Dose-finding Study of SPK-8016 Gene Therapy in Patients With Hemophilia A to Support Evaluation in Individuals With FVIII Inhibitors
Phase: PHASE1
Conditions: Adeno-Associated Virus (AAV), Blood Coagulation Disorder, Blood Coagulation Disorders, Inherited, Coagulation Protein Disorders, Factor VIII (FVIII), Factor VIII (FVIII) Deficiency, Factor VIII (FVIII) Gene, Factor VIII (FVIII) Protein, Genetic Diseases, Inborn, Genetic Diseases, X-Linked, Gene Therapy, Gene Transfer, Hematologic Diseases, Hemorrhagic Disorders, Recombinant, Vector, Inhibitors
Sponsor: Spark Therapeutics, Inc.

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TITLE PAGE 
PROTOCOL TITLE: 
Dose-finding study of SPK-8016 gene therapy in 
patients with hemophilia A to support future evaluations 
in individuals with FVIII inhibitors 
PROTOCOL NUMBER: 
SPK-8016-101 
INVESTIGATIONAL 
PRODUCT/NUMBER: 
SPK-8016 
PHASE OF DEVELOPMENT 
1/2a 
INDICATION: 
Hemophilia A 
SPONSOR: 
Spark Therapeutics 
SPONSOR ADDRESS: 
3737 Market Street, Suite 1300 
Philadelphia, PA 19104 
USA 
REGULATORY AGENCY 
IDENTIFYING NUMBER(S): 
 
ORIGINAL PROTOCOL 
DATES: 
18 September 2018 (Version 1.0) 
29 October 2018 (Version 2.0, Amendment 1) 
VERSION DATE: 
19 March 2021 
This study will be conducted in accordance with the standards of Good Clinical Practice (as defined by the 
International Conference on Harmonization), the ethical principles that have their origin in the Declaration of Helsinki 
and all applicable national and local regulations. 
This protocol includes information and data that contain trade secrets and privileged or confidential information, which 
is the property of Spark Therapeutics, Inc. (“Spark”). This information must not be made public without written 
permission from Spark. These restrictions on disclosure will apply equally to all future information supplied to you. 
This material may be disclosed to and used by your staff and associates as may be necessary to conduct the clinical 
study. 
NCT #: NCT03734588

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SPONSOR SIGNATORY FORM 
 
Protocol Title: Dose-finding study of SPK-8016 gene therapy in patients with 
hemophilia A to support future evaluations in individuals with FVIII inhibitors 
 
Protocol No: SPK-8016-101 
 
This study protocol was subject to critical review and has been approved by the appropriate 
protocol reviewers of the Sponsor. The information contained in this protocol is consistent with: 
 
• The current benefit-risk evaluation of the investigational product. 
• The moral, ethical, and scientific principles governing clinical research as set out in the 
Declaration of Helsinki, International Conference on Harmonization Good Clinical Practice 
(ICH GCP) guidelines and according to applicable local requirements. 
 
The Investigator will be supplied with details of any significant or new findings, including adverse 
events, relating to treatment with the investigational product. 
 
Spark Therapeutics: 
 
 
 
 
 
Spark Therapeutics 
 
 
 
 
Signature 
  
Date (DD-MMM-YYYY) 
 

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INVESTIGATOR STATEMENT 
I have read the protocol, including all appendices, and I agree that it contains all of the necessary 
information for my staff and I to conduct this study as described. I will conduct this study as 
outlined herein, in accordance with Good Clinical Practice: Consolidated Guideline approved by 
the International Conference on Harmonization (ICH), and all applicable local and federal 
regulatory requirements and will make a reasonable effort to complete the study within the time 
designated. 
I will provide all study personnel under my supervision copies of the protocol and any 
amendments, and access to all information provided by Spark or specified designees. I will discuss 
the material with them to ensure that they are fully informed about the study. 
 
 
Institution Name 
 
 
Principal Investigator (PI) Name/Site Number 
 
 
Principal Investigator’s (PI) Signature 
Date (DD-MMM-YYYY) 
 

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LIST OF PERSONNEL AND ORGANIZATIONS RESPONSIBLE FOR 
CONDUCT OF THE STUDY 
A list of personnel and organizations responsible for the conduct of the study will be supplied to 
study sites as part of the Investigator’s Study File. This list will be updated by Spark (or delegate) 
and provided to study sites as needed. 

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PROTOCOL DOCUMENT HISTORY 
 
DOCUMENT HISTORY 
Document 
Date 
Amendment 2 
19-Mar-2021 
Amendment 1 
29-Oct-2018 
Original Protocol 
18-Sep-2018 
 
A summary of the changes made in this protocol amendment is located in Section 16. 
 
 
 
 
 

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TABLE OF CONTENTS 
TITLE PAGE ................................................................................................................................. 1 
SPONSOR SIGNATORY FORM ................................................................................................. 2 
INVESTIGATOR STATEMENT ................................................................................................. 3 
LIST OF PERSONNEL AND ORGANIZATIONS RESPONSIBLE FOR CONDUCT OF THE 
STUDY .............................................................................................................................. 4 
PROTOCOL DOCUMENT HISTORY ........................................................................................ 5 
TABLE OF CONTENTS ............................................................................................................... 6 
LIST OF FIGURES ..................................................................................................................... 12 
LIST OF TABLES ....................................................................................................................... 12 
1 
PROTOCOL SUMMARY ............................................................................................... 13 
2 
INTRODUCTION ........................................................................................................... 30 
2.1 
Background ................................................................................................................... 30 
2.1.1 
Hemophilia A ............................................................................................................. 30 
2.1.2 
Clinical Manifestations .............................................................................................. 31 
2.1.3 
Current Therapies and Prevention for Hemophilia A ................................................ 32 
2.1.3.1 
Current Therapies.................................................................................................... 32 
2.1.3.2 
Current Prevention .................................................................................................. 33 
2.1.4 
Alternative Therapy for Hemophilia A ...................................................................... 34 
2.1.4.1 
Factor VIII and Protein ........................................................................................... 34 
2.1.4.2 
Biology of Adeno-Associated Virus (AAV) Vectors ............................................. 35 
2.1.4.3 
Gene Therapy as an Alternative Approach ............................................................. 36 
2.2 
Rationale ....................................................................................................................... 36 
2.2.1 
Description of SPK-8016 ........................................................................................... 37 
2.2.2 
Summary of Non-Clinical Experience with SPK-8016 or Other Relevant Spark  AAV 
Vector ......................................................................................................................... 38 
2.2.3 
Summary of Clinical Experience with SPK-8016 or Other Relevant Spark  AAV 
Vector ......................................................................................................................... 40 
2.2.3.1 
Summary of Overall Clinical Experience with AAV-Mediated Gene Therapy ..... 41 
2.2.4 
Summary of Non-clinical and Clinical Experience with 
 ...................... 42 
2.2.5 
Summary of Non-Clinical and Clinical Experience with 
 ... 43 
2.3 
Benefit/Risk Assessment .............................................................................................. 44 

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2.3.1 
Risk Assessment ........................................................................................................ 44 
2.3.1.1 
Allergic Reaction or Anaphylaxis ........................................................................... 44 
2.3.1.2 
Inhibitor Development ............................................................................................ 45 
2.3.1.3 
Elevation of Hepatic Transaminases ....................................................................... 45 
2.3.1.4 
Anti-AAV Neutralizing Antibody Development .................................................... 46 
2.3.1.5 
Bleeding Episodes ................................................................................................... 46 
2.3.1.6 
Possible Side Effects from Corticosteroids............................................................. 46 
2.3.1.7 
Risks Associated with Additional Immune Modulating Agents............................. 47 
2.3.2 
Potential Benefits ....................................................................................................... 52 
2.4 
Rationale ....................................................................................................................... 52 
2.5 
Rationale for Dose and Schedule Selection .................................................................. 54 
3 
OBJECTIVES AND ENDPOINTS ................................................................................. 56 
3.1 
Primary Objective ......................................................................................................... 56 
3.2 
Secondary Objectives.................................................................................................... 56 
3.3 
Primary Endpoints ........................................................................................................ 56 
3.4 
Secondary Endpoints .................................................................................................... 56 
3.5 
Exploratory Endpoints .................................................................................................. 56 
4 
STUDY DESIGN............................................................................................................. 58 
4.1 
Overall Design .............................................................................................................. 58 
4.1.1 
Sequence of Enrollment ............................................................................................. 58 
4.1.1.1 
Dose-Level and Cohort Expansion ......................................................................... 60 
4.1.2 
FVIII Incremental Recovery ...................................................................................... 61 
4.1.3 
Corticosteroids ........................................................................................................... 61 
4.1.3.1 
Corticosteroid Regimen .......................................................................................... 63 
4.1.3.2 
Other Immunomodulatory Considerations ............................................................. 64 
4.1.3.3 
 
4.1.3.4 
Experience with Long Term Immunomodulation................................................... 65 
4.1.4 
Three to Seven Days Prior to Day 0 .......................................................................... 66 
4.1.5 
Day -2......................................................................................................................... 66 
4.1.6 
Dosing Period (Days 0, 1) .......................................................................................... 67 
4.1.7 
Follow-Up Observation Period .................................................................................. 67 

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4.2 
Study Duration, Enrollment and Number of Sites ........................................................ 67 
4.2.1 
Duration of Study Participation ................................................................................. 67 
4.2.2 
Total Number of Participants; Sites Projected and Geographic Regions .................. 68 
4.3 
Study Stopping Rules .................................................................................................... 68 
5 
STUDY POPULATION .................................................................................................. 70 
5.1 
Inclusion Criteria .......................................................................................................... 70 
5.2 
Exclusion Criteria ......................................................................................................... 71 
5.3 
Screen Failures .............................................................................................................. 73 
5.4 
Enrollment of Participants ............................................................................................ 73 
5.5 
Randomization .............................................................................................................. 73 
5.6 
Blinding Procedures ...................................................................................................... 73 
6 
STUDY PROCEDURES/ASSESSMENTS AND SCHEDULE ..................................... 74 
6.1.1 
Clinical Safety Assessments ...................................................................................... 74 
6.1.2 
Laboratory Safety Assessments ................................................................................. 74 
6.2 
Additional Assessments ................................................................................................ 74 
6.2.1 
Joint Assessments ...................................................................................................... 74 
6.2.2 
Hemophilia Joint Health Score .................................................................................. 75 
6.2.3 
Activity Assessments (Hemophilia Activities List) ................................................... 75 
6.2.4 
Participant Questionnaires ......................................................................................... 75 
6.2.5 
Health-economic Assessment .................................................................................... 75 
6.2.6 
Archived Bio-samples ................................................................................................ 75 
6.3 
Clinical Procedures ....................................................................................................... 76 
6.4 
Screening Period ........................................................................................................... 78 
6.4.1 
Screening Assessments .............................................................................................. 79 
6.5 
Three to Seven Days Prior to Day 0 Assessments ........................................................ 80 
6.6 
Day -2............................................................................................................................ 81 
6.7 
Dosing Day Assessments (Day 0)................................................................................. 81 
6.7.1 
FVIII Dosing .............................................................................................................. 81 
6.7.2 
........................................................................................ 81 
6.7.3 
Vector Dosing ............................................................................................................ 82 
6.7.4 
Day 1 .......................................................................................................................... 82 

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6.8 
Follow-up Observation Period (Weeks 1-52) ............................................................... 83 
6.8.1 
Days 3, 7, 10, 14, 17, 21, 24, 28, 31, 35, 38, 42, 45, 49, 52, 56, 59, 63, 66, 70, 73, 77, 
84 (± 2 days) .............................................................................................................. 83 
6.8.2 
Weeks 14, 16, 18, 22, 26, 30, 34, 40, 46, 52/End of Study (± 2 weeks).................... 84 
7 
STUDY INTERVENTION .............................................................................................. 86 
7.1 
Description of Study Drug ............................................................................................ 86 
7.2 
Study Drug Doses ......................................................................................................... 86 
7.3 
Dose Schedule and Administration ............................................................................... 87 
7.4 
Treatment Compliance .................................................................................................. 87 
7.5 
Study Drug Storage ....................................................................................................... 87 
7.6 
Study Drug Preparation, Handling and Disposal .......................................................... 88 
7.6.1 
Study Drug Preparation.............................................................................................. 88 
7.6.2 
Study Drug Handling and Disposal ........................................................................... 88 
7.6.3 
Accountability and Destruction ................................................................................. 88 
7.7 
Labeling ........................................................................................................................ 89 
7.8 
Study Compliance ......................................................................................................... 89 
7.9 
Prior and Concomitant Medications ............................................................................. 89 
7.9.1 
Concomitant Therapy................................................................................................. 89 
7.9.2 
Permitted Therapy ...................................................................................................... 89 
7.9.3 
Prohibited Therapy..................................................................................................... 90 
7.9.4 
Concomitant Procedures ............................................................................................ 90 
8 
DISCONTINUATION OF STUDY INTERVENTION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL........................................................................ 91 
8.1 
Participant Discontinuation/Withdrawal from the Study .............................................. 91 
8.2 
Early Termination Study Visit ...................................................................................... 91 
8.3 
Lost to Follow Up ......................................................................................................... 91 
9 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS ........................................ 93 
9.1 
Definitions..................................................................................................................... 93 
9.1.1 
Adverse Event ............................................................................................................ 93 
9.1.2 
Definition of SAE ...................................................................................................... 94 
9.1.3 
Adverse Events of Special Interest (AESI) ................................................................ 95 
9.2 
Recording of AE and/or SAE ....................................................................................... 95 

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9.3 
Safety Classifications .................................................................................................... 96 
9.3.1 
Assessment of Intensity ............................................................................................. 96 
9.3.2 
Assessment of Causality ............................................................................................ 96 
9.4 
Follow-up and Reporting Requirements ....................................................................... 97 
9.4.1 
Follow-up of AEs and SAEs ...................................................................................... 97 
9.4.2 
Reporting of SAEs ..................................................................................................... 97 
9.5 
Time Period and Frequency for Collecting AE and/or SAE Information .................... 97 
9.5.1 
Time Period and Frequency for Collecting of AEs and SAEs ................................... 97 
9.5.2 
Collection of AEs and SAEs information after conclusion of the study ................... 98 
9.5.3 
Regulatory Reporting Requirements for SAEs .......................................................... 98 
9.5.4 
Pregnancy ................................................................................................................... 98 
9.6 
Treatment of Overdose ................................................................................................. 99 
10 
STATISTICAL CONSIDERATIONS........................................................................... 100 
10.1 
Statistical Hypotheses ................................................................................................. 100 
10.2 
Sample Size Determination......................................................................................... 100 
10.3 
Populations for Analyses ............................................................................................ 100 
10.4 
Demography and Baseline Disease Characteristics .................................................... 100 
10.5 
Primary and Secondary Endpoints .............................................................................. 100 
10.5.1 
Safety Analysis ........................................................................................................ 100 
10.5.2 
Efficacy Analysis ..................................................................................................... 101 
10.5.3 
Pharmacokinetics Analysis ...................................................................................... 101 
10.6 
Exploratory Endpoints Analysis ................................................................................. 102 
10.7 
Interim Analyses ......................................................................................................... 103 
10.8 
End of Study Definition .............................................................................................. 103 
11 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 104 
11.1 
Regulatory, Ethical and Study Oversight Considerations........................................... 104 
11.1.1 
Regulatory and Ethical Considerations .................................................................... 104 
11.1.2 
Financial Disclosure................................................................................................. 104 
11.1.3 
Informed Consent Process ....................................................................................... 105 
11.1.4 
Data Protection......................................................................................................... 105 
11.1.5 
Committee Structure ................................................................................................ 105 

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11.1.6 
Dissemination of Clinical Study Data...................................................................... 106 
11.1.7 
Data Quality Assurance ........................................................................................... 106 
11.1.8 
Source Documents ................................................................................................... 106 
11.1.9 
Study and Site Closure ............................................................................................. 107 
11.1.10 
Publication Policy .................................................................................................... 107 
12 
REFERENCES .............................................................................................................. 108 
13 
APPENDIX 1:  FACTOR VIII INFUSION LOG ......................................................... 121 
14 
APPENDIX 2:  HEMOPHILIA ASSESSMENTS ........................................................ 122 
14.1 
HEMOPHILIA ACTIVITIES LIST ........................................................................... 122 
14.2 
HAEM-A-QOL QUESTIONNAIRE .......................................................................... 133 
14.3 
EQ-5D-5L QUESTIONNAIRE .................................................................................. 141 
14.4 
HEMOPHILIA JOINT HEALTH SCORE ................................................................. 144 
15 
APPENDIX 3: ABBREVIATIONS .............................................................................. 148 
16 
APPENDIX 4: SUMMARY OF CHANGES FROM THE PREVIOUS PROTOCOL 
VERSION ...................................................................................................................... 154 
 

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LIST OF FIGURES 
Figure 1: 
Study Schema....................................................................................................... 25 
Figure 2: 
The Blood Coagulation Cascade .......................................................................... 31 
Figure 3: 
Schematic of SPK-8016 vector genome .............................................................. 38 
 
LIST OF TABLES 
Table 1: 
Schedule of Assessments ..................................................................................... 26 
Table 2: 
............................................ 55 
Table 3: 
Clinical Procedures: Study Assessments ............................................................. 76 
 

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1 
PROTOCOL SUMMARY 
Protocol Title:  
Dose-finding study of SPK-8016 gene therapy in patients with 
hemophilia A to support future evaluations in individuals with FVIII 
inhibitors. 
Protocol 
Number: 
SPK-8016-101 
Sponsor: 
Spark Therapeutics 
Development 
Phase: 
Phase 1/2a 
Name of 
Investigational 
Product: 
SPK-8016 
Study Indication: Hemophilia A 
Rationale:  
The primary aim of hemophilia care is to prevent and treat bleeding due 
to deficient clotting factors. Both hemophilia A and B can be treated with 
recombinant factor replacement leading to significant improvement in 
morbidity and mortality. However, such treatment is extremely costly 
and is often still associated with clinical complications that include 
bleeding, particularly bleeding into the joints (hemarthrosis). Moreover, 
this treatment involves a life-long need for recombinant factor infusions 
2 to 3 times per week to maintain minimal therapeutic levels; thus, it is 
demanding and invasive and may negatively impact an individual’s 
quality of life. 
Since genes were initially cloned more than 30 years ago, the curative 
treatment of hemophilia via gene therapy has been a primary goal.  As a 
novel alternative to the current treatment for hemophilia A, gene therapy 
could potentially reduce short-term disability and long-term hemophilic 
arthropathy, reduce incidence of central nervous system (CNS) bleeding, 
eliminate the need for indwelling intravenous catheters or frequent factor 
infusion, and improve patients’ overall quality of life (QoL) and 
functional independence (Colvin, 2008). Indeed, recent studies have 
shown that liver mediated gene therapy with adeno-associated virus 
(AAV) vectors can safely and consistently achieve sustained clinically 
meaningful levels of coagulation factor VIII (FVIII) activity and can 
potentially eliminate spontaneous hemarthrosis. In the Western world, 
 

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the hemophilia population with the greatest unmet need are patients who 
develop inhibitors, or neutralizing antibodies, to FVIII. For these 
patients, a cornerstone of treatment is the eradication of these 
neutralizing antibodies after they develop, and data in animal models of 
hemophilia support the possibility of using gene therapy to carry out this 
eradication.  
SPK-8016 (adeno-associated viral vector with a 
 
) is being developed by Spark Therapeutics for 
the treatment of hemophilia A in patients including those with FVIII 
inhibitors. This initial clinical study (SPK-8016-101) will evaluate the 
safety, efficacy, and tolerability of SPK-8016 in adult males with 
clinically severe hemophilia A and no measurable inhibitor against 
FVIII. The data obtained from this study will inform the design and dose 
selection for subsequent studies of SPK-8016 in other patient 
populations (e.g., those with inhibitors).  
In this SPK-8016-101 study, 4 participants have already received SPK-
8016 at a dose level of 5x1011 vg/kg. Reactive corticosteroids have been 
administered to avoid the loss of factor VIII transgene expression. This 
reactive treatment was initiated ~ 3-6 weeks after vector administration 
in response to a change in the alanine aminotransferase (ALT), FVIII, or 
ELISpot.  Three of the participants required prolonged tapering of daily 
oral corticosteroids (total courses of 43-48 weeks prednisone) to avoid 
and treat late recurrences of apparent hepatocyte-targeted immune 
response. For 2 of these participants, it was clinically necessary to 
initiate a steroid sparing regimen (i.e., 
) to 
aid in the prednisone taper.  
Additional investigation will be needed to determine whether there is a 
short course immunosuppressive regimen that will reliably control 
immune responses in all patients and allow long-term expression of the 
donated gene. Going forward, administration of SPK-8016, beginning at 
the 1x1012 vg/kg dose, will be explored in 2 cohorts chosen based on 
prior clinical studies, non-clinical studies, and input from clinical experts 
in rheumatology, organ transplantation, viral immunology, and bone 
marrow transplantation. 
 
 
 
 
 
 
 
 
 prior to vector infusion in an effort to prevent the immune 
response. In the follow up period, corticosteroids will only be 
administered if triggered by the clinical observation of increased liver 
transaminases or decreased circulating FVIII. The ELISpot will not be 
considered a predictive tool independent of other immune triggers.  
 
 

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  Oral 
corticosteroids will be used when clinically indicated. 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
Study Design: 
SPK-8016-101 is a Phase 1/2a, open-label, non-randomized, dose-
escalation study designed to evaluate the safety, efficacy, and 
tolerability of SPK-8016 in adult males with clinically severe 
hemophilia A and no measurable inhibitor against FVIII.  The data 
obtained from this study will inform the study design and dose 
selection for subsequent studies of SPK-8016 in other patient 
populations (e.g., those with inhibitors).  
In this study, approximately 40 evaluable participants will be dosed 
with a single intravenous infusion of SPK-8016 at one of the following 
doses (up to n=10 per dose cohort and group):   
• Dose group 1 (starting dose): 5x1011 vg/kg   
• Dose group 2 (middle dose): 1x1012 vg/kg   
• Dose group 3 (high dose): 2x1012 vg/kg   
Based on FVIII activity levels observed after any of the planned dose 
levels, a cohort and dose level may be expanded to up to 10 
participants.  
Study SPK-8016-101 is composed of 3 study periods: Screening (up to 
16 weeks), Dosing (24 hours), and Follow-up Observation (52 weeks). 
The total duration of the study for an individual participant is up to 
68 weeks (including up to 16 weeks of screening). A schematic of the 
study design is presented in Figure 1. 
After completion of Study SPK-8016-101, participants will be consented 
to enroll in a long-term follow-up (LTFU) study for an additional 4 years 

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of post-treatment follow-up.  If the LTFU study is not enrolling at the 
time of a participant’s planned Week 52 visit, the participant may remain 
in study SPK-8016-101. The Week 52 procedures are to be performed 
only at the EOS Visit.  The procedures from Week 46, except for 
collection of immunology samples for enzyme-linked immunospot assay 
(ELISpot), will be performed at Week 52 and every 12 weeks until the 
LTFU study is open. At that time, the week 52/EOS visit should occur. 
Study Objectives 
and Endpoints: 
Objectives 
Primary 
• To evaluate the safety and tolerability of SPK-8016. 
• To evaluate the efficacy as evidenced by prevention of 
bleeds and level of FVIII expression with SPK-8016. 
Secondary 
• To evaluate additional parameters associated with SPK-
8016 directed FVIII expression and vector shedding. 
• To characterize the immune response to the vector and 
transgene product. 
Endpoints 
Primary 
• For safety and tolerability: 
o Incidence of adverse events, including 
clinically significant abnormal laboratory 
values. 
o Occurrence of hepatic transaminase elevation 
requiring immunosuppression. 
• For efficacy: 
o Peak and steady-state FVIII activity levels 
assessed by coagulation clotting assays. In this 
study, steady-state levels are based on FVIII:C 
measurements starting 12 weeks post vector 
administration and without the use of 
exogenous FVIII products since vector 
administration.  
o Number of bleeding events (spontaneous and 
traumatic) since vector administration. 
o Number of FVIII infusions since vector 
administration. 

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Secondary 
• Additional kinetic assessments including, but not limited to:  
o Time to achieve peak and steady-state FVIII activity 
levels. 
o Vector shedding of SPK-8016 in bodily fluids. 
• Incidence of immune responses to AAV capsid 
protein and BDD-hFVIII transgene. 
Exploratory 
• Joint assessments 
• Activities assessments 
• Quality-of-life assessments 
• Health-economic parameters 
• Exploratory inflammatory profiling of plasma and 
immune function gene expression of Peripheral Blood 
Mononuclear 
Cells 
(PBMCs) 
after 
vector 
administration (ELISpot, and other exploratory 
biomarkers) 
Study 
Population: 
Adult males with clinically severe hemophilia A 
Inclusion 
Criteria: 
Participants must meet all the following eligibility criteria at screening 
and prior to dosing of SPK-8016 (Day 0) to be eligible for the study: 
1. Be able to understand the purpose and risks of the study and provide 
signed and dated informed consent and authorization to use protected 
health information (PHI) in accordance with national and local 
privacy regulations  
2. Be male and ≥18 years of age  
3. Have clinically severe hemophilia A, defined as: 
a)  <1% (<1 IU/dL) endogenous FVIII activity levels as historically 
documented by a certified laboratory or screening data results; 
OR 
b) 1-2% (1-2 IU/dL) endogenous FVIII activity levels and >10 
bleeding events per year (in the last 52 weeks prior to screening); 
OR 
c) 1-2% (1-2 IU/dL) endogenous FVIII activity levels and on 
prophylaxis  

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4. Have had >150 prior exposure days to any recombinant and/or 
plasma-derived FVIII concentrates or cryoprecipitates based on 
historical data from medical records/history 
5. Have no prior history of hypersensitivity or anaphylaxis associated 
with any FVIII or IV immunoglobulin administration 
6. Have no measurable inhibitor against FVIII at screening (i.e., <0.6 
Bethesda Units); no confirmed history of clinically significant FVIII 
inhibitor and no clinical signs or symptoms of decreased response to 
FVIII administration (Note: Family history of inhibitors is not 
exclusionary, nor is remote documentation (greater than 5 years) of 
a single measurement of Bethesda titer of >0.6 BU that is not 
accompanied by clinical evidence of failure to respond to infused 
FVIII concentrate 
7. Have acceptable laboratory values sampled at screening and 
reviewed prior to Day 0: 
a. Hemoglobin ≥11 g/dL; 
b. Platelets ≥100,000 cells/μL; 
c. Aspartate aminotransferase (AST), ALT, alkaline phosphatase  
< upper limit of normal (ULN); 
d. Bilirubin ≤1.5x ULN (Bilirubin levels above the laboratory’s 
normal range are acceptable in individuals with a documented 
history or laboratory evidence of Gilbert’s Disease);  
e. Creatinine ≤2.0 mg/dL; 
f. Absolute neutrophil count (ANC) ≥ 2000 per mm3;  
g. Fibrinogen antigen ≥ 180 mg/dL for 
in Cohort 2 
8. Agree to use reliable barrier contraception after the administration of 
SPK-8016 until notified by the Investigator or designee. 
 
Exclusion 
Criteria: 
Participants who meet any of the following criteria at screening or prior 
to dosing of SPK-8016 (Day 0) are not eligible for the study: 
1. Have active hepatitis B or C. All participants must be screened for 
both active hepatitis B and C regardless of prior known history.   
a. Screening for hepatitis B: All participants must have a single 
sample collected at screening for each of the following tests: 
HBsAg (hepatitis B surface antigen), anti-HBc (total Hepatitis B 
core antibody), and Hepatitis B virus (HBV)-DNA viral assay 
(nucleic acid test for hepatitis B virus DNA). 
i. 
A participant is not eligible if either HBsAg is positive or 
HBV-DNA is positive/detectable. 

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ii. 
A participant is eligible if the anti-HBc is positive and 
both HBsAg and HBV-DNA are negative, as this would 
be consistent with a prior infection of hepatitis B. Anti-
HBc must be obtained in all participants to discriminate 
between acute infection and possible reactivation of 
hepatitis B during the trial (e.g., in participants with no 
prior history of hepatitis B). 
b. Screening for hepatitis C: All participants, including those 
who have never been treated or who have completed anti-viral 
therapy for chronic hepatitis C, must have a single Hepatitis C 
virus (HCV)-ribonucleic acid (RNA) load assay (also 
referred to as a nucleic acid test [NAT] for HCV RNA) at 
screening.  
i. 
A participant is not eligible if his HCV-RNA load assay 
is positive/detectable.  
ii. 
A participant treated with anti-viral therapy for chronic 
hepatitis C must have completed anti-viral therapy at 
least 6 months prior to screening and must have a 
negative HCV-RNA at the time of screening. 
iii. 
A participant with a documented or self-reported history 
of HCV must have a single negative HCV-RNA at the 
time of screening. 
2. Are currently on antiviral therapy to treat their hepatitis B or C 
3. Have significant underlying liver disease. A participant is not eligible 
with any of the following documented diagnoses, indicative of 
significant underlying liver disease: 
a. Portal hypertension; or 
b. Splenomegaly; or  
c. Hepatic encephalopathy. 
Any participant without any of these pre-existing diagnoses must 
have the following performed at screening: 
a. 
Serum albumin measurement.  A participant is not eligible if 
the serum albumin level is below the lower limit of normal of 
the laboratory;  and  
b. 
Diagnostic 
test 
for 
liver 
fibrosis 
(e.g., 
FibroScan, 
FibroTest/FibroSURE, or AST-to-Platelet Ratio Index 
[APRI]).  A participant is not eligible if any of the following 
findings, which are indicative of fibrosis ≥stage 3, are present: 
i. 
FibroScan score >8.3 kPa units; or 
ii. 
FibroTest/FibroSURE >0.48; or 

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iii. 
APRI >1 
If more than 1 diagnostic test result is available, then the 
FibroScan score will be used as the primary consideration for 
eligibility. 
4. Have serological evidence of human immunodeficiency virus (HIV)-
1 or HIV-2 with CD4 counts ≤200/mm3.  
i. 
Participants who are HIV-positive and stable, with an 
adequate CD4 count (>200/mm3) and undetectable 
viral load (<50 gc/mL) measured at screening, and 
who are on an antiretroviral drug regimen are eligible 
to enroll    
5. Have 
 neutralizing antibody titers ≥1:1 
6. Have a history of active cancer in the past 6 months, chronic 
infection, latent or active tuberculosis, uncontrolled immune disorder 
or other chronic disease that the Investigator and/or Sponsor 
considers to constitute an unacceptable risk 
7. Have been dosed in a previous gene therapy research trial within the 
last 52 weeks or have participated in a clinical study with an 
investigational drug within the last 12 weeks prior to signing the 
informed consent 
8. Have a history of diverticulitis, diverticulosis requiring antibiotic 
treatment, or chronic ulcerative lower G.I. disease that might 
predispose a patient to perforations 
9. Have any concurrent clinically significant major disease (such as 
liver abnormalities, type I diabetes, uncontrolled hypertension, or 
vertebral compression) or any other condition such as active 
infections or COVID-19 or any other unspecified reasons that, in the 
opinion of the Investigator and/or Sponsor, makes the participant 
unsuitable for participation in the study.  
i. 
At the time of screening, the Investigator will consider 
the local geographic and institutional epidemiology of 
coronavirus disease caused by severe acute respiratory 
syndrome coronavirus (SARS-CoV 2) (COVID-19) 
and other infectious pathogens when determining 
suitability of the participant for participation in the 
study, including considering the potential clinical 
relevance of additional screening 
10. Have a planned surgical procedure in the next 12 months requiring 
FVIII prophylactic treatment 

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11. Are unable or unwilling to comply with the schedule of visits and 
study assessments described in the clinical protocol. 
Study Duration: 
The total duration of the study is approximately 68 weeks, including up 
to 16 weeks for screening.  The study will include the following phases: 
a) Screening period (up to a maximum of 16 weeks);  
b) Study Intervention/Dosing day (Day 0); 
Follow-up observation period (52 [±2] weeks).   
Planned Number 
of Participants: 
Up to 40 evaluable participants will be dosed with a single intravenous 
infusion of SPK-8016. 
Number and 
Location of 
Study Sites: 
Sequence of 
Enrollment: 
The study is planned to be conducted at approximately 15 study centers 
(vector-administration centers and/or follow-up centers) worldwide. 
 
Two staggering strategies are employed in this study: 
a) 
The first 2 participants in each dose level will be infused with SPK-
8016 at least 6 weeks apart to mitigate acute safety risk; and 
b) There will be at least 6 weeks of staggering between each dose level.  
The Data Monitoring Committee (DMC) will review at least 6 
weeks of follow-up data from up to 4 participants who have received 
SPK-8016 at a given dose level prior to infusing the first participant 
in the next dose level. 
c) 
Continued enrollment will alternate between Cohort 1 and Cohort 2 
beginning at 1x1012 vg/kg. A stagger of at least 4 weeks will occur 
between the first and the second participants in Cohort 2 (See Figure 
1). 
The dose level for Cohorts 1 & 2 will begin at 1x1012 dose to explore 
immunomodulation.  Cohort 1 will utilize 
 
 begun approximately 48 hours prior to and for 13 weeks 
following vector infusion. Cohort 2 will utilize
 administered prior 
to vector infusion. 
HIV positive individuals with stable CD4+ count >200 mm3 are eligible 
provided all other inclusion criteria are met. However, they will not be 
enrolled as the first 2 participants in Cohort 2. 
Criteria for 
Evaluation: 
Safety Evaluation: 
Safety will be assessed by physical examination, vital signs, adverse 
events, measurement of antibodies against FVIII, immune responses 
against transgene product and/or vector, vector shedding of bodily fluids, 

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laboratory parameter changes over time, and the use of any 
immunosuppressive therapy.  
The infusion of SPK-8016 to the first 2 participants at each dose level 
will be staggered by at least 6 weeks to mitigate acute safety risk.  
Additionally, at least 6 weeks of follow up data from up to 4 participants 
in a given dose level will undergo review by the DMC prior to infusing 
the first participant in the next dose level. 
Pharmacokinetic Evaluation: 
The responses to SPK-8016 will consist of peak and steady-state values 
of circulating vector-derived FVIII activity levels after SPK-8016 
infusion. Additional pharmacokinetic (PK) evaluation of SPK-8016 is 
carried out by analysis of serum urine, semen, saliva for the presence of 
vector DNA.  
Efficacy Evaluation: 
The following information will be collected for efficacy evaluations: 
• Vector-derived factor VIII in circulation (FVIII:C) activity levels 
(including additional peak and steady-state levels) 
• FVIII antigen level  
• Number of FVIII infusions (prophylaxis and/or on-demand) 
- Number of bleeding episodes (spontaneous and traumatic) 
- Annualized FVIII usage (AFU) 
- Annualized bleeding rate (ABR) (spontaneous and traumatic) 
• Joint assessments 
• Activities assessments 
• Quality-of-life assessments 
• Health-economic assessments 
Investigational 
Product 
Description: 
 
 
 
 
 
 
 
 
 
 

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Dose Expansion/ 
Escalation Plan: 
Each dose level is planned to enroll and dose a minimum of 2 
participants. The first 2 participants at each dose level will be dosed at 
least 6 weeks apart to ensure the safety and tolerability of the vector 
administration. 
Dose-Level and Cohort Expansion: 
A. Dose-Level Expansion - After 2 participants are dosed at a given 
dose-level, it may be possible to expand that cohort to up to 10 
participants if there is evidence of FVIII:C increases above 5% of 
normal in either participant by Week 4 post-vector administration.  
This initial expansion will provide additional information about the 
variability of responses within the same dose-level.  
B. New Cohort Expansion – For Cohorts 1 and 2 of the 5 x 1011 vg/kg 
dose, the first 2 participants in each cohort will be infused at least 6 
weeks apart.  After 2 participants are dosed in a cohort, an initial 
expansion of up to 3 participants may occur and, thereafter, an 
additional expansion for up to a total of 10 participants may occur. 
The additional expansion may occur in 1 or more cohorts.  
Dose Escalation:  
The decision to dose escalate will be made by the Sponsor in consultation 
with the DMC to ensure safety. 
Dose escalation to the next dose level may occur under the following 
scenarios, provided there are no safety concerns after at least 6 weeks of 
follow-up data have been reviewed by the DMC. Dose escalation 
through the first 3 dose levels (5x1011 vg/kg, 1x1012 vg/kg, 2x1012 vg/kg) 
may occur in, but is not restricted to, the following scenarios. 
Dose escalation may be considered: 
a) If neither of the first 2 participants achieve FVIII:C above 5% of 
normal by Week 6 post vector-administration; or 
b) If at least 2 participants in a given dose-cohort achieve FVIII:C ≤ 
40% of normal by Week 6 post vector-administration; or 
c) If 3 participants in a given dose-cohort achieve ‘steady-state’ 
FVIII:C  50% of normal by Week 6 post vector-administration. 

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There will be no dose escalation if at least 3 participants in any dose 
level achieve ‘steady-state’ FVIII:C    80% of normal. Steady-state 
levels are based on at least 2 separate FVIII:C measurements, at least 2 
weeks apart, starting 8-12 weeks post vector administration while not 
receiving daily corticosteroid therapy and without use of exogenous 
FVIII product since vector administration. 
After escalation through the first 3 planned dose levels, a fourth 
dose level may be explored in consultation with the DMC. Additionally, 
the Sponsor may decide to further expand the starting dose cohort 
(5x1011 vg/kg), the middle dose cohort (1x1012 vg/kg), or the high dose 
(2x1012 vg/kg) to better evaluate the safety, efficacy, and variability of 
response within a given cohort.   Any decision to add a fourth dose 
level or further expand one of the existing protocol-defined dose levels 
will be made in consultation with the DMC. In consultation with the 
DMC, further dose exploration may be considered if effective 
immunomodulation has been demonstrated in any cohort and/or FVIII 
expression is < 80% of normal 12 weeks following gene transfer in at 
least 2 participants. 
Data and Safety 
Monitoring Plan: 
The independent DMC is composed of at least 3 independent experts in 
hemophilia or immunology. The independent DMC will be responsible 
for reviewing safety and efficacy data, as well as other data, as needed, 
on a periodic basis during the course of the study.  The specifics 
regarding the DMC organization and procedures will be outlined in the 
DMC Charter.  
Statistical 
Methods: 
Statistical analyses will be primarily descriptive in nature.  Summary 
statistics will be presented for all safety endpoints and vector-derived 
FVIII:C activity levels over time after SPK-8016 administration. No 
formal statistical hypothesis testing will be performed.  Various 
exploratory statistical tests may be applied to data generated from this 
study to generate hypotheses to be tested in subsequent trials. 
In general, descriptive statistics including number of observations, mean, 
standard deviation, median, minimum, and maximum will be presented 
for continuous parameters.  Categorical parameters will be presented 
using counts and percentages within each category. 
Interim analyses may be performed after at least 2 participants in a given 
dose cohort complete Week 12.  
 

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Figure 1: 
Study Schema  
 
1. 
 
 
2. The dose level for Cohort 1 and Cohort 2 will begin at 1x1012 vg/kg.  Dose escalation is possible per 
Section 4.1.1. 
 

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Table 1: 
Schedule of Assessments  
 
Screen  
Period  
Three to
Seven 
Days 
prior to 
Day 025 
Day -2 
Dosing Period 
Follow-Up Observation Period (52 Weeks)  
Tests and Assessments 
Screen1 
Weeks -
16 to -1 
Day 0 
Day 
1 
Study Days 3-84 (± 2 days)3, 11 
Study Weeks 14-52 (± 2 weeks) 2, 14 
 
FVIII dose
 
Dose 
Pre-
SPK 
8016 
SPK8016 
Dose 
30 (± 2) min,
2 and 5 hrs 
(±10 mins) 
from stop of 
SPK-8016 
infusion 
 
3 
7 
10 14 17 21 24 28 31 35 38 42 45 49 52 56 59 63 66 70 73 77 84 14 16 18 22 26 30 34 40 46 
52 / 
 EOS2, 23 
Informed Consent 4 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Review Inclusion / Exclusion 
criteria 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics, Medical and 
Hemophilia History 5 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Genotype, HLA, if not known5 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Target joints, Joint Health 
Assessment, Hemophilia Joint 
Health Score 
X6 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 X 
 
 
 
 
X 
Physical Exam, Height, 
Weight3,8 
X7 
 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 X  
 
 
 
 
 
 
 
X 
 
 X  X 
 X X  
X 
Vital Signs 
X 
 
 
 
 
X9 
 
X9 
X9 
X 
 
 
 
 
 
 
 
 
 
 
 
 X  
 
 
 
 
 
 
 
X X X X X X X X X X 
X 
α-fetoprotein (CL) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Liver ultrasound (if indicated)13 
X6 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
HBsAg, anti-HBc, HBV-DNA 
(CL)10 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 X29  
 
 
 
 
 
 
 
 
 
HCV-RNA load assay (CL)10 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV-1/HIV-2 Ag/Ab (CL)10 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CD4+ count, HIV-1, or HIV-2 
viral load (CL)10 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hematology (CL)26 
X 
X 
 
 
 
 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 X  
 
 
 
 
 
 
 
X X X X  X 
 
 
 
 
X 
Clinical Chemistry (CL) 
X 
 
 
 
 
 
 
 
 
X             X         X   X  X     X 
Urinalysis (CL) (using dipstick) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 X 
 
 
 
 
X 
Coagulation – aPTT and FVIII 
Activity (CL, LL), FVIII antigen 
(CL) 
X 
 
 
 
 
X17 
 
 
X 
X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 
X 
Fibrinogen Antigen, Thrombin 
Time, D-Dimer35 (LL) 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
X  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
FVIII inhibitor (CL, LL at 
Screening) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 X  
 
 
 
 
 
 
 
X X  
 
 X 
 
 X  
X 

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Screen  
Period  
Three to
Seven 
Days 
prior to 
Day 025 
Day -2 
Dosing Period 
Follow-Up Observation Period (52 Weeks)  
Tests and Assessments 
Screen1 
Weeks -
16 to -1 
Day 0 
Day 
1 
Study Days 3-84 (± 2 days)3, 11 
Study Weeks 14-52 (± 2 weeks) 2, 14 
 
FVIII dose
 
Dose 
Pre-
SPK 
8016 
SPK8016 
Dose 
30 (± 2) min,
2 and 5 hrs 
(±10 mins) 
from stop of 
SPK-8016 
infusion 
 
3 
7 
10 14 17 21 24 28 31 35 38 42 45 49 52 56 59 63 66 70 73 77 84 14 16 18 22 26 30 34 40 46 
52 / 
 EOS2, 23 
VWF Activity & VWF Antigen 
(CL) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Liver function tests and CRP 
(LL and CL)11 
X11 
X 
 
 
 
 
 
 
X 
X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 
X 
Immunology (CL)25  
 
X 
 
 
 
X 
 
 
 
X 
 X  X  X  X  X  X  X  X  X  X  
X X X X X X X X X X 
X 
ECG (if indicated)12 
X 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Liver fibrosis diagnostic test13 
X 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Lipid panel, if indicated 
(CL)14,26 
X 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
AAV Neutralizing Antibody 
(CL) 
X 
 
 
 
 
 
 
 
 
X 
 X  X  X  X  
 
 
 X  
 
 
 
 
 
 
 
X X  
 
 X 
 
 X  
X 
Vector shedding (CL)15 
X6 
 
 
 
 
 
 
 
 
X 
 X  X  
<<< if indicated >>> 
Immune Profiling30,32 
 
X 
 
 
 
X 
 
X30 
X 
 
X 
 
X  X 
 
X  
 
 
 
 
 
 X  
 
 
 
 
 
 
 X  
 
 
 
 
 
 
 
PAX Gene31,32 
 
X 
 
 
 
X 
 
X31 
X 
 
 
 
X   
 
X  
 
 
 
 
 
 X  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Spare Plasma (CL)21 
X 
 
 
 
 
 
 
 
X 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 X 
 
 X  
X 
Thiopurine Methyltransferase 
(TPMT) (LL) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TB Testing (LL) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SARS-CoV2 Testing (LL)33 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 X  
 
 
 
 
 
 
Infusion with FVIII product16 
 
 
 
X 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Infusion with SPK-801618  
 
 
 
 
 
 
X 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
regimen, Cohort 
226,27 
 
 
 
 
X27 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
regimen, Cohort 126,28 
 
 
X28 
 
 
 
 
 
 
 
 
 
 
PK Profile34 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hem Activities List, Health-
economic Assessment19 
X6 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 X  
 
 
 
 
 
 
 
X X  
 
 X 
 
 X  
X 
Haem-A-QoL, EQ-5D-5L19 
X6 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 X 
 
 
 
 
X 

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Screen  
Period  
Three to
Seven 
Days 
prior to 
Day 025 
Day -2 
Dosing Period 
Follow-Up Observation Period (52 Weeks)  
Tests and Assessments 
Screen1 
Weeks -
16 to -1 
Day 0 
Day 
1 
Study Days 3-84 (± 2 days)3, 11 
Study Weeks 14-52 (± 2 weeks) 2, 14 
 
FVIII dose
 
Dose 
Pre-
SPK 
8016 
SPK8016 
Dose 
30 (± 2) min,
2 and 5 hrs 
(±10 mins) 
from stop of 
SPK-8016 
infusion 
 
3 
7 
10 14 17 21 24 28 31 35 38 42 45 49 52 56 59 63 66 70 73 77 84 14 16 18 22 26 30 34 40 46 
52 / 
 EOS2, 23 
Dispense/Review FVIII Infusion 
Log20 
X6 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 X  
 
 
 
 
 
 
 
X X  
 
 X 
 
 X  
X 
Adverse Events 
X 
 
 
 
 
<<< throughout the study >>> 
Prior and Concomitant 
Therapies and Procedures24 
X 
 
 
 
 
<<< throughout the study >>> 
Unscheduled visit or safety test22 
 
 
 
 
 
<<< as needed >>> 
AAV=adeno-associated virus vector; aPTT=activated partial thromboplastin time; anti-HBc=total hepatitis B core antibody; CD4= cluster of differentiation 4; CL=central laboratory; CRP=c-reactive protein; 
ECG=electrocardiogram; EOS=end of study; EQ-5D-5L=Euro Quality-of-Life Five Dimensions Questionnaire; FVIII=coagulation factor VIII; Haem-A-QoL=Hemophilia Quality of Life Questionnaire; HBV-
DNA=hepatitis B virus-deoxyribonucleic acid; HBsAg= hepatitis B surface antigen; HCV-RNA=hepatitis C virus-ribonucleic acid; HIV-1=human immunodeficiency virus 1; HIV-2= human immunodeficiency virus 
2; HIV-1/HIV-2 Ag/Ab=human immunodeficiency virus 1/human immunodeficiency virus 2 antigen/antibody; HLA= human leukocyte antigen; LL=local laboratory; PK=pharmacokinetics; SARS-CoV-2= severe 
acute respiratory syndrome coronavirus 2;  SPK-8016=study drug in this protocol; TB=tuberculosis; 
; TPMT=thiopurine methyltransferase; VWF=von Willebrand factor 
1. The Screening period may be up to 16 weeks. If the screening period exceeds 16 weeks, then the participant must repeat all screening procedures.   
2. End of Study (EOS) is at the Week 52 visit. If a participant discontinues the study prior to week 52, EOS procedures must be performed within the timeframe of the next scheduled visit.  If EOS does not occur at 
Week 52, see footnote 23. 
3. Any follow-up visit requiring a physical exam must be performed at the study center. All other visits without a physical exam may be performed by a qualified and trained in-home service provider.  
4. Informed consent must be obtained prior to any study-related procedures.   
5. Screening genotype and/or human leukocyte antigen (HLA) samples collected only if not known. Investigator and participant to review age-appropriate vaccinations during the screening period.  
6. Assessments are completed between Screening visit and prior to Dose Day 0.  Lab collection for vector shedding is allowed on Day 0 pre-SPK-8016 dosing. 
7. Height measured only at Screening.  
8. At Day 0, weight obtained from Screening (or the weight obtained from the most recent visit prior to infusion) will be used to calculate the dose of FVIII product and SPK-8016.  Physical Exam is 
comprehensive (not targeted), at all designated visits. 
9. At Days 0 and 1, vital signs (i.e., blood pressure, pulse, respiratory rate, and oral/temporal temperature), are measured after the participant has been resting upright or supine for approximately 5 mins at the 
following timepoints: SPK-8016: Pre-infusion and post-infusion of SPK-8016 (i.e., ±2 min, 2 hrs (±10 mins), and 24 (±1) hr).  
10. Screening serology will be performed as follows:  For all participants: HBsAg, anti-HBc, HBV-DNA; HCV-RNA load assay, HIV-1/HIV-2 Ag/Ab; For HIV-positive participants: CD4+ count and HIV-1/HIV-2 
viral load. LL results may be used for eligibility assessment. 
11. Screening liver function tests (LFTs) and CRP performed by CL and LL.  LL results may be used for eligibility assessment. LFTs on 3-7 days Prior to Day 0 are performed by the local lab only. 
Twice weekly monitoring of AST, ALT, ALP, GGT, LDH, FVIII:C, and FVIII Ag will be collected during Days 3 through 77 post vector-infusion visits.  
12. Screening ECG required for participants > 50 years of age, or if clinically indicated.    

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13. Screening Fibroscan (LL), FibroTest/Fibrosure (CL), or AST-to-Platelet Ratio Index (LL) is required for participants without known pre-existing diagnosis of portal hypertension, splenomegaly, or hepatic 
encephalopathy. For Liver Ultrasound, "if indicated" means "if, in the judgment of the investigating site or the Sponsor, the liver ultrasound is indicated to aid interpretation of the screening evaluation of liver 
fibrosis”. 
14. Lipid panels (total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), triglycerides) for participants with a history of dyslipidemia or 
hypercholesterolemia, or if clinically indicated. 
15. Vector shedding:  polymerase chain reaction (PCR) analysis on serum, saliva, urine, and semen will be collected at Screening or Day 0 (prior to vector infusion) and weekly starting at Day 3 post vector infusion 
and continuing until 3 consecutive samples are negative (classified as: at or below the limit of detection of the assay).   
16. The morning of Day 0, or prior to 
for cohort 2, participants will administer a single prophylactic IV infusion of approximately 50 IU/kg of FVIII product. This may be self-administered and recorded in the 
subject infusion log.  Site assistance is permitted for the FVIII infusion 
17. Day 0 blood samples for FVIII activity, FVIII antigen, and aPTT will be collected pre-SPK-8016 infusion. 
18. Participants will receive a single IV infusion of SPK-8016 for approximately 60 minutes. 
19. Quality-of-life questionnaires (Hemophilia Activities List, Haem-A-QoL, EQ-5D-5L) will be completed by the participants. 
20. Training and dispensation of FVIII Infusion Log at Day 0, collect, review, and dispense at subsequent visits. 
21. Spare plasma will be collected for further coagulation assays, future research, or for clarification of any clinical or laboratory AEs. 
22. Unscheduled visits or safety tests may be performed for safety monitoring purposes or repeat safety assessments.   
23. If the long-term follow-up (LTFU) study is not enrolling at the time of a participant’s planned Week 52 visit, the participant may remain in SPK-8016-101. The procedures from Week 46, except for collection of 
immunology samples for ELISpot, will be performed at Week 52 every 12 weeks until the LTFU study is open. At that time, the week 52/EOS visit should occur. The Week 52 procedures are to be performed 
only at the EOS Visit. 
24. Any concomitant medication that has known hepatoxicity should be discontinued in the first 12 weeks following vector infusion. 
25. Local hematology, local liver function, and central immunology and immune profiling must be obtained 3 to 7 days prior to Day 0. A remote service provider may be used for this visit. Contact the participant to 
verify no changes in the general health status prior to first dose of
 
26. Hematology should be monitored weekly and lipid profile monthly for Cohort 1 and 2 until 
 dosing has stopped. The frequency may be altered based on clinical response. 
27. In Cohort 2, 
 
 following Day 0 and may be considered based on clinical/immune response in consultation with the Sponsor. See Section 4.1.1 
28. In Cohort 1, 
  
29. Participants in cohort 1 and 2 will be retested at Day 84 for HBsAg and anti-HBc.  
30. Immune profiling will be collected prior to the immunomodulation 
 3-7 days prior to Day 0, pre-administration of SPK-8016, and 30 (± 2) minutes, 2 hr (± 10 min), 5 hr 
(±10 min), and 24 (± 1) hours post vector infusion, and Days 7, 14, 21, 28, 56, and Week 16. 
31. PAX gene will be collected prior to the immunomodulation 
, pre- administration of SPK-8016, 5 hr (± 10 min) and 24 (± 1) hours post vector infusion, and Days 14, 28, and 
56. 
32. If an apparent CTL immune response/liver inflammation is observed and triggers the initiation of reactive corticosteroids, please collect additional samples for “Immune Profiling” and “PAX gene” at the 
following times: prior to initiating 
 corticosteroids or oral corticosteroids (if possible, without delaying the initiation of corticosteroid therapy) and then week 1 (± 2 days) and Week 2 (± 2 days) 
following initiation of corticosteroids.  
33. Anti-SARS-CoV2 serology testing at baseline and at week 18. At the discretion of the Investigator, Nucleic acid amplification testing (NAAT) for infection with SARS-CoV2 may be performed 3 to 7 days prior 
to Day 0 (Cohort 2 only) and prior to the initiation of reactive corticosteroids. 
34. If the PK Profile is not available in historical records on the current FVIII product, the participant must undergo a PK analysis locally at Screening or Day 0. 
35. Fibrinogen antigen, Thrombin Time (TT), D-dimer are required at Screening, Day 10 and Day 24 for participants enrolled in Cohort 2. 

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2 
INTRODUCTION 
2.1 
Background 
SPK-8016 is an investigational gene therapy medicinal product that contains 
 
 
 
SPK-8016 is being developed by Spark Therapeutics for the treatment of hemophilia A.  
This clinical study (SPK-8016-101) will evaluate the safety, efficacy, and tolerability of SPK-
8016 in adult males with clinically severe hemophilia A and no measurable inhibitor against 
FVIII.  Data obtained from this study will inform the design and dose selection for subsequent 
studies of SPK-8016 in other patient populations (e.g., those with inhibitors). 
2.1.1 
Hemophilia A 
Hemophilia A is an X-chromosome linked bleeding disorder that primarily affects 1 in 5,000 
male births (Giangrande, 2005; Srivastava, 2013). The severity of disease is characterized by the 
endogenous level of FVIII measured in the plasma.  Severe hemophilia A is defined as a 
coagulation activity of FVIII in plasma (FVIII:C) of <1% (<1 IU/dL) of normal levels.  Factor 
VIII, a single chain glycoprotein pro-cofactor, is composed of 6 putative domains arranged in the 
order of a heavy chain (A1, A2, and B regions) and a light chain (A3, C1, and C2 regions).  The 
light chain (Lollar, 1988) contains binding sites for von Willebrand factor (VWF), activated 
protein C (Walker, 1990), activated factor IX (FIXa), and phospholipid (Foster, 1990).  FVIII 
and VWF circulate in plasma as a non-covalently-linked complex that stabilizes the intrinsically 
unstable FVIII protein.  Observations that the FVIII-VWF complex is dissociated by 
phospholipids and that VWF prevents FVIII from binding to phospholipids and platelets suggest 
antagonism between phospholipids and VWF for binding to FVIII.  Although VWF and FVIII 
are glycoproteins, they have different roles in the initiation and regulation of hemostasis.  VWF 
is necessary for mediating platelet-vessel interactions at the site of vascular injury.  Thrombin 
and FIXa each activate FVIII functions by cleaving the light chain at amino acid residue 1689. 
This cleavage releases FVIII from VWF and allows binding of FVIII to phospholipid resulting in 
the formation of the tenase-activated factor X (FXa) complex. This is the central step of the 
coagulation cascade.  The coagulation cascade has 2 pathways: the Activation Pathway (Intrinsic 
Pathway) and the Tissue Factor Pathway (Extrinsic Pathway) (Makaryus, 2013). The plasma 
factors normally circulate in inactive forms and are activated in a cascade or “waterfall” of 
amplifying reactions (Macfarlane, 1964; Davie, 1964) one after the other until the soluble plasma 
protein, fibrinogen, is transformed into a fibrinous clot.  The blood coagulation cascade is 
illustrated in Figure 2. 

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Figure 2: 
The Blood Coagulation Cascade  
 
Although platelets are critical to the formation of the hemostatic plug, an effective clot cannot be 
formed without adequate levels of pro-coagulant factors.  In normal individuals, the level of 
coagulation factors in the plasma ranges from 50 - 150% (or 50 -150 IU/dL) of the level in 
normal pooled plasma.  Therefore, in hemophilia, clinical features and factor coagulant activity 
define the severity of the disease. 
2.1.2 Clinical Manifestations  
About 70% of newborn babies with hemophilia have a positive family history.  When the 
diagnosis is not suspected based on a positive family history, affected children present with 
bleeding from the umbilical stump, prolonged bleeding after circumcision, bleeding following 
intramuscular immunization, excessive bruising, or rarely with intracranial 
hemorrhage. Individuals with FVIII:C ≤ 2% of normal experience frequent life-threatening 
spontaneous and traumatic bleeding, particularly in joints, soft tissues, and muscles.  When 
inadequately managed, musculoskeletal hemorrhages can lead to recurrent hemarthroses (chronic 
arthropathy) and the development of target joints (the generally accepted criterion is a minimum 
of 3 bleeds into a single joint within a consecutive 6-month period (Blanchette, 2014). The 
inevitable result of such bleeding events is progressive joint damage, leading to disabling 
arthritis with major effects on physical and psychosocial quality-of-life (QoL) and socio-
economic parameters for hemophilia patients (Fogarty, 2011).  Intracranial hemorrhage is a 
leading cause of death among individuals with hemophilia, with a mortality rate of up to 50% in 
adults, as well as in children.  An intracranial hemorrhage can occur after trauma, but as many as 
50% of cases occur spontaneously (Stieltjes, 2005).   
Development of alloantibodies to FVIII (i.e., inhibitors) is the main complication of any factor 
replacement therapy (Goudemand, 2006; Kessler, 1991; White, 2005).  Inhibitory antibodies to 

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FVIII (usually immunoglobulin G subclass 4 [IgG4] antibodies that neutralize the procoagulant 
activity of FVIII or coagulation factor IX [FIX]) are estimated to occur in 20 to 35% of 
individuals with severe and mild-to-moderate hemophilia A, respectively (Gouw, 2013; van den 
Berg, 2013; Mancuso, 2012; Hay, 2011), following exposure to factor replacement therapy.  In 
the presence of these inhibitory antibodies, replacement of the missing clotting factor by infusion 
of FVIII becomes less effective.  Once replacement therapy becomes ineffective, the acute 
management of bleeding requires agents that bypass FVIII activity.  Long-term management of 
inhibitors in hemophilia A typically consists of eradicating the inhibitor through immune 
tolerance.  Therefore, development of inhibitors significantly adds to patients’ disease burden.   
2.1.3 Current Therapies and Prevention for Hemophilia A 
2.1.3.1 Current Therapies 
There is no available cure for hemophilia A.  Factor replacement therapy, purified from human 
plasma, first became available more than 4 decades ago.  Although these FVIII products 
dramatically improved life expectancy and QoL in the U.S.A. and Western Europe, they also 
resulted in exposure of individuals with hemophilia to blood borne viruses – most significantly 
hepatitis B, hepatitis C, and the human immunodeficiency virus (HIV).  HIV sero-conversion 
studies documented that most individuals with hemophilia were infected between 1978 and 1984 
(Eyster, 1985; Ragni, 1986; Ragni, 1987).  In the U.S.A., most patients with severe hemophilia 
who were born before 1987 are HIV positive, and many have already died from complications 
related to the acquired immune deficiency syndrome (AIDS). The majority of persons with 
hemophilia A born before 1987 are affected by hepatitis C virus (HCV) disease which can cause 
chronic and progressive hepatitis, with eventual development of cirrhosis.  Late complications of 
HCV are an increasing cause of death in adults who have been infected for decades (Brettler, 
1990; Darby, 1997).   
In the 1990’s, concerns about viral contamination (Mannucci, 2001) prompted the development 
of high-purity virus-inactivated plasma-derived products and genetically engineered recombinant 
factors with no animal- or human-plasma-derived proteins to minimize the risk of disease 
transmission (Roth, 2001; White, 1998).  Indeed, since the introduction of effective virus 
inactivation procedures, there have been no documented transmissions of hepatitis B and C 
viruses, HIV, West Nile, or malaria (Tabor, 1999),  although no pathogen inactivation process 
has been shown to eliminate all pathogens (such as rare reports of infectious prions and 
parvovirus transmission).  However, these products have not circumvented all the problems of 
protein-based therapies (Mannucci, 1993a; Mannucci, 1993b).  
Current treatment of the disease is based on venipuncture and intravenous administration of 
either plasma-derived or recombinant FVIII (rFVIII) protein replacement home therapy to raise 
the circulating FVIII (FVIII:C) activity level to the lowest effective dose to achieve either 
resolution of bleeding (on-demand treatment) or prevention of bleeding (prophylaxis treatment) 
(Roberts, 1993; Srivastava, 2013; National Hemophilia Foundation, 2007). Venous access via 
peripheral veins remains the preferred option for the administration of FVIII products 
(Komvilaisak, 2006) because it allows a large amount of product to be administered frequently 
as a short infusion using small needles (23-25 gauge).  The frequency of administration of FVIII 
products varies among individuals and is tailored to the individual’s clinical status, taking into 

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consideration the type of bleeding, frequency of bleeding, and goal of treatment for the 
participant.  Both the U.S. National Hemophilia Foundation and the World Federation of 
Hemophilia (Srivastava, 2013) established recommendations of plasma factor levels and duration 
of administration for different types of bleeds based on observations over the years.  
Improvements in FVIII replacement therapy have vastly increased the QoL and life expectancy 
of individuals with hemophilia A; and a recently licensed modified FVIII agent with an extended 
half-life (EHL) (Mahlangu, 2014) provided more convenient dosing options (Lambert, 2007).   
2.1.3.2 Current Prevention 
Chronic arthropathy is the major morbidity of hemophilia resulting from recurrent spontaneous 
bleeds into the joints.  Many studies have shown that, even at high doses, on-demand therapy is 
not effective in preventing arthropathy (Petrini, 1991; Aledort, 1994). Thus, scheduled protein 
replacement to prevent bleeding (prophylactic treatment) as the standard of care for hemophilia 
rather than on-demand treatment was facilitated by the introduction of rFVIII concentrates in the 
early 1990’s.  Prophylactic treatment (regular IV infusions ranging from twice a week up to 
every other day) aims to maintain plasma FVIII levels ≥ 1%, thereby changing the expected 
phenotype from severe to moderate hemophilia (Nilsson, 1992).  Observations of individuals 
with moderate hemophilia, and a generation of clinical research in hemophilia patients treated 
prophylactically with clotting factor replacement, have documented that minimal elevations in 
the levels of normal circulating clotting factor activity >1% are sufficient to prevent bleeding, 
and this has been demonstrated in the Swedish prophylactic studies (summarized in Löfqvist, 
1997; Ljung, 1998; and Nilsson, 1992).   
Prophylactic therapy, which aims to convert the severe phenotype to moderate through regular 
infusions of clotting factor, with 100% adherence to dosing regimen, is more effective than on-
demand treatment at preserving joint health (Manco-Johnson 2007; Gringeri, 2011). It has 
revolutionized health outcomes in hemophilia by enabling affected individuals to participate in 
physical activities (Wang, 2016; Negrier, 2013), and natural history data suggests that FVIII:C 
around 12% of normal levels may be sufficient to protect from spontaneous joint bleeds (Mahdi, 
2015; den Uijl, 2011).  However, regular replacement therapy poses significant challenges for 
the hemophilia community, including the frequency of IV infusions required, necessity to adhere 
rigorously to the prophylactic regimen, variability in individual pharmacokinetics (PK) requiring 
personalized regimens, potential development of neutralizing alloantibodies (“inhibitors”), and 
cost-effectiveness.   
Prophylactic therapy for hemophilia has gradually increased among the adult population in the 
U.S.A. As of 2014, an estimated 85% of children and 63% of adults with hemophilia were on 
prophylactic regimens (World Federation of Hemophilia (WFH) Annual Global Survey 2014, 
2015). However, it is still not universally practiced for several reasons. The expense of 
prophylaxis may be prohibitive; the cost of prophylactic treatment for an adult (70 kg) can be as 
high as $630,630 annually using the currently available EHL FVIII fusion proteins (Croteau, 
2015). Furthermore, the burden of treatment on patients is great.  Prophylaxis with either regular 
or EHL rFVIII protein (half-lives range from 10.8 -19.7 hours) typically requires 2-3.5 infusions 
per week (National Hemophilia Foundation - Medical and Scientific Advisory Council [NHF-
MASAC] Recommendations Document #240). In a 2001 survey of 34 hemophilia patients on 
prophylaxis, only 60% reported infusing at least three fourths of the recommended factor, 

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commonly missing doses due to time commitment and complexity (Hacker, 2001).  Recent 
results from a multi-center study assessing adherence to prophylaxis and clinical outcomes in the 
Netherlands revealed only 43% of the patients adhered to the prophylactic regimen (Schrijvers, 
2016), despite the fact that poor adherence resulted in significantly greater numbers of bleeding 
episodes and lower physical health status (Krishnan, 2015).   
2.1.4 Alternative Therapy for Hemophilia A 
In the case of hemophilia, where a cure is currently not attainable and lifelong therapy is needed, 
QoL is an essential outcome parameter.  All hemophilia prophylaxis studies using health-related 
quality-of-life (HR-QoL) score as an outcome reported a decreased HR-QoL compared with the 
general population and an improvement with prophylactic treatment (Royal, 2002; Fischer, 
2003a; Fischer, 2003b).  Although the World Health Organization (WHO) has advised the 
continuation of prophylactic treatment for life, its establishment has economic and practical 
hurdles.  The goals of an alternative approach are to reduce short-term disability and long-term 
joint damage and to improve patients’ overall QoL and functional independence (Colvin, 2008).   
2.1.4.1 Factor VIII and Protein 
The human FVIII gene is located on the long arm of the X chromosome, at Xq28 (Poustka, 1991; 
Freije, 1992).  It spans 186 kb and consists of 26 exons separated by 25 introns (Gitschier, 1984; 
Toole, 1984). Most of the exons that make up the 9 kb mRNA are small (69-262 bp), with the 
exception of exon 14 (3.1 kb) and exon 26 (1.9 kb), which primarily consist of the 3’ 
untranslated sequence. The resulting 7 kb coding sequence encodes a 2351 residue single chain 
precursor protein. Following cleavage of a 19 amino acid signal peptide, the mature 2332 amino 
acid protein is produced, which has a domain structure of A1-A2-B-A3-C1-C2.  The 3 
homologous A domains bind Ca+2 and are essential for FVIII catalytic cofactor activity 
(Tagliavacca, 1997). There are short acidic sequences (a1, a2, a3) between A1 and A2 and at the 
A2-B and B-A3 junctions, respectively.  These regions are close to important proteolytic 
cleavage sites, contain tyrosine residues that are sulfated, and may affect the interaction of FVIII 
with other components of the coagulation pathway (Mumford, 2002; Pittman, 1992). The large 
central B domain is encoded by exon 14, is heavily glycosylated and is not necessary for activity 
(Pittman, 1993; Eaton, 1986). The acidic region (a3) that follows the B domain contains a major 
VWF binding site (Foster, 1988), and the 2 C domains are responsible for FVIII binding to 
phospholipids (Arai, 1989).  
Single chain FVIII is proteolytically processed to generate a heavy chain that is composed of 
domains A1-A2-B and a light chain that is composed of domains A3-C1-C2.  These chains 
circulate in an inactive state bound to VWF as a heterodimer. Activation of FVIII occurs 
following thrombin cleavages between domains A1-A2 and A2-B, resulting in release of the B 
domain and formation of a heterotrimer containing the A1, A2 and A3-C1-C2 domains.  
The full length FVIII protein is encoded by a 7 kb DNA sequence, which exceeds the packaging 
limit of an AAV vector (~4.7 kb). However, since the 
 is not required for FVIII activity 
(Toole, 1986), the 3.1 kb DNA sequence encoding the 
 can be eliminated, resulting in 
an ~4 kb coding sequence that produces a 
 but active FVIII protein. 
 

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The natural site for the biosynthesis of FVIII has been the subject of debate for decades. 
Although it has been clear that liver cells contribute significantly to circulating FVIII levels, 
since liver transplantation corrects FVIII levels in hemophilia patients (Bontempo, 1987), the 
specific cell type responsible for biosynthesis and secretion of the protein has been controversial. 
Early after isolation of the gene, it was thought that FVIII mRNA and protein were co-localized 
in hepatocytes (Wion, 1985; Zelechowska, 1985). However, based on improved cell separation 
and sorting techniques, it is now clear that liver sinusoidal endothelial cells (LSECs) are the 
natural site of FVIII biosynthesis (Shahani, 2014). At the present time, no rAAV vectors that 
efficiently target LSECs are available. Therefore, 
 
 
  
2.1.4.2 Biology of Adeno-Associated Virus (AAV) Vectors 
AAV is a non-enveloped, replication-defective parvovirus that has not been associated with 
human disease.  AAV vectors are derived from the parent virus by removing all of the viral 
elements except for the inverted terminal repeats (ITR) and inserting the gene or genes of interest 
and their associated regulatory elements (Samulski, 1982; Samulski, 1987). The long-term safety 
of these vectors in humans is unknown; however, AAV vectors have been delivered to hundreds 
of human participants in trials for cystic fibrosis, rheumatoid arthritis, inherited retinal 
degeneration due to autosomal-recessive retinal pigment epithelium 65 (RPE65) gene mutations, 
α1-antitrypsin deficiency, as well as hemophilia, and have been remarkably free of vector-related 
adverse events (Mingozzi, 2011a; Mingozzi, 2013a). Thus, AAV vectors are one of the most 
efficient in vivo gene delivery platforms.  In October 2012, the European Commission granted 
marketing authorization for Glybera® under exceptional circumstances as a treatment for adult 
patients diagnosed with familial lipoprotein lipase deficiency (LPLD) confirmed by genetic 
testing and suffering from severe or multiple pancreatitis attacks despite dietary fat restrictions.  
This was the first AAV-based vector product granted marketing authorization in an International 
Conference on Harmonisation (ICH) region. 
AAV vectors do not require actively dividing target cells to achieve efficient transduction, as 
demonstrated in post-mitotic cells of brain, muscle, liver, and retina in vivo (Maguire, 2008; 
Maguire, 2009; Mingozzi, 2011b; Mingozzi, 2013b; Mingozzi, 2011a).  Also, at least in animal 
studies, there is no immune response directed against the transduced cell, likely because all the 
viral genes have been removed. This absence of immune response accounts, at least in part, for 
prolonged (months to years) transgene expression observed in animals following a single 
administration of an AAV vector.  Several groups have established that AAV efficiently 
transduces hepatocytes following a single administration via the portal vein, hepatic artery, or IV 
route, resulting in long-term (more than 10 years), dose-dependent transgene expression in large 
animals including dogs and NHPs (Snyder, 1997; Nakai, 1998; Xiao, 1998; Jiang, 2006; 

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Niemeyer, 2009; Nathwani, 2011a). A memory CD8+ T cell response to AAV in humans would 
be expected to respond more efficiently to partially degraded AAV capsid peptides displayed on 
the cell surface of transduced hepatocytes in the context of major histocompatibility complex 
(MHC) Class I molecules (Manno, 2006). Indeed, studies document expansion of a population of 
AAV-capsid specific CD8+ T cells after AAV vector infusion into the hepatic artery in humans, 
whereas no such expansion is seen in animals receiving an AAV vector (Mingozzi, 2007).  Thus, 
humans’ previous exposure to wild-type AAV likely accounts for the differences between 
humans and all other species in immune response to capsid.  Although NHPs also have prior 
exposure to wild-type AAV, they fail to mount recall T cell responses to AAV capsid antigens in 
spite of readily detectable AAV capsid-specific T cells at baseline.  Ertl and colleagues have 
shown that AAV capsid-specific T cells from rhesus macaques show marked differences in 
function and differentiation status compared to those found in healthy human adults (Li, 2011), 
which may explain their differential reactivation by AAV vectors. 
2.1.4.3  Gene Therapy as an Alternative Approach 
As a proposed alternative approach, gene therapy may potentially reduce short-term disability 
and long-term hemophilic arthropathy, reduce incidence of central nervous system (CNS) 
bleeding, eliminate the need for indwelling intravenous catheters or frequent factor infusion, and 
improve participants’ overall QoL and functional independence (Colvin, 2008).  Preliminary 
results of a hemophilia B study, sponsored by St. Jude Children’s Research Institute-University 
College London, demonstrate a reduction in the prophylactic use of factor replacement therapies 
in 10 participants who received gene transfer.  Additionally, at least 5 of these participants have 
reduced factor consumption by greater than 90% while remaining free of spontaneous bleeding 
episodes (Nathwani, 2011b; Nathwani, 2014).  
Several features make hemophilia A good model for gene therapy. First, the precise regulation of 
transgene expression is not required.  The therapeutic range is remarkably wide, from >1% to 
150% of normal.  Data from administration of FVIII products into patients with hemophilia A 
demonstrate clearly that levels ≤150% are not associated with ill-effects since the protein 
circulates as a zymogen (inactive precursor).  Second, as stated, >1% circulating FVIII levels 
may provide protection against chronic arthropathy and CNS bleeding.  Patients with levels >5% 
have mild severity and only rarely experience spontaneous bleeding episodes (although they 
exhibit abnormal bleeding in response to hemostatic challenges such as surgery or trauma).  
Third, hemophilia A has available both large and small animal models of the human disease, a 
major asset in efforts to establish an experimental basis for gene therapy. There are well 
characterized, naturally occurring canine models of the disease and genetically engineered 
hemophilic mice (Evans, 1989a; Evans, 1989b; Connelly, 1996; Bi, 1995; Lin, 1997). Fourth, 
determination of therapeutic efficacy is straightforward and unequivocal in the case of 
hemophilia A since plasma levels of FVIII are easy to measure and correlate well with clinical 
disease severity. 
2.2 
 
Rationale 
The primary aim of hemophilia care is to prevent and treat bleeding due to deficient clotting 
factors. Both hemophilia A and B can be treated with recombinant factor replacement leading to 
significant improvement in morbidity and mortality. However, such treatment is extremely costly 

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and is often still associated with clinical complications that include bleeding, particularly 
bleeding into the joints (hemarthrosis). Moreover, this treatment requires recombinant factor 
infusions 2 to 3 times per week to maintain minimal therapeutic levels; thus, it is demanding and 
invasive.  
Since genes were initially cloned more than 30 years ago, gene therapy has been the goal for 
curative treatment of hemophilia.  As a novel alternative to the current treatment for hemophilia 
A, gene therapy could potentially reduce short-term disability and long-term hemophilic 
arthropathy, reduce incidence of CNS bleeding, eliminate the need for indwelling intravenous 
catheters or frequent factor infusion, and improve patients’ overall QoL and functional 
independence (Colvin, 2008). Indeed, recent studies have shown that liver mediated gene therapy 
with AAV vectors can safely and consistently achieve sustained clinically meaningful levels of 
FVIII activity and can potentially eliminate spontaneous hemarthrosis.   
SPK-8016 (adeno-associated viral vector with a 
 is 
being developed by Spark Therapeutics for the treatment of hemophilia A. This clinical study 
(SPK-8016-101) will evaluate the safety, efficacy, and tolerability of SPK-8016 in adult males 
with clinically severe hemophilia A and no measurable inhibitor against FVIII. The data obtained 
from this study will inform the design and dose selection for subsequent studies of SPK-8016 in 
other patient populations (e.g., those with inhibitors).  
In the Western world, the hemophilia population with the greatest unmet need are patients who 
develop inhibitors, or neutralizing antibodies, to FVIII. For these patients, a cornerstone of 
treatment is the eradication of these neutralizing antibodies after they develop, and data in animal 
models of hemophilia support the possibility of using gene therapy to carry out this eradication. 
2.2.1 Description of SPK-8016 
SPK-8016 is a rAAV vector composed of 
 and  
 
. The full length FVIII protein is 
encoded by a 7 kb cDNA sequence, which exceeds the packaging limit of an AAV vector 
(~4.7kb).  Thus, the Sponsor has chosen 
 
 
 
A number of measures have been taken to improve the expression and safety of the transgene 
product compared with the previous vector iterations tested by the Sponsor or by others (Figure 
3). 

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a) Capsid: 
• 
 
   
b) Cassette: 
• 
 
 
• 
 
 
 
 
Figure 3: 
Schematic of SPK-8016 vector genome 
It has been previously demonstrated that excess empty capsids may adsorb low-level neutralizing 
antibodies and non-neutralizing antibodies, permitting liver transduction after peripheral vector 
infusion even in their presence (Mingozzi, 2013b). This may be particularly important at lower 
vector doses.  This hypothesis is consistent with clinical data from previous hemophilia B gene 
therapy trials in which the presence of empty capsids in the formulation correlated with higher 
expression levels at low vector doses (Monahan, 2015; Nathwani, 2014). For this reason, 
 
, comparable to the Sponsor’s recent clinical trial (Investigational New 
Drug [IND] 
) for hemophilia B (High, 2016), as well as the Sponsor’s ongoing SPK-8011-
101 clinical study in patients with severe hemophilia A. 
SPK-8016 is manufactured according to good manufacturing practices (GMP) guidelines and 
will be administered to male individuals with severe hemophilia A (Section 5.1). 
2.2.2 Summary of Non-Clinical Experience with SPK-8016 or Other Relevant Spark  
AAV Vector 
, another vector developed by the Sponsor 
and currently under clinical investigation in a Phase 1/2 study for the treatment of hemophilia A 
(NCT03003533, 
). 
 
 

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 Given the 
similarities between the 2 vectors, non-clinical studies with SPK-8011 are relevant to support the 
safety and potential efficacy of SPK-8016 in humans and are described below. 
As discussed in the Investigator’s Brochure (Section 3.1), the non-clinical testing for SPK-8016, 
as well as other Spark gene therapy vectors for hemophilia A (e.g., SPK-8011), has been 
complicated by 2 major observations:  
• 
 transduces mouse hepatocytes poorly, yielding extremely 
low levels of liver-directed transgene expression following intravenous administration. 
Consequently, mouse models are inappropriate to evaluate the safety of hepatic transgene 
expression derived from vectors of this AAV serotype.   
• 
 
 
 
 
 
Based on these findings, it was necessary to adopt a nonconventional testing strategy that 
involved evaluating the safety of sustained expression of the transgene (hFVIII) products using 
 in 1 species (mice) and the safety of the novel 
 in 
another species, NHPs, as it is not possible to evaluate the safety of both sustained hFVIII 
expression and capsid with a single vector in a single species. 
Specifically, for SPK-8016, 2 non-GLP safety and pharmacology studies were performed in 
cynomolgus macaques to evaluate the safety of the vector capsid and compare short-term 
potency of different Spark AAV vectors.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Preliminary information on the biodistribution of the 
 capsid in NHPs 
demonstrates that the vector disseminates mostly to the liver and spleen, with more than 1000-
fold less vector observed in other tissues.  A separate study designed to evaluate the potential for 
germline transmission of the 
 
was performed in rabbits using a vector 
encoding the hFIX transgene.  The 
 showed very limited distribution to 
semen, even at doses as high as 1x1013 vg/kg. This pattern differs from other vectors investigated 
including AAV2, AAV8, and 
 potentially making this rAAV capsid safer, in 
terms of genotoxicity, than the others. 
2.2.3 
Summary of Clinical Experience with SPK-8016 or Other Relevant Spark  
AAV Vector 
This study (SPK-8016-101) is the first clinical study to administer SPK-8016 to humans. 
However, 
 
, has been administered to 17 
patients with hemophilia A in an ongoing clinical study (SPK-8011-101; a summary of 
preliminary results is provided in Section 2.2.3.1). Additionally, gene therapy with rAAV vectors 
has been administered to humans in more than 100 gene transfer clinical trials for a wide range 
of indications, including multiple studies in hemophilia A and B (George, 2017; Rangarajan, 
2017; Miesbach, 2017; Pasi, 2020; Nathwani, 2014; Nathwani, 2011b; Manno, 2006; Monahan, 
2015; Mingozzi, 2011a; Mingozzi, 2011b).  
 
 

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. 
 
 
 
 
 One participant did not require immunosuppressive 
therapy. No participant developed FVIII inhibitors. No serious adverse events (SAEs) or increase 
in liver transaminases above the upper limit of normal (ULN) related to the vector were 
observed. 
2.2.3.1 
Summary of Overall Clinical Experience with AAV-Mediated Gene Therapy 
Based on early experience with SPK-8016, SPK-8011, as well as other AAV-mediated gene 
transfer trials for hemophilia A and B, the following preliminary observations can be made:  
• AAV-based strategies for hemophilia have resulted in sustained expression of clotting 
factor at levels sufficient to reduce or eliminate the need for clotting factor infusions after 
a single IV infusion of the AAV-mediated gene. 
• Neutralizing antibodies against FVIII or FIX have not been reported for any participant to 
date. 
• 
 
 
 
 
 
. 
• 
 
 
 
 
 
 
 
 
 
 
• 
 
 
 
 
 
  

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Although the early results are promising, additional investigation is needed to determine whether 
there is a transient immunosuppressive regimen that will reliably control immune responses in all 
patients and allow long-term expression of the donated gene.  Based on prior clinical studies, 
non-clinical studies summarized here, and input from clinical experts in rheumatology, organ 
transplantation, viral immunology, and bone marrow transplantation, the 2 prophylactically 
administered immunomodulatory regimens shown in Figure 1 will be evaluated. 
 
 
 
 
 
 
 
 
  
2.2.4 Summary of Non-clinical and Clinical Experience with 
 
In order to test whether 
 
 
 a non-good laboratory practice (GLP) study in NHPs (Study# 
IMM_PC2019_001/Covance 8400655) was performed. One of the aims of the study was to rule 
out the possibility of 
blockade increasing the percent of animals developing anti-transgene 
antibodies, as observed with other immunosuppressive drugs (Mingozzi, 2007). However, since 
virtually all NHPs will develop neutralizing antibodies against human FVIII following gene 
transfer (McIntosh, 2013), this assessment would be precluded if a FVIII cassette were used. To 
this end, FIX was selected as the transgene of choice, as only approximately one third of NHPs 
develop antibodies against AAV-derived human FIX. An AAV vector serotype, 
 
was used to express the human coagulation factor IX (hFIX) transgene in the livers of healthy 
male cynomolgus macaques under the control of a liver-specific promoter. Ten Spark100-
seronegative animals were divided into 2 experimental groups (5 per group). One of the groups 
received 
 1 day prior to vector infusion. 
 
 
 
 
 
  
No animals demonstrated hypersensitivity or anaphylactic reactions following administration of 
either 
 or 
.  Animals were bled every week for analysis of safety 
endpoints and hFIX transgene expression levels, along with monitoring 
 
 The study had a follow up duration of 13 weeks post vector infusion. All animals survived 
to their scheduled sacrifice.  The development of subacute decreases in neutrophil counts 
following single dose 
 has been observed in human healthy volunteers; no animals in this 
study developed neutropenia or thrombocytopenia below baseline. Two of 5 animals developed 
isolated transient elevations of liver transaminases (<2 x upper limit of normal (ULN)) which 
resolved without intervention without evidence of hepatic obstructive disease 

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(hyperbilirubinemia) or hepatic insufficiency. Importantly, clinical and anatomic pathology 
examination of the liver and a panel of 40 organs and tissues showed no macroscopic or 
microscopic findings in animals receiving the 
 vector alone or in 
combination with 
.  Hepatic transduction as assessed by vector genome copy number in liver 
(at sacrifice) and by circulating hFIX antigen levels (weekly throughout the study) was not 
significantly different in the animals that received 
 compared to controls. 
, and in general innate immunity driven by the AAV capsid (Hösel, 2012; Kuranda, 2018) 
and DNA payload (Martino, 2011; Rogers, 2017), are key triggers of AAV vector 
immunogenicity including adaptive humoral and cellular responses.  Several observations from 
this study suggest that animals that received 
 
 
 
 
 
 
 
  
With regard to cellular immune responses, prior studies performed using human CD8+ and 
CD4+ T lymphocytes identify TNFα as one of the main cytokine signatures of T cell activation 
in response to AAV (Kuranda, 2018). Thus, peripheral blood mononuclear cells (PBMC) from 
control animals and 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2.2.5 
Summary of Non-Clinical and Clinical Experience with 
 
Pre-clinical studies of hepatic artery administration of AAV8-hAAT-hFIX16 were performed in 
rhesus macaques; in these studies, vector was given alone or together with a short course of 

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immunomodulation to assess the potential safety and efficacy of the approach. 
 
 
The 
 
 and contained the wild type copy of the hFIX cDNA. 
 
 
 
 
The regimen of 
 
 
 
The side effect profile was acceptable and there were no SAEs on this regimen. The 
side effects of immunomodulatory drugs are well described (Post, 2005; Perry, 2005) and 
include the risk of hepatitis virus reactivation (Savas, 2007; Melon, 2005; Lalazar, 2007; 
Francisci, 2006; Calabrese, 2006) and the occurrence of B cell lymphoproliferative disorders 
(Gross, 1999; Pascual, 2007; Bakker, 2007). 
 
 
 
 
 
 
 
 
 
  
 
  
2.3 
Benefit/Risk Assessment 
2.3.1 Risk Assessment 
The following are potential risks with the administration of any rAAV gene therapy for 
hemophilia and they will be monitored in this study.  Participants should be advised to notify the 
Investigator(s) immediately and/or seek immediate emergency care, depending on the severity of 
the reaction, if any symptoms occur. 
2.3.1.1 Allergic Reaction or Anaphylaxis 
There has been 1 report of symptoms associated with SPK-8011 (rAAV-FVIII) infusion, 
occurring following SPK-8011 vector infusion at a dose of 2x1012 vg/kg. Nonserious events 
including vomiting, pyrexia, back pain, and myalgia were reported in 1 participant; events started 
to resolve within 12 hours (per Investigator), all completely resolved in 1 to 3 days with 
acetaminophen (High, 2018).  Participants should be informed of early symptoms and signs of 

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hypersensitivity reactions, including hives, generalized urticaria, angioedema, chest tightness, 
dyspnea, wheezing, faintness, hypotension, tachycardia, and anaphylaxis.  If such an event 
occurs, the participant should be instructed to seek immediate medical care. 
2.3.1.2 
Inhibitor Development 
There is a possibility that participants infused with SPK-8016 will develop inhibitors to FVIII, 
although inhibitor development has not occurred in any participants who have received AAV2-
hFIX, AAV8-hFIX (Nathwani, 2014), AAV8-hFIX19 
 or BAX-335 (under 
National Clinical Trial (NCT) 01687608).  In addition, inhibitor development has not been 
reported in any of the 17 participants in the Sponsor initiated Hemophilia A gene therapy study 
SPK-8011-101. Moreover, no inhibitor development has been reported in the recent BioMarin 
Hemophilia A gene therapy trial of an AAV5-BDD-hFVIII (BMN-270) (Rangarajan, 2017; Pasi, 
2020).  Inhibitor development most often occurs within the first 20 infusions of FVIII 
replacement therapies.   The likelihood of participants in this study developing inhibitors will be 
minimized by including only individuals who have not made inhibitor antibody to FVIII, despite 
prior heavy exposure to the FVIII infused protein (i.e., those who have had greater than 150 
exposure days to FVIII concentrates).  Blood samples will be taken at regular intervals to 
monitor for inhibitor formation during the study and archives will be maintained for possible 
future analysis should inhibitor development occur. 
2.3.1.3 Elevation of Hepatic Transaminases 
None of the 4 enrolled participants in study SPK-8016-101 experienced adverse events of 
transaminitis related to SPK-8016.  Of the 17 participants enrolled in the Sponsor’s similar 
hemophilia A gene therapy trial SPK-8011-101, 5 (29%) reported SPK-8011-related adverse 
events of increase of ALT/aspartate aminotransferase (AST) with severity ranging from mild to 
moderate in the higher dose groups (2 in the 1.5x1012 vg/kg dose cohort, and 3 in the 2x1012 
vg/kg dose cohort); 1 of the events in the 2x1012 vg/kg dose was reported as a SAE. All of them 
were resolved with steroid intervention. The cause of a transient and asymptomatic hepatic 
transaminase elevation (i.e., elevated ALT and/or AST) observed in earlier liver-directed AAV-
mediated gene transfer clinical trials has not been established.   However, some individuals have 
developed dose-dependent T cell responses to AAV capsids, in the context of elevation in 
transaminases, and the presumptive mechanism is a memory CD8+ T cell response to peptides 
derived from the AAV capsid of the vector (Mingozzi, 2007).  
 
 
 
 
 
 
  
In a recent AAV-based gene therapy Phase 1/2 clinical trial investigating AT132 for X-linked 
myotubular myopathy (XLMTM, NCT03199469 [ASPIRO]), 3 child participants who were 
older age and heavier weight, had pre-existing liver disease, and were from the highest tested 
dose cohort of 3x1014 vg/kg AT132 experienced SAEs of hyperbilirubinemia. All 3 SAEs 

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resulted in fatal outcomes caused by progressive liver dysfunction followed by bacterial 
infections, sepsis, and GI bleeding. The high AT132 dose of 3x1014 vg/kg is the highest tested 
dose for any AAV-based gene therapy to date. Among the 6 child participants treated at  
1x1014 vg/kg, including 4 with a previous history of hepatobiliary disease, none have yet 
developed hepatic SAEs despite being years out from treatment (High-dose AAV, 2020; 
Audentes Therapeutics, 2020a; Audentes Therapeutics, 2020b). Note that the patient population 
(adults in the SPK-8016-101 study versus children in the ASPIRO study) and disease severity 
(Hemophilia A is a less severe disease than XLMTM) are quite different between Study SPK-
8016-101 and the ASPIRO study. Moreover, individuals with medically significant underlying 
liver disease will be excluded from participating in the SPK-8016-101 study and the starting dose 
that was tested in the study (5x1011 vg/kg) is 3 orders of magnitude lower. 
2.3.1.4 Anti-AAV Neutralizing Antibody Development 
As is expected after systemic administration of AAV vectors, all 4 participants (100%) in study 
SPK-8016-101 who were treated with SPK-8016 developed neutralizing antibodies to the AAV 
capsid. This risk seems to have no immediate impact on the expression of FVIII produced by the 
current AAV gene therapy, but development of neutralizing antibodies to AAV could potentially 
preclude the chance of a participant receiving another AAV gene therapy in the future. 
2.3.1.5 
Bleeding Episodes 
The proposed dose levels of SPK-8016 may not be sufficient to raise the vector-derived FVIII:C 
levels to a therapeutic level for controlling and preventing bleeding episodes when the 
participants halt their prophylactic (factor or non-factor replacement) treatment after vector 
administration.  Clinical experience suggests most patients can stop routine prophylaxis 
approximately 2 to 4 weeks after receiving SPK-8016 based on the expression of FVIII from the 
transgene.  Participants will be advised to treat their bleeding events with their usual FVIII 
products during the follow-up observation period.  Although bleeding (the nature of the disease) 
is not considered an adverse event, participants will record the dates of bleeding events, the type 
and location of the bleed, and the FVIII product and dosage used to treat the bleeding episode in 
the infusion log (Section 13). 
2.3.1.6 
Possible Side Effects from Corticosteroids 
To mitigate the potential for harm due to cytotoxic T-lymphocyte (CTL) response against the 
capsid and to maintain endogenous FVIII expression, corticosteroids are allowed in the current 
protocol for participants who develop elevated transaminases and/or declining FVIII activity.  
Corticosteroids for the interruption of autoimmune-mediated inflammation most commonly have 
been given as daily oral therapy (e.g., prednisone or prednisolone) or as pulses of intravenously 
dosed 
(Gordon, 2018).  
Participants with elevated hepatic transaminases will be monitored closely to minimize the risk 
of side effects, to utilize the lowest effective dose and to shorten the duration of the 
corticosteroid therapies by tapering as soon as there is evidence of resolution of hepatic 
transaminase elevation and stabilization of FVIII. The disappearance of capsid-specific T cells, 
as measured by ELISpots, from the peripheral blood is an exploratory assay and may or may not 

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signal an immune response. While on corticosteroids, participants will also be monitored for side 
effects including opportunistic infections.   
Corticosteroids such as 
, prednisolone, and prednisone have a number of 
well-described side effects including: hypertension, edema and swelling, tachycardia, congestive 
cardiac insufficiency, alterations in serum electrolytes, hyperglycemia, pain, increased blood 
urea nitrogen (BUN) and creatinine, osteoporosis and avascular necrosis of bone, decreased 
resistance to infection, cataract development, dizziness, trembling, emotional instability, 
insomnia, nausea, vomiting, weight gain, and elevated intraocular pressure.  
Populations at special risk for side effects from prolonged and/or high dose corticosteroid use 
include individuals with pre-existent osteoporosis (in particular vertebral osteoporosis), brittle 
diabetes, labile hypertension, obesity, and emotional instability. The use of alternative or 
combined immune modulating agents may allow lower exposure to corticosteroids (also 
discussed below in Section 2.3.1.7). 
Steroid-associated and nonserious adverse events (e.g., cushingoid appearance, candida 
infection, swelling face, tooth infection, increased blood glucose, and fatigue) have been reported 
in 3 of the enrolled participants in study SPK-8016-101 due to excessive long-term use of 
corticosteroids. The planned 
 in future enrolled 
participants are aimed to reduce the need for corticosteroids to avoid these side effects.  
2.3.1.7 
Risks Associated with Additional Immune Modulating Agents 
The potential side effects of corticosteroids increase in prevalence as treatment is prolonged. For 
this reason, inflammatory conditions that respond to corticosteroid monotherapy are frequently 
treated instead with combined immune modulating agents to achieve anti-inflammatory efficacy 
while limiting the quantity and/or duration of corticosteroid exposure. An example of such a 
“steroid-sparing approach” that has been clinically validated is provided in the guidelines for the 
treatment of autoimmune hepatitis recommended by the American Association for the Study of 
Liver Disease (AASLD) and by the European Association for the Study of the Liver (EASL).  
Both the AASLD and the EASL provide recommendations 
 
to prednisone/prednisolone therapy to provide therapeutic benefit while using lower steroid 
doses and/or to successfully taper off corticosteroids. Additional immune modulating agents, 
including 
, have been used to overcome suboptimal response to 
corticosteroid monotherapy or 
 combined therapy in the setting of 
immune-mediated hepatic inflammation. Participants who are treated with 
, 
, or other immune suppressive agents will be monitored closely as described above 
(Section 2.3.1.6) to assess side effects and to enable use of the lowest dose and shortest course of 
immune modulating agents.  
Use of any of these drugs with AAV, concomitant medications and/or other immunomodulatory 
drugs may increase the risk of hepatotoxicity.   
Use of any immunosuppressants may increase the risk of infection. This may include community 
acquired viruses such as influenza and COVID-19, as well as opportunistic infections.  
Immunosuppressive therapy may enhance adverse/toxic effects of live vaccines and may 
diminish the protective effect of vaccines. Due to the potential that trial participants may receive 

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an immunosuppressive medication during the SPK-8016-101 trial, it is suggested that the 
Investigator and participant review and complete all age-appropriate vaccinations during the 
screening period and at least 4 weeks prior to the planned day of administration of SPK-8016. If 
that patient is currently receiving immunosuppressive medication, timing of vaccinations could 
be adjusted around the patient’s course of immune suppressant treatment. 
2.3.1.7.1 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
  
2.3.1.7.2 
 
 
 
 
 
 
 
 
 

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2.3.1.7.3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
. 
 
 
 
 
 
 
 

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2.3.1.7.4 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  

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2.3.1.7.5 
Immune Suppressive Medications and Risk of Infections, including COVID-
19 Risk 
Immune suppressive medications including 
 
 used alone or in combination, may increase the risk of bacterial, mycobacterial, 
fungal and viral infection. This may include community acquired viruses such as influenza and 
COVID-19 as well as opportunistic infections. Extensive information regarding the risk of 
contracting COVID-19 and the clinical course of COVID-19 for individuals taking immune 
suppressant medications and individuals with immune suppression is currently unknown (Guan, 
2020; Richter, 2016; Ni, 2019; Bello, 2012; Russell, 2020a; Russell, 2020b; Favalli, 2020). The 
chronic use of corticosteroids and biological immune suppressive agents has been associated 
with increased rates of influenza infection (an example of epidemic pneumonia), so increased 
vigilance regarding diagnosis is particularly important because symptoms of infection, including 
fever, may be masked by the medications (Favalli, 2020).  The clinical course of COVID-19 
infection including morbidity and mortality for individuals taking immune suppressant 
medication is the subject of prospective investigation in clinical trials (Richter, 2016; Russell, 
2020a; Russell, 2020b; Favalli, 2020). Corticosteroid use in the treatment of the Acute 
Respiratory Distress Syndrome (ARDS) of influenza has been associated with increased 
mortality and increased morbidity including secondary bacterial and fungal infections (Ni, 2019; 
Favalli, 2020; Russell, 2020a; Russell, 2020b). Although corticosteroids were employed as a 

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potential supportive therapy in the treatment of the ARDS associated with coronavirus outbreaks, 
SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV), clearance 
of the virus and steroid-associated complications resulted, without improved mortality. Several 
clinical trials are currently examining the potential therapeutic value of 
 and other biological 
immune suppressive drugs in suppressing the cytokine release syndrome caused by SARS-CoV2 
that drives ARDS, however, results are not yet available to guide current management (Russell, 
2020a; Russell, 2020b).  
As clinical evidence accrues, it is advised to consider participants using immune suppressive 
medication in the SPK-8016 clinical trial program to be at increased risk of infection (clinically 
susceptible) including pneumonia infection with community acquired influenza and COVID-19. 
Increased vigilance for signs and symptoms consistent with a diagnosis of COVID-19 is 
warranted because symptoms of infection, including fever, may be less clinically evident 
(Favalli, 2020) while taking immune suppressive medication. Careful clinical management 
should include instruction regarding COVID-19 preventive measures, e.g., social distancing and 
hand washing hygiene (Public Health England, 2020; National Health Service, 2020). 
Investigators along with their SPK-8016-101 clinical trial participants should consider 
individualized approaches to social/behavioral interactions as well as clinical visits and 
treatments consistent with national, local, and institutional COVID-19 prevention guidance (US 
Food and Drug Administration, 2020).   
2.3.2 Potential Benefits 
The main purpose of study SPK-8016-101 is to evaluate the safety of SPK-8016 at 3 or 4 
different dose levels.  It is not known if higher dose levels will raise the activity level of FVIII in 
humans, but, based on prior clinical experience in hemophilia B and non-clinical experience in 
NHPs with SPK-8016, the dose levels to be tested may result in detectable FVIII:C.  Current 
commercially available FVIII concentrates exhibit an in vivo half-life (ranging from 10.8 to 19.7 
hours) that requires IV injections approximately every 2 to 3.5 days for effective prophylactic 
treatment (NHF-MASAC Recommendations Document #240).  SPK-8016 has the potential to 
drastically reduce this frequency of factor administration in the prevention of bleeding episodes. 
Additionally, data generated in this study will generate important scientific insights about rAAV-
mediated gene transfer for hemophilia. 
2.4 
Rationale 
Published data from Nathwani and colleagues (Nathwani, 2014) of an AAV8-mediated self-
complementary hFIX gene transfer trial for severe hemophilia B demonstrated long-term 
expression of FIX activity levels with mean (±SD) of 5.1 ±1.7% of normal in 6 participants at 
the highest dose level of 2x1012 vg/kg, over a median period of 3.2 years after vector 
administration.  No vector- and procedure-related safety concerns were observed in the initial 
study or the 3-year LTFU.  Four of these 6 participants developed an asymptomatic rise in 
hepatic transaminases between weeks 7 and 10 after vector administration, which required a 
short, approximately 8-week tapering course of prednisolone therapy to prevent loss of FIX 
transgene expression.  The recent results from an AAV5-mediated 
 
 gene transfer trial for severe hemophilia A 
demonstrated FVIII activity levels that were sub-therapeutic (0.6% of normal) over 34 weeks in 

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1 participant dosed at 6x1012 vg/kg (low dose-level),  levels of 2.3% of normal over 26 weeks in 
1 participant dosed at 2x1013 vg/kg (middle dose-level), and levels of 5 - 271% of normal in 7 
participants at  6x1013 vg/kg (high dose-level)  in the first year after vector infusion (Rangarajan, 
2017). However, longer-term follow-up has shown a gradual decline in FVIII levels in the 
circulation. Thus, at year 1, 6 out of 7 participants showed FVIII within the normal range, and 1 
out of 7 displayed mild hemophilia. At year 3, 1 out of 7 remains in the normal range, 5 out of 7 
are in the mild hemophilia range, and 1 out of 7 is in the moderately severe hemophilia range.  
At the year 3 median of 19.9%, half the participants are either at or approaching expression 
levels associated with bleeding events. In that trial, in order to avoid the asymptomatic rise in 
hepatic transaminases concomitant with the loss of transgene FVIII activity levels, all 7 
participants receiving the highest dose of BMN-270 received a course of steroid therapy. Six 
participants received prophylactic corticosteroid regimens starting 3 weeks after vector 
administration and had exposure from 15 weeks to at least 35 weeks in 1 participant (Pasi, 2020).  
In the Sponsor’s similar gene therapy trial SPK-8011-101, 17 participants have been dosed with 
SPK-8011 at the following dose levels: 5x1011, 1x1012, 2 x 1012, and 1.5x1012 vg/kg. Two 
participants in the low dose cohort, 5x1011 vg/kg, did not mount an immune response and have 
sustained FVIII expression of ~5% of normal. Three participants in the second dose cohort, 
1.0x1012 vg/kg, express FVIII ~ 4-23% of normal, with 2 of these participants receiving 
corticosteroids post vector infusion for a declining FVIII level. In the 2x1012 vg/kg dose cohort, 
7 out of 9 participants demonstrate FVIII expression ranging from ~ 3-22% of normal. The 2 
participants who lost expression have returned to prophylaxis uneventfully. Prophylactic 
corticosteroids were explored in 2 participants at 2x1012 vg/kg. In both, early initiation of 
corticosteroids resulted in a transient supraphysiologic increase in FVIII levels to greater than 
200% of normal. Based on discussions with the study’s independent DMC, an intermediate dose 
cohort (1.5x1012 vg/kg) was initiated.  Reduction of the vector genome dose was successful in 
avoiding supraphysiologic levels of FVIII, but in all participants where corticosteroids have been 
started prior to 6 weeks post vector infusion, tapering off has proven difficult, and consequently, 
prolonged courses have been required, with associated steroid related side effects. In these 
participants, steroid sparing agents have allowed a taper of the prednisone. Because of the need 
for a prolonged steroid taper when steroids are administered beginning at 4 weeks, which is not 
observed with later initiation of steroids, prophylactic steroids will be avoided going forward. 
Optimal vector dose and immune suppression regimens, including alternatives to daily oral 
steroid use, are being explored to optimize predictable, safe, efficacious and durable FVIII 
expression (George, 2019).  
A vector that can safely and consistently achieve sustained high levels of FVIII activity and 
potentially eliminate spontaneous hemarthrosis are the goals for the incorporation of gene 
transfer into hemophilia care. 
The objective of this study is to determine the safety, tolerability, and efficacy as evidenced by 
prevention of bleeds and level of FVIII expression of a single IV infusion of SPK-8016 in 
individuals with hemophilia A. 

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2.5 
Rationale for Dose and Schedule Selection 
 
 
 
 In non-clinical studies, conducted in both NHPs and in cell culture, SPK-8016 resulted in 
increased expression of B domain-deleted FVIII, ranging from 2.6- to 4.4-fold higher in in vivo 
experiments, and 3.7- to 6.6-fold higher in in vitro experiments. With this promising nonclinical 
data support, the Sponsor hypothesizes that SPK-8016 will be able to deliver the lowest dose 
possible with the lowest toxicity and with sufficient FVIII expression for hemophilia A patients. 
Table 2 provides Factor VIII plateau levels in Phase 1 Study SPK-8011-101 from 5 participants 
infused at doses of 5x1011 vg/kg or 1x1012 vg/kg. All factor levels are measured by one stage 
clotting assay in the central laboratory. A chromogenic assay using the Biophen FVIII:C 
chromogenic assay (Hyphen BioMed) gives levels that are similar to or lower than the one stage 
assay. Plateau levels are determined by averaging all values obtained over the period from 12 
weeks following vector infusion until the most recent value (range 142-182 weeks).   

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Table 2: 
Factor VIII Plateau Levels in Study SPK-8011-101 
Participant number 
Dose 
Date infused 
Plateau FVIII by one stage 
clotting assay central lab 
5x1011 vg/kg 
11% 
5x1011 vg/kg 
7.3% 
1x1012 vg/kg 
4.4% 
1x1012 vg/kg 
9.9% 
1x1012 vg/kg 
22% 
Of note, the range is similar for the 2 doses, which is not surprising since there is only a 2-fold 
difference in dose. However, the data at the 2 doses provides evidence for the estimate of levels 
of expression that can be obtained at the 5x1011 vg/kg dose in the SPK-8016-101 study.  The 
highest-fold increase from the in vitro experiments comparing SPK-8011 and SPK-8016, and the 
higher plateau level obtained at the 5x1011 vg/kg dose with SPK-8011 are used as references to 
predict that the plateau FVIII level would be approximately 7 x 11% = 77%, comfortably within 
a normal range.   
Thus, planned dose levels include 5x1011, 1x1012, and 2x1012 vg/kg of SPK-8016. 
At the starting clinical dose of 5x1011 vg/kg, FVIII activity levels around 6% of normal were 
achieved in 3 participants. One participant has FVIII expression of >20% one-year post SPK-
8016 infusion. Although the FVIII levels achieved have reduced the annualized bleeding rates 
for these participants, FVIII expression remains below the normal range. Optimal vector dose 
and immune suppression regimens are needed.  
Therapeutic rAAV-based gene transfer is hampered in individuals who develop neutralizing 
antibodies against AAV capsids.  Even if switching to a different vector serotype, participants 
who develop these are unlikely to qualify for future gene transfer clinical studies.  For this 
reason, the Sponsor proposed the starting dose level of 5x1011 vg/kg that could provide a 
reasonable therapeutic level while offering an ample safety margin for the participants. 
Safety of these dose levels is supported by previous and ongoing human clinical trials of 
 
 
, as well as the NHP studies with SPK-8011 in which dose levels up to 2x1013 vg/kg were 
infused without evidence of adverse effects. Notably, the NHP dose level is ~40-fold higher than 
the proposed starting dose level of 5x1011 vg/kg in this clinical study. 
The Sponsor has previously demonstrated that the presence of an excess of empty capsids may 
adsorb low-level and non-neutralizing antibodies, permitting liver transduction after peripheral 
vector infusion even in their presence (Mingozzi, 2013b). This hypothesis is consistent with the 
clinical data from previous AAV-mediated hemophilia B gene transfer trials, in which the 
presence of empty capsids in the formulation correlated well with higher expression levels at low 
vector doses (Monahan, 2015; Nathwani, 2014). 
 

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3 
 
OBJECTIVES AND ENDPOINTS 
3.1 
Primary Objective 
Primary objectives for this study are: 
• To evaluate the safety and tolerability of SPK-8016. 
• To evaluate the efficacy as evidenced by prevention of bleeds and level of FVIII 
expression with SPK-8016. 
3.2 
Secondary Objectives 
Secondary objectives for this study are: 
• To evaluate additional parameters associated with SPK-8016 directed FVIII expression 
and vector shedding. 
• To characterize the immune response to the vector and transgene product. 
3.3 
Primary Endpoints 
The primary endpoints include the following: 
• For safety and tolerability: 
− Incidence of adverse events, including clinically significant abnormal laboratory 
values. 
− Occurrence of hepatic transaminase elevation requiring immunosuppression. 
• For efficacy: 
− Peak and steady-state FVIII activity levels assessed by coagulation clotting assays. In 
this study, steady-state levels are based on FVIII:C measurements starting 12 weeks 
post vector administration and without the use of exogenous FVIII products since 
vector administration.  
− Number of bleeding events (spontaneous and traumatic) since vector administration. 
− Number of FVIII infusions since vector administration. 
3.4 
Secondary Endpoints 
The secondary endpoints include the following: 
• Additional kinetic assessments including, but not limited to:  
− Time to achieve steady-state FVIII activity levels;  
− Vector-shedding of SPK-8016 in bodily fluids. 
• Incidence of immune responses to AAV capsid protein and BDD-hFVIII transgene. 
3.5 
Exploratory Endpoints 
The exploratory endpoints will assess changes from baseline in the following: 
36. Joint assessments:  

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• Number of target joints 
• Hemophilia Joint Health score 
37. Activities assessments:  
• Hemophilia Activities List  
• Change in Level of Activity questionnaire 
38. Quality-of-life assessments:  
• Haem-A-QoL questionnaire  
• Euro Quality-of-Life Five Dimensions (EQ-5D-5L) questionnaire 
39. Health-economic parameters including, but not limited to, collection of information on the 
following: 
• Number of hospitalizations (excluding pre-planned hospitalizations documented at 
screening) 
• Number of hospitalization days 
• Number of emergency room visits 
• Number of physician visits, excluding study visits 
• Number of days off school or work 
40. Exploratory inflammatory profiling of plasma and immune function gene expression of 
PBMCs after vector administration (ELISpot, and other exploratory biomarkers) 

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4 
STUDY DESIGN 
4.1 
Overall Design 
SPK-8016-101 is a Phase 1/2a, open-label, non-randomized, dose escalation study to evaluate 
the safety, tolerability, and efficacy of a single IV infusion of SPK-8016 in men with clinically 
severe hemophilia A, and no measurable inhibitor against FVIII.  A maximum of 40 evaluable 
(i.e., dosed) participants will be dosed with a single IV infusion of SPK-8016. 
4.1.1 Sequence of Enrollment 
The following 2 staggering strategies are used in this study based on the recommendation from 
The Guidance for Industry: Considerations for the Design of Early-Phase Clinical Trials of 
Cellular and Gene Therapy Products (Center for Biologics Evaluation and Research, Office of 
Cellular, Tissue, and Gene Therapies, Food and Drug Administration; US Dept. of Health and 
Human Services 2015):   
• The first 2 participants in each dose-level (5x1011 vg/kg, 1x1012, 2x1012 vg/kg) will be 
infused with SPK-8016 at least 6 weeks apart to mitigate acute safety risk; and  
• An independent DMC will review at least 6 weeks of follow-up data from a minimum of 
2 participants who have received SPK-8016 at a given dose level prior to dosing the first 
participant in the next dose level.  
After escalation through the first 3 planned dose levels, a fourth dose level may be explored in 
consultation with the DMC. Additionally, the Sponsor may decide to further expand the starting 
dose cohort (5x1011 vg/kg), the middle dose cohort (1x1012 vg/kg), or the high dose (2x1012 
vg/kg) to better evaluate the safety, efficacy, and variability of response within a given 
cohort.   Any decision to add a fourth dose level or further expand one of the existing protocol-
defined dose levels will be made in consultation with the DMC. 
The dose level for Cohorts 1 & 2 will begin at 1x1012 to explore immunomodulation.   
Cohort 1 will utilize 
 
 
 
 
 should a steroid sparing regimen be indicated, based on the clinical judgement of the 
Investigator and in consultation with the Sponsor. The steroid sparing approach will include 
consideration of individual risk including potential toxicity that may result from interactions with 
concomitant medications.  
 
 
 
 The clinical 
course of these 2 participants will be monitored including findings potentially consistent with a 
cellular immune response targeting transduced hepatocytes (e.g., acute decrease in plasma factor 
VIII activity and/or acute increase in liver ALT transaminase). The clinical course of these first 2 
participants who receive 
will be reviewed by the Sponsor in consultation 

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with the DMC, including any additional immune monitoring parameters that are available from 
among the Immune Profiling assays (e.g., serum cytokines, acute phase reactants e.g., CRP, 
fibrinogen). Based upon the Sponsor and DMC review of clinical observations, a decision will be 
reached whether to allow 
 administered up to 4 weeks after vector 
infusion. In any event, 
 will not be administered later than 4 weeks following vector 
administration, due to concern that incidence of 
-related transaminitis may increase with 
repeat dosing, which could in turn confound the effort to monitor vector-associated hepatocyte 
loss (which is most likely to occur 6 weeks or later following rAAV dosing).  Due to the same 
safety consideration, a second dose of 
 will not be administered to any individual if the 
participant’s current ALT is ≥ 1.5 x the participant’s baseline ALT value (assayed prior to vector 
administration) or greater than the Upper Limit of Normal for the ALT (whichever of these 2 
values is lower). Therefore, following the review of the data from the first 2 participants who 
receive a single Day 0 dose of 
, the criteria that would be considered a potential trigger for a 
second dose of 
 are: decline in factor VIII activity (e.g., decline of >30-40% of FVIII 
activity over a period of ≤ 1 week) with or without AAV IFN-gamma ELISpot positive. In 
addition, the presence of any of the following clinical observations, probably or possibly a 
complication of the initial dose of 
, are a contraindication to a second dose of 
: 
a. Hypersensitivity/Anaphylaxis in response to 
 
b. Transaminitis. Do not give a second dose of 
 if the ALT greater than the Upper Limit of 
the Normal Range or greater than 1.5 times the study subject’s baseline ALT value (if the 
latter degree of elevation remains within normal limits). 
c. Neutropenia. Do not give a second dose of 
 if the ANC <2000 per mm2 
d. Thrombocytopenia. Do not give second dose of 
 if platelet count is <100,000 cells/μL 
e. Hypofibrinogenemia. Do not give a second dose of 
 if fibrinogen antigen is <180 mg/dl 
Cohort  
Number of 
Participants  
SPK-8016 Dose  
Immunomodulatory Regimen  
1  
3-5  
1x1012 vg/kg  
2x1012 vg/kg  
 
 
2  
3-5  
1x1012 vg/kg  
2x1012 vg/kg  
 
 
 
 
. 
Enrollment will begin in either Cohort 1 or 2 at a dose of 1 x 1012 vg/kg then alternate between 
cohorts. A stagger of 4 weeks will occur between the first and second participants in Cohort 2.  If 
in the opinion of the Investigator and Sponsor, there are individual or site-specific circumstances 
that might preclude enrollment in either cohort in an otherwise eligible participant, enrollment in 
the alternate Cohort may be considered (i.e., HIV status, timely access for IV steroid 
administration based on geographic location). The Sponsor has a similar investigational gene 
therapy, SPK-8011-101, evaluating immunomodulation prior to vector infusion (NCT03003533). 

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These data will be informative to SPK-8016 enrollment and shared with the DMC.  If safety 
signals are observed in any cohort, the Sponsor in consultation with the DMC will decide 
whether and how the safety findings will impact future cohort assignment.  
4.1.1.1 
Dose-Level and Cohort Expansion 
Dose-cohort expansion may occur under the following scenarios: 
Dose Level Expansion - After 2 participants are dosed in a given dose level, it may be possible 
to expand a cohort to up to 10 participants if there is evidence of FVIII:C increases above 5% of 
normal in either participant by Week 4 post vector-administration.  This initial expansion will 
provide additional information about variability of response within the same dose level. 
New Cohort Expansion - After 2 participants are dosed in a cohort, an initial expansion of up to 
3 participants may occur and, thereafter, an additional expansion for up to a total of 10 
participants may occur. The additional expansion may occur in 1 or more cohort(s). For Cohort 2 
of the 1 x 1012 dose, the first 2 participants will be infused at least 4 weeks apart.  
If either of the immunomodulatory regimens is deemed clinically superior and if agreed to by the 
DMC, the cohort evaluating the less effective regimen will be closed to enrollment.  
Dose Escalation 
The decision to dose-escalate will be made by the Sponsor in consultation with the DMC to 
ensure safety. 
Dose escalation to the next dose level may occur under the following scenarios, provided there 
are no safety concerns after at least 6 weeks of follow-up data have been reviewed by the DMC. 
Dose escalation through the first 3 dose levels (5.0x1011 vg/kg, 1.0x1012, 2.0x1012 vg/kg) may 
occur in, but is not restricted to, the following scenarios. 
Dose escalation may be considered if: 
1. If neither of the first 2 participants achieve FVIII:C above 5% of normal by Week 6 post 
vector-administration; or 
2. If at least 2 participants in a given cohort achieve FVIII:C ≤ 40% of normal by Week 6 post 
vector-administration; or 
3. If 3 participants in a given cohort achieve ‘steady-state’ FVIII:C ≤ 50% of normal. 
4. There will be no dose escalation if at least 3 participants in any dose level achieve steady-
state FVIII:C  80% of normal.  Steady-state levels are based on at least 2 separate FVIII:C 
measurements, at least 2 weeks apart, starting 8-12 weeks post vector administration while 
not receiving daily corticosteroid therapy and without use of exogenous FVIII products since 
vector administration. 
Corticosteroid therapy may increase the transgene expression from the SPK-8016 expression 
cassette, similar to SPK-8011, as observed in both animal models and in our clinical observations 
using both early prophylactic and the reactive application of corticosteroids. In SPK-8011, this 
effect appears to occur in a dose-dependent fashion; in clinical observation the effect has 
appeared somewhat greater with initiation of daily oral corticosteroids in a prophylactic fashion 
in the early weeks after vector delivery when compared to reactive corticosteroids given at later 
time points. The Primary Endpoint for efficacy for this study is the FVIII activity at steady state, 

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which Spark does not interpret while any subject remains on corticosteroid treatment. Instead, 
expression level of steady-state FVIII:C as an efficacy endpoint is evaluated after daily oral 
corticosteroids or pulse steroids have been discontinued for at least 2 weeks. Note that if the 
concomitant use of steroids is to have an effect to artifactually increase the FVIII expression to a 
level greater than the eventual steady state expression (after discontinuation of steroids), this 
would not trigger an inappropriate escalation of the SPK-8016 dose (which would be a safety 
concern) but instead might result in an expansion of the present dose cohort or a delay in the 
decision to dose escalate until after the observation of steady state while off steroid therapy.  
 
 
 
 
 
 
   
In consultation with the DMC, further dose exploration may be considered if effective 
immunomodulation has been demonstrated in any cohort and/or FVIII expression is less than 
80% of normal 12 weeks following gene transfer in at least 2 participants. 
4.1.2 FVIII Incremental Recovery 
If FVIII levels resulting from endogenous FVIII production from transduced hepatocytes begin 
to decline without evidence of elevation of transaminases, the participant will be evaluated for 
evidence of anti-FVIII antibody formation. This evaluation will include FVIII inhibitor assays 
and FVIII incremental recovery with blood sample collection consistent with the Investigator’s 
clinical practice after receiving 50 IU/kg of FVIII product. Blood sampling will be done at 
appropriate time-points to enable acceptable determination of PK parameters (Iorio, 2016). 
4.1.3 Corticosteroids  
Based on observation and experience from earlier clinical studies of liver-directed AAV gene 
transfer, including the SJ-UCL trial (Nathwani, 2011b; Nathwani, 2014), the Sponsor’s earlier 
clinical studies 
, and the Baxalta BAX 335 trial 
(NCT#01687608), participants may develop an immune response to the vector capsid, as 
evidenced by a transient rise in transaminases (AST and/or ALT) and/or a loss in FVIII activity 
in the peripheral blood, as measured by IFN-gamma ELISpot (Manno, 2006; Mingozzi, 2007; 
Nathwani, 2011b).  In the Sponsors’ similar gene therapy trial, SPK-8011, 12 participants have 
required corticosteroids following vector infusion. Two participants in the low dose cohort, 5x 
1011 vg/kg, did not mount an immune response. Two of the 3 participants in the second dose 
cohort, 1.0x1012 vg/kg, received corticosteroids starting at 5 and 11-weeks post vector infusion; a 
declining FVII level was the trigger. Of the 7 initial participants treated in the third dose cohort 
with 2x1012 vg/kg SPK-8011, 5 required corticosteroids, starting at 6 to 11-weeks post vector 
infusion for 1 or more of the following triggers: declining FVIII levels, rise in ALT above 
participant baseline or elevated IFN-ɣ ELISpots to AAV capsid. Steroid initiation normalized 

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ALT levels and extinguished the ELISpot signal in all cases. Two of the 5 participants who 
received corticosteroids in the 2x1012 vg/kg dose group, however, showed loss of transgene 
expression likely due to the immune response. Both of these individuals eventually returned to 
hemophilia prophylaxis regimens, one with recombinant FVIII, the other with emicizumab. 
Given that 5 of 7 participants in the 2x1012 vg/kg group required corticosteroids, including 2 
participants who lost FVIII expression, the institution of a consistent, standardized short course 
of prophylactic corticosteroids appeared warranted in an attempt to pre-emptively manage 
potential immune responses. In the first 2 participants administered SPK-8011 at 2x1012 vg/kg 
using prophylactic oral corticosteroids (1 mg/kg or 60-80 mg), the first was started on 
corticosteroids on Day 27 due to falling FVIII and the second was started on Day 26 due to rising 
ALT and decreasing FVIII expression. In both of these participants, early initiation of 
corticosteroids resulted in a transient supraphysiologic increase in FVIII levels to greater than 
200%.  An intermediate dose cohort (1.5x1012 vg/kg) was initiated.  Reduction of the vector 
genome dose was successful in terms of avoiding supraphysiologic levels of FVIII upon 
initiation of corticosteroids, but in all participants where corticosteroids have been started prior 
to 6 weeks post vector infusion, tapering off has been challenging, and consequently, prolonged 
courses have been required, with associated steroid related side effects. 
 
 
 Because of the prolonged steroid taper when steroids 
were administered beginning at 4 weeks, which was not observed with later initiation of steroids, 
prophylactic steroids will be avoided going forward.  
Based on discussions with expert immunology consultants, the Sponsor proposes to again 
investigate administration of steroids only in response to one of the previously described triggers, 
i.e., declining FVIII levels or rising transaminases, 
 
 Oral corticosteroids may be utilized, if clinically indicated.  ELISpot is an exploratory 
assay and not predictive as a trigger for immunomodulation.   
In this SPK-8016-101 study, 4 participants have received SPK-8016 at dose level  
5x1011 vg/kg. Three of the 4 participants developed an apparent immune response to the AAV 
capsid, characterized by transient elevations in liver transaminases and a decrease in FVIII 
expression, and/or persistent positive IFN-γ ELISpot results to the 
, which 
have prompted initiation of corticosteroid therapy (prednisone). Prednisone was started week 3-6 
post infusion. Prolonged tapering of daily oral corticosteroids has been required in all 
participants (total courses of 43-48 weeks prednisone) to avoid and treat late recurrences of 
apparent hepatocyte-targeted immune response.
 
 
 
 
 
 
 
 
 
 

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. 
Significant elevations of transaminases (≥5-10x ULN) will be treated as a safety issue and 
appropriate medical care will be instituted according to the standard of care of the medical 
facility involved.  
Based on the data observed to date in this trial as well as in Hemophilia B trials (Manno, 2006; 
Nathwani, 2014), a course of corticosteroids may be initiated in participants, depending on the 
clinical judgement of the Investigator and in consultation with the Sponsor, after a trigger is 
identified in the transaminases or FVIII post vector transfusion and may be continued in a pulse 
or tapering regimen, IV and/or oral, until clinical concern of hepatic inflammation is resolved 
and per Investigator/Sponsor discretion. The ELISpot assay is considered exploratory and not a 
predictive trigger for reactive immunomodulation. 
4.1.3.1  Corticosteroid Regimen   
If transaminases become elevated or the FVIII declines, 
 
 
 
 
  
 
 
 
 
 
  
 
 
 
. Treatment will continue until resolution of the 
immune response occurs and may require the use of additional immunomodulatory agents. Daily 
oral corticosteroids given in a tapering dose regimen may be utilized instead of 
 
corticosteroids, if in the estimation of the Investigator an oral dosing regimen is clinically 
indicated. The Investigator in collaboration with the Sponsor will have flexibility in 
implementing the corticosteroid regimen, since the exact regimen and course will depend on 
clinical circumstances. 
If reactive corticosteroids 
 or oral dosing) are triggered by an apparent immune 
response, additional blood samples for “Immune Profiling” (cytokines) and “PAX gene” should 
be collected 1) prior to the initiation of corticosteroids (if this is possible without delaying the 
initiation of corticosteroid therapy) and 2) at 1 week (± 2 days) and at 2 weeks (± 2 days) 
following the first dose of corticosteroids. At the discretion of the Investigator, nucleic acid 
amplification testing (NAAT) for infection with SARS-CoV-2 (the infectious agent in COVID-
19) may be performed at this time for individuals who do not have symptoms consistent with 
upper or lower respiratory tract infection or COVID-19. NAAT testing should be performed at 

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this time for individuals with any of the following symptoms: cough, shortness of breath or 
difficulty breathing, fever, chills, sore throat, new loss of taste or smell; new persistent/recurrent 
muscle pain or headache. For individuals who do not have any of these symptoms, the initiation 
of reactive corticosteroids should not be delayed pending results of NAAT testing. For 
individuals who do display any of these symptoms, initiation of corticosteroids may be delayed 
at the discretion of the Investigator until after results of NAAT testing are known. Note: The 
Infectious Disease Society of America panel suggests collecting nasopharyngeal, or mid-
turbinate or nasal swabs rather than oropharyngeal swabs or saliva alone for SARS-CoV-2 RNA 
testing in symptomatic individuals with upper respiratory tract infection (URTI) or influenza like 
illness (ILI) suspected of having COVID-19. 
4.1.3.2 Other Immunomodulatory Considerations 
Prolonged courses of high dose oral corticosteroids are undesirable and associated with adverse 
events. In the AASLD and the EASL guidelines for the treatment of autoimmune hepatitis, an 
alternative regimen of corticosteroids and steroid sparing agents, 
, is 
outlined for use in individuals who cannot tolerate high doses of prednisone or are experiencing 
difficulty tapering. A steroid-sparing agent administered concurrently with lower (compared to 
prednisone monotherapy) and tapering doses of prednisone may be utilized based on individual 
clinical circumstances. Both 
 have been utilized as steroid-sparing 
agents in the most recent iteration of this study, to good effect in both cases, and are described 
below. 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
The ultimate determination of steady state FVIII expression and determination of expression 
above a threshold FVIII:C activity value are interpreted by evaluating serial FVIII:C activity 
over multiple timepoints examined after discontinuation of corticosteroids and of steroid-sparing 
immune suppressive agents.  

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4.1.3.3 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
  
4.1.3.4 Experience with Long Term Immunomodulation 
There is extensive experience with corticosteroids and immunosuppressive regimens in 
hemophilia: first, as a maneuver to eradicate antibodies to factor VIII or IX, clinically termed 
inhibitors (Nilsson, 1976; Hultin, 1976; Hay, 2006) and second, in the setting of liver 
transplantation due to the high prevalence of hepatitis C among adults with haemophilia 
(Gordon, 1998; Wilde, 2002).  Many individuals with hemophilia have been maintained on 
standard liver transplant immunosuppression regimens for years. Corticosteroids have been used 
to treat people with asthma, idiopathic thrombocytopenic purpura, organ injury from 
autoimmune diseases including autoimmune hepatitis and systemic lupus erythematosus 
nephritis and lupus CNS manifestations, and other medical conditions. For participants with 
severe hemophilia A, the benefit of long-term expression of a modest level of clotting factor far 
outweighs the risk of receiving a course of corticosteroids or other immunomodulatory agents. 
The long-term side effects of the immunomodulatory drugs expected to be used in this study are 
well-characterized.  Participants who develop immune hepatitis will be monitored closely to 
minimize the risk of the side effects. Pulse dosing of steroids has been shown to be efficacious in 
reducing an immediate immune response with a reduction in corticosteroid related adverse 
events (when compared to daily high dose oral corticosteroids) in several autoimmune diseases 
(Fanouriakis, 2019; Gordon, 2018; Wei, 2016). The use of oral prednisone in 
 
 may reduce the reactivation of the inflammatory process or 

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rebound of the immune response; however, a standard regimen has not been determined 
(Gordon, 2018; Torres, 2018).  To utilize the lowest effective dose and shorten the duration of 
the immunosuppressive therapies, tapering of the regimen will start as soon as there is evidence 
of resolution of hepatic transaminase elevation and disappearance from the peripheral blood of 
capsid-specific T cells. While on immunomodulatory regimens, participants will also be 
monitored for side effects, including opportunistic infections. Subjects should receive 
Pneumocystis jiroveci pneumonia prophylaxis while on immunosuppression. Antibiotics or other 
medications to minimize the risk of opportunistic infection may be prescribed at the discretion of 
the Investigator. All events related to the use of immunomodulatory drugs (e.g., hyperglycemia, 
weight gain, infections, acute exacerbation of hypertension or mood disorder, insomnia) will be 
recorded as AEs and attributed to immunomodulation, and medications may be prescribed at the 
discretion of the Investigator.   
Individual participant recommendations regarding the use of alternative and/or additional 
immunomodulatory regimens or therapies (other than corticosteroids); e.g., 
 
 will be discussed with the Investigator(s) and the Sponsor and will involve a 
thorough review of the participants medical history, labs, concomitant medications, and clinical 
presentation. Depending on the agent selected, additional blood draws for therapeutic or 
laboratory monitoring and subsequent dosage adjustment may be required. The Investigator will 
discuss the risks and benefits of any immunomodulatory therapy with the participant and will 
consult with additional experts as needed (i.e., transplant surgeon, hospital pharmacist, infectious 
disease specialist, endocrinologist etc.).  
Screening/Baseline:  Participants who have provided consent will undergo the screening 
assessments described in  Table 1 up to a maximum of 16 weeks prior to the infusion of SPK-
8016. If there is no historical ≤1% FVIII activity levels documented by the certified laboratory, 
then the screening data must be generated documenting ≤2% FVIII activity at baseline.     
Re-screening: Re-screening is permitted once, if the participant is found to be eligible for the 
study but the screening period is prolonged greater than 16 weeks. A participant may be re-
screened into the study using the same participant ID; however, all screening assessments must 
be repeated. 
4.1.4  Three to Seven Days Prior to Day 0  
Participants in Cohorts 1 and 2 will require a laboratory assessment within 3 to 7 days prior to 
Day 0 to verify continued eligibility prior to starting immunomodulatory agents (i.e., 
 
).  This visit may be conducted by a remote provider. The study staff must contact the 
participant to verify and document no changes in the general health status prior to first dose of 
 
4.1.5 
Day -2 
 
 

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4.1.6 Dosing Period (Days 0, 1) 
Assessments and procedures to be performed on Dosing Day (Day 0) are described in Table 1.  
All participants will administer a prophylactic infusion of 50 IU/kg of their current FVIII 
concentrate the morning of Day 0.  This may be self-administered and recorded in the subject 
infusion log.  Site assistance is permitted for the FVIII infusion. On Day 0, participants in Cohort 
2 will receive a dose of 
 intravenously over 60 minutes, with the 
 infusion to 
be completed at least 4 hours (up to 24 hours) prior to the start of vector administration. The 
dose will not exceed 
 (Please see 
prescribing information [Genentech, 
2019]). Participants will receive a single IV infusion of SPK-8016 at the vector-administration 
center.  The complete dose of SPK-8016 will be infused via infusion pump over approximately 
60 minutes.  Vital signs will be taken at various time-points from the start of infusion (see Table 
1). 
Blood samples for immune profiling will be collected pre-administration of 
 (3-7 
Days Prior), and SPK-8016, and 30 (±2) minutes, 2 hours (± 10 minutes), 5 hours (± 10 
minutes), and 24 (±1) hours from the completion of the SPK-8016 infusion. PAX Gene will be 
collected pre-administration of 
, pre-administration of SPK-8016, and 5 hours (± 10 
minutes) and 24 (±1) hours from the completion of the SPK-8016 infusion. Additional blood 
samples, including FVIII activity levels, will also be collected pre-infusion of SPK-8016. 
4.1.7 
Follow-Up Observation Period 
Participants who have received a dose of SPK-8016 will report to either the vector-
administration center or follow-up center for follow-up evaluations, according to the protocol 
assessments for 52 (±2) weeks after the infusion of SPK-8016.  During the follow-up observation 
period, a qualified/trained in-home service provider may be utilized, if needed, for remote-
phlebotomy and sample-collection services during the visits which do not require physical 
examination. For participants in Cohorts 1 and 2, 
 
 
 
4.2 
Study Duration, Enrollment and Number of Sites 
4.2.1 Duration of Study Participation 
The study will consist of the following periods 
• Screening period (up to a maximum of 16 weeks)  
• Study Intervention: Assignment into cohort with possible pre-infusion 
immunosuppressive medication or administration of oral immunosuppressive medication, 
and then study product infusion 
• Follow-up observation period (52 [±2] weeks post-infusion of SPK-8016)   
The total duration of the study for an individual participant is up to 68 weeks (including up to a 
maximum of 16 weeks of screening).   

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4.2.2 Total Number of Participants; Sites Projected and Geographic Regions 
It is estimated that approximately 50 potential participants may be enrolled for a maximum of 40 
evaluable (i.e., dosed) participants.  
The study is planned to be conducted at approximately 15 study centers (vector-administration 
centers and/or follow-up centers) located worldwide.  
4.3 
Study Stopping Rules  
The Sponsor may terminate this study at any time.  The Sponsor, or designee, will notify the 
Investigator(s) and appropriate regulatory authorities if the study or any given study cohort is 
suspended, terminated, or completed.  If the study or any given study cohort is suspended or 
terminated, the following instructions should be followed, unless the DMC and/or the Sponsor 
advise otherwise: 
• Participants who have already received SPK-8016 will continue to maintain the protocol 
schedule;  
• Participants who are in the screening phase of the study or study cohort but have not 
received SPK-8016 will wait for DMC recommendation.  The scheduled date for SPK-
8016 infusion may be postponed or cancelled. 
Any of the following occurrences may result in suspension of further enrollment into the study or 
any given study cohort while the events are under investigation: 
• Any SPK-8016 related death during the study; 
• The development, in any participant, of SPK-8016 related Grade III-IV toxicity, 
including, but not limited to confirmed persistent FVIII inhibitor, allergic reaction 
(bronchospasm and anaphylaxis), excluding elevated transaminases;  
• The development, in any participant, of SPK-8016 related (>10x ULN) elevated 
transaminases; 
• The development, in any participant, of SPK-8016 related (>2.5x ULN) elevated 
transaminases that fail to resolve to less than 2.5x ULN within 4 weeks on an 
immunomodulatory regimen;   
• The occurrence, in any participant, of a medically important event that warrants further 
evaluation; 
• Any occurrence of a malignancy at any point after vector infusion that is related to SPK-
8016 or one of the immune modulating agents.   
* It is important to note that AAV vector-mediated insertional mutagenesis, if it should occur, is 
not likely to be observed in the initial year following gene transfer; therefore, the Sponsor 
intends to provide long-term safety monitoring of participants in a - LTFU study. 
In addition to halting enrollment, such an event will be handled as a SAE and reported in the 
timeframe according to Section 9.4.2.  The DMC will review data relevant to the event and will 
receive information from the Sponsor and/or Investigator before providing appropriate 
recommendations.  The event and the DMC’s recommendation will be discussed with the U. S. 
Food & Drug Administration (FDA) and other regulatory authorities prior to re-initiation of 

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enrollment.  All participants who were infused with the study drug will continue to comply with 
the follow-up schedule according to the protocol. 

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5 
STUDY POPULATION 
Prospective approval of protocol deviations to recruitment and enrollment criteria, also known as 
protocol waivers or exemptions, is not permitted. 
5.1 
Inclusion Criteria 
Participants must meet all the following criteria at screening and prior to dosing of SPK-8016 
(Day 0) to be eligible for the study: 
1. Be able to understand the purpose and risks of the study and provide signed and dated 
informed consent and authorization to use protected health information (PHI) in accordance 
with national and local privacy regulations  
2. Be male and ≥18 years of age  
3. Have hemophilia A with: 
a) < 1% (<1 IU/dL) endogenous FVIII activity levels as historically documented by 
a certified laboratory or screening data results; OR  
b) 1-2% (1-2 IU/dL) endogenous FVIII activity levels and >10 bleeding     events 
per year (in the last 52 weeks prior to screening); OR 
c) 1-2% (1-2 IU/dL) endogenous FVIII activity levels and on prophylaxis  
4. Have had >150 prior exposure days to any recombinant and/or plasma-derived FVIII protein 
products or cryoprecipitates based on historical data from medical records/history 
5. Have no prior history of hypersensitivity or anaphylaxis associated with any FVIII or IV 
immunoglobulin administration 
6. Have no measurable inhibitor against FVIII at screening (i.e., <0.6 Bethesda Units); no 
confirmed history of clinically significant FVIII inhibitor and no clinical signs or symptoms 
of decreased response to FVIII administration (Note: Family history of inhibitors is not 
exclusionary, nor is remote documentation (greater than 5 years) of a single measurement of 
Bethesda titer of >0.6 BU that is not accompanied by clinical evidence of failure to respond 
to infused FVIII concentrate) 
7. Have acceptable laboratory values sampled at screening and reviewed prior to Day 0: 
a) Hemoglobin ≥11 g/dL; 
b) Platelets ≥100,000 cells/μL; 
c) AST, ALT, alkaline phosphatase < ULN; 
d) Bilirubin ≤1.5x ULN (Bilirubin levels above the laboratory’s normal range are 
acceptable in individuals with a documented history or laboratory evidence of 
Gilbert’s Disease); 
e) Creatinine ≤2.0 mg/dL;  
f) Absolute neutrophil count (ANC) ≥ 2000 per mm3;  

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g) Fibrinogen antigen ≥ 180 mg/dL for participants in Cohort 2 
8. Agree to use reliable barrier contraception after the administration of SPK-8016 until 
notified by the Investigator or designee. 
5.2 
Exclusion Criteria 
Participants who meet any of the following criteria at screening or prior to dosing of SPK-8016 
(Day 0) are not eligible for the study:  
1. Have active hepatitis B or C. All participants must be screened for both active hepatitis B and 
C regardless of prior known history.   
a. Screening for hepatitis B: All participants must have a single sample collected at 
screening for each of the following tests: HBsAg (hepatitis B surface antigen), anti-HBc 
(total Hepatitis B core antibody), and HBV-DNA viral assay (nucleic acid test for 
hepatitis B virus DNA). 
i. A participant is not eligible if either HBsAg is positive or HBV-DNA is 
positive/detectable. 
ii. A participant is eligible if the anti-HBc is positive and both HBsAg and HBV-DNA 
are negative, as this would be consistent with a prior infection of hepatitis B. Anti-
HBc must be obtained in all participants to discriminate between acute infection and 
possible reactivation of hepatitis B during the trial (e.g., in participants with no prior 
history of hepatitis B). 
b. Screening for hepatitis C: All participants, including those who have never been treated 
or who have completed anti-viral therapy for chronic hepatitis C, must have a single 
HCV-RNA load assay (also referred to as a nucleic acid test [NAT] for HCV RNA) 
at screening.  
i. A participant is not eligible if his HCV-RNA load assay is positive/detectable.  
ii. A participant treated with anti-viral therapy for chronic hepatitis C must have 
completed anti-viral therapy at least 6 months prior to screening and must have a 
negative HCV-RNA at the time of screening. 
iii. A participant with a documented or self-reported history of HCV must have a single 
negative HCV-RNA at the time of screening. 
2. Are currently on antiviral therapy to treat their hepatitis B or C 
3. Have significant underlying liver disease. A participant is not eligible with any of the 
following documented diagnoses, indicative of significant underlying liver disease: 
• Portal hypertension; or 
• Splenomegaly; or  
• Hepatic encephalopathy. 
Any participant without any of these pre-existing diagnoses must have the following 
performed at screening: 

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a. 
Serum albumin measurement.  A participant is not eligible if the serum albumin level is 
below the lower limit of normal of the laboratory; and  
b. 
Diagnostic test for liver fibrosis (e.g., FibroScan, FibroTest/FibroSURE, or AST-to-
Platelet Ratio Index [APRI]).  A participant is not eligible if any of the following 
findings, which are indicative of fibrosis ≥ stage 3, are present: 
i. FibroScan score > 8.3 kPa units; or  
ii. FibroTest/FibroSURE > 0.48; or 
iii. APRI > 1 
If more than 1 diagnostic test result is available, then the FibroScan score will be used 
as the primary consideration for eligibility 
4. Have serological evidence of HIV-1 or HIV-2 with CD4 counts ≤200/mm3.  
a. Participants who are HIV-positive and stable, with an adequate CD4 count 
(>200/mm3) and undetectable viral load (<50 gc/mL) , measured at screening, and 
who are on an antiretroviral drug regimen are eligible to enroll    
5. Have 
neutralizing antibody titers ≥1:1 
6. Have a history of active cancer in the past 6 months, chronic infection, latent or active TB, 
uncontrolled immune disorder or other chronic disease that the Investigator and/or Sponsor 
consider to constitute an unacceptable risk 
7. Have been dosed in a previous gene therapy research trial within the last 52 weeks or have 
participated in a clinical study with an investigational drug within the last 12 weeks prior to 
signing the informed consent          
8. Have a history of diverticulitis, diverticulosis requiring antibiotic treatment, or chronic 
ulcerative lower G.I. disease that might predispose a patient to perforations              
9. Have any concurrent clinically significant major disease (such as liver abnormalities, type I 
diabetes, uncontrolled hypertension, or vertebral compression) or any other condition such as 
active infections or COVID-19 or any other unspecified reasons that, in the opinion of the 
Investigator and/or Sponsor, makes the participant unsuitable for participation in the study.  
a. At the time of screening, the Investigator will consider the local geographic and 
institutional epidemiology of COVID-19 and other infectious pathogens when 
determining suitability of the participant for participation in the study, including 
considering the potential clinical relevance of additional screening 
10. Have a planned surgical procedure in the next 12 months requiring FVIII prophylactic 
treatment 
11. Are unable or unwilling to comply with the schedule of visits and study assessments 
described in the clinical protocol. 
Participants who do not meet all of the enrollment inclusion criteria may not be enrolled.  Any 
violations of these criteria must be reported in accordance with Sponsor and Internal Review 
Board (IRB) Policies and Procedures. 

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5.3 
Screen Failures 
Screen failures are defined as participants who consent to participate in the clinical study but are 
not subsequently administered study drug. A minimal set of information will be collected for 
screen failures: demography, screen failure details, eligibility criteria, and, if applicable, any 
SAE. 
Individuals who do not meet the criteria for participation in this study (screen failure) may not be 
rescreened unless previous approval has been received from the Sponsor. 
5.4 
Enrollment of Participants 
Each participant will be assigned a unique participant identification (ID) number after providing 
written informed consent.  The participant ID number will be used to identify the participant for 
the duration of the study.  Participant ID numbers will not be reassigned or reused. No 
participant may be dosed prior to obtaining the unique ID number.  The Investigator must 
confirm and verify the inclusion/exclusion criteria, following the review of screening results 
from the laboratory and other documents.  
5.5 
Randomization 
This is a non-randomized study; therefore, there is no randomization of patients. 
5.6 
Blinding Procedures 
This is an open-label study; therefore, there is no study blinding. 

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6 
STUDY PROCEDURES/ASSESSMENTS AND SCHEDULE 
Planned timepoints for all safety assessments are provided in Table 1. 
6.1.1 Clinical Safety Assessments 
The following clinical assessments will be performed to assess the safety profile of SPK-8016: 
• Physical examination  
• Vital signs  
• AEs 
• Concomitant therapy and procedures. 
6.1.2 Laboratory Safety Assessments 
The following laboratory tests will be performed to assess the safety profile of SPK-8016 
• Hematology 
• Clinical chemistry 
• Urinalysis 
• Liver Function Tests   
• Coagulation  
• Neutralizing antibody development against FVIII by Bethesda assay (FVIII inhibitor)  
• Neutralizing antibody to 
 
• Immunology to assess cellular immune responses 
• Immune profiling of plasma after vector administration 
• Vector shedding (serum, saliva, urine, semen) 
6.2 
Additional Assessments 
6.2.1 Joint Assessments 
Baseline clinical status of joints and identification of target joint(s) will be conducted during 
Screening or pre-vector infusion.   
Joint health and identification of target joint(s) will be assessed during this study. A target joint 
is defined as a major joint (e.g., hip, elbow, wrist, shoulder, knee, and ankle) into which 3 or 
more spontaneous bleeds occurs in a single joint within a consecutive 6-month period as 
documented in the medical or home treatment records. Symptoms of pre-existing target joint 
involvement or hemophilic arthropathy (e.g., synovitis, persistent swelling, effusion, limitation 
of range of motion) should be documented as part of the Joint assessment.    
The Investigator will assess the participant’s target joint(s) identified at baseline by location of 
target joint(s), location of joint bleeding, and frequency of bleeding from each joint. 

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6.2.2 Hemophilia Joint Health Score 
Joint assessment will be conducted using a Hemophilia Joint Health Score (HJHS) (See Section 
14.4).  HJHS will be performed to evaluate the clinical status of the joints at screening or prior to 
Day 0, Week 26, and Week 52.  This assessment is based on the scoring system used in a joint 
scoring reliability study in boys with hemophilia (Hilliard, 2006).  It has been used as a tool to 
evaluate musculoskeletal outcomes in a cohort of 20 boys, aged 4 to 17 years (Saulyte 
Trakymiene, 2010).   
Six joints (left ankle [LA], right ankle [RA], left elbow [LE], right elbow [RE], left knee [LK], 
and right knee [RK]) will be scored according to the following criteria:  swelling, muscle 
atrophy, crepitus, flexion loss, extension loss, instability, joint pain, and strength.  Gait will be 
scored based on walking and climbing stairs.   
6.2.3 Activity Assessments (Hemophilia Activities List) 
Participants will complete the Hemophilia Activities List (HAL) questionnaire (See Section 
14.1) (van Genderen, 2004; van Genderen, 2006).  In the questionnaire, several activities are 
listed that could be difficult for patients with hemophilia. 
6.2.4 Participant Questionnaires 
The following questionnaires will be administered at selected visits: 
• Quality-of-Life (See Section 14.2): The Haem-A-QoL Questionnaire, a hemophilia-
specific QoL tool for hemophilia patients who are 17 years old and above, consists of 46 
items covering 10 dimensions to assess a patient’s health-related quality-of-life (von 
Mackensen, 2005). 
• Health Assessment (See Section 14.3): The EQ-5D-5L Questionnaire is a simple generic 
measure, containing 5 dimensions (mobility, self-care, usual activities, pain/discomfort, 
and anxiety/depression).  It is a standardized instrument that was established by the 
EuroQol Group, providing a simple descriptive profile and a single index value for health 
status (EuroQol Group, 1990). 
6.2.5 Health-economic Assessment 
The following information will be captured during the study as part of a health-economic 
assessment: 
a) Number of hospitalizations (excluding pre-planned hospitalizations documented at screening) 
b) Number of hospitalization days 
c) Number of emergency room visits 
d) Number of physician visits, excluding study visits 
e) Number of days off from school or work 
6.2.6 Archived Bio-samples 
At each time-point where samples are collected for FVIII activity, spare plasma will also be 
collected.  Samples will be archived for testing (if required) of further coagulation assays, or for 

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clarification of any clinical or laboratory AEs.  If a participant provides consent, samples may be 
used for genetic analyses and future research.  
6.3 
Clinical Procedures 
The clinical procedures that will be conducted during this study related to the evaluation of 
population demographics, medical and hemophilia history, safety, and efficacy are provided in 
Table 3. 
Table 3: 
Clinical Procedures: Study Assessments 
Assessment 
Description 
Demographics 
Date of birth, age, sex, ethnicity, and race  
Medical History 
Relevant medical history 
Prior/concomitant therapies within the past 30 days from signing the informed 
consent 
Hemophilia History 
Hemophilia A diagnosis and status: 
• 
Documented date of and age at diagnosis 
• 
FVIII activity level 
• 
Blood type and Rh factor 
• 
FVIII Genotype and HLA Genotyping 
• 
History of inhibitors to FVIII or allergic reactions to FVIII products 
• 
Bleed history in the past 52 weeks 
• 
Previous therapy in the past 52 weeks 
PK profile 
• 
Current FVIII product.  
o 
Recovery (maximum drug concentration [Cmax] following a 
known IU/kg dose of FVIII)  
o 
Half-life calculations 
Joint Assessments 
Target joint assessment in the past 26 weeks: 
• 
Location of target joint(s) 
• 
Location of joint bleeding 
• 
Frequency of bleed per joint 
• 
Target joint history 
o 
Hemophilic Arthropathy Assessment  
o 
HJHS 
Physical Exam 
• 
Full physical examination 
• 
Height and body weight 
Vital Signs 
• 
BP (systolic and diastolic) 
• 
Respiratory rate  
• 
Pulse rate 
• 
Temporal or Oral temperature (oC) 
Electrocardiogram 
• 
ECG 
Quality of Life 
questionnaires 
Haem-A-QoL 
EQ-5D-5L 
Activity Assessments and 
Health Economics 
Hemophilia Activities List 

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Assessment 
Description 
Health Economic Assessment 
Review of AEs and 
Concomitant Therapies 
Review of AEs and concomitant therapies throughout the study 
Hematology (CL) 
WBC count with Differential 
o 
Neutrophils 
o 
Lymphocytes 
o 
Monocytes 
o 
Eosinophils 
o 
Basophils 
RBC 
o 
Hemoglobin 
o 
Hematocrit 
o 
Platelet count 
Clinical Chemistry (CL) 
• 
Sodium 
• 
Potassium 
• 
Chloride 
• 
Bicarbonate 
• 
Glucose 
• 
Phosphate 
• 
Serum creatinine 
• 
BUN 
Liver Fibrosis Diagnostic 
Test 
• 
Fibroscan (LL)  
• 
FibroTest/Fibrosure (CL)  
o 
Apolipoprotein A1 
o 
Alpha 2 Macroglobulin 
o 
Haptoglobin 
• 
AST-to-Platelet Ratio  
• 
Liver Ultrasound 
• 
α-fetoprotein 
 
Urinalysis (CL) 
• 
pH 
• 
Glucose 
• 
Protein 
• 
Blood 
• 
Ketones 
Liver Function Tests and 
CRP (CL and LL) 
• 
Albumin,  
• 
Total bilirubin 
• 
Direct bilirubin 
• 
Indirect bilirubin 
• 
ALP 
• 
AST 
• 
ALT 
• 
Total protein 
• 
GGT 
• 
LDH 
• 
CRP 
Coagulation  
(CL, LL as noted) 
• 
FVIII activity, aPTT (CL and LL) 
• 
FVIII antigen (CL) 
• 
FVIII inhibitor (Nijmegen Bethesda) (CL, LL at screening), VWF antigen, 
VWF activity (CL) 
• 
Spare Plasma  
Neutralizing Antibody (CL) 
Neutralizing Antibody 
Immunology (CL) 
• 
Immun PBMC for ELISpot 
• 
Immun PBMC for Inflamm 
Immune Profiling (CL) 
• 
Cytokine/Chemokine Analysis 
• 
Tryptophan metabolites 
• 
Complement activation 
PAX Gene (CL) 
• 
PAX Gene (RNA) 
Vector Shedding (CL) 
• 
Urine 
• 
Serum 
• 
Saliva 
• 
Semen 
 

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Assessment 
Description 
HBV, HCV (CL) 
• 
Hepatitis B surface 
antigen  
• 
Hepatitis B core 
antibody total  
• 
Hepatitis B core 
antibody total  
• 
HCV RNA 
Ampliprep Taqman 
2.0 
• 
HBV DNA 
Ampliprep Taqman 
2.0 
• 
HBV DNA 
Ampliprep Taqman 
2.0DIL 
HIV Serology (CL) 
HIV-1/HIV-2 Antibody Screen 
HIV-1/HIV-2 Antigen Screen 
Viral load and CD4 (CL) 
HIV-1/HIV-2 Viral Load, CD4+ Count  
TPMT (LL) 
TPMT activity or genotype 
Lipid Profile (LL) 
Lipid profile (Cholesterol LDL, VLDL, and HDL, triglycerides) 
TB (LL) 
Tuberculosis Interferon-gamma Release Assay 
SARS-CoV-2 Testing (LL) 
anti-SARS-CoV-2 serology  
NAAT for infection with SARS-CoV-2 
Fibrinogen (LL) 
Fibrinogen Antigen, D-Dimer, Thrombin Time 
AE=adverse event; ALP=alkaline phosphatase; ALT=alanine aminotransferase; AST=aspartate aminotransferase; anti-
HBc=total hepatitis B core antibody; aPTT=activated partial thromboplastin time; BP=blood pressure; BUN=blood urea 
nitrogen; CD4=cluster of differentiation 4; CL=central laboratory; CRP=c-reactive protein; ELISpot=enzyme-linked 
immunospot assay; EQ-5D-5L=Euro Quality-of-Life Five Dimensions Questionnaire; FVIII=coagulation factor VIII; GGT= 
gamma-glutamyl transferase; Haem-A-QoL=Hemophilia Quality of Life Questionnaire; HBV-DNA=hepatitis B virus-
deoxyribonucleic acid; HBsAg= hepatitis B surface antigen; HCV-Ab=hepatitis C virus antibody; HCV-RNA=hepatitis C 
virus- ribonucleic acid; HDL=high-density lipoprotein; HIV-1=human immunodeficiency virus 1; HIV-2= human 
immunodeficiency virus 2; HJHS=hemophilia joint health score; HLA= human leukocyte antigen; LDH= lactate 
dehydrogenase; LDL=low-density lipoprotein; LL=local laboratory; NAAT=nucleic acid amplification testing; 
PBMC=peripheral blood mononuclear cells; RBC=red blood cell; Rh factor= rhesus factor; RNA=ribonucleic acid; SARS-
CoV-2=severe acute respiratory syndrome coronavirus 2; TB=tuberculosis; 
; TPMT=thiopurine 
methyltransferase; VWF=von Willebrand factor; WBC=white blood cell. 
 
6.4 
Screening Period 
All participants must provide written informed consent before any study-specific procedures or 
assessments are performed.  All screening evaluations must be completed and reviewed to 
confirm that potential participants meet all eligibility criteria. The Investigator will maintain a 
screening log to record details of all participants screened and to confirm eligibility or record 
reasons for screening failure, as applicable.  
Immunosuppressive therapy may enhance adverse/toxic effects of live vaccines and may 
diminish the effect of inactivated vaccines. Due to the potential that trial participants may receive 
an immunosuppressive medication during the SPK-8016-101 trial, it is highly recommended that 
the Investigator and participant review and complete all age-appropriate vaccinations during the 
screening period, at least 4 weeks prior to the planned day of administration of SPK-8016.  

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Anti-SARS-CoV-2 serology testing will be conducted at baseline and at Week 18. This result 
will be informative only and does not affect trial Inclusion/Exclusion. Multiple effects of 
COVID-19 on the hemostatic system have been described, including the development of anti-
phospholipid antibodies (Lupus-like anticoagulants), which might confound interpretation of 
one-stage clotting factor VIII assays.  
6.4.1 Screening Assessments 
The following procedures will be performed during the screening period, which may occur over 
a period up to a maximum of 16 weeks: 
• Obtain written informed consent 
• Review inclusion/exclusion criteria 
• Review prior and concomitant therapy 
• Record information on demographic and baseline characteristics  
• Record medical history and hemophilia history, including FVIII treatment history and 
bleeding history 
• Record Target Joints, Hemophilia Joint Health Score, and Joint Health Assessment – May 
be done at Screening or prior to Day 0 
• Measure vital signs (blood pressure (systolic and diastolic), pulse, respiratory rate, and 
oral/temporal temperature) 
• Perform physical examination, including height and weight 
• Haem-A-QoL, EQ-5D-5L – May be done at Screening or prior to Day 0 
• Electrocardiogram (ECG) (for participants >50 years of age, or if clinically indicated) 
• Obtain samples for central laboratory evaluation, unless otherwise noted: 
− Hematology 
− Clinical Chemistry 
− α-fetoprotein 
− Vector shedding (Serum, saliva, urine, semen) – May be done at Screening or Day 0 
(prior to vector infusion) 
− Spare Plasma 
− AAV Neutralizing Antibody 
− HBsAg, anti-HBc, HBV-DNA  
− HCV-RNA load assay 
− Urinalysis (using dipstick) 
− Coagulation: 
o aPTT, FVIII Activity, FVIII Inhibitor (local and central labs) 
o FVIII Antigen, VWF Activity, VWF antigen 
− Liver function and CRP tests (local and central lab) 
− Liver fibrosis diagnostic test, i.e., FibroScan (LL), FibroTest (CL), or AST-to-Platelet 
Ratio Index (LL) 
− Fibrinogen Antigen (local lab)  
o D-Dimer 

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o Thrombin Time  
− Thiopurine methyltransferase (TPMT) (local lab)  
− TB Interferon-gamma Release Assay (local lab) 
− Anti-SARS-CoV-2 serology (local lab) 
− If indicated: 
o HIV1/HIV2 viral load and CD4+ T cell count (for participants who are HIV-
positive), 
 (local lab or central lab), 
o Lipid panel (for participants with a history of dyslipidemia or 
hypercholesterolemia, or if clinically indicated) 
o Liver ultrasound ("if indicated" means "if, in the judgment of the investigating site 
or the Sponsor”, the liver ultrasound is indicated to aid interpretation of the 
screening evaluation of liver fibrosis”.) 
o FVIII Genotyping and HLA Genotyping (Optional sample if genotype is 
unknown) * 
− Dispense and train on FVIII Infusion Log (May be done at Screening or Day 0 (prior 
to vector infusion) 
− Provide PK profile using participants’ current FVIII product. This can be obtained 
from historical values if available and must include recovery (maximum drug 
concentration [Cmax] following a known IU/kg dose of FVIII) and half-life 
calculations. Population PK analysis using 2-4 samples is acceptable. If this is not 
available on the current FVIII product, the participant must undergo a PK analysis 
locally – May be done at Screening or Day 0 (prior to vector infusion) 
*For participants, whose FVIII genotype is unknown, a sample may be drawn for analysis at 
screening.  This is not an inclusion or exclusion criterion; the participant’s refusal to have this 
FVIII genotype sample taken would not exclude the participant from the study unless the 
severity of the disease is otherwise unable to be verified. FVIII genotyping may provide 
information regarding the predisposition of genotypic subpopulations to experience different 
bleeding frequencies and/or to experience different immunologic responses to FVIII following 
gene therapy. 
6.5 
 Three to Seven Days Prior to Day 0 Assessments 
Laboratory assessments should occur within 3 to 7 days prior to Day 0 for Cohorts 1 and 2. 
Results must be consistent with inclusion/exclusion criteria.  
• Liver Function (local lab)  
• Hematology (local lab)   
• Immunology (central lab)  
• Immune Profiling (central lab)  
• PAX Gene (RNA) (central lab)  
• SARS-CoV-2 NAAT Testing (local lab) 

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At the discretion of the Investigator, NAAT for infection with SARS-CoV-2 (the infectious agent 
in COVID-19) may be performed at this time for asymptomatic individuals. NAAT testing 
should be performed at this time for individuals with any of the following symptoms: cough, 
shortness of breath or difficulty breathing, fever, chills, sore throat, new loss of taste or smell, 
new persistent or recurrent muscle pain, and/or headache. Note: The Infectious Disease Society 
of America panel suggests collecting nasopharyngeal, or mid-turbinate or nasal swabs rather than 
oropharyngeal swabs or saliva alone for SARS-CoV-2 RNA testing in symptomatic individuals 
with URTI or ILI suspected of having COVID-19. 
The participants in Cohort 1 should be contacted prior to Day -2 to verify no changes in the 
general health status and confirm the prescribed 
 regimen. 
6.6 
Day -2 
 
6.7 
Dosing Day Assessments (Day 0) 
If a participant has a bleeding event between the screening period and the start of Day 0, he 
should be treated with his previous FVIII product. All bleeding events and related treatment 
should be recorded in the electronic case report form (eCRF).   
All participants will administer a prophylactic infusion of 50 IU/kg of their current FVIII 
concentrate the morning of Day 0. This can be self-administered and recorded in the subject 
infusion log.  Supervision from the site staff is permitted.  
 
 
  
All participants will be infused with a single IV infusion of SPK-8016 at the vector-
administration center.  The complete dose of SPK-8016 will be infused via infusion pump over 
approximately 60 minutes.   
6.7.1 FVIII Dosing 
At FVIII dosing: 
• Verify the administration (self-infused or assisted by site staff) of a single prophylactic 
IV infusion of 50 IU/kg of FVIII product on the morning of Day 0. 
6.7.2 
 
 
 
 
 

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6.7.3 Vector Dosing 
Assessments before SPK-8016 Vector infusion: 
• Review Inclusion/ Exclusion Criteria 
• Measure vital signs (blood pressure, pulse, respiratory rate, and oral/temporal temperature)  
• Perform physical examination, including weight  
• Hemophilia Activities List, Health Economic Assessment  
• Record AEs and concomitant therapies  
• Vector shedding, if not done at screening 
• Immunology 
• Immune Profiling 
• PAX Gene 
Assessments during Vector infusion: 
• Administer a single IV infusion of SPK-8016 via infusion pump over approximately 60 
minutes 
• Record AEs and concomitant therapies 
Assessments after completion of Vector infusion: 
• Vital signs immediately following the infusion (± 2 minutes), and 2 hours (±10 min) after the 
end of vector infusion 
• Obtain blood samples for central laboratory evaluation at 30 (±2) min, 2 hours (±10 min), 
and 5 hours (±10 min) after the completion of the vector infusion: 
- Immune Profiling 
- PAX Gene (RNA) (at 5 hours [±10 min]) 
• Record AEs and concomitant therapies 
6.7.4 Day 1 
Measure vital signs 24 (± 1 hr) hours post-SPK-8016 dose (blood pressure, pulse, respiratory 
rate, and oral/temporal temperature).  
Obtain blood samples for central laboratory evaluation at all visits, unless otherwise noted: 
• Immune profiling (CL) (24 (±1) hours post vector infusion) 
• PAX Gene (CL) (24 (±1) hours post vector infusion) 
• Hematology (CL and LL) 
• Coagulation: aPTT and FVIII Activity (CL and LL), FVIII Antigen (CL) 
• Liver Function tests and CRP (CL and LL) 
• Spare plasma 
• Record AEs and concomitant therapies 

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6.8 
Follow-up Observation Period (Weeks 1-52) 
Visits during the follow-up period may occur at either the vector-administration or follow-up 
center.  Any visit requiring a physical exam must be performed at the study center. All other 
visits without a physical exam may be performed by a qualified and trained in-home service 
provider. 
U.S. Centers for Disease Control and Prevention (CDC) guidance on hygiene, travel, and social 
interactions will be considered by the Investigator and Sponsor to minimize the risk of 
community acquired viral infections. This may include recommending a shelter in place and/or 
other social distancing measures during immunomodulation. FDA Guidance for Industry, 
Investigators, and Institutional Review Boards on Conduct of Clinical Trials of Medical 
Products during COVID-19 Pandemic (2020) will provide considerations to assist in assuring the 
safety of trial participants. International and local health guidelines may be observed for sites 
outside of the United States. 
6.8.1 
Days 3, 7, 10, 14, 17, 21, 24, 28, 31, 35, 38, 42, 45, 49, 52, 56, 59, 63, 66, 70, 73, 77, 
84 (± 2 days) 
A minimum of twice weekly visits is required from Weeks 1-11 to monitor for a potential rise in 
hepatic transaminases and/or loss of FVIII transgene expression. Visits at Weeks 1-11 may be 
performed by an in-home service provider.  
If an apparent immune response/liver inflammation is observed and triggers the initiation of 
reactive corticosteroids, additional samples are to be collected for Immune Profiling and PAX 
Gene prior to corticosteroid administration, if possible, and then Week 1 (± 2 days) and Week 2 
(± 2 days)  following initiation of corticosteroids (see Table 1 and Section 4.1.3.1). 
If immunomodulation is initiated, weekly visits should continue to collect transaminases, FVIII 
(local and central lab), Immunology. Weekly hematology and monthly lipid profile results 
should also be monitored locally for Cohorts 1 and 2 until 
 dosing has stopped, and 
this may be altered based on clinical response. Social distancing may be recommended for the 
duration of immunomodulation. 
The following procedures will be performed at all visits unless otherwise noted: 
• Perform physical examination, including weight (Days 3, 49, 84) 
• Measure vital signs (Days 3, 49, 84) 
• Hemophilia Activities List, Health Economic Assessment (Days 49 and 84) 
• Review FVIII Infusion Log (Days 49, 84) 
• Obtain samples for central laboratory evaluation at all visits, unless otherwise noted: 
- Coagulation 
o FVIII Inhibitor (Days 49, 84) 
o aPTT, FVIII activity (local and central labs)  
o FVIII antigen (central lab) 
- Liver function tests and CRP (local and central labs) 
- Hematology (Days 3, 49, 84)  
- Clinical Chemistry (Days 3, 49, 84) 

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- AAV Neutralizing antibody (Days 3, 10, 17, 24, 31, 49, 84) 
- Immunology (Days 3, 10, 17, 24, 31, 38, 45, 52, 59, 66, 73, 84) 
- Immune profiling (Days 7, 14, 21, 28, 56) 
- PAX Gene (RNA) (Days 14, 28, 56) 
- Spare plasma (Day 84) 
- Vector shedding samples (serum, saliva, urine, semen) 
NOTE: Vector shedding samples are collected only until the results of 3 consecutive 
samples are negative and received by the Investigator 
• Fibrinogen (Days 10, 24) (local) 
- Fibrinogen Antigen 
- D-Dimer 
- Thrombin Time 
• Record AEs and concomitant therapies 
6.8.2 Weeks 14, 16, 18, 22, 26, 30, 34, 40, 46, 52/End of Study (± 2 weeks) 
Visits will occur every 2 weeks from Weeks 12-16 and every 4 weeks from Weeks 18-34, then 
every 6 weeks from Weeks 40-52/ EOS. If immunomodulation is initiated, weekly visits should 
continue to collect transaminases, FVIII, and Immunology. Weekly Hematology and monthly 
lipid profile results should also be monitored regularly in the local lab for Cohorts 1 and 2 until 
 dosing has stopped, and this may be altered based on clinical response.  
The following procedures will be performed at all visits unless otherwise noted: 
• Perform physical examination, including weight (Weeks 18, 26, 34, 40, 52/EOS) 
• Measure vital signs 
• Perform liver ultrasound (Week 52/EOS, if indicated) 
• Record Target Joints and Joint Health Assessment (Week 26, 52/EOS) 
• Hemophilia Activities List, Health Economic Assessment (Weeks 14, 26, 40, 52/EOS) 
• Haem-A-QoL, EQ-5D-5L (Weeks 26, 52/EOS) 
• Review FVIII Infusion Log (Weeks 14, 26, 40, 52/EOS) 
• Obtain samples for central laboratory evaluation, unless otherwise noted: 
- Coagulation: 
o aPTT, FVIII activity (local and central labs)  
o FVIII antigen  
o FVIII Inhibitor (Weeks 14, 26, 40, 52)  
o VWF activity, VWF antigen (Week 52/EOS only) 
- Liver function tests and CRP (local and central labs) 
- Immunology 
- Immune profiling (Week 16) 
- Lipid panel (Week 52, if indicated) 
- Vector shedding samples (serum, saliva, urine, semen) 
NOTE: Vector shedding samples are collected only until 3 consecutive samples are 
negative 
- Hematology (Weeks 14, 16, 18, 26, 52/EOS)  

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- Clinical Chemistry (Weeks 18, 26, 52/EOS) 
- AAV Neutralizing antibody (Weeks 14, 26, 40, 52/EOS) 
- Spare plasma (Weeks 26, 40, 52/EOS) 
- Urinalysis (Weeks 26, 52/EOS) 
- Anti SARS-CoV-2 serology (Week 18) 
- α-fetoprotein (Week 52/EOS) 
• Record AEs and concomitant therapies 
If the LTFU study is not enrolling at the time of a participant’s planned Week 52/EOS visit, the 
participant may remain in SPK-8016-101. The Week 52 procedures are to be performed only at 
the EOS Visit.  The procedures from Week 46, except for collection of immunology samples for 
ELISpot, will be performed at Week 52 and every 12 weeks until the LTFU study is open. At 
that time, the Week 52/EOS visit should occur. 

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7 
STUDY INTERVENTION 
7.1 
Description of Study Drug 
Study Drug Name: 
SPK-8016 
Formulation 
(including dosage 
form and strength): 
 
 
Route of 
Administration: 
IV infusion 
Manufacturing 
Process 
 
 
Packaging and 
Labeling: 
1. SPK-8016 will be provided as a 
sterile frozen (< -60°C) liquid at a 
volume of 1.0 mL in a 
polypropylene sterile 1.5 mL 
cryogenic screw cap vial.  
2. SPK-8016-EC (
) 
will be provided as a sterile 
frozen (< -60°C) liquid at a 
volume of 1.0 mL in a 
polypropylene sterile 1.5 mL 
cryogenic screw cap vial.  
3. Study drug will be labeled as 
required per country 
requirements. 
1. SPK-8016 
will 
be 
provided 
as 
a 
sterile 
frozen (≤ -65°C) liquid at 
a volume of 1.0 mL in a 2 
mL Crystal Zenith® vial 
with stopper and flip-off 
seal.  
2. Study drug will be labeled 
as required per country 
requirements. 
Immunomodulators may be provided by the Sponsor. Refer to the most recent version of the 
Investigator’s and Pharmacy Brochures for further details on the study drug and 
immunomodulators. 
7.2 
Study Drug Doses 
The proposed SPK-8016 doses for this study are:  
• Starting dose (5x1011 vg/kg)  
• Middle dose (1x1012 vg/kg) 
• High dose (2x1012 vg/kg) 
Any decision to add a fourth dose level or further expand one of the existing protocol-defined 
dose levels will be made in consultation with the DMC. 
Once at least 2 participants of a given dose level complete 6 weeks of safety evaluation and the 
safety data have been reviewed by the DMC without any safety concern, then the first participant 
at the next dose level can be infused with SPK-8016.  Dosage for each participant will be 

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calculated according to the dose calculation worksheet and verified by the Sponsor. Weight 
obtained at Screening may be used for dose calculations. 
For participants with body mass index (BMI) exceeding 30 kg/m2, the study dose will be 
calculated based on an alternative body weight determination that assumes a maximum 
permissible BMI of 30 kg/m2.  For example, a participant who is 6’2” and weighs 370 pounds 
(BMI 47.5 kg/m2) would receive a vector dose based on an alternative body weight of 234 
pounds (which is the body weight associated with a BMI of 30 kg/m2 for a 6’2” individual). 
7.3 
Dose Schedule and Administration 
 
 
 
 
 
 
7.4 
Treatment Compliance 
Compliance with the infusion of FVIII protein product and SPK-8016 on the morning of Day 0 
will be verified and recorded by trained site staff. 
Participants will self-infuse with 50 IU/kg of their usual FVIII protein product per the product 
insert. Supervision from the site staff is permitted.  Number of vials, total volume, and total 
dosage will be monitored and recorded by the site staff.   
Cohort 1 will initiate 
 approximately 48 hours prior to Day 0. The time of administration 
will be monitored and recorded by the study staff. Participants in Cohort 2 will be infused with 
 after FVIII administration and will be supervised by the 
study staff. The relevant information will be monitored and recorded by the site staff.   
 
 
 
  
 
7.5 
Study Drug Storage 
SPK-8016 must be stored in a secure, environmentally controlled, and monitored (manual or 
automated) area in accordance with the labeled storage conditions, as outlined in the Pharmacy 
Brochure, with access limited to the Investigator and authorized site staff.  Sponsor-provided 
immunomodulators must be stored in a controlled environment under conditions outlined in the 
Pharmacy Brochure.   

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7.6 
Study Drug Preparation, Handling and Disposal 
7.6.1 Study Drug Preparation 
SPK-8016 is derived from a virus and should be considered and handled as an infectious agent. 
A qualified pharmacist with specific training on this protocol will be responsible for vector 
receipt from the Sponsor, storage, documentation of traceability of investigational product at the 
investigational site, and preparation on the day of administration.   
Prior to preparing the study drug and immunomodulators, the responsible pharmacy staff should 
first carefully review the instructions provided in the Pharmacy Brochure and/or relevant product 
labeling.  The investigational pharmacy personnel preparing the study drug for infusion will use 
universal precautions and appropriate personal protective equipment. The study drug dilution 
will be performed using aseptic techniques in a Class II biosafety cabinet.  Just prior to use, the 
frozen study drug product will be thawed at room temperature and diluted according to the 
verified dose calculation worksheet.  The diluted SPK-8016 product (i.e., for IV infusion), will 
be stored at room temperature and infusion must be initiated within 6 hours of thawing to assure 
maximum potency.  
7.6.2 Study Drug Handling and Disposal 
The Investigator or designee must confirm appropriate temperature conditions have been 
maintained during transit and on-site storage for all study drug and immunomodulators received, 
and any discrepancies are reported and resolved before use of the study drug or 
immunomodulator. Immediately after receipt, the study drug and immunomodulators should be 
stored as described in Section 7.5. Refer to the Pharmacy Brochure for management and 
reporting of any out-of-range temperatures. 
Only participants enrolled in the study may receive study drug or immunomodulator and only 
trained and authorized site staff may prepare, dispense and/or administer (except self-
administered medications) the study drug and immunomodulators. Study drug must only be 
thawed after confirmation that the participant is eligible, if applicable. Vials are for single use 
only.  
The site should store all used and unused vials of SPK-8016 and immunomodulators, as 
instructed by the Sponsor and as per the institution’s procedures.  
7.6.3 Accountability and Destruction 
The Investigator, institution, or the head of the medical institution (where applicable) is 
responsible for study drug accountability, reconciliation, and record maintenance (i.e., receipt, 
reconciliation, and final disposal records). Accountability must be maintained for SPK-8016 and 
immunomodulators. 
Participant returns of self-administered (take home) immunomodulators provided by the Sponsor 
must be documented by site personnel and retained at the site until reconciliation, where 
allowable per institutional policy, by the study monitor. At study completion, reconciliation must 
be made between the amount of study drug and immunomodulators supplied, dispensed, and 
subsequently destroyed or returned to the Sponsor, or its designee.   

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Further guidance and information for the final disposal of used/unused study drug and Sponsor-
provided immunomodulators are provided in the Pharmacy Brochure.  
7.7 
Labeling 
The SPK-8016 product label includes investigational product name; manufacturer; specific lot 
number; date of manufacture; vial number; storage instructions; and investigational product 
warning. The immunomodulator product labels include similar information. The Pharmacy 
Brochure will contain copies of all approved labels.  
7.8 
Study Compliance 
Following the administration of SPK-8016, participants will be followed according to the 
protocol schedule of assessments.  Participants will be encouraged to follow-up completely and 
according to study endpoints.  Non-adherence to the protocol will be reported to the relevant 
regulatory groups overseeing the study. 
7.9 
Prior and Concomitant Medications 
The use of concomitant therapies or procedures, as defined below, must be recorded on the 
participant’s eCRF. AEs related to administration of these therapies or procedures must be 
documented on the appropriate eCRF. 
7.9.1 Concomitant Therapy 
Any medication or vaccine (including over-the-counter or prescription medicines, vitamins, 
and/or herbal supplements) that the participant is receiving 30 days prior to screening, at the time 
of enrollment or during the study must be recorded in the eCRF along with: 
• Reason for use 
• Dates of administration including start and end dates 
• Dosage information including dose and frequency 
Participants taking medication routinely for a pre-existing condition should be on a regimen, 
which has been stable for at least 3 weeks, and dosage changes should not be anticipated during 
the post-infusion period for this study.  Any dose change during the study must be documented 
in the participant’s record and eCRF. The Investigator or designee should review the list of all 
current medications, over the counter medications, vitamins, and herbal supplements. Any 
concomitant medication that has known hepatoxicity should be discontinued in the first 12 weeks 
following vector infusion.  The Medical Monitor should be contacted if there are any questions 
regarding concomitant or prior therapy. 
7.9.2 Permitted Therapy 
During the study, participants are requested to suspend their prophylaxis regimen, but 
participants are permitted to take: 
• FVIII product, as needed for bleeding.  Usage of clotting factors (product, date, dosage, 
reason) will be recorded in the infusion log. The use of FVIII for prophylaxis is allowed 

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during the first 14 days post SPK-8016 infusion if deemed medically necessary by the 
Principal Investigator. 
• Non-steroidal anti-inflammatory drugs, except ibuprofen and acetylsalicylic acid 
(aspirin). 
Participants should be instructed to discuss any new medications, including non-prescription 
drugs and herbal preparations, with the Investigator prior to taking them.  
Other therapies considered necessary for the participant’s welfare may be given at the discretion 
of the Investigator.  All such therapies must be recorded in the eCRF.  
The CDC recommends a limit of 2 alcoholic drinks per day or fewer for adult males. While this 
is a general guideline, the Sponsor recommends not having any alcohol drinks during the first 12 
weeks after infusion of the study product. Alcohol has the potential to inflame the liver, which 
may make it difficult to monitor the status of liver health and has the potential to interact 
negatively with the study product or other medications. 
7.9.3 Prohibited Therapy 
The following concomitant medications are not permitted during the study: 
• Acetylsalicylic acid (aspirin) or ibuprofen; however, other non-steroidal anti-
inflammatory drugs are permitted. 
• Routine prophylactic treatment with FVIII product after Day 14 post vector infusion 
unless clinically indicated and in discussion with the Investigator and/or Sponsor. 
• Routine prophylactic treatment with emicizumab unless clinically indicated and in 
discussion with the Investigator and/or Sponsor 
• Any other investigational therapies prior to screening as defined in the exclusion criteria 
or used during the study 
Any medication or herbal supplement that may cause liver toxicity should be avoided. If a 
medication with known liver toxicity cannot be avoided, the Investigator should discuss it with 
the Sponsor.  
7.9.4 Concomitant Procedures 
A concomitant procedure is any therapeutic intervention (e.g., surgery/biopsy, physical therapy, 
tooth extraction) or diagnostic assessment (e.g., blood gas measurement, bacterial cultures) 
performed between the time the participant is enrolled (at screening) and the last study visit  
(Week 52/EOS). The use of concomitant procedures must be recorded in the eCRF. 

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8 
DISCONTINUATION OF STUDY INTERVENTION AND 
PARTICIPANT DISCONTINUATION/WITHDRAWAL 
8.1 
Participant Discontinuation/Withdrawal from the Study 
Participants may withdraw from the study at any time at their own request or may be withdrawn 
at any time at the discretion of the Investigator for safety, behavioral, compliance, or 
administrative reasons. If the participant withdraws consent for disclosure of future information, 
the Sponsor may retain and continue to use any data collected before such a withdrawal of 
consent. If a participant withdraws from the study and also withdraws consent for disclosure of 
future information, the Sponsor may retain and continue to use any data collected before such 
withdrawal of consent.  
The Investigator should record the reason and date of withdrawal in the eCRF and in the 
participant’s medical records. If possible, the participant should confirm his decision in writing. 
Participants who withdraw from the study after being dosed should return for a final visit within 
the timeframe of their next scheduled visit and complete all EOS assessments (see Table 1). 
A participant may permanently withdraw from the study at any time. An investigator and the 
Sponsor may permanently remove a participant from the study at their discretion for any of the 
following reasons: 
• The participant withdraws consent.   
• At the discretion of the Investigator for medical reasons. 
• At the discretion of the Investigator or Sponsor for non-compliance. 
• The participant is unwilling or unable to attend study visits and undergo safety 
assessments as per the protocol. 
If a participant withdraws from the study after enrollment, but before receiving a dose of the 
study drug (SPK-8016), then follow-up beyond the screening evaluations is not required.  
Withdrawn participants that did not receive a dose of the study product will be replaced. 
8.2 
Early Termination Study Visit 
If the participant withdraws after receiving the investigational product, the early termination 
study procedures should be identical to those of the EOS visit.  If the participant withdraws 
before receiving the investigational product, early termination study procedures are not 
necessary. 
8.3 
Lost to Follow Up 
A participant will be considered lost to follow-up if he/she repeatedly fails to return for 
scheduled visits and there is documentation that he/she has been unable to be contacted by the 
study site. 
The following actions must be taken if a participant fails to return to the clinic for a required 
study visit: 

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• The site must document the attempt to contact the participant and reschedule the missed 
visit as soon as possible.  The site should counsel the participant on the importance of 
maintaining the assigned visit schedule and ascertain if the participant wishes to and/or 
should continue in the study. 
• Before a participant is deemed lost to follow up, the Investigator or designee must make 
every effort to regain contact with the participant.  If the participant provided a telephone 
contact number, 3 telephone call attempts will be made to the participant’s last known 
phone number.  In addition, a certified letter will be sent to the participant’s last known 
mailing address (or local equivalent methods). These contact attempts will be 
documented in the participant’s study record. 
• Should the participant continue to be unreachable, he/she will be considered to have 
withdrawn from the study.  

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9 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
The definitions of an AE or SAE can be found in the following sections. 
An AE will be reported by the participant (or, when appropriate, by a caregiver, surrogate, or the 
participant's legally authorized representative).  
The Investigator and any qualified designees are responsible for detecting, documenting, and 
recording events that meet the definition of an AE or SAE and remain responsible for pursuing 
follow-up information for AEs or SAEs (see Section 9.4.1). 
9.1 
Definitions 
9.1.1 Adverse Event 
An AE is any untoward medical occurrence in a patient or clinical investigation participant 
administered a pharmaceutical product and which does not necessarily have a causal relationship 
with this treatment. An AE can therefore be any unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease temporally associated with the use of a 
medicinal (investigational) product, whether or not related to the medicinal (investigational) 
product.   
All AEs must be documented regardless of causality. 
Events meeting the AE definition include: 
• Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or 
other safety assessments (e.g., ECG, radiological scans, vital signs measurements), 
including those that worsen from baseline, considered clinically significant in the medical 
and scientific judgment of the Investigator (i.e., not related to progression of underlying 
disease). 
• Exacerbation of a chronic or intermittent pre-existing condition including either an 
increase in frequency and/or intensity of the condition. 
• New conditions detected or diagnosed after investigational product administration even 
though it may have been present at one time before the start of the study. 
• Signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction. 
• Signs, symptoms, or the clinical sequelae of a suspected overdose of either 
investigational product or a concomitant medication. Overdose per se will not be reported 
as an AE/SAE unless it is an intentional overdose taken with possible suicidal/self-
harming intent. Such overdoses should be reported regardless of sequelae. 
Lack of perceived efficacy, failure of expected pharmacological action, or similar assessment 
will not necessarily be reported as an AE. Such instances will be captured in the efficacy 
assessments.  However, if signs, symptoms and/or clinical sequelae resulting from lack of 
efficacy meet the definition of an SAE, they will be reported as such. 
Events that do NOT meet the AE definition include: 
• In this population, bleeding episodes are not considered AEs.  All bleeding episodes will 
be captured in the eCRF.  If serious criteria apply (see Section 9.1.2), the event should be 
reported as an SAE. 

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• Clinically significant abnormal laboratory findings or other abnormal safety assessments 
which are associated with the underlying disease, unless judged by the Investigator to be 
more severe than expected for the participant’s condition. 
• The disease/disorder being studied or expected progression, signs, or symptoms of the 
disease/disorder being studied, unless more severe than expected for the participant’s 
condition. 
• Medical or surgical procedures (e.g., endoscopy, appendectomy) should not be reported 
as AEs, but should be reported as the condition that led to the procedure as the AE. 
• Situations in which an untoward medical occurrence did not occur (social and/or 
convenience admission to a hospital). 
• Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present or 
detected at the start of the study that do not worsen. 
9.1.2 Definition of SAE 
If an event is not an AE per definition above, then it cannot be an SAE even if serious conditions 
are met (e.g., hospitalization for signs/symptoms of the disease under study, death due to 
progression of disease). 
An SAE is defined as any untoward medical occurrence that, at any dose meets the following 
criteria: 
• Results in death 
• Is life-threatening 
The term 'life-threatening' in the definition of 'serious' refers to an event in which the 
participant was at risk of death at the time of the event. It does not refer to an event which 
hypothetically might have caused death if it were more severe. 
• Requires inpatient hospitalization or prolongation of existing hospitalization 
In general, hospitalization signifies that the participant has been detained (usually involving 
at least an overnight stay) at the hospital or emergency department for observation and/or 
treatment that would not have been appropriate in the physician’s office or outpatient setting. 
Complications that occur during hospitalization are AEs. If a complication prolongs 
hospitalization or fulfills any other serious criteria, the event is serious. When in doubt as to 
whether “hospitalization” occurred or was necessary, the AE should be considered serious. 
• Results in persistent or significant disability/incapacity 
The term disability means a substantial disruption of a person’s ability to conduct normal life 
functions. 
This definition is not intended to include experiences of relatively minor medical significance 
such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, and accidental 
trauma (e.g., sprained ankle) which may interfere with or prevent everyday life functions but 
do not constitute a substantial disruption. 
• Is a congenital anomaly/birth defect 
• Other Medically Important conditions:  

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Medical or scientific judgment should be exercised in deciding whether SAE reporting is 
appropriate in other situations such as important medical events that may not be 
immediately life-threatening or result in death or hospitalization but may jeopardize the 
participant or may require medical or surgical intervention to prevent one of the other 
outcomes listed in the above definition. These events should usually be considered serious. 
A prescheduled, elective procedure, or a routinely scheduled treatment that requires 
hospitalization is NOT considered as to be an SAE; the study site must document all the 
following: 
• The prescheduled, or elective procedure, or routinely scheduled treatment was scheduled 
(or participant was on a waiting list to be scheduled) prior to obtaining the participant’s 
consent to participate in the study. 
• The condition requiring the prescheduled, or elective procedure, or routinely scheduled 
treatment was present before and did not worsen or progress, in the opinion of the 
Investigator, between the participant’s consent to participate in the study and the time of 
the procedure or treatment. The prescheduled, or elective procedure, or routinely 
scheduled treatment is the sole reason for the intervention or hospital admission. 
9.1.3 Adverse Events of Special Interest (AESI) 
There are several AEs that will be monitored closely during the study as adverse events of 
special interest (AESIs) to enable an adequate risk-benefit evaluation of SPK-8016 versus 
standard therapy during the study and additional data may be requested for these events. The 
AESIs are: 
• Any AEs associated with FVIII inhibitor formation 
• The development, in any participant, of SPK-8016 related (>10x ULN) elevated 
transaminases  
• The development, in any participant, of SPK-8016 related (≥2.5x ULN) elevated hepatic 
transaminases that fails to resolve to less than 2.5x ULN within 4 weeks with 
immunomodulatory agents 
• Any occurrence of a malignancy at any point after vector infusion that is related to SPK-
8016 or one of the immune modulating agents 
• Any thrombotic and/or embolic events (TEE) 
9.2 
Recording of AE and/or SAE 
When an AE/SAE occurs, it is the responsibility of the Investigator to review all documentation 
(e.g., hospital progress notes, laboratory reports, and diagnostics reports) related to the event. 
• The Investigator will then record all relevant AE/SAE information in the eCRF. 
• It is not acceptable for the Investigator to send photocopies of the participant’s medical 
records to Sponsor/designee in lieu of completion of the AE/SAE eCRF page. 
• There may be instances when copies of medical records for certain cases are requested by 
Sponsor/designee.  In this case, all participant identifiers, with the exception of the 
participant number, will be redacted on all copies of the medical records before 
submission to Sponsor/designee. 

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9.3 
Safety Classifications 
9.3.1 Assessment of Intensity 
The Investigator will assess the intensity for each AE and SAE reported during the study and 
assign it to one of the following categories: 
• Mild: An event that is easily tolerated by the participant, causing minimal discomfort and 
not interfering with everyday activities. 
• Moderate: An event that causes sufficient discomfort and interferes with normal everyday 
activities. 
• Severe: An event that prevents normal everyday activities. An AE that is assessed as 
severe should not be confused with an SAE. Severe is a category utilized for rating the 
intensity of an event; and both AEs and SAEs can be assessed as severe. 
An event is defined as ‘serious’ when it meets at least 1 of the predefined criteria as described in 
the definition of an SAE (see Section 9.1.2), NOT when it is assessed as having an intensity of 
severe.  
Other measures to evaluate AEs and SAEs (especially for abnormal laboratory changes) may be 
utilized (e.g., National Cancer Institute Common Terminology Criteria for Adverse Events [NCI-
CTCAE v5.0]). 
9.3.2 
Assessment of Causality 
The Investigator is obligated to assess the relationship between investigational product (or 
administration procedure or study required concomitant therapy) and occurrence of each 
AE/SAE. 
The causality assessment is one of the criteria used when determining regulatory reporting 
requirements. The causality assessment will be categorized either as: 
• Related – there are facts (evidence) or arguments to suggest a "reasonable possibility" for 
a causal relationship between the investigational product (or administration procedure or 
study required concomitant therapy) and the event. 
• Not Related – there is no reasonable temporal sequence or known pattern of response 
after administration of the investigational product and/or the AE could have been 
produced by the participant’s clinical state, environmental or toxic factors, or other 
therapy administered to the participant. 
The Investigator will use clinical judgment to determine the relationship. Alternative causes, 
such as underlying disease(s), concomitant therapy, and other risk factors, as well as the 
temporal relationship of the event to investigational product administration will be considered 
and investigated. 
The Investigator will also consult the Investigator’s Brochure (IB) and/or Product Information, 
for marketed products, in his/her assessment. 
For each AE/SAE, the Investigator must document in the medical notes that he/she has reviewed 
the AE/SAE and has provided an assessment of causality. 

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There may be situations in which an SAE has occurred, and the Investigator has minimal 
information to include in the initial report to Spark or designee. However, it is very important 
that the Investigator always assess the causality for every event before the initial transmission of 
the SAE data to Spark or designee. 
9.4 
Follow-up and Reporting Requirements 
9.4.1 Follow-up of AEs and SAEs 
After the initial AE/SAE report, the Investigator is required to proactively follow each 
participant at subsequent visits/contacts. All AE/SAEs will be followed until resolution, 
stabilization, the event is otherwise explained, or the participant is lost to follow-up. 
New or updated information will be recorded in the originally completed eCRF and SAE form. 
The Investigator will submit any updated SAE data to the Sponsor within 24 hours of receipt of 
the information. 
9.4.2 Reporting of SAEs 
Reporting to the Sponsor/designee via a SAE form will be performed as follows:  
• All SAEs must be recorded in the eCRF and reported in the SAE form to the Sponsor 
within 24 hours of site awareness via email to Spark@primevigilance.com.  
• In rare circumstances where SAE information is discussed during a phone conversation 
with the medical monitor, this SAE information must be documented and immediately 
sent via email to Spark PV at sparkpv@sparktx.com. A copy of the SAE form must be 
submitted following the telephone conversation to the SAE reporting email address. 
• Initial notification via telephone does not replace the need for the Investigator to 
complete and sign the SAE form pages within the designated reporting time frames. 
• Contacts for SAE reporting can be found on the SAE form 
The minimum reporting requirements for immediate reporting of SAEs include:  
• Participant ID 
• Event description 
• Identifiable reporting source 
• Investigator causality assessment 
9.5 
Time Period and Frequency for Collecting AE and/or SAE Information 
9.5.1 Time Period and Frequency for Collecting of AEs and SAEs 
All AEs and SAEs will be recorded during the period from the signing of the informed consent 
form (ICF) until the EOS visit at the time points specified in the Schedule of Events. 
Medical occurrences that begin before signing of the informed consent form will be recorded in 
the Medical History section of the eCRF not in the AE section. 

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9.5.2 
Collection of AEs and SAEs information after conclusion of the study 
Investigators are not obligated to actively seek AE or SAE information after conclusion of the 
study participation. However, if the Investigator learns of any SAE, including a death, at any 
time after a participant has been discharged from the study, and he/she considers the event to be 
reasonably related to study intervention or study participation, the Investigator must promptly 
notify the Sponsor. 
The method of recording, evaluating, and assessing causality of SAEs and the procedures for 
completing and transmitting SAE reports are provided in Section 9.2, Section 9.3, and Section 
9.4. 
9.5.3 
Regulatory Reporting Requirements for SAEs 
• Prompt notification by the Investigator to the Sponsor of a SAE is essential so that legal 
obligations and ethical responsibilities towards the safety of participants and the safety of 
a study intervention under clinical investigation are met.  
• The Sponsor has a legal responsibility to notify both the local regulatory authority and 
other regulatory agencies about the safety of a study intervention under clinical 
investigation. The Sponsor will comply with country-specific regulatory requirements 
relating to safety reporting to the regulatory authority, IRB/Independent Ethics 
Committees (IEC), and investigators. 
• Investigator safety reports must be prepared for suspected unexpected serious adverse 
reactions (SUSAR) according to local regulatory requirements and sponsor policy and 
forwarded to investigators as necessary. 
• An Investigator who receives an investigator safety report describing a SAE or other 
specific safety information (e.g., summary or listing of SAEs) from the Sponsor will 
review and then file it along with the Investigator’s Brochure and will notify the 
IRB/IEC, if appropriate according to local requirements. 
9.5.4 Pregnancy 
Participants included in this study are exclusively male.  Should a female partner of the 
participant become pregnant during the study, the Investigator must notify Spark within 14 days 
of the Investigator becoming aware of the pregnancy. The participant and female partner must 
sign an IRB approved pregnant partner release of medical information/ICF before the 
Investigator can collect medical information about the pregnancy.  
Whenever possible, and after a pregnant partner’s release of medical information/ICF is signed, a 
pregnancy in a female partner of a male participant exposed to SPK-8016 should be followed to 
term so as to assess any potential occurrence of congenital anomalies or birth defects. Any 
follow-up information, including premature termination and the status of the mother and child 
after delivery, should be reported by the Investigator to Spark using a Pregnancy 
Reporting/Outcome Form.  

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9.6 
Treatment of Overdose 
The chance of overdosing is remote as the SPK-8016 gene therapy product is a one-time 
administration with no participant access to the study drug.  Furthermore, SPK-8016 will be 
prepared by trained pharmacy staff and verified by a trained pharmacist before administration to 
the participant.  Nevertheless, for this study, an overdose is any dose given to a participant that is 
2 X greater than the intended dose of the study drug.  If an adverse event(s) is associated with 
(“results from”) the overdose, the adverse event(s) and overdose will be reported as serious 
adverse events, even if no other seriousness criteria are met.  If the overdose did not result in any 
associated clinical symptoms or abnormal laboratory results, the overdose will be reported as a 
non-serious adverse event as “accidental overdose without adverse effect”.  All overdoses with 
and without an adverse event should be recorded in the eCRF and only overdose with associated 
AEs should be reported in an SAE form and sent to the Sponsor/designee within 24 hours.  The 
participant should be monitored and should be treated as medically indicated based on their 
condition. 
In the event of an overdose, the Investigator should: 
1. Contact the Medical Monitor immediately. 
2. Closely monitor the participant for any AE/SAE and LFT abnormalities. LFTs should be 
monitored as per protocol and any abnormalities should be immediately reported to the 
Medical Monitor. 
3. Document the quantity of the excess dose as well as the duration of the overdose in the 
eCRF. 
 

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10 
STATISTICAL CONSIDERATIONS 
10.1 
Statistical Hypotheses 
No statistical hypotheses will be tested as part of this protocol.  This study will be used to 
establish an initial safety and efficacy profile of SPK-8016.  
10.2 
Sample Size Determination 
The sample size is based on the need to establish the initial safety and efficacy profile of SPK-
8016.  Up to 40 eligible participants will be dosed.  If more than 40 eligible participants are to be 
dosed, regulator(s) will be informed along with DMC recommendation. 
Because the size of the hemophilia A population is limited (an estimated incidence of 1 in 
5,000 male births), the number of participants available for study is correspondingly limited.  
Thus, the sample size is based on clinical, rather than statistical, considerations.  
10.3 
Populations for Analyses 
The Full Analysis Set (FAS) is defined as all participants who receive the infusion of SPK-8016. 
The analyses of efficacy will be performed in this population, including the evaluation of vector-
derived FVIII:C activity for estimation of peak and steady-state activity levels. As this is an 
open-label study, additional subgroup analyses may be specified following clinical review. 
The Safety Population will include all participants who receive any study-mandated medication 
and will be used to summarize all safety data.  
10.4 
Demography and Baseline Disease Characteristics 
All participants who receive the infusion of SPK-8016 will be included in the analysis of 
demography and baseline disease characteristics. 
Demographic and other baseline characteristics will be summarized using descriptive statistics.  
Data to be tabulated will include, but not be limited to age, race, medical/hemophilia history, and 
other disease-specific measures.  All data will be summarized by the overall study population 
and by dose level. 
10.5 
Primary and Secondary Endpoints 
10.5.1 
 Safety Analysis 
For the analysis of safety, the incidence and severity of AEs will be tabulated.  Changes from 
baseline in clinical laboratory variables (including FVIII inhibitor and laboratory parameters for 
thrombotic potential), vital signs, and physical examination findings will be summarized by 
descriptive statistics.   
All adverse events will be tabulated by occurrence, grouped by body system, and summarized by 
dose group. The rate of occurrence for each adverse event will be summarized by body system, 
severity, and relation to the administration of SPK-8016. All SAEs will be summarized 
separately.  

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Physical examination and clinical laboratory value abnormalities will be summarized by time 
point. Values and changes from baseline of key safety parameters (e.g., LFTs) at each time point 
will be tabulated. Exploratory analysis of safety parameters may be done by tabulating 
transitions from baseline to the EOS visit at Week 52 (e.g., normal to normal, normal to 
abnormal, etc.).  
Descriptive statistics will be used to evaluate the occurrence of hepatic transaminase elevation 
requiring immunosuppression and the incidence of immune responses to AAV capsid protein and 
. 
10.5.2 
Efficacy Analysis 
Summary statistics will be created by SPK-8016 dose group for the following parameters: 
• Peak and steady-state FVIII activity levels assessed by coagulation clotting assays. In this 
study, steady-state levels are based on FVIII:C measurements starting 12 weeks post 
vector administration and without the use of exogenous FVIII products since vector 
administration. See Section 10.5.3 for additional details.  
• The number of bleeding events expressed as the ABR per participant (spontaneous and 
traumatic combined) following SPK-8016 administration and beginning 28 days 
following SPK-8016 administration.  
• Absolute change in ABR for all bleeding events and for bleeding events observed 
beginning 28 days following SPK-8016 administration from the hemophilia history-based 
ABR for the 52 weeks prior to SPK-8016 administration. 
• Proportion of participants with a post SPK-8016 administration ABR of 0 or 1 (separately 
for all bleeding events and for bleeding events reported beginning at least 28 days 
following SPK-8016 administration).  
• Number of recorded FVIII infusions per participant (referred to in the protocol as 
annualized FVIII usage) following SPK-8016 administration and beginning 28 days 
following SPK-8016 administration. 
• Absolute change in the number of recorded FVIII infusions following SPK-8016 
administration (all infusions and infusions reported beginning 28 days following SPK-
8016 administration) from the prior 52-week hemophilia history. 
10.5.3 
Pharmacokinetics Analysis 
FVIII:C activity level (one stage; central lab-recorded) at nominal weeks 4, 8, 12, 16, 20, 24, 28, 
32, 36, 40, 44, 48 and 52 following SPK-8016 administration will be summarized descriptively 
by SPK-8016 dose group. For each participant, the difference between the lab visit date and the 
SPK-8016 infusion date will be obtained as the number of days of follow-up. The nominal week 
of follow-up will then be computed as the ceiling value of the number of days of follow-up 
divided by 7. Thus, as an example, nominal week 4 will be assigned to days 22 through 28 of 
follow-up. An average value will then be computed for all recorded data for the 4 nominal weeks 
up to a specific time point as defined above, e.g., the nominal week 16 activity level will be the 
average for all values obtained across nominal weeks 13, 14, 15 and 16. In the event of missing 
information, the available non-missing data will be used in this calculation, e.g., a participant 

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with no calculated nominal week 14 FVIII:C value would have the available nominal week 13, 
15 and 16 data used to estimate the nominal week 16 result. 
Steady-state FVIII:C activity (central lab-recorded) from nominal week 12 through nominal 
week 52 following SPK-8016 administration will be summarized descriptively by SPK-8016 
dose group. Using the same 4-week block construct already identified, an average value for each 
participant across all time points beginning with nominal week 12 will be computed, i.e., the 
average FVIII:C level across the individual participant’s averages obtained across nominal 
weeks 12, 16, 20, 24, 28, 32, 36, 40, 44, 48 and 52. The proportion of participants who maintain 
a FVIII:C activity level ≥ 12% across nominal weeks 12, 16, 20, 24, 28, 32, 36, 40, 44, 48 and 52 
will also be obtained. Depending on the observed distribution, additional exploratory 
calculations may be performed to evaluate the proportion of participants with a FVIII:C activity 
level ≥ 12% across, for example, at least 9 of these 11 timepoints to account for expected random 
variability. It is expected that corticosteroid therapy will increase the transgene expression from 
the SPK-8016 expression cassette; this effect is transient and dose-dependent so that as the 
corticosteroid is tapered and discontinued, the steady state FVIII expression from transduced 
hepatocytes becomes evident (not influenced by concurrent steroids). The assay of the FVIII 
activity that is measured while on corticosteroids (as assayed by one-stage and by chromogenic 
FVIII activity) appears not to be confounded, i.e., while on corticosteroids the observed 
hemostatic protection afforded by the transgene-derived FVIII is consistent with the measured 
FVIII activity and the increased measured level of FVIII is not an assay artifact. In contrast, 
 
is expected not to influence levels of expression of the transgene product, as evaluated in pre-
clinical animal studies (summarized in Section 2.2.4) and is expected not to have ongoing 
activity in circulation after week 12 (the timepoint after which the steady state FVIII:C activity is 
examined). For this reason, the treatment effect, i.e., determination of the ultimate steady state of 
FVIII:C activity resulting from the SPK-8016 dose, is not estimated while participants remain on 
corticosteroid therapy, and is evaluated after corticosteroids have been discontinued completely 
for at least 2-4 weeks. Additionally, the evaluation of the peak vector-derived FVIII:C activity 
may require careful interpretation if the participant is receiving corticosteroids at the time of 
peak activity, whereas 
 is not expected to have direct action on transgene expression and 
peak FVIII:C activity. One potential outcome at the conclusion of the study is that the peak 
FVIII:C activity may demonstrate a positive association with eventual steady state FVIII:C 
activity for participants who are not receiving reactive corticosteroids at time of peak activity (in 
Cohort 1 and Cohort 2) but may fail to demonstrate an association for participants whose peak 
FVIII:C activity occurs during concurrent treatment with corticosteroids. This potential 
confounding effect will be considered in the descriptive statistical review.  
Vector-shedding of SPK-8016 or time to Below Quantifiable Limits (BQL) will be summarized 
descriptively by dose group and by PBMC and bodily fluids. 
10.6 
Exploratory Endpoints Analysis 
All data on the new target joints, joint health score, changes in level of activity, hemophilia 
activities list, and Haem-A-QoL and EQ-5D-5L questionnaires, as described in Section 6.2, will 
be summarized descriptively by SPK-8016 dose group and provided in participant listings.  The 
questionnaires will be scored according to the recommendations of the questionnaire authors.  

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Each domain score and change from baseline in domain scores will be summarized by visit.  Full 
methods of analysis for this data will be presented in the Statistical Analysis Plan (SAP). 
All data on the joints and health-economic parameters will be summarized for exploratory 
purposes only.  Data will be analyzed according to related recommendations and guidelines.  
Continuous variables will be summarized by descriptive statistics and categorical variables will 
be presented with the number and percentage in each category.  No imputation of data will be 
performed.   
Exploratory inflammatory profiling of plasma and immune function gene expression of PBMC 
after vector administration (ELISpot, and other exploratory biomarkers) are described in Section 
3 and will be summarized descriptively by SPK-8016 dose group. 
10.7 
Interim Analyses 
Interim analyses – may be performed after at least 2 participants from a given dose cohort 
complete Week 12. 
The SAP will describe the planned interim analyses in greater detail. 
10.8 
End of Study Definition 
A participant is considered to have completed the study if he has completed all phases of the 
study including the EOS visit in Table 1. 
The EOS is defined as the date of the last participant’s last visit (LPLV). 

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11 
SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS 
11.1 
Regulatory, Ethical and Study Oversight Considerations 
The Sponsor and the Investigator(s) will comply with all instructions, regulations, and 
agreements in this protocol, and with applicable ICH GCP guidelines, and will conduct the study 
according to applicable local regulations.  The Sponsor and the Investigator(s) must adhere to the 
principles set forth by the Declaration of Helsinki dated October 2008. 
11.1.1 Regulatory and Ethical Considerations 
This study will be conducted in accordance with the protocol and with the following: 
• Consensus ethical principles derived from international guidelines including the 
Declaration of Helsinki and Council for International Organizations of Medical Sciences 
(CIOMS) International Ethical Guidelines 
• Applicable ICH GCP Guidelines 
• Applicable laws and regulations 
The protocol, protocol amendments, ICF, Investigator’s Brochure, and other relevant documents 
(e.g., advertisements) must be submitted to an IRB/IEC by the Investigator and reviewed and 
approved by the IRB/IEC before the study is initiated.  
Any amendments to the protocol will require IRB/IEC approval before implementation of 
changes made to the study design, except for changes necessary to eliminate an immediate 
hazard to study participants.  
The Investigator will be responsible for the following: 
• Providing written summaries of the status of the study to the IRB/IEC annually or more 
frequently in accordance with the requirements, policies, and procedures established by 
the IRB/IEC 
• Notifying the IRB/IEC of SAEs or other significant safety findings as required by 
IRB/IEC procedures 
• Providing oversight of the conduct of the study at the site and adherence to requirements 
of 21 CFR, ICH guidelines, the IRB/IEC, European regulation 536/2014 for clinical 
studies (if applicable), and all other applicable local regulations 
11.1.2 Financial Disclosure 
Investigators and sub-investigators will provide the Sponsor with sufficient, accurate financial 
information as requested to allow the Sponsor to submit complete and accurate financial 
certification or disclosure statements to the appropriate regulatory authorities. Investigators are 
responsible for providing information on financial interests during the course of the study and for 
1 year after completion of the study. 

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11.1.3 Informed Consent Process 
• The Investigator or his/her designee will explain the nature of the study to the participant 
or his legally authorized representative and answer all questions regarding the study.  
• Participants must be informed that their participation is voluntary. Participants or their 
legally authorized representative will be required to sign a statement of informed consent 
that meets the requirements of 21 CFR 50, local regulations, ICH guidelines, Health 
Insurance Portability and Accountability Act (HIPAA), where applicable, and the 
IRB/IEC or study center.  
• The medical record must include a statement that written informed consent was obtained 
before the participant was enrolled in the study and the date the written consent was 
obtained. The authorized person obtaining the informed consent must also sign the ICF. 
• Participants must be re-consented to the most current version of the ICF during their 
participation in the study.  
• A copy of the ICF(s) must be provided to the participant or the participant’s legally 
authorized representative. 
• Pregnant partners of male participants will be asked to sign a separate pregnant partner 
release of medical information/ICF to obtain pregnancy outcomes.  
11.1.4 
Data Protection 
Prior to any testing under this protocol, including screening tests and assessments, candidates 
must also provide all authorizations required by local law (e.g., HIPAA authorization in North 
America). 
The participant will not be identified by name in the eCRF or in any study reports and these 
reports will be used for research purposes only.  Participants will be assigned a unique identifier 
by the Sponsor. Any participant records or datasets that are transferred to the Sponsor will 
contain the identifier only; participant names or any information which would make the 
participant identifiable will be redacted.  
The participant must be informed that his medical records may be examined by Clinical Quality 
Assurance (CQA) auditors or other authorized personnel appointed by the Sponsor, by 
appropriate IRB/IEC members, and by inspectors from regulatory authorities. Every effort will 
be made to keep the participant’s personal medical data confidential. 
The participant must be informed that his personal study-related data will be used by the Sponsor 
in accordance with local data protection laws. The level of disclosure must also be explained to 
the participant.  
11.1.5 
Committee Structure 
The independent DMC is composed of at least 3 independent experts in hemophilia or 
immunology.  The independent DMC will be responsible for reviewing safety and efficacy 
periodically over the course of the study.  The specifics regarding the DMC organization and 
procedures will be outlined in the DMC Charter.  

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11.1.6 
Dissemination of Clinical Study Data 
The Sponsor will register the study and post study results, regardless of outcome, on a publicly 
accessible website (e.g., www.ClinicalTrials.gov), in accordance with the applicable laws and 
regulations. The Sponsor may also provide study information for inclusion in national registries 
according to local regulatory requirements. Results of this study will be disclosed according to the 
relevant regulatory requirements. 
11.1.7 
Data Quality Assurance 
• All participant data relating to the study will be recorded using an eCRF unless 
transmitted to the Sponsor or designee electronically (e.g., laboratory data). The 
Investigator is responsible for verifying that data entries are accurate and correct by 
signing the eCRF.  
• The Investigator must maintain accurate documentation (source data) that supports the 
information entered in the eCRF.   
• The Investigator must permit study-related monitoring, audits, IRB/IEC review, and 
regulatory agency inspections and provide direct access to source data documents.  
• The Sponsor or designee is responsible for the data management of this study including 
quality checking of the data.  
• Study monitors will perform ongoing source data verification to confirm that data entered 
into the eCRF by authorized site personnel are accurate, complete, and verifiable from 
source documents; that the safety and rights of participants are being protected; and that 
the study is being conducted in accordance with the currently approved protocol and any 
other study agreements, ICH GCP, and all applicable regulatory requirements.  
• Records and documents, including signed ICFs, pertaining to the conduct of this study 
must be retained by the Investigator for a period of 2 years following the date a marketing 
application is approved for the drug for the indication for which it is being investigated; 
or, if no application is to be filed or if the application is not approved for such indication, 
until 2 years after the investigation is discontinued and the FDA is notified; or, for a 
longer retention period if local regulations or institutional policies so require.  No records 
may be destroyed during the retention period without the written approval of the Sponsor. 
No records may be transferred to another location or party without written notification to 
the Sponsor.  
11.1.8 Source Documents 
Source documents provide evidence for the existence of the participant and substantiate the 
integrity of the data collected. Source documents are filed at the Investigator’s site. 
Data entered in the eCRF that are transcribed from source documents must be consistent with the 
source documents or the discrepancies must be explained. The Investigator may need to request 
previous medical records or transfer records, depending on the study. Also, current medical 
records must be available. 

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11.1.9 Study and Site Closure 
Study completion is defined as the date that the final participant was examined or received an 
intervention for the purposes of final collection of data for the primary outcome.  A study site is 
considered closed when all eCRFs have been signed by the Investigator and locked, all required 
documents and study supplies have been collected and a study-site closure visit has been 
performed. The Sponsor reserves the right to close the study site or terminate the study at any 
time for any reason at the sole discretion of the Sponsor. 
The Investigator may initiate study-site closure at any time, provided there is reasonable cause 
and sufficient notice is given in advance of the intended termination. 
Reasons for the early closure of a study site by the Sponsor or Investigator may include but are 
not limited to: 
• Failure of the Investigator to comply with the protocol, the requirements of the IRB/IEC 
or local health authorities, the Sponsor's procedures, or GCP guidelines 
• Inadequate recruitment of participants by the Investigator 
• Discontinuation of further study development 
11.1.10 
Publication Policy 
All study data and intellectual property rights in the results derived from the study are the 
property of the Sponsor.   The Sponsor may utilize the data in various ways, such as submission 
to government regulatory authorities or disclosure to other Investigators.  The Sponsor 
recognizes that the rights of the Investigator, although he/she is free to utilize data derived from 
the study for scientific purposes or publish the results in recognized scientific journals, are 
subject to the provisions of the Clinical Trial Agreement (CTA) upon completion of the study.  
Unless otherwise specified in the CTA, the Investigator’s institution and Investigator(s) shall not 
publish or present data from an individual study center until after publication of the results of the 
complete multicenter study. Subsequent publications must refer to the multicenter findings. The 
institution and Investigator shall submit reports, abstracts, manuscripts, and/or other presentation 
materials to the Sponsor for review and approval before submission for publication or 
presentation. The Sponsor shall have 30 days in advance of submission to respond with any 
requested revisions, including, without limitation, the deletion of confidential information. The 
Investigator(s) shall act in good faith upon requested revisions and shall delete any confidential 
information from such proposed publication. 

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12 
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contain factor VIII. Journal of Thrombosis and Haemostasis : JTH, 12(1), 36-42.  
139. 
Snyder, R.O., Miao, C.H., Patijn, G.A., Spratt, S.K., Danos, O., Nagy, D., ... & Kay, 
M.A. (1997). Persistent and therapeutic concentrations of human factor IX in mice 
after hepatic gene transfer of recombinant AAV vectors. Nature Genetics, 16(3), 
270-6. 
140. 
Srivastava, A., Brewer, A.K., Mauser-Bunschoten, E.P., Key, N.S., Kitchen, S., Llinas, 
A., ... & Street, A. (2013). Guidelines for the management of hemophilia. 
Haemophilia: The Official Journal of the World Federation of Hemophilia, 19(1), 
e1-47.  
141. 
Stieltjes, N., Calvez, T., Demiguel, V., Torchet, M.F., Briquel, M.E., Fressinaud, E., ... 
& Chambost, H. (2005). Intracranial haemorrhages in French haemophilia patients 
(1991-2001): Clinical presentation, management and prognosis factors for 
death. Haemophilia: The Official Journal of the World Federation of 
Hemophilia, 11(5), 452-8. 
142. 
Tabor E. (1999).  The epidemiology of virus transmission by plasma derivatives: clinical 
studies verifying the lack of transmission of hepatitis B and C viruses and HIV type 
1.  Transfusion, 39(11-12): 1160-1168. 
143. 
Tagliavacca, L., Moon, N., Dunham, W.R., & Kaufman, R.J. (1997). Identification and 
functional requirement of Cu(I) and its ligands within coagulation factor VIII. The 
Journal of Biological Chemistry, 272(43), 27428-34. 
 
 
 
145. 
Toole, J.J., Pittman, D.D., Orr, E.C., Murtha, P., Wasley, L.C., & Kaufman, R.J. 
(1986). A large region (approximately equal to 95 kDa) of human factor VIII is 
dispensable for in vitro procoagulant activity. Proceedings of the National 
Academy of Sciences of the United States of America, 83(16), 5939-42. 
146. 
Toole, J.J., Knopf, J.L., Wozney, J.M., Sultzman, L.A., Buecker, J.L., Pittman, D.D., ... 

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& Orr, E.C. (1984). Molecular cloning of a cDNA encoding human antihaemophilic 
factor. Nature, 312(5992), 342-7. 
147. 
Saulyte Trakymiene, S., Ingerslev, J., & Rageliene, L. (2010). Utility of the 
Haemophilia Joint Health Score in study of episodically treated boys with severe 
haemophilia A and B in Lithuania. Haemophilia: The Official Journal of the World 
Federation of Hemophilia, 16(3), 479-86. 
148. 
Torres, R.P.A., Torres, R.F.A., Torres, R.A., & Torres, R.S.L.A. (2018). Pulse therapy 
combined with oral corticosteroids in the management of severe rheumatic carditis 
and rebound. Cardiology in the Young, 28(2), 309-14. 
149. 
U.S. Food and Drug Administration. 2020.  FDA Guidance on Conduct of Clinical 
Trials of Medical Products during COVID-19 Public Health Emergency. 
Washington, DC.  Retrieved 04May2020 from 
https://www.fda.gov/media/136238/download 
150. 
van den Berg, H.M., Gouw, S.C., & van der Bom, J.G. (2013). Factor VIII products and 
inhibitors in severe hemophilia A. The New England Journal of Medicine, 368(15), 
1457. 
151. 
van Genderen, F.R., van Meeteren, N.L., van der Bom, J.G., Heijnen, L., de Kleijn, P., 
van den Berg, H.M., & Helders, P.J. (2004). Functional consequences of 
haemophilia in adults: the development of the Haemophilia Activities 
List. Haemophilia: The Official Journal of the World Federation of 
Hemophilia, 10(5), 565-71. 
152. 
van Genderen, F.R., Westers, P., Heijnen, L., de Kleijn, P., van den Berg, H.M., 
Helders, P.J., & van Meeteren, N.L. (2006). Measuring patients' perceptions on their 
functional abilities: validation of the Haemophilia Activities List. Haemophilia: The 
Official Journal of the World Federation of Hemophilia, 12(1), 36-46. 
153. 
von Mackensen S, Gringeri A, Ravera S. (2005). Validation of the haemophilia-specific 
quality of life questionnaire for adult patients with haemophilia (Haem-A-QoL). 
Haematologica. 90, 115-6. 
154. 
Walker, F.J., Scandella, D., & Fay, P.J. (1990). Identification of the binding site for 
activated protein C on the light chain of factors V and VIII. The Journal of 
Biological Chemistry, 265(3), 1484-9. 
155. 
Wang, M., Álvarez-Román, M.T., Chowdary, P., Quon, D.V., & Schafer, K. (2016). 
Physical activity in individuals with haemophilia and experience with recombinant 
factor VIII Fc fusion protein and recombinant factor IX Fc fusion protein for the 
treatment of active patients: a literature review and case reports. Blood Coagulation 
& Fibrinolysis : An International Journal in Haemostasis and Thrombosis, 27(7), 
737-44. 
156. 
Wei, Y., Ji, X.B., Wang, Y.W., Wang, J.X., Yang, E.Q., Wang, Z.C., ... & Hou, M. 
(2016). High-dose dexamethasone vs prednisone for treatment of adult immune 
thrombocytopenia: a prospective multicenter randomized trial. Blood, 127(3), 296-
302; quiz 370. 
157. 
White, G.C., Kempton, C.L., Grimsley, A., Nielsen, B., & Roberts, H.R. (2005). 
Cellular immune responses in hemophilia: why do inhibitors develop in some, but 
not all hemophiliacs. Journal of Thrombosis and Haemostasis : JTH, 3(8), 1676-81.  
158. 
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S. (1998). Clinical evaluation of recombinant factor IX. Seminars in 
Hematology, 35(2 Suppl 2), 33-8. 
159. 
Wilde, J., Teixeira, P., Bramhall, S.R., Gunson, B., Mutimer, D., Mirza, D.F. (2002). 
Liver transplantation in haemophilia.” British Journal of Haematology 117 (4): 952–
56. 
160. 
Wion, K.L., Kelly, D., Summerfield, J.A., Tuddenham, E.G., & Lawn, R.M. (1985). 
Distribution of factor VIII mRNA and antigen in human liver and other 
tissues. Nature, 317(6039), 726-9. 
161. 
World Federation of Hemophilia. (2015). World Federation of Hemophilia Report on 
the Annual Global Survey 2014. http://www1.wfh.org/publications/files/pdf-
1627.pdf  
162. 
Xiao, W., Berta, S.C., Lu, M.M., Moscioni, A.D., Tazelaar, J., & Wilson, J.M. (1998). 
Adeno-associated virus as a vector for liver-directed gene therapy. Journal of 
Virology, 72(12), 10222-6. 
163. 
Zelechowska, M.G., van Mourik, J.A., & Brodniewicz-Proba, T. (1985). Ultrastructural 
localization of factor VIII procoagulant antigen in human liver 
hepatocytes. Nature, 317(6039), 729-30.

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13 
APPENDIX 1:  FACTOR VIII INFUSION LOG 
 

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APPENDIX 2:  HEMOPHILIA ASSESSMENTS 
14.1 
HEMOPHILIA ACTIVITIES LIST  
 

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14.2 
HAEM-A-QOL QUESTIONNAIRE 
 

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14.3 
EQ-5D-5L QUESTIONNAIRE 
 

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14.4 
HEMOPHILIA JOINT HEALTH SCORE 
 

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15 
APPENDIX 3: ABBREVIATIONS 
AAV2 
Adeno-associated virus vector, serotype 2 
AAV5 
Adeno-associated virus vector, serotype 5 
 
 
AAV8 
Adeno-associated virus vector, serotype 8 
AAVhu37 
Adeno-associated virus vector, serotype hu37 
AAVrh10 
Adeno-associated virus vector, rhesus serotype 10 
 
 
 
 
ABR 
Annualized bleeding rate 
AE 
Adverse event  
AESI 
Adverse event of special interest 
AFU 
Annualized factor usage 
AIDS 
Acquired immunodeficiency syndrome 
ALP 
Alkaline phosphatase 
ALT 
Alanine aminotransferase 
ANC 
Absolute neutrophil count 
Anti-HBc 
Total Hepatitis B core antibody 
APRI 
AST-to-Platelet Ratio Index 
aPTT 
Activated partial thromboplastin time 
ARDS 
Acute Respiratory Distress Syndrome 
AST 
Aspartate aminotransferase 
 
 
 
 
BID 
Twice daily 
BMI 
Body mass index 
  
 
 
bp 
Base pair 
BP 
Blood pressure 
BU 
Bethesda units  

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BUN 
Blood urea nitrogen  
BQL 
Below Quantifiable Limits 
˚C 
Celsius  
CD4 
Cluster of differentiation 4 
CD8 
Cluster of differentiation 8 
CDC 
U.S. Centers for Disease Control and Prevention 
cDNA 
Complimentary deoxyribonucleic acid 
CIOMS 
Council for International Organizations of Medical Sciences 
Cl 
Chlorine 
CL 
Central laboratory 
Cmax 
Maximum drug concentration 
CNS 
Central nervous system 
COVID-19 
Coronavirus disease caused by SARS-CoV-2 
CRP 
C-reactive protein 
CQA 
Clinical Quality Assurance 
CTA 
Clinical trial agreement 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTL 
Cytotoxic T lymphocyte 
DMC 
Data Monitoring Committee 
DNA 
Deoxyribonucleic acid 
EASL 
European Association for the Study of the Liver 
EC 
Empty capsid 
ECG 
Electrocardiogram 
CRF/eCRF   Case report form/electronic case report form 
ED 
Exposure day 
EDC 
Electronic Data Capture 
EHL 
Extended half-life 
ELISpot 
Enzyme-linked immunospot assay 
EOS 
End of study 
EQ-5D-5L 
Euro quality-of-life five dimensions questionnaire 
FAS 
Full analysis set 
FDA 
U.S. Food and Drug Administration  

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FIX 
Coagulation factor IX 
FIXa 
Activated factor IX 
FVIII 
Coagulation factor VIII 
FVIII:C 
Factor VIII in circulation 
 
 
 
FXa 
Activated factor X 
GGT 
Gamma-glutamyl transferase 
GI 
Gastrointestinal  
GLP  
Good Laboratory Practice 
GMP 
Good Manufacturing Practice 
HAART 
Highly active anti-retroviral therapy 
Haem-A-
QoL 
Hemophilia Quality of Life Questionnaire 
HAL 
Hemophilia activities list 
HBsAg 
Hepatitis B surface antigen 
HBV 
Hepatitis B virus 
HCV 
Hepatitis C virus  
HDL 
High-density lipoprotein 
hFIX39-
Padua 
Human coagulation factor IX Padua variant 
hFVIII 
Human coagulation factor VIII 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human immunodeficiency virus  
HLA 
Human leukocyte antigen 
HR-QoL 
Health-related quality-of-life 
IB 
Investigator’s Brochure 
ICF 
Informed consent form 
ICH 
Intracranial hemorrhage 
ICH GCP 
International Conference on Harmonization Good Clinical Practice 
ID 
Identification 
IEC 
Institutional Ethics Committee 
IFN-γ 
Interferon gamma 

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IgG 
Immunoglobulin G 
IgG4 
Immunoglobulin G subclass 4 
IgM 
Immunoglobulin M 
IL 
Interleukin  
ILI 
Influenza like illness 
IND 
Investigational new drug application  
IRB 
Institutional Review Board 
ITR 
Inverted terminal repeats 
IU 
International units  
IV 
Intravenous 
LDH 
Lactate dehydrogenase  
LDL 
Low-density lipoprotein 
LFT 
Liver function test 
LL 
Local laboratory 
LPLD 
Lipoprotein lipase deficiency 
LPLV 
Last Participant’s Last Visit 
LSEC 
Liver sinusoidal endothelial cells 
LTFU 
Long-term follow-up 
MERS-
CoV 
Middle East respiratory syndrome-related coronavirus 
MHC 
Major histocompatibility complex  
 
 
mRNA 
Messenger ribonucleic acid 
NAb 
Neutralizing antibody 
NAAT 
Nucleic acid amplification testing 
NCT 
National Clinical Trial 
NHF-
MASAC 
National Hemophilia Foundation’s Medical and Scientific Advisory Council 
NHP 
Non-human primate  
PBMC 
 
Peripheral blood mononuclear cells  
PCR 
Polymerase chain reaction 
PHI 
Protected health information 
PI 
Principal Investigator 

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PK 
Pharmacokinetics 
PML 
Progressive multifocal leukoencephalopathy 
PV 
Pharmacovigilance  
QoL 
Quality of life 
rAAV 
Recombinant adeno-associated viral vectors  
RBC 
Red blood cells 
rFVIII 
Recombinant factor VIII 
RNA 
Ribonucleic acid 
RPE65 
Retinal pigment epithelium 65 gene 
SAE 
Serious adverse event  
SAP 
Statistical Analysis Plan 
SARS-
CoV-2 
Severe acute respiratory syndrome coronavirus 2 
SD 
Standard deviation 
SPK-8016  
Study drug in this protocol 
 
 
SPK-9001  
  
 
SUSAR 
Suspected Unexpected Serious Adverse Reactions 
TB 
Tuberculosis  
 
 
TEE 
Thrombotic and/or embolic events 
TNFα 
Tumor Necrosis Factor alpha 
TPMT 
Thiopurine methyltransferase 
TTR 
Transthyretin 
TTRm 
Modified transthyretin 
ULN 
Upper limit of normal  
URTI 
upper respiratory tract infection 
vg 
Vector genome  
VLDL 
Very low-density lipoprotein 
VWF 
von Willebrand factor 
WBC 
White blood cell 

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WFH 
World Federation of Hemophilia 
WHO 
World Health Organization 
XLMTM 
X-linked myotubular myopathy 

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16 
APPENDIX 4: SUMMARY OF CHANGES FROM THE PREVIOUS 
PROTOCOL VERSION 
 
AMENDMENT 2 
SECTION 
SUMMARY OF REVISIONS MADE 
RATIONALE 
1.1 Protocol 
Synopsis 
Protocol Synopsis has been updated. 
Updates reflect changes made 
throughout Amendment 2. 
Study Schema 
Figure 
Updated Study Schema Figure 1. 
The study schematic was updated 
to include the two cohorts. 
Schedule of 
Assessments 
Updated Schedule of Assessments Table 1.  
 
Additional screening and follow 
up is required. This includes 
screening labs, three to seven days 
prior to vector, Day -2 infusion of 
 and weekly follow up while 
on immunomodulation. In 
addition, the FVIII levels on Day 0 
were eliminated for all 
participants.  
 
2.2.4 & 2.2.5 
Added Summary of Non-clinical 
Experience with 
 
The preclinical study results 
provide evidence to support the 
design of cohort. 
2.2.5 
Added preclinical experience with 
 
The preclinical study results 
provide evidence to support the 
design of cohort 1. 
2.3.1 
Added risks associated with 
 
 
The risks associated with 
 
were provided to support the 
design of cohort 2. 
2.3.1 
Added risks associated with 
  
 
The risks associated with 
 
were provided to support the 
design of cohort 1  
2.4 
Updated Study Rationale with most recent 
published data. 
Results from gene therapy in 
hemophilia have been published 
since the previous amendment. 
Data from the Spark participants to 
date provides the rationale for this 
amendment. 
4.1.1 
Added cohorts 1 and 2 to the study design 
and provided instruction on sequence of 
enrollment. 
 
Details for the expansion and 
enrollment sequence of the two 
cohorts are included. A description 
of the expansion of cohorts up to 

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10 participants is described if 
additional data is needed. 
4.1.1 
Refined dose cohort expansion. 
Details for the expansion and 
enrollment sequence of the 3 doses 
into two cohorts are included. A 
description of the expansion of 
cohorts up to 10 participants is 
described if additional data is 
needed.  
4.1.3.1 
Added SARS-coV2 testing  
Multiple effects of COVID-19 on 
the hemostatic system have been 
described, including the 
development of anti-phospholipid 
antibodies (Lupus-like 
anticoagulants), which might 
confound interpretation of one-
stage clotting factor VIII assays 
4.2.2 
Updated study design to include up to 40 
participants. 
The addition of one additional 
dose and expansion of all dose 
levels into two cohorts will require 
an increase in the number of 
participants in the trial. 
5.2 
Provided clarification in eligibility for 
assessing liver fibrosis. 
On occasion, discordant results as 
it relates to liver health have been 
found in review of the medical 
record. The Fibro scan is 
considered the gold standard in 
determining liver 
elasticity/fibrosis. This protocol 
was clarified to elevate this test as 
the determinant for eligibility. 
5.2 
Exclusion criteria updated to included 
gastrointestinal disease, cancer, and latent 
infectious disease due to the addition of 
immunomodulation in the amendment. 
The use of immunomodulation 
agents has been included in the 
study design. The exclusion 
criteria changes are reflective of 
contraindications identified in the 
package insert for 
 
6.2.6 
Added ability to perform genetic analyses 
on archived samples   
Archived samples may be used for 
genetic sequencing if the 
participant provides consent. 
6.4.1 
Changed the FVIII PK analysis from Day 0 
to historic or during the screening period.  
 
The PK analysis added to the 
complexity of Day 0. Historic PK 
results provide sufficient 
information should it be needed to 
compare FVIII response post gene 
therapy.  

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6.7.1 
Changed the Day 0 FVIII infusion to a 
routine prophylactic treatment. 
 
The PK analysis is no longer 
needed on Day 0. Patients with 
hemophilia administer 
prophylactic treatments routinely. 
This will provide bleed protection 
for a portion of week one.  
6.5 & 6.6 
Added additional laboratory assessments 
for Cohort 2 prior to dosing day and 
following vector infusion.  
 
Additional laboratory assessments 
are required for participants in 
cohort 2 due to the planned use of 
 The testing three to seven 
days prior to dosing day is 
required to confirm that the critical 
laboratory values have not 
changed during the screening 
period.  Weekly follow up while 
on immunomodulation is 
recommended in the clinical 
management of participants 
receiving immunomodulation. 
NAAT testing for infection with 
SARS-coV2 in asymptomatic 
study participants prior to immune 
suppression and to recommend 
NAAT testing in symptomatic 
study participants prior to immune 
suppression has been included for 
participant safety.  
7 
Added information on description, storage, 
preparation, handling, disposal, 
accountability, and labeling of Sponsor-
provided immunomodulators 
Language was added to support 
Sponsor supply of 
immunomodulators. 
9.1.1 
Updated AE language 
 
AE language was updated to 
include standard toxicology 
reporting. Clarification of the 
timeframe to collect AEs was 
provided. 
11.1.9 
Added requirements for eCRF data to be 
locked before a site is closed.  
Clarification was provided to 
ensure eCRF data for a site is 
locked prior to that site being 
closed. 
Throughout 
Updated text and references as they relate 
to the Immunomodulatory clinical 
experience and the cohort design in this 
trial. 
Literature in the gene therapy is 
regularly published. Data and 
references were updated in all 
areas of the protocol to maintain 
relevant resources.  Data from the 

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Spark participants to date provides 
the rationale for this amendment. 
 
Document 
Description of Main Changes 
Amendment 1 
1. Incorporated minor edits for readability, corrected formatting issues, and 
corrected typographical errors to improve accuracy and clarity and updated 
abbreviations, citations and references. Changes and updates from the 
Sponsor’s latest protocol template were incorporated.  
2. Updated study schematic figure 1. 
3. Removed language requiring prophylactic steroid administration on Week 4. 
4. 
 
corticosteroids in response to an immune response trigger. 
5. Updated language to allow the Investigator in collaboration with the Sponsor 
to use other immunomodulatory agents, should they be required, to reduce 
long term and high dose steroid exposure 
6. Inserted Thiopurine methyltransferase (TPMT) as a screening requirement. 
7. Added risks associated with 
. 
8. Updated text and references as it relates to immunomodulatory clinical 
experience in Hemophilia Gene Therapy trials.