, and outer regions of the cell 
membrane, such as sarcoglycan and dystroglycan, and are interrelated to form a whole inside 
and outside the cell membrane. It maintains material exchange and connection inside and 
outside the cell membrane, protects the integrity and stability of the cell membrane structure, 
and thus protects muscles from damage and/or promotes muscle repair and regeneration. 
Therefore, SPOT-mRNA03 to DMD patients can theoretically alleviate the clinical 
symptoms and improve the quality of life of DMD patients. 
Since SPOT-mRNA03 is completely different from existing gene therapy products on the 
market, it is the first to achieve in vivo targeted delivery of full-length dystrophin mRNA 

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molecules, which can generate full-length and complete dystrophin in the body. Therefore, 
SPOT-mRNA03 based on dystrophin mRNA loaded on EVs can provide a new and effective 
protein replacement therapy for DMD patients, making clinical cure of DMD possible. 
4. Study Objectives and Endpoints 
Table 4 The objectives and endpoints of this study 
Objectives 
Endpoints 
Primary objective: 
Primary endpoints: 
• To evaluate the safety and 
tolerability 
of 
SPOT-
mRNA03 administered by 
intravenous infusion (IV) 
to DMD patients. 
• Collection and quantification of adverse events 
(description of event, time of onset and resolution, 
assessment of severity and causal relationship to 
the investigational drug), as well as the data of 
physical examination, vital signs, 12-lead ECG, 
clinical laboratory test results (hematology, 
biochemistry, urinalysis) in this study. 
Secondary objectives: 
Secondary endpoints: 
• To evaluate the changes 
of dystrophin mRNA in 
serum and muscles of 
DMD patients after IV 
infusion 
of 
SPOT-
mRNA03. 
• Changes of dystrophin mRNA concentration in 
serum before and different timepoints after the 
initial administration (see Table 5 for schedule) as 
well as before and after the 2nd, 4th, 6th, and 8th 
administration and during follow-up. And the 
changes in dystrophin mRNA concentration in 
muscles at baseline and D29 after the completion 
of the last dose (if the biopsy time is between 12-
24 hours after the last dose) (by qPCR). 
• To evaluate the changes 
in dystrophin expression 
and 
engraftment 
in 
muscles of DMD patients 
after IV infusion of 
SPOT-mRNA03. 
• Changes in the expression and engraftment level 
of dystrophin in muscle biopsies at baseline and 
D29 (after the completion of the last dose) (by 
Western Blot and IHC methods). 
• To evaluate the changes 
in serum anti-dystrophin 
antibodies and cytokines 
in DMD patients after IV 
infusion 
of 
SPOT-
mRNA03. 
• Changes in anti-dystrophin antibodies and 
cytokines in the serum at baseline and before the 
2nd, 4th, 6th, and 8th administration and during 
follow-up (see Table 5 for schedule). 
5. Study Design 
5.1. Overall Design 
This is a FIH, open-label, single-arm, and single-center exploratory clinical study of SPOT-
mRNA03 administered via IV infusion for DMD patients. The primary objective of this study 
is to evaluate the safety and tolerability of SPOT-mRNA03 for DMD patients. And the 

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secondary objectives are to preliminarily investigate the concentration change of dystrophin 
mRNA, the expression and engraftment level changes of dystrophin protein, as well as 
changes in cytokines and immunogenicity. The study has two ascending dose cohorts: 
The study has two ascending dose cohorts: 
Ⅰ cohort：5.0 × 109 CN Dystrophin mRNA / kg 
Ⅱ cohort：5.0 × 1010 CN Dystrophin mRNA / kg 
The study will have a screening period of 30 days, during which patients or their legal 
guardian written informed consent will be obtained before screening assessments and 
eligibility will be determined. A total of 6 DMD patients will participate in the study. there 
are 3 patients in each dose cohort [No previous treatment with corticosteroids]. All subjects 
started taking 0.05-0.1mg/kg/d (adjusted according to the actual clinical situation)  tacrolimus 
or sirolimus orally at D-3 (3 days before initial dose of SPOT-mRNA03) for 4 weeks. All 
subjects are first administered via intravenous infusion on D1, and then administered twice a 
week (once every 4 days) for a total of 8 doses. Four weeks after the initial administration of 
the subjects in the previous dose cohort, if there are no serious adverse events related to the 
treatment, it will be determined that the subjects in next dose cohort could be administered 
after discussion between the investigators and the sponsor. 
Safety evaluations on subjects are conducted during each administration and follow-up. 
Muscle biopsies are performed at baseline and D29 after administration (see Table 5 for 
details). Samples for muscle biopsy will be taken from gastrocnemius/biceps brachii muscle 
at baseline and from the gastrocnemius and biceps brachii muscles on D29. Western blot (WB) 
is 
used 
to 
detect 
the 
expression 
and 
engraftment 
of 
dystrophin 
protein. 
Immunohistochemistry (IHC) is used to detect fiber intensity and percentage of dystrophin 
positive fibers, and qPCR is used to detect changes in dystrophin mRNA at baseline and D29 
(if the biopsy time is between 12~24 hours after the last dose). 
The blood samples are collected for detection of dystrophin mRNA, cytokines and 
immunogenicity (see Table 5 for details) besides for laboratory testing. Perform MRI and 
EMG examinations at baseline and at week 16 after administration. 
5.2. Duration of Study 
The screening period is 0-30 days, with 4 weeks of treatment period and a follow-up period 
of 6 months.  
5.3. Termination Criteria 
1) According to CTCAE v5.0 criteria, during the dose escalation process, more than 

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1/2 of the subjects have grade 2 or above liver, kidney, cardiac and hematological 
TRAEs, or more than 1/3 of the subjects have other system AEs of grade 3 or above 
occur, the trial should be terminated.  
2) The collaborator requests to terminate the study due to financial or management 
reasons, etc. 
3) Termination of the trial is requested by IRB/EC. 
5.4. Schedule of Events  

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Table 5 Schedule of Events 
Study Period 
Screeni
ng 
Baseli
ne 
Treatment 
Follow-up 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
Visit Timea  
D-
30~D-8 
D-
7~D-1 
D1 
D5 
D9 
D13 
D17 
D21 
D25 
D29 
W8 
W12 
W16 
W20 
W24 
W28 
Visit Window 
(Days) 
 
 
 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±3 
±3 
±5 
 
 
 
Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical 
Historyb 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital Signsc 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical 
Examinationd 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
ECHOe  
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
12-lead ECG f 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
PFTg 
X 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Chest X-Ray 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Haematologyh 
X 
X 
 
X  
 
X  
 
X 
 
X 
 
 
X 
 
 
X 
Biochemistryi 
X 
X 
 
X  
 
X  
 
X 
 
X 
 
 
X 
 
 
X 
Urinalysisj 
X 
X 
 
X  
 
X  
 
X 
 
X 
 
 
X 
 
 
X 
Hepatitis B & 
C, HIVk 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Coagulationl 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SPOT-
mRNA03 
Administration 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
EMG 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
MRIm 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
PKn 
 
 
X 
X 
 
X 
 
X 
 
X 
X 
X 
X 
 
 
 
Muscle 
Biopsyo 
 
X 
 
 
 
 
 
 
 
X  
 
 
 
 
 
 
Photographyp 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 

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Study Period 
Screeni
ng 
Baseli
ne 
Treatment 
Follow-up 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
Visit Timea  
D-
30~D-8 
D-
7~D-1 
D1 
D5 
D9 
D13 
D17 
D21 
D25 
D29 
W8 
W12 
W16 
W20 
W24 
W28 
Visit Window 
(Days) 
 
 
 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±3 
±3 
±5 
 
 
 
BiomarkerTesti
ngq 
 
 
X 
X 
 
X 
 
X 
 
X 
X 
X 
X 
 
 
 
Immunogenicit
yr 
 
X 
X 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
NSAAs 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
Administration 
of 
tacrolimus/sirol
imus 
 
Starting from D-3, take 0.05-0.1mg/kg tacrolimus/sirolimus orally daily for 4 
weeks 
 
 
 
 
 
 
Adverse Events 
Continuous monitoring records from initial administration to last visit 
Concomitant 
Medications 
Continuous recording from signing informed consent to the last follow-up 
ABBREVIATIONS: D = study day; W = study week; ECG = electrocardiogram; ECHO = echocardiogram; PFT = pulmonary function test; EMG = electromyography; 
MRI = magnetic resonance imaging; EOS = end of study; AE = adverse events. Unscheduled tests may be performed at the discretion of the Investigator if clinically 
indicated and required for safety. 
a) Study drug will be started administration on Day 1 (t = 0). Day 1 is defined as the day of first administration of study drug. The time of the study drug first 
administration is defined as t = 0. Baseline will be the last-performed assessment before the first administration of study drug. There is no Day 0; the day before 
Day 1 is Day -1. 
b) A full medical history will be obtained from subjects to determine eligibility. If AEs to be recorded from time of dosing rather than from signing consent to be 
determined Any medical events occurring in the Screening period until first administration of study drug will be recorded as medical history. Current medical 
history related to DMD (gene detection results and corresponding treatments information), as well as other treatment history within 6 months will be collected. 
c) Vital signs include blood pressure, pulse, respiration, and body temperature. During the treatment period, vital sign test will be performed within 1 hour before 
each administration. In addition, vital sign testing will also be conducted 30 minutes (± 10 minutes), 2 hours, and 4 hours (± 30 minutes) after the initial 
administration. 
d) Physical examination includes height, weight, skin and mucous membranes, lymph nodes, head and neck, chest, abdomen, spine, limbs and joints, 
musculoskeletal system, and nervous system. It will be performed before administration in each cycle. Calculate the individual dosage based on the baseline 
body weight and height values. Only the height and weight values measured during the screening period are used to calculate BMI and determine whether 
participants can be enrolled. BMI = weight (kg)/height 2 (m2). 

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e) ECHO will be performed during screening and EOS. 
f) 
12-lead ECG testing is required before and 2 hours (± 1 hour) after D1 administration, and others are before administration. When the ECG measurement results 
are abnormal (including QTcF interval abnormalities, such as QTcF>500 ms or relative baseline increase>60 ms) or other abnormalities have clinical significance, 
an additional 2 ECG measurements are required, with a minimum interval of about 2 minutes between repeated measurements. 
g) Pulmonary function tests include forced vital capacity (FVC), maximum inspiratory pressure (MIP), and maximum expiratory pressure (MEP). It will be 
performed during screening, baseline, and at W16 visits).。 
h) Haematology includes white blood cell count (WBC), neutrophil count (NEUT #), eosinophil count (EO #), basophil count (BASO #), lymphocyte count 
(LYMPH #), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), and hematocrit (HCT) (the test results within 7 days before administration 
can be accepted). 
i) 
Biochemistry includes bilirubin (total, indirect and direct), total protein, albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), 
and gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), creatinine (Cr), blood urea (Urea), blood glucose (GLU), serum sodium (Na+), serum 
potassium (K+), serum calcium (Ca2+), serum magnesium (Mg2+), serum chloride (CL -), lactate dehydrogenase (LDH), serum creatine kinase (CK), serum 
creatine kinase MB isoenzyme (CKMB) and c-reactive protein (CRP). Calculation of eGFR [CKD-EPI]. (the test results within 7 days before administration can 
be accepted).  
j) 
Urinalysis includes Urinary bilirubin (UBG), urinary bilirubin (BIL), urinary ketone body (KET), urinary occult blood (BLD), urinary protein (PRO), urinary 
nitrite (NIT), urinary glucose (GLUU), urinary specific gravity (SG), urinary pH (pH), red blood cells (RBC), and white blood cells (WBC). (the test results 
within 7 days before administration can be accepted). 
k) Hepatitis B & C and HIV serology tests at Screening will include hepatitis B surface antigen (HBs Ag), anti-hepatitis B surface antibodies (anti-HBs Ab) anti-
hepatitis B core antibodies (anti-HBc Ab), hepatitis B e antigen (HBe Ag), anti-hepatitis B e antibodies (anti-HBe Ab), hepatitis C antibodies, anti-HIV1 and 
anti-HIV2 antibodies. Any positive result is exclusionary. (the test results within 14 days before screening can be accepted). 
l) 
Coagulation includes prothrombin time (PT), activated partial thromboplastin time (APTT) and international normalized ratio (INR). The examination results of 
coagulation within 14 days before screening can be accepted. 
m) Only conscious sedation will be used during the cardiac MRI. If general anesthesia is required, the cardiac MRI will not be performed. 
n) The blood sample for the PK test will be collected before and 0h+10min after the 1st, 2nd, 4th, 6th, and 8th administration. Intensive blood collection will be 
performed before and 1h ± 10min, 2h ± 20min, 8h ± 1h, 24h ± 1h, 48h ± 2h, and 72h ± 3h after the initial administration. PK blood samples will also be collected 
at each follow-up. If dystrophin mRNA cannot be detected at W8 follow-up, blood samples may not be collected for this test in subsequent follow-up. 
o) Three subjects from each cohort will undergo muscle biopsy at baseline and on D29 (after administration). Perform biopsy sampling from the 
gastrocnemius/biceps brachii muscle at baseline and from the gastrocnemius and biceps brachii muscles on D29 (after the last dose). 
p) Photography: Take photos of the biopsy site before and after each biopsy. Take photos of the infusion site approximately 1 hour after each administration and 
observe the reaction at the infusion site. 
q) Cytokine test: TNF-α, INF-γ, IL-2, IL-6, and IL-10. Collect blood samples before the 1st, 2nd, 4th, 6th, and 8th administration. If the cytokine levels return to 
normal at W8 follow-up, blood samples may not be collected for this test during subsequent follow-up. 

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r) 
Immunogenicity: anti-dystrophin antibody test. Collect blood samples at baseline/before the initial dose, before the 2nd, 4th, 6th, and 8th dose (if no more than 
7 days from baseline to the initial dose, no additional collection is required before the initial dose) and during each follow-up. If the anti-dystrophin antibody 
cannot be detected in the follow-up samples at week 8, blood samples may not be collected for this test during subsequent follow-up. 
s) 
NSAA：North Star Ambulatory Assessment, conducted at baseline, W16, and W28 to assess differences in ambulatory function before and after dosing. The 
NSAA examination includes: standing, walking, rising from a chair, stepping onto a raised surface, jumping, running, standing on one leg, heel walking, 
descending stairs, ascending stairs, lifting a leg, and hopping on one leg.

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6. Study Population 
6.1 Inclusion criteria 
Only those who meet all the following criteria can be enrolled: 
1) According to the requirements of the region/country and/or IRB/IEC, the patient 
and/or legal guardian have signed a written informed consent form and are aware of 
all relevant study content. 
2) Ambulatory boys aged 2 to 6 years, inclusive. 
3) Confirmed diagnosis of DMD through multiplex ligation-dependent probe 
amplification (MLPA) and whole-exome sequencing. 
4) Tolerance for muscle biopsy under anesthesia with no absolute contraindications to 
the procedure 
5) Heart, liver, lung, and kidney functions are sufficient: 
a) The left ventricular ejection fraction (LVEF) should be ≥ 50%; 
b) Forced vital capacity (FVC) ＞ 50% of the expected value, and do not require 
nighttime ventilation; 
c) Patient's glomerular filtration rate (GFR)>30 mL/min/1.73 m2 
6.2 Exclusion Criteria 
Those who meet any one of the following criteria must be excluded: 
1) Complications other than DMD that may cause muscle weakness and/or motor 
dysfunction. 
2) There are severe intellectual disabilities (such as severe autism, severe cognitive 
impairment, and severe behavioral disorders) that, according to the investigator's 
judgment, can affect the study. 
3) Hospitalization for respiratory failure within 8 weeks prior to screening. 
4) Asthma or underlying lung diseases that are poorly controlled, such as bronchitis, 
bronchiectasis, emphysema, or recurrent infectious pneumonia that investigator 
believes may affect respiratory function. 
5) Severe uncontrolled heart failure (NYHA III-IV), including any of the following 
conditions： 

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a) Intravenous administration of diuretics or positive inotropic drugs is required 
within 8 weeks prior to screening. 
b) Hospitalization due to worsening heart failure or arrhythmia within 8 weeks prior 
to screening. 
6) Abnormal laboratory values considered clinically significant: 
a) GGT > 3 × upper limit of normal 
b) Bilirubin ≥ 3.0 mg/dL 
c) Creatinine ≥ 1.8 mg/dL 
d) Hemoglobin < 8 or > 18 g/dL 
e) White blood cell count > 18,500/μL 
7) There are arrhythmias that require antiarrhythmic treatment. 
8) Subjects who are undergoing immunosuppressive therapy. 
9) Has used other gene therapy, investigational drugs, or any treatment aimed at 
increasing dystrophin expression. 
10) Subjects with a history of major surgeries within 12 weeks prior to the initial infusion 
or planning to undergo major surgeries (such as scoliosis surgery) during this study. 
11) Subjects who are allergic to investigational products or local anesthetic drugs or have 
a history of severe allergies or genetic allergic reactions. 
12) Within 6 months prior to the initial infusion, the subjects are exposed to another 
investigational drug or are participating in an intervention clinical trial. 
13) Subjects with positive of hepatitis B core antibody or hepatitis C antibody or HIV 
antibody during screening. 
14) Investigator believes that the presence of any other serious diseases, medical 
conditions, or chronic drug treatment needs can pose unnecessary risks to gene 
transfer. 
7. Subject Coding and Grouping 
This study is an open-label, single-arm study. 
There are two ascending dose cohorts in this study: 
Ⅰ cohort：5.0 × 109 CN Dystrophin mRNA / kg 
Ⅱ cohort：5.0 × 1010 CN Dystrophin mRNA / kg 

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Four weeks after the initial administration of the subjects in the previous dose cohort, if 
there are no serious adverse events related to the treatment, it will be determined that the 
subjects in next dose cohort could be administered after discussion between the 
investigators and the sponsor. 
This study is a first-in-human, open-label study of SPOT-mRNA03, with only 6 subjects, 
so the subjects will be coded, grouped, and dosed only based on the time of participant 
screening and enrollment. Once the subjects sign the informed consent form, they will 
receive a unique screening number 01, 02, 03 ┉ in order, and so on. After enrollment, 
obtain the unique enrollment numbers AZ01, AZ02, AZ03 ┉ in order, and so on. 
8. Study Procedures 
8.1. Treatment and Follow-up  
Those subjects who are eligibility will receive an enrollment number and participate in 
subsequent clinical items. The detailed items that subjects need to participate in at each 
visit are as follows. 
8.1.1. Screening (D-30  ~ -8) 
1) Sign informed consent form: explain the study process to the subjects or legal 
guardian and obtain a written informed consent form from the subjects. 
2) Assign screening numbers in the order of obtaining written informed consent. 
3) Demographics: gender, age, race, height, weight. 
4) Disease history：medical history and surgical history within the 6 months prior 
to screening. 
5) Medical history：drug allergy history and medication history within the 6 months 
prior to screening. 
6) Physical examination: height, weight, skin and mucous membranes, lymph nodes, 
head and neck, chest, abdomen, spine, limbs and joints, musculoskeletal system, 
and nervous system. 
7) Vital signs: blood pressure, pulse, respiration, and body temperature. 
8) ECHO and 12-lead ECG：the ECHO and ECG records will be checked by an 
investigator to determine if the patient meets the inclusion and exclusion criteria. 
9) Pulmonary function tests: forced vital capacity (FVC), maximum inspiratory 
pressure (MIP), and maximum expiratory pressure (MEP). 
10) Chest X-Ray. 

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11) Lab tests: the items include Haematology, Biochemistry, Urinalysis, Coagulation, 
and Hepatitis B & C, HIV examination. 
12) Collect concomitant medication information. 
Determine the eligibility of subjects based on the test results during the screening period. 
Eligible subjects will participate in the baseline examinations listed below. 
8.1.2. Baseline (D-7 ~ -1) 
1) Vital signs and physical examination 
2) 12-lead ECG 
3) PFT 
4) Lab tests: Haematology, Biochemistry and Urinalysis (if the screening period test 
is conducted within 7 days before administration, this test may not be performed 
here). 
5) Starting from D-3, take 1mg/kg tacrolimus/sirolimus orally daily. 
6) EMG 
7) MRI  
8) Sampling for muscle biopsy is performed from the gastrocnemius/biceps brachii 
muscle, and the biopsy site is photographed before and after biopsy. 
9) Collect blood samples required for immunogenicity testing.  
10) Collect concomitant medication information. 
11) Collect adverse events information (due to the use of tacrolimus/sirolimus, collect 
AEs from D-3). 
12) NSAA: To evaluate subjects' motor function and ambulatory capacity. It’s 
included 12 functional activities: standing, walking, rising from a chair, stepping 
onto a raised surface, jumping, running, standing on one leg, heel walking, 
descending stairs, ascending stairs, active straight leg raise, and hopping on one 
leg. 
8.1.3. Treatment (D1 ~ 29) 
The subjects participating in the baseline will receive treatments of SPOT-mRNA03 via 
IV on D1. 
Before the initial administration on D1 
1) Vital signs and physical examination. 

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2) 12-lead ECG  
3) Collect blood samples for PK and Biomarker tests, if blood samples for 
immunogenicity testing were not collected during the baseline, they should also 
be collected at this time. 
4) Take 0.05-0.1mg/kg tacrolimus/sirolimus orally daily. 
5) Collect concomitant medication information. 
6) Collect adverse events information. 
Calculate the dosage of SPOT-mRNA03 based on the concentration of SPOT-mRNA03, 
subject weight, and dose cohort participating in the study.  
After the initial administration on D1 
1) Take photos of the infusion site and observe the infusion response within 4 hours. 
2) Vital signs: testing will be conducted 30 min (± 10 min), 1 h (± 10 min), 2 h (± 
30 min), and 4 h (± 30 min) after the initial infusion. 
3) The blood samples required for PK testing will be collected densely at timepoints 
of 0h +10min, 1h ± 10min, 2h ± 20min, 8h ± 1h, 24h ± 1h, 48h ± 2h, 72h ± 3h 
after the initial infusion, respectively. 
4) 12-lead ECG needs to be tested 2 hours (± 1 hour) after the end of infusion. 
5) Collect adverse events information. 
Other treatment cycles 
1) Vital signs: vital signs should be tested within 1 hour before and after each 
administration. 
2) Physical examination should be performed prior to each administration. 
3) 12-lead ECG should be performed prior to each administration. 
4) Lab tests: perform before the 2nd, 4th, 6th, and 8th administration, respectively, 
including Haematology, Biochemistry and Urinalysis. 
5) Take photos of the infusion site, within 4 hours after each administration, closely 
observe whether the subjects have any infusion reactions. 
6) Take 1mg/kg tacrolimus/sirolimus orally daily for 4 weeks. 
7) Samples for muscle biopsy will be taken from the gastrocnemius muscle and 
biceps brachii muscle, and the sampling site will be photographed before and after 
sampling.  
8) Blood samples for PK, Biomarker and Immunogenicity testing will be collected 

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before the 2nd, 4th, 6th, and 8th administration, respectively. And blood samples 
for PK test will also be collected after 0h + 10min of the 2nd, 4th, 6th, and 8th 
administration. 
9) Collect concomitant medication information. 
10) Collect adverse events information. 
Inform the subjects and guardians of the specific time for the next follow-up at the end of 
each follow-up. 
8.1.4. Follow-up 
The subjects will be followed up for various tests and sample collection at weeks 8, 12,  
16, 20, 24 and 28 after the initial administration, as follows: 
1) Perform vital signs, physical examination, and 12-lead ECG at each follow-up. 
2) Blood sample collection for PK, biomarkers, and immunogenicity testing (if any 
one of dystrophin mRNA, cytokine, or anti-dystrophin antibody cannot be 
detected in the blood sample of W8 or can be detected but returns to normal, 
further blood sample collection for this test is not necessary in subsequent follow-
up). 
3) Lab tests (Haematology, Biochemistry and Urinalysis) and NSAA at W16 and 
W28 
4)  ECHO, EMG, MRI, and PFT tests will be conducted at W16. 
5) Collect concomitant medication information. 
6) Collect adverse events information. 
The study will be ended after completing follow-up at W28. 
8.2. Supply of Investigational drugs/treatments 
8.2.1. Name, Source and Strength 
【Generic Name】Dystrophin mRNA Injection 
【Product code】SPOT-mRNA03 
【Strength】****E10 Dystrophin mRNA / 10mL / vial 
【Expiry Date】3 month 
【Active Pharmaceutical Ingredients】Dystrophin mRNA-loaded EVs 
【Pharmaceutical excipients】CryoStorCS10 frozen storage solution (containing 10% 
dimethyl sulfoxide (DMSO)), phosphate-buffered saline (PBS), human serum 

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albumin (HSA) 
【Storage】-60℃~ -90℃ 
8.2.2. Preparation and Storage 
SPOT-mRNA03 needs to be transported on dry ice and stored at -60 ℃ to -90 ℃. It is 
expected to be stored for 3 months.  
8.2.3. Route of Administration and Dosage Adjustment 
Route of Administration 
Intravenous infusion: twice a week (once every 4 days). Each subject will receive a total 
of 8 treatments. 
Each bag of SPOT-mRNA03 needs to slowly thaw at 4 ℃/ice before use. If there are no 
special requirements, SPOT-mRNA03 after fusion should be used as soon as possible. 
Dosage Adjustment 
Calculate the dosage of SPOT-mRNA03 based on the concentration of SPOT-mRNA03, 
subject weight, and dose cohort participating in the study. The total volume of infusion is 
40 mL.  
Choose an appropriate intravenous infusion site based on the subject's personal and venous 
condition, with an infusion time of 60~120 minutes. If the subject experiences mild allergic 
reactions such as dizziness, chills, rash or extravasation during the infusion process, the 
infusion should be immediately suspended. After recovery, the investigator will determine 
whether to continue the administration. The infusion speed can be appropriately reduced 
(as determined by the investigator), and the infusion time can be correspondingly extended 
(as determined by the investigator). After completion of infusion, record the actual dosage 
and start and end time of infusion. 
Photograph 
Take photos for the infusion site before and after each infusion, and carefully observe the 
infusion reaction. 
8.3. Concomitant medication 
The newly generated dystrophin will be recognized by the immune system of the subjects 
as an exogenous protein, triggering an immune response. Any immune response to 
Dystrophin can affect its engraftment and potentially induce further muscle damage, 

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reversing any benefits that patients should have received. Therefore, the subjects should 
begin to orally take 0.05-0.1 mg/kg tacrolimus or sirolimus daily for 4 weeks at D-3 before 
SPOT-mRNA03 administration. 
Tacrolimus/Sirolimus are both potent immunosuppressants, and there is a possibility of 
clinical side effects after use, which require experienced clinical physicians to handle. 
Along with its needed effects, tacrolimus may cause some unwanted effects. Although not 
all of these side effects may occur, if they do occur, they may need medical attention. Check 
with your doctor immediately if any of the following side effects occur while taking 
tacrolimus/sirolimus. 
1) Cardiovascular system symptoms: including hypertension, angina, palpitations, 
heart failure, etc. 
2) Sensory system symptoms: Some patients have tremors, headaches, insomnia, 
depression, hallucinations, forgetfulness, etc. 
3) Digestive system symptoms: including constipation, diarrhea, nausea, vomiting, 
weight changes, difficulty swallowing, stomach pain, etc. 
4) Respiratory symptoms: Some patients have asthma, respiratory failure, lung 
function damage, pulmonary fibrosis, etc. 
5) Skin symptoms: may cause itching, sweating, rash, hirsutism, etc. 
6) Symptoms 
of 
blood 
and 
lymphatic 
system: 
leukopenia, 
anemia, 
thrombocytopenia, bone marrow suppression, etc. Daily manifestations can 
include sudden high fever, pale complexion, mental fatigue, limb weakness, 
memory loss, bleeding, etc. 
7) Renal dysfunction: renal failure, tubular necrosis, oliguria, etc. Absence of urine 
and uremia are rare. 
8) Other: blurred vision, photophobia, tinnitus, edema, local pain, urinary and fecal 
incontinence, thyroid abnormalities, etc. 
8.4. Patient Compliance and Withdrawal 
8.4.1. Screening Failure 
Screening failure is defined as the subject who signed the informed consent form not being 
eligibility for the study according to the results of various examination during screening 

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period. Subjects who failed the screening should not participate in this trial, and the reasons 
for the screening failure must be recorded in the original records. 
8.4.2. Withdrawal Criteria 
1) Considering the safety, the investigator believes that withdrawing from the study is in 
the best interest of the subjects. 
2) Subjects with poor compliance cannot insist on completing the trial as planned or have 
other circumstances that may affect the judgment of the research results. 
3) The subject voluntarily requested to withdraw from the trial and withdraw the 
informed consent. 
8.4.3. Management of Subjects During the Study 
Participants in this study should follow the process arrangement of the study. At each visit, 
participants should be arranged to participate in relevant study items as required. Inform 
the subjects and guardians of the specific time and the testing items to be implemented for 
the next follow-up at the end of each follow-up. At each visit, the study logs of the subjects 
should be checked. 
Participants can withdraw from this study at any time during the trial process. investigators 
should understand the reasons for subjects withdrawing from the trial and whether any 
adverse events have occurred. If any adverse events occur, follow-up should be conducted. 
Participants who withdraw early should complete the corresponding experimental 
procedures (including physical examinations, vital signs, ECG, and laboratory tests) as 
much as possible according to the protocol requirements. 
Subjects who withdrew from the study due to adverse events or abnormal safety 
evaluations with clinical significance should be followed up by the investigators until the 
adverse events are resolved or stabilized until the investigators deems follow-up 
unnecessary or lost. 
The date and reason for early withdrawal from the study must be recorded in the 
corresponding case report form (CRF). Adverse events, concomitant medications, tracking 
of abnormal laboratory test results, and the end page of CRF must be completed as much 
as possible. 

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9. Study Assessment  
9.1 PK Assessment 
9.1.1 Assessment Indicators 
Application of qPCR method to detect changes in dystrophin mRNA concentration in the 
serum of subjects. Intensive collection of blood samples after the initial administration for 
detecting single-dose PK. Subsequent blood collection is used to detect steady-state PK 
after multiple administration. 
PK indicators: Cmax (peak concentration), Tmax (peak time), C trough, ss (steady-state 
minimum blood drug concentration), Css,max (steady-state maximum blood drug 
concentration), AUC0-t (area under the curve of the last measurable concentration time 
point) and t1/2 (elimination phase half-life).  
9.1.2 Timepoints of Sampling 
The blood samples for PK test will be collected before and 0 h +10 min after the 1st, 2nd, 
4th, 6th, and 8th infusion. Intensive blood collection will be carried out at 1 h ± 10 min, 2 
h ± 20 min, 8 h ± 1 h, 24 h ± 1 h, 48 h ± 2 h, and 72 h ± 3 h after the initial infusion. PK 
blood sample collection is also performed during the follow-up (if dystrophin mRNA 
cannot be detected in the blood samples at W8, blood samples for PK testing do not need 
to be collected again during subsequent follow-up). 
9.2 Pharmacodynamic Assessment 
9.2.1 Dystrophin Expression 
1) Assessment Indicators 
Quantity of dystrophin is measured by western blot of biopsied muscle. 
Immunohistochemistry (IHC) is used to detect fiber intensity and percentage of dystrophin 
positive fibers. Levels are described as change from baseline, in order to remove any 
background signal in the assay. Levels are expressed as a percent of control (i.e., as a 
percent of the level of normal, wild-type dystrophin present in muscle tissue of healthy 
individuals without DMD or Becker muscular dystrophy).  
2) Sampling for Muscle Biopsy 
Select a suitable location of the gastrocnemius muscle/biceps brachii muscle, confirm the 
puncture site, disinfect with iodine, and after local anesthesia, use puncture needle to 

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penetrate the tissue and muscle, extract muscle tissue with a size of 0.5 cm × 1 cm × 0.5 
cm and sent to a pathological laboratory for biopsy. Camera the biopsy site before and after 
each muscle biopsy. 
3) Timepoints of Sampling 
Samples of each subject for biopsy will be taken from gastrocnemius or biceps brachii 
muscle at baseline (one site) and from the gastrocnemius and biceps brachii muscles on 
D29 (two sites). 
4) Detection Method 
The expression of dystrophin muscle tissue is detected by the following methods: 
• Evaluate expression level of dystrophin (measured by western blot) at baseline and 
D29 (after the completion of the last dose). 
• 
Evaluate engraftment of dystrophin (measured by immunofluorescence for fiber 
intensity and percentage of dystrophin positive fibers) at baseline and D29 (after 
the completion of the last dose). 
The concentration of dystrophin mRNA in muscle tissue will be detected: 
• 
Evaluate changes in dystrophin mRNA (detected by qPCR) at baseline and D29 
dystrophin protein (if the biopsy time is between 12~24 hours after the last dose).  
9.2.2 Electromyography 
Perform electromyography at baseline and at W16 after the initial administration to assess 
muscle health status.  
9.2.3 MRI 
Perform electromyography at baseline and at W16 after the initial administration to assess 
muscle health status. 
9.3 Immunogenicity Assessment 
9.3.1 Assessment Indicators 
1) Changes of concentration of cytokine including TNF-α, INF-γ, IL-2, IL-6, and 
IL-10 will be detected. 
2) Changes of anti-dystrophin antibody level can be measured. 

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9.3.2 Timepoints of Sampling 
Cytokine test: TNF-α, INF-γ, IL-2, IL-6, and IL-10. Collect blood samples before the 
initial administration and before the 1st, 2nd, 4th, 6th, and 8th administration. If the 
cytokine levels return to normal at W8 follow-up, blood samples may not be collected for 
this test during subsequent follow-up. 
Anti-dystrophin antibody test. Collect blood samples at baseline/before the initial dose, 
before the 2nd, 4th, 6th, and 8th dose (if no more than 7 days from baseline to the initial 
dose, no additional collection is required before the initial dose) and at each follow-up. If 
the anti-dystrophin antibody cannot be detected in the follow-up samples at week 8, blood 
samples may not be collected for this test during subsequent follow-up. 
9.3.3 Detection Method 
Immunogenicity and cytokine tests will be detected by ELISA method. 
9.4 Safety Assessment  
9.4.1 Safety Parameters 
All AEs and clinically significant laboratory abnormalities will be graded according to 
Common Terminology Criteria for AEs, Version 5.0 dated 27 November 2017. 
Safety parameters include vital signs (including temperature, pulse, respiratory rate, blood 
pressure), physical examination (including general condition, mucous membranes, lymph 
nodes, head and neck, abdomen, musculoskeletal, respiratory rate, cardiovascular, nervous 
and psychiatric conditions) and laboratory tests (including hematology, urinalysis, blood 
chemistry) and 12-lead ECG. In case of any abnormality, the corresponding indicators shall 
be rechecked. 
Evaluate the TRAEs occurred during the clinical study, record its clinical manifestation, 
severity, occurrence time, duration, treatment method and prognosis, and determine its 
correlation with the investigational drug. 
9.4.2 Baseline Signs and Symptoms 
The baseline value serves as the starting point for clinical evaluation, and a series of 
physical examinations or laboratory tests are conducted to confirm the premedication 
status of the subjects, in order to facilitate comparison between post-treatment and pre- 
treatment. All subjects are required to undergo baseline measurement, which should be 

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done before randomization. However, if the examination is invasive (such as biopsy), it 
should be performed after chemical examination as much as possible to reduce harm to the 
subjects. In addition, the baseline value should be as close as possible to the initial 
treatment. Before the study begins, conduct a comprehensive physical and laboratory 
examination of the study subjects, and record relevant data. Inquire and record in detail the 
lifestyle and past medication history of the study subjects. Based on the collected data, 
determine the baseline status of each study subject and record it in the CRF. 
9.4.3 Laboratory Safety Assessment  
According to the study Schedule in Table 5, list the Hematology, blood biochemistry, 
Urinalysis, coagulation, and serum pathogen test for each subject. If applicable, it will 
also be marked as above or below the corresponding normal range. Evaluate changes in 
laboratory results before and after administration. We will also compare the indicators 
before and after the study to discover whether the parameters have returned to the pre study 
level. 
1) Haematology: white blood cell count (WBC), neutrophil count (NEUT #), 
eosinophil count (EO #), basophil count (BASO #), lymphocyte count (LYMPH 
#), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), and 
hematocrit (HCT) (the test results within 7 days before administration can be 
accepted). 
2) Urinalysis: Urinary bilirubin (UBG), urinary bilirubin (BIL), urinary ketone body 
(KET), urinary occult blood (BLD), urinary protein (PRO), urinary nitrite (NIT), 
urinary glucose (GLUU), urinary specific gravity (SG), urinary pH (pH), red 
blood cells (RBC), and white blood cells (WBC). 
3) Biochemistry: bilirubin (total, indirect and direct), total protein, albumin (ALB), 
alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-
glutamyl transferase (GGT), alkaline phosphatase (ALP), creatinine (Cr), blood 
urea (Urea), blood glucose (GLU), serum sodium (Na+), serum potassium (K+), 
serum calcium (Ca2+), serum magnesium (Mg2+), serum chloride (CL -), lactate 
dehydrogenase (LDH), serum creatine kinase (CK), serum creatine kinase MB 
isoenzyme (CKMB) and c-reactive protein (CRP). Calculation of eGFR [CKD-
EPI]. 
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(APTT) and international normalized ratio (INR). 
5) Hepatitis B & C and HIV serology tests: hepatitis B surface antigen (HBs Ag), 
anti-hepatitis B surface antibodies (anti-HBs Ab) anti-hepatitis B core antibodies 
(anti-HBc Ab), hepatitis B e antigen (HBe Ag), anti-hepatitis B e antibodies (anti-
HBe Ab), hepatitis C antibodies, anti-HIV1 and anti-HIV2 antibodies. 
9.4.4 Vital Signs and Physical Examination 
Perform vital signs and physical examinations at each visit and record any clinically 
significant changes. The changes in vital signs from baseline to the EOS will be provided, 
and the incidence of significant abnormalities in all vital signs will be summarized and 
listed. The safety assessment of vital signs and physical examinations will only be provided 
in the list.  
Vital signs: temperature, resting pulse, blood pressure, and respiratory rate. Vital 
signs will be recorded at every visit. 
Physical examination: Including general condition, mucous membranes, lymph 
nodes, head and neck, abdomen, musculoskeletal, respiratory rate, cardiovascular, nervous 
and psychiatric conditions. Weight and height (Screening visit only) should be recorded 
and calculated for BMI. BMI=weight (kg) /height2 (m2). 
9.4.5 12-Lead ECG 
A 12-lead ECG will be recorded measuring the RR, PR, QRS, and QT interval durations. 
Summarize the changes in ECG parameters during the study period relative to baseline 
values and present abnormal results. All ECG data will also be provided in the subject list. 
9.4.6. NSAA Scale 
NSAA (North Star Ambulatory Assessment) is a validated rating scale used to assess the 
impact of Duchenne Muscular Dystrophy on patients' ambulatory function. Assessment 
items 
include: 
standing/walking/rising from 
a chair/stepping onto 
a raised 
surface/jumping/running/standing on one leg/heel walking/descending stairs/ascending 
stairs/lifting a leg/hopping on one leg. 
Subjects complete NSAA at baseline to record pre-dosing motor function scores.Post-
dosing, subjects complete NSAA twice at Week 16 and Week 28 to record scores again. 
Clinical performance is based on NSAA scores. Higher scores indicate better functional 
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improvement, maintenance, or decline in motor function over time. 
Scoring and Results: 
Score 0: Unable to complete task 
Score 1: Completes with assistance, or completes independently with difficulty 
Score 2: Completes independently without difficulty 
Age-Specific Maximum Scores: 
Ages 2-3: 8 items, maximum 16 points 
Ages 3-4: 13 items, maximum 26 points 
Ages 4-6: Maximum 45 points 
NSAA Assessed Skills 
Patient scores 
Stand  
Stand barefoot for as long and still as 
possible without external support. 
 
Walk  
Walk forward for at least 10 steps (about 
8-10 feet) with a consistent heel-to-toe 
gait. 
 
Rise from chair 
Begin seated with arms crossed over 
chest, then stand up from the chair without 
uncrossing arms. 
 
Climb step (right leg) 
Step onto a box step at least 15cm high 
with right (or dominant) foot. 
 
Climb step (left leg) 
Step onto a box step at least 15cm high 
with left (or non-dominant) foot, joining 
the other. 
 
Gets to sitting  
Lay flat on the floor with arms by side and 
move to a sitting position without turning 
towards the floor or using both hands to 
get up. Using one hand is permissible to 
achieve the top score. 
 
Jump 
Stand on the floor with both feet together 
and jump as high as possible with minimal 
forward movement. 
 
Run 
Run as fast as possible for about 32 feet. 
To achieve the highest score, both feet 
must clear the ground when running. 
 
Stand on right leg 
Run as fast as possible for about 32 feet. 
To achieve the highest score, both feet 
must clear the ground when running. 
 
Stand on left leg 
Stand on left leg, with arms down, for as 
long as possible. 
 

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Descend box step 
(right leg) 
Facing forward, step down from the box 
with right (or dominant) foot. 
 
Descend box step (left 
leg) 
Facing forward, step down from the box 
with left (or non-dominant) foot, joining 
the other. 
 
Stand on heels 
Lean back onto heels for three counts 
while barefoot. To achieve a top score, 
both feet must be lifted at the same time 
using clear dorsiflexion (raising the foot 
towards the shin). 
 
Rise from floor 
Lay flat on back and stand up as quickly 
as possible without rolling into a four-
point kneeling or prone position (Gower’s 
maneuver). 
 
Lift head 
Lay flat on the floor with arms crossed 
across the chest and hands resting below 
the shoulder. Then, lift head, touching 
chin to chest, while keeping arms folded. 
 
Hop on right leg 
Stand on right leg and hop one-legged 
without landing on both feet. 
 
Hop on left leg  
Stand on left leg and hop one-legged 
without landing on both feet. 
 
NSAA TOTAL SCORE 
 
10. Adverse Event Reporting 
10.1. Definition of Adverse Event  
10.1.1. Definition 
Adverse Event：An adverse event (AE) is defined as any reaction, side effect, or untoward 
event that occurs during the course of the clinical trial whether or not the event is 
considered related to the study drug.  
Serious adverse events: A serious adverse event (SAE) is defined as any serious adverse 
medical events such as death, life-threatening, permanent or severe disability or loss of 
function of the subject after receiving the investigational drug, the subject's need for 
hospitalization or extended hospitalization time, as well as congenital abnormalities or 
birth defects. 
10.1.2. Categorization of AEs 
All AEs and clinically significant laboratory abnormalities will be graded according to 
Common Terminology Criteria for AEs, Version 5.0 dated 27 November 2017. For any 

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term that is not specifically listed on the CTCAE scale, intensity will be assigned a grade 
of 1 through 5 using the CTCAE guidelines Table 6。 
Table 6 The CTCAE displays grade 1 through 5 criteria of adverse event 
Grades 
Criteria 
Grade 1 
Mild； asymptomatic or mild symptoms; clinical or diagnostic observation only; 
intervention not indicated 
Grade 2 
Moderate; minimal, local or noninvasive intervention indicated; limiting age-
appropriate instrumental activities of daily living (ADL)*. 
Grade 3 
Severe or medically significant but not immediately life-threatening; hospitalization or 
prolongation of hospitalization indicated; disabling; limiting self care ADL
△. 
Grade 4 
Life-threatening consequences; urgent intervention indicated. 
Grade 5 
Death related to AE. 
*Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the 
telephone, managing money, etc. 
△Self care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, taking 
medications, and not bedridden. 
10.1.3. Causes of AEs 
According to the standards formulated by the Adverse Drug Reaction Monitoring Center 
of the Ministry of Health, the five-level classification is "Definitely related, Probably 
related, Possibly related, Unlikely related, Unrelated". The first three levels were judged 
to be related to the investigational drug. The judgment criteria of the five-level 
classification method on AEs are shown in Table 7 below： 
(1) Whether there is a reasonable order of time. 
(2) Whether it is consistent with the known AR type of the investigational drug. 
(3) Whether it can be explained by the volunteers' clinical status, concomitant 
medication, combined therapy and previous therapy. 
(4) If the investigational drug is stopped or the dosage is reduced, whether it is 
reduced or disappeared. 
(5) Whether the same reaction occurs again after repeated exposure to the same drug. 
Table 7 Causality or relatedness assessment criteria in AEs and investigational drugs 
Analysis results 
Indicators 
1 
2 
3 
4 
5 
Definitely Related 
+ 
+ 
- 
+ 
+ 
Probably Related 
+ 
+ 
- 
+ 
? 
Possibly Related 
+ 
+ 
± 
± 
? 
Unlikely related 
+ 
- 
± 
± 
? 
Unrelated 
- 
- 
+ 
- 
- 

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The relationship between AEs (including SAEs) and drugs should be determined as far as 
possible. If it is believed that it is “Definitely related, Probably related and Possibly 
related”, it should be considered as an adverse reaction caused by investigational drug, and 
whether it is a SAE should be considered according to the severity. Adverse reactions 
should be listed and classified according to organ system or adverse event syndrome. 
10.2. Adverse Event Recording and Reporting 
The investigator should tell the subject who is required to truthfully feedback the changes 
after the medication but should avoid inductive questions. While observing the curative 
effect, the investigator should pay close attention to the AEs or unexpected toxic and side 
effects (including symptoms, signs and laboratory tests), analyze the causality, make 
judgments, track the observation and record, and count the incidence of adverse events. 
For the AEs occurred during the trail, the investigator should record the time, severity, 
duration, treatment measures, outcome, etc. of the occurrence in the CRF, judge the 
correlation with the investigational drug, sign and indicate the date. In case of SAE in the 
trail, the investigator must immediately take measures to protect the safety of the subjects, 
and report to the department responsible for adverse drug reaction reporting in the study 
site in a timely manner. Then the adverse drug reaction report form should carefully be 
filled in according to their opinions. Finally, continue reporting should follow the site's 
process. 
10.3. Risk Prevention and Handling 
When AEs occur, the investigator can decide whether to terminate the trial based on 
the severity, and the researcher will provide corresponding treatment in a timely 
manner according to the subject's condition. In case of SAEs, Emergency handling 
measures must be taken to protect the safety of the subjects. All AEs should be tracked 
and followed up, with detailed records of the handling process and results, until they 
are properly resolved or the condition stabilizes. If there are laboratory abnormalities, 
they should be traced back to normal or baseline levels. The follow-up can be selected 
based on the severity of AEs, including hospitalization, outpatient, home visits, phone 
calls, mail, and other forms.  

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11. Statistical Analysis 
11.1. Sample Size  
The study is an IIT and no sample size estimation is conducted. The number of subjects is 
determined based on the study objectives and relevant guiding principles. Six healthy 
subjects are planned to be enrolled, and those who have not been administered after 
randomization will be replaced. 
11.2. Populations for Analysis 
Ambulatory boys with confirmed DMD by clinical diagnosis and genetic testing, aged 
2~6years. 
The age, gender, and race of the subjects, as well as their height and weight, should be 
recorded. Information about allergies/drug sensitivity or drug abuse should be obtained. 
All the information of these subjects should be recorded in descriptive language in the table. 
11.2.1 Full Analysis Set 
Full analysis set (FAS): FAS will be used to report medication compliance and to 
summarize demographic characteristics (age, gender, race, height and weight) and 
background characteristics (medical history, concomitant medications, and physical 
examination, etc.) for all randomized subjects by treatment group. 
11.2.2 Per-Protocol Set  
Per-protocol set (PP)：Per-protocol analysis is a comparison of treatment groups that 
includes only those patients who completed the treatment originally allocated. 
11.2.3 Safety Analysis Set 
Safety analysis set (SAS): All subjects who have been randomized into cohorts, have used 
the investigational drug, and have safety evaluation data post-treatment constitute the 
safety population of this study. The safety population will be used for safety analysis. 
11.2.4 Pharmacokinetic Dataset 
The pharmacokinetic dataset (PKS) consists of all enrolled subjects who have received at 
least one study drug and have post-treatment PK evaluation data, forming the PK analysis 
set for this study. In the PK analysis set, different pharmacokinetic parameters may include 

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different numbers of subjects based on the actual completion of the study. 
11.3. Efficacy Analysis and Statistical Methods 
Due to the small sample size and open-label design of this study, only descriptive statistics 
will be conducted for all endpoints, including the number of subjects, mean and standard 
deviation, minimum and maximum values of continuous variables, and the number and 
percentage of categorical variables. 
Using descriptive statistical methods, describe the number and proportion of subjects 
entering each statistical analysis set by treatment group, the number and proportion of 
participants who completed the trial and withdrew midway, and the reasons for 
withdrawing midway (and their proportion). Describe demographic and other baseline 
characteristics by treatment group. 
11.3.1 Analysis of Primary Endpoints  
The primary endpoint is safety. Using a safety analysis set, summarize the baseline data, 
post-treatment data, and change from baseline data by follow-up and treatment group based 
on laboratory tests, vital signs, electrocardiograms, and other safety data. A cross table will 
be used to describe the changes from baseline to each follow-up after administration 
regarding the normality and clinical significance of various examination results used as 
categorical data. 
11.3.2 Analysis of Secondary Endpoints  
1) PK Parameter Indicators and Assessment 
The main pharmacokinetic parameters include t1/2, AUC, Vss, CL, Cmax, Tmax, etc. 
Descriptive statistical analysis of pharmacokinetic parameters for different dose groups or 
queues, calculating the arithmetic mean, standard deviation, coefficient of variation, 
median, maximum, minimum, and geometric mean of pharmacokinetic parameters for 
each dose group or queue. 
2) PD Assessment 
Statistically describe each pharmacodynamic indicator according to its data characteristics, 
and compare the changes after administration with the baseline characteristics. Analyze 
pharmacodynamic parameters according to actual needs.  

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3) Immunogenicity Assessment 
Descriptive statistical analysis was conducted to compare the changes in cytokine 
production and anti-dystrophin antibodies in subjects at different time points after 
treatment with the baseline period. 
11.4. Safety Analysis and Statistical Methods 
Using the safety analysis set, AEs, ARs, and SAEs of each treatment group will be 
summarized and utilized. Compare the incidence rates among different treatment groups 
using the χ2-test/Fisher exact probability method. Adverse events and reactions will also 
be standardized medical codes based on System Organ Class and Preferred Term. 
All completed examination items including physical examination, 12-lead ECG, lab tests 
(Haematology, Urinalysis, Biochemistry, coagulation function, etc.), and descriptive 
statistics are listed in the form of a cross tabulation of pre- and post- treatment (based on 
the judgment of clinical doctors). The items of abnormal ECG, physical examination and 
laboratory test at each visit shall be listed in the form of a list. Descriptive recording of 
vital sign data, including values at each timepoint and their relative changes relative to 
baseline. 
List all AEs, including subject number, dosage group, AE name, description, start date, 
stop date, NCI-CTCAE level, severity of AEs, relationship with study drug, impact on 
study drug, outcome, etc.  
11.5. Mid-term Analysis 
Due to the small sample size of this study, which is only an exploratory IIT clinical study, 
no criteria for mid-term analysis/stop analysis are set. Both parties can obtain study data. 
If there are SARs during the study, both parties can negotiate and decide on the continued 
implementation of the study. 
11.6. Data Monitoring Committee 
Due to the small sample size and short study duration, which is only an exploratory IIT 
clinical study, no data monitoring committee was established. 
12. Data Collection and Management 
12.1. CRF/EDC 
Due to the small sample size in this study, the data will be entered into a paper CRF. 

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Clinical investigators or data entry personnel (clinical coordinators) designated by the 
investigators shall promptly and accurately input source data into CRF. The data entry 
personnel or investigators can modify the data after verifying it, and the modified data 
needs to be filled in the modification reason on the CRF. The investigators has signature 
permission for all final data. 
12.2. Data Management 
1) Investigators should keep all detailed original files of the subjects to ensure that 
the data is accurate, complete, and timely. The original documents, medical 
records, etc. should be clear, detailed, and easily identifiable by personnel 
participating in this clinical trial. 
2) The data in CRF can only be modified by investigators or authorized personnel. 
3) During the monitoring visit, the inspector can contact the staff of the research 
center, obtain source documents, and provide an appropriate environment to 
complete the review of study related documents. The monitor will meet regularly 
with the investigators during the study to provide feedback on the progress of the 
study. 
4) The inspector will compare the CRF data with hospital records (source files). The 
nature and location of all source files will be clarified to ensure understanding of 
all sources of raw data required for filling out CRF. Supervisors can also contact 
the study center to review these data sources. 
5) After the study is completed, archive the CRF as needed. The experimental data 
should be retained for 5 years after the end of the study. But if there are 
requirements in current regulations or agreements with partners, these materials 
should be kept for a longer period of time. The collaborators will notify the 
investigators in writing when these materials will no longer need to be preserved. 
13. Ethical Considerations 
13.1. Ethics Committee/Institutional Review Board  
This protocol, written informed consent form, and materials directly related to the subjects 
must be submitted to the ethics committee for written approval before the study can be 
officially conducted. Investigators must submit their annual study report to the ethics 
committee at least annually (if applicable). When the study is terminated and/or completed, 

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the investigators must notify the ethics committee in writing. Investigators must promptly 
report all changes that have occurred in their study work to the ethics committee (such as 
revisions to relevant study documents such as protocols and/or informed consent forms), 
and these changes must not be implemented without approval from the ethics committee, 
unless they are made to eliminate obvious and direct risks to the subjects. When such 
situations occur, the ethics committee will be notified. 
13.2. Informed Consent 
In this study, all subjects are required to voluntarily sign a written informed consent form.  
Investigators must provide informed consent forms that are easy to understand and 
approved by the ethics committee to the subjects or their guardians and give them sufficient 
time to consider this study. Before obtaining a signed written informed consent form from 
the subject, the subject is not allowed to join the study. During the study, all updated 
versions of the informed consent form and written information will be provided to the 
participants. The informed consent form should be kept as an important document for 
clinical trials for future reference. 
13.3. Protocol Amendment 
The protocol amendment may affect the legal and ethical status of the study, as well as the 
statistical evaluation and the possibility of achieving the primary purpose of the study. 
The terms of the protocol and its annexes must be strictly observed, except in emergency 
situations. If amendment is required, it must be provided in writing according to the 
corresponding SOP of the study site and approved. The protocol amendment will be 
presented to the investigator and explained. All protocol amendments must be submitted 
to the EC for review and approval before implementation, as well as to the management 
department for approval/notification when appropriate. Major amendments to the protocol 
must obtain the approval of Haining Spot Biotech Co., Ltd., the site’s EC and the 
management department (if required) before implementation. 
14. Confidentiality 
Only investigators and monitors participating in clinical trials may have access to the 
personal medical records of the subjects, and they will sign a "Researcher Declaration" or 

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"Confidentiality Commitment" that includes confidential information. Data processing 
will adopt a "data anonymity" approach, omitting information that can identify the 
individual identity of the subjects. The medical records of the subjects will be kept in the 
data archive room of the clinical trial center. 
The results of this study may be published in medical journals, but we will keep patient 
information confidential in accordance with legal requirements, unless personal 
information of patients is not disclosed due to relevant legal requirements. When necessary, 
government management departments, hospital ethics committees, and their relevant 
personnel may access patient information in accordance with regulations. 
 

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