A Phase I/II, Open-label, Uncontrolled, Single-dose, Dose-ascending, Multi-centre Trial Investigating an Adeno-associated Viral Vector Containing a Codon-optimized Human Factor IX Gene (AAV5-hFIX) Administered to Adult Patients With Severe or Moderately Severe Hemophilia B
Phase: PHASE1
Conditions: Hemophilia B
Sponsor: CSL Behring

Clinical Trial Protocol 
Trial ID: CT-AMT-060-01 
 
 
uniQure biopharma B.V. 
 
Version 7.0, 20 April 2021 
Proprietary and Confidential 
 
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Clinical Trial Protocol 
 
Investigational Medicinal 
Product (IMP): 
AAV5-hFIX (adeno-associated viral vector containing a 
codon-optimized human factor IX gene) 
Indication: 
Haemophilia B 
Phase: 
I/II 
Title: 
A phase I/II, open-label, uncontrolled, single-dose, 
dose-ascending, multi-centre trial investigating an 
adeno-associated viral vector containing a codon-optimized 
human factor IX gene (AAV5-hFIX) administered to adult 
patients with severe or moderately severe haemophilia B 
Short Title: 
Phase I/II trial of AAV5-hFIX in severe or moderately severe 
haemophilia B 
Trial ID: 
CT-AMT-060-01 
EudraCT No.: 
2013-005579-42 
Date: 
20 April 2021 
Version: 
7.0 
Original Protocol 
20 June 2014 
Protocol Amendment 1 
17 July 2014 
Protocol Amendment 2 
24 November 2014 
Protocol Amendment 3 
12 December 2014 
Protocol Amendment 4 
12 January 2015 
Protocol Amendment 5 
27 March 2015 
Protocol Amendment 6 
23 September 2015 
Protocol Amendment 7 
22 December 2015 
Protocol Amendment 8 
20 April 2021 
Sponsor: 
uniQure biopharma B.V. 
Paasheuvelweg 25A 
1105 BP Amsterdam 
The Netherlands 
GCP Statement: 
This protocol has been designed and will be conducted, 
recorded, and reported in compliance with the principles of 
Good Clinical Practice (GCP) guidelines (as are stated in 
U.S. federal regulations as well as in “Guidance for Good 
Clinical Practice”, International Council for Harmonisation 
of Technical Requirements for Pharmaceuticals for Human 
Use), as well as with the detailed guidelines on good clinical 
practice specific to advanced therapy medicinal products (as 
issued by the European Commission on 03 December 2009). 
 
Confidentiality statement 
This uniQure biopharma B.V. document is confidential. The information within this document may not be 
reproduced or otherwise disseminated without approval of uniQure biopharma B.V. 
uniQure 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Date: 06 January 2022
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Clinical Trial Protocol 
Trial ID: CT-AMT-060-01 
uniQure biopharma B.V. 
Version 7.0, 20 April 2021 
Proprietary and Confidential 
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Sponsor Information 
Research & Development 
uniQure, Inc. 
113 Hartwell Avenue 
Lexington, MA 02421 
USA 
Phone: 
E-mail:
uniQure, Inc. 
113 Hartwell Avenue 
Lexington, MA 02421 
USA 
Phone: 
E-mail:
uniQure 
 
 
 
 
 
 
 
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Clinical Trial Protocol 
Trial ID: CT-AMT-060-01 
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Version 7.0, 20 April 2021 
Proprietary and Confidential 
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EMERGENCY CONTACT INFORMATION 
Any SAE, as well as any AE qualifying for special notification, occurring in a subject 
from the first visit until the end of trial participation must be reported on the paper SAE 
report form.  
In the event of a serious adverse event (SAE) or an adverse event (AE) qualifying for special 
notification, the Investigator must complete the Clinical Trial Serious Adverse Events Form 
in the paper SAE report from. 
The Investigator will ensure that the form is completed and faxed or scanned/e-mailed to 
 within 24 hours of becoming aware of the event meeting the 
protocol definition for an SAE or an AE qualifying for special notification. The paper SAE 
form and all other relevant documents supporting the reported SAE must be faxed or 
scanned/e-mailed to: 
24 hour Hotline: 
Fax: 
E-mail:
 
More details can be found in Section 13 of this protocol. 
uniQure 
 
 
 
 
 
 
 
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Table of Contents. 
Sponsor Information .................................................................................................................. 2
Emergency Contact Information ................................................................................................ 3
Table of Contents. ...................................................................................................................... 4
List of Tables ............................................................................................................................. 9
List of Figures .......................................................................................................................... 10
List of Abbreviations ............................................................................................................... 11
1.
Sponsor’s Approval of the Clinical Trial Protocol ......................................................... 15
2.
International Coordinating Investigator’s Approval of the Clinical Trial Protocol ....... 16
3.
Principal Investigator’s Agreement to the Clinical Trial Protocol ................................. 17
4.
Protocol Summary .......................................................................................................... 18
4.1 
Primary Objective ................................................................................................. 18
4.2 
Secondary Objectives ............................................................................................ 18
4.3 
Trial Design ........................................................................................................... 18
4.4 
Trial Population ..................................................................................................... 18
4.5 
Intervention ........................................................................................................... 18
4.6 
Interim Analyses ................................................................................................... 19
5.
Schedule of Trial Procedures.......................................................................................... 20
6.
Introduction and Rationale ............................................................................................. 29
6.1 
Introduction to Disease Area ................................................................................. 29
6.2 
Treatments ............................................................................................................. 29
6.3 
Limitations of Current Treatment Approach: Medical Need ................................ 30
Reliance on Central Venous Access Devices ......................................... 30
Risk of FIX Inhibitors ............................................................................. 30
Non-adherence Resulting in Increased Risk of Spontaneous Bleeds ..... 30
Burden of the Disease ............................................................................. 31
6.4 
Scientific Rationale for Gene Therapy in Haemophilia B .................................... 31
6.5 
Rationale for AAV Serotype 5 Vector in Haemophilia B ..................................... 32
6.6 
Investigational Medicinal Product ........................................................................ 32
Description of IMP ................................................................................. 32
Non-clinical Experience ......................................................................... 32
Clinical Experience with Similar Products ............................................. 37
6.7 
Risk/Benefit Considerations .................................................................................. 40
6.8 
Accommodations Due to the COVID-19 Pandemic ............................................. 42
7.
Purpose of the Trial ........................................................................................................ 44
8.
Trial Objectives and Endpoints ...................................................................................... 45
8.1 
Objectives .............................................................................................................. 45
Primary Objective ................................................................................... 45
Secondary Objectives ............................................................................. 45
6.3.1 
6.3.4 
6.6.1 
6.6.2 
6.6.3 
8.1.1 
8.1.2 
uniQure 
6.3.4 
8.1.1
6.3.1
6.3.3
6.3.2
6.6.1 
8.1.2
6.6.2 
6.6.3 
 
 
 
 
 
 
 
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Clinical Trial Protocol 
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8.2 
Endpoints ............................................................................................................... 45
Primary Endpoint .................................................................................... 45
Secondary Endpoints .............................................................................. 45
9.
Trial Population .............................................................................................................. 47
9.1 
Population ............................................................................................................. 47
9.2 
Inclusion Criteria ................................................................................................... 47
9.3 
Exclusion Criteria .................................................................................................. 48
9.4 
Withdrawal of Individual Subjects ........................................................................ 48
Replacement of Individual Subjects Withdrawn .................................... 49
10.
Trial Design .................................................................................................................... 50
10.1 Overall Trial Design .............................................................................................. 50
Considerations Due to the COVID-19 Pandemic ................................... 52
10.2 Trial Periods .......................................................................................................... 52
Screening Visit (Visit 1) ......................................................................... 52
Dosing Visit (Visit 2) .............................................................................. 53
Follow-up Period (Visits 3 – 35) ............................................................ 54
Additional Visits ..................................................................................... 54
Follow-Up after End of Trial .................................................................. 54
10.3 Use of Subject e-Diary .......................................................................................... 54
Training in the Use of Subject e-Diary ................................................... 54
10.4 Rationale for Trial Design ..................................................................................... 55
10.5 Dose Rationale ...................................................................................................... 56
10.6 Rationale for Proposed Intra- and Inter-cohort Staggering Intervals .................... 56
10.7 Rationale for Stopping Prophylactic FIX Replacement Therapy ......................... 57
11.
Trial Treatment ............................................................................................................... 58
11.1 Investigational Medicinal Product (IMP) .............................................................. 58
Description of IMP ................................................................................. 58
Packaging, Labelling and Storage of the IMP ........................................ 58
IMP Accountability, Traceability and Disposition ................................. 58
Preparation of the IMP for Intravenous Infusion .................................... 59
11.2 IMP Administration .............................................................................................. 59
Dose Levels and Allocation to Dosing ................................................... 59
Method of IMP Administration .............................................................. 59
Recommendation of Treatment in Case of Increased Liver 
Transaminases ......................................................................................... 60
11.3 Concomitant Medication/Therapy ........................................................................ 61
Allowed Medication ............................................................................... 61
Disallowed Medication ........................................................................... 61
Reporting of Concomitant Medication/Therapy ..................................... 61
8.2.1 
8.2.2 
10.1.1 
10.2.1 
10.2.2 
10.2.3 
10.2.4 
10.2.5 
10.3.1 
11.1.1 
11.1.2 
11.1.3 
11.1.4 
11.2.1 
11 .2.2 
11.2.3 
11.3.1 
11.3.2 
11.3.3 
uniQure 
8.2.1
8.2.2
9.4.1
10.1.1
10.2.1
10.2.2
10.2.3
10.2.4
10.2.5
10.3.1
11.1.1
11.1.2
11.1.3
11.1.4
11.2.1
11.2.2
11.2.3
11.3.1
11.3.2
11.3.3
 
 
 
 
 
 
 
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11.4 Tapering and Subsequent Withholding of Prophylactic FIX Replacement 
Therapy ................................................................................................................. 61
12.
Assessments .................................................................................................................... 64
12.1 Efficacy Assessments ............................................................................................ 64
FIX Activity for Efficacy Evaluation ..................................................... 64
Bleeding Episodes ................................................................................... 64
Prophylactic FIX Replacement Therapy ................................................. 66
 ............................................................................... 66
12.2 Safety Assessments ............................................................................................... 67
Adverse Events ....................................................................................... 67
Physical Examination ............................................................................. 67
Blood Pressure, Pulse and Body Temperature ........................................ 67
Serum Chemistry, Haematology, Coagulation and Urine Parameters 
for Central Lab ........................................................................................ 68
Vector DNA ............................................................................................ 69
Neutralizing Antibodies to AAV5 .......................................................... 69
Total Antibodies to AAV5 ...................................................................... 69
AAV5 capsid-specific T cells ................................................................. 70
Anti-FIX Antibodies ............................................................................... 70
FIX Inhibitors ......................................................................................... 70
FIX Recovery .......................................................................................... 70
Inflammatory Markers ............................................................................ 71
12.3 Other Assessments ................................................................................................ 71
Local Laboratory Sampling for Management of Subjects and 
Eligibility Check ..................................................................................... 71
FIX Protein Concentration ...................................................................... 72
Demographics and Disease Characteristics ............................................ 73
FIX Gene Mutation ................................................................................. 73
Medical History and Concomitant Illnesses ........................................... 73
History and Status of Bleeding ............................................................... 74
Information on Haemophilia B Disease Related Surgeries and Joint 
Status ....................................................................................................... 74
Body Mass Index .................................................................................... 75
Laboratory Parameters for Evaluation of Subject Eligibility ................. 75
12.3.10 Further Testing of Subjects with a Suspected FIX Inhibitor .................. 76
12.3.11 Laboratory Samples for Future Research ............................................... 76
12.4 General Information Regarding Laboratory Sampling and Results ...................... 76
12.5 Total Blood Volume .............................................................................................. 77
13.
Safety Reporting ............................................................................................................. 78
13.1 Adverse Event Definitions .................................................................................... 78
13.2 Adverse Event Assessment Definitions ................................................................ 78
12.1.l 
12.1.2 
12.1.3 
12.1.4 
12.2.1 
12.2.2 
12.2.3 
12.2.4 
12.2.5 
12.2.6 
12.2.7 
12.2.8 
12.2.9 
12.2.10 
12.2.11 
12.2.12 
uniQure 
12.1.1
12.1.2
12.1.3
12.1.4
12.2.1
12.2.2
12.2.3
12.2.4
12.2.5
12.2.6
12.2.7
12.2.8
12.2.9
12.2.10
12.2.11
12.2.12
12.3.1
12.3.2
12.3.3
12.3.4
12.3.5
12.3.6
12.3.7
12.3.8
12.3.9
12.3.10
12.3.11
 
 
 
 
 
 
 
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Severity ................................................................................................... 78 
Relationship to IMP ................................................................................ 79 
Outcome .................................................................................................. 79 
13.3 Reporting of Adverse Events ................................................................................ 80 
13.4 Prompt Reporting of SAEs and Other Events to uniQure ..................................... 80 
13.5 Regulatory Reporting Requirements for SAEs and Other Events ........................ 81 
13.6 Adverse Events Qualifying for Special Notification ............................................ 81 
13.7 Follow-up on Adverse Events ............................................................................... 82 
13.8 Reporting Requirements for Disease-Related Bleeding Episodes ........................ 82 
13.9 Pregnancies ........................................................................................................... 83 
13.10 Occupational Exposure ......................................................................................... 83 
14.
Statistical Methods ......................................................................................................... 84 
14.1 Sample Size Calculation ....................................................................................... 84 
14.2 Statistical Methods ................................................................................................ 84 
Primary Efficacy Endpoint Analysis ...................................................... 84 
Secondary Efficacy Endpoint Analysis .................................................. 85 
Patient Reported Outcome Data ............................................................. 85 
14.3 Subject Population(s) for Analysis ........................................................................ 86 
14.4 Significance Level ................................................................................................. 86 
14.5 Termination Criteria .............................................................................................. 86 
14.6 Handling of Missing Data ..................................................................................... 86 
Handling of Missing Data for Bleeding Episodes .................................. 86 
14.7 Deviation Reporting .............................................................................................. 86 
14.8 Interim Analysis .................................................................................................... 86 
15.
Data Monitoring Committee........................................................................................... 87 
16.
Ethics .............................................................................................................................. 88 
16.1 Independent Ethics Committee / Institutional Review Board (IEC/IRB) ............. 88 
16.2 Subject Information and Informed Consent .......................................................... 88 
16.3 Subject Treatment Cards ....................................................................................... 88 
17.
Quality Control and Quality Assurance ......................................................................... 89 
17.1 Compliance with Good Clinical Practice .............................................................. 89 
17.2 Monitoring, Audit and Inspection ......................................................................... 89 
17.3 Source Data Verification ....................................................................................... 89 
18.
Data Handling and Record Retention ............................................................................. 90 
18.1 Data Handling ....................................................................................................... 90 
18.2 Data from Clinical Trial Sites ............................................................................... 90 
18.3 Coding of Data ...................................................................................................... 90 
18.4 Subject Confidentiality .......................................................................................... 91 
18.5 Record Keeping ..................................................................................................... 91 
19.
Changes to Trial Conduct ............................................................................................... 92 
13.2.1 
13.2.2 
13.2.3 
14.2.1 
14.2.2 
14.2.3 
14.6.1 
uniQure 
13.2.1
13.2.2
13.2.3
14.2.1
14.2.2
14.2.3
14.6.1
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Clinical Trial Protocol 
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19.1 Protocol Amendments ........................................................................................... 92
19.2 Premature Termination of the Trial or Dosing Temporarily on Hold ................... 92
Trial Stopping Rules ............................................................................... 92
20.
Reporting of Results ....................................................................................................... 94
20.1 Integrated Clinical Study Report ........................................................................... 94
20.2 Public Disclosure and Publication Policy ............................................................. 94
21.
Financing and Insurance ................................................................................................. 96
22.
Administrative Aspects .................................................................................................. 97
22.1 Investigators .......................................................................................................... 97
International Coordinating Investigator .................................................. 97
22.2 Clinical Trial Sites ................................................................................................. 97
22.3 Vendors ................................................................................................................. 97
23.
References ...................................................................................................................... 98
23.1 Publications ........................................................................................................... 98
23.2 Guidelines ........................................................................................................... 101
23.3 Other .................................................................................................................... 101
uniQure 
19.2.1 
22.1.1 
19.2.1
22.1.1
 
 
 
 
 
 
 
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Clinical Trial Protocol 
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List of Tables 
Table 5-1, Flow Chart for Safety and Efficacy Evaluation, Visits 1 - 21 ................................ 21 
Table 5-2, Flow Chart for Laboratory Parameters, Visits 1 – 21 ............................................ 23 
Table 5-3, Flow Chart for Visit 2 (Dosing Visit)..................................................................... 25 
Table 5-4, Flow Chart for Safety and Efficacy Evaluation, Visits 22 – 35 ............................. 26 
Table 5-5, Flow Chart for Laboratory Parameters, Visits 22 – 35 .......................................... 27 
Table 6-1, Overview of Non-clinical Safety and Pharmacology Studies and Key Findings ... 34 
Table 6-2, Clinical Trials with Gene Therapy for Haemophilia A and B ................................ 37 
Table 6-3, AAV8 Vector Approach in Haemophilia B Patients .............................................. 38 
Table 6-4, AAV5 Vector Approach in Acute Intermittent Porphyria ..................................... 39 
Table 11-1, Components of AAV5-hFIX Drug Product ......................................................... 58 
Table 11-2, Use of Prednisolone for the Treatment of Transaminase Elevation ..................... 60 
Table 11-3, Actions During the First Period of Withholding Prophylactic FIX Replacement 
Therapy .................................................................................................................................... 63 
Table 12-1, Safety Laboratory Parameters .............................................................................. 68 
Table 12-2, Sampling for Serum Chemistry ............................................................................ 68 
Table 12-3, Sampling for Haematology, Coagulation and Urine Parameters ......................... 68 
Table 12-4, Local Laboratory Sampling .................................................................................. 72 
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Clinical Trial Protocol 
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List of Figures 
Figure 10-1, Inter-cohort Staggering Interval .......................................................................... 50 
Figure 10-2, Intra-cohort Staggering Interval .......................................................................... 51 
 
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List of Abbreviations 
Abbreviation 
Definition 
AASLD 
American Association for the Study of Liver Diseases 
AAV 
Adeno-Associated Virus 
AAV2 
Adeno-Associated Viral vector serotype 2 
AAV5 
Adeno-Associated Viral vector serotype 5 
AAV8 
Adeno-Associated Viral vector serotype 8C 
ADR 
Adverse Drug Reaction 
AE 
Adverse Event 
AIP 
Acute Intermittent Porphyria 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Aminotransferase 
aPTT 
activated Partial Thromboplastin Time 
AST 
Aspartate Aminotransferase 
ATMP 
Advanced Therapy Medicinal Product 
cDNA 
complementary Deoxyribonucleic Acid 
CHMP 
Committee for Medicinal Products for Human Use 
Cmax 
Maximum concentration 
COVID-19 
Coronavirus Disease 2019 
CRP 
C-Reactive Protein
CSR 
Clinical Study Report 
CTL 
Cytotoxic T Lymphocyte 
DIBD 
Development International Birth Date 
DIC 
Disseminated Intravascular Coagulation 
DNA 
Deoxyribonucleic Acid 
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Clinical Trial Protocol 
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Abbreviation 
 
Definition 
DSUR 
 
Development Safety Update Report 
eCRF 
 
electronic Case Report Form 
EMA 
 
European Medicines Agency 
EU 
 
European Union 
FDA 
 
Food and Drug Administration 
FIX 
 
coagulation Factor IX 
ɣGT 
 
gamma-Glutamyl Transpeptidase 
gc 
 
genome copies 
GCP 
 
Good Clinical Practice 
GLP 
 
Good Laboratory Practice 
GMP 
 
Good Manufacturing Practice 
HBeAg 
 
Hepatitis B extracellular Antigen 
HBsAg 
 
Hepatitis B surface Antigen  
HBV DNA 
 
Hepatitis B Virus Deoxyribonucleic Acid  
HCV RNA 
 
Hepatitis C Virus Ribonucleic Acid 
HD-Ad 
 
Helper Dependent Adenovirus 
hFIX 
 
human coagulation Factor IX 
hFVIII 
 
human coagulation Factor VIII 
HIV 
 
Human Immunodeficiency Virus 
ICH 
International Council for Harmonisation of Technical 
Requirements for Pharmaceuticals for Human Use 
IEC 
 
Independent Ethics Committee 
IgG 
 
Immunoglobulin G 
IgM 
 
Immunoglobulin M 
IL-1β 
 
Interleukin-1beta 
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Abbreviation 
 
Definition 
IL-2 
 
Interleukin-2 
IL-6 
 
Interleukin-6 
IMP 
 
Investigational Medicinal Product 
IND 
 
Investigational New Drug 
INFγ 
 
Interferon gamma 
INR 
 
International Normalized Ratio 
IRB 
 
Institutional Review Board 
IU 
 
International Unit 
MCP-1 
 
Monocyte Chemotactic Protein-1 
MedDRA 
 
The Medical Dictionary for Regulatory Activities 
mL 
 
millilitre 
mRNA 
 
messenger Ribonucleic Acid 
NCI 
 
National Coordinating Investigator 
nr LAM PCR 
non-restrictive Linear Amplification Mediated Polymerase Chain 
Reaction 
OTC 
 
Over the Counter 
PBGD 
 
Porphobilinogen Deaminase 
PCR 
 
Polymerase Chain Reaction 
PT 
 
Prothrombin Time 
 
 
 
Q-PCR 
 
Quantitative Polymerase Chain Reaction 
qs 
 
sufficient quantity 
rAAV 
 
recombinant Adeno-Associated Virus 
SAE 
 
Serious Adverse Event 
SAP 
 
Statistical Analysis Plan 
uniQure 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Abbreviation 
Definition 
SARS-CoV-2 
Severe acute respiratory syndrome coronavirus 2 
SD 
Standard Deviation 
SDV 
Source Data Verification 
SPC 
Summary of Product Characteristics 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
US 
United States of America 
VG 
Vector Genomes 
WHO-DD 
World Health Organisation Drug Dictionary 
WNL 
Within Normal Limits 
w/w 
weight per weight 
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1.
Sponsor’s Approval of the Clinical Trial Protocol
uniQure biopharma B.V. 
Signature 
Date 
uniQure biopharma B.V. 
Signature 
Date 
uniQure 
 
 
 
 
 
 
 
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Clinical Trial Protocol 
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2. 
International Coordinating Investigator's Approval of the Clinical 
Trial Protocol 
International Coordinating Investigator 
 
Hamophiliezentrum, Johann 
Wolfgang Goethe-Universitat, 
Frankfurt am Main, Germany 
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Date 
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3.
Principal Investigator’s Agreement to the Clinical Trial Protocol
I confirm that I have read and understood this protocol and agree to conduct this study in 
compliance with the Declaration of Helsinki, the International Council for Harmonisation 
(ICH) Guideline for GCP and applicable regulatory requirements. I confirm agreement to 
conduct the study in compliance with the protocol. I fully understand that any changes from 
the protocol constitute a deviation and will be notified to the sponsor. 
Name and title in print 
Signature 
Date 
uniQure 
 
 
 
 
 
 
 
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Clinical Trial Protocol 
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4.
Protocol Summary
4.1 
Primary Objective 
The primary objective of this trial is to investigate the safety of systemic administration of 
AAV5-hFIX, an adeno-associated viral (AAV) vector containing a codon-optimized human 
coagulation Factor IX (hFIX) gene, to adult patients with severe or moderately severe 
haemophilia B. 
The primary objective will be assessed based on adverse events (AEs). 
4.2 
Secondary Objectives 
Secondary objectives will be addressing efficacy and safety of systemic administration of 
AAV5-hFIX. 
4.3 
Trial Design 
This trial has an open-label, uncontrolled, single-dose, dose-ascending design and consists of 
2 cohorts. The trial will be conducted at multiple centres in multiple countries.  
At study completion, subjects will be invited to participate in a separate long-term follow-up 
study that will collect efficacy and safety data up to 10 years from the IMP dosing date. 
4.4 
Trial Population 
Male patients, aged ≥ 18 years, with severe or moderately severe haemophilia B with a severe 
bleeding phenotype defined as:  
-
Known coagulation Factor IX (FIX) deficiency with plasma FIX activity level < 1%
(severe) or plasma FIX activity level ≥ 1% and ≤ 2% (moderately severe) and
-
currently on prophylactic FIX replacement therapy for a history of bleeding, or
-
currently on on-demand FIX replacement therapy with a current or past history of
frequent bleeding (defined as 4 or more bleeding episodes in the last 12 months or
chronic haemophilic arthropathy [pain, joint destruction, and loss of range of motion]
in one or more joints).
Patients should have had more than 150 previous exposure days of FIX replacement therapy. 
4.5 
Intervention 
The Investigational Medicinal Product (IMP) is a recombinant adeno-associated viral vector 
of serotype 5 (AAV5) containing the codon-optimized hFIX complementary 
deoxyribonucleic acid (cDNA) under the control of a liver-specific promoter. The IMP is 
identified as AAV5-hFIX. The pharmaceutical form of AAV5-hFIX is a solution for 
intravenous infusion. 
Subjects will be allocated to one of 2 cohorts with the following planned dose levels: 
-
Cohort 1 (5 subjects): AAV5-hFIX 5 × 1012 gc/kg
-
Cohort 2 (5 subjects): AAV5-hFIX 2 × 1013 gc/kg
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Clinical Trial Protocol 
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The process for cohort allocation is described in Section 11.2.1. Subjects will receive the 
IMP on only one occasion and will thereafter be followed for 5 years with respect to safety 
and with respect to efficacy measured as levels of FIX, bleeding patterns and consumption 
of FIX replacement therapy. 
4.6 
Interim Analyses 
Interim analyses will be performed at the request of the data monitoring committee and at the 
request of the sponsor to enable reporting of adequate data at selected time points during the 
course of the trial. Descriptive statistics alone will be used for the interim analyses. 
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Clinical Trial Protocol 
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5.
Schedule of Trial Procedures
Subjects will attend a screening visit (Visit 1) at a maximum of 6 weeks prior to the 
anticipated dosing visit (Visit 2). When the screening visit blood sample analysis results from 
the local and central laboratory are available, and it is confirmed that the subject is fulfilling 
the trial eligibility criteria, the dosing visit can occur provided that the intra- and inter-cohort 
staggering principles are adhered to (see Section 10.1). Sufficient time should be allowed for 
IMP shipment and preparation.  
In case the subject no longer fulfils the eligibility criteria at the time of the dosing visit, 
re-screening is allowed as detailed in Section 10.2.1. 
After IMP administration, subjects will be monitored for tolerance to the IMP and detection 
of potential immediate AEs at the clinical trial site for 24 hours (overnight stay). Thereafter, 
subjects will be followed with respect to safety and efficacy parameters for 5 years 
(260 weeks) as follows: 
-
Twice weekly up to week 12
-
Every 2nd week from week 12 to week 26
-
Every 13th week from week 26 to week 156 (6 months to 3 years)
-
Every 26th week from week 156 to week 260 (3 years to 5 years)
Clinical procedures to be performed in the period up to week 26 are listed in Table 5-1. 
Clinical procedures to be performed after week 26 and up to week 260 are listed in Table 5-4. 
Laboratory parameters to be measured in the period up to week 26 are listed in Table 5-2. 
Laboratory parameters to be measured after week 26 and up to week 260 are listed in 
Table 5-5. 
Clinical procedures to be performed in relation to IMP administration and the subsequent 
24 hour observation period at the clinic (Visit 2) are listed in Table 5-3. 
Adjustments to the visit location or schedule may be made to accommodate safety concerns 
and restrictions due to the Coronavirus Disease 2019 (COVID-19) pandemic. In all cases, 
subjects will be kept informed as much as possible, of changes to the study and monitoring 
plans that could impact them. 
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Clinical Trial Protocol 
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Table 5-1, Flow Chart for Safety and Efficacy Evaluation, Visits 1 - 21 
Visit Number 
1 
Screening 
2 1) 
Day 1 
2 1) 
Day 2 
3 to 14 
(every week) 
3b to 14b  
(every week) 
15 to 20 
(every 2nd week) 
21 
Additional visits 2) 
Weeks 4) 
-6  
0 
0 
1, 2, 3, 4, 5, 6, 7, 8, 
9, 10, 11, 12 
1, 2, 3, 4, 5, 6, 7, 8, 9, 
10, 11, 12 
14, 16, 18, 20, 22, 
24 
26 
 
Visit window 4) 
 
 
 
±2 days 
1-4 days after main 
weekly visit 
±5 days 
±5 days 
 
Informed consent 
x 
 
 
 
 
 
 
(x) 3) 
Inclusion/exclusion criteria 
x 
x 
 
 
 
 
 
(x) 3) 
24 hour stay at clinic 
 
x -------------- x 
 
 
 
 
 
Administration of IMP 
 
x 
 
 
 
 
 
 
Tapering prophylactic FIX replacement 
therapy (see Section 11.4) 5) 
 
 
 
X (Visits 8-14 / 
weeks 6-12) 
 
 
 
 
Demographics and medical history 6) 
x 
 
 
 
 
 
 
 
History and status of bleeding 
x 
 
 
 
 
 
 
 
History of surgery 
x 
 
 
 
 
 
 
 
Joint status 
x 
 
 
 
 
 
 
(x) 2) 
Subject e-diary instruction 
x 
x 
 
 
 
 
 
 
Subject e-diary interview 
(including re-instruction, if needed) 
 
x 
x 
x 
 
x 
x 
(x) 2) 
Review of e-diary data (bleeding 
episodes, FIX replacement therapy) 
 
x 
x 
x 
 
x 
x 
(x) 2) 
Registration of severity of bleeding 
episode(s) 
 
x 
x 
x 
 
x 
x 
(x) 2) 
Registration of concomitant treatment 
other than FIX replacement therapy 
x 
x 
x 
x 
 
x 
x 
(x) 2) 
Blood pressure, pulse, body temperature 
x 
x 
x 
x 
 
x 
x 
(x) 2) 
Physical examination 
x 
x 
x 
x 
 
x 
x 
(x) 2) 
Height 
x 
 
 
 
 
 
 
 
Body weight 
x 
x 
 
 
 
 
x 
(x) 2) 
Blood and urine sampling  
(see Table 5-2) 
x 
x 
x 
x 
x 
x 
x 
(x) 2) 
Adverse events 
x 
x 
x 
x 
 
x 
x 
(x) 2) 
 
x 
 
 
 
 
 
x 
 
Footnotes are given on next page.
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Date: 06 January 2022
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Footnotes to Table 5-1: 
1)
Time of procedures in relation to IMP administration is detailed in Table 5-3. 
2)
An additional visit can be performed at any time for the purpose of conducting one or more procedures listed in the column “Additional Visits” according to the choice of the Investigator. Hence, “(x)” refers 
to a procedure that can be performed, if judged relevant by the Investigator. 
3)
An additional visit can be performed at any time in advance of the screening visit (i.e. Visit 1), after obtaining written informed consent, for the purpose of pre-determination of subject eligibility. For this 
purpose a selection of the procedures listed in the column “Additional Visits” (i.e. neutralizing antibodies to AAV5, HIV, viral load, CD4+, HBsAg, HBeAg, HBV DNA, HCV RNA) can be performed.
4)
Each visit is scheduled in relation to date of IMP administration, not in relation to the date of its previous visit. 
5)
Tapering of prophylactic FIX replacement therapy may be started prior to 6 weeks after IMP administration as detailed in Section 11.4. 
6)
Including available information on FIX gene mutation. If FIX gene mutation information is not available and if separate informed consent for FIX gene sequencing analysis is given by the subject, a blood 
sample for the purpose of FIX gene sequencing analysis should be drawn (see Table 5-2). 
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Clinical Trial Protocol 
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Page 23 of 101 
 
Table 5-2, Flow Chart for Laboratory Parameters, Visits 1 – 21 
Visit Number 
1 
Screening 
2 
Day 1 
2 
Day 1 
2 
Day 2 
3 to 14 
(every week) 
3b to 14b  
(every week) 
15 to 20 
(every 2nd week) 
21 
Additional 
visits 1) 
Weeks 3) 
-6  
0 
Pre IMP 
0 
3h post IMP 
0 
24h post IMP 
1, 2, 3, 4, 5, 6, 7, 8, 
9, 10, 11, 12 
1, 2, 3, 4, 5, 6, 7, 
8, 9, 10, 11, 12 
14, 16, 18, 20, 
22, 24 
26 
 
Visit window 3) 
 
 
±15 minutes 
±60 minutes 
±2 days 
1-4 days after 
main weekly visit 
±5 days 
±5 days 
 
Local Laboratory 
 
FIX: one-stage aPTT or 
chromogenic/amidolytic assay for FIX 
activity for local monitoring 4) 
x 
x 
 
 
x 
x 
x 
x 
(x) 1) 
FIX inhibitors (Bethesda assay or 
Nijmegen modified Bethesda assay) for 
local monitoring and eligibility check 4) 
x 
x 
 
 
x: Visit 14 
 
 
x 
(x) 1) 
Liver enzymes (AST/ALT) 
x 
x 
 
x 
x 
x 
x 
x 
(x) 1) 
Central Laboratory 
 
FIX: one-stage aPTT and 
chromogenic/amidolytic assay for FIX 
activity, and FIX protein concentration 
x 
x 
 
 
x 
 
x 
x 
(x) 1) 
Anti-FIX antibodies 
 
x 
 
 
 
 
 
x 
 
FIX inhibitors (Nijmegen modified 
Bethesda assay) 
x 
x 
 
 
x: Visit 14 
 
 
x 
(x) 1) 
FIX recovery 5) 
 
x 
 
 
 
 
 
 
(x) 1) 
Total (IgM and IgG) antibodies to AAV5 
x 
x 
 
 
x: Visits 3, 4, 5, 6 
 
 
x 
 
Neutralizing antibodies to AAV5 
x 
x 
 
 
x: Visits 3, 4, 5, 6 
 
 
x 
(x) 1,2) 
AAV5 capsid-specific T cells 
 
x 
 
 
x 
 
x 
x 
 
Sampling for vector genome detection: 
 
 
 
 
 
 
 
 
 
- Blood, urine, saliva, nasal 
secretions 6) 
 
x 
 
x 
x 
 
x 
x 
 
- Faeces, semen 6) 
 
x 
 
 
x: Visits 3, 5, 8, 11, 
14 
 
x 
x 
 
Inflammatory markers IL-1β, IL-2, IL-6, 
INFγ, MCP-1 
 
x 
 
 
x 
 
x: Visits 15, 16, 
17 
 
 
Haematology parameters 7) 
x 
x 
 
 
x: Visits 6, 10, 14  
 
x: Visits 16, 18, 
20 
x 
(x) 1) 
Coagulation parameters 8) 
x 
x 
 
 
x: Visits 6, 10, 14 
 
x: Visits 16, 18, 
20 
x 
(x) 1) 
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Clinical Trial Protocol 
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Visit Number 
1 
Screening 
2 
Day 1 
2 
Day 1 
2 
Day 2 
3 to 14 
(every week) 
3b to 14b 
(every week) 
15 to 20 
(every 2nd week) 
21 
Additional 
visits 1) 
Weeks 3) 
-6 
0 
Pre IMP 
0 
3h post IMP 
0 
24h post IMP 
1, 2, 3, 4, 5, 6, 7, 8, 
9, 10, 11, 12 
1, 2, 3, 4, 5, 6, 7, 
8, 9, 10, 11, 12 
14, 16, 18, 20, 
22, 24 
26 
Visit window 3) 
±15 minutes 
±60 minutes 
±2 days 
1-4 days after 
main weekly visit 
±5 days 
±5 days 
Serum chemistry parameters 9) 
x 
x 
x 10) 
x 10) 
x 
x: Visits 16, 18, 
20 
x 
(x) 1)
Urine parameters 11 
x 
x 
x: Visits 6, 10, 14 
x: Visits 16, 18, 
20 
x 
(x) 1)
HIV, viral load, CD4+ 
x 
(x) 1,2)
HBsAg, HBeAg, HBV DNA and HCV 
RNA 
x 
(x) 1,2)
FIX gene sequencing 12 
x 
Blood sample for future research 13 
x 
x 
x: Visits 7, 12 
x 
1)
An additional visit can be performed at any time for the purpose of conducting one or more procedures listed in the column “Additional Visits” according to the choice of the Investigator. Hence, “(x)” refers 
to a procedure that can be performed, if judged relevant by the Investigator. 
2)
An additional visit can be performed at any time in advance of the screening visit (i.e. Visit 1), after obtaining written informed consent, for the purpose of pre-determination of subject eligibility. For this 
purpose a selection of the procedures listed in the column “Additional Visits” (i.e. neutralizing antibodies to AAV5, HIV, viral load, CD4+, HBsAg, HBeAg, HBV DNA, HCV RNA) can be performed.
3)
Each visit and time point is scheduled in relation to date of IMP administration, not in relation to the date of its previous visit or time point.
4)
Preferably, the same type of assay is used consistently for the individual subject throughout the entire trial period. 
5)
At Visit 2 and additionally at suspicion of FIX inhibitor or at increase in bleeding frequency, as judged by the Investigator. At each occasion a FIX challenge dose of 40 U/kg should be administered. A blood 
sample should be drawn just prior FIX dosing and at 30 minutes after FIX dosing. 
6)
Sampling applicable for the individual subject and for a specific matrix, only until 3 consecutive negative samples have been detected for the subject for that particular type of matrix. Based on the wish of the
subject, faeces and semen samples can be collected at home prior to attending the visit (at the visit day or at the day before the visit day). 
7)
Haemoglobin, haematocrit, platelet count, red blood cells, white blood cells with differential count (all expressed in % as well as in absolute numbers). 
8)
aPTT and PT (INR). In addition, lupus anticoagulant and antithrombin will be measured at Visit 1. 
9)
Serum electrolytes (sodium, potassium), creatinine, gamma-glutamyltransferase (ɣGT), aspartate aminotransferase (AST), alanine aminotransaminase (ALT), alkaline phosphatase (ALP), C-reactive protein 
(CRP), albumin, total bilirubin, glucose (non-fasting). 
10) Blood sample only for CRP.
11) pH, protein, blood, leucocyte esterase, glucose. 
12) Only if FIX gene mutation information is not available and if separate informed consent for FIX gene sequencing analysis is given by the subject. Preferably at Visit 1, but otherwise at a later time point during 
the subject’s trial participation. 
13) Only if separate informed consent is given by the subject, see also Section 12.3.11. 
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Table 5-3, Flow Chart for Visit 2 (Dosing Visit) 
Visit 
2 
Day 1 
2 
Day 2 
Pre IMP 
Hours after IMP infusion completed 
0 
0.5 
1 
2 
3 
4 
6 
8 
12 
24 
Inclusion/exclusion criteria 
x 
Admission for overnight stay 
x 
IMP infusion 1) 
x 
Subject e-diary instruction 
x 
Subject e-diary interview (including re-instruction, if needed) 
x 
x 
Review of e-diary data (bleeding episodes, FIX replacement 
therapy) 
x 
x 
Registration of severity of bleeding episode(s) 
x 
x 
Registration of concomitant treatment other than FIX 
replacement therapy 
x 
x 
Blood pressure, pulse, body temperature 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Body Weight 
x 
Physical examination 
x 
x 
Blood and urine sampling 
x 
x2) 
x2) 
Sampling for vector genome detection: 
-
Blood, urine, saliva, nasal secretions
x 
x 
-
Faeces, semen 3)
x 
Adverse events 
x ----------------------------------------------------------------------------------------------------------------------------------- x 
Completion of 24 hour stay 
x 
1)
Start time and stop time of infusion will be recorded.
2)
Blood sample only for CRP. 
3)
Based on the wish of the subject, the pre-dose faeces and semen samples can be collected at home prior to attending the visit (at the visit day or at the day before the visit day). 
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Table 5-4, Flow Chart for Safety and Efficacy Evaluation, Visits 22 – 35 
Visit Number 
22 
23 
24, 25, 26 
27 
28, 29, 30 
31 
32 
33 
34 
Additional 
visits 1) 
35 
End of 
Trial 2)
Weeks 3) 
39 
52 
(1 year) 
65, 78, 91 
104 
(2 years) 
117, 130, 
143 
156 
(3 years) 
182 
208 
(4 years) 
234 
260 
(5 years) 
Visit window 3) 
±2 weeks 
±2 weeks 
±2 weeks 
±2 weeks 
±2 weeks 
±2 weeks 
±4 weeks 
±4 weeks 
±4 weeks 
±4 weeks 
Joint status 
x 
x 
x 
x 
(x) 1)
x 
Subject e-diary interview 
(including re-instruction, if needed) 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
Review of e-diary data 4) 
(bleeding episodes, FIX replacement therapy) 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
Registration of severity of bleeding episode(s) 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
Registration of concomitant treatment other 
than FIX replacement therapy 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
Blood pressure, pulse, body temperature 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
Physical examination 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
Body Weight 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
Blood and urine sampling (see Table 5-5) 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
Adverse events 4) 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
x 
x 
x 
x 
x 
1)
An additional visit can be performed at any time for the purpose of conducting one or more procedures listed in the column “Additional Visits” according to the choice of the Investigator. Hence, “(x)” refers 
to a procedure that can be performed, if judged relevant by the Investigator. 
2)
Since this is a gene therapy trial in which the IMP is administered to human subjects for the first time, the Investigator should make all reasonable attempts to maintain the subjects in the trial to allow long-
term follow-up on safety. However, should a subject wish to withdraw from the trial, the Investigator should call the subject for the End of Trial Visit (Visit 35) to have all Visit 35 procedures conducted 
(unless the subject has withdrawn consent to perform any further visits). 
3)
Each visit is scheduled in relation to date of IMP administration, not in relation to the date of its previous visit.
4)
After Visit 21 and until Visit 31 the visit frequency is every 13 weeks, and after Visit 31 and until Visit 35 (End of Trial Visit) the visit frequency is every 26 weeks. In these periods, AEs and e-diary data 
review will be performed at least monthly, i.e. in addition to the review at each visit. Subjects who are on continuous routine FIX prophylaxis during the long-term follow-up phase of the trial are required to 
contact the site staff immediately in case of a bleed and/or FIX use different from their routine FIX prophylaxis, in addition to completing the questions/information requested on the e-diaries to capture all 
information. 
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Table 5-5, Flow Chart for Laboratory Parameters, Visits 22 – 35 
Visit Number 
22 
23 
24, 25, 26 
27 
28, 29, 30 
31 
32 
33 
34 
Additional 
visits 1) 
35 
End of 
Trial 
Weeks 2) 
39 
52 
(1 year) 
65, 78, 91 
104 
(2 years) 
117, 130, 143 
156 
(3 years) 
182 
208 
(4 years) 
234 
260 
(5 years) 
Visit window 2) 
±2 weeks 
±2 weeks 
±2 weeks 
±2 weeks 
±2 weeks 
±2 weeks 
±4 weeks 
±4 weeks 
±4 weeks 
±4 weeks 
Local Laboratory 
FIX: one-stage aPTT or 
chromogenic/amidolytic assay for FIX 
activity for local monitoring 3) 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
FIX inhibitors (Bethesda assay or 
Nijmegen modified Bethesda assay) for 
local monitoring and eligibility check 3) 
x 
x 
x 
x 
(x) 1)
x 
Liver enzymes (AST/ALT) 
(x) 1)
Central Laboratory 
FIX: one-stage aPTT and 
chromogenic/amidolytic assay for FIX 
activity, and FIX protein concentration 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
Anti-FIX antibodies 
x 
x 
x 
x 
x 
FIX inhibitors (Nijmegen modified 
Bethesda assay) 
x 
x 
x 
x 
(x) 1)
x 
FIX recovery 4) 
(x) 1)
Total (IgM and IgG) antibodies to AAV5 
x 
x 
x 
x 
x 
Neutralizing antibodies to AAV5 
x 
x 
x 
x 
x 
Sampling for vector genome detection: 
Blood, urine, saliva, nasal secretions 5) 
Faeces, semen 5) 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Haematology parameters 6) 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
Coagulation parameters 7) 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
Serum chemistry parameters 8) 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
Urine parameters 9) 
x 
x 
x 
x 
x 
x 
x 
x 
x 
(x) 1)
x 
Blood sample for future research 10) 
x 
Footnotes are given on next page. 
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Clinical Trial Protocol 
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Footnotes to Table 5-5: 
1) 
An additional visit can be performed at any time for the purpose of conducting one or more procedures listed in the column “Additional Visits” according to the choice of the Investigator. Hence, “(x)” refers 
to a procedure that can be performed, if judged relevant by the Investigator. 
2) 
Each visit and time point is scheduled in relation to date of IMP administration, not in relation to the date of its previous visit or time point. 
3) 
Preferably, the same type of assay is used consistently for the individual subject throughout the entire trial period. 
4) 
At suspicion of FIX inhibitor or at increase in bleeding frequency, as judged by the Investigator. At each occasion a FIX challenge dose of 40 U/kg should be administered. A blood sample should be drawn 
just prior FIX dosing and at 30 minutes after FIX dosing. 
5) 
Sampling applicable for the individual subject and for a specific matrix, only until 3 consecutive negative samples have been detected for the subject in that particular type of matrix. Based on the wish of the 
subject, faeces and semen samples can be collected at home prior to attending the visit (at the visit day or at the day before the visit day). 
6) 
Haemoglobin, haematocrit, platelet count, red blood cells, white blood cells with differential count (all expressed in % as well as in absolute numbers). 
7) 
aPTT and PT (INR). 
8) 
Serum electrolytes (sodium, potassium), creatinine, gamma-glutamyltransferase (ɣGT), aspartate aminotransferase (AST), alanine aminotransaminase (ALT), alkaline phosphatase (ALP), CRP, albumin, total 
bilirubin, glucose (non-fasting). 
9) 
pH, protein, blood, leucocyte esterase, glucose. 
10) Only if separate informed consent is given by the subject, see also Section 12.3.11. 
 
 
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6.
Introduction and Rationale
6.1 
Introduction to Disease Area 
Congenital haemophilia B is characterized by an increased bleeding tendency due to either a 
partial or complete deficiency of the essential blood coagulation FIX. Haemophilia B is an 
X-linked, recessive condition. Approximately 1 in 25,000 live male newborns have
haemophilia B (Bolton-Maggs & Pasi, 2003). The number of diagnosed haemophilia B
patients globally is about 34,000 (World Federation of Haemophilia, 2018). Individuals with
severe haemophilia B are usually diagnosed during the first year of life. The severity of
symptoms can vary and the severe forms become apparent early in life. Bleeding is the main
symptom of the disease and usually increases when the infant becomes mobile. Internal
bleeding may occur anywhere and bleeding into joints is common.
Haemophilia B is caused by a variety of genetic anomalies distributed throughout the gene on 
the long arm of the X chromosome, with the most common being single base-pair changes 
that result in missense, frame shift, or nonsense mutations (Cooper et al., 2009). With a 
deficiency or absence of FIX, activation of coagulation factor X becomes severely impaired 
leading to delayed and insufficient thrombin burst for normal haemostasis. The haemostatic 
plug formed in these patients is fragile and easily dissolved by normal fibrinolytic activity 
with impaired haemostasis, prolonged bleeding episodes and re-bleeding as the 
consequences.  
About 1/3 of individuals with haemophilia B have a severe disorder characterised by 
functional FIX levels that are less than 1% of normal (Kessler & Mariani, 2006). Mild and 
moderate haemophilia B are each observed in about 1/3 of patients (Kessler & Mariani, 
2006). 
FIX is synthesized as a single polypeptide chain that undergoes extensive posttranslational 
modifications (Furie & Furie, 1992; Mann, 1999). The liver is the primary site of FIX 
synthesis and hepatocytes directly secrete FIX into the plasma (Furie & Furie, 1992).  
The classical laboratory findings in haemophilia B include a prolonged activated partial 
thromboplastin time and a normal prothrombin time (Bowyer et al., 2011). 
6.2 
Treatments 
Haemophilia B is currently managed by intravenous injections of clotting factor in various 
forms such as recombinant hFIX concentrates and recombinant or plasma derived FIX 
concentrates, prothrombin complex concentrates or plasma, or via plasma and whole blood 
transfusions. Fresh frozen plasma is still the only product available for treatment of 
haemophilia B in several developing countries across the world. Plasma and whole blood 
products are less effective, since they contain lower concentrations of FIX. They also convey 
risks of circulation overload and heart failure but mainly carry the risk of viral 
contaminations. In the 1970s and 1980s, thousands of people with haemophilia around the 
world were infected with human immunodeficiency virus (HIV) and hepatitis C by 
contaminated plasma-derived products. Although safer products have emerged from this 
tragedy, the risk of contamination of blood products with known and unknown potentially 
infectious agents remains.  
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Because they are devoid of such risks, recombinant hFIX concentrates are widely accepted as 
the optimal treatment.  
Current treatment for haemophilia B in the western world involves intravenous infusions of 
recombinant FIX concentrates at the time of a bleed (“on-demand” therapy). This is highly 
effective at arresting haemorrhages, but cannot prevent spontaneous bleeds or any chronic 
damage that ensues after a bleed. In patients with severe haemophilia B, spontaneous 
bleeding episodes can be dramatically reduced when plasma FIX levels are maintained 
continuously at, or are above 1% of normal (0.01 IU/mL) by prophylactic administration of 
FIX protein. However, the relatively short half-life of hFIX (approximately 19 hours 
according to the Summary of Product Characteristics (SPC) for BeneFIX® and approximately 
25 hours according to Prescribing Information for RIXUBIS®) necessitates frequent 
intravenous administration of concentrates (2-3 times a week) at 40 U/kg or even higher to 
maintain minimum trough levels of ≥ 1%. Frequent intravenous injections are invasive, 
inconvenient and highly problematic for children. Since prophylaxis can reduce the risk of 
spontaneous bleeds and help reduce or prevent joint damage (Manco-Johnson et al., 2007; 
Valentino et al., 2014), it is becoming the standard of care in countries with access to 
adequate quantities of clotting factor concentrates. For Western countries, standard of care is 
to start prophylaxis at 1 year of age, or after first joint bleed. 
6.3 
Limitations of Current Treatment Approach: Medical Need 
 
Reliance on Central Venous Access Devices 
The treatment by prophylactic regular intravenous injections is not curative and is very 
demanding. To make regular injections easier a port-a-cath, or implantable venous access 
device, can be implanted under the skin, usually in the upper chest. Use of port-a cath has 
made prophylactic treatment easier as they provide venous access, however these devices are 
also associated with a risk of bacterial infections. Studies differ, but some show an infection 
rate as high as 50%. These infections can usually be treated with intravenous antibiotics, but 
sometimes the device must be removed (World Federation of Haemophilia). Also, the same 
source cites some studies showing a risk of clots forming at the tip of the catheter.  
 
Risk of FIX Inhibitors 
Approximately 3-5% of patients with severe haemophilia B develop alloantibody inhibitors 
that can neutralize both recombinant and plasma-derived administered FIX. These FIX 
inhibitors are usually immunoglobulins G (IgGs) and often appear after approximately 
10 infusions of FIX concentrate (Chitlur et al., 2009; DiMichele, 2007; Warrier, 1999). 
However, they may appear at any time in the patient’s life. Inhibitor development to FIX is 
considered the most severe problem in haemophilia care today as it affects the efficacy of 
patient treatment, increases the risk of developing joint disease, increases the cost of 
haemophilia care, and leads to increased morbidity. 
 
Non-adherence Resulting in Increased Risk of Spontaneous Bleeds 
Therapy adherence drops sharply when patients reach the teenage years and begin 
self-infusing and remains low thereafter (Geraghty et al., 2006). As many as 41% of patients 
report that they do not always take factor replacement according to their prescribed regimen. 
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6.3.2 
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This is of serious concern considering that as few as 1 to 2 bleeds can trigger progressive, 
irreversible joint disease (Gater et al., 2011). 
 
Chronic debilitating joint disease results from recurrent bleeding into the joint, synovial 
membrane inflammation, hypertrophy, and, eventually, destructive arthritis. Chronic joint 
deformities that need to be managed by an orthopaedic specialist may occur. Furthermore, 
joint replacement(s) may be needed.  
 
The overall life expectancy in severe haemophilia is approximately 72 years in patients 
without HIV/hepatitis C (Plug et al., 2006). In the United Kingdom Haemophilia Centre 
Doctors' Organisation database there were, from 1977 to 1998, a total of 10 deaths from 
intracranial haemorrhage in children younger than 5 years as compared with only 0.013 
deaths expected in this age population in general (Darby et al., 2007). 
 
 
Burden of the Disease 
The burden of the disease is high - both for the individual patient and their families and for 
society. Patients may not be able to participate in certain activities (e.g. contact sports), and 
they encounter long-term impairments in mobility and functional status leading to absence 
from school or work. Issues may surface with social participation and peer integration, 
particularly when children are growing up. Haemophilia patients are less likely to proceed 
into full-time employment and occupational disability is more frequent (Fischer et al., 2013). 
Living with haemophilia can have a substantial effect on mental wellbeing, particularly 
among young people and signs of major depressive disorder are not uncommon. The 
economic burden for the society is significant. Haemophilia patients are accredited with 
requiring 2-3 times the health care resources per inhabitant in developed countries (Schramm 
& Berger, 2003). The described use of FIX replacement therapy varies considerably across 
national economies, even among the wealthiest of countries. Trends suggest that the reported 
FIX usage increases with increasing economic capacity and has been increasing over time. 
Trends also suggest that consumption of FIX has been increasing at a greater rate in high-
income countries. However, approximately 70–80% of the world’s haemophilia B 
population, primarily in the developing world, receives inadequate or no treatment because of 
unavailable and/or unaffordable factor concentrates (Stonebraker et al., 2011). 
6.4 
Scientific Rationale for Gene Therapy in Haemophilia B 
Somatic gene therapy for haemophilia B offers the potential for a shift of the disease severity 
from severe to a moderate or mild haemophilia phenotype through continuous endogenous 
production of FIX after a single administration of vector, especially since a small rise in 
circulating FIX can substantially ameliorate the bleeding phenotype. 
 
AAV vectors of serotypes 2 and 8 (AAV2 and AAV8), coupled to hFIX have been used in 
previous clinical trials of haemophilia B (Manno et al., 2003; Manno et al., 2006; Nathwani 
et al., 2011). The clinical experience with liver-directed AAV gene therapy includes more 
than 20 subjects with severe haemophilia B (Manno et al., 2003; Manno et al., 2006; 
Nathwani et al., 2011). The most recently published clinical trial (Nathwani et al., 2014) 
demonstrated that sufficient FIX expression can be achieved through intravenous infusion of 
AAV8-hFIX in haemophilia B subjects thus converting severe haemophilia B phenotypes to 
moderate/mild haemophilia B. 
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6.5 
Rationale for AAV Serotype 5 Vector in Haemophilia B 
Over the years, several clinical trials testing different AAV vectors have guided the 
understanding towards development of optimal configuration of an adequate gene therapy 
approach for haemophilia B. The overall progress of liver-directed gene transfer for 
haemophilia and other diseases has revealed that the risk of immune-mediated toxicity may 
be the most important concern and that it is multifaceted. Early clinical trials using an AAV 
serotype 2 elicited immune responses, hypothetically leading to loss of expression. Newer 
AAV serotype 8 based vectors have significantly reduced this phenomenon, but not 
eliminated it.  
The liver-directed AAV5 vector product to be used in haemophilia B is based on the same 
technology as used in the ongoing clinical trial of Acute Intermittent Porphyria (AIP). The 
AAV5 vector is produced by uniQure, and is manufactured using uniQure’s baculovirus 
based expression vector system, which provides a consistent and robust product quality and 
thereby allows for mitigating any risks linked to product quality. The lowest prevalence of 
neutralizing antibodies is found in the adult European population for AAV5 (3.2%) followed 
by AAV8 (19%) and is highest for AAV2 (59%) and AAV1 (50.5%) (Boutin et al., 2010). 
Furthermore, among individuals seropositive for AAV5 and AAV8, around 70-100% present 
low titres (Boutin et al., 2010). Since even modest neutralizing antibody titres are thought to 
be able to preclude efficient liver transduction (Mingozzi and High, 2011) it is of importance 
to limit any effects on efficacy due to pre-existing immunity to AAV. In an effort to 
circumvent any inhibitory effects of possible pre-existing immunity, uniQure has chosen to 
use the less common AAV5. However, the prevalence of neutralizing antibodies may differ 
depending on the region, the age and the medical history of the patients (e.g. seroconversion 
due to blood transfusion) and some authors have reported differing numbers (Li et al., 2012; 
Boutin et al., 2010; Moskalenko et al., 2000). Both aspects may contribute to optimization of 
the risk/benefit for liver-directed AAV based gene therapy products in haemophilia (see also 
Investigator’s Brochure). 
6.6 
Investigational Medicinal Product 
 
Description of IMP 
The IMP is a recombinant AAV5 containing the codon-optimized hFIX cDNA under the 
control of a liver-specific promoter. The IMP is identified as AAV5-hFIX. The 
pharmaceutical form of AAV5-hFIX is a solution for intravenous infusion. 
 
Non-clinical Experience 
An overview of the non-clinical evaluation of AAV5-hFIX is given in Table 6-1. Further 
details can be found in the Investigator’s Brochure. 
A range of in vivo studies in wild-type and haemophilia B mice and in non-human primates 
have been performed to characterize the safety and pharmacology of AAV5-hFIX. 
Infusion of AAV5-hFIX to mice and non-human primates resulted in vector dose-dependent 
circulating levels of (human) FIX protein. To verify that AAV5-hFIX mediates expression of 
biologically active hFIX, and to confirm that the hFIX produced and secreted into the 
circulation can ameliorate the clotting defect inherent to FIX deficiency, a dose-range study 
has been performed in a murine model of haemophilia B (for details, see the Investigator’s 
Brochure).  
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In both Rhesus and Cynomolgus macaques, injected at various doses with AAV5-hFIX the 
resulting hFIX expression correlated with the dose and was sustained for the duration of the 6 
months follow-up (Cynomolgus macaque) and 90 days follow-up (Rhesus monkey). 
 
None of the animals presented elevated liver enzymes levels or other signs of toxicity during 
the whole observation period, and after sacrifice, no abnormalities were observed in the liver. 
Further details are available in the Investigator’s Brochure. 
 
These non-clinical data suggest that intravenous administration of AAV5-hFIX is able to 
mediate sustained levels of FIX, and that such administration is not associated with any 
significant safety concerns.  
 
 
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Table 6-1, Overview of Non-clinical Safety and Pharmacology Studies and Key Findings 
Study 
Endpoint 
Status 
Key findings 
Biological activity of AMT-060 
assessed in C57Bl/6 mice for 28 days. 
(Non-GLP) (Study NR-060-11-007) 
Detection of vector DNA in liver 
Detection of hFIX in blood 
Dose-response (FIX levels) 
Completed 
Targeting of vector to the liver. 
Dose dependent levels of vector DNA in 
liver. 
Dose-dependent circulating human FIX 
protein levels over 4 weeks period. 
90-days toxicity and biodistribution 
study in Rhesus monkeys (non-GLP) 
(Study 520665 and NR-060-11-009) 
Local tolerance 
Human FIX levels at the chosen dose 
Clinical chemistry and haematology 
Analysis of vector DNA after 3 months in 
selected organs 
Gross necropsy and histopathology 
Completed 
Some weight loss in the treated animals. 
No further signs of any toxicities / No 
histopathological findings. 
Human FIX is expressed in the liver and 
circulating levels of the protein are detected 
over the 3 months period. 
180-days toxicity and biodistribution 
study in Cynomolgus macaques 
(GLP) (Study 522156 and NR-060-
14-006) 
Local tolerance, Effect on cytokines, 
Dose-response (FIX levels), 
Clinical chemistry and haematology, 
Biodistribution of vector DNA after 
6 months, 
mRNA 
levels 
in 
liver, 
Antibodies to human FIX 
Gross necropsy and histopathology 
Completed 
Some weight loss in the treated animals. 
No further signs of any toxicities / No 
histopathological findings. 
At 6 months, vector DNA in liver and 
adrenals. 
Dose-dependent human FIX expression, 
circulating hFIX protein levels over the 6 
months observation period and hFIX in the 
liver.  
One animal with significant levels of 
xenogeneic antibodies against human FIX. 
hFIX circulating levels decreased in direct 
relation with the antibody formation. 
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Study 
Endpoint 
Status 
Key findings 
180-days toxicity and biodistribution 
study in C57Bl/6 mice (GLP) (Study 
522657 and NR-060-13-006) 
Local tolerance, Acute immune reactions, 
Dose-response (FIX levels), 
Clinical chemistry and haematology, 
Biodistribution 
of 
vector 
DNA 
and 
persistence over 6 months, mRNA in liver 
Gross necropsy and histopathology 
In-life and histopathology 
completed. 
Biodistribution ongoing 
No weight loss or signs of any toxicities / No 
histopathological findings. 
Pending Q-PCR analysis. 
Dose-dependent human FIX expression, 
circulating hFIX protein levels over the 6 
months observation period. 
No immune response against human FIX. 
Prednisone has a negative effect on the 
levels of circulating hFIX. 
Germ line transmission / Breeding 
study in C57Bl/6 mice (GLP) (Study 
496402) 
Biodistribution 
of 
vector 
DNA 
to 
reproductive organs and to foetuses via 
paternal line.  
In-life completed, tissues 
analysis ongoing 
No specific observations so far. 
Pending Q-PCR analysis. 
Biological activity of AAV5-hFIX in 
mFIX deficient (haemophilia B) mice 
(Non-GLP) (Study NR-060-13-007) 
Dose-response (FIX levels) 
Evaluation of the biological activity of the 
expressed human FIX in a murine FIX 
deficient haemophilia B mouse model 
Completed 
Direct 
correlation 
in 
vivo 
between 
circulating human FIX protein levels and 
human FIX activity 
In vitro biological activity (potency) 
in Huh-7 cells. (non-GLP) (Study 
NR-060-12-001) 
Assay to determine the biological activity 
of AMT-060 in vitro in human hepatocyte-
derived cell line 
Completed 
Direct correlation in vitro between the 
expressed hFIX protein levels and the 
measured FIX activity  
Integration site analysis on liver 
tissue obtained from C57B/l6 mice 
and non-human primates (Macaca 
fascicularis) injected with AAV5-
hFIX vector (AMT-060) (Non-GLP) 
(Study NR-060-14-007) 
Liver samples from studies 522657 and 
522156 (4 animals of each dose group for 
mice, all animals for non-human primates) 
sacrificed at Day 180. Integration site 
analysis by nrLAM-PCR and high 
throughput sequencing. 
Completed 
No preferred integration in the genes 
MECOM (MDS1-EVI1), in LMO2 or in 
HMGA2. In mice CIS were found in active 
genes such as Alb, Ttc39c, Esp38, Lrrc4c.  
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Study 
Endpoint 
Status 
Key findings 
Validation of Q-PCR method (GLP) 
(Study 314899) 
Validation of the Q-PCR method to 
determine vector DNA levels in tissues, 
body fluids and excreta from cynomolgus 
monkeys and assess biodistribution and 
shedding 
Completed 
Method validated in a representative panel 
of matrices 
Validation of Q-PCR method (GLP) 
(Study 20041333) 
Validation of the Q-PCR method to 
determine vector DNA levels in tissues and 
body fluids from C57Bl/6 mice and assess 
the biodistribution and PK of the vector 
DNA 
Completed 
Method validated in a representative panel 
of matrices 
Evaluation of the long-term 
expression of hFIX in new-born, 
juvenile and adult mice after single 
injection of AAV5-hFIX (Non-GLP) 
(Study NR-060-14-008) 
Local tolerance, Body weight (gain), 
Circulating human FIX levels, Gross 
necropsy and histopathology 
Ongoing 
No weight loss or signs of any toxicity so far. 
Circulating hFIX levels are stable but 
depend on the age and liver size at the 
moment of injection. 
GLP: Good Laboratory Practice
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Clinical Experience with Similar Products 
Emerging AAV Gene Therapy for Haemophilia A and B 
Patients with haemophilia are ideal candidates for gene therapy, as even a small increase in 
protein production may lead to significantly decreased bleeding diathesis. Over the years 
several clinical trials testing different vectors have guided the understanding toward 
development of optimal configuration of an adequate gene therapy approach for 
haemophilia B (Cancio et al., 2013), see Table 6-2. 
Table 6-2, Clinical Trials with Gene Therapy for Haemophilia A and B 
Reference 
Year 
Vector 
Gene 
delivered 
Subjects 
(n) 
Administration 
Outcome/comments 
Roth et al. 
2001 
Plasmid 
DNA 
hFVIII 
6 
Laparoscopic 
injection of ex vivo 
genetically altered 
fibroblasts in the 
omentum 
Safe and well tolerated. 
No benefit long term 
Powell et 
al. 
2003 
𝜸retrovi
ral 
hFVIII 
13 
Intravenous infusion 
Safe and well tolerated. 
Short lived circulating 
FVIII 
White & 
Monahan 
2005 
HD-Ad 
vector 
hFVIII 
1 
Intravenous infusion 
Severe hepatotoxicity 
and DIC. No detectable 
FVIII expression 
Manno et 
al. 
2003 
AAV2 
hFIX 
8 
Intramuscular 
injection 
Safe and well tolerated. 
First parenteral 
administration of rAAV 
in humans. Circulating 
FIX levels < 1.5% 
Manno et 
al. 
2006 
AAV2 
hFIX 
7 
Hepatic artery 
infusion 
Transient transaminase 
elevation. FIX 
expression detected but 
only transiently with 
loss of expression 
concurrent with 
transaminase elevation 
Nathwani 
et al. 
2014 
AAV8 
hFIX 
10 
Intravenous infusion 
Mild, transient 
transaminase elevation 
in 4 out of 6 high dose 
subjects. Stable FIX 
expression > 1% for up 
to 48 months. Effect on 
transient transaminase 
elevation on FIX levels 
unclear 
The first 2 AAV gene therapy clinical trials for haemophilia B consisted of an AAV2 
encoding FIX. The route of administration was via intramuscular injection and hepatic artery 
infusion, respectively (Manno et al., 2003; Manno et al., 2006). 
The hepatic artery approach produced the most promising results. However, some of the 
findings from this recombinant AAV administration procedure were the observation of 
transient, apparently Cytotoxic T Lymphocyte (CTL) mediated liver enzyme perturbations 
that peaked 2-4 weeks after vector infusion. An inverse correlation between pre-existing 
' 
-
-
-
-
I 
I 
I 
J 
 
 
 
 
 
 
 
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neutralizing antibody titres towards the vector, and the ability to achieve peak FIX expression 
was observed. These results led to the consideration that the selection of AAV vector 
serotype may be an important differentiator for both safety and efficacy of gene therapy. 
The third AAV gene therapy clinical trial used an alternate AAV serotype (i.e. AAV8), which 
seems to be less frequently found in humans as compared to AAV2 (Calcedo et al., 2009), 
less efficiently transduces antigen-presenting cells as compared to AAV2 (Vandenberghe et 
al., 2006) and has increased liver tropism as compared to AAV2 (Nathwani et al., 2007), 
which enabled peripheral vein infusion of the vector. Results on the first 6 subjects from the 
clinical trial were published in 2011 (Nathwani et al., 2011). The 6 adult subjects, all 
suffering from severe haemophilia B, were intravenously infused via a peripheral vein with 
1 × 1011 gc/kg (low dose), 6 × 1011 gc/kg (medium dose), or 2 × 1012 gc/kg (high dose) of 
AAV8-hFIX (Nathwani et al., 2011). An additional 4 subjects have subsequently been treated 
with the high dose (Nathwani et al., 2014). The population in the clinical trial by Nathwani 
comprised severe haemophilia patients both on on-demand treatment and on continuous 
prophylaxis. The clinical trial demonstrated that stable FIX expression can be achieved 
through intravenous infusion of AAV8-hFIX in haemophilia B patients thus potentially 
converting severe haemophilia B phenotypes to mild haemophilia B (see Table 6-3). 
Table 6-3, AAV8 Vector Approach in Haemophilia B Patients 
Vector 
FIX expression level 
S-ALT levels 
No 
Serotype 
Dose 
(VG/kg) 
Steady state FIX level 
Peak (IU/L) 
Time 
Steroids 
1 
AAV8 
2x1011
2.17% 
WNL 
- 
No 
2 
AAV8 
2x1011 
1.4% 
WNL 
- 
No 
3 
AAV8 
6x1011 
2.86% 
WNL 
- 
No 
4 
AAV8 
6x1011 
2.17% 
WNL 
- 
No 
5 
AAV8 
2x1012 
3.56% 
202 
8 weeks 
Prednisolone 
6 
AAV8 
2x1012 
7.21% 
36 
9 weeks 
Prednisolone 
7 
AAV8 
2x1012 
5.00% 
43 
7 weeks 
Prednisolone 
8 
AAV8 
2x1012 
6.67% 
WNL 
- 
No 
9 
AAV8 
2x1012 
5.23% 
WNL 
- 
No 
10 
AAV8 
2x1012 
2.89% 
64 
9 weeks 
Prednisolone 
Adapted from Nathwani et al., 2014. 
The intravenous administration of AAV8-hFIX did not cause any unexpected or serious 
adverse events (SAEs). A single intravenous infusion of vector in all 10 subjects with severe 
haemophilia B resulted in a dose-dependent increase in circulating FIX to a level that was 
1 to 6% of the normal value over a median period of 3.2 years, with observation ongoing. In 
the high-dose group, a consistent increase in the FIX level to a mean (± standard deviation 
[SD]) of 5.1 ± 1.7% was observed in all 6 subjects, which resulted in a reduction of more 
than 90% in both bleeding episodes and the use of prophylactic FIX concentrate (Nathwani et 
al., 2014). Of the 7 subjects who were receiving FIX concentrate prophylactically at the time 
of vector infusion, 4 were able to discontinue therapy without having spontaneous bleeding. 
Vector DNA was found in excretions and secretions, but was cleared around 6 weeks after 
administration. A transient increase in the mean alanine aminotransferase level occurred 
between week 7 and week 10 in 4 of the 6 subjects in the high-dose group but resolved over a 
median of 5 days (range, 2 to 35) after prednisolone treatment. None of the 10 subjects have 
developed inhibitors to FIX. 
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The elevation of serum aminotransferase levels appeared to be different to what had occurred 
in the previous liver-directed AAV clinical trial, by manifesting itself later following vector 
administration. The causal relationship and the significance of the liver enzyme perturbations 
currently remain unclear. It has been speculated that the liver enzyme increase is related to a 
specific T cell reactivity (CTL). Two subjects at the lower dose level of AAV8 presented 
with high levels of CTL reactivity to capsid as observed in peripheral blood mononuclear 
cells without concurrent elevation of liver enzyme levels. 
 
The clinical results published by Nathwani (Nathwani et al., 2011 and Nathwani et al., 2014) 
has invigorated the field and, currently, 3 ongoing clinical trials that also apply AAV based 
liver-directed gene therapy approaches for the treatment of haemophilia B are registered on 
www.clinicaltrials.gov (NCT01620801; NCT00979238; NCT01687608). 
 
Optimized AAV gene therapy for treatment of haemophilia B 
AAV5-hFIX has been designed to further optimize the risk/benefit balance for haemophilia B 
by balancing adequate potency and longevity of expression with any potential risk, see 
Section 6.5. 
 
A clinical trial with a similar AAV5 vector produced by uniQure for the treatment of AIP, 
displayed promising safety data at similar dose levels as planned to be used for the treatment 
of haemophilia B (Table 6-4). Eight subjects with AIP were treated with 4 escalating dose 
levels with 2 subjects dosed in each cohort. The maximum dose was 2 x 1013 gc/kg. Liver 
biopsies were performed in 6 out of 8 subjects.  
 
Table 6-4, AAV5 Vector Approach in Acute Intermittent Porphyria 
 
Vector 
PBGD expression level 
S-ALT levels 
 
No 
Serotype 
Dose 
(gc/kg) 
Liver 
biopsy 
Detectable 
PBGD DNA 
Peak 
(IU/L) 
Time 
Steroids 
1 
AAV5 
5 x 10
11 
Yes 
+ 
WNL 
6 months 
No 
2 
AAV5 
5 x 10
11 
Yes 
++ 
WNL 
6 months 
No 
3 
AAV5 
2 x 10
12 
Yes 
+ 
WNL 
6 months 
No 
4 
AAV5 
2 x 10
12 
Yes 
++ 
WNL 
6 months 
No 
5 
AAV5 
6 x 10
12 
No 
N/A 
WNL 
6 months 
No 
6 
AAV5 
6 x 10
12 
Yes 
+++ 
WNL 
6 months 
No 
7 
AAV5 
2 x 10
13 
Yes 
++ 
WNL 
6 months 
No 
8 
AAV5 
2 x 10
13 
No 
N/A 
WNL 
6 months 
No 
 
A 6 months interim analysis of the clinical safety data has been performed. For a period of 
6 months after administration of the liver-directed AAV gene therapy based on a similar 
AAV5 approach as AAV-FIX, no safety concerns have been identified. In particular, no liver 
enzyme perturbations have been noted at any dose level and no rescue administration of 
corticosteroids has been administered. 
 
As described earlier, the liver-directed AAV5 vector product to be used in this trial is based 
on the same technology as the product used in an ongoing clinical trial in AIP. uniQure has 
licensed the liver gene expression cassette for hFIX used in the clinical trial published by 
Nathwani et al. (Nathwani, 2011). In this clinical trial the gene cassette demonstrated the 
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ability to achieve adequate and prolonged expression of human FIX protein levels (Nathwani 
et al., 2014). 
In summary, although AAV5-FIX has not yet been tested in subjects with haemophilia B, a 
significant number of subjects have already been exposed in clinical trials to similar products 
containing relevant components of AAV5-hFIX, such as the capsid and the hFIX gene 
cassette, although not used together. 
Furthermore, significant amount of published human safety information and experience has 
been established with AAV gene therapy approaches in general and for liver-directed AAV 
gene therapy in haemophilia B in particular (AAV2 and AAV8). 
6.7 
Risk/Benefit Considerations 
Gene therapy for haemophilia B offers the potential benefit for a shift of the disease severity 
from severe to a moderate or mild haemophilia phenotype or complete amelioration of the 
haemophilic state through continuous endogenous production of FIX after a single 
administration of vector.  
The identified risks are considered low and manageable and not affecting the risk/benefit 
balance in an unfavourable way. uniQure’s optimized AAV5 approach has the potential to 
further limit the risks currently associated with AAV gene therapy approaches.  
The following risks have to be considered: 
Risk of infusion-related toxicity 
To date no infusion related toxicities have been reported in clinical trials with liver-directed 
AAV gene therapy approaches and at dose levels comparable with dose levels planned to be 
applied in this first human AAV5-hFIX clinical trial. This potential risk is being addressed by 
administering the infusion of AAV5-FIX during hospitalisation combined with in-hospital 
post-administration surveillance of up to 24 hours. 
Risk of immune mediated neutralisation of the AAV5 gene transfection 
-
Pre-existing neutralizing antibodies against the AAV5 capsid potentially result in
significantly reduced bioavailability of AAV5-hFIX, and hence significantly reduced
efficacy. Excluding subjects with pre-existing neutralizing antibodies against the AAV5
capsid minimizes this potential risk. Subjects who in spite of this precaution do not
achieve adequate expression will be able to continue their current regimen with FIX
replacement therapy.
-
Subjects administered the vector will develop antibodies against the viral capsid proteins
and these antibodies are likely to persist. This is unlikely to cause any symptoms or lack
of expression of FIX, but prevents any future administration or second dose of the same
vector, since these antibodies may neutralize newly infused vector capsids. The rationale
for the starting dose level is addressing this risk by increasing the likelihood of achieving
a therapeutic benefit also at the lowest dose level.
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Risk of immune mediated liver toxicity 
Intravenous administration of a liver-directed AAV vector might lead to transaminase 
elevation. Previous clinical trials have shown that potential elevations in liver enzymes react 
promptly with normalization after administration of glucocorticoids. Subjects in the trial will 
be monitored twice weekly during the first 12 weeks after infusion of AAV5-hFIX for the 
occurrence of transaminase elevations, which may warrant the initiation of a corticosteroid 
treatment protocol. 
Risks to third parties and the environment related to shedding via body fluids 
The AAV vector will distribute systemically and small amounts of vector DNA have been 
observed in previous non-clinical and clinical studies in secreta and excreta, especially saliva 
and urine, for a limited period of time. The vector is non-pathogenic and cannot replicate. 
Therefore, the risk for third parties such as family and health care personnel is considered 
marginal. Due to the incapacity of replication, the non-infectious nature of the shed DNA and 
the negligible amounts shed, the risk to the environment can be considered negligible. No 
specific containment or protection measures are deemed necessary. 
Risk of vector DNA integration into the host genome 
AAV vector genomes remain mainly episomal after transduction of the target cells with 
minimal integration into the mammalian genome and with no association with oncogenicity. 
Integration site analysis was carried out by non-restrictive Linear Amplification Mediated 
Polymerase Chain Reaction (nr LAM PCR) and subsequent high throughput sequencing on 
DNA extracted from the livers of both mouse and cynomolgus macaques after administration 
of AAV5-hFIX at various doses. There was no preferred integration in genes known to 
mediate malignant transformation or clonal dominance. Both episomal (concatemeric) and 
integrated forms of AAV5-hFIX were retrieved, but the sequences were present almost 
exclusively as non-integrated episomal forms. The retrieved integrants were randomly 
distributed throughout the host genome. No specific clustering was seen in cynomolgus 
macaque genome, while some level of clustering around active genes was seen in the mouse. 
There were no signs of in vivo clone selection in the animals. The risk of oncogenicity will be 
assessed clinically during the duration of the trial, see Section 10.2.5. 
Risk of germ-line transmission of vector DNA 
The risk of germ line transmission is considered negligible for AAV-based vectors due to the 
marginal integration level of the vector DNA into the host genome. Any potential risk is 
addressed by requiring the use of a condom during the trial in the period from administration 
of the investigational drug until the AAV5 vector has been cleared from semen, as evidenced 
by negative analysis results for AAV5 vector for at least 3 consecutively collected semen 
samples. 
Risk of off-target expression of the transgene 
The vector will distribute systemically to all tissues thereby potentially infecting other cells 
than liver cells resulting in off-target gene expression. This risk is addressed by the use of a 
liver-specific promoter in the gene cassette. In other clinical trials using similar vector 
approaches, no AEs have been reported that could be related to potential off-target 
expression.  
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Risk of inhibitor formation to FIX expressed from the transgene 
There is a risk of inhibitor/antibodies development against the expressed FIX protein. No FIX 
inhibitor formation was seen in any of the previous clinical trials where subjects were 
exposed to hFIX gene transfer and where the expressed levels of FIX were measurable 
(Manno et al., 2003; Manno et al., 2006; Nathwani et al., 2011 and 2014). This risk is being 
addressed by using the same hFIX gene cassette as was used in the Nathwani study which has 
shown to result in successful and adequate FIX expression levels without development of 
FIX inhibitors. In addition, subjects will be selected on the basis of a low risk of FIX 
inhibitor development by choosing subjects with more than 150 exposure days to a FIX 
product as well as omitting subjects with a previous FIX inhibitor. Subjects will be regularly 
monitored for FIX inhibitor development. 
 
Risk of breakthrough bleeding 
The scope of the liver-directed AAV gene therapy approach is to establish a stable and 
durable expression of FIX and to convert a severe haemophilia phenotype to a mild 
phenotype. Previous clinical trials with similar AAV vectors and the identical gene cassette 
have shown that stable and year-long FIX expression can be achieved (Nathwani et al., 
2014). In order to reduce the risk of breakthrough bleedings during the early course of the 
clinical trial, a tapering approach is implemented regarding reduction in prophylactic 
treatment and including close monitoring of plasma FIX concentrations before stop of 
prophylactic therapy. 
 
Furthermore, subjects are monitored closely for 5 years after administration of IMP and the 
need for renewed prophylactic therapy will be clinically assessed by the treating physician 
according to local standard of care for haemophilia subjects. 
6.8 
Accommodations Due to the COVID-19 Pandemic 
In the first quarter of 2020, a pandemic was announced for COVID 19, which is caused by 
the virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The pandemic 
impacted the conduct of clinical trials due to quarantines, site closures, travel limitations, 
diversion of resources and general interruptions in study related procedures, leading to 
protocol deviations. This study protocol includes contingency measures to manage 
disruptions due to COVID 19 illness and/or public health control measures; see 
Section 10.1.1 for details on measures related to adjustments to visit location/method and 
schedule. In all cases, subjects will be kept informed as much as possible, of changes to the 
study and monitoring plans that could impact them. The impacts of these implemented 
contingency measures on the outcomes of this study, including any protocol deviations that 
ultimately result from COVID-19 illness and/or COVID-19 public health control measures 
will be discussed in the Clinical Study Report (CSR). 
 
The decision to test a subject in the study for COVID-19 should be based on the site’s current 
guidelines and at the discretion of the Investigator. If a subject participating in the study is 
identified as a person under investigation for possible COVID-19 infection, the Investigator 
should notify appropriate authorities as per site’s regulations and notify uniQure’s Medical 
Directors. As co-infections can occur, the site should consider all subjects for COVID-19 
virus testing regardless of whether another respiratory pathogen is found.  
 
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If a subject is confirmed positive for COVID-19 at any time during the trial, the medical care, 
isolation, and management should be according to national, local, institutional, and public 
health guidelines. 
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7.
Purpose of the Trial
The trial is designed to investigate the safety and efficacy of AAV5-hFIX in patients with 
haemophilia B. In addition, the trial is intended to provide data to enable decisions regarding 
the further development of AAV5-hFIX in haemophilia B. 
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8.
Trial Objectives and Endpoints
8.1 
Objectives 
 
Primary Objective 
To investigate the safety of systemic administration of AAV5-hFIX, an adeno-associated 
viral vector containing a codon-optimized hFIX gene, to adult patients with severe or 
moderately severe haemophilia B 
 
Secondary Objectives 
Secondary objectives will be addressing the efficacy and safety of systemic administration of 
AAV5-hFIX to adult patients with severe or moderately severe haemophilia B: 
Efficacy Objectives 
-
To investigate the effect of AAV5-hFIX on FIX activity level
-
To investigate the effect of AAV5-hFIX on the use of FIX replacement therapy
-
To investigate the effect of AAV5-hFIX on bleeding episodes
-
To investigate the effect of AAV5-hFIX on 
 parameters 
Safety Objectives 
-
To monitor shedding of the vector in various body matrices (i.e. fluids/excretions)
-
To monitor the immune responses against AAV5 capsid proteins in response to
AAV5-hFIX
-
To monitor for immune responses against FIX protein after administration of
AAV5-hFIX
-
To investigate the effect of AAV5-hFIX on inflammatory markers
8.2 
Endpoints 
 
Primary Endpoint 
Adverse events 
 
Secondary Endpoints 
Secondary endpoints will be addressing efficacy and safety: 
Confirmatory Secondary Efficacy Endpoint 
-
FIX-replacement-therapy-free FIX activity1, 2
Supportive Efficacy Endpoints 
-
Bleeding rate
-
Total consumption of FIX replacement therapy
- 
 scores 
1 Definition of FIX-replacement-therapy-free FIX activity: FIX activity measured any time from 72 hours after 
latest FIX replacement therapy administration and until next administration of FIX replacement therapy, if any 
2 FIX activity will be measured with both the one-stage aPTT assay and the amidolytic/chromogenic assay. 
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8.1.1 
8.1.2 
8.2.1 
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Safety Endpoints 
-
Vector DNA in semen, blood, saliva, nasal secretions, urine and faeces
-
Neutralizing antibodies to AAV5
-
Total (IgM and IgG) antibodies to AAV5
-
AAV5 capsid-specific T cells
-
Antibodies to FIX
-
FIX inhibitors
-
Inflammatory markers: IL-1β, IL-2, IL-6, INFγ, MCP-1
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9.
Trial Population
9.1 
Population 
Subjects will be adult patients with haemophilia B and will be recruited from haemophilia 
centres in multiple countries. A minimum of 10 subjects will be enrolled: 5 subjects into each 
of 2 dose cohorts. The Sponsor may approve additional enrolment of up to 2 subjects in 
Cohort 2. The numbers of enrolled subjects projected for each clinical trial site will be 
specified in other document(s) such as the Clinical Trial Application forms. 
The subjects’ either severe or moderately severe haemophilia B should be of a severe 
bleeding phenotype as defined in inclusion criterion no. 3 below. 
Recruitment will be controlled such that a maximum of 2 subjects with moderately severe 
haemophilia B phenotype are enrolled per cohort. 
9.2 
Inclusion Criteria 
1.
Male
2.
Age ≥ 18 years
3.
Patients with congenital haemophilia B classified as one of the following:

Known severe FIX deficiency with plasma FIX activity level < 1% and a severe
bleeding phenotype defined by one of the following:
o
Currently on prophylactic FIX replacement therapy for a history of bleeding
o
Currently on on-demand FIX replacement therapy with a current or past history
of frequent bleeding defined as 4 or more bleeding episodes in the last
12 months or chronic haemophilic arthropathy (pain, joint destruction, and loss
of range of motion) in one or more joints

Known moderately severe FIX deficiency with plasma FIX activity level between
≥ 1% and ≤ 2% and a severe bleeding phenotype defined by one of the following:
o
Currently on prophylactic FIX replacement therapy for a history of bleeding
o
Currently on on-demand FIX replacement therapy with a current or past history
of frequent bleeding defined as 4 or more bleeding episodes in the last
12 months or chronic haemophilic arthropathy (pain, joint destruction, and loss
of range of motion) in one or more joints
4.
More than 150 previous exposure days3 of treatment with FIX protein.
5.
Acceptance to use a condom during sexual intercourse in the period from IMP
administration until AAV5 has been cleared from semen, as evidenced by the central
laboratory from negative analysis results for at least 3 consecutively collected semen
samples (this criterion is applicable also for subjects who are surgically sterilized)
6.
Following receipt of verbal and written information about the trial, the subject has
provided signed informed consent before any trial related activity is carried out.
3 Regarding exposure days: Prophylaxis, prevention, on-demand and treatment during surgery counts as 
exposure days. If it is not possible to count the actual number of exposure days based on the medical record, the 
Investigator should make a written statement with an estimate based on e.g. subject’s age, treatment frequency, 
medical history, discussion with previous doctors, transfer notes and other relevant information. This statement 
should be filed in the subject’s medical record. 
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9.3 
Exclusion Criteria 
1.
History of positive FIX inhibitor test
2.
Positive FIX inhibitors test at Screening (measured by the local laboratory)
3.
Neutralizing antibodies against AAV5 at Screening (measured by the central laboratory)
4.
Screening laboratory values (measured by the central laboratory):

ALT > 2 times upper normal limit

AST > 2 times upper normal limit

total bilirubin > 2 times upper normal limit

ALP > 2 times upper normal limit

creatinine > 1.5 times upper normal limit
5.
Positive HIV serological test at Screening, not controlled with anti-viral therapy as
shown by CD4+ counts ≤ 200 per µL or by a viral load of > 200 copies per mL
(measured by the central laboratory)
6.
Active infection with Hepatitis B or C virus as reflected by Hepatitis B Surface
Antigen (HBsAg), Hepatitis B extracellular Antigen (HBeAg), Hepatitis B Virus
deoxyribonucleic acid (HBV DNA) or Hepatitis C Virus ribonucleic acid (HCV RNA)
positivity, respectively, at Screening (measured by the central laboratory).
7.
History of Hepatitis B or C exposure, currently controlled by antiviral therapy
8.
Any coagulation disorder other than haemophilia B
9.
Thrombocytopenia, defined as a platelet count below 50 × 109/L, at Screening (measured
by the central laboratory)
10. Body mass index < 16 or ≥ 35 kg/m2
11. Planned surgery for the initial 6 months after IMP administration in this trial
12. Previous arterial or venous thrombotic event (e.g. acute myocardial infarction,
cerebrovascular disease and venous thrombosis)
13. Active severe infection or any other significant concurrent, uncontrolled medical
condition including, but not limited to, renal, hepatic, haematological, gastrointestinal,
endocrine, pulmonary, neurological, cerebral or psychiatric disease, alcoholism, drug
dependency or any other psychological disorder evaluated by the Investigator to interfere
with adherence to the protocol procedures or with the degree of tolerance to the IMP
14. Known significant medical condition including disseminated intravascular coagulation,
fibrinolysis and liver fibrosis which, in the opinion of the Investigator, may confound,
contraindicate or limit the interpretation of either safety or efficacy data
15. Known history of an allergic reaction or anaphylaxis to FIX products
16. Known uncontrolled allergic conditions or allergy/hypersensitivity to any component of
the IMP excipients
17. Previous gene therapy treatment
18. Receipt of an experimental agent within 60 days prior to Visit 1
19. Current participation or anticipated participation within one year after IMP
administration in this trial in any other interventional clinical trial involving drugs or
devices.
9.4 
Withdrawal of Individual Subjects 
Since this is a gene therapy trial in which the IMP is administered to human subjects for the 
first time, the Investigator should make all reasonable attempts to maintain the subjects in the 
trial after IMP administration to allow long-term follow-up of safety. However, a subject can 
withdraw from the trial, at any time, if it is the wish of the subject or in the best interest of the 
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subject as per the Investigator’s assessment. The standard of care of the subject will not be 
affected in any way. 
Should a subject wish to withdraw from the trial at his own request or based on a decision of 
the Investigator the Investigator should call the subject for the End of Trial Visit (Visit 35) to 
have all Visit 35 procedures conducted, unless the subject has withdrawn consent to perform 
any further visits. 
 
Replacement of Individual Subjects Withdrawn 
If a subject withdraws from the trial prior to IMP administration or withdraws from the trial 
within the first 12 weeks after IMP administration, the subject will be replaced. Possible 
additionally enrolled subjects number 6 and 7 in Cohort 2 will not be replaced. 
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10.
Trial Design
10.1 Overall Trial Design 
This trial has an open-label, uncontrolled, single-dose, dose-ascending design and consists of 
2 cohorts, each of a minimum of 5 subjects. Due to the low prevalence of severe and 
moderately severe haemophilia B patients with a severe bleeding phenotype, the trial will be 
conducted at multiple centres in multiple countries. Since this is a first-in-man single dose 
escalation trial with a high-risk medicinal product, an adequate information communication 
system between the sites will be in place (details will be described in the Trial Procedures 
Manual).  
Subjects fulfilling the eligibility criteria will be allocated to either Cohort 1 or Cohort 2 as 
described in Section 11.2.1. Each subject will receive a single dose of IMP (AAV5-hFIX) 
and will thereafter be followed for 5 years with respect to safety and with respect to efficacy 
measured as levels of FIX, bleeding patterns, and consumption of FIX replacement therapy.  
Cohort 2 will be initiated based on the recommendation of the data monitoring committee 
after review of safety data from Cohort 1. These data will include 12-week follow-up for the 
first 3 subjects dosed as well as 4 weeks follow-up for the remaining 2 subjects in Cohort 1 
(see Figure 10-1). If transaminase elevations requiring corticosteroid treatment have occurred 
in one or more of the first 3 subjects in Cohort 1, the data monitoring committee may 
recommend to extend the follow-up period, before sponsor decision to initiate Cohort 2 is 
taken. Inter-cohort stopping criteria are described in Section 19.2.1. 
Figure 10-1, Inter-cohort Staggering Interval 
Within each cohort, there will be an observation period between IMP administration to the 1st 
and 2nd subject and between IMP administration to the 2nd subject and subsequent subjects 
(Figure 10-2). Intra-cohort stopping criteria are described in Section 19.2.1. 
-
After the 1st subject has been dosed in a cohort, the data monitoring committee will
evaluate available safety data collected during a period of minimum 24 hours after
IMP administration to this first subject. The data monitoring committee will
recommend if and when dosing of the 2nd subject can be initiated
-
After the 2nd subject has been dosed in a cohort, the data monitoring committee will
evaluate safety data collected during a period of minimum 24 hours after IMP
Day 1 
IMPadm. 
I 
I 
, , ;• 
, 
Day 1 
IMPadm. 
uniQure 
5 years 
End ofTrial 
Cohort2 
l 
l 
5 subjects 
t---"----------//------
( 2.0 x 1013 gc/kg) 
5 years 
End ofTrial 
I 
Cohort 1 
l 
11 
l 
Ssubjects 
f-L----_,_-------//------~ 
(5.0 x 1012 gc/kg) 
Review of minimum 
U week safety data for 
1", 2nd and 3"' subject 
and miminum4week 
safety data for 4°' and 
5°' subject 
 
 
 
 
 
 
 
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administration to this second subject. The data monitoring committee will recommend 
if and when dosing of the subsequent 3 subjects can be initiated 
-
The dosing of the 3rd, 4th and 5th, and possibly 6th and 7th subject within a cohort must
be separated by a minimum of 24 hours to ensure observation of any acute reactions.
Details on an adequate information communication system with and between the sites to 
ensure the timings of subject dosing within a cohort are adhered to will be described in the 
Trial Procedures Manual. 
Figure 10-2, Intra-cohort Staggering Interval 
After IMP administration, subjects will remain at the clinical trial site for 24 hours for 
monitoring of tolerance to IMP and for detection of potential immediate AEs. Hereafter, 
subjects will be followed with respect to safety and efficacy parameters for 5 years 
(260 weeks): 
-
Twice weekly up to week 12
-
Every 2nd week from weeks 12 to 26
-
Every 13th week from weeks 26 to 156 (6 months to 3 years)
-
Every 26th week from weeks 156 to 260 (3 years to 5 years).
Subjects on prophylactic FIX replacement therapy will be tapered of their prophylactic FIX 
replacement therapy in the period from Visit 8 to Visit 14 (weeks 6-12). If the Investigator 
determines that FIX replacement therapy should be re-initiated, based on FIX levels or for 
other reasons, prophylactic FIX replacement therapy may be tapered again at a later time 
point. In relation to the withholding of FIX replacement therapy, additional visits may be 
required for the purpose of additional monitoring of FIX activity levels (see Table 5-2). For 
further details, please see Section 11.4.  
During the entire trial period, blood samples will be drawn regularly for measurements of 
FIX activity and FIX protein concentration in plasma. For subjects on prophylactic FIX 
replacement therapy, attempts should be made to allow blood sampling at time points where 
FIX activity and FIX protein concentration are expected to be at lowest justifiable levels. For 
further information, please see Section 12.1.1.  
uniQure 
Day 1 • 
5 years 
IMP adm. 
End ofTrial 
1
3"', 4th, 5th subject I i 
// 
! 
, ..-> in cohort 
1-. - - - - - - - - - - - - - -
_______ 
__, 
I 
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, 
Day 1 
,' 
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in cohort 
Review of minimum 
24 hour safety data 
Review of minimum 
24 hour safety data 
II 
5 years 
End 1Trial 
• Day 1 of 3'", 4th and 5th subject must be separated bya minimumof24 hours 
 
 
 
 
 
 
 
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 Considerations Due to the COVID-19 Pandemic 
Due to the COVID-19 pandemic, adjustments to the visit location/method or schedule may be 
made to accommodate safety concerns and restrictions experienced by individual subjects 
and sites. In all cases, subjects will be kept informed, via the site staff, as much as possible, 
of changes to the study and monitoring plans that could impact them. 
Discontinuation of subjects from the study post-treatment with AMT-060 is not considered to 
be in the best interest of the subject, due to the irreversible nature of the IMP. Wherever 
possible, every effort is to be made to have the subject visit the clinic for the study visits 
according to schedule. Should a clinic visit not be possible, options that may be considered 
include site nurses travelling to a subject’s home, local laboratory use, or home nursing 
services (for certain visits, if pre-approved by the Investigator and uniQure). These options, if 
used, will be supplemented with a phone call or telemedicine/telehealth safety follow-up call. 
A (temporary) transfer of a subject to an alternate clinical trial sites may also be considered in 
order to continue on-site visits, only if this does not pose undue burden to the subject and/or 
"new" site. 
In some instances, it may not be possible to conduct any type of visit at all. Where none of 
the above options are feasible, subject visits may be moved beyond the maximum visit 
window permitted. Such delays will be assessed on a case by case basis. Until visits are 
rescheduled, supplemental phone calls or telemedicine/telehealth contact between 
Investigator/study staff and subject are to be arranged. 
Supplemental phone calls or telemedicine/telehealth safety follow-up calls will be used to 
confirm the subject’s status and wellbeing. At these calls, safety information should be 
gathered (i.e. AEs, concomitant medication use), subjects should be asked about any new 
unreported bleeds or FIX consumption and confirm their use of the e-diary (as applicable), 
and there should be continued discussions with the subject on the importance of a healthy 
liver. These discussions will be documented in the source documents. 
All deviations from the study protocol are to be documented, with rationale. If a protocol 
deviation is due to the COVID-19 pandemic, this will be noted. 
10.2 Trial Periods 
 Screening Visit (Visit 1) 
The screening visit, Visit 1, will take place at a maximum of 6 weeks prior to the anticipated 
IMP dosing visit (Visit 2). 
Before any trial related activity takes place, signed informed consent must be obtained from 
the subject (see Section 16.2). 
All subjects undergoing screening procedures will be allocated a subject ID number and will 
be listed in a log. The subject ID number will consist of a 2-digit number for the clinical trial 
site followed by a consecutive 2-digit number for the subject. 
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If pre-determination of subject eligibility is desired by the Investigator in advance of the 
conduct of the full screening visit (Visit 1), an additional visit can be performed during which 
a selection of laboratory parameters can be measured (see Table 5-2, additional visits). When 
pre-determination of subject eligibility is established and the subject continues into the trial, a 
full screening visit (Visit 1) needs to be conducted (see Table 5-1 and Table 5-2, Visit 1), 
however if the timing of the additional visit is within 2 weeks of Visit 1 there is no need to 
repeat the laboratory parameters previously measured. 
Eligibility according to the trial inclusion and exclusion criteria will be evaluated at 
Screening and during the period up to IMP administration at Visit 2. 
During the period up to Visit 2, laboratory results from blood and urine samples taken at 
Screening (either at Visit 1 or at an additional visit for pre-determination of subject 
eligibility) will be provided by the central laboratory and the local laboratory and will be 
decisive for the evaluation of a number of exclusion criteria (see Section 9.3).  
At visits where the 
 is to be completed, the subject will be asked to 
complete this prior to any other visit procedure is initiated. For Visit 1, this applies, however, 
after informed consent has been obtained. 
Subjects qualifying as screen failures (i.e. subjects who fail to meet the inclusion criteria 
and/or fail to not meet any of the exclusion criteria during screening) may be re-evaluated for 
participation in the trial once. The definition of screen failure also applies to subjects who 
experience a bleeding episode, surgery or another event after Visit 1 that warrants a delay of 
Visit 2. In this situation, where the screening period exceeds the maximum of 6 weeks 
re-screening will require a renewed informed consent to be obtained from the subject, a new 
electronic Case Report Form (eCRF) should be started and the subject should be allocated a 
new subject ID number. 
 Dosing Visit (Visit 2) 
Visit 2 should be planned to take place as an overnight stay at the clinic and should take place 
within a maximum of 6 weeks after Visit 1. 
At Visit 2 the trial inclusion and exclusion criteria assessed at Visit 1 should be re-assessed, 
including any laboratory analysis results (see Section 9.2 and 9.3). 
Pre-IMP samples for laboratory analysis will be collected. For details, please see Table 5-2 
(under “Visit 2, Day 1, pre-IMP”). 
Hereafter, IMP should be administered according to the procedures described in 
Section 11.2.2 and in the IMP Handling Manual. 
The clinical assessments and laboratory sampling procedures to be followed in relation to 
IMP administration are given in Table 5-3.  
The subject may leave the clinic after all assessments at 24 hours after IMP administration 
have been performed. 
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 Follow-up Period (Visits 3 – 35) 
The procedures to be followed at Visits 3 – 35 are given in Table 5-1 and Table 5-4 and the 
laboratory samples to be collected are given in Table 5-2 and Table 5-5.  
At Visits 21 (26 weeks), 23 (1 year), 27 (2 years), 31 (3 years), 33 (4 years) and 35 (5 years) 
the subject should complete the 
. At these visits, the subject will be 
asked to complete the 
 prior to any other visit procedure is initiated. 
Options for how visits in the follow-up period may occur to accommodate safety concerns 
and restrictions due to COVID-19 are described in Section 10.1.1. 
 Additional Visits 
The subject may be called in for additional visits, at the discretion of the Investigator. The 
subject may also contact the clinical trial site for an additional visit. 
An extra visit may include additional assessments, as deemed necessary by the Investigator, 
such as blood sampling for FIX activity (see Section 11.4), blood sampling for FIX inhibitors 
(Section 12.3.10), repetition of blood sampling due to erroneous results (Section 12.4), 
pre-determination of subject eligibility (see Section 10.2.1), conduct of measurements that 
were missed at the previous visit or repetition of instructions to the subject regarding subject 
diaries (Section 10.3.1).  
 Follow-Up after End of Trial 
The individual subjects will be followed for 5 years after administration of IMP. This is in 
line with the guideline on follow-up of subjects administered with a gene therapy medicinal 
product (EMEA/CHMP/GTWP/60436/2007).  
At study completion, subjects will be invited to participate in a separate long-term follow-up 
study that will collect efficacy and safety data up to 10 years from the IMP dosing date. 
10.3 Use of Subject e-Diary 
From Visit 1 (after completion of all screening assessments) and throughout the entire trial 
participation period, subjects will be requested to document use of FIX replacement therapy 
and record information on bleeding episodes in a dedicated electronic subject diary (e-diary). 
 Training in the Use of Subject e-Diary 
At Visit 1, subjects will be trained in the use of the e-diary system by the Investigator or 
study nurse. Furthermore, the subject will be provided with a written instruction. Training 
will be repeated at a minimum at Visit 2 as well as at any subsequent visit as judged 
necessary by the Investigator or study nurse. 
At each visit after Visit 1, subjects will be interviewed with respect to problems, if any, 
related to e-diary data entry. Furthermore, the Investigator and study nurse will have access to 
the data entered in e-diaries. At each subject visit, e-diary data will be reviewed. Based on 
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10.2.4 
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interviews and e-diary data review the Investigator and/or study nurse will evaluate if training 
needs to be repeated. In case e-diary training is repeated, this will be reported in the subject’s 
medical record.  
After Visit 21 and until Visit 31 the visit frequency is every 13 weeks, and after Visit 31 and 
until Visit 35 (End of Trial Visit) the visit frequency is every 26 weeks. In these periods, 
e-diary data review will be performed at least monthly, i.e. in addition to the review at each
visit (as described in Section 12.1.2 and 12.1.3). The Investigator or study nurse will review
the data entered in e-diaries and if judged needed based on this review, the Investigator or
study nurse will call in the subject for an additional visit for the purpose of a repeated training
in the use of the subject e-diary. Subjects who are on continuous routine FIX prophylaxis
during the long-term follow-up phase of the trial are required to contact the site staff
immediately in case of a bleed and/or FIX use different from their routine FIX prophylaxis, in
addition to completing the questions/information requested on the e-diaries to capture all
information.
10.4 Rationale for Trial Design 
A dose ascending design is chosen to determine the safety and tolerability of 2 dose levels. 
As a secondary objective, the trial design supports the scope to explore the maximum 
achievable bioactivity of AAV5-hFIX as assessed by FIX-replacement-therapy-free FIX 
activity levels at the 2 dose levels. 
The objective for the first cohort is to explore whether 5 × 1012 gc/kg can achieve 
therapeutically relevant bioactivity levels of >2% of FIX-replacement-therapy-free FIX 
activity without safety concerns. 
The objective for the second cohort is to explore whether 2 × 1013 gc/kg can achieve an 
expression level of FIX in the range of 5-10% of FIX-replacement-therapy-free FIX activity 
without safety concerns. 
The design is based upon the non-clinical data (see Section 6.6.2) and the available data from 
prior clinical experience with similar AAV-vector based gene therapy products (see 
Section 6.6.3) that supports that safe and therapeutically relevant expression levels of FIX 
may be anticipated with AAV5-hFIX at one or both of the 2 dose levels (see also the dose 
rationale in Section 10.5). 
To support subject safety, the trial is designed with 2 consecutive dose arms with ascending 
doses and data monitoring committee review before start of dosing in the next cohort. 
Subjects are therefore not randomly assigned to the 2 arms, which introduces a risk of 
selection bias. Part of this risk will be mitigated by strict adherence to inclusion and exclusion 
criteria and in addition only 2 subjects with moderately severe haemophilia will be allowed in 
each cohort thereby preventing that the moderately severe subjects are allocated to the lowest 
dose level only.  
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Due to the nature of the disease in question it is not ethical to perform a placebo-controlled 
trial and no relevant active comparators exist. Based on this the trial is designed as an open-
label and uncontrolled trial. 
10.5 Dose Rationale 
The dose selection is based on a combination of safety and efficacy data from non-clinical 
studies and from prior in-human experience with similar AAV based products (see 
Section 6.6.3). 
 
The non-clinical safety for AAV5-hFIX has been examined in rodents and non-human 
primates using single doses up to 2.3 × 1014 and 0.9 × 1014 gc/kg, respectively, without 
showing any adverse reaction and the No Observed Adverse Effect Level was set in both 
species as the highest tested dose. These data establish an adequate safety margin for the 
highest proposed dose level of 2 × 1013 gc/kg. 
 
The non-clinical pharmacology studies suggest that hFIX expression levels of 5-10% of 
normal human level can be achieved and that a dose relationship can be expected. In rhesus 
macaques 5-10% expression level was achieved with a dose of 5 x 1012 gc/kg. In cynomolgus 
macaques the expression levels achieved with similar dose levels was lower, however clear 
dose response from 5×1011 to 1×1014 was established. Despite observed differences between 
rhesus and cynomolgus monkeys in FIX expression levels at similar dose levels, the levels of 
hFIX expressed are dose dependent in all tested species and it is anticipated that this also 
applies to humans. The study in cynomolgus macaques indicates that a dose of 5 × 1011 gc/kg 
may be sub-therapeutic in humans and the proposed Minimum Anticipated Biological Effect 
Level is therefore 5 × 1012 gc/kg body weight. Despite anticipated differences between non-
human primates and humans, a starting dose level at 5 × 1012 gc/kg is justified in order to 
minimize the risk of exposing haemophilia B patients to sub--therapeutic dose levels in the 
first cohort. 
 
The combination of the AAV5 capsid and the hFIX gene cassette of AAV5-hFIX has not yet 
been administered to humans in clinical trials. However, the serotype 5 capsid and the gene 
cassette have separately been used in previous clinical trials without safety concerns (see 
Section 6.6.3). 
 
In conclusion, the available non-clinical and clinical data support the safety of both the 
starting dose and the top dose. Furthermore, the data support the expectation that safe and 
therapeutically relevant expression levels of FIX may be anticipated with AAV5-hFIX at one 
or both of the 2 dose levels. 
10.6 Rationale for Proposed Intra- and Inter-cohort Staggering Intervals 
The specification of the intra- and inter-cohort staggering intervals is based on a combination 
of generic safety surveillance principles applicable for a first in human clinical trial, and the 
absence of acute and sub-acute AEs in the preclinical studies with AAV5-hFIX and the 
timing, frequency and severity of expected AEs based upon clinical studies with similar 
liver-directed AAV products that contain the AAV vector serotype and gene cassette 
components of AAV5-hFIX. 
 
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The data monitoring committee will evaluate available safety data collected during the initial 
24 hours after IMP administration prior to dosing of the second subject in a cohort and prior 
to the decision to dose the subsequent subjects in a cohort in order to adequately assess the 
risk for subsequent subjects of any acute AEs observed. The dosing of the 3rd, 4th and 5th, and 
possibly 6th and 7th subject will be separated with at least 24 hours. The intra – and inter-
cohort stopping criteria are described in Section 19.2.1. 
The data monitoring committee will evaluate all available safety data collected during the 
period of a minimum of 12 weeks after IMP administration in the first 3 subjects of Cohort 1 
and for a minimum of 4 weeks after IMP administration in subjects 4 and 5. 
-
The requirement to follow 3 subjects for 12 weeks is a precautionary measure to
secure that the low risk or even absence of liver enzyme perturbations requiring
corticosteroid treatment demonstrated at the highest dose level with
AAV5-Porphobilinogen Deaminase (PBGD) will be reproduced with the lowest dose
level of AAV5-FIX
-
The rationale for the 12 weeks in the first 3 subjects is based on the data from the
AAV8-hFIX clinical trial described in Section 6.6.3, where mild liver enzyme
perturbations occurred during a window of 7-9 weeks after intravenous administration
-
The rationale for the 4 weeks is based on the consideration to allow the data
monitoring committee to adequately assess potential unexpected and significant
sub-acute AEs.
10.7 Rationale for Stopping Prophylactic FIX Replacement Therapy 
In the clinical trial with AAV8-hFIX, measurements of FIX activity showed an initial burst 
lasting up to 4 weeks after administration of AAV8-hFIX (Nathwani et al., 2011; Nathwani et 
al., 2014). In the clinical trial, it was possible to stop or reduce prophylactic FIX replacement 
therapy in 4 of 7 subjects once evidence of sustained FIX activity at 3-5% was achieved. The 
time point for stopping or reducing prophylactic FIX replacement therapy varied between 
6 and 15 weeks. 
In this trial, a similar attempt to stop or reduce prophylactic FIX replacement therapy has 
been implemented, following an algorithm based on FIX activity measurements, as outlined 
in Section 11.4. 
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11.
Trial Treatment
11.1 Investigational Medicinal Product (IMP) 
 Description of IMP 
The IMP is a recombinant AAV5 containing the codon-optimized hFIX cDNA under the 
control of a liver-specific promoter. The IMP is identified as AAV5-hFIX. 
The pharmaceutical form of AAV5-hFIX is solution for intravenous infusion. 
AAV5-hFIX will be supplied in 2 mL glass vials filled with a total volume of 1.2 mL IMP 
to guarantee at least 1 extractable mL of IMP. The components of the AAV5-hFIX drug 
product are given in Table 11-1. 
For further information, please see the Investigator’s Brochure. 
 Packaging, Labelling and Storage of the IMP 
Packaging and labelling of the IMP for all clinical trial sites will be coordinated by uniQure. 
IMP used in this trial will be prepared and labelled according to GMP, GCP and local 
regulatory requirements. 
For further information, including information on labelling of vials and boxes, please see the 
IMP Handling Manual. 
 IMP Accountability, Traceability and Disposition 
The Investigator must ensure that a designated person (e.g. pharmacist) receives the IMP 
from the sponsor. Upon receipt of the IMP, the Investigator or designee must maintain 
adequate records of all IMP received, dispensed, administered and returned or destroyed 
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using a drug accountability log. This drug accountability log must be available for inspection 
at any time.  
In addition, an in-clinic traceability record is maintained for the prepared IMP infusion bag to 
collect information about infusion bag details (e.g. expiry date/time), IMP administration 
details (e.g. date of infusion and start/stop time of infusion), waste disposal details (e.g. type 
of materials disposed), as well as details by whom the infusion bag dispensed, collected and 
returned. 
The Investigator or designee must ensure that the IMP is maintained under required storage 
conditions in a secure area with restricted access. The IMP may only be used in accordance 
with this approved protocol and must not be used for any other purpose. The IMP may only 
be used for subjects who have provided written informed consent to participate in this trial 
and meet all trial inclusion criteria and none of the exclusion criteria. 
Return/Destruction authorization forms must be completed by the Investigator or designee 
each time IMP is returned or destroyed, but only after written authorization is provided by the 
sponsor. The drug accountability log will be reviewed by the trial monitor. Upon completion 
of review, the trial monitor will provide the site with the written authorization by the sponsor 
to destroy the IMP or return it to the sponsor. 
Disposal of hazardous material (e.g. syringes, needles, etc.) must be conform applicable laws 
and regulations. See the IMP Handling Manual for further instructions on accountability and 
disposition of IMP.  
 Preparation of the IMP for Intravenous Infusion 
The IMP will be supplied in 2 mL vials containing 1.2 mL of IMP as a solution for 
intravenous infusion. Based on the assigned cohort and the subject’s body weight the 
volume of IMP to be administered will be calculated. The solution for intravenous infusion 
will be drawn from the vials to prepare the final total volume. Detailed preparation 
instructions will be provided in the IMP Handling Manual and must be followed. 
11.2 IMP Administration 
 Dose Levels and Allocation to Dosing 
Subjects will be allocated to the cohorts with the following planned dose levels based on the 
chronological order the subjects complete the screening visit (Visit 1): 
-
Cohort 1 (5 subjects): AAV5-hFIX 5 × 1012 gc/kg
-
Cohort 2 (5 subjects): AAV5-hFIX 2 × 1013 gc/kg
Dosing will take place once (at Visit 2). No other medication will be provided in the trial. 
 Method of IMP Administration 
AAV5-hFIX will be administered as an intravenous infusion with a common intravenous 
infusion device. The required volume of AAV5-hFIX will depend on the cohort and the 
subject’s body weight. 
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Detailed IMP administration instructions and instructions for the recording of the IMP 
administration details will be provided in the IMP Handling Manual and must be followed. 
 Recommendation of Treatment in Case of Increased Liver Transaminases 
Background Information 
The combination of the AAV5 capsid and the hFIX gene cassette of AAV5-hFIX has not 
previously been administered to humans in clinical trials. However, the capsid serotype 5 
and the gene cassette have separately been used in previous clinical trials without safety 
concerns (see Section 6.6.3). 
Two gene therapy clinical trials targeting the liver with other AAV serotypes have been 
published (Nathwani et al., 2011 and 2014; Manno et al., 2006), and in both of the studies a 
transient transaminase elevation was observed in some subjects (see Section 6.6.3 for further 
details). 
Although the risk of encountering significant liver enzyme perturbations with AAV5-hFIX 
is considered low, it cannot be ruled out that short-term treatment with corticosteroids may 
be required. 
In case the Investigator judges that treatment with corticosteroids may be required, it is 
recommended to follow the scheme below. However, the Investigator - based on local 
clinical practice and medical judgement - may decide to deviate from the outlined 
recommendation. 
Recommended approach and treatment 
Treatment is recommended to be instituted if, in the absence of alternative aetiology, the ALT 
level increase is greater than 1.5 – 2-fold the baseline level (Day 1, pre-IMP).  
Based on previous clinical trials (Nathwani et al., 2011 and 2014; Manno et al., 2006) where 
liver enzymes elevations were observed, corticosteroids could be used for a maximum of 
6 months (see Table 11-2). The Investigator is recommended to initiate treatment with 
prednisolone as soon as possible after detection of ALT elevation. A combined 
immunosuppressant regimen or the use of other products can also be considered in case of 
prednisolone treatment failure or contraindication.  
Table 11-2, Use of Prednisolone for the Treatment of Transaminase Elevation 
Timeline 
Prednisolone dose (mg/day) 
Week 1 
60 
Week 2 
40 
Week 3 
30 
Week 4 
30 
Maintenance until ALT returns to baseline level (Day 1, pre-IMP) 
20 
After pre-IMP level has been reached 
Reduce daily dose with 5 mg/week 
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11.3 Concomitant Medication/Therapy  
 Allowed Medication 
Subjects will continue their usual FIX replacement therapy (on-demand and prophylactic), as 
applicable. However, for subjects on prophylactic FIX replacement therapy, prophylaxis will 
be tapered when endogenous production of FIX is anticipated to have reached adequate levels 
(see Section 11.4). 
 
 Disallowed Medication 
The following treatments will not be allowed during trial participation: 
 Treatment in another interventional clinical trial involving drugs or devices within 1 year 
from IMP administration in this trial 
 Another gene therapy treatment and/or participation in another gene therapy clinical trial 
Apart from the above listed treatments, no protocol restrictions will apply with respect to 
concomitant treatment. 
 
 Reporting of Concomitant Medication/Therapy 
Concomitant medication/therapy is any medication/therapy being continued by the subject at 
trial entry and any new medication received during the trial. 
 
At every visit the Investigator or a qualified designee will ask the subject about concomitant 
medication. The Investigator should record the use of all medication (including over the 
counter [OTC] medication, vitamin and/or mineral supplements, homeopathic remedies and 
herbal preparations) used and changes in the use of medication, with the exception of FIX 
replacement therapy for which only the prescribed regimen should be recorded. Information 
on actual FIX replacement therapy taken will be reported separately by the subject in an 
e-diary (see Sections 12.1.2 and 12.1.3). The Investigator should also record other 
concomitant treatments/therapy, e.g. physiotherapy.  
 
The following information will be recorded: 
- Drug name (generic name preferred) 
- Indication 
- Dosing regimen (dose, unit, route, frequency) 
- Route of administration 
- Start date (if started ≥ 3 months prior to Visit 1, then this can be stated instead of 
recording the specific start date) 
- Stop date (or ongoing, if ongoing at end trial participation). 
11.4 Tapering and Subsequent Withholding of Prophylactic FIX Replacement Therapy 
In the period of 6 to 12 weeks after IMP administration (i.e. Visits 8-14, both visits included) 
subjects who are on prophylactic FIX replacement therapy will be tapered of their 
prophylactic therapy. The tapering of prophylactic therapy should only be initiated if the FIX 
activity level is ≥ 2.0% at the previous 2 consecutive visits (as measured by the local 
laboratory). 
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The exact date on when the tapering of prophylactic FIX replacement therapy is initiated 
within the 6 to 12-week period will be decided by the Investigator. The tapering of 
prophylactic FIX replacement therapy may be started prior to 6 weeks after IMP 
administration at the discretion of the Investigator (in consultation with the subject), but only 
after consultation with the Sponsor’s Medical Officer. 
The Investigator will record the date of initiating the tapering of prophylactic FIX 
replacement therapy and the reason for the date chosen. 
Tapering of prophylactic FIX replacement therapy 
The tapering of prophylactic FIX replacement therapy will take place in 3 steps (over 
2 weeks).  
1. During the first week, the subject will receive 50% of his usual prophylactic dose per
infusion. The number of infusions per week should not be changed.
2. During the second week, the subject will receive 25% of his usual prophylactic dose
per infusion. Again, the number of infusions per week should not be changed. The
decision to go from 50% to 25% of the usual prophylactic dose will be made based on
an individual assessment of the subject by the Investigator, and will include the
requirement that the subject is able to maintain a FIX activity level ≥ 2.0% (as
measured by the local laboratory).
3. From the third week onwards, no further prophylactic dosing should be administered
except in situations of e.g. major surgery. The decision to go from 25% of the usual
prophylactic dose to no prophylactic dose will be made based on an individual
assessment of the subject by the Investigator, and will include the requirement that the
subject is able to maintain a FIX activity level ≥ 2.0% (as measured by the local
laboratory).
Alternatively, the frequency of FIX prophylaxis infusions may be decreased, or the 
prophylactic FIX replacement therapy may be stopped in one step at the discretion of the 
Investigator (in consultation with the subject). 
Actions during the first period of withholding prophylactic FIX replacement therapy 
(variable timespan) 
The decision to continue to withhold the prophylactic FIX replacement therapy will be made 
based on an individual assessment of the subject by the Investigator, and will include the 
requirement to document that the subject is able to maintain a FIX activity level ≥ 2.0% (as 
measured by the local laboratory) for at least 2 consecutive measurements.  
The choice for the time points of the 2 consecutive measurements is guided by an algorithm 
that is described in Table 11-3, and is used to 
-
Decide to either to continue to withhold the prophylactic FIX replacement therapy, or
to re-start prophylactic FIX replacement therapy
-
Determine the time point for next FIX activity level assessment
If per the algorithm, the next time point would not be on a business day, it may be moved to a 
business day at the discretion of the Investigator. 
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Table 11-3, Actions During the First Period of Withholding Prophylactic FIX 
Replacement Therapy 
FIX activity level (%) a) 
 
Action (re-measurement or re-start of prophylactic therapy) b) 
≥ 8.0 
Continue to withhold prophylactic FIX replacement therapy and assess FIX 
activity level again in 4-5 days 
5.0-7.9 
Continue to withhold prophylactic FIX replacement therapy and asses FIX 
activity level again in 3-4 days 
3.0-4.9 
Continue to withhold prophylactic FIX replacement therapy and asses FIX 
activity level again in 2-3 days 
2.0-2.9 
Continue to withhold prophylactic FIX replacement therapy and asses FIX 
activity level again in 1-2 days 
< 2.0 
Re-start prophylactic FIX replacement therapy according to local practices 
a) 
FIX activity will be measured by a central laboratory using the one-stage aPTT assay for FIX and the 
chromogenic/amidolytic assay for FIX, and by the local laboratory at each clinical trial site using the one-stage aPTT 
assay for FIX or the chromogenic/amidolytic assay for FIX. Analysis results from the local laboratory will be regarded 
as decisive for actions related to withholding FIX therapy. 
b) 
In case the required time for next FIX activity measurement (according to Table 11-3) does not correspond with time 
for next clinic visit (according to Table 5-2), then one or more additional visits should be scheduled. 
 
Second period of withholding prophylactic FIX replacement therapy (4 weeks timespan) 
If FIX activity levels are ≥ 2.0% at both of the 2 measurements during the first period of 
withholding prophylactic FIX replacement therapy, the withholding can be continued and the 
subject is followed with weekly FIX activity measurements for an additional 4 weeks. In case 
the required time points for these 4 weekly FIX activity measurements do not correspond 
with time points for the next clinic visits (according to Table 5-2), then one or more 
additional visits should be scheduled.  
 
After the second period of confirming FIX activity levels has been completed, the FIX 
activity levels will be assessed at the per protocol scheduled visits, as listed in Table 5-1 
(Visits 1 - 21) and Table 5-4 (Visits 22 - 31). 
 
Re-starting prophylactic FIX replacement therapy after (or during) tapering 
It is the intention that FIX replacement therapy is tapered for all subjects, provided the 
requirement of FIX activity level continuously being ≥ 2.0% is met. Prophylactic FIX 
replacement therapy is re-started should the FIX activity level be < 2.0%. In addition, the 
Investigator may at any time decide for medical reasons to re-start prophylactic FIX 
replacement therapy. 
 
Subsequent re-initiating of tapering the prophylactic FIX replacement therapy may be done if 
clinically justified. The same requirements for minimum FIX activity level and the following 
up on the FIX activity levels as described above (i.e. first and second period of withholding 
prophylactic FIX replacement therapy) should be adhered to. 
 
In case of any bleeding episode (spontaneous, traumatic, recurrent, related to surgical 
intervention, or other), on-demand FIX replacement therapy should be administered at the 
discretion of the Investigator, according to local practice. 
 
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12.
Assessments
12.1 Efficacy Assessments 
 FIX Activity for Efficacy Evaluation 
Blood samples for FIX activity will be collected at all visits, including at the additional visits 
as required during or after the tapering of prophylactic FIX replacement therapy as described 
in Section 11.4. At Visit 2, blood sampling will take place prior to IMP administration. 
Throughout the entire trial period (from Visit 1 and onwards) where a subject is on 
prophylactic FIX replacement therapy, it will be the aim to draw blood samples at time points 
where FIX activity is expected to be at the lowest levels. To the extent visit windows allow, 
the Investigator and/or study nurse will collaborate with the subject to schedule visits to take 
place on days where prophylactic FIX replacement therapy is planned to be administered. At 
these visits, blood sampling will then take place just prior to administration of prophylactic 
FIX replacement therapy, which will then be administered at the clinic instead of at home. 
The following FIX activity assays will be applied at a central laboratory: 
-
one-stage activated Partial Thromboplastin Time (aPTT) assay
-
chromogenic/amidolytic assay
 Bleeding Episodes
From Visit 1 and throughout the entire trial participation period, subjects will be asked to 
record information on bleeding episodes in an e-diary. Regarding training of subjects in the 
use of the e-diary, please see Section 10.3.1. 
The subject e-diary will include questions regarding each bleeding episode with respect to: 
-
Date and time of onset of bleed
-
Date and time of stop of bleed
-
Location of bleed
-
Circumstances of bleed (spontaneous, traumatic, minor surgery), see definitions at the
end of this section for further details)
-
FIX replacement treatment of bleed (medication, dose, date and time of
administration)
-
Other therapy (pain relieving medication, compression, ice)
-
Evaluation of effect of treatment (excellent, good, moderate, none), see definitions at
the end of this section for further details.
At each visit, the Investigator will review e-diary entries on bleeding episodes and the 
subject’s medical/hospital records on bleeding episodes (if any). After Visit 21, e-diary data 
review will be performed at least monthly in addition to the review at each visit. E-diary 
entries and medical/hospital records on bleeding episodes will be considered source data. The 
Investigator will record his/her assessment of the severity of the bleed (see definitions 
below).  
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In case of bleeding episodes fulfilling a seriousness criterion (i.e. the bleeding episode is a 
SAE), information on the bleeding episodes is not required to be entered in the e-diary but in 
the medical/hospital records only. In those cases, the Investigator will also record his/her 
evaluation of effect of treatment of bleed (excellent, good, moderate, none), if applicable, see 
definitions at the end of this section for further details. Safety reporting requirements for 
disease-related bleeding episodes are described in Section 13.8. 
 
Definitions 
 
Minor surgery 
Any invasive operative procedure for which only the skin, the mucous membranes or 
superficial connective tissue is manipulated. Examples include implanting pumps or ports in 
subcutaneous tissue, skin biopsies and simple dental procedures. 
 
Circumstances of bleed  
Only spontaneous bleeds, traumatic bleeds and minor surgery are collected in the e-diary and 
eCRF. Data on bleeds related to major surgery requiring in-patient hospitalization are 
collected via SAE forms. Classification of re-bleed will be performed by the trial statistician 
at the time of analysis according to the following criteria: A re-bleed is defined as a 
worsening of symptoms in the same location after an initial period of improvement, either on 
treatment or within 72 hours after stopping treatment. If a bleed occurs in the same location 
later than 72 hours after stopping, the bleed is considered a new bleed. 
 
Subject’s evaluation of effect of treatment of bleed 
Excellent: Abrupt pain relief and/or equivocal improvement in objective signs of bleeding 
within approximately 8 hours of a single infusion 
Good:  
Definite pain relief and/or improvement in signs of bleeding with approximately 
8 hours of one infusion, but possibly requiring more than one infusion for 
complete resolution 
Moderate: Probable or slight beneficial effect approximately 8 hours after the first infusion; 
usually requiring more than one infusion 
None: 
No improvement, or worsening of symptoms 
 
Investigator’s evaluation of severity of bleed 
Mild/moderate bleeds: 
- Uncomplicated joint bleeds 
- Muscular bleeds without compartment syndrome 
- Mucosal bleeds 
- Subcutaneous bleeds 
Severe bleeds: 
- Complicated joint bleeds 
- All intracranial, retroperitoneal, iliopsoas or neck bleeds 
- Muscle bleeds with compartment syndrome 
- Bleeds associated with a decrease in haemoglobin levels of more than 2 g/dL 
 
Investigator’s evaluation of effect of treatment of bleed in case of bleeding episodes being a 
SAE 
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Excellent: Abrupt pain relief and/or equivocal improvement in objective signs of bleeding 
within approximately 8 hours of a single infusion  
Good: 
Definite pain relief and/or improvement in signs of bleeding with approximately 
8 hours of one infusion, but possibly requiring more than one infusion for 
complete resolution  
Moderate: Probable or slight beneficial effect approximately 8 hours after the first infusion; 
usually requiring more than one infusion 
None: 
No improvement, or worsening of symptoms 
 Prophylactic FIX Replacement Therapy 
From Visit 1 and throughout the entire trial participation period, subjects will be asked to 
document use of FIX replacement therapy in an e-diary. Regarding training of subjects in the 
use of the e-diary, please see Section 10.3.1. 
The subject e-diary will include questions with respect to 
-
Date and time of administration
-
Drug name
-
Dose
At each visit, the Investigator will review e-diary entries on prophylactic FIX replacement 
therapy and the subject’s medical/hospital records on prophylactic FIX replacement therapy 
(if any). After Visit 21, e-diary data review will be performed at least monthly in addition to 
the review at each visit. E-diary entries and medical/hospital records on prophylactic FIX 
replacement therapy will be considered source data. Subjects who are on continuous routine 
FIX prophylaxis during the long-term follow-up phase of the trial are required to contact the 
site staff immediately in case of a bleed and/or FIX use different from their routine FIX 
prophylaxis, in addition to completing the questions/information requested on the e-diaries to 
capture all information. 
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. 
12.2 Safety Assessments 
 Adverse Events 
Information on AEs will be reported at each visit. After Visit 21, AEs will be assessed at least 
monthly. 
Details regarding definitions of AEs, definitions of AE assessments, how to obtain 
information on AEs, how to report AEs and how to follow-up on AEs are described in 
Section 13. 
AEs and SAEs that the Investigator becomes aware of should continue to be reported to 
 following the completion of the study for a period of up to 
10 years following gene therapy dosing, until/unless the subject is enrolled in the extension 
protocol CT-AMT-060-04. 
 Physical Examination 
A physical examination will be performed at all visits. At Visit 2, the physical examination 
will be performed both prior to IMP administration and at 24 hours after IMP administration. 
The physical examination will include general appearance and bedside examination of the 
following body systems: Lymph nodes, eyes and ears, mouth and throat, lungs, abdomen, 
extremities, musculoskeletal system, neurological system, cardiovascular system and skin. 
The evaluation of each body system will be recorded as “normal” or “abnormal”. 
Abnormalities will also be recorded. 
Any abnormality judged by the Investigator as a clinically relevant worsening since Visit 1 
should be reported as an AE. 
 Blood Pressure, Pulse and Body Temperature 
Blood pressure, pulse and body temperature will be measured at all visits. At Visit 2 (dosing 
visit with overnight stay) blood pressure, pulse and body temperature will be measured prior 
to IMP administration and at the following approximate time points after IMP administration: 
0.5, 1, 2, 3, 4, 6, 8, 12 and at 24 hours. 
Before measurement of blood pressure and pulse the subject should rest for at least 5 minutes. 
For the individual subject, all measurements should be performed while the subject is in the 
same position (i.e. sitting or lying) throughout the trial. 
Body temperature should be measured using the same method (e.g. an ear thermometer) for 
the individual subject throughout the trial.  
Any abnormality judged by the Investigator as a clinically relevant worsening since Visit 1 
should be reported as an AE. 
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12.2.1 
12.2.2 
12.2.3 
 
 
 
 
 
 
 
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 Serum Chemistry, Haematology, Coagulation and Urine Parameters for Central Lab 
Table 12-1 provides the safety laboratory parameters to be measured: 
Visits at which samples should be drawn as well as a specification of the parameters to be 
measured are given in Table 12-2 and Table 12-3. 
Table 12-1, Safety Laboratory Parameters 
Serum Chemistry 
Serum electrolytes (sodium, potassium), creatinine, ɣGT, AST, ALT, ALP, CRP, 
albumin, total bilirubin, glucose (non-fasting) 
Haematology 
Haemoglobin, haematocrit, platelet count, red blood cells, white blood cells with 
differential count (band forms, segment forms, neutrophils, eosinophils, basophils, 
monocytes, lymphocytes) (all expressed in % as well as in absolute numbers) 
Coagulation 
aPTT, PT (or INR [International Normalized Ratio]), lupus anticoagulant, antithrombin 
Urine parameters 
pH, protein, blood, leucocyte esterase, glucose 
Table 12-2, Sampling for Serum Chemistry 
Visit number 
Week in relation to IMP administration 
Time point at Visit 
Parameter to be 
measured 
1 
Maximum 6 weeks prior to anticipated 
IMP administration 
Any time at visit 
All parameters 
2 
- 
Prior to IMP 
administration 
All parameters 
2 
- 
3 and 24 hours after 
IMP administration 
CRP only 
3 - 14 
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 
Any time at visit 
All parameters 
16, 18, 20, 21 
16, 20, 24, 26 
Any time at visit 
All parameters 
22 - 35 
39, 52, 65, 78, 91, 104, 117, 130, 143, 
156, 182, 208, 234, 260 
Any time at visit 
All parameters 
Table 12-3, Sampling for Haematology, Coagulation and Urine Parameters 
Visit number 
Week in relation to IMP administration 
Time point at Visit 
Parameter to be 
measured 
1 
Maximum 6 weeks prior to anticipated 
IMP administration 
Any time at visit 
All parameters a)  
2 
- 
Prior to IMP 
administration 
All parameters a)  
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6, 10, 14 
4, 8, 12 
Any time at visit 
All parameters a)  
16, 18, 20, 21 
16, 20, 24, 26 
Any time at visit 
All parameters a)  
22 - 35 
39, 52, 65, 78, 91, 104, 117, 130, 143, 
156, 182, 208, 234, 260 
Any time at visit 
All parameters a)  
a) Lupus anticoagulant and antithrombin will be measured only at Visit 1 
 Vector DNA 
Sampling of the following types of matrix will be performed to determine Vector DNA 
levels: Blood, saliva, nasal secretions, urine, faeces and semen. Sampling will be initiated at 
Visit 2 and will continue at the time points given in Table 5-2 (Visits 1 - 21), Table 5-3 
(Visit 2, overnight stay), and Table 5-5 (Visits 22 – 35). 
Sampling should continue for the individual subject and for a specific matrix until 3 
consecutive negative samples have been detected for the subject for that particular type of 
matrix. 
Based on the wish of the subject, faeces and semen samples can be collected at home prior to 
attending the visit (at the visit day or at the day before the visit day). 
 Neutralizing Antibodies to AAV5 
Blood sampling for measurement of neutralizing antibodies to AAV5 will take place at the 
following time points: 
-
at Visit 1 (Screening Visit)
-
at Visit 2 (prior to IMP administration)
-
at Visits 3 - 6 (weekly in weeks 1 - 4)
-
at Visit 21 (week 26)
-
at Visit 23 (week 52 / year 1)
-
at Visit 27 (week 104 / year 2)
-
at Visit 31 (week 156 / year 3)
-
at Visit 33 (week 208 / year 4)
-
at Visit 35 (week 260 / year 5)
The Investigator will be informed of the value at Screening for the purpose of evaluation of 
exclusion criterion no. 3. 
 Total Antibodies to AAV5 
Blood sampling for measurement of total (IgG and IgM) antibodies to AAV5 will take place 
at the following time points: 
-
at Visit 1 (Screening Visit)
-
at Visit 2 (prior to IMP administration)
-
at Visits 3 - 6 (weekly in weeks 1 - 4)
-
at Visit 21 (week 26)
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12.2.5 
12.2.6 
12.2.7 
 
 
 
 
 
 
 
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- at Visit 23 (week 52 / year 1) 
- at Visit 27 (week 104 / year 2) 
- at Visit 31 (week 156 / year 3)  
- at Visit 33 (week 208 / year 4) 
- at Visit 35 (week 260 / year 5) 
 
 AAV5 capsid-specific T cells 
Sampling for the measurement of AAV5 capsid-specific T cells will take place at the 
following time points: 
- at Visit 2 (prior to IMP administration) 
- at Visits 3 - 14 (weekly in weeks 1 - 12) 
- at Visits 15 - 21 (bi-weekly in weeks 14 - 26) 
 
 Anti-FIX Antibodies 
Blood sampling for measurement of anti-FIX antibodies will take place at the following time 
points: 
- at Visit 2 (prior to IMP administration) 
- at Visit 21 (week 26) 
- at Visit 23 (week 52 / year 1) 
- at Visit 27 (week 104 / year 2) 
- at Visit 31 (week 156 / year 3)  
- at Visit 33 (week 208 / year 4) 
- at Visit 35 (week 260 / year 5) 
 
 FIX Inhibitors 
Blood sampling for measurement of FIX inhibitors (Nijmegen modified Bethesda assay at a 
central laboratory) will take place at the following time points: 
- at Visit 1 (Screening Visit) 
- at Visit 2 (prior to IMP administration) 
- at Visit 14 (week 12) 
- at Visit 21 (week 26) 
- at Visit 23 (week 52 / year 1) 
- at Visit 27 (week 104 / year 2) 
- at Visit 31 (week 156 / year 3)  
- at Visit 33 (week 208 / year 4) 
- at Visit 35 (week 260 / year 5) 
 
 FIX Recovery 
Measurement of FIX recovery (Cmax) and incremental recovery measured as increase in 
activity per unit infused (IU/ml per U/kg) at 30 min after infusion of a dose of FIX will be 
performed at Visit 2. Additionally, measurement of FIX recovery and incremental recovery 
should be done at suspicion of FIX inhibitor (see also Section 12.3.10) or at increase in 
bleeding frequency, as judged by the Investigator. 
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12.2.8 
12.2.9 
12.2.10 ----
12.2.11 ----
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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At each occasion, a FIX challenge dose of 40 U/kg should be administered while at the 
clinical trial site. A blood sample should be drawn just prior to FIX dosing and at 30 minutes 
after FIX dosing. The blood sample should preferably be drawn from a vein different from 
the vein used for FIX infusion. 
Date of sampling, times of blood sampling (pre and post FIX administration) and time of FIX 
administration will be recorded. 
FIX activity will be measured at a central laboratory using the one-stage aPTT assay and the 
chromogenic/amidolytic assay. 
  Inflammatory Markers 
Blood sampling for measurement of IL-1β, IL-2, IL-6, INFγ and MCP-1 will take place prior 
to IMP administration and in the period up to 18 weeks after IMP administration: 
-
at Visit 2 (prior to IMP administration)
-
at Visits 3 - 14 (weekly in weeks 1 - 12)
-
at Visits 15 - 17 (bi-weekly in weeks 14, 16, 18)
12.3 Other Assessments  
12.3.1 Local Laboratory Sampling for Management of Subjects and Eligibility Check 
Table 12-4 provides the laboratory parameters to be measured by the local laboratory, and 
specifies which samples should be drawn at each visit.  
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Table 12-4, Local Laboratory Sampling 
Visit number 
Week in relation to IMP 
administration 
Time point at Visit 
Parameter to be measured 
1 
Maximum 6 weeks prior to anticipated 
IMP administration 
Any time at visit1) 
FIX activity2) 
FIX inhibitors3) 
Liver enzymes (AST/ALT) 
2 
- 
Prior to IMP 
administration 
FIX activity2) 
FIX inhibitors3) 
Liver enzymes (AST/ALT) 
2 
- 
24 hours after IMP 
administration 
Liver enzymes (AST/ALT) 
3 - 14 
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 
Any time at visit1) 
FIX activity2) 
FIX inhibitors3) (v14 only)  
Liver enzymes (AST/ALT) 
3b – 14b  
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 
Any time at visit1) 
FIX activity2) 
Liver enzymes (AST/ALT) 
15 - 20 
14, 16, 18, 20, 22, 24 
Any time at visit1) 
FIX activity2) 
Liver enzymes (AST/ALT) 
21 
26 
Any time at visit1) 
FIX activity2) 
FIX inhibitors3) 
Liver enzymes (AST/ALT) 
22 - 35 
39, 52, 65, 78, 91, 104, 117, 130, 143, 
156, 182, 208, 234, 260 
Any time at visit1) 
FIX activity2) 
FIX inhibitors3) (v23, 27, 31, 
33, 35 only) 
1)
For subjects on prophylactic FIX replacement therapy, attempts should be made to allow blood sampling for FIX 
activity at time points where FIX activity is expected to be at lowest justifiable levels (see Section 12.1.1). 
2)
The analysis method applied at the local laboratory for FIX activity should be one of the following:
- 
One-stage aPTT assay
- 
Amidolytic/chromogenic assay
Preferably, the same type of assay is applied consistently for the individual subject throughout the entire trial period. 
3)
The analysis method applied at the local laboratory for FIX inhibitors should be one of the following:
- 
Nijmegen modified Bethesda assay
- 
Bethesda assay
Preferably, the same type of assay is applied consistently for the individual subject throughout the entire trial period. 
The Investigator should arrange with the local laboratory that analysis results are provided on 
the same day, or the day after, blood sampling has taken place. 
The Investigator should enter analysis results, as well as related reference ranges and analysis 
method applied (if applicable) in the eCRF. In addition, the local laboratory result reports 
should be kept in the subject’s medical record. 
12.3.2 FIX Protein Concentration 
Blood samples for FIX protein concentration (FIX:Ag) will be collected at all visits. At 
Visit 2, blood sampling will take place prior to IMP administration. 
Throughout the entire trial period (from Visit 1 and onwards) where a subject is on 
prophylactic FIX replacement therapy, it will be the aim to draw blood samples at time points 
where FIX protein concentration is expected to be at the lowest levels. To the extent visit 
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windows allow, the Investigator and/or study nurse will collaborate with the subject to 
schedule visits to take place on days where prophylactic FIX replacement therapy is planned 
to be administered. At these visits, blood sampling will then take place just prior to 
administration of prophylactic FIX replacement therapy, which will then be administered at 
the clinic instead of at home. 
 
FIX protein concentration in plasma will be measured at a central laboratory. 
 
12.3.3 Demographics and Disease Characteristics 
At Visit 1 (Screening Visit), date of birth, race, ethnic group and gender will be recorded 
according to local regulations. In addition, medical history and concomitant illness(es), 
(disease related) surgical history, haemophilia B history, and history and status of FIX 
medication and bleeding will be recorded. 
 
12.3.4 FIX Gene Mutation 
Available information on FIX gene mutation will be collected at Visit 1 (Screening Visit) and 
recorded. 
 
If information on FIX gene mutation is not available, a blood sample for the purpose of FIX 
gene sequencing analysis will be collected (preferably at Visit 1, but otherwise at a later time 
point during the subject’s trial participation), but only if separate informed consent is given 
by the subject.  
 
Subjects who do not wish to participate in the FIX gene sequencing analysis may still 
participate in the trial and will not be required to withdraw from the trial if they withdraw 
consent for the FIX gene sequencing analysis. 
 
Gene sequencing analysis will be performed at a central laboratory.  
 
12.3.5 Medical History and Concomitant Illnesses 
Medical history is any previous medical condition or surgical event, i.e. a condition/event that 
started prior to Visit 1 (Screening Visit), but is not ongoing at Visit 1. A concomitant illness 
is a medical condition that is ongoing at Visit 1. Information on bleeding history and 
haemophilia B disease related surgical events and joint status will be reported separately (see 
Sections 12.3.6 and 12.3.7, respectively). 
 
At Visit 1, information on relevant medical history will be obtained and recorded. The 
following conditions and events will be considered relevant (bleeding events excluded): 
- Any surgical event or any chronic or ongoing medical condition, regardless if it 
requires/required therapy or not 
- Any medical condition or surgical event that has resulted in sequelae 
- Any isolated or one-off medical condition or surgical event that has occurred within 
1 year prior to Visit 1 irrespective of the outcome of the event. 
- Any isolated or one-off medical condition or surgical event that has resolved without 
sequelae and occurred more than 1 year prior to Visit 1 if judged relevant by the 
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Investigator (for example conditions that the Investigator evaluates could re-emerge 
over time, e.g. cancers). 
Furthermore, at Visit 1 information on any medical condition ongoing at Visit 1 will be 
obtained and recorded. 
12.3.6 History and Status of Bleeding 
At Visit 1 (Screening Visit) information on the following bleeding history data will be 
collected: 
For all subjects: 
-
Number of exposure days prior to trial entry. An exposure day is a day where the
subject received one or more infusion(s) of FIX replacement therapy. Additional
information will be recorded for FIX replacement therapy administered during the last
7 days prior to trial entry (trade name of FIX medication, date & time of
administration, units per administration).
For subjects receiving prophylactic FIX replacement therapy: 
-
Number of months on prophylactic FIX replacement therapy for the last 12 months
-
Dose and frequency of dosing; in case the subject is on intermittent prophylactic FIX
replacement therapy, each regimen followed in the last 12 months should be reported
-
Recombinant or plasma FIX product
-
Number of treatment requiring bleeding episodes in the last 12 months during each
specific prophylactic treatment regimen
-
Average number of units to treat a bleed during each specific prophylactic treatment
regimen during the last 12 months
-
Number of bleeding episodes prior to initiation of prophylactic FIX replacement
therapy.
For subjects receiving on-demand FIX replacement therapy only: 
-
Number of months on on-demand FIX replacement therapy for last 12 months
-
Number of treatment requiring bleeding episodes in the last 12 months during each
specific treatment regimen
-
Recombinant or plasma FIX product
-
Average number of units to treat a bleed during each specific on-demand FIX
replacement therapy regimen during the last 12 months.
12.3.7 Information on Haemophilia B Disease Related Surgeries and Joint Status 
Information on history of haemophilia B disease related surgery will for all subjects be 
collected at Visit 1. 
Information on joint status, target joints4 and range of motion will for all subjects be recorded 
at Visit 1 as well as every 12 months after IMP administration, i.e. at Visit 23, 27, 31, 33 and 
35. 
4 Definition of target joint: A joint which the subject has bleed into in the last 6 months at least 3 times 
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Data include: 
- Haemophilia B disease related surgery 
o Date of surgery 
o Surgical event 
o Preventive treatment during surgery: Recombinant or plasma product 
- Joint status (Haemophilia Joint Health Score version 2.1). The instruction manual for 
this assessment will be contained in the Trial Procedures Manual and must be 
followed. Preferably, the same assessor will consistently perform the assessment on 
the individual subject throughout the entire trial period 
- Number and location of target joints 
- Range of motion of joints. 
 
12.3.8 Body Mass Index 
At Visit 1 (Screening Visit), height (without shoes) will be measured and recorded, rounded 
to the nearest centimetre. 
 
At Visits 1, 2 and 21-35, body weight (without overcoat and shoes) will be measured and 
recorded, rounded to the nearest kilogram. Body mass index will be calculated automatically, 
based on eCRF entries of height and body weight. 
 
12.3.9 Laboratory Parameters for Evaluation of Subject Eligibility 
At Visit 1 (Screening Visit) blood samples will be drawn for the purpose of measurement of 
the following laboratory parameters: HIV, CD4+, HIV viral load, HBsAg, HBeAg, HBV 
DNA and HCV RNA.  
 
As described in Section 12.3.1, a blood sample will be drawn at Visit 1 (Screening Visit) for 
the purpose of measurement of FIX inhibitor by the local laboratory.  
 
As described in Section 12.2.6 at Visit 1 (Screening Visit) a blood sample will be drawn for 
the purpose of measurement of neutralizing antibodies to AAV5. 
 
Furthermore, as described in Section 12.2.4, Visit 1 blood and urine samples will be drawn 
for analysis of serum chemistry, haematology, coagulation and urine parameters. 
 
For the purpose of evaluation of Exclusion Criterion 2, the Investigator will use the Visit 1 
(Screening) laboratory result from the local laboratory. The Investigator will be informed by 
the central laboratory about the Screening laboratory results for the purpose of evaluation of 
Exclusion Criteria 3, 4, 5, 6, 8 and 9. 
 
If pre-determination of subject eligibility is desired by the Investigator in advance of the 
conduct of the full screening visit (Visit 1), an additional visit can be performed during which 
a selection of laboratory parameters (neutralizing antibodies, HIV, CD4+, HIV viral load, 
HBsAg, HBeAg, HBV DNA and HCV RNA) can be measured (see Table 5-2, additional 
visits). When pre-determination of subject eligibility is established and the subject continues 
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into the trial, a full screening visit (Visit 1) needs to be conducted (see Table 5-1 and 
Table 5-2, Visit 1), however if the timing of the additional visit is within 2 weeks of Visit 1 
there is no need to repeat the laboratory parameters previously measured. 
12.3.10 Further Testing of Subjects with a Suspected FIX Inhibitor 
A subject is said to suffer from FIX inhibitors if tested positive for FIX inhibitors at 
2 consecutive tests (as measured by the local laboratory), performed preferably within 
2 weeks. 
If a subject has confirmed FIX inhibitor and continues with no change to treatment type for 
6 weeks and the FIX inhibitor test is negative after that time, the FIX inhibitor is classified as 
transient. 
12.3.11 Laboratory Samples for Future Research 
Additional blood samples for the purpose of potential future research in the haemophilia B 
disease area (including development and validation of assays to support efficacy assessments) 
will be drawn at screening (Visit 1), baseline (i.e. pre-IMP time point during Visit 2) and at 
the following time points after IMP administration: 5 weeks, 10 weeks, 26 weeks and 
52 weeks (Visit 7, 12, 21 and 23). 
These additional blood samples will only be drawn if separate informed consent is given by 
the subject. Subjects who do not wish to donate blood samples for the purpose of potential 
future research may still participate in the trial and will not be required to withdraw from the 
trial if they withdraw consent for the potential future research. 
The procedures for the collection, processing, storage and shipment of these blood samples 
are described in the Laboratory Manual. 
12.4 General Information Regarding Laboratory Sampling and Results 
All laboratory assessments will be conducted at a central laboratory, except FIX activity 
assay for monitoring of subjects (Section 12.3.1), FIX inhibitor assay for monitoring of 
subjects and for eligibility check (Section 12.3.1) and liver enzymes (AST and ALT) 
(Section 12.3.1) which will be conducted locally. 
Dates and times of sampling will be recorded. 
Detailed procedures for the collection, processing, storage and shipment of laboratory 
samples are described in the Laboratory Manual. This manual as well as all material such as 
test tubes and labels will be provided by the coordinating central laboratory. 
After the laboratory samples have been analysed, they will remain stored for potential 
re-analysis at any time during the trial to a maximum of up to 1 year after the trial has been 
completed, before being destroyed. Exceptions are the future research blood samples, which 
will be stored and used for medical research until there is no sample remaining. 
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The Investigator will be provided with laboratory results at regular intervals for review and 
sign-off. Any abnormality, judged by the Investigator as a clinically relevant worsening since 
the first measurement, i.e. at Visit 1 or Visit 2, should be reported as an AE, unless the 
laboratory abnormality is associated with an already reported AE. 
Any report of erroneous results in the following parameters from Visit 21 and onwards 
should prompt that the subject is called in for an Additional Visit to have blood sample(s) 
drawn for the purpose of re-measurement:  
-
FIX activity measured at local laboratory
-
FIX activity measured at central laboratory
-
FIX inhibitors measured at local laboratory
-
FIX inhibitors measured at central laboratory
-
Coagulation parameters
-
AST, ALT
-
FIX recovery and incremental recovery.
The Additional Visit should preferably take place within 1 week after the report of the 
erroneous result(s). 
12.5 Total Blood Volume 
Blood samples will be collected for efficacy, safety and screening assessments. For each 
subject, the total blood volume in the first half year, including screening, is expected to be 
1906 mL (excluding any additional visits). For the remaining 4.5 years of the study, the total 
blood volume is expected to be 684 mL. Therefore, the total blood volume for subjects 
completing the study will be 2590 mL. The maximum blood volume per additional visit 
(during tapering phase or any other time) is 130 mL. 
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13.
Safety Reporting
13.1 Adverse Event Definitions 
An adverse event (AE), an adverse drug reaction (ADR) and a serious adverse event (SAE) 
are defined according to ICH Guideline E2A. 
An AE is any untoward medical occurrence in a subject administered the IMP and which 
does not necessarily have a causal relationship with this IMP or the IMP administration 
procedure. An AE can therefore be any unfavourable and unintended sign (including an 
abnormal laboratory finding, see Section 12.4), symptom, or disease temporally associated 
with the use of the IMP including the IMP administration procedure. 
An ADR is an untoward and unintended response to the IMP related to any dose 
administered. A causal relationship between the IMP and the AE is at least a reasonable 
possibility. 
An SAE is any untoward medical occurrence that at any dose: 
-
Results in death
-
Is life-threatening (this refers to an event in which the subject was at risk of death at
the time of the event; it does not refer to an event that hypothetically might have
caused death if it was more severe)
-
Requires in-patient hospitalization or prolongation of existing hospitalization
-
Results in persistent or significant disability or incapacity
-
Is a congenital anomaly or birth defect
-
Is judged medically important by the Investigator (this refers to an event, not resulting
in any of the outcomes listed above, but may jeopardize the subject or may require
intervention to prevent one of the other outcomes listed)
In the following situations, events are not defined as an AE: 
-
Medical or surgical procedure (e.g. endoscopy, appendectomy); the condition that
leads to the procedure is an AE
-
Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present
or detected at the start of the trial that do not worsen
-
Condition(s) for which pre-planned procedure(s) have been recorded at Visit 1,
including hospitalization(s), unless the condition(s) for which the procedure and/or
hospitalization was planned has worsened from the first trial related activity after the
subject has signed the informed consent form
-
Concomitant illness identified during the screening procedures will be recorded as
medical history. However, whenever symptoms for these condition(s) worsen and/or
become serious, then these events must be reported as an AE or SAE, as applicable.
13.2 Adverse Event Assessment Definitions 
 Severity 
The Investigator should assess the severity of all AEs according to the following definitions: 

Mild: Awareness of symptoms, sign, illness or event that is easily tolerated.
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
Moderate: Discomfort sufficient to cause interference with usual activity.

Severe: Incapacitating with inability to work or undertake further normal activities.
Note the distinction between seriousness and severity: The term severe is used to describe the 
intensity of the event and a severe event is not necessarily serious (e.g. a severe headache 
would probably not constitute an SAE; however a mild myocardial infarction could constitute 
an SAE). The seriousness criteria serve as a guide for defining regulatory reporting 
obligations. 
If an AE changes severity over time, the severity of maximum intensity should be reported. 
 Relationship to IMP 
Assessment of causality is based on the following considerations: associative connections 
(time and/or place), pharmacological explanations, previous knowledge of the drug, presence 
of characteristic clinical or pathological phenomena, exclusion of other causes, and/or 
absence of alternative explanations. 
The Investigator will be asked to assess causal relationship to the trial treatment according to 
the following classifications: 

Probably related: An AE with a reasonable time sequence to administration of the
IMP, unlikely to be attributed to concurrent disease or other drugs or chemicals, and
which follows a clinically reasonable response on withdrawal (de-challenge). Re-
challenge information is not required to fulfil this definition.

Possibly related: An AE with a reasonable time sequence to administration of the
IMP, but which could also be explained by concurrent disease or other drugs or
chemicals. Information on drug withdrawal may be lacking or unclear.

Unlikely related: An AE with a temporal relationship to IMP administration which
makes a causal relationship improbable, and in which other drugs, chemicals or
underlying disease provide plausible explanations.

Not applicable: This assessment can be used e.g. in cases where the subject did not
receive any treatment with IMP.
 Outcome 
The Investigator will be asked to record the most appropriate outcome of the following: 
-
Recovered
-
Recovered with sequelae
-
Recovering
-
Not recovered
-
Fatal
-
Unknown
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13.3 Reporting of Adverse Events 
All events meeting the definition of an AE must be reported in the period starting at the first 
visit during which any trial related activity takes place until the end of trial participation. At 
each visit the subject will be asked about AEs in an objective manner, e.g.: “Have you 
experienced any problems since the last visit?”. Only medically qualified personnel 
(Investigators) must assess AEs. 
AEs must be reported in the source data and the eCRF. The diagnosis will be recorded, if 
available and applicable. If no diagnosis is available each sign and symptom will be recorded 
as individual AEs. 
Recurring AEs should be reported separately, i.e. with separate start date and time and stop 
date and time. 
13.4 Prompt Reporting of SAEs and Other Events to uniQure 
SAEs, AEs qualifying for special notification and disease-related bleeding episodes related to 
the IMP and/or with fatal outcome, must be reported as described in the following table once 
the Investigator determines that the event meets the protocol definition for that event.  
Initial Reports 
Follow-up Information on a 
Previous Report 
Type of Event 
Time Frame 
Documents 
Time Frame 
Documents 
All SAEs 
24 hours 
SAE form 
72 hours* 
SAE form 
All AEs qualifying for 
special notification as 
defined in Section 13.6 
24 hours 
SAE form 
72 hours* 
SAE form 
Disease-related bleeding 
episodes related to the 
IMP and/or with fatal 
outcome as defined in 
Section 13.8 
24 hours 
SAE form 
72 hours* 
SAE form 
* If however, in the opinion of the Investigator, the follow-up information may have implications for the 
safety of other subjects, the follow-up information is to be reported immediately (i.e. within 24 hours).
The information will be reported on a paper SAE form and will include assessment of 
severity, causal relationship to the IMP or trial procedures, action taken, outcome, and a 
narrative description of the course of the event. Additional information may be subsequently 
provided.  
The paper SAE form and all other relevant documents supporting the reported SAE or AE 
qualifying for special notification (e.g. diagnostic procedures, hospital records, autopsy 
reports) must be faxed or scanned/e-mailed to 
24 hour Hotline:
F: 
E-mail:
 
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The Independent Ethics Committees/Institutional Review Boards (IECs/IRBs) and regulatory 
authorities will be notified of SAEs according to current regulation and local requirements. 
SAEs occurring to a subject after the subject has completed the clinical trial and for which a 
reasonable possibility of a causal relationship is assessed by the Investigator, should be 
reported by the Investigator to the sponsor if the Investigator becomes aware of them 
regardless of the time that has elapsed (post-trial events). 
13.5 
Regulatory Reporting Requirements for SAEs and Other Events 
Prompt notification by the Investigator to uniQure of SAEs, AEs qualifying for special 
notification and disease-related bleeding episodes related to the IMP and/or with fatal 
outcome is essential, so that legal obligations and ethical responsibilities towards the safety of 
subjects are met.  
uniQure has a legal responsibility to notify both the local regulatory authority and other 
regulatory agencies about the safety of a product under clinical investigation. uniQure will 
comply with ICH/Food and Drug Administration (FDA)/European Medicines Agency (EMA) 
and country-specific regulatory requirements relating to safety reporting to the regulatory 
authority, IECs/IRBs and Investigators. Details are laid down in the safety reporting plan 
which will comply with these regulatory requirements (e.g. 21CFR 312.32). 
Expedited reporting of suspected unexpected serious adverse reactions will be taking place 
within 7 or 15 days depending on fatal/life threatening status, seriousness, expectedness and 
causality from all clinical trials globally (suspected unexpected serious adverse reaction 
[SUSAR] reporting in EU and IND safety reports in the US). Any suspected adverse reaction 
that is both serious and unexpected according to the Investigator’s Brochure will be reported 
provided there is evidence to suggest a causal relationship between the drug and the AE. 
Relevant follow-up information to an SAE IND safety report will be submitted as soon as the 
information is available.  
Information about SUSARs is also forwarded to Investigators and IECs/IRBs as necessary. 
An Investigator who receives an SUSAR describing an SAE(s) or other specific safety 
information (e.g. summary or listing of SAEs) from uniQure will file it with the 
Investigator’s Brochure and will notify the IEC/IRB, if appropriate according to local 
requirements. 
In addition to submission of SAEs, an annual Development Safety Update Report (DSUR) 
will be prepared and submitted to the EMA, FDA, and locally if applicable according to the 
development international birth date (DIBD). 
13.6 Adverse Events Qualifying for Special Notification 
New AEs potentially related to the conduct of the trial or the development of the Advanced 
Therapy Medicinal Products (ATMP) and likely to affect the safety of the subjects should be 
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reported and followed in the same manner as SAEs (see Section 13.3 and Section 13.7). Note 
that the AEs may be serious or non-serious by definition (see Section 13.1). 
 
In addition, the following (S)AEs qualify for special notification as they are seen as safety 
issues of particular concern for ATMPs (ENTR/F/2/SF/dn D(2009) 35810. Brussels, 
03/12/2009) and gene therapy medicinal products (EMEA/CHMP/GTWP/60436/2007): 
 
- AEs related to the IMP administration procedure (intravenous infusion) 
- Suspected or confirmed cases of opportunistic or serious infections that in the 
Investigator’s opinion might be related to the IMP 
- Unexpected reactions (e.g. hypersensitivity, immunological, toxic or other as 
consequence of a change in the construction or function of the viral vector [e.g. 
generation of replication competent virus]) 
- AEs related to product failure (including lack of efficacy) 
- Confirmed FIX inhibitors 
- Thromboembolic events 
- Transaminase elevation > 1.5 times baseline value 
- Allergic reactions 
- AEs that could be related to potential off-target expression  
- Development of any new/recurrent cancer.  
13.7 Follow-up on Adverse Events 
All AEs should be followed until they have reached a “final outcome” (recovered, recovered 
with sequelae, recovering, not recovered, fatal or unknown) or the subject’s participation in 
the trial ends, whichever comes first. 
 
Severe, non-serious AEs assessed as “Probably related” or “Possibly related” to IMP and all 
SAEs and AEs qualifying for special notification (regardless of their relationship to IMP) still 
ongoing after ended trial participation, should be followed on a regular basis according to the 
Investigator’s clinical judgment until a “final outcome” has been established. 
 
The outcome “recovering” can be used as the “final outcome” for events that are stabilized 
(i.e. no further worsening is expected) and expected by the Investigator to resolve over time. 
 
The outcome “not recovered” can be used as the “final outcome” for events that are not 
expected to resolve over time (e.g. cancer).  
 
If a subject dies during the reporting period attempts should be made to obtain biopsy 
material to perform assay for replication competent retrovirus or other relevant part of the 
gene therapy medical product and to ascertain the cause of the death 
(EMEA/CHMP/GTWP/60436/2007). 
13.8 Reporting Requirements for Disease-Related Bleeding Episodes 
Disease-related bleeding episodes evaluated by the Investigator as part of the underlying 
disease should not be reported as AEs unless related to the IMP (including bleeding episodes 
due to lack of efficacy of the IMP) or if the bleeding has worsened as compared to the 
subject’s normal bleeding pattern, as evaluated by the Investigator. In case a bleeding episode 
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is meeting one or more of the SAE criteria or is related to major surgery requiring in-patient 
hospitalization, it must be reported as an SAE. 
13.9 Pregnancies 
Pregnancies of the subject’s sexual partner will be recorded separately from AEs, but will be 
reported in a manner identical to the reporting of SAEs, however via the use of a pregnancy 
reporting form instead of a SAE form. 
All attempts will be made to record all pregnancies occurring during this trial for which the 
subject received IMP and where the subject’s sexual partner has become pregnant after IMP 
administration.  
All attempts will be made to follow-up the pregnancy until the outcome of the pregnancy has 
been determined and to capture information on the development of the infant in the period up 
until and including the age of 1 year. This information will only be collected if separate 
informed consent is given by the subject and the subject’s sexual partner. 
Any report of a congenital abnormality/birth defect is an SAE and should be reported as such. 
Any complication of a pregnancy occurring during this trial, including elective termination 
for medical reasons, must be reported with the pregnancy reporting form. 
13.10  Occupational Exposure 
Occupational exposure refers to the exposure to a medicinal product as a result of one’s 
professional or non-professional occupation and will be reported in a manner identical to the 
reporting of SAEs, however via the use of an occupational exposure form instead of a SAE 
form.  
Occupational exposure with or without an AE, should be reported within 24 hours of 
becoming aware of the exposure. 
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14.
Statistical Methods
A Statistical Analysis Plan will be prepared prior to the first interim analysis. Overall 
principles are described below. 
14.1 Sample Size Calculation 
No formal sample size calculation was made. The choice of 5 to 7 subjects in each cohort is 
considered sufficient to capture commonly occurring AEs (Percy et al., 2010). 
14.2 Statistical Methods 
All reporting will be made for the Full Analysis Set. Reporting may be made for other 
analysis populations, or subgroups of subjects based on for example genotype. 
Data will be presented in tables and listings following the principles in ICH E3 guideline 
regarding structure and content of clinical reports. Data will also be presented graphically by 
subject and for selected endpoints all data will be presented.  
Summary tables will show number of values and when applicable number of missing values. 
Categorical data will be presented with number of cases and percentage of total number of 
cases. Continuous data will be presented with mean and SD, median, minimum and 
maximum as appropriate. 
Continuous data will be shown in listings with the same precision as captured. 
Subject level summaries will be presented adjusted for observation time. This includes but is 
not limited to annualized rate of selected signs and symptoms, annualized bleeding rate, 
average value for laboratory parameter or area under the time-profile curve normalized with 
time.  
Laboratory parameters will be presented by visit and include categorization of parameters 
into low/normal/high where applicable. 
Prophylactic and on-demand FIX replacement therapy will be summarized according to type 
of medication. Other concomitant treatment will be presented by therapeutic grouping 
presenting number of subjects and number of treatments. 
No formal statistical hypothesis will be tested. All confidence intervals represent exploratory 
analyses and will not be related to formal statistical hypothesis tests. No correction for 
multiplicity will be applied. 
 Primary Efficacy Endpoint Analysis 
AEs will be coded using Medical Dictionary for Regulatory Activities (MedDRA) and 
summarized by system organ class and preferred term presenting the number of subjects and 
number of events. Furthermore, summary tables will be made presenting severity for each 
event, and using the maximal severity for event. Separate summaries will be made for 
selected AEs. 
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 Secondary Efficacy Endpoint Analysis 
FIX Levels in Plasma 
Data will be presented by subject, time point and dose level as summary tables and 
graphically.  
Adjustment for time since most recent administration of FIX replacement therapy may be 
made, for example reporting by time less than 72 hours versus more than 72 hours after 
(reflecting prophylaxis free assessment). If applicable, the presentation will include summary 
statistics and confidence intervals for difference in FIX concentration between time-periods. 
If applicable, the FIX level will be analysed using Non-Linear Mixed Models, possibly after 
transforming the data, and may include subject level random effect or subject level covariate 
to describe dependency between measurements. The analysis may include information on 
dose and timing of FIX replacement therapy. If relevant, the analysis may be extended to 
include modelling of values below lower level of quantification. The modelling may include 
subject level information on type of FIX replacement therapy, half-life, and if available 
previous knowledge of subject specific half-life for the FIX replacement therapy. 
Number, Severity and Type of Bleeding Episodes 
Bleeding episodes will be summarized by subject and overall, and will include number, 
duration and severity. Separate summaries may be made for bleeds depending on type and 
location, including target joints, and also summaries excluding recurrent bleeds.  
Annualized bleeding rates overall and by severity will be presented. If relevant, summaries 
will be made using timing representing subject specific periods for pre-tapering, tapering 
period, and post tapering period until 26 weeks, and post 26 weeks.  
Furthermore, the number of bleedings may be analysed using models for count data adjusting 
for time at risk using generalized linear model with Poisson or Negative Binomial 
distribution. Where description of timing and inter-event duration is of interest, models for 
time-to-event analysis may be used, such as Cox Proportional models or other parametric 
model, possibly including subject level frailty parameter. The analysis may include 
information on dose and timing of FIX replacement treatment, and measured FIX 
concentration. 
Prophylactic and On-demand FIX Replacement Therapy 
The use of prophylactic and on-demand FIX replacement therapy in general will be 
summarized by subject and overall. If relevant, summaries will be made using timing 
representing subject specific periods for pre-tapering, tapering period, and post tapering 
period until 26 weeks, and post 26 weeks.  
 Patient Reported Outcome Data 
For 
, the scale scores will be listed by subject and visit and 
summarized as appropriate. 
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14.3 Subject Population(s) for Analysis 
The Full Analysis Set will consist of all dosed subjects and will be used for reporting. If 
applicable, further analysis sets will be defined, such as a Per Protocol Analysis set. 
14.4 Significance Level 
No formal statistical testing will be performed. Unless otherwise noted statistical tests will be 
reported using a significance level of 0.05. Confidence intervals will be presented as 95% 
2-sided confidence intervals. 
14.5 Termination Criteria 
Not applicable, as no formal procedure based on statistical decision rules is defined. 
14.6 Handling of Missing Data 
All data will be listed, including unused and spurious data. Imputation of missing or partial 
data will not be done unless explicitly described. In case of imputation of missing data, both 
the original missing or partial data and the imputed values will be listed. 
 
 Handling of Missing Data for Bleeding Episodes 
Bleeding episodes will be recorded with start and end date and time. A bleeding episode is 
classified as a re-bleed, if there is less than 72 hours between start and stop of previous 
episode (definition of re-bleed in Section 12.1.2). In case of partial start and stop date, the 
classification will be made using a conservative approach.  
14.7 Deviation Reporting 
Deviation(s) from the planned statistical analysis plan will be described and justified in the 
protocol and/or in the final CSR.  
14.8 Interim Analysis 
Interim analyses will be performed at the request of the data monitoring committee and at the 
request of the sponsor to enable reporting of adequate data at selected time points during the 
course of the trial. Descriptive statistics alone will be used for the interim analyses. 
 
Formal interim analyses will occur after all subjects have been followed for at least 12 weeks 
(i.e. completed Visit 14) and at least 52 weeks (i.e. completed Visit 23). The data from the 
52-week analyses will be reported in an interim CSR. 
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15. 
Data Monitoring Committee 
A data monitoring committee will be established to:  
- perform on-going safety surveillance throughout the entire trial period 
- evaluate available safety data from the first subject in each cohort and provide a 
recommendation to the sponsor regarding dosing of the second subject 
- evaluate available safety data from the second subject in each cohort and provide a 
recommendation to the sponsor regarding dosing of the subsequent subjects in a 
cohort 
- evaluate available safety data from subjects in Cohort 1 and provide a 
recommendation to the sponsor regarding dosing of subjects in Cohort 2 
 
The data monitoring committee’s review of available data prior to dosing of subjects within 
each cohort as well as between cohorts may reveal safety issues requiring further 
investigation prior to dosing of the next subject(s). The data monitoring committee will align 
their recommendation(s) with the Trial Stopping Rules as given in Section 19.2. 
 
The data monitoring committee will consist of at least 2 external and independent experts, of 
which one will be the chairman. In addition, at least one of the external independent experts 
should be medically qualified.  
 
Details on the composition, tasks and responsibilities of the data monitoring committee will 
be described in the data monitoring committee charter. 
 
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16. 
Ethics 
16.1 Independent Ethics Committee / Institutional Review Board (IEC/IRB) 
Before implementing this trial, the protocol, the proposed information sheet and informed 
consent form, the subject diaries and other documents as required, will be reviewed by a 
properly constituted IEC / IRB.  
 
A signed and dated statement stating that the documents have been approved by the 
IEC / IRB and regulatory authority will be obtained before initiation of the trial. 
 
The name of the IEC / IRB chairman and the members of the IEC / IRB will be collected as 
well as a statement that the IEC / IRB works in accordance with the principles of GCP. The 
IEC / IRB will receive updates on the trial progress according to local regulations. 
16.2 Subject Information and Informed Consent 
Prior to or no later than at start of the screening visit, i.e. before any trial related activity takes 
place, the Investigator or a qualified designee will explain to the potential subject the aims, 
methods, reasonably expected benefits and potential hazards of the trial and any discomfort 
participation in the trial may entail. Subjects will be informed that participation in the trial is 
voluntary and that the subject may withdraw from the trial at any time and for any reason. 
Subjects will be informed that if they choose not to participate, this will not affect the care the 
subject will receive for the treatment of his or her disease. Finally, subjects will be informed 
that their records may be accessed by regulatory authorities and authorized sponsor staff 
without violating the confidentiality of the subject, to the extent permitted by the applicable 
laws or regulations. 
 
The informed consent will also contain any additional required information with regards to 
the nature of the IMP, i.e. being an ATMP (ENTR/F/2/SF/dn D(2009) 35810. Brussels, 
03/12/2009). 
 
All explanations will be in layman’s language. The subject will be given sufficient time to 
consider the trial before deciding whether to participate or not. All subjects will be given 
opportunity to ask questions and will be given sufficient time to consider before consenting. 
The subjects may choose to be accompanied, e.g. by a family member, during the information 
process. 
 
After having consented, a copy of the information sheet and the signed informed consent 
form will be given to the subject. 
16.3 Subject Treatment Cards 
All subjects will receive a subject treatment card, which has been approved by the sponsor 
and the IEC / IRB, containing at a minimum: 
 The name of the subject 
 The Investigator’s contact number 
 Information regarding the IMP received 
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17. 
Quality Control and Quality Assurance 
17.1 Compliance with Good Clinical Practice 
This protocol is written to be in compliance with the guideline produced by the ICH on the 
topic GCP (ICH Guideline E6 (R1)), and with the related detailed guidelines on GCP specific 
to advanced therapy medicinal products, as well as with applicable regulatory requirements in 
the countries where the trial will take place. 
 
The overall responsible Investigator at each clinical trial site (i.e. the principal Investigator) 
will agree to conduct the trial in adherence to the instructions and procedures described in the 
protocol, to the Declaration of Helsinki, to the principles of GCP and to applicable regulatory 
requirements by signing the Principal Investigator’s Agreement page of this protocol. 
17.2 Monitoring, Audit and Inspection 
Monitoring visits to the clinical trial site will be made periodically in person or virtually 
during the trial to ensure adherence to the protocol, to the principles of GCP and to applicable 
regulatory requirements. 
 
The Investigator will ensure that the trial monitor is given direct access to source data (in 
paper and/or electronic format), enabling the trial monitor to perform data validation. 
 
The clinical trial site may be audited by the sponsor or inspected by regulatory agencies. The 
Investigator will make sure that he/she and his/her relevant clinical trial site personnel make 
themselves available during monitoring visits, audits and inspections. 
17.3 Source Data Verification 
Source data must be available to document the existence of the subject and to substantiate 
integrity of study data collected. Source data must include the original information related to 
the study (e.g. electrocardiogram traces, laboratory prints), to the medical treatment, and 
history of subject. Source data are contained in source documents (original records or 
certified copies). E-diary entries, 
 entries and medical/hospital records on bleeding 
episodes will be considered source data. 
 
All data entered in the eCRF should be verifiable in source documents in the subject’s 
medical record, work sheets, or in other records at the clinical trial site. 
For each clinical trial site, the location of the individual type of source data will be defined 
and documented. 
 
The trial monitor will check the eCRF for accuracy and completeness and perform Source 
Data Verification (SDV). The trial monitor will document SDV of all reviewed sections of 
the eCRF. 
 
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18. 
Data Handling and Record Retention 
18.1 Data Handling 
Data from clinical trial sites will be entered in an eCRF. 
 
The Investigator will sign relevant eCRF sections. Only an Investigator (i.e. medically 
qualified site personnel) can sign data on medical assessments. Any corrections made by the 
Investigator or authorized staff to the eCRF after original entry will be recorded in an audit 
trail. The person making the change and the date, time and reason for the change will be 
identified in the audit trail. Changes to data already approved/signed by an Investigator will 
require re-signature by the Investigator. The Investigator (principal Investigator or 
co-Investigator) will sign all subject data in the eCRF by an electronic signature. 
18.2 Data from Clinical Trial Sites 
In the trial an eCRF system will be used for data capture. The system is fully validated and 
access at all levels to the system is granted/revoked following vendor procedures, in 
accordance with regulatory requirements and system requirements (e.g. FDA 21CFR11). 
 
Trial data has to be entered into the eCRF system in a timely manner and no later than 5 
working days after the subject visit has taken place. 
 
 data will be collected on paper, and subsequent data entry into the eCRF system 
will be performed by local clinical trial personnel. 
 
Subject reported diary data will be collected in an electronic diary system, with the capability 
to upload this data to the eCRF system, to facilitate Investigator assessment. 
 
Local laboratory data of FIX activity, inhibitors and liver enzymes will be entered into the 
eCRF system by clinical trial personnel. Other laboratory data will be added to the trial 
database directly, prior to database lock.  
 
To aid in CSR reporting of missed visits due to COVID-19, the eCRFs will capture if a visit 
is missed and reason(s) why. 
 
After the trial database is declared clean and is released to the statistician, a final copy of the 
database, including metadata, closed queries and the audit trail report, will be stored at the 
sponsor. The Investigator will receive a copy of the clinical trial site’s final and locked data, 
including metadata, closed queries and the audit trail report, as write-protected pdf-files, 
directly from the database vendor, before their access to the eCRF system is revoked. 
18.3 Coding of Data 
Medical history, concomitant illnesses, signs and symptoms will be coded using MedDRA. 
 
Prophylactic and on-demand FIX replacement therapy and other concomitant treatment will 
be coded using WHO Drug Dictionary (WHO-DD). 
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18.4 Subject Confidentiality 
Subject data will be collected anonymously and the subjects will be identified only by subject 
ID number consisting of a 2-digit number for the clinical trial site followed by a consecutive 
2-digit number for the subject. 
18.5 Record Keeping 
The trial monitor will instruct the Investigator to maintain source documents and the signed 
informed consent form(s) for each subject. Furthermore, the trial monitor will instruct the 
Investigator to maintain essential documents during the trial and to archive essential 
documents for the duration defined in the ICH Guideline E6, or for 15 years, whichever 
comes first. 
 
The duration of archiving defined in the ICH Guideline E6 is as follows: Essential documents 
should be retained until at least 2 years after the last approval of a marketing application in an 
ICH region and until there are no pending or contemplated marketing applications in an ICH 
region or at least 2 years have elapsed since the formal discontinuation of clinical 
development of the investigational product. These documents should be retained for a longer 
period however if required by the applicable regulatory requirements or by an agreement with 
the sponsor. The sponsor will notify the Investigator when retention of the trial-related 
records is no longer required. 
 
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19. 
Changes to Trial Conduct 
19.1 Protocol Amendments 
Prospective protocol waivers are prohibited. Any changes to the protocol will be described in 
a protocol amendment. Any protocol amendment will be agreed upon (signed) by the 
principal Investigator and the sponsor prior to its implementation. In addition to Investigator 
and sponsor signature, substantial amendments will be submitted to the IECs / IRBs and 
regulatory authorities for approval, prior to their implementation. 
 
The Investigator may implement a deviation from, or a change of, the protocol to eliminate 
an immediate hazard(s) to trial subjects without prior IEC / IRB approval/favourable opinion. 
As soon as possible, the implemented deviation or change, the reasons for it, and, if 
appropriate, a protocol amendment(s) should be submitted to the IEC / IRB for review and 
approval/favourable opinion and, if required, to the regulatory authority(ies). 
 
The sponsor will decide if an amendment is to be considered substantial or non-substantial, in 
adherence with “Detailed guidance for the request for authorization of a clinical trial on a 
medicinal product for human use to the competent authorities, notification of substantial 
amendments and declaration of the end of the trial” (CT1, 2010/C 82/01, issued by the 
European Commission, 2010). 
19.2 Premature Termination of the Trial or Dosing Temporarily on Hold 
The data monitoring committee will perform review of available safety data prior to dosing of 
subjects within each cohort as well as between cohorts (see Section 15). This review may 
reveal safety issues requiring further investigation prior to dosing of the next subject(s) or 
safety issues requiring the trial being terminated prematurely (see Trial Stopping Rules in 
Section 19.2.1). 
 
The trial may be terminated prematurely by the sponsor. The reasons may be: 
- Safety alert or safety issue that prevents further dosing (see Trial Stopping Rules in 
Section 19.2.1) 
- Recruitment not feasible 
- A decision to discontinue development of the IMP. 
 
If the trial is terminated prematurely, the sponsor will notify the IECs/IRBs and regulatory 
agencies immediately (in writing), including the reason for early termination. 
 
A clinical trial site may be terminated prematurely by the sponsor. The reasons may be:  
- Insufficient compliance with trial procedures 
- Failure to recruit subjects at an acceptable rate. 
 
 Trial Stopping Rules 
As the trial is a one-dose trial, stopping rules are relevant only for the recruitment period. 
Independently of any changes to the recruitment, subjects who have already been dosed will 
be followed as per protocol to the end of trial. 
 
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The data monitoring committee will prior to dosing review data as detailed below and 
recommend whether: 
- The next subject could be dosed 
- Further data would be required before the next dosing 
- No further dosing should take place i.e. subject recruitment is discontinued.  
 
The following events may lead to a decision to pause dosing, i.e. stop subject recruitment to 
allow for evaluation and assessment of the implications of the event for further subject 
dosing: 
1. A more than 5-fold increase in ALT, AST or both in one or more subjects after IMP 
administration which is not manageable by steroid rescue treatment implemented 
according to American Association for the Study of Liver Diseases (AASLD) 
guidelines. (Manns et al. 2010) 
2. An SAE judged as probably or possibly related to the IMP and which pose either an 
immediate risks to subject’s health or is likely to adversely affect the subject’s health 
long term. This includes events classified as AEs qualifying for special notification 
(Brussels, 03/12/2009,ENTR/F/2/SF/dn D(2009) 35810; 
EMEA/CHMP/GTWP/60436/2007) as listed in Section 13.6, if these are judged as 
probably or possibly related to the IMP. The development of an inhibitor towards FIX 
in one subject is not a cause for stop of subject recruitment, however the development 
of an FIX inhibitor in more than one subject should lead to pausing of dosing and 
further investigations of causal relationship 
3. Death of a subject, after having received the IMP, that is judged as related to the IMP 
4. The occurrence of a malignancy at any point after gene transfer that is judged as 
probably or possibly related to the IMP.  
 
Decision to start Cohort 2 
If none of the events listed above have occurred the data monitoring committee may 
recommend that Cohort 2 can be initiated. If one or more of these events have occurred, 
dosing will be paused while the event(s) is/are further investigated including the implications 
for further dosing and dose level. 
 
Decision to dose the next subject within a cohort 
In addition to the above criteria (and as detailed in Section 10.1), the data monitoring 
committee will evaluate the available safety data collected during a period of minimum 
24 hours after IMP administration to the 1st subject and recommend if and when dosing of the 
2nd subject can be initiated. Similarly, after the 2nd subject has been dosed in a cohort, the data 
monitoring committee will evaluate available safety data collected during a period of 
minimum 24 hours after IMP administration to this second subject and recommend if and 
when dosing of the subsequent subjects can be initiated. The dosing of the 3rd, 4th and 5th and 
possibly 6th and 7th subject within a cohort will be separated by a minimum of 24 hours (see 
Section 10.1) to allow evaluation of any acute, unexpected AEs considered related to the IMP 
as well as any clinically relevant changes in CRP. 
 
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20. 
Reporting of Results 
20.1 Integrated Clinical Study Report 
An interim analysis will be performed after all enrolled subjects have completed at least 
52 weeks of follow-up after IMP administration (i.e. completed Visit 23). A database freeze 
will be performed. Collected efficacy and safety data will be analysed and reported in an 
interim CSR. The data will be considered locked at that point and will not be changed. 
 
After all subjects have completed the 5-year visit (i.e. Visit 35), all collected safety and 
efficacy data will be locked, analysed, and reported in a full (5 year) CSR.  
 
The full (5 year) CSR will also summarize contingency measures implemented to manage 
study conduct due to COVID-19 control measures, protocol deviations due to COVID-19, 
and the impact COVID-19 had on visit schedules, missed visits, and missing information. 
 
The sponsor will notify the accredited IEC/IRBs and the regulatory authority of the end of the 
trial within a period of 90 days. The end of the trial is defined as the last subject’s last visit.  
 
In case the trial is ended prematurely, the sponsor will notify the IEC/IRB and the regulatory 
authority within 15 days, including the reasons for the premature termination. Within one 
year after the end of the trial, the Investigator/sponsor will submit the interim CSR and full 
(5 year) CSR with the results of the trial, including any publications/abstracts of the trial, to 
the accredited IEC/IRB and the regulatory authority. 
20.2 Public Disclosure and Publication Policy 
Basic information of this trial will be posted by the sponsor on the website 
www.clinicaltrials.gov before the first subject enters the trial. 
 
The sponsor is committed to secure that the results of the trial will be published and presented 
at (scientific) symposia and/or congresses, whether positive or negative and the Investigators 
will have free access to review and publish the data generated from the trial. The criteria for 
authorship as set out by the Committee of Medical Journal Editors (www.icmje.org) will be 
applied. 
 
The contributorship model will be applied and contributors who do not meet the criteria for 
authorship will be listed in an acknowledgments section with descriptions of the role of each 
contributor in order to ensure indexation in the National Library of Medicine. 
 
Publications are subject to the following conditions: 
- All information concerning the product and the sponsor’s operations (such as patent 
applications, formulae, manufacturing processes, basic scientific data or formulation 
information supplied to the Investigator by the sponsor and not previously published) 
is considered confidential by the sponsor and cannot be disclosed by the Investigator 
to any third party without the sponsor’s prior written approval. The Investigator 
agrees to use this information only in accomplishing the trial and will not use it for 
other purposes without the written approval of the sponsor. 
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- It is understood by the Investigator that the sponsor will use the information 
developed in this trial in connection with the development of the compound and 
therefore, may disclose it as required to other clinical Investigators, the licensing 
authority, or to regulatory agencies of other governments. In order to allow for the use 
of the information derived from this trial, the Investigator understands that he/she has 
an obligation to provide and disclose test results and all data developed during this 
trial to the sponsor. 
- Any data from the trial are the property of the sponsor and cannot be published 
without prior authorization from the sponsor. 
- Publications must be drafted with protection of individual privacy, intellectual 
property and contract rights in mind, and also conform to legislation and current 
national practices in patent and other laws. 
- Publications may not disclose any sponsor confidential information or property 
without prior written consent. Therefore, any final version of a manuscript based on 
data derived from this protocol must be submitted to the sponsor prior to submission 
to the publisher and provide the sponsor with at least 45 days of full review of the 
manuscript. This is to secure that the sponsor can submit relevant intellectual property 
information prior to public disclosure of intellectual property right sensitive 
information.  
- The primary publication should be published before, or in parallel with, any 
secondary publications.  
 
The sponsor will organize the publication process to ensure  
- Publication of the results of the trial (primary and secondary publications) in a timely 
and responsible manner. 
- That any publication is written according to appropriate guidelines (Wager et al., 
2003; Graf et al., 2009). 
 
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21. 
Financing and Insurance 
The financial aspects of the trial will be addressed in a separate agreement between the 
sponsor and the Investigator/clinical trial site. 
 
The sponsor will cover this trial by means of an adequate insurance of the subject, which will 
be in place prior to the start of the trial. As evidence of this insurance, a copy of the insurance 
statement will be provided to the Investigator for storage in the Investigator site file. 
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22. 
Administrative Aspects 
22.1 Investigators 
One principal Investigator and one or more sub-Investigators will be appointed for each 
clinical trial site. Name and title of the Investigator(s) who is (are) responsible for conducting 
the trial, and the address and telephone number(s) of the trial site will be contained in other 
documents such as the Trial Procedures Manual and Clinical Trial Application forms. 
 
One National Coordinating Investigator (NCI) will be appointed for each participating 
country. The NCI will be responsible for national issues relating to the trial. 
 
 International Coordinating Investigator 
The International Coordinating Investigator is responsible for approval of the protocol and for 
approval of the CSR on behalf of all trial Investigators. 
22.2 Clinical Trial Sites 
Since this is a first in-human trial, the clinical trial sites should be appropriate clinical 
facilities and the trial should be conducted by trained Investigators who have acquired the 
necessary expertise and experience in conducting early phase trials and medical staff with 
appropriate level of training and previous experience of first in-human trials. They should 
also understand the IMP, its target and mechanism of action. 
 
Clinical trial sites should also have immediate access to equipment and staff for resuscitating 
and stabilising individuals in an acute emergency (such as cardiac emergencies, anaphylaxis, 
cytokine release syndrome, convulsions, hypotension), and ready availability of Intensive 
Care Unit facilities. Procedures should be established between the clinical trial site and its 
nearby Intensive Care Unit regarding the responsibilities and undertakings of each in the 
transfer and care of subjects. 
22.3 Vendors 
The sponsor will engage vendors to perform the following services: 
- Laboratory analysis 
o Individual central laboratories will be applied for specific laboratory analyses. 
o One coordinating central laboratory will prepare the Laboratory Manual, 
sampling kits, perform training of clinical trial site personnel, arrange for 
courier shipments, manage central preparation and storage of samples. 
- Clinical monitoring 
- Pharmacovigilance 
- Data management 
- Statistics 
- The provision of the 
 
- The provision of the e-diaries 
 
Details on the vendors will be contained in other document(s) such as the Trial Procedures 
Manual and Clinical Trial Application forms. 
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23. 
References 
23.1 Publications 
Bolton-Maggs P, Pasi K (2003). Haemophilias A and B. Lancet 361:1801-1809. 
Boutin S, Monteilhet V, Veron P, et al. (2010). Prevalence of serum IgG and neutralizing 
factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy 
population: implications for gene therapy using AAV vectors. Hum Gene Ther 21:704-712. 
Bowyer A, Smith J, Woolley A, Kitchen S, Hampton K, Maclean R, Van Veen J, Makris M 
(2011). The investigation of a prolonged APTT with specific clotting factor assays is 
unnecessary if an APTT with Actin FS is normal. Int J Lab Hematol 33:212-218. 
Calcedo R, Vandenberghe L, Gao G, Lin J, Wilson J (2009). Worldwide Epidemiology of 
Neutralizing Antibodies to Adeno-Associated Viruses. J Infect Dis 199:381-90. 
Cancio M, Reiss U, Nathwani A, Davidoff A, Gray J (2013). Developments in the treatment 
of hemophilia B: focus on emerging gene therapy. Appl Clin Genet 18:91-101.  
Chitlur M, Warrier I, Rajpurkar M, Lusher J (2009). Inhibitors in factor IX deficiency a 
report of the ISTH-SSC international FIX inhibitor registry (1997-2006). 
Haemophilia 15:1027-1031. 
Cooper M, Nayak S, Hoffman B, Terhorst C, Cao O, Herzog R (2009). Improved induction 
of immune tolerance to factor IX by hepatic AAV-8 gene transfer. 
Hum Gene Ther 20:767-776. 
Darby S, Kan S, Spooner R, Giangrande P, Hill F, Hay C, Lee C, Ludlam C, Williams M 
(2007). Mortality rates, life expectancy, and causes of death in people with hemophilia A or 
B in the United Kingdom who were not infected with HIV. Blood 110:815-825. 
DiMichele D (2007). Inhibitor development in haemophilia B: an orphan disease in need of 
attention. Br J Haematol 138:305-315. 
Fischer K, Steen Carlsson K, Petrini P, et al. (2013). Intermediate-dose versus high-dose 
prophylaxis for severe hemophilia: comparing outcome and costs since the 1970s. Blood 
122(7):1129-1136. 
Furie B, Furie B (1992). Molecular and cellular biology of blood coagulation. N Engl J Med 
326:800–806.  
Gater A, Thomson T, Strandberg-Larsen M (2011). Haemophilia B: impact on patients and 
economic burden of disease. Thromb Haemost 1:1349-1355. 
Geraghty S, Dunkley T, Harrington C, Lindvall K, Maahs J, Sek J. (2006). Practice patterns 
in haemophilia A therapy - global progress towards optimal care. Haemophilia 12:75-81. 
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Graf C, Battisti W, Bridges D, Bruce-Winkler V, Conaty J, Ellison J, et al. (2009). Good 
publication practice for communicating company sponsored medical research: the GPP2 
guidelines. BMJ 339:b4330. 
Kessler C, Mariani G (2006). Clinical manifestations and therapy of the haemophilias. 
In: Colman R, Hirsh J, Marder V, Clowes A, George J, eds. Hemostasis and Thrombosis: 
Basic Principles and Clinical Practice. 5 ed. Philadelphia: Lippincott-Raven, 887-904. 
Li C, Narkbunnam N, Samulski R, et al. (2012). Neutralizing antibodies against 
adeno-associated virus examined prospectively in pediatric patients with hemophilia. Gene 
Therapy 19(3):288-294 
Manco-Johnson MJ, Abshire T, Shapiro A, et al. (2007). Prophylaxis versus Episodic 
Treatment to Prevent Joint Disease in Boys with Severe Hemophilia. N Engl J Med 
357(6):535-544. 
Mann K (1999). Biochemistry and physiology of blood coagulation. Thromb Haemost 
82:165–174. 
Manno C, Chew A, Hutchison S, et al. (2003). AAV-mediated factor IX gene transfer to 
skeletal muscle in patients with severe haemophilia B. Blood 101:2963-2972. 
Manno C, Pierce G, Arruda V, et al. (2006). Successful transduction of liver in haemophilia 
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Nathwani A, Tuddenham E, Rangarajan S, et al. (2011). Adenovirus-associated virus 
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gene therapy in haemophilia B. N Engl J Med 371:1994-2004. 
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focused on safety, efficacy and selected patient populations: A report from the Clinical Trial 
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Design Task Force of the National Cancer Institute Investigational Drug Steering Committee. 
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Powell J, Ragni M, White G 2nd, et al. (2003). Phase 1 trial of FVIII gene transfer for severe 
hemophilia A using a retroviral construct administered by peripheral intravenous infusion. 
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Study Group. Nonviral transfer of the gene encoding coagulation factor VIII in patients with 
severe hemophilia A. N Engl J Med 344:1735-1742. 
Schramm W, Berger K (2003). Economics of prophylactic treatment. 
Haemophilia 9(suppl s1):111-116  
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reported factor IX use around the world. Haemophilia 17:446-455. 
Valentino L (2014). The role of Rixubis™ in the treatment of hemophilia B. Immunotherapy 
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Vandenberghe L, Wang L, Somanathan S, et al. (2006). Heparin binding directs activation of 
T cells against adeno-associated virus serotype 2 capsid. Nat Med 12:967-971. 
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Warrier I (1999). Factor IX antibody and immune tolerance. Vox Sang 77 Suppl 1:70-71. 
White G, Monahan P (2005). Gene therapy for hemophilia A. In Lee, C., Berntrop, E. and 
Hoots, K. (eds) Textbook of Hemophilia, Blackwell Publishing, Oxford, UK, pp. 226–228. 
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global survey 2017. dated 2018 October [cited 2018 November 23]. 
http://www1.wfh.org/publications/files/pdf-1714.pdf  
 
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23.2 Guidelines 
Committee for Medicinal Products for Human Use (CHMP). Guideline on Follow-up of 
Patients Administered with Gene Therapy Medicinal Products. 
EMEA/CHMP/GTWP/60436/2007. London, 22 October 2009. 
Detailed guidelines on good clinical practice specific to advanced therapy medicinal 
products. ENTR/F/2/SF/dn D(2009) 35810. Brussels, 03/12/2009. 
European Commission. Detailed guidance for the request for authorisation of a clinical trial 
on a medicinal product for human use to the competent authorities, notification of substantial 
amendments and declaration of the end of the trial (CT1), 2010/C 82/01. 
ICH Guideline E2A: Clinical Safety Data Management: Definitions and Standards for 
Expedited Reporting. Finalised under Step 4 in October 1994. 
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E2A/Step
4/E2A_Guideline.pdf. 
ICH Guideline E3: Structure and Content of Clinical Study Reports. Finalised under Step 4 in 
November 1995. 
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E3/E3_G
uideline.pdf. 
ICH Guideline E6(R2): Integrated Addendum to ICH E6(R1): Guideline for Good Clinical 
Practice. Finalised under Step 4 in November 2016. 
https://database.ich.org/sites/default/files/E6_R2_Addendum.pdf. 
23.3 Other 
Investigator’s Brochure AAV5-hFIX and AAV5-hFIXco-Padua (AMT-060 and AMT-061 
[Etranacogene dezaparvovec) Version 2.0; February 2021. 
Summary of Product Characteristics BeneFIX, 2020 October [cited 2021 Jan 11]. Available 
from: http://www.medicines.org.uk/emc/medicine/20376/SPC/benefix. 
Prescribing Information RIXUBIS, June 2020 June [cited 2021 Jan 11]. Available from: 
https://www.shirecontent.com/PI/PDFs/RIXUBIS_USA_ENG.pdf. 
World Federation of Haemophilia. http://www.wfh.org/.  
 
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