Clinical trial NCT01852071
Conditions: ADA-SCID
Keywords: gene therapy hematopoietic stem cell ADA-SCID lentiviral vector
Sponsor: University of California, Los Angeles
Start date: 2013-08-02; Completion date: 2018-08-27
Enrollment: 46 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The aim of this study is to assess the safety and efficacy of autologous transplantation of hematopoietic stem cells (CD34+ cells) from the bone marrow (BM) of ADA-deficient SCID infants and children following human ADA cDNA transfer by the EFS-ADA lentiviral vector. The level of gene transfer in blood cells and immune function will be measured as endpoints.

Detailed description:
The study is open to twenty (20) infants and children diagnosed with ADA-deficient SCID who did not have a medically eligible, human leukocyte antigen (HLA)-identical sibling donor for bone marrow transplantation. The EFS-ADA lentiviral vector with the human ADA cDNA will be used to transduce autologous CD34+ cells from the bone marrow of these subjects. The subjects will receive 4 mg/kg busulfan prior to re-infusion of their gene-modified cells. Safety is the primary endpoint. During the follow-up phase, the investigators aim to determine whether the cells could engraft and produce mature cells that contain and express the corrected ADA gene in the absence of pegademase bovine (PEG-ADA) enzyme replacement therapy (ERT), which will be withheld at Day +30 following transplant. Efficacy studies to evaluate the level of immune reconstitution, will be performed in the first and second years of the study.

Arms: arm 1: Infusion of autologous EFS-ADA Lentiviral (LV) CD34+ cells
Interventions: intervention 1: Infusion of autologous EFS-ADA LV CD34+ (OTL-101) intervention 2: busulfan intervention 3: PEG-ADA ERT
Intervention descriptions: intervention 1: autologous EFS-ADA LV CD34+ cells (OTL-101) are infused intravenously intervention 2: Busulfan is used for non-myeloablative conditioning intervention 3: PEG-ADA ERT is discontinued at Day +30 (-3/+15 days) after successful engraftment

Protocol documents:
Prot_000.pdf: 
COVER PAGE
Official Study Title: Autologous Transplantation of 
Bone Marrow CD34+ Stem/Progenitor Cells After 
Addition of a Normal Human ADA Complementary 
DNA (cDNA) by the EFS-ADA Lentiviral Vector for 
Severe Combined Immunodeficiency Due to 
Adenosine Deaminase Deficiency (ADA-SCID)
EFS-ADA Protocol v12.0. dated 28-Mar-2018
NCT01852071


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Autologous Transplantation of Bone Marrow CD34+ Stem/Progenitor Cells 
after Addition of a Normal Human ADA cDNA by the EFS-ADA Lentiviral 
Vector for Adenosine Deaminase (ADA)-Deficient Severe Combined 
Immunodeficiency (SCID) 
IND Sponsor: Orchard Therapeutics, Ltd. 
BB IND # 15440 
Other Identifying Numbers: NIH OBA RAC #0910-1006 
UCLA IRB # 11-002303  
Version Number: 12.0 –post NIH RAC, FDA IND, and NHLBI DSMB reviews 
Date:  28 March 2018 
Confidentiality Statement 
This document is confidential and is to be distributed for review only to investigators, potential investigators, 
consultants, study staff, and applicable independent ethics committees or institutional review boards.  The 
contents of this document shall not be disclosed to others without written authorization from NIAID (or 
others, as applicable), unless it is necessary to obtain informed consent from potential study participants. 


 
 
 
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Statement of Compliance: 
 
(i) Name of IND Drug: Autologous bone marrow CD34+ cells transduced with the 
lentiviral vector, EFS-ADA (OTL-101).  
(ii) This is a Phase I/II Study.  
(iii) This clinical investigation will not begin until an IND is in effect.  
(iv) The Institutional Review Board (IRB) of the Office for Human Research 
Protection Program (OHRPP) at the University of California, Los Angeles (UCLA) 
will be responsible for the initial and continuing review and approval of this study. 
Any proposed changes will be reported to the IRB.  
(v) The investigation will be conducted in accordance with all other applicable 
regulatory requirements.  
(vi) The CLINICAL TRIAL Company, Ltd. (TCTC) will be responsible for monitoring 
the conduct and progress of the clinical investigation.  
(vii) Orchard Therapeutics, Ltd. will be responsible under 312.32 for review and 
evaluation of information relevant to the safety of the drug.  
 
     The signature below constitutes the approval of this protocol and the attachments, and 
provides the necessary assurances that this trial will be conducted according to all stipulations of 
the protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable U.S. federal regulations and ICH guidelines. 


 
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Table of Contents 
 
LIST OF ABBREVIATIONS ............................................................................................................................ VIII 
1 
OVERVIEW ..................................................................................................................................................... 11 
1.1 
PROTOCOL SUMMARY ............................................................................................................................... 11 
1.2 
SCHEMATIC OF STUDY DESIGN: ................................................................................................................ 14 
1.3 
KEY ROLES ................................................................................................................................................ 16 
1.3.1 
Performance sites: ........................................................................................................................... 16 
 
 
2 
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ....................................................... 19 
2.1 
BACKGROUND INFORMATION ..................................................................................................................... 19 
2.1.1 
Severe combined immunodeficiency (SCID) ............................................................................. 19 
2.1.2 
Adenosine deaminase (ADA)-deficient SCID ............................................................................. 19 
2.1.3 
Treatment Options for ADA-Deficient SCID ............................................................................... 20 
2.1.3.1 
Allogeneic Bone Marrow Transplantation (BMT) ............................................................................. 20 
2.1.3.2 
ADA Enzyme Replacement Therapy ................................................................................................... 23 
2.1.4 
Gene Therapy for ADA-Deficient SCID ........................................................................................ 24 
2.1.4.1 
ADA Gene Therapy Using T Lymphocytes ........................................................................................ 24 
2.1.4.2 
Gene Therapy for ADA-Deficient SCID using Hematopoietic Stem Cells .................................. 25 
2.1.5 
Summary of Previous Clinical Studies with -Retroviral Vectors by Investigators ......... 27 
2.1.5.1 
CHLA/NIH trial of gene transfer into umbilical cord blood CD34+ cells from ADA-deficient 
SCID infants (1993) ..................................................................................................................................................... 27 
2.1.5.2 
Phase I trial of gene transfer to CD34+ cells from the bone marrow CD34+ cells from ADA-
deficient SCID infants and children (2001-2009) ................................................................................................. 28 
2.1.5.3 
Phase II trial of gene transfer to CD34+ cells from the bone marrow CD34+ cells from ADA-
deficient SCID infants and children (2009-2014) ................................................................................................. 33 
2.1.6 
Summary of Relevant Clinical Studies ........................................................................................ 35 
2.1.6.1 
Complications from Vector Integration with -Retroviral Vectors ............................................... 35 
2.1.6.2 
Lentiviral Vectors May be More Effective and Safer ....................................................................... 36 
2.1.7 
Description of the Study Agent(s)/Intervention(s) ................................................................... 38 
2.1.8 
Summary of Pre-Clinical Studies with Study Agent/Intervention......................................... 39 
2.1.8.1 
Evaluation of ADA Lentiviral Vectors ................................................................................................. 39 
2.1.8.2 
Transduction of Human CD34+ cells with EFS-ADA ....................................................................... 40 
2.1.8.3 
In vitro immortalization (IVIM) assay ................................................................................................... 44 
2.2 
RATIONALE ................................................................................................................................................. 45 
2.3 
POTENTIAL RISKS AND BENEFITS .............................................................................................................. 46 
2.3.1 
Potential Risks ................................................................................................................................... 46 
2.3.2 
Potential Benefits .............................................................................................................................. 50 
3 
STUDY OBJECTIVES ................................................................................................................................... 50 
3.1 
PRIMARY OBJECTIVES ............................................................................................................................... 50 
3.2 
SECONDARY OBJECTIVES .......................................................................................................................... 50 
4 
STUDY DESIGN ............................................................................................................................................. 51 
4.1 
DESCRIPTION OF THE STUDY DESIGN ....................................................................................................... 51 
4.2 
STUDY ENDPOINTS .................................................................................................................................... 51 
4.2.1 
Primary Endpoints ............................................................................................................................ 51 
4.2.1.1 
Evaluation of Safety ................................................................................................................................ 51 
4.2.1.2 
Evaluation of Efficacy ............................................................................................................................. 52 
4.2.2 
Secondary Endpoints ...................................................................................................................... 52 
5 
STUDY POPULATION .................................................................................................................................. 54 
5.1 
DESCRIPTION OF THE STUDY POPULATION ............................................................................................... 54 


 
 
 
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5.1.1 
Participant Inclusion Criteria ......................................................................................................... 54 
5.1.2 
Participant Exclusion Criteria ........................................................................................................ 55 
5.1.2.1 
Co-enrollment Guidelines ...................................................................................................................... 57 
5.2 
STRATEGIES FOR RECRUITMENT AND RETENTION ................................................................................... 57 
6 
STUDY AGENT/INTERVENTIONS ............................................................................................................. 57 
6.1 
STUDY AGENT ACQUISITION ...................................................................................................................... 58 
6.1.1 
Study Agent/Intervention ................................................................................................................ 62 
6.1.1.1 
Formulation, Packaging, and Labeling ............................................................................................... 62 
6.1.1.2 
Preparation, Administration, Storage, and Dosage of Study Agent(s)/Intervention(s) .......... 63 
6.1.1.3 
Study Agent Accountability Procedures ............................................................................................ 64 
6.2 
ASSESSMENT OF PARTICIPANT COMPLIANCE WITH STUDY AGENT(S)/INTERVENTION(S) ....................... 64 
6.3 
CONCOMITANT MEDICATIONS AND PROCEDURES .................................................................................... 64 
6.4 
PRECAUTIONARY AND PROHIBITED MEDICATIONS AND PROCEDURES .................................................... 64 
6.4.1 
Prohibited Medications and Procedures .................................................................................... 64 
6.4.2 
Precautionary Medications and Procedures .............................................................................. 65 
6.5 
PROPHYLACTIC MEDICATIONS AND PROCEDURES ................................................................................... 65 
6.6 
RESCUE MEDICATIONS .............................................................................................................................. 65 
7 
STUDY PROCEDURES/EVALUATIONS................................................................................................... 66 
7.1 
OVERALL STUDY PLAN ........................................................................................................................ 66 
7.2 
PRE-TREATMENT PHASE: ........................................................................................................................... 66 
7.3 
TREATMENT PHASE: .................................................................................................................................. 67 
7.4 
POST-TREATMENT PHASE: ........................................................................................................................ 67 
7.5 
EXTENDED FOLLOW-UP PHASE: ................................................................................................................ 69 
7.6 
CLINICAL AND LABORATORY EVALUATIONS .............................................................................................. 69 
7.6.1 
Clinical and Research Laboratory Evaluations and Specimen Collection ........................ 69 
8 
STUDY SCHEDULE ...................................................................................................................................... 72 
8.1 
SCREENING ................................................................................................................................................ 72 
8.2 
ENROLLMENT/BASELINE ............................................................................................................................ 73 
8.3 
TREATMENT ................................................................................................................................................ 74 
8.4 
FOLLOW-UP ................................................................................................................................................ 75 
8.5 
FINAL STUDY VISIT .................................................................................................................................... 76 
8.6 
EARLY TERMINATION VISIT ........................................................................................................................ 76 
8.7 
PREGNANCY VISIT ..................................................................................................................................... 77 
8.8 
UNSCHEDULED VISITS ............................................................................................................................... 77 
9 
ASSESSMENT OF SAFETY ........................................................................................................................ 77 
9.1 
SPECIFICATION OF SAFETY PARAMETERS ................................................................................................ 77 
9.2 
DEFINITION OF AN ADVERSE EVENT (AE) ................................................................................................. 77 
9.3 
DEFINITION OF A SERIOUS ADVERSE EVENT (SAE) ................................................................................. 77 
9.4 
METHODS AND TIMING FOR ASSESSING, RECORDING, AND ANALYZING, MANAGING SAFETY 
PARAMETERS ............................................................................................................................................. 78 
9.4.1 
Methods and Timing for Assessment .......................................................................................... 78 
9.4.2 
Recording/Documentation.............................................................................................................. 79 
9.4.3 
Analysis/Management ..................................................................................................................... 80 
9.5 
REPORTING PROCEDURES ........................................................................................................................ 80 
9.5.1 
General Details for Reporting Procedures ................................................................................. 80 
9.5.2 
Specific Serious Adverse Event Requirements ........................................................................ 81 
9.5.2.1 
PI to Sponsor ............................................................................................................................................ 81 
9.5.2.2 
Sponsor to FDA ........................................................................................................................................ 81 
9.5.2.3 
UCLA to DSMB .......................................................................................................................................... 81 


 
 
 
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9.5.2.4 
UCLA to RAC ............................................................................................................................................. 81 
9.6 
REPORTING PREGNANCY........................................................................................................................... 82 
9.7 
TYPE AND DURATION OF THE FOLLOW-UP OF PARTICIPANTS AFTER ADVERSE EVENTS ........................ 82 
9.8 
MODIFICATION OF STUDY AGENT(S)/INTERVENTION(S) FOR A PARTICIPANT .......................................... 82 
9.8.1 
Dose / Schedule Modifications for a Participant ...................................................................... 82 
9.9 
HALTING RULES FOR THE PROTOCOL ....................................................................................................... 82 
9.10 
STOPPING RULES FOR AN INDIVIDUAL PARTICIPANT/COHORT ................................................................. 83 
9.11 
PREMATURE WITHDRAWAL OF A PARTICIPANT ......................................................................................... 83 
9.12 
REPLACEMENT OF A PARTICIPANT WHO DISCONTINUES STUDY TREATMENT ......................................... 84 
10 CLINICAL MONITORING STRUCTURE .................................................................................................... 84 
10.1 
SITE MONITORING PLAN ............................................................................................................................ 84 
10.2 
SAFETY MONITORING PLAN ....................................................................................................................... 84 
10.2.1 Safety Review Plan by the DSMB / SMC ..................................................................................... 84 
11 STATISTICAL CONSIDERATIONS ............................................................................................................ 84 
11.1 
BACKGROUND ............................................................................................................................................ 84 
11.2 
DESIGN CONSIDERATIONS AND DATA ....................................................................................................... 85 
11.3 
ENDPOINTS ................................................................................................................................................ 86 
11.3.1 Safety ................................................................................................................................................... 86 
11.3.2 Primary Efficacy ................................................................................................................................ 86 
11.3.3 Secondary Efficacy........................................................................................................................... 86 
11.4 
SAMPLE SIZE CONSIDERATIONS ................................................................................................................ 86 
11.5 
ANALYSIS SETS .......................................................................................................................................... 87 
11.5.1 Safety Analysis Set........................................................................................................................... 87 
11.5.2 Full Analysis Set ............................................................................................................................... 87 
11.6 
STATISTICAL METHODS ............................................................................................................................. 87 
11.6.1 Overview ............................................................................................................................................. 87 
11.6.2 Interim Look ....................................................................................................................................... 88 
11.6.2.1 
Subject Disposition ................................................................................................................................. 88 
11.6.2.2 
Baseline Characteristics ........................................................................................................................ 88 
11.6.2.3 
Safety Analyses ........................................................................................................................................ 88 
11.6.2.4 
Efficacy Analyses ..................................................................................................................................... 88 
11.6.3 Primary Analysis .................................................................................................................................... 89 
11.6.4 End of study Analysis ............................................................................................................................ 89 
11.7 
SAFETY REVIEW ......................................................................................................................................... 89 
11.7.1 Stopping Rules for the Protocol ................................................................................................... 89 
11.7.2 Stopping Rules for an Individual Participant/Cohort ............................................................... 90 
11.7.3 Immunogenicity or Efficacy Review............................................................................................. 90 
12 QUALITY CONTROL AND QUALITY ASSURANCE .............................................................................. 91 
13 ETHICS/PROTECTION OF HUMAN SUBJECTS .................................................................................... 91 
13.1 
INSTITUTIONAL REVIEW BOARD/ETHICS COMMITTEE ............................................................................... 91 
13.2 
INFORMED CONSENT PROCESS ................................................................................................................ 91 
13.2.1 Assent or Informed Consent Process (in Case of a Minor) ................................................... 92 
13.3 
EXCLUSION OF WOMEN, MINORITIES, AND CHILDREN (SPECIAL POPULATIONS) .................................... 92 
WOMEN, MINORITIES AND CHILDREN WILL BE INCLUDED. ....................................................................................... 92 
13.4 
PARTICIPANT CONFIDENTIALITY ................................................................................................................ 92 
13.5 
STUDY DISCONTINUATION ......................................................................................................................... 92 
14 DATA HANDLING AND RECORD KEEPING ........................................................................................... 93 
14.1 
DATA MANAGEMENT RESPONSIBILITIES .................................................................................................... 93 
14.2 
DATA CAPTURE METHODS ........................................................................................................................ 94 


 
 
 
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14.3 
TYPES OF DATA ......................................................................................................................................... 95 
14.4 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS ...................................................... 95 
14.5 
TIMING/REPORTS ....................................................................................................................................... 95 
14.6 
STUDY RECORDS RETENTION ................................................................................................................... 96 
14.7 
PROTOCOL DEVIATIONS ............................................................................................................................ 96 
15 PUBLICATION POLICY ................................................................................................................................ 96 
16 SCIENTIFIC REFERENCES ........................................................................................................................ 97 
17 APPENDICES ............................................................................................................................................... 108 
 
 
 
 
17.2 
APPENDIX B: SCHEDULE OF PROCEDURES/EVALUATIONS ..................................................................... 117 
17.3 
APPENDIX C:  EFS-ADA TRIAL BUSULFAN PHARMACOKINETIC ANALYSIS ........................................... 127 
17.4 
APPENDIX D:  SOP FOR HEMATOPOIETIC STEM CELL PRODUCT AND PATIENT SPECIMEN TRANSPORT
 ................................................................................................................................................................. 128 
17.5 
APPENDIX E:  SOP FOR TRANSDUCTION ................................................................................................ 133 
17.6 
APPENDIX F:  MONITORING FOR MONOCLONAL EXPANSION OR LEUKOPROLIFERATION DUE TO 
INSERTIONAL ONCOGENESIS ................................................................................................................... 134 
17.7 
APPENDIX G:  TOXICITY TABLE FOR GRADING SEVERITY OF ADVERSE EXPERIENCES (>3 MONTHS OF 
AGE)......................................................................................................................................................... 138 
17.8 
APPENDIX H:  DETAILS OF HISTORICAL HSCT CONTROL GROUPS......................................................... 139 
17.9 
APPENDIX I:  LIST OF PROTOCOL CHANGES ........................................................................................... 142 
 
 


Lenitviral Vector ADA Gene Transfer for ADA-Deficient SCID (Protocol ver. 12.0, dated 28 March 2018) 
 
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List of Abbreviations 
This list should be modified to include protocol-specific terms. 
 
ADA 
Adenosine Deaminase 
ADA-SCID 
ADA Deficient SCID 
AE 
Adverse Event/Adverse Experience 
ANC 
Absolute Neutrophil Count 
BMT 
Bone Marrow Transplant 
CBC 
Complete Blood Count 
cDNA 
Complementary Deoxyribonucleic Acid 
CD 
Cluster of Differentiation 
CI 
Clinical Investigator 
CID 
Combined Immunodeficiency 
CRF 
Case Report Form 
dAXP 
Deoxyadenosine 
DCC 
Data Coordinating Center 
DSMB 
Data and Safety Monitoring Board 
EFS 
Elongation Factor 1 alpha promoter (short) 
EvFS 
Event free survival 
ERT  
Enzyme Replacement Therapy  
eLTCIC 
Extended Long-Term Culture Initiating Cells 
FDA 
Food and Drug Administration 
FWA 
Federal-Wide Assurance 
GALV 
Gibbon Ape Leukemia Virus 
GCP 
Good Clinical Practice 
GMP 
Good Manufacturing Practice 
GOSH 
Great Ormond Street Hospital 
GVHD 
Graft Versus Host Disease 
HCG 
Human Chorionic Gonadotropin 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency Virus 
HLA 
Human Leukocyte Antigen 
HSC 
Hematopoietic Stem Cell 
HSCT 
Hematopoietic Stem Cell Transplant 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonization 
ICP 
Intracranial Pressure 
IDE 
Investigational Device Exemption 
IEC 
Independent or Institutional Ethics Committee 
Ig 
Immunoglobulin 
IL 
Interleukin 


Lenitviral Vector ADA Gene Transfer for ADA-Deficient SCID (Protocol ver. 12.0, dated 28 March 2018) 
 
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List of Abbreviations 
IND 
Investigational New Drug 
IO 
Insertional Oncogenesis 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
IV 
Intravenously 
LAM-PCR 
Linear Amplification-Mediated PCR 
LPD 
Lymphoproliferative Disorder 
LTR 
Long Terminal Repeat 
MLV 
Moloney Murine Leukemia Virus 
MOI 
Multiplicity of Infection 
MFD 
Matched Family Donor 
MSD 
Matched Sibling Donor 
MUD 
Matched Unrelated Donor 
N 
Number (typically refers to participants) 
NCI 
National Cancer Institute, NIH 
NIAID 
National Institute of Allergy and Infectious Diseases, NIH 
NIH 
National Institutes of Health 
NK 
Natural Killer Cells 
NMDP 
National Marrow Donor Program 
NOD 
Non-Obese Diabetic 
OHRP 
Office for Human Research Protections 
PBMC 
Peripheral Blood Mononuclear Cells 
PCR 
Polymerase Chain Reaction 
PEG-ADA 
Pegylated Adenosine Deaminase 
PGK 
Phosphoglycerate Kinase 
PHA 
Phytohemagglutinin Stimulation Test 
PHI 
Protected Health Information 
PI 
Principal Investigator 
PK 
Pharmacokinetics 
Plt 
Platelet count 
PO 
Per os, by mouth 
PRBC 
Packed Red Blood Cells 
PT 
Prothrombin Time 
PTT 
Partial Thromboplastin Time 
QA 
Quality Assurance 
QC 
Quality Control 
RBC 
Red Blood Cells 
RCL 
Replication Competent Lentivirus 
RCR 
Replication Competent Retrovirus 
SAE 
Serious Adverse Event/Serious Adverse Experience 


Lenitviral Vector ADA Gene Transfer for ADA-Deficient SCID (Protocol ver. 12.0, dated 28 March 2018) 
 
Orchard Therapeutics, Ltd. 
 
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List of Abbreviations 
SCID 
Severe Combined Immune Deficiency 
SCF 
Stem Cell Factor 
SOP 
Standard Operating Procedure 
TNC 
Total Nucleated Cells 
UCBC 
Umbilical Cord Blood Cells 
UCBT 
Umbilical Cord Blood Transplant 
UCL 
University College London 
VSV 
Vesicular Stomatitis Virus 
WB 
Western Blot 
WBC 
White Blood Cells 
WNL 
Within normal limits 
 
 
 
 
 


Lenitviral Vector ADA Gene Transfer for ADA-Deficient SCID (Protocol ver. 12.0, dated 28 March 2018) 
 
Orchard Therapeutics, Ltd. 
 
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1 
OVERVIEW 
1.1 
Protocol Summary 
 
Full Title 
Autologous Transplantation of Bone Marrow CD34+ Stem/Progenitor Cells 
after Addition of a Normal Human ADA cDNA by the EFS-ADA Lentiviral 
Vector for Adenosine Deaminase (ADA)-Deficient Severe Combined 
Immunodeficiency (SCID) 
Short Title 
Lentiviral Vector ADA Gene Transfer for ADA-Deficient SCID 
Clinical Trial Phase I/II 
IND Sponsor  
Orchard Therapeutics, Ltd. 
Conducted By 
University of California, Los Angeles and the National Institutes of Health 
 
 
 
Sample Size 
n=20 
Study Population 
Children older than 1.0 months who have ADA-deficient SCID 
Accrual Period 
5 years 
Study Design 
This is a controlled, prospective, non-randomized Phase I/II clinical trial to 
assess the safety (as primary end-point) and efficacy (as secondary 
endpoints) of autologous transplantation of CD34+ cells from the bone 
marrow (BM) of ADA-deficient SCID infants and children following human 
ADA cDNA transfer by the EFS-ADA lentiviral vector. Following provision 
of informed consent, enrolled subjects will be screened to determine full 
eligibility for participation over 1-2 weeks.  Eligible subjects will be admitted 
to undergo harvesting of autologous BM under general anesthesia, with 
placement of a central venous access line, if not already present.  If 
sufficient BM cells were collected to cryopreserve a “back-up sample” of at 
least 5x107 total nucleated cells/kg (or 3x107 mononuclear cells/kg) and 
also yield ≥1x106 CD34+ cells/kg, the CD34+ cells will be cultured for EFS-
ADA lentiviral vector-mediated transduction.  The subjects will receive a 
single dose of busulfan (4 mg/kg) IV.  At the end of transduction, the final 
cell product (autologous BM CD34+ cells transduced with the EFS-ADA 
lentiviral vector, OTL-101) will be assessed to determine if it meets release 
criteria, and if so, it will be infused IV.  For subjects who have successfully 
received the final cell product, PEG-ADA ERT will be stopped at day +30 
after transplant. The initial portion of the study will require the subject to be 
treated in the hospital for a typical duration of 10 days-6 weeks.  After 
discharge from the hospital, the subject will be seen for interval history and 
examination by either their home physician, the PI or a CI and have blood 


Lenitviral Vector ADA Gene Transfer for ADA-Deficient SCID (Protocol ver. 12.0, dated 28 March 2018) 
 
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drawn at months 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 21, and 24.  Any 
medically-indicated interventions will be made at those visits. Subjects will 
then be consented for a long-term follow-up study with semi-annual to 
annual visits and lab draws over the subsequent 13 years. 
Study Duration 
Each patient will be followed for 24 months. There will be a separate study 
for long-term follow-up for these patients for years 3-13.  The total follow-
up period for these subjects will be up to 15 years. 
Study 
Agent/Intervention 
Description 
The Investigational New Drug to be tested is autologous BM CD34+ cells 
transduced ex vivo with a self-inactivating (SIN) HIV-1 based lentiviral 
vector: EFS-ADA (also referred to as OTL-101). The vector has been 
produced as cell-free lentiviral supernatant from the 293T cell line, and has 
an envelope pseudotype from the vesicular stomatitis virus (VSV).  The 
vector will be used for ex vivo transduction of CD34+ progenitor cells from 
the BM of infants or children with adenosine deaminase (ADA)-deficient 
severe combined immunodeficiency (SCID). OTL-101 will be subsequently 
infused intravenously after a single non-myeloablative dosage of busulfan 
(4 mg/kg intravenously x1). 
Primary Objective 
 
The primary objectives are: 
•  to examine the safety of autologous transplantation of bone marrow 
CD34+ cells transduced with the EFS-ADA lentiviral vector (OTL-101).  
The safety review will include the identification of grade III/IV serious 
adverse events (SAE).  Subjects will also be monitored for clinical 
toxicities, replication competent lentivirus, and monoclonal expansion 
at regular intervals by a clinician, as well as standard blood counts and 
chemistries. 
• to estimate overall and event-free survival by 12 months where failure 
is defined by one of the following endpoints:  death; reinstitution of 
PEG-ADA; or performance of an allogeneic BMT.   
Secondary 
Objective 
The secondary objectives will assess the efficacy of OTL-101, engraftment 
of transduced cells and the extent of ADA expression and immune 
reconstitution. 
The secondary outcomes will:  
1. Assess overall and event free survival at 24 months, 
2. Compare overall survival and event free survival at 24 months 
between patients treated with OTL-101 and patients treated 
with allogeneic HSCT, 
3. Assess the extent of gene transfer in peripheral blood cells,  
4. Assess ADA gene expression by measuring ADA enzymatic 
activity and adenine nucleotides,  
5. Examine the effects of reconstituting ADA gene expression 
on immune function through serial examination of peripheral 
blood leukocytes and myeloid cells,  
6. Assess immune reconstitution and infection rates, and 
7. Assess use of immunoglobulin replacement therapy. 


Lenitviral Vector ADA Gene Transfer for ADA-Deficient SCID (Protocol ver. 12.0, dated 28 March 2018) 
 
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Endpoints 
Primary Safety Endpoint:   Incidence and grade of Serious Adverse 
Events (SAE). 
Serious adverse events may be manifest as:   
• 
clinical toxicities 
• 
exposure to replication-competent lentivirus (RCL)  
• 
development of monoclonal expansion or leukoproliferative 
complications from vector insertional effects. 
Primary Efficacy Endpoint:  Overall/event-free survival (survival without 
need for BMT or PEG-ADA). Overall/event-free survival will be determined 
for each subject by 12 months where failure is defined by one of the 
following endpoints: death; reinstitution of PEG-ADA; or performance of an 
allogeneic BMT. 
Secondary Endpoints include:   
• 
Overall survival and event free survival at 24 month 
• 
Efficacy of gene transfer/engraftment of HSC  
• 
ADA expression, enzyme activity and detoxification  
• 
Effects on ADA reconstitution on immune phenotype and  function 
(the need for IgRT will be assessed under this category) 
• 
Immune reconstitution  
• 
Infection rates 
 
 


Lenitviral Vector ADA Gene Transfer for ADA-Deficient SCID (Protocol ver. 12.0, dated 28 March 2018) 
 
Orchard Therapeutics, Ltd. 
 
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1.2 
Schematic of Study Design: 
 
ADA GENE TRANSFER WITH MARROW CYTOREDUCTION 
 
 
 
ADA-Deficient SCID Patient 
 
 
Child with confirmed diagnosis of ADA deficient SCID 
 
Medically eligible, HLA-matched sibling ---> Offer allogeneic BMT 
 
No medically eligible HLA-matched sibling 
 
 
Institute/Continue PEG-ADA therapy     Decision to forego PEG-ADA 
  
      
PI or CI meets with family to provide Experimental Subject's Bill of Rights 
Describe protocol, give consent document for consideration 
 
No    Parents agree to participate 
 
Yes 
 
Obtain informed consent.  Enroll in study 
 
Perform pre-treatment screening studies 
 
No  Patient meets inclusion criteria, no exclusions 
 
Yes 
 
Perform pre-operative screening studies 
 
Patient continues to meet inclusion criteria 
 
 
 
 
Continued on Page 15 
 


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Cryopreserve 
and offer 
second harvest 
yes 
no 
Harvest cells→ Bone Marrow and Stem Cell Processing 
5x107 TNC/kg from whole bone marrow or 3 x 107 MNC/kg from 
ficolled bone marrow retained as back-up  
Isolate CD34 cells from remainder 
< 1 x106/kg CD34+ 
≥ 1x106/kg CD34+ 
yes 
no 
Infuse back-up cells, if not given 
Patient follow-up 
Patient follow-up 
yes 
no 
No serious infection and evidence of immune reconstitution (>25% of baseline 
absolute T cell count, or detectable PBMC ADA 6 months post-transplant) 
Patient follow-up 
Start PEG-ADA, 
Patient follow-up 
CD34+ cells enter 2 day 
transduction process in GMP lab 
Busulfan IV (4 mg/kg) 
Cells meet specs at end of culture 
Infuse≤15x106cells/kg; 
if ≤0.5x106/kg, add 
back-up cells 
Infuse back-up cells; 
Continue PEG-ADA 
Patient follow-up 
Discontinue PEG-ADA 
ERT on Day +30 
ANC < 200/l or platelets < 20,000/l 
on 3 independent determinations 
after day +42 from cell infusion 


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2 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
2.1 
Background Information 
2.1.1 Severe combined immunodeficiency (SCID)  
      ADA acts on both adenosine and deoxyadenosine, producing inosine and deoxyinosine 
(Hershfield and Mitchell, 1995; Hirschhorn, 1993).  In the absence of ADA, deoxyadenosine can 
be phosphorylated, particularly by lymphoid cells, which have high levels of the enzyme 
deoxycytidine kinase.  The resulting dATP pool expansion has been shown to inhibit DNA 
replication and repair, and to induce apoptosis in immature thymocytes.  In addition, 
deoxyadenosine inactivates the enzyme S-adenosylhomo-cysteine (SAH) hydrolase; SAH 
accumulation inhibits transmethylation reactions, which may contribute to immunodeficiency.  It 
has been speculated that effects of extracellular accumulated adenosine acting through its 
various receptors may also play a role in pathogenesis. 
     A minority (10-15%) of ADA-deficient patients does not present with the classical phenotype 
of “severe combined immunodeficiency” (SCID), but with a milder and variable degree of immune 
dysfunction that delays their diagnosis beyond the first year of life (delayed onset) or the first 
decade (late onset).  Although abnormal, immunity is partially conserved in these patients who 
are commonly classified as being affected with “combined immunodeficiency” (CID) until a 
definitive diagnosis is made.  The clinical presentation of these patients is characterized mostly 
by recurrent lung infections that occur at higher frequency than in normal subjects, but that usually 
respond to therapy, which can mask the true underlying cause for many years.  The  
immunodeficiency of these patients may be adequately controlled with antibiotics and immune 
globulin prophylaxis, or they may be treated in the same manner as early onset ADA-Deficient 
SCID. 
2.1.2 Adenosine deaminase (ADA)-deficient SCID 
     Severe combined immunodeficiency (SCID) has been recognized for over four decades as an 
inherited disorder in which patients have profoundly defective immunity with essentially no T 
lymphocyte function and minimal to absent B cell function (Kohn and Weinberg, 1996; Buckley, 
2004).  SCID patients typically present for medical attention between 3-9 months of age with 
multiple, recurrent severe infections with common childhood pathogens (encapsulated bacteria, 
respiratory syncytial virus, parainfluenza virus) or opportunistic organisms (Candida albicans, 
Pneumocystis jirovecii, cryptococcus).  Related findings often include failure to thrive and wasting, 
persistent diarrhea, and delayed growth and dentition.  
     The clinical syndrome of SCID is due to a variety of specific genetic lesions, each of which 
impairs normal T cell development and function.  The first specific genetic cause of SCID to be 
identified was deficiency of the enzyme adenosine deaminase (ADA) (Giblett et al, 1972).  It is 
estimated that ADA deficiency accounts for 15-20% of cases of SCID.  Other known genetic 
lesions resulting in SCID include:  mutations in the IL-2 receptor gamma chain (γc), (responsible 


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for X-linked SCID); defects in Jak3 kinase and other components of T cell intracellular signaling 
pathways; the inability to produce interleukin-2 (IL-2); defects in components of the T cell antigen 
receptor complex; defects in the recombinase system which is responsible for rearrangement of 
immunoglobulin and T cell receptor genes; and impaired expression of HLA antigens (bare 
lymphocyte syndrome). Once a definitive diagnosis of ADA deficiency is made, patients may be 
treated with a hematopoietic stem cell transplant (HSCT), which includes HLA-identical matched 
sibling donor bone marrow transplant (BMT)  or  haplo-identical parental T cell-depleted BMT,  or 
in some cases a matched unrelated adult donor  BMT or matched unrelated cord blood HSCT.   
However, if a matched sibling donor is unavailable, most ADA-deficient patients are treated with 
ADA enzyme replacement therapy (ERT).  These treatment options and outcomes are discussed 
in detail below. 
2.1.3 Treatment Options for ADA-Deficient SCID 
2.1.3.1 Allogeneic Bone Marrow Transplantation (BMT) 
a. Matched sibling donor BMT      
     Bone marrow transplantation was first successfully applied in the treatment of a patient with 
SCID in 1968 (Gatti, et al, 1968).   ADA deficiency was recognized as a cause of SCID in 1972 
and patients with ADA-deficient SCID were successfully transplanted at that time (Giblett, et al, 
1972; Parkman, et al, 1975).   For SCID patients with an HLA-matched sibling donor, BMT is the 
treatment of choice.  Barring patients with severe pre-existing infections, greater than 90% of 
SCID patients (with or without ADA deficiency) can be cured with BMT from a matched sibling.  
Due to the lack of immune reactivity, a matched sibling BMT for SCID is accomplished quite 
easily; a modest amount of donor bone marrow (e.g. 5 x 107 cells/ kg) is taken directly from the 
donor and given to the SCID recipient without the use of cytoablation or immune suppression.  
The majority of patients treated by matched BMT will develop a normal immune system comprised 
of donor-derived T lymphocytes.  
b. Haplo-identical, parental T cell depleted BMT 
    However, only ~20% of SCID patients will have a matched sibling donor.  An alternative 
treatment which has been developed is the use of haplo-identical (parental) bone marrow, 
depleted of mature T lymphocytes.  The results with T cell-depleted BMT for SCID patients have 
gradually improved, so that 60-80% of patients will survive with gain of improved-to-normal 
immune function (Fischer, et al, 1990; O'Reilly, et al, 1989; Buckley, et al, 1999; Filipovich, 2008; 
Cowan, 2008, Griffith, 2009).  However, despite T cell depletion of the donor marrow, patients 
can suffer graft versus host disease (GVHD), requiring further immunosuppressive therapy.   
Some patients will recover only moderate immune function, with B cell deficiency and 
hypogammaglobulinemia seen in up to half of the patients.  Additionally, immune recovery is a 
slow process, taking 4-12 months, during which time the patients are vulnerable to opportunistic 
infections. 
     Buckley and co-workers have reported their results using haplo-identical T-depleted BMT to 
treat 77 SCID patients, of which 60 of 77 survived (78%).  Of the total transplants:  74 of 77 of 


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these received the transplants without the use of cytoablative therapy or GVHD prophylaxis; 3 
received unrelated donor cord blood transplants (2 with pre-conditioning and post-GVHD 
prophylaxis) after unsuccessful haplo transplants; 19 required at least a second cell infusion (with 
a few subjects getting three or four transplants); 21 of 60 were stated to have "some donor B 
cells,"; and 45 of the total 72 survivors continue to receive IVIg. 
     Specifically, 13 subjects had ADA-deficiency as the genetic basis of their SCID and 11 of these 
survived.  Of the 13 patients, 9 received haplo-identical T cell-depleted transplants and 4 received 
matched sibling transplants.  Of the 9 receiving haplo-identical T cell-depleted transplants, 7 were 
alive.  Of the 7 surviving patients, 6 have donor engraftment (66%) and one subject is receiving 
PEG-ADA after rejection of two paternal haplo-identical T-depleted transplants.  Of the 11 
surviving ADA-deficient subjects, only 3 had evidence of donor B cell production and 5 were still 
receiving IVIg.  From the publication, it is not possible to determine how many of the ADA-deficient 
subjects were infants, defined as less than 3 months of age at the time of transplant.   
c. Unrelated donor BMT 
     Using unrelated or haplo-identical (parent) bone marrow from adult donors, the results for 
ADA-deficient SCID are consistently significantly worse than for other genetic forms of SCID.  For 
example, in a large retrospective review of the pan-European experience, survival for other types 
of SCID patients was 77% with HLA-identical donors compared to 54% for HLA-mismatched 
donors.  However for ADA-deficient SCID patients, survival was 88% with HLA-identical donors 
compared to only 29% with HLA-mismatched donors (Antoine, et al, 2003).   
     A consensus conference held in 2006 accumulated the following outcome data on allogeneic 
HSCT for adenosine deaminase-deficient SCID from 4 major centers in Europe and 1 US center 
(Booth, et al, 2007).  
Table 1. HSCT transplant outcome for ADA-SCID 
 
Total  
 
MSD/MFD 
 
MUD 
 
Haploidentical 
 
 
 
Number 
Alivea 
Number 
Alivea 
Number 
Alive 
Alive + T engraftment 
London 
31 
13 
11 
5 
3 
13 
3 
3 
Ulm 
15b 
7 
7 
2 
1 
6 
4 
4 
Brescia 
4 
3 
3 
1 
1 
0 
0 
 
Paris 
7 
3 
2 
0 
0 
4 
0 
 
Dukec 
23 
4 
4 
0 
0 
19 
14 
7 
Total 
80 
30 
27 (90%) 
8 
5 (63%) 
42 
21 (50%) 
14 (33%) 
MSD—matched sibling donor. 
 
MFD—matched family donor. 
 
MUD—matched unrelated donor. 
 
a All patients alive with T-cell engraftment. 


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b Two patients from Munich. 
 
c All patients transplanted without conditioning.  
     More recently, these results were supplemented by ascertainment of the outcomes in 
additional cases of allogeneic BMT for ADA-deficient SCID (Gaspar, et al, 2009) (see Figure 1).  
These results demonstrate that allogeneic BMT other than with a matched related sibling donor 
produce sub-optimal rates of survival and immune reconstitution.  
Figure 1. Survival of patients receiving HSCT for ADA-SCID 
 
 
d. Umbilical cord blood HSCT 
   There are few published series reporting the results of unrelated umbilical cord blood 
hematopoietic stem cell transplant (UCBT) for SCID patients of any genetic types and only four 
for ADA-SCID.  Knutsen et al. (2010) performed 3 UCBT for non-ADA SCID patients. Other than 
grade I GVHD, they are all doing well.  In another study, of 9 SCID patients that were treated with 
UCBT, 4 had ADA-deficient SCID. 2/4 of the ADA-deficient patients died and one of the surviving 
patients had grade III GVHD.  All of the other 5 patients, who had other forms of SCID, survived 
(Bhattacharya, et al, 2005).  This series was listed in a recent review and a fifth ADA-SCID patient 
had since been transplanted with umbilical cord blood as the donor source.  This brought the 
Survival of Patients Receiving HSCT for ADA-SCID 
(preliminary results from an international multicenter retrospective study)
Gaspar, Aiuti, Porta, Candotti, Hershfield, Notarangelo. 
Blood114:3524-3532. 2009


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overall survival to 40% (Gennery and Gant, 2007).  Another series of 8 non-ADA-deficient SCID 
had 2/8 survive, but 7/8 experienced GVHD and 2 were grade 3-4.      
     Altogether, these reports show that following unrelated umbilical cord blood transplants, 14/16 
(87.5%) SCIDs (non-ADA) are alive, but only 2/5 (40%) ADA-deficient SCIDs are alive.   These 
are small numbers of patients, but the results are consistent with the larger numbers of SCID 
recipients of MUD adult products, showing worse outcome for the ADA-deficient form of SCID 
when compared to the other forms SCID.   Furthermore, the recipients of unrelated cord blood 
receive high dose chemotherapy conditioning (e.g. busulfan 16 mg/kg plus cyclophosphamide 
200 mg/kg and ATG) and require post-transplant GVHD prophylaxis (Cyclosporine A and 
methylprednisolone, despite which, cord blood HSC recipients often have frequent low grade 
GVHD and occasional high grade GVHD. 
e. Recent multinational report on outcomes from allogeneic HSCT for ADA- deficient SCID: 
[Hassan et al. Blood e-pub July 2012]. (Paraphrased from the Abstract): 
     In this multicenter retrospective study, the authors analyzed outcome of HCT in 106 patients 
with ADA-SCID who received a total of 119 transplants. HCT from matched sibling and family 
donors (MSD, MFD) had significantly better overall survival (OS) (86% and 81%) in comparison 
to HCT from matched unrelated (66%; p<0.05) and haploidentical donors (43%; p<0.0001). 
Superior OS was also seen in patients who received unconditioned transplants in comparison to 
myeloablative procedures (81% vs. 54%; p<0.003) although in unconditioned haploidentical 
donor HCT, non-engraftment was a major problem. Long term immune recovery showed that 
regardless of transplant type, overall T cell numbers were similar although a faster rate of T cell 
recovery was observed following MSD/MFD HCT. Humoral immunity and donor B cell 
engraftment was achieved in nearly all evaluable surviving patients and was seen even after 
unconditioned HCT. These data detail for the first time the outcomes of HCT for ADA-SCID and 
demonstrate that if patients survive HCT, long term cellular and humoral immune recovery is 
achieved.   These outcomes confirm many previous smaller reports on the rates of success and 
complications for different types of allogeneic HSCT for ADA-deficient SCID.   
     Thus, while T cell-depleted or unrelated adult donor or cord blood HSCT have become useful 
treatment modalities for ADA-deficient SCID patients, they are still beset by a number of serious 
limitations.  Because of these limitations, haploidentical or matched unrelated BMT are generally 
not offered as options for ADA-SCID.  The two conventional treatment options are HLA-identical 
related BMT for those with available matched siblings and ADA enzyme replacement therapy in 
the form of PEG-ADA for most others.  
2.1.3.2 ADA Enzyme Replacement Therapy 
     In the mid-1970's, it was observed that transfusion of packed red blood cells from normal 
donors into ADA-deficient patients could cause a partial improvement in immune function, 
including a rise in the lymphocyte count (Polmar, et al, 1976).   It was postulated that ADA within 
the donor RBC was acting to detoxify adenosine and deoxyadenosine metabolites, thereby 
protecting developing T lymphocytes.  However, the responses to the RBC transfusions were only 
seen in a minority of patients and were typically modest in magnitude and duration.   


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     Based upon the experience with RBC transfusions, a far more effective means of ADA enzyme 
replacement was developed.  Bovine ADA may be isolated in relatively large quantities, but has 
a half-life of only minutes when injected parenterally and may be immunogenic upon repeated 
exposures.  It had previously been shown that conjugation of proteins to polyethylene glycol 
(PEG) leads to greatly prolonged serum survival time and decreased immunogenicity.  A 
pharmaceutical preparation of bovine ADA conjugated with polyethylene glycol (PEG-ADA, 
ADAGEN™) was approved by the US FDA in 1990 for treating ADA deficiency.  It has been used 
to treat over 130 patients, often those considered to be too ill to safely undergo haplo-identical 
bone marrow transplantation (Hershfield et al, 1992; updated in Gaspar, 2009).   PEG-ADA avoids 
the risks of red cell transfusion, and the amount of ADA enzyme activity provided by one or two 
weekly intramuscular injections of PEG-ADA exceeds by as much as 10 fold the amount that can 
be supplied by repeated RBC transfusions.  PEG-ADA has been well tolerated, with no reports of 
allergic reactions thus far. 
      Treatment with PEG-ADA consistently corrects metabolic abnormalities due to ADA 
deficiency.  Lymphocyte counts and immune function begin to improve after 2 to 4 months of 
treatment.  Most patients have done well clinically (overall mortality is <20%, and ~10% in patients 
treated more than 6 months) (Hershfield & Mitchell, 1995; Gaspar 2009).  Recovery of 
immunologic function on PEG-ADA therapy has been variable, and about 20% of patients, 
primarily those with the most severely compromised immune function, have had minimal 
improvement in lymphocyte counts and function. We reported long-term (4-11 years) follow-up on 
a cohort of ADA-deficient SCID patients treated with ADA-GEN at Children’s Hospital Los Angeles 
and observed that T lymphocyte counts never reached into the normal range and had 
progressively declined to values of 100-400 over the duration of treatment (Chan et al, 2005).  A 
few patients on PEG-ADA developed autoimmune hemolytic anemia, which was eventually fatal.  
Autoimmune disorders are seen in some patients with incomplete immune function and may 
represent the presence of immune effector cells without an appropriate level of immune regulatory 
cells.  Others have died from progressive pulmonary insufficiency and/or have developed 
lymphoproliferative disorders or hepatocellular carcinoma.    
     The other major limitation to PEG-ADA therapy is the high cost.  Treatment of a child with 
weekly PEG-ADA injections may cost between $200,000-500,000 per year.  Because PEG-ADA 
therapy is palliative and not curative, injections must be continued throughout the life of the 
patient.  The high cost of this treatment may limit access and create severe financial burdens on 
patients and their families.    
2.1.4 Gene Therapy for ADA-Deficient SCID 
2.1.4.1 ADA Gene Therapy Using T Lymphocytes 
     Gene therapy as a new alternative treatment for ADA-deficient SCID has been under 
investigation for more than two decades.  The first clinical trial of human gene therapy for ADA-
deficient SCID targeted the corrective ADA gene to peripheral blood T cells.  Pre-clinical studies 
performed at the NIH in the mid-1980's demonstrated that introducing a normal human ADA cDNA 
(using a retrovirus as the gene delivery vector) into cultured T lymphocytes from patients with 


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ADA-deficient SCID resulted in the production of normal levels of ADA enzyme (Kantoff et al, 
1986).  In cell culture, the "cured" cells were able to survive in the presence of levels of 
deoxyadenosine that were toxic to the parental ADA-deficient cells.  Studies performed in Italy 
demonstrated that ADA-corrected human T lymphocytes survived significantly longer in vivo in 
immune deficient mice compared to ADA-deficient cells (Ferrari et al, 1991).   
     Based upon these experimental results, a trial of gene therapy for ADA deficiency was 
performed in two patients at the NIH, beginning in 1990 (Culver et al, 1991; Blaese et al, 1995; 
Mullen et al, 1996).  Peripheral blood T lymphocytes were collected by leukopheresis, transduced 
in the laboratory with the ADA gene, expanded in numbers by stimulating with growth factors and 
then re-infused into the patients.  The procedures of leukopheresis, transduction and reinfusion 
were repeated at 1-2 month intervals over the course of two years.  Two children were treated by 
this method with no evidence of toxicity.  The presence of cells containing the inserted ADA gene 
was documented in both patients, although one patient had a significantly greater number of 
transduced cells than the other.   In at least one of the patients, ADA-transduced T lymphocytes 
remain at a relatively stable level of 30-50% of total PBL, six years after the last cell infusion;  this 
observation shows that T lymphocytes subjected to ex vivo transduction are capable of extended 
in vivo survival (Muul et al, Blood 2003).  Both patients had significant increases in numbers of 
circulating T lymphocytes and some evidence of improved immune function.  However, they 
remain on PEG-ADA enzyme replacement, making it difficult to attribute immunologic functions 
to the gene transfer.  An ADA-deficient SCID patient in Japan was treated under this same 
protocol with good ADA gene transfer achieved (Onodera et al, 1998; Misaki, 2001). 
2.1.4.2 Gene Therapy for ADA-Deficient SCID using Hematopoietic Stem Cells 
a.  Gene Transfer into Hematopoietic stem cells 
    One drawback to gene therapy using T lymphocytes is that the immunologic repertoire of the 
transduced lymphocytes may be restricted to those specificities present in the population of cells 
which were treated; responses to newly encountered antigens may not be possible.    Additionally, 
these cells may have a finite life-span (months to years), which may necessitate repeated ADA 
gene transfers to be performed to sustain a therapeutic level of transduced cells. 
     Insertion of the ADA gene into hematopoietic stem cells (HSC), rather than mature T 
lymphocytes, may produce a long-lived effect.  HSC are cells which produce all blood cells by a 
complex process of proliferation and differentiation.  HSC are long-lived and can function for the 
life of the individual after transplantation.  For children and adults, HSC are found primarily in the 
bone marrow, but HSC are also present in the umbilical cord blood at birth.  Genetic correction of 
a patient's HSC may provide a continuous, enduring source of gene corrected mature blood cells, 
including T lymphocytes.   
     From more than twenty years, γ-retroviral-mediated gene transfer into hematopoietic stem 
cells can routinely transduce the majority of stem cells in murine gene transfer/BMT models.  
However, work in large animal models (canine and rhesus) in the 1990’s could only achieve 0.1-
1.0% stem cell transduction (Kohn, 1995; Dunbar, 1996).  The initial sets of clinical trials of gene 
transfer into autologous hematopoietic cells for ADA-deficient SCID and other disorders 


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performed to that time had also shown extremely limited abilities to transduce long-lived human 
stem cells, with marking of only 0.1-1% of cells seen.  The reasons for the greater difficulty in 
performing gene transfer into stem cells from the large animals and human subjects were not fully 
understood; possible explanations include lower numbers of receptors for the amphotropic virus 
envelopes used, intracellular blocks to virus integration, or a lower percentage of stem cells in 
active cell cycle which can be transduced by γ-retroviral vectors. 
      Over that time period, incremental improvements in γ-retroviral-mediated gene transfer into 
human hematopoietic stem cells (HSC) were achieved, using GALV pseudotypes, "mobilized 
BM," recombinant fibronectin support, new cytokines (Flt-3 ligand, thrombopoietin), and 
manipulation of cell cycle kinetics (Kiem et al, 1997; Dunbar et al, 1996; Hannenberg et al, 1996; 
Shah et al, 1996;  Dao et al, 1997; Dao et al, 1998; Piacibello et al, 1997).  Combinations of these 
techniques resulted in modest, yet significant, increases in gene marking in primate stem cell 
transplant models (e.g. 10%, up from the previous ceiling of 0.1-1.0%) (Kiem et al, 1998; Tisdale 
et al, 1998).  Based on these methodological advances, a second generation of clinical trials of 
gene therapy for both ADA-deficient SCID as well as XSCID and other disorders were begun in 
this decade.  
b.  Gene therapy for ADA deficiency using hematopoietic stem cells 
     At least 50 ADA-deficient SCID patients have now been treated by insertion of the ADA gene 
into their bone marrow cells (see Table 2, below).   
Table 2. Clinical Trials of Gene Therapy for ADA-deficient SCID (as of June 2011) 
Year 
Site 
Cell 
Type Vector 
PEG-ADA 
Pre-transplant 
Conditioning 
Clinical Benefit 
(#benefited/ 
#treated) 
1990 
Bethesda, U.S. 
T 
RV 
Continued 
n.a. 
No (0/2) 
1993 
Milan, Italy 
T 
& 
BM 
RV 
Continued 
No 
No (0/2) 
1993 
Netherlands/UK 
BM 
RV 
Continued 
No 
No (0/3) 
1993 
L.A./Bethesda, U.S. 
UCB 
RV 
Continued 
No 
No (0/3) 
1995 
Milan, Italy 
T 
RV 
Continued 
n.a. 
No (0/3) 
1996 
Hokkaidō, Japan 
T 
RV 
Continued 
n.a. 
No (0/1) 
2000 
Milan, Italy 
BM 
RV 
No 
Busulfan  
(4 mg/kg) 
Yes 
(15/18) 
2004 
Hokkaidō, 
Japan 
(Otsu et al) 
BM 
RV 
No 
None 
No (2/2) 
2002 
London, UK 
BM 
RV 
No 
Melphalan  
(140 mg/m2) 
Yes (3/6) 
2001 
L.A./Bethesda, U.S. 
BM 
RV 
Continued 
None 
No (0/4) 
No 
Busulfan    
(75-90 mg/m2) 
Yes (3/6) 


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2009 
L.A./Bethesda, U.S. 
BM 
RV 
No 
Busulfan 
(4 mg/kg) 
Yes (4/5) 
T = peripheral blood T lymphocytes, BM = bone marrow, UCB = umbilical cord blood, RV = γ-retroviral 
vector, n.a. = not applicable 
 
An initial set of clinical trials performed in Europe and the US in the 1990’s in which γ-retroviral 
vectors were used to introduce the human ADA cDNA into bone marrow CD34+ cells from ADA-
deficient patients were ineffective.  All of the studies only were able to achieve very low numbers 
of ADA-gene corrected blood cells, generally at the limits of detection by PCR, and in most cases 
these cells were only present for the initial months after the procedure (Ferrari et al 1992; 
Bordignon et al, 1993; Hoogerbrugge et al, 1992; Kohn 1993).  In all of these trials, patients 
continued to receive PEG-ADA enzyme therapy because of the ethical concerns about stopping 
an approved efficacious medication to test an unproven approach.  While the PEG-ADA sustained 
the immunity of these patients, it may have blunted the putative selective survival advantage for 
ADA-expressing lymphocytes that may support the amplification of lymphoid cells produced by 
gene-transduced stem/progenitor cells. Additionally, the patients did not receive any pre-
transplant conditioning with chemotherapy, which may have facilitated the engraftment of the 
transplanted gene-modified stem/progenitor cells, again due to the potential risks and unknown 
prospects of benefit. 
     A clinical trial of gene therapy for ADA deficiency carried out in Italy starting in ~2000, initially 
reported on two patients who did not have access to PEG-ADA and who were treated with gene-
corrected autologous CD34+ bone marrow cells after receiving chemotherapy with Busulfan at 
the dose of 4 mg/kg (Aiuti, 2002).  In this case, normalization of T cell numbers and function was 
observed as soon as 6 months after treatment in the first patient and partial improvement of T cell 
counts was observed in the second patient.  Both patients are well with normal growth and 
development (Aiuti et al 2001; Aiuti et al 2002).  An additional 10 subjects have been enrolled in 
Milan and six in London, and early reports demonstrate that most of them have reconstituted 
immunity (Aiuti A et al., 2009; Gaspar HB, et al, 2006).  These results are unprecedented for gene 
therapy of ADA deficiency and represent a dramatic improvement over previous clinical trials.   In 
the Italian and London trials, chemotherapy was administered to aid engraftment of gene-
corrected cells.  We have recapitulated these results in our trials, as described below and will be 
discussed in detail in section 2.1.2.  No adverse reactions from the vectors have been noted to 
date in these ADA deficient patients.    
2.1.5 Summary of Previous Clinical Studies with -Retroviral Vectors by 
Investigators 
2.1.5.1 CHLA/NIH trial of gene transfer into umbilical cord blood CD34+ cells from 
ADA-deficient SCID infants (1993) 
    We have performed a clinical gene therapy study with direct relevance to the studies proposed 
here (Kohn et al, 1995).  Three infants were diagnosed prenatally with adenosine deaminase 
(ADA)-deficiency, in families with previous affected infants with severe combined 
immunodeficiency (SCID).  In 1993, these infants were infused with γ-retroviral vector-transduced 


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umbilical cord blood CD34+ cells on their fourth day of life.  Each infant was immediately started 
on enzyme replacement therapy with PEG-ADA. As a result, they had normalization of the levels 
of deoxyadenosine metabolites, which were elevated at birth, and improvements of their T cell 
numbers and function over the first few months.  One patient was kept on a fixed dose of PEG-
ADA so that the effective dose/kg decreased as he grew; the other two patients were maintained 
on a constant dose/kg (adjusted as they grew) for the first 18 months (through Dec 94) and were 
then lowered to half this dose for the subsequent time.  
In summary, this trial of gene transfer to ADA-deficient CD34+ cells showed that: 
1. Long-lived hematopoietic progenitor (stem) cells among the CD34+ cells from umbilical cord 
blood can be transduced by γ-retroviral vectors, engraft without prior administration of cytoablative 
conditioning, and produce gene-containing peripheral blood leukocytes for at least 10 years. 
2. There is selective accumulation of gene-containing T cells upon reduction of the PEG-ADA 
dosage. 
3. Expression by the LASN vector in resting CD3+ T lymphocytes is poor (<1% of that seen in 
Jurkat cells).  Expression increases significantly upon stimulation of the T cells with PHA (to 
approximately the same level as in Jurkat cells).  
4. While there was no evidence that either B lymphocytes or NK cells containing the ADA gene 
selectively accumulate, both of these cell types were rapidly lost upon withdrawal of PEG-ADA. 
5. In one subject, complete cessation of PEG-ADA for two months did not demonstrate that he 
had protective immune function solely from gene-corrected cells. Thus, the low level of 
transduced progenitors present in this patient was not sufficient to sustain immune function 
following PEG-ADA withdrawal during the observation period.  Potentially, higher frequencies of 
transduced progenitor cells may allow long-lasting, protective immune function independently of 
exogenous enzyme replacement. 
6. No abnormal lymphoproliferation was detected in these 3 umbilical cord blood enrollees. 
7.  Retrospective analysis by LAM-PCR showed that one of the subjects had a nearly-monoclonal 
pattern of marking, with a single predominate vector integrant present and stable over more than 
eight years, without cell expansion.  This predominant integrant was seen in peripheral blood T 
cell clones with different patterns of T cell receptor rearrangement, indicating that transduction 
was of a pre-thymic stem or progenitor cell (Schmidt et al, 2003).  The stable benign monoclonal 
marking provides an important contrast to the unstable monoclonal lymphoproliferation seen in 
the X-SCID studies.   
2.1.5.2 Phase I trial of gene transfer to CD34+ cells from the bone marrow CD34+ 
cells from ADA-deficient SCID infants and children (2001-2009) 
     A Phase I trial of gene therapy for ADA-deficient SCID was performed by investigators at 
Childrens Hospital Los Angeles and the NIH Clinical Center (FDA BB IND# 8556) in a total of ten 
(10) subjects. This trial compared two slightly different γ-retroviral vectors, MND-ADA and GCsap-
ADA, with half of the CD34+ cells in each patient getting one vector and the other half of their 
Cd34+ cells getting the other vector.  The study was performed in two stages: 1. Four subjects 


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underwent the procedure in 2001-2002 and remained on PEG-ADA and did not receive busulfan 
chemotherapy and 2. Six subjects underwent the procedure in 2005-2009 and had PEG-ADA 
withdrawn and received non-myeloablative marrow conditioning with busulfan.  Patient 
demographics, results of CD34+ transduction, and peripheral blood mononuclear cell marking are 
shown (see Table 3, below). 
 
First stage without busulfan and on PEG-ADA: 
     In the first stage, the two younger subjects (202N and 204C at 4 years of age) had sustained 
gene marking, albeit at a low level, whereas the two older subjects (201C at 15 and 203N at 20 
years of age, respectively) did not have detectable long-term marking.  This finding suggests that 
younger subjects may be more likely to derive benefit from this approach, either due to better 
thymic function or a greater content of transducible stem cells in their bone marrow. There has 
not been any significant effect on immune reconstitution in these four subjects, and the patients 
have remained on PEG-ADA since participating in the study.  Patient toxicity for these patients 
who did not receive busulfan and continued on PEG-ADA has been non-serious (class 1 or 2, 
primarily due to anemia from bone marrow harvest), and there has not been evidence of either 
lymphoproliferation or the presence of RCR.  The subjects were treated while remaining on PEG-
ADA enzyme replacement therapy and they did not receive pre-transplant conditioning 
chemotherapy.   
     It has now been shown in multiple studies (including our own described below) that it 
is necessary to “make space” in the marrow of patients undergoing autologous BMT with 
gene transfer for ADA-deficient SCID to appreciate significant engraftment of the gene-
corrected cells. The initial experiences from Milan (n=10) and London (n=1) have been published 
(Aiuti 2002, Aiuti, 2007; Aiuti, 2009; Gaspar, 2009) documenting clinical benefit to subjects pre-
treated with a non-myeloablative dosage of the chemotherapy agent busulfan prior to reinfusion 
of gene-modified CD34+ cells. Subsequent clinical trials of gene therapy for Chronic 
Granulomatous Disease, X-adrenoleukodystrophy, and beta-thalassemia have all used busulfan 
marrow conditioning to enhance engraftment of gene-modified stem cells (Ott, 2006; Cartier, 
2009; Kaiser, 2009).  ADA deficiency is a systemic metabolic disease, so engraftment of a larger 
amount of gene-corrected stem cells may be needed than in X-linked SCID, where pathology is 
confined to the immune system and a profound selective advantage exists for lymphocytes that 
have been corrected to express the common cytokine receptor gamma protein.  Because of these 
considerations, we amended our clinical trial protocol to include the pre-administration of a non-
myeloablative dosage of busulfan and this approach then comprised a second stage of the trial. 
 
Second stage with busulfan and with PEG-ADA withdrawn: 
   We adapted this type of approach (marrow cytoreduction with Busulfan in patients off PEG-
ADA) in an amendment to the original CHLA/NIH research protocol (approved 2005) which led to 
the second stage of the trial.  We have enrolled 6 ADA-deficient SCID infant subjects under this 
amended protocol, between 2005 – 2009 (Table 3).  
 
 


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Safety:  
   Subject 302 had prolonged pancytopenia after administration of busulfan and administration 
of gene-modified CD34+ cells, persisting even after subsequent infusion of the non-processed 
“back-up” autologous marrow.  She was subsequently found to have trisomy 8 mosaicism pre-
existing in her marrow, which is known to lead to marrow dysfunction (Engel et al, Blood   
109:503-6 2007). She had a successful unrelated donor HSCT and has been well.  The 
protocol was amended to require demonstration of normal cytogenetics in blood or marrow 
cells as an eligibility criterion.   
   Subject 304 developed an infection at 4 months after treatment, and she was put back on 
PEG-ADA at the clinician’s decision and she has continued to receive PEG-ADA since that 
time (2007). She also has several other medical problems, including obesity, type II diabetes, 
hepatic steatosis, reactive airway disease, sensorineural hearing loss, and developmental 
delay. She continues to have gene-containing and ADA-expressing PBMC, belying the 
putative blunting of selective advantage for ADA gene corrected cells by ERT. 
   Subject 306N developed an adenoviral infection at 5-6 months after transplant and was put 
back on PEG-ADA.  ERT was stopped again in March 2011 and he is currently being followed 
at the NIH. 
Table 3.   Clinical Trials of Gene Therapy for ADA-Deficient SCID (As of June 2011) 
Gender PEG-ADA 
Busulfan 
mg/m2 
CD34+ 
Cells/kg 
Long-Term 
Gene 
Marking 
PBMC 
ADA 
Activity 
(nl >58 U) 
Length 
of 
Follow-up 
(ca. 6/21/11)
usulfan, remain on PEG-ADA 
F 
not withdrawn 
None 
6.6 x 105 
No 
< 5 U 
removed 
F 
not withdrawn 
None 
9.75 x 106 
Yes 
< 5 U 
10 yr 
F 
not withdrawn 
None 
1.1 x 106 
No 
< 5 U 
removed 
M 
not withdrawn 
None 
1.88 x 106 
Yes 
< 5 U 
9.5 yr 
ulfan administered, PEG-ADA withdrawn 
M 
withdrawn 
75 
4.77 x 106 
Yes 
30-60 U 
4.5 yr 
F 
withdrawn/ 
restarted 
75 
1.5 x 105 
Non-engraft 
Trisomy 8**  
n.e. 
S/P MUD BMT 
N/A* 
F 
withdrawn 
75 
1.9 x 106 
Yes 
10-30 U 
4 yr 
F 
withdrawn/ 
restarted 
64.6 
1.6 x 106 
Yes 
50-110 U 
3.5 yr 
M 
withdrawn 
90 
9.75 x 106  
Yes 
60-120 U 
2.7 yr 
M 
Withdrawn/ 
restarted 
90 
9.5 x 106  
Yes 
10-20 U 
2.2 yr 
 
  * (C= CHLA, N=NIH) 


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** 302C had trisomy 8 mosaicism and failed gene therapy (Engel, BC et al. Blood, 2007) 
 
     Besides the expected myelosuppressive effects of busulfan (moderate neutropenia with nadir 
ANC’s of >200-300/ul3 and mild thrombocytopenia with nadir platelet counts of >100,000/ul3) (see 
Figure 2), there were grade I-II elevations of AST and ALT in most subjects (2-5x ULN at 
approximately 40 days after stopping PEG-ADA ERT and 30 days after administration of 
busulfan). There were no discernible clinical toxicities from the administration of busulfan (no 
seizures, N+V, diarrhea, mucositis, anorexia, or hair loss), with dilantin and ondansterone given 
prophylactically.   
Figure 2. Representation of the myelosupressive effects of busulfan. 
 
   The kinetics of myelosuppression are different in this setting using only a single non-
myeloablative dose of busulfan, compared to a typical conditioning regimen for allogeneic HSCT 
that combines busulfan with other drugs such as cytoxan or fludarabine. In a typical allogeneic 
transplant with multi-drug conditioning over the course of 5-7 days, neutropenia may be severe 
already by the day of transplant and generally recovers 2-3 weeks later.  In this current study, we 
have observed slow progressive decline in neutrophils and platelet counts after the single dose 
of busulfan, with moderate neutropenia (ANC<500) being reached by 10-14 days. Cell counts 
reach nadirs 25-35 days after busulfan with relatively slow recovery of neutrophils for up to 3 
months. No subject has had later problems with neutropenia or bacterial infections. A possible 
explanation for the different kinetics is that busulfan is uniquely active for the elimination of 
quiescent pluripotent stem cells to make space for donor engraftment, whereas most other 
chemotherapeutic agents are active against cycling progenitor cells and may lead to quicker cut-
off of the supply of peripheral blood neutrophils and platelets.  
   Dr. Candotti and his group have identified a high frequency of abnormalities in the myeloid cells 
in ADA-deficient SCID patients, including spontaneous moderate neutropenia, unusual sensitivity 
to antibiotics and multiple cytological abnormalities, such as hyper-segmented nuclei (Sokolic, 
2011). Inherent deficiencies of myelopoiesis in ADA-SCID may explain the slower recovery of 
neutrophils observed.  
   Clonality of vector integrants has been monitored at the National Gene Vector Biorepository 
using LAM-PCR; no evidence of clonal dominance or expansion has been observed.  
Efficacy:   
   Subjects 301, 303, and 305 remain well with laboratory evidence of immune reconstitution 
without PEG-ADA therapy over 52, 48, and 32 months after gene therapy (as of June, 2011).   


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The gene marking in subjects 301, 303, 304, and 305 has been in the range of 1-10% in the 
PBMC fraction, with overall higher marking with the MND-ADA vector than the GCsap-M-ADA 
vector.  All of the 5 evaluable subjects show the production of ADA enzyme activity in their PBMC 
(see Figure 3). This Phase I study is closed to accrual after enrolling the proposed total of 10 
subjects, all of whom have been enrolled in a long-term follow-up study at UCLA or NIH to 
complete another 13 years of follow-up. These results are encouraging in terms of safety and 
efficacy.  A Phase II trial was developed under the same IND (BB# 8556) to extend the studies to 
an additional cohort.  
 
Figure 3. Gene Marking in +Bu/-ERT Subjects. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
0.000001
0.00001
0.0001
0.001
0.01
0.1
1
0
5
10
15
20
25
Vector Copy/Cell
Month After Transplant
GCsapM-ADA PBMC
301N
303N
304C
305C
306N
0.000001
0.00001
0.0001
0.001
0.01
0.1
1
0
5
10
15
20
25
Vector Copy/Cell
Month After Transplant
MND-ADA PBMC
301N
303N
304C
305C
306N
0.000001
0.00001
0.0001
0.001
0.01
0.1
1
0
5
10
15
20
25
Vector Copy/Cell
Month After Transplant
GCsapM-ADA Grans
301N
303N
304C
305C
306N
0.000001
0.00001
0.0001
0.001
0.01
0.1
1
0
5
10
15
20
25
Vector Copy/Cell
Month After Transplant
MND-ADA Grans
301N
303N
304C
305C
306N


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Figure 4. ADA Enzymatic Activity 
 
 
 
 
 
 
 
 
 
   
Results of this clinical trial were published (Candotti et al, Blood 2012) 
2.1.5.3 Phase II trial of gene transfer to CD34+ cells from the bone marrow CD34+ 
cells from ADA-deficient SCID infants and children (2009-2014)  
   A Phase II clinical trial evaluating the efficacy of a single γ-retroviral vector (MND-ADA) in a total 
of 10 ADA-deficient SCID subjects was performed at UCLA and the NIH, and funded by an FDA 
Oprhan Product Development Grant (1 R01 FD003005).  In addition to only using a single vector 
(MND-ADA), the Phase II trial had two modifications from the prior Phase I study. An amendment 
to this Phase II Trial was approved in June 2009 (1) to lower the minimum age limit for enrollment 
from 6 months to 3.5 months of age and (2) to consolidate the administration of the busulfan to a 
single dose (90 mg/m2, ~ 4 mg/kg).  In March 2010, we submitted an amendment to further lower 
the age of entry to 1.0 month and this was accepted by the FDA, the IRB at UCLA and NIH and 
the NHBLI DSMB.  The lower limit of 6 months old was imposed in the Phase I trial in 2004 under 
FDA guidance following the first two cases of T lymphoproliferative disease in the XSCID study. 
However, subsequent cases were in subjects older than 6 months at the time of treatment, and 
thus, young age does not appear to be a significant risk factor.  Opening the study to younger 
subjects may allow them to be treated prior to acquisition of infections.   
   Administration of IV busulfan once daily has been shown to have essentially identical effects as 
giving it divided into 4 doses q 6 hours as was done with the oral preparation (e.g. Almog, BBMT, 
2011).  The single dosing schedule will allow an easier treatment regimen and will allow a single 
set of blood samples to be taken for pK determination, rather than two complete sets. 
-10
10
30
50
70
90
110
130
ADA activity (U)
Month After Transplant
ADA Enzyme Activity
301N
303N
304C
305C
306N
Normal 
Range


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   Ten subjects were enrolled and have undergone the procedure between 2009-2014, with seven 
subjects at Mattel Children’s Hospital at UCLA, and three at the NIH (Table 4). 
Table 4. Enrolled Subjects since 2009 to Phase II Study. 
 
   There have been no severe adverse events in these subjects, besides busulfan induced 
transient moderate neutropenia and mild thrombocytopenia. PBMC ADA enzyme activity and 
absolute lymphocyte counts have remained low in subjects 401 and 403, who were the two oldest 
subjects in this series (15 and 8 y/o, resp.) (see Figure 5). Subject 401 was re-started on PEG-
ADA ERT after 6 months due to absence of efficacy. Subjects 402 and 404 have had recovery of 
lymphocytes (ALC> 500 by 6 months) and PBMC ADA enzyme activity in the normal range. 
Subject 405U was treated only 5 months ago and 406N one and they are not yet evaluable.    
Figure 5. Levels of absolute lymphocyte counts and PBMC ADA enzyme activity. 
 
 
 
 
 
 
 
 
 
 
 
 
 
Age at 
Entry 
Gender 
PEG-ADA 
Busulfan 
mg/ 
 
m2 
CD34+ 
Cells/kg 
ALC at 
6 Months 
PBMC ADA 
Activity 
(nl>58U) 
Length 
of 
Follow-Up 
(Oct. 2016) 
ministered, PEG-ADA withdrawn 
15 yr 
M 
withdrawn/ 
restarted 
90 
0.6x106 
96 
< 5 U 
72 mo 
4 mo 
F 
withdrawn 
90 
1.7x106 
480 
120-180 U 
72 mo 
8 yr 
M 
withdrawn 
90 
1.4x106 
70 
10-20 U 
72 mo 
3 mo 
F 
withdrawn 
90 
7.1x106 
699 
70-120 U 
72 mo 
8 mo 
M 
withdrawn 
90 
7.6 x 106 
306 
39.8 
60 mo 
13 mo 
F 
withdrawn 
90 
8.0 x 106 
120 
176.6 
60 mo 
14 mo 
M 
Withdrawn 
90 
5.6 x 106 
50 
16.9 
60 mo 
3 mo 
F 
withdrawn 
90 
6.8 x 106 
1050 
272 
60 mo 
19 mo 
F 
withdrawn 
90 
2.9 x 106 
160 
18.9 
42 mo 
3 mo 
F 
withdrawn 
90 
8.4 x 106 
2200 
527.8 
42 mo 
-100
0
100
200
300
400
500
600
700
800
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32
Cells/mm3
Months After Gene Therapy
Absolute CD3+ T cells
401U
402U
403N
404U
405U
406N
407N
408U
15 y/o
8 y/o
ERT


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In total, our study and those from Milan and London confirm the potential efficacy from autologous 
transplantation using γ-retroviral-mediated ADA gene transfer to bone marrow CD34+ cells with 
non-myeloablative cytoreductive conditioning. There appears to be a difference in responses to 
this procedure between younger (< 2 years) and older (> 5 years) subjects, when combining 
results from the Phase I and Phase II studies.  
 
Table 5. Summary of ADA-Deficient SCID Patients. 
 
 
2.1.6 Summary of Relevant Clinical Studies 
2.1.6.1 Complications from Vector Integration with -Retroviral Vectors 
   Stable and persistent gene correction of hematopoietic stem cells has required the use of gene 
delivery vectors that can covalently integrate the transferred gene into cellular chromosomal DNA 
so that the sequences may be replicated and passed on to the billions of progeny blood cells that 
derive from the stem cells.  The relatively random insertion of γ-retroviral vectors poses risks of 
“insertional oncogenesis” (IO), in which vectors integrated in the vicinity of critical cellular genes 
may alter their expression; changes in the expression of genes that affect cell proliferation, 
survival or other biological properties may initiate a cascade of cell proliferation, initially manifest 
as a monoclonal expansion which may ultimately culminate in frank malignant transformation.  
Complications from vector-mediated IO have occurred in several clinical trials of gene transfer 
using γ-retroviral, and more recently lentiviral vectors.  
   After immune reconstitution in 9 of 10 subjects participating in an X-SCID gene transfer study 
in Paris, France four of these subjects developed leukemia ≥ 2½ years after receiving the gene-


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transduced CD34+ cells (Cavazzana-Calvo M et al 2000; Hacein-Bey-Abina S et al 2002, Hacein-
Bey-Abina, S et al., 2003A; Hacein-Bey-Abina, S et al., 2003B; Hacein-Bey-Abina S et al., 2008).  
In a similar trial of gene therapy for X-SCID performed in London, England, one of 10 subjects 
also developed T lymphoproliferation (Howe J et al, 2008).  One of these five patients who 
developed T lymphoproliferative disease (LPD) has died and the other four are in leukemia 
remission and continue to have restored immunity from the gene therapy.  For three of the 
subjects with leukemia, the proviral insertion sites have been mapped within or near (3 kb) of the 
LMO-2 gene (Hacein-Bey-Abina, S., et al, 2003), a human proto-oncogene associated with T-cell 
leukemia (Neale et al. 1997; Mead et al. 2001; Rabbitts, T. H. et al. 1998).  In a fourth case, two 
integrations sites were described:  LMO-2 and BMI1, which is also a proto-oncogene.  In a fifth 
subject, vector had integrated near a third proto-oncogene, CCND2 (Hacein-Bey-Abina S et al., 
2008).  These are the first reported cases of leukemia developing in subjects participating in a 
clinical study of γ-retroviral-mediated gene transfer to CD34+ cells.  The probability of leukemia 
developing in other participants of the French trial, or in participants of other trials, is not known.   
   Two young men with X-linked Chronic Granulomatous Disease were treated by γ-retroviral 
vector-mediated transfer of the relevant oxidase cDNA to G-CSF mobilized peripheral blood stem 
cells (Ott, 2006).  The vector had viral long-terminal repeats (LTR) from the murine Spleen Focus 
Forming Virus, which has very potent enhancer/promoter activity in myeloid cells to drive high 
level expression of the transferred gene. Both subjects had oligoclonal expansion of 
stem/progenitor cells containing vector integrants near a few cellular genes involved in myeloid 
cell proliferation, which progressed to myelodysplasia. 
   In Germany, 7 of 10 children receiving -retroviral-mediated gene transfer for WAS, developed 
an acute T-cell leukemia (Braun, CJ et al., 2014). 
   Of note, to date (October 2016), at least 40 ADA-deficient SCID patients have undergone these 
protocols using γ-retroviral vectors with intact LTR in Milan, London and in our trials at CHLA/NIH 
and UCLA/NIH.  No cases of T LPD or other adverse consequences of vector insertion have 
occurred in any of the ADA subjects, in sharp contrast to the X-SCID results (Aiuti, 2007). 
2.1.6.2 Lentiviral Vectors May be More Effective and Safer 
     Lentiviral vectors were developed in the mid 1990’s from components of the HIV-1 lentivirus. 
Lentiviral vectors have been found to have several attributes that make them potentially more 
effective and safe than γ-retroviral vectors (Naldini, et al, 1996; Zufferey, et al, 1997; Zufferey, et 
al,1998).  Lentiviruses, but not retroviruses, can transduce non-dividing cells, a property of the 
most primitive and long-lived HSC. Lentiviral vectors are more effective than γ-retroviral vectors 
when compared using multiple surrogate assays for human HSC, including in vivo growth in 
NOD/SCID mice and eLTCIC assays of quiescent CD34+/CD38- cells (Miyoshi, et al, 1999; Case, 
et al,1999). In our studies, only the lentiviral vector could transduce CD34+/CD38- cells, a 
phenotype that greatly enriches for quiescent cells of higher proliferative and differentiation 
potential; MLV vectors showed essentially no transduction of CD34+/CD38- cells. Lentiviral 
vectors can perform effective transduction during just 1-2 days of culture (in contrast to the 3-5 
days needed using γ-retroviral vectors); this shorter time of culture has been clearly proven to 
increase the survival of pluripotent stem cells (Mazurier, 2004; Kennedy, 2009). Transduction by 


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lentiviral vectors could be achieved even with a single “hit” on the first day of cell isolation.  
Transduction could also be achieved in the absence of recombinant cytokines, although the 
efficiency was approximately 2-fold less than in the presence of factors such as IL-3/IL-6/SCF.  
Ideally, this will lead to more effective transduction of quiescent HSC with preservation of stem 
cell function. 
   Thus, lentiviral vectors show greatly improved ability to transduce human HSC which 
may lead to increased gene modification in vivo. More effective gene transfer to long-term 
reconstituting pluripotent hematopoietic stem cells may lead to more robust and rapid immune 
reconstitution than with using γ-retroviral vectors.   
   Additionally, lentiviral vectors configured to lack strong the strong enhancer elements of γ-
retroviral long-terminal repeats, present in typical γ-retroviral vectors, have been shown to have 
lower risks for causing insertional oncogenesis in a number of in vitro and in vivo models.   This 
may translate into a lower risk for causing leukoproliferative disorders in gene therapy subjects, 
However, one subject of a clinical trial using a lentiviral vector for gene therapy of beta-
thalassemia had monoclonal expansion from a stem/progenitor cell in which the integrated vector 
altered the expression of the HMGA2 gene, although there have been no hematological 
abnormalities observed to date (in fact, the subject has had a significant clinical benefit).  The 
vector used contained elements from the β-globin gene cluster locus control region, which has 
strong enhancer activity, which may play a role in over-expression of the HMGA2 gene. But, the 
mechanism for clonal expansion may be due to a novel gene disruption event, with the vector 
integrated into the 3rd intron of the HMGA2 gene leading to aberrant splicing, production of a 
truncated HMGA2 gene transcript and protein, with loss of 3’ regulatory sequences involved in 
expression repression by a microRNA (Kaiser, 2009).   
   In contrast, a recently reported clinical trial using a lentiviral vector for gene therapy of X-
adrenoleukodystrophy has not encountered any adverse vector insertional effects, despite high 
level of engraftment of gene-transduced HSC (Cartier, 2009).   There are pre-clinical data which 
indicate that lentiviral vectors per se have moderately lower risks for causing IO, compared to γ-
retroviral vectors (Montini, 2009).  Additionally, lentiviral vectors that have “self-inactivated” (SIN) 
LTR with deletion of their enhancers and instead use cellular promoters lacking strong enhancers 
(as in the EFS-ADA vector to be used in this Protocol), have essentially undetectable IO activity 
in current assays (Modlich, 2009). 
   Lentiviral vectors have entered the clinic in a trial with HIV-1-infected subjects (performed by 
investigators from VirXSys Corp. and the University of Pennsylvania) (Levine, 2006).  This study 
targeted mature T cells collected by apheresis for ex vivo transduction with a lentiviral vector 
encoding antisense RNA to HIV-1 env sequences.  There was relatively effective gene delivery 
to the T cells, with ongoing detection of gene-modified T cells for more than one year in two of 
the subjects.  Follow-up over two years did not detect adverse clinical effects.  
   Lentiviral vectors to transduce CD34+ HSC are only recently entering human clinical trials.  One 
trial to use a lentiviral vector to introduce a shRNA into G-CSF mobilized PBSC to down-regulate 
the HIV-1 co-receptor CCR5 yielded only very low levels of gene-containing peripheral blood cells 
(Zaia, 2009).  Importantly, in a recently reported clinical trial of gene therapy for X-linked 


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adrenoleukodystrophy, the first to use a lentiviral vector to transduce CD34+ PBSC and which 
used full cytoablative conditioning, 10-20% gene marking was obtained in all blood cell lineages 
both subjects for at least one year (Cartier, 2009).  One subject in a clinical trial in France using 
a lentiviral vector carrying a human beta-globin expression cassette attained up to 10% gene 
marking and clinical improvement to the point of becoming transfusion-independent (Cavazzana-
Calvo, 2010). 
   As of October 2016, 20 patients have been treated at UCLA with OTL-101, and the trial is closed 
to enrollment.  At last analysis cut-off date (31 March 2016), 18 evaluable patients have 100% 
overall survival and 100% event-free survival (defined as not returning to ERT or requiring a 
rescue HSCT).  Additionally, 15 evaluable patients (of 18 patients total, including compassionate 
use patients) have been treated with the related product at Great Ormond Street Hospital 
(GOSH)/University College London (UCL) with 100% overall survival and 93.3% event free 
survival.  At UCLA all of the patients with sufficient follow-up have also stopped immunoglobulin 
replacement therapy (IgG RT) and all but one has stopped at GOSH/UCL.    
 
2.1.7 Description of the Study Agent(s)/Intervention(s) 
   The EFS-ADA lentiviral vector: The EFS-ADA lentiviral vector (see Figure 6) was constructed 
in the research laboratory of Adrian Thrasher at University College London, United Kingdom. The 
key goals of this vector are to attain efficient ADA gene transfer with sufficient expression 
with highest safety.  The vector backbone is the pCCL vector, an HIV-1 lentiviral-based, self-
inactivating (SIN) vector, described by Zufferey, et al (1998). SIN vectors have the enhancer and 
promoter elements deleted from the long-terminal repeat (LTR), eliminating the most significant 
vector factor in causing insertional oncogenesis (IO); vector of this design may have lower risks 
for causing IO, improving the outcomes for patients.  Instead of using the vector’s LTR 
enhancer/promoter to drive transcription of the ADA transgene, a codon-optimized human ADA 
cDNA was cloned downstream of the “short” human elongation factor alpha-1 promoter (EFS), 
which lacks the intron and enhancers of the larger EF1-α sequences used in many expression 
plasmids. The “EFS” promoter has been shown to direct high level transcription of reporter genes 
in murine hematopoietic cells and to have significantly reduced trans-activation potential 
compared to γ-retroviral LTR (Zychlinski, et al, 2008).  A WPRE element with the open-reading 
frame eliminated is present to boost titer and gene expression.  
 
 
 
 
 
 
 


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Figure 6. Lentiviral Vector:  EFS-ADA 
 
_______________________________________________________________________ 
huADA cDNA = human ADA cDNA; EFS =  elongation factor 1α short (see text for description); WPRE = woodchuck 
hepatitis post-transcriptional regulatory element. 
   The EFS-ADA lentiviral vector is packaged by co-transfection of its encoding plasmid into 
human 293T cells along with a plasmid encoding the HIV-1 virion and enzymatic polyproteins 
GAG and POL, another plasmid encoding the HIV-1 REV protein to facilitate vector RNA genome 
nuclear export, and the VSV-G protein to pseudotype the vector for stability to allow vector 
concentration and high efficiency transduction of human CD34+ cells.  The concentrated GMP-
grade EFS-ADA vector will be produced at the Indiana University Vector Production Facility under 
contract through the NHLBI Gene Therapy Resource Program. It will be fully characterized to 
meet Release Criteria agreed upon with FDA CBER, including purity (microbial, absence of 
replication competent lentivirus), potency (titer, transduction of human bone marrow CD34+ cells, 
expression of ADA enzymatic activity) and identity (sequence of vector provirus). Lentiviral 
vectors produced by this method of transient transfection have been used in several clinical trials 
without incident. 
 
2.1.8 Summary of Pre-Clinical Studies with Study Agent/Intervention 
2.1.8.1 Evaluation of ADA Lentiviral Vectors 
a. Human hematopoietic and lymphoid cell lines: 
   ADA gene transfer and expression by a series of lentiviral vectors was studied in a panel of 
human cell lines (HT29 colon carcinoma, K562 erythroleukemia, CEM T lymphoblastic, and 
HTVL-1 immortalized ADA-deficient SCID T cells).  The transduced cells were assayed to quantify 
the vector copies (VC)/cell, using qPCR, and the ADA enzymatic activity, using a colorimetric 
biochemical assay.  Expressed ADA enzymatic activity per vector copy was then calculated.  The 
EFS-ADA lentiviral vector (here called CCL-c-sEF1a-hADA – far right bars) expressed ADA 
enzyme activity well (~1-2 U/VC).  It yields a single band on Southern Blot (not shown).   
b. Human CD34+ cord blood cells in vitro:         
   Studies of these lentiviral vectors have also been performed using human CD34+ cells isolated 
from normal umbilical cord blood (see Figure 7).  The CD34+ cells were transduced under the 
culture conditions to be used for this clinical trial (see Section 5.1), testing a range of vector 
concentrations (106, 107 and 108 TU/ml). Additionally, the MMA-hADA (MND-MFG-ADA) γ-
retroviral vector that is currently being used in our ongoing clinical trial was also compared. The 
clinical MND-MFG-ADA vector supernatant produced from a stable PG13 cell clone has a titer of 
approximately 1.8x105 TU/ml.  The transduced CD34+ cells were grown in culture for two weeks, 
HuADA
EFS
U3
R  U5
WPRE
R  U5
U3


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and then cell samples assayed to quantify the vector copies/cell, using qPCR, and the ADA 
enzymatic activity, using a colorimetric biochemical assay.  Expressed ADA enzymatic activity 
per vector copy was then calculated. 
   These studies show that transduction, as measured by vector copy per cell, is directly related 
to the concentration of vector during transduction, with 107 TU/ml leading to approximately 2 
copies/cell for each of the lentiviral vectors.  In contrast, the MMA-hADA γ-retroviral vector 
currently being used in our clinical trial, has a lower titer (1.8x105 TU/ml) and led to only ~0.2 
copies/cell.  Expression of the transferred ADA cDNA was at a similar level by the EFS-ADA 
lentiviral vector to be used in this clinical trial, the MMA-hADA γ-retroviral vector currently in trial 
and a lentiviral vector with the MND LTR (pCSO-re-MCU3-hADA), (~1-2 U/vector copy). 
Figure 7.  ADA activity and vector copy number results of human CD34+ cord blood cells 
trasduced with lentiviral vectors. 
 
2.1.8.2 Transduction of Human CD34+ cells with EFS-ADA 
a.  Human CD34+ bone marrow cells in vivo: 
   Human CD34+ cells from normal donor bone marrow were transduced with the EFS-ADA 
lentiviral vector and transplanted by intravenous injection into neonatal NOD/SCID/γc(null) (NSG) 
mice. After four months, the mice were euthanized and their spleens (Spl), bone marrow (BM), 
and thymuses (Thy) were harvested.  Portions of each organ were analyzed by flow cytometry 
and used to make lysates for ADA enzyme assay and qPCR measurement of vector copies (VC) 
0
2
4
6
8
10
12
14
CSO-MCU3-hADA CCL-sEF1a-hADA CCL-PGK-hADA-
WPRE 
MND-MFG-ADA
Vector copy number per cell
1.0 x 10^8 TU/ml
1.0 x 10^7 TU/ml
1.0 x 10^6 TU/ml
1.8 x 10^5 TU/ml
Lentiviral Vector Transduction of Human CD34+ Cord Blood Cells
(2 week in vitro culture)
0
2
4
6
8
10
12
14
16
18
CSO-MCU3-hADA
CCL-sEF1a-hADA CCL-PGK-hADA-WPRE
MND-MFG-ADA
ADA Activity (Units)
1.0 x 10^8 TU/ml
1.0 x 10^7 TU/ml 
1.0 x 10^6 TU/ml
1.8 x 10^5 TU/ml


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/cell and other portions were used to isolate specific human cell populations by immunomagnetic 
bead separate (CD3+ T cells from spleen, and CD45+ human leukocytes from the bone marrow) 
and these sub-populations were analyzed by FACS and extracted for ADA enzyme assay and 
qPCR for VCN.  
Figure 8. Vector copy number and ADA activity results in human bone marrow CD34+ 
cells into NSG mice. 
 
 
 
 
 
 
 
 
 
   There was a dose-dependent relationship between lentiviral vector concentration during CD34+ 
cell transduction and both the ADA enzyme activity measured (above right) and the vector copy 
per cell (above, left).  Human cells contained approximately 0.1-1 VC/cell and this yielded 1-3 fold 
net ADA enzyme activity compared to the endogenous activity in the mock-transduced cells (from 
normal, not ADA-deficient human donors). The ADA activity per vector copy number was in the 
range of 1-2 U/VC. 
 
 
 
 
 
Human Bone Marrow CD34+ Cells into NSG Mice:
ADA Enzyme Activity After 4 Months
Human Bone Marrow CD34+ Cells into NSG Mice:
Vector Copy Number After 4 months 
Human Bone Marrow CD34+ Cells into NSG Mice:
ADA Activity Per Vector Copy After 4 Months
ADA Activity per Vector Copy


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b.  Human ADA-deficient SCID bone marrow in vitro and in vivo. 
   Small aliquots of bone marrow were obtained from two of the ADA-deficient SCID subjects 
enrolled in the γ-retroviral vector-mediated ADA gene transfer trial during marrow harvest 
(samples 1 and 2B) or during a diagnostic marrow aspiration (sample 2A) under UCLA IRB #09-
04-012-01.  CD34+ cells were isolated from the marrow samples and they were transduced with 
the EFS-ADA vector using the proposed clinical methods, with a final vector concentration in the 
cultures of 3 x 10e7 TU/ml.   Following the single exposure to vector, a portion of the cells was 
injected into NSG mouse neonates and the remaining portion was expanded in short-term culture 
for two weeks and then assayed for ADA enzyme activity and vector copy numbers per cell. 
   In the analysis of the aliquots that were grown in vitro for two weeks, ADA activity in the mock-
transduced cells from the ADA-deficient donors was essentially undetected and was significantly 
increased in the transduced cells. Vector copy numbers per cell ranged from 2-3.5.  The 
calculated ADA activity per vector copy was 1-2.5 U/VC. 
 
Figure 9 
 
 
 
 
0
1
2
3
4
5
6
7
8
9
1
2A
2B
ADA Activity (Units/10^8 cells)
ADA Deficient SCID Bone Marrow Samples
ADA Activity of EFS-ADA Transduced 
ADA-deficient SCID BM CD34+ Cells
(2-week post transduction)
efs-ADA 3x10^7
mock
0
0.5
1
1.5
2
2.5
3
3.5
1
2A
2B
Vector Copies per Cell
ADA-deficient SCID Bone Marrow Samples
Vector Copy Per Cell in EFS-ADA Transduced 
ADA-deficient SCID BM CD34+ Cells 
(2-week post transduction)
efs-ADA 3x10^7
mock


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  The NSG mice were analyzed four months after neonatal injection and analyzed as described 
above (see figures below).   After 4 months, the EFS-ADA transduced cells human ADA-deficient 
cells isolated from the murine tissues had 3-10 fold more ADA enzyme activity than the mock 
transduced cells with 0.1-5.0 vector copies per cell, averaging 1-2 U/VC. 
 
 Figure 10 
 
c.  GMP-comparable batch of EFS-ADA manufactured at the IUVPF.   In a separate study, a 
cryopreserved aliquot of CD34+ cells from sample 2B (above) was used to assess the activity of 
a GMP-comparable batch of EFS-ADA manufactured at the IUVPF.  A sample of EFS-ADA vector 
manufactured at UCLA with less extensive purification was used in comparison and equivalent 
TU/ml of the two preparations were used, following the clinical transduction methods, based on 
titer determination  at UCLA.    
 
 
 
 
 
 
 
 


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Figure 11 
 
 
 
   ADA activity increased proportionately to vector concentration above the low background of the 
mock-transduced ADA-deficient cells, although the rise was steeper with the IUVPF preparation. 
Similar dose-related effects on vector copy number were seen.  The net ADA activity per vector 
copy was between 1-2 U/VC.  These data indicate that the vector preparation from the IUVPF 
was more effective at transducing the human CD34+ cells, but ADA enzyme expression per vector 
copy was similar.   
2.1.8.3 In vitro immortalization (IVIM) assay 
To measure the insertional oncogenesis activity of the EFS-ADA vector, n vitro insertional 
mutagenesis (IVIM) assays were performed with using murine bone marrow depleted of cells 
0
1
2
3
4
5
6
7
UCLA vector
Indiana Univ. Vector
ADA Activity (Units/10^8 cells)
ADA Enzyme Activity of EFS-ADA Transduced 
ADA-deficient SCID BM (ADA-/-) CD34+ Cells
(16 days post transduction)
efs-ADA 3x10^6
efs-ADA 1x10^7
efs-ADA 3x10^7
mock


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expressing mature lineage markers (Lin-) transduced by the EFS-ADA lentiviral vector, the 
SR91GFP positive control gamma-retroviral vector with intact SFFV LTRs previously shown to 
cause a high frequency of progenitor immortalization (Modlich. 2006), the MND-ADA γ-retroviral 
vector or mock-transduced. Cells were cultured for two weeks after transduction under conditions 
which lead to HSC depletion unless a vector integrant causes clonal immortalization. A sample of 
the cells was taken for VCN determination, and then cells were plated by limiting dilution at 100 
cells/well and 1,000 cells/well.  After 2-3 weeks, replating frequency was determined based on 
scoring wells with vigorous growth of cells.   
   The SF91GFP and MMA gamma-retroviral vectors produced abundant clones, with replating 
frequencies between 1x 10e4-10e2.  The frequency of replating by marrow transduced with the 
EFS-ADA lentiviral vector was significantly lower than by the cells transduced with either of the 
gamma-retroviral vectors, with no colonies formed by the EFS-ADA-transduced cells across all 4 
assays performed, similar to mock-transduced marrow. 
  
Figure 12 
 
   In total, these data support the use of the EFS-ADA vector to achieve ADA expression in 
vivo in human cells derived from the transduced CD34+ cells.  It can efficiently and 
reproducibly transfer and express the human ADA cDNA in human CD34+ cells from ADA-
deficient SCID subjects.  
  
2.2 
Rationale 
   Because a small number of ADA-corrected T lymphocytes may have a selective survival 
advantage and lead to immune reconstitution, the current levels of gene transfer using γ-retroviral 


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vectors have been sufficient for clinical benefit in some subjects.  In this Protocol, with the 
improved gene transfer and engraftment of HSC that may be expected using a lentiviral 
vector, we anticipate a more marked clinical benefit as measured by higher gene marking 
and improved immune reconstitution status without PEG-ADA, which will form the 
Secondary Objectives of this study. Pre-clinical studies suggest that the lentiviral vector 
configuration to be used (enhancer-deleted LTR and internal EFS promoter) has a significantly 
lower risk for causing insertional perturbations of cellular genes than the γ-retroviral vectors we 
have used before, although there are no human clinical data.   
   There have been no clinical trials to date utilizing lentiviral vectors for gene-correction of ADA-
SCID.  We will use the EFS-ADA lentiviral vector with the human ADA cDNA to transduce 
autologous CD34+ cells from the bone marrow of ADA-deficient SCID patients.  We will assess 
safety (as primary end-point) and also evaluate the production of gene-containing peripheral 
blood cells, ADA enzyme levels in peripheral blood cells, and immune function (as secondary 
end-points) in twenty (n=20) subjects.  This Phase I/II clinical trial will be conducted at Mattel 
Children’s Hospital at UCLA and at the NIH.  [A parallel study will be performed in the United 
Kingdom at Great Ormond Street Hospital (GOSH)/University College of London (UCL) using the 
same lentiviral vector and a similar clinical trial.]  The study is open to infants and children 
diagnosed with ADA-deficient SCID who do not have a medically eligible, HLA-identical sibling 
donor for bone marrow transplantation.  This trial will evaluate the safety (as primary endpoint) 
and efficacy (as secondary end-points) of a lentiviral vector (EFS-ADA).   
   We hypothesize that the EFS-ADA lentiviral vector will result in increased transduction efficiency 
of the CD34+ cells, compared to the γ-retroviral vectors used previously.  We anticipate that this 
increased transduction will result in better engraftment of gene-corrected stem cells, which will in 
turn result in increased ADA gene marking in peripheral blood cells, more rapid and robust 
production of gene-corrected lymphocytes that express ADA, and a reconstituted immune system 
compared to the current standard of care, hematopoietic stem cell transplant (HSCT).  
 
2.3 
Potential Risks and Benefits 
2.3.1 Potential Risks 
Withholding of PEG-ADA: 
   As part of the treatment protocol, subjects are withdrawn from their PEG-ADA (if they are on it) 
30 days after receiving their gene-modified cells to select for the propagation of gene-modified 
cells after they have been re-infused.  Withdrawal of PEG-ADA can have adverse effects on 
immunity, as well as on other organ systems.  Once PEG-ADA is withdrawn, the numbers of 
circulating T, B, and NK lymphocytes will decline and may remain low for several months until the 
autologous transplant of gene-corrected CD34+ cells leads to immune reconstitution.  It is 
possible that an infection of high severity may develop during this time and this could even be 
fatal if it does not respond to therapy. If immunity is severely decreased after discontinuation of 
enzyme replacement therapy and the subject experiences multiple or serious infections, then 
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Other Risks of infection: 
   It is possible that, because of the trips that are required to participate in the trial, patients may 
get exposed to infections that they could have avoided by not traveling.  This will primarily be a 
consideration during the follow-up after discharge from the hospital.  These patients then will not 
be in isolation and therefore exposed to the potential pathogens found in the public venues they 
visit.  For these patients, it is difficult to clearly assess whether or not traveling constitutes an 
additional risk, and its potential significance.  For patients whose clinical condition is not judged 
stable enough to travel (that is, hospitalized or at home with clinical signs of infection), screening 
or follow-up visits will be rescheduled after the resolution of the clinical problem.      
Busulfan toxicity: 
   Busulfan has been widely used for myeloablation in bone marrow transplantation and has a 
well-described toxicity profile.  When Busulfan is used at high doses (16 mg/kg, which translates 
to approximately 40 mg/m2 x 16 doses) along with cyclophosphamide, as administered prior to 
allogeneic bone marrow transplantation, significant toxicity including thrombocytopenia and 
neutropenia have occurred, Busulfan-related seizures and emesis may occur and are largely 
preventable with prophylactic medication.  Rarely, veno-occlusive disease and pulmonary fibrosis 
(the latter two toxicities seen with Busulfan/cyclophosphamide combinations) have been 
described. However, the nature and extent of toxicity resulting from low-dose Busulfan (e.g. 4 
mg/kg IV x 1) in a pediatric population are not known.  A recent report in ten ADA-SCID children 
demonstrated evidence of neutropenia but suggested no limiting toxicity with 4 mg/kg (Aiuti, A et 
al., 2002, 2009).  These complications of high dose busulfan in combination with other 
chemotherapy drugs has not been seen in any of the more than 25 ADA-deficient patients treated 
using low dose busulfan.   
Busulfan is mutagenic (a clastogenic agent, which induces DNA damage) and another potential 
cause for cancer in this study group.  Although Busulfan is frequently used in combination with 
cyclophosphamide in the clinical setting, there is the risk that it independently can cause cancer, 
even with the low dose proposed in this study.  Also, there is the possibility that a subject will be 
exposed to busulfan without receiving the gene-modified cells.  
Insertional oncogenesis (IO): 
   Risks from IO have occurred in clinical trials of gene transfer using γ-retroviral vectors. After 
immune reconstitution in 9 of 10 subjects participating in an X-SCID gene transfer study in Paris, 
France (Cavazzana-Calvo M et al 2000; Hacein-Bey-Abina S et al 2002), four  of these subjects 
developed leukemia ≥ 2½ years after receiving the gene-transduced CD34+ cells (Hacein-Bey-
Abina, S et al., 2003A; Hacein-Bey-Abina, S et al., 2003B; Hacein-Bey-Abina S et al., 2008).  In 
a similar trial of gene therapy for X-SCID performed in London, England, one of 10 subjects also 
developed T lymphoproliferation (Howe, 2008).  One of these five patients who developed T 
lymphoproliferative disease (LPD) has died and the other four are in leukemia remission and 
continue to have restored immunity from the gene therapy.  For three of the subjects with 
leukemia, the proviral insertion sites have been mapped within or near (3 kb) of the LMO-2 gene 
(Hacein-Bey-Abina, S., et al, 2003), a human proto-oncogene associated with T-cell leukemia 
(Neale et al. 1997; Mead et al. 2001; Rabbitts, T. H. et al. 1998).  In a fourth case, two integrations 


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sites were described:  LMO-2 and BMI1, which is also a proto-oncogene.  In a fifth subject, vector 
had integrated near a third proto-oncogene, CCND2 (Hacein-Bey-Abina S et al., 2008).  These 
are the first reported cases of leukemia developing in subjects participating in a clinical study of 
γ-retroviral-mediated gene transfer to CD34+ cells.  Additionally, a monkey infused with 
hematopoietic progenitors exposed to a γ-retroviral vector died from granulocytic leukemia, 
probably due to vector insertional oncogenesis (Seggeweis et al, 2006). The probability of 
leukemia developing in other participants of the French trial, or in participants of other trials, is not 
known.  To date (10/08/12), 17 ADA-deficient SCID patients have undergone these protocols in 
Milan and six in London (by personal communication from site PIs) and 14 in the CHLA/NIH and 
UCLA/NIH trials.  No cases of T LPD have occurred in the ADA subjects, in sharp contrast to the 
X-SCID results.  
   Currently, there have been no reported cases of insertional mutagenesis in clinical trials using 
a lentiviral vector for gene therapy.  Themis et al (2005) observed oncogenesis using lentiviral 
vector-mediated gene therapy in fetal and neonatal mice, but this has not been shown in humans 
nor primate models.  Modlich (2009) showed that the risks for insertional mutagenesis are lower 
with lentiviral vectors that lack strong γ-retroviral LTR *(as in the EFS-ADA vector) compared to 
γ-retroviral vectors with intact LTR. 
Bone marrow harvest: 
   The principal risk of the bone marrow removal is associated with having general anesthesia.  
General anesthesia can cause problems, such as allergic reactions, breathing or heart problems 
and, rarely, the possibility of death.  Other risks include bleeding, pain, or infection at the bone 
marrow removal sites.  Subjects will receive pain medication to relieve pain as necessary. 
Antibiotics  will be administered as soon as possible if an infection is detected.  Blood tests will 
be done 1-3 days prior to the bone marrow harvest to be sure that subjects do not have a greater 
risk for bleeding.  There is a possibility that the bone marrow harvest will make subjects anemic 
enough to require a blood transfusion. 
CD34+ cell infusion: 
   At the time the transduced cells are infused back into the bloodstream, the subjects may 
experience fever, chills, bronchospasm, and rarely, an anaphylactic reaction, which could be fatal. 
Replication-competent lentivirus (RCL) exposure or other microbial contaminants: 
   The specific preparations of lentiviral vector supernatant used to transduce the CD34+ cells will 
have been screened to ensure the absence of replication-competent lentivirus (RCL).   
Replication-defective lentiviral vectors do not cause any known disease in human beings. 
However, there is a small possibility and risk from this treatment that undetected RCL are present, 
which could cause an active infection in subjects.  However, the residual HIV-1 sequences without 
any open reading frames remaining in the lentiviral vector backbone would not be expected to be 
able to cause pathology.  
   It is also possible that the cells could become infected with bacteria or fungus during the growth 
period in the laboratory.  If there is any evidence of infection of the cells with unwanted agents 
and Busulfan has already been administered, the cells will not be returned to the subject, and the 


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back-up bone marrow will be infused.  However, there is a remote chance that subjects could 
acquire a bacterial or fungal infection from the re-infused cells (this also applies to the back-up 
cells, if administered).   
Allergic/immunological responses to cell processing excipients: 
   Stem Cell Factor (SCF) and Megakaryocyte Growth and Development Factor (MGDF), also 
called thrombopoietin (TPO) are used to maintain stem cell viability and to promote gene transfer 
during the ex vivo transduction process.  When these factors are administered parenterally, they 
may have untoward immunologic or allergic consequences such as thrombocytopenia or allergic 
reactions. The CD34+ cells cultured in these cytokines are thoroughly washed following ex vivo 
exposure.  Since small amounts of residual factors may remain, it is possible that the presence 
of these may result in an adverse immunologic or allergic event.  This hypothetical complication 
has not been reported from any of the clinical trials using these factors for ex vivo transduction.  
Germ-line transmission of vector sequences: 
   Issues of contraception and pregnancy are moot in infants and children before puberty.  Since 
the effects of the lentiviral vector on the fetus is unknown, females of child-bearing potential are 
to use an effective means of birth control during the treatment phase of the study and for at least 
six months after the cell infusion.  Similarly, males are to use at least a barrier method of 
contraception. Females of child-bearing potential must have had a negative pregnancy test within 
three days prior to bone marrow harvest, the morning before receiving Busulfan and again the 
morning of and prior to, cell reinfusion.  Because we will only be putting the EFS-ADA vector into 
the CD34+ cells from bone marrow ex vivo, we do not anticipate that they can enter the germ 
cells. 
Other adverse clinical consequences:  
   Participation in this protocol may have deleterious effects on subsequent attempts at allogeneic 
HSC transplantation.  These adverse effects could be due to "immunization" to normal human 
ADA produced from the transgene.  Most patients, however, will have received bovine PEG-ADA, 
which is more likely to be immunogenic than human ADA; the development of significant inhibitory 
antibodies has been relatively rare in patients treated with PEG-ADA alone.  It is possible that 
non-PEGylated human ADA could be more immunogenic or its expression in cells could induce 
more T cell responses than injected recombinant protein.  Additionally, this protocol could lead to 
restoration of partial immunity, insufficient for complete protection from infections and yet 
sufficient to increase the risks of rejections of allogeneic cells.  This partial immunity may also 
result from PEG-ADA alone, although it may be possible to eradicate this residual activity by 
complete PEG-ADA cessation.  For patients who have not received PEG-ADA the gene transfer 
procedure would introduce these risks de novo, although it is difficult to know a priori the potential 
likelihood of these hypothetical risks. 
   As in any new form of therapy, there may be risks which are unknown or not anticipated.  More 
importantly, the prior 16 patients that were treated by a similar protocol by these investigators did 
not experience any permanently debilitating nor deleterious side effects. 
 


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2.3.2 Potential Benefits 
At this stage, the procedure is experimental.  However, similar studies using γ-retroviral vectors 
to transfer an ADA cDNA into bone marrow CD34+ cells done in performed in Italy, the UK and 
by our group in the US resulted in clinical benefits in the majority of subjects.  It is too early to 
determine whether these patients will have durable, life-long ADA gene expression/enzyme 
production and sustained immune function.  If the gene transfer using the EFS-ADA lentiviral 
vector is more effective than with the γ-retroviral vectors, then it is possible that the subjects will 
develop improved immune function and not require either a bone marrow transplant or further 
PEG-ADA therapy. 
 
3 
STUDY OBJECTIVES 
3.1 
Primary Objectives 
The primary objectives are;  
• to examine the safety of autologous transplantation of bone marrow CD34+ cells transduced 
with the EFS-ADA lentiviral vector (OTL-101).  The safety review will include the identification 
of grade III/IV serious adverse events (SAE).  Subjects will also be monitored for clinical 
toxicities, replication competent lentivirus, and monoclonal expansion at regular intervals by 
a clinician, as well as standard blood counts and chemistries. 
• to estimate overall and event-free survival by 12 months where failure is defined by one of the 
following endpoints:  death; reinstitution of PEG-ADA; or performance of an allogeneic BMT.    
3.2 
Secondary Objectives 
The secondary objectives will assess the efficacy of OTL-101, engraftment of transduced cells 
and the extent of ADA expression and immune reconstitution. The secondary outcomes will:  
1. Assess overall and event free survival at 24 months, 
2. Compare overall survival and event free survival at 24 months between patients 
treated with OTL-101 and patients treated with allogeneic HSCT, 
3. Assess the extent of gene transfer in peripheral blood cells,  
4. Assess ADA gene expression by measuring ADA enzymatic activity and adenine 
nucleotides,  
5. Examine the effects of reconstituting ADA gene expression on immune function 
through serial examination of peripheral blood leukocytes and myeloid cells,  
6. Assess immune reconstitution and infection rates, and 
7. Assess use of immunoglobulin replacement therapy. 
 


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4 
STUDY DESIGN 
4.1 
Description of the Study Design 
   This is a controlled, non-randomized Phase I/II clinical trial to assess the safety and efficacy of 
autologous transplantation of CD34+ cells from the bone marrow of ADA-deficient SCID infants 
and children following human ADA cDNA transfer by the EFS-ADA lentiviral vector.  
   Following provision of informed consent, enrolled subjects will be screened to determine full 
eligibility for participation over 1-2 weeks.  Eligible subjects will undergo bone marrow harvest 
under general anesthesia. The marrow will be processed to isolate CD34+ cells and transduce 
them with the EFS-ADA lentiviral vector.  If sufficient cells are obtained, the subjects will undergo 
marrow cytoreduction with busulfan (4 mg/kg).  If OTL-101 meets all release criteria, the cells will 
be infused IV.  PEG-ADA ERT will be discontinued at day +30.  The initial portion of the study will 
require the subject to be treated in the hospital for a typical duration of 10 days-6 weeks.  After 
discharge from the hospital, the subject will be seen for interval history and examination by either 
their home physician, the PI or a CI and have blood drawn at months 1, 2, 3, 4, 5, 6, 8, 10, 12, 
15, 18, 21, and 24.  Subjects will then be consented for a long-term follow-up study that consists 
of semi-annual to annual visits and lab draws over the subsequent 13 years. 
   The initial purpose of this study is to determine the safety of the procedure in which CD34+ cells 
from bone marrow of ADA-deficient SCID patients are transduced by lentiviral-mediated transfer 
of a normal human ADA cDNA, infused intravenously, and engrafted to produce mature peripheral 
blood leukocytes containing and expressing the ADA cDNA.  Secondary objectives will assess 
the efficacy of this procedure.  Potentially, the development of lymphocytes expressing ADA could 
restore functional immunity in the absence of exogenous PEG-ADA enzyme replacement therapy. 
4.2 
Study Endpoints 
4.2.1 Primary Endpoints 
4.2.1.1 Evaluation of Safety 
Primary Endpoint:   Incidence and grade of Serious Adverse Events (SAE). 
Serious adverse events may be manifest as:   
a) clinical toxicities 
b) exposure to replication-competent lentivirus (RCL)  
c) development of monoclonal expansion or leukoproliferative complications from vector 
insertional effects.  
a)  To detect potential clinical toxicities from the treatment, patients will have interval medical 
histories taken, complete physical examinations, and phlebotomy to obtain peripheral blood for 
laboratory studies  on day +1, and then at months 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 21 and 24 (+/- 
4 weeks).    Laboratory studies performed at these time-points will include CBC with differential 
and platelet count and a chemistry panel and electrolytes.  Toxicities relative to the clinical 
parameters, the CBC with differential, and the chemistry panel are defined by an adapted 
Pediatric Clinical Toxicity Scale from the NIAID, Division of AIDS. (Appendix G) 


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   Patients will also be monitored by clinical examination and laboratory studies in the immediate 
period (3 to up to 6 weeks, depending on clinical course) after infusion of OTL-101 to monitor 
myelosuppression expected from the administered busulfan.  
b)  To detect exposure to replication-competent lentivirus:  
i.  PBMC will be assayed by PCR to detect RCL.  This test will be performed by the National Gene 
Vector Biorepository on samples obtained at baseline (pre-infusion), 3, 6, 12 and 24 months post-
infusion.  Samples will also be collected annually thereafter, and cells archived for PCR assay, to 
be run if any samples are positive. 
ii. Serum for measurement of antibody to RCL will be archived for the presence of antibody to 
VSV-G envelope at baseline (pre-infusion), 3, 6, 12 and 24 months post-infusion (and annually 
thereafter).   
c)  To monitor for monoclonal expansion or leukoproliferative complications, LAM-PCR 
will be performed, as described in Appendix F. 
4.2.1.2 Evaluation of Efficacy 
Primary Efficacy:  Overall/event-free survival (survival without need for BMT or PEG-ADA). 
   Overall/event-free survival will be determined for each subject by 12 months where failure is 
defined by one of the following endpoints: death; reinstitution of PEG-ADA; or performance of an 
allogeneic BMT. 
4.2.2 Secondary Endpoints 
The secondary endpoint is overall survival and event free survival at 24 month. 
The other Secondary efficacy falls under the following outcomes or categories: 
1. 
Efficacy of gene transfer/engraftment of HSC  
2. 
ADA expression, enzyme activity and detoxification  
3. 
Effects on ADA reconstitution on immune phenotype and function (the need for IgRT will be 
assessed under this category) 
4. 
Immune reconstitution  
5. 
Infection rates 
1.  Efficacy of gene transfer/engraftment of HSC  
Secondary Efficacy Endpoint 1:   Determine the frequency of gene marking in PB cells. 
   To assess the efficacy of stem cell transduction/engraftment by serial examination of peripheral 
blood lymphocytes and myeloid cells to quantify the percentages of cells containing the ADA 
cDNA by quantitative DNA-PCR.  We will obtain serial samples of peripheral blood and fractionate 
the leukocytes on ficoll-hypaque to obtain peripheral blood mononuclear cells (PBMC) and 
granulocytes. Genomic DNA isolated from each cell population will be assayed for the frequency 
of cells containing the EFS-ADA vector by quantitative PCR (qPCR).  If sufficient cells are 
available, then the PBMC cells may be sub-fractionated by immunoaffinity or FACS sorting into T 


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cells (CD3+), B cells  (CD19+), NK cells (CD16+/CD56+) and myeloid cells (CD13+/CD14+, or 
CD33+) and DNA from these cell sub-populations assayed by qPCR for vector marking.   
   The presence of transduced peripheral blood leukocytes will demonstrate the transduction and 
engraftment of the manipulated CD34+ cells.  Absence of transduced cells will indicate failure of 
transduction and/or engraftment.  Because of the rapid turnover of mature myeloid cells, the 
continued presence (>6 months) of transduced granulocytes and monocytes, will be taken as 
evidence of transduced/engrafted long-lived stem cells.  Increased levels of transduced T and B 
lymphocytes, above the level of transduced myeloid cells, will indicate a selective advantage to T 
and B progenitors expressing the normal ADA gene.  
Secondary Efficacy Endpoint 2:  Quantitate clonal diversity of vector integrants. 
   The clonal diversity will be quantitated and used as an index of the numbers of vector 
transduced HSC that engrafted in the subjects.  The number of retroviral vector (for the prior and 
current trials) and lentiviral vector (for the proposed trial)  integration sites will be amplified by a 
modified nrLAM-PCR protocol (Paruzynski, et al, 2010) and sequenced via Illumina GAIIx high-
throughput sequencers.  Custom software code has been written to process raw sequence reads 
and determine the genomic positions of integration sites.  We expect to identify >1x 105 integration 
sites per experiment, which will lend significant statistical power to downstream analysis. 
2.  ADA expression and enzyme activity 
Secondary Efficacy Endpoint 3:  ADA enzyme activity in erythrocytes. 
   ADA enzymatic activity in erythrocytes will be measured by Michael Hershfield’s CLIA-certified 
laboratory at Duke University, Durham, North Carolina as a clinical test.  Attainment (yes/no) of 
levels compared to the normal reference range (63±41 nmol/h/mg) will be evaluated. 
Secondary Efficacy Endpoint 4:  Total adenine nucleotides in erythrocytes.  
   The levels erythrocyte deoxyadenosine nucleotides will be determined at each time point (at 
the laboratory of Dr. Michael Hershfield, Duke University).  The level of deoxyadenine nucleotides 
in erythrocytes provides an indirect assessment of systemic detoxification by ADA enzyme 
activity.  While not a primary study end-point, these parameters will be measured to provide 
additional information on the effects of the gene transfer. 
3.  Effects on ADA reconstitution on immune phenotype and function 
   These immunological assays will provide a laboratory assessment of immune function after the 
gene transfer procedure.  
Secondary Efficacy Endpoint 5:  Absolute lymphocytes on CBC 
Absolute lymphocyte counts (ALC) will be determined by routine clinical laboratory complete 
blood counts (CBC). 
Secondary Efficacy Endpoint 6:  Absolute number T, B, and NK lymphocytes  
Absolute number T, B, NK lymphocytes in peripheral blood will be determined by flow cytometry. 
Secondary Efficacy Endpoint 7:  Lymphocyte mitogenic proliferation  


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T lymphocyte proliferative responses to mitogen (phytohemagglutinin – PHA) and to antigens 
(tetanus toxoid after vaccination and Candida albicans) will be determined by tritiated thymidine 
incorporation or carboxyfluorescein succinimidyl ester (CFSE) labeling by routine clinical 
laboratory assay. 
Secondary Efficacy Endpoint 8:  Quantitative immunoglobulins by class  
Serum immunoglobulin levels (IgG, IgA and IgM) will be determined by routine clinical laboratory 
assays. Requirement of immune replacement therapy will be assessed 
Secondary Efficacy Endpoint 9: Specific Antibody responses  
Serum titers of isohemagglutinin antibodies and antibodies to vaccine antigen tetanus toxoid and 
to polyribosylphosphate {PRP} will be determined by routine clinical laboratory tests if 
vaccinations have been administered.  
Secondary Efficacy End-point 10:  T lymphocyte reconstitution 
T lymphocyte production and diversity will be analyzed by measuring PBMC TREC by qPCR and 
vector integration sites by LAM-PCR, and TCR Vβ family usage by FACS. 
Secondary Efficacy End-point 11:  Infection rates will be also measured under immune 
reconstitution as part of the efficacy assessment of a competent immune system 
 
5 
STUDY POPULATION 
5.1 
Description of the Study Population 
Number of subjects to be studied: 
   A total of twenty (20) subjects will be treated under this protocol.  Eligible subjects will be 
children ≥ 1.0 months of age with a diagnosis of ADA-deficient SCID, lacking a medically eligible 
HLA-matched sibling donor and meeting defined inclusion/exclusion criteria related to medical 
status and organ function (vide infra). 
5.1.1 Participant Inclusion Criteria 
Inclusion criteria:  
Participants must satisfy Inclusion Criteria I, II, and III. 
  I.  Children ≥ 1.0 months of age with a diagnosis of ADA-deficient SCID based on: 
A. Decreased ADA enzymatic activity in erythrocytes, leukocytes, skin fibroblasts, or in 
cultured fetal cells to levels consistent with ADA-deficient SCID as determined by 
reference laboratory or confirmed ADA gene mutation(s) known to cause disease 
 
AND  
 
B. Evidence of severe combined immunodeficiency based on either:  
1. Family history of first order relative with ADA deficiency and clinical and laboratory 
evidence of severe immunologic deficiency,      
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  2.  Evidence of severe immunologic deficiency in subject prior to institution of immune 
restorative therapy, based on 
a.  lymphopenia (absolute lymphocyte count <400 cells/mcL) OR absence or low 
number of T cells (absolute CD3+ count <300 cells/mcL) 
OR 
b. severely 
decreased 
T 
lymphocyte 
blastogenic 
responses 
to 
phytohemagglutinin (either <10% of lower limit of normal controls for the 
diagnostic laboratory, <10% of the response of the normal control of the day, 
or stimulation index <10) 
 
II. 
Ineligible for matched sibling allogeneic bone marrow transplantation: 
Absence of a medically eligible HLA-identical sibling, with normal immune function, who may 
serve as an allogeneic bone marrow donor. 
III. Signed written informed consent according to guidelines of the IRB (UCLA Office of 
Human Research Protection Program and National Human Genome Research Institute 
(NHGRI) Institutional Review Board 
5.1.2 Participant Exclusion Criteria 
Exclusion criteria:   
1. Age ≤ 1.0 months 
 Appropriate organ function as outlined below must be observed within 60 days of entering this 
trial.  
2. Hematologic 
   a.   Anemia (hemoglobin < 10.5 g/dl at < 2 years of age, or < 11.5 g/dl at > 2 years of age). 
   b. Neutropenia:  absolute granulocyte count <500/mm3. 
   c.    Thrombocytopenia (platelet count < 150,000/mm3, at any age). 
   d.    INR or PT > 2X the upper limit of normal or PTT > 2.33X the upper limits of normal (patients 
with a correctable deficiency controlled on medication will not be excluded).  
   e.    Cytogenetic abnormalities on peripheral blood or bone marrow or amniotic fluid (if available). 
   f.    Prior allogeneic HSCT with cytoreductive conditioning.   
 
3. Infectious 
   a. Evidence of infection with HIV-1, hepatitis B, Hepatitis C, or parvovirus B 19 by DNA PCR 
within 90 days prior to bone marrow harvest.  If other infection is present, it must be under control 
(e.g. stable or decreasing viral load) at the time of screening. 
4. Pulmonary 


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   a. Resting O2 saturation by pulse oximetry < 95% on room air.  
   b. Chest x-ray indicating active or progressive pulmonary disease. 
5. Cardiac 
   a. Abnormal electrocardiogram (EKG) indicating cardiac pathology. 
   b. Uncorrected congenital cardiac malformation with clinical symptomatology. 
   c. Active cardiac disease, including clinical evidence of congestive heart failure, 
       cyanosis, hypotension.     
   d. Poor cardiac function as evidenced by LV ejection fraction < 40% on echocardiogram.  
6. Neurologic 
   a. Significant neurologic abnormality by examination.   
 
   b. Uncontrolled seizure disorder. 
7. Renal 
   a. Renal insufficiency: serum creatinine  1.2 mg/dl, or  3+ proteinuria.    
   b. Abnormal serum sodium, potassium, calcium, magnesium, phosphate at grade III or IV by 
Division of AIDS Toxicity Scale.  
8. Hepatic/GI: 
   a. Serum transaminases > 5X the upper limit of normal (ULN). 
   b. Serum bilirubin > 2X ULN. 
   c. Serum glucose >  1.5x ULN. 
   d. Intractable severe diarrhea. 
9. Oncologic* (see below) 
   a. Evidence of active malignant disease other than dermatofibrosarcoma protuberans (DFSP) 
b. Evidence of DFSP expected to require anti-neoplastic therapy within the 5 years following 
the infusion of genetically corrected cells 
c. Evidence of DFSP expected to be life limiting within the 5 years following the infusion of 
genetically corrected cells 
10. Known sensitivity to Busulfan 
11. General 
a. Expected survival < 6 months. 
b. Pregnant. 
c. Major congenital anomaly. 


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d. Ineligible for autologous HSCT by the criteria at the clinical site. 
e. Other conditions which in the opinion of the principal investigator and/or co-investigators, 
contra-indicate the bone marrow harvest, the administration of busulfan, infusion of OTL-
101 or indicate the patient or patient’s parents/primary caregivers inability to follow 
protocol.     
* DFSP is a rare, locally invasive tumor with low metastatic potential.  Patients receiving active 
anti-neoplastic therapy for any cancer, including DFSP, are not eligible. Patients with DFSP who 
are not being treated with active anti-neoplastic therapy at the time of enrollment AND have no 
plan to receive active anti-neoplastic therapy in the absence of progressive malignant disease 
AND whose DFSP is not expected to be life-limiting within the five years following the infusion of 
genetically corrected cells are eligible.  
   Patients with DFSP, for whom radiation or chemotherapy has been chosen, would remain 
ineligible during treatment, as the interaction of busulfan and the experimental gene transfer 
vectors with active anti-neoplastic therapy is difficult to predict and could reasonably be expected 
to be deleterious due to overlapping toxicities. When anti-neoplastic therapy is concluded, 
patients with ADA-SCID and a history of DFSP can be included. 
5.1.2.1 Co-enrollment Guidelines 
Co-enrollment: 
   We will request that subjects do not enroll in any other gene therapy for 2 years after the 
autologous infusion.  Since this is a Phase I/II clinical trial, we do not have enough information to 
predict any interactions with other study agents.  We will allow enrollment in other data collection, 
sample banking, and registry studies. 
5.2 
Strategies for Recruitment and Retention 
Recruitment:  
   We will promote recruitment by posting the study on the NIH website for clinical trials, 
http://www.clinicaltrials.gov.  We will also make our best efforts to present our trial at national 
meetings for Immunology, Gene and Stem Cell Therapies, and Bone Marrow Transplantation.  
Retention: 
   This patient population will require close clinical monitoring after the initial admission for the 
harvest, transduction, and re-infusion of cells to observe their immune function status.  To attempt 
to retain subjects for follow-up evaluations, after completing the informed consent process, we 
will also have multiple discussions with the patient’s parent(s)/legal representative (the patients 
are usually minors) and primary physician so that they have a good grasp of the treatment plan 
and what it entails and the rationale for the proposed follow-up studies.  
6 
STUDY AGENT/INTERVENTIONS 
   The Investigational New Drug to be tested is autologous CD34+ cells transduced ex vivo with a 
lentiviral vector based upon the self-inactivating (SIN) lentivirus (HIV-based LV): EFS-ADA 


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(referred in this document as OTL-101).  The vector has been produced as cell-free lentiviral 
supernatant from the 293T cell line, and has an envelope pseudotype from the vesicular stomatitis 
virus (VSV).  The vector will be used for ex vivo transduction of CD34+ progenitor cells from the 
bone marrow of infants or children with adenosine deaminase (ADA)-deficient severe combined 
immunodeficiency (SCID).  OTL-101 will be subsequently infused intravenously after a single non-
myeloablative dosage of busulfan (4 mg/kg intravenously x1). 
   This is a Phase I/II Clinical Trial to evaluate the safety (as primary endpoint) and efficacy (as 
secondary end-points) of the addition of a normal human ADA cDNA by the EFS-ADA lentiviral 
vector to bone marrow CD34+ stem/progenitor cells.  Due to the natural course of the disease, 
these patients require some form of treatment and with that in mind, a placebo group would be 
unethical.  At the current time, the standard of care is a matched sibling allogeneic bone marrow 
transplantation, when a medically-suitable donor exists.  Since our agent is still under 
investigation, if a patient has an HLA matched sibling and qualifies for a bone marrow 
transplantation, they will be excluded from our study.  The other current treatment options have 
their drawbacks and limitations, including high financial cost, need for regular IM injections (PEG-
ADA) or GVHD, dysfunctional immune system, and death from unrelated or mismatched HSCT 
(described in more details above).  
   An “Appendix M” has been submitted to the Recombinant DNA Advisory Committee (RAC) as 
per the Office of Biological Activities (OBA), RAC guidelines and unanimous RAC 
recommendation for approval was obtained on Dec 2, 2009 (#0910-1006). 
6.1 
Study Agent Acquisition 
   The study agent is autologous bone marrow CD34+ cells transduced with the EFS-ADA 
lentiviral vector (OTL-101). The CD34+ cells will be isolated from the bone marrow of each 
subject.  The EFS-ADA lentiviral vector preparation will be produced at the Indiana University 
Vector Production Facility (IUVPF), Indianapolis IN.  The vector supernatant will be shipped to 
UCLA and the NIH on dry ice under UN 2814 (infectious substances) and UN 1845 (dry ice).   
   Cytokines used for ex vivo stimulation of the CD34+ cells during transduction will be purchased 
from commercial sources and qualified for use following guidelines of the FDA.  Recombinant 
fibronectin fragment CH-296 will be supplied from Takara Shuzo, Co, Otsu Japan. CD34+ cells 
will be isolated using the CliniMacs from Miltenyi (with cross-reference to their IDE). 
A.   CD34+ Cell Isolation and Transduction with EFS-ADA 
CD34+ Cell Isolation     
   The bone marrow will be collected from the subjects in an operating room in the Ronald Reagan 
Medical Center (RRMC).  The bone marrow will be transported from the surgical collection site to 
the Bone Marrow/Hematopoietic Progenitor Stem Cell (BM/HPSC) Laboratory (46-126 CHS), 
according to RRMC SOP, for initial processing (sterility sampling and cell count). The back-up 
bone marrow (5 × 107 TNC/kg, or 3 x 107 ficolled MNC/kg) will be removed, if necessary*, and 
cryopreserved according to the SOP of the BM/HPSC Lab.  The bone marrow will be transported 


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to the GMP facility (14-246 Factor), according to the Clinical Gene Therapy Laboratory SOP 
(#7000), for CD34+ cell isolation and transduction.   
   At NIH, all steps for the bone marrow processing for CD34+ cell isolation and EFS-ADA lentiviral 
vector transduction are performed at the Cell Processing Section (CPS) of the Department of 
Transfusion Medicine in the NIH Clinical Center. 
   Bone marrow mononuclear cells will be separated using the standard ficoll-hypaque 
centrifugation method.  The buffy coat layer will be collected and processed to isolate CD34+ 
cells, using the Miltenyi CliniMACS®, following the manufacturer's instructions.  The collected 
CD34+ cell fraction will be characterized for total cell number, % of CD34+ cells, and CFU-GM 
content by CFU assay in methylcellulose.  If the resulting cell number is >5x105 and <1x106 
CD34+ cells/ kg, the isolated CD34+ cells will be cryopreserved as additional back-up, and the 
subject will be offered the option for a second marrow harvest at least one month later.  If sufficient 
CD34+ cells are obtained from the second harvest (≥1x106/kg), the procedure may proceed. If 
insufficient cells are obtained from both harvest(s), the subject will be withdrawn from further study 
participation.  If sufficient CD34+ cells are isolated (≥1x106 / kg), the cells will be processed for 
gene transduction. 
* If the subject’s umbilical cord blood (UCB) was cryopreserved by a clinical stem cell lab at the 
time of birth, the back-up cells may be allocated from the UCB.  If no UCB was collected or if less 
than 5x107/kg TNC are contained in the UCB at the time of cryopreservation, all (if no UCB) or 
the remainder (if insufficient TNC from the UCB) of the back-up cells will be obtained from the 
bone marrow at the time of harvest. 


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B. Certificate of Analysis for Final cell Product 
CHARACTERIZATION OF OTL-101 
Assays during transduction: 
 
 
 
 
Results    
In-process daily bacterial culture  
 
 
 
no growth* 
In-process daily fungal culture  
 
 
 
 
no growth* 
 
 
 
 
 
 
 
 
 
Assay of culture medium at completion of transduction:  
 
Assay for RCL (archived and done if necessary)  
 
no RCL detected   
 
Assays of cells at completion of transduction: 
Cell number1 
 
 
 
 
 
 
≥1x10e6 nucleated cells/kg* 
Viability 
 
 
 
 
 
 
 
≥70% trypan blue exclusion* 
Gram stain of OTL-101   
 
 
no bacteria seen* 
Endotoxin assay of final cell product 
 
 
 
less than 5 EU/kg/hr* 
Bacterial culture of final cell product 
 
 
 
no growth,  14 days 
Fungus culture of final cell product  
 
 
 
no growth, Δ14 days 
Mycoplasma PCR¥ 
 
 
 
 
 
not detected 
Assay for RCL by PCR on cell pellet DNA & co-cultivation on 
1% final cell product (archived & done if necessary) 2 
 
no RCL detected 
FACS analysis of %CD34+ 
 
 
 
 
to be determined 
Vector copy number3   
 
 
 
 
 
to be determined 
Identity assay by PCR 
 
 
 
 
 
presence of expected band 
 
* Release criteria.  Other values will be known after cell infusion. 
¥Performed by Labs, Inc (Centennial, CO) or Rapid mycoplasma detection method of BREL 
Method Number 300200GMP.BSV 
  1Cell counts and viability done by trypan blue/hemacytometer or automated cell counter in 
clinical lab (UCLA). 
  2 Cells and medium will be archived for assay of RCL; if any PBMC samples are positive by 
qPCR for VSV-G sequences, then RCL assays would be run on these archived samples 
3  Performed by ddPCR or qPCR on post-transduction bulk cells after 2 weeks culture 
      
For female subjects of child-bearing age, a serum pregnancy test performed the morning prior 
to infusion must be negative. 
 
C. Action plan for positive culture result from transduction 
     The final cell product is tested by doing gram stain, endotoxin testing for bacteria, and 
cultures for bacteria and fungi and mycoplasma PCR.  If the gram stain or endotoxin is 
positive, then the cells will not be infused and the stored back-up will be infused.  Should any 
of the culture results return positive after the product has been infused, the PI would be 
immediately notified.  The subject will most likely still be hospitalized and will be checked for 
constitutional symptoms (fever, chills, malaise, fatigue).  If any signs or symptoms of bacterial 


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infection exist, the subject would have complete cultures of blood, urine, sputum done and 
chest X-ray performed, if clinically indicated, and broad-spectrum antibiotics initiated.  Should 
any cultures return positive, antimicrobial therapy would be tailored to the sensitivity of the 
specific organism.  If cultures of the final product or of components used in the gene transfer 
matched the culture results of the subject’s samples, the FDA and the IRB would be notified 
for further action plans, and the trial put on hold until the source of contamination is confirmed. 
D. Dosage Calculation 
    After subtraction of the back-up (5 x 107 whole BM TNC / kg, or 3 x 107 ficolled MNC/kg), 
the criteria for sufficient cell number to proceed with Busulfan conditioning is: 
     The successful isolation of CD34+ cells from treatment phase bone marrow harvest is 
defined as ≥ 1 x 106 /kg.  If between 5x105 and 1x106 cells are obtained, they will be 
cryopreserved as additional back-up and the subject offered a second marrow harvest.  If the 
CD34+ cell yield from the second harvest is ≥ 1x106/kg, the cells will be transduced with the 
EFS-ADA lentiviral vector.  If < 1 x 106 /kg cells are obtained from the second harvest, the 
subject will be withdrawn from further participation in the study.  
This number is derived as follows: 
     A standard estimate of the minimal numbers of allogeneic bone marrow total nucleated 
cells used for transplant is at least 5 x 107 cells/kg of recipient weight.  This cell number (or 3 
x 107 ficolled MNC/kg) will be frozen as a back-up.  The back-up will be used if OTL-101 does 
not meet infusion specifications, or if there is engraftment failure evident after +42 days post 
cell infusion  [ANC < 200 or platelet count < 20,000, repeated and confirmed x3 total 
determinations).  With CD34+ cells comprising approximately 1.0% of the mononuclear cells 
in marrow, this translates to an average number of back-up CD34+ cells being approximately 
0.5 x 106/kg.  CD34+ cells will be isolated from bone marrow TNC in excess of 5 x 107/kg or 
ficolled MNC in excess of 3 x 107/kg.  [NOTE:  If the subject’s umbilical cord blood (UCB) was 
cryopreserved by a clinical stem cell lab at the time of birth, the backup cells may be allocated 
from the cord blood.  If less than 5x107/kg TNC are contained in the cord blood at the time of 
cryopreservation, the remainder of the backup cells will be obtained from the bone marrow at 
the time of harvest.  If 5x107/kg TNC are obtained from the UCB for the backup, the volume 
obtained from the bone marrow harvest at the time of treatment, less 2 ml required for 
characterization, will be used to isolate CD34+ cells for the transduction procedure.]  The cells 
will then be transduced with the vector and, after washing, given back to the subject pending 
meeting the release criteria.  If cells do not meet release criteria, they will not be infused.  If 
the subject has already received Busulfan, the subject will be administered only the non-
transduced back-up cells.           
     Therefore, these dosage calculations will be used for assessment of our ability to achieve 
the end-points.  It is difficult to specify the minimal number of transduced CD34+ cells to be 
infused, because it is possible that successful engraftment of transduced stem cells may be 
achieved with lower numbers of engrafting, transduced CD34+ cells than those set by arbitrary 
criteria.  We consider the minimal acceptance criteria for infused, transduced cells to be 0.5 x 
106 total cells / kg.  


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     It should be noted that since the subjects will receive prior non-myeloablative conditioning, 
we will cryopreserve non-transduced cells.  These will be infused if the final product contains 
< 0.5 x 106 cells / kg.  If necessary, these back-up bone marrow cells would be re-infused to 
overcome more severe myelosuppressive effects of the conditioning regimen (persistent ANC 
< 200/µl or platelets < 20,000/µl after day +42  from the day of cell infusion on three 
independent and consecutive determinations over at least ten days).  It is recognized that 
there is no specific information available to extrapolate from the traditional, but empirically-
based, numbers of cells needed to overcome allogeneic transplant barriers to the number of 
ex vivo-transduced, autologous cells needed for engraftment and therapeutic effects.  
Similarly, it should be recognized that transduction of clonogenic progenitors poorly predicts 
transduction of pluripotent stem cells, which can only be assessed by transplantation into 
recipients and observation of cells produced over subsequent months and years.   
     Therefore, these observations of the numbers of CD34+ cells obtained and the 
percentages that become transduced will be recorded. However, if < 1 x 106 CD34+ cells/kg 
are isolated after two harvests, subjects will be withdrawn from the study and not receive 
busulfan.  We will limit total infusion dose to 15 x 106 cells per kg, as approved by the 
FDA/CBER.  If more than 15 X 106 cells per kg are isolated after the culturing period, the 
gene-modified cells that remain after the total maximum dose has been given will be disposed 
or stored for research. 
 
6.1.1 Study Agent/Intervention 
 
6.1.1.1 Formulation, Packaging, and Labeling 
   The study agent is manufactured for individual-specific use with the individual’s (subject’s) 
autologous cells.  Manufacturing will take place in the GMP facility of the Human Gene Medicine 
Program (HGMP) at UCLA or at the Cell Processing Section of the Department of Transfusion 
Medicine (CPS, DTM) of the NIH Clinical Center (FDA registration number 1174694).  Both sites 
will use the same transduction protocol and the same lots of reagents (e.g. vector supernatant, 
cytokines, retronectin).  With the exception of vector DNA PCR and ADA enzyme activity, which 
will be the same at both sites, tests for the Certificate of Analysis (e.g. cell counts, viability, sterility, 
endotoxin, CD34 enumeration, and CFU analysis) will be according to each sites’ SOPs.  As 
UCLA is the lead site, the NIH site will be qualified by the Protocol Manager on behalf of the 
Sponsor to ensure that operations at the NIH are comparable.   Additionally, the NIH will perform 
validation runs to ensure that the endpoints of the Final Certificate of Analysis can be met. 
   Following the transduction procedure, the cells are washed and resuspended in Plasma-lyte A 
with 1% human serum albumin (HSA).  This “final product” is loaded into a sterile syringe, capped, 
and labeled using the following example label or similar: 
 


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   The study agent is stable indefinitely at room temperature and is administered within 3 hours of 
its preparation.  The entire dose of cells that is manufactured for each subject (minus ~10% for 
quality control) will be administered to the subject by I.V. injection. 
6.1.1.2 Preparation, 
Administration, 
Storage, 
and 
Dosage 
of 
Study 
Agent(s)/Intervention(s) 
   The study agent is prepared on the day of administration.  Following the transduction procedure, 
in which the CD34+ cells have been in culture for approximately 36-48 hours, the cells are 
harvested, washed with  Hank’s Balanced Salt Solution (HBSS) + 1% HSA and resuspended in 
25 mL of Plasma-lyte A + 1% HSA and placed in a sterile syringe.  This “final product” is kept at 
room temperature and transported to the infusion site (usually hospital room) in a small “Igloo”-
type cooler, or equivalent per site SOP.  The study agent will be administered within 3 hours of 
its preparation via I.V. injection over 5-15 minutes.  During the administration period and 
afterwards, the subject will be monitored for any adverse events, such as allergic reaction(s).  
TylenolTM and BenadrylTM will be administered approximately ½ hour prior to administration of the 
study agent.   
   The entire contents of the syringe will be administered; there will be no need to store and re-
use “left-over” cells.  The dose of OTL-101 that will be given is a one-time administration of 1 X 
106 to 15 X 106 total cells/kg.  If the number of cells harvested after culturing exceeds 15 X 106 
cells/kg, the remaining cells with be frozen and used for research or discarded, but they will not 
be thawed at a later date to give to the subject.  Because the study agent is a biologic, care must 
be taken in handling this product, such as using gloves.  After the cells have been administered, 
the empty syringe will be disposed of as biohazardous waste. 


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6.1.1.3 Study Agent Accountability Procedures 
   The study agent is manufactured on an individual basis for subject-specific use.  All of the 
manufactured product (less ~10% for QC) will be administered to the subject one time only.  A 
clinical investigator or registered nurse will administer the study agent by I.V. infusion.  Thus, the 
study agent will not be distributed to subjects for self-administration. 
   The reagents used to produce the final cell product (EFS-ADA lentiviral vector, recombinant 
cytokines, recombinant fibronectin) will be kept under locked restricted access only to the study 
investigators.  
6.2 
Assessment 
of 
Participant 
Compliance 
with 
Study 
Agent(s)/Intervention(s)  
   The only time the subjects will be exposed to the study agent is the day they receive their gene-
modified cells.  The subjects are required to make follow-up visits, either to us or to their personal 
physician, at day 1 and months 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 21 and 24 post-infusion of gene-
modified cells for a physical exam and blood draw only (i.e. no more administration of study 
agent). These follow-up visits are scheduled by the physician’s office by actively contacting the 
subject/subject’s parents/legal representative. 
6.3 
Concomitant Medications and Procedures 
   Entry into this study will not affect concomitant treatments for ADA-deficiency, such as 
prophylactic antibiotics or intravenous gamma globulin therapy.  However, the patient will be 
withdrawn from TMP/SMX and/or dapsone by day -2 at least and may not receive these 
medications until ANC > 500 x 3 consecutive days post nadir (i.e., myeloid engraftment).  Use of 
monthly aerosolized or intravenous pentamidine, monthly oral atovaquone or other appropriate 
prophylaxis for Pneumocystis jirovecii must be started by Day + 30 after cell product infusion.     
6.4 
Precautionary and Prohibited Medications and Procedures 
6.4.1 Prohibited Medications and Procedures 
Criteria for PEG-ADA administration 
PEG-ADA will be continued through marrow harvest, conditioning and cell product infusion.  PEG-
ADA will be stopped on day +30 post cell product infusion if the patient received busulfan and the 
final cell product, and does not have active infections or other major medical problems.   
We will recommend restarting PEG-ADA if: 
By 12 months after the cell product infusion there is any one of the following:   
a) no evidence of PBMC or erythrocyte ADA enzyme activity above baseline/pre-
treatment level,  
 
b) no evidence of immune reconstitution (absolute # CD3 cells <200/mm3),   
 
c) no detectable gene-marked granulocytes by ddPCR/qPCR (<1/10,000 cells). 


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The patient will be put back on PEG-ADA, and be followed-up medically. 
PEG-ADA may be re-started if:  
 
 
 
 
  
 
The PI deems it in the best interest of the subject on clinical grounds e.g. multiple, 
 
serious or unresponsive infections or sub-normal immune reconstitution.   
PEG-ADA may be re-stopped (after it has been re-started for above reasons) if: 
Criteria for being off are present: no active infections, PBMC or erythrocyte ADA 
>baseline/pre-treatment levels, absolute CD3+ T cells counts ≥200/mm3, and peripheral 
blood samples are positive for vector sequences by ddPCR/qPCR. 
6.4.2 Precautionary Medications and Procedures 
We also prefer for patients to abstain from myelosuppressive medications, including bactrim for 
Pneumocystis jirovecii prophylaxis.  However, if medications that fit this description are clinically 
warranted, and there are no viable alternatives, then the subject may receive the medication. 
6.5 
Prophylactic Medications and Procedures 
Prophylactic medications: 
   Subjects will receive acetaminophen and diphenhydramine prior to the infusion of OTL-101.  
   As a part of standard care for autologous hematopoietic stem cell transplantation, patients will 
be provided coverage for Pneumocystis jirovecii and mucosal yeast infection until adequate 
immune 
reconstitution 
has 
occurred 
(see 
Section 
7.4). 
Medications 
may 
include 
trimethoprim/sulfamethoxazole, pentamidine, atovaquone, or other effective PJP prophylaxis 
started by Day + 30 after cell product infusion and p.o. fluconazole or nystatin for mucosal thrush 
prophylaxis. Prophylactic anti-viral medications (e.g. acyclovir) may be given for clinical care 
considerations, but are not required in the absence of evidence of infection or exposure to herpes 
or other viruses.  
   Seizures are a rare, but known side effect of busulfan.  To prevent this occurrence, we will 
prophylactically treat our subjects with oral or intravenous Keppra prior to starting busulfan until 
at least 24 hours after completion.  Nausea and vomiting are other side effects of busulfan 
chemotherapy.  Subjects will receive oral or intravenous Ondansetron prior to starting busulfan to 
prevent nausea and vomiting.  
   Standard treatment for patients with SCID includes regular immunoglobulin administered either 
subcutaneously or intravenously. We will continue this through the process.  Patients will not 
receive any other prophylactic medications unless clinically warranted.    
6.6 
Rescue Medications 
   Rescue medications may be needed as a part of standard treatment for autologous 
hematopoietic stem cell transplantation.  If the subjects experience fever while neutropenic, they 
will receive broad spectrum intravenous antimicrobials as per the guidelines at UCLA or NIH.  The 
antimicrobials will be tailored based on clinical status and for any culture sensitivity results.  
Subjects will receive oral acetaminophen and/or oral/intravenous narcotics for pain that may occur 


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as a result of the bone marrow harvest.  Subjects may have a reaction (e.g. rash, hypertension, 
hypotension, dyspnea) to the infusion of autologous cells for which they will be treated per the 
guidelines at UCLA or NIH. Patients may be placed back on PEG-ADA if they fail to achieve 
immune reconstitution and have unresponsive infections.  
7 
STUDY PROCEDURES/EVALUATIONS 
7.1 
OVERALL STUDY PLAN 
   These studies will seek to determine the safety (as primary end-point) and efficacy (as 
secondary end-points) of transplantation of OTL-101to provide functional immunity without 
exogenous enzyme replacement therapy.   Overall, the study procedures are within one of four 
distinct phases: 
1. Pre-treatment 
2. Treatment 
3. Post-treatment  
4. Extended Follow-up 
The following is a brief overview of the Study Plan.  The detailed study plan is described in 
Appendix B:  Schedule of Procedures/Evaluations  
7.2 
Pre-treatment phase: 
Baseline Screening: The first part of the study will involve performing laboratory tests to 
determine whether the subject meets the inclusion criteria and does not have a contra-indication 
causing exclusion.  
Continuation/Cessation of Medications/Procedures:  If enrolled into this study and meeting 
all eligibility criteria, the subject will continue PEG-ADA ERT and prophylactic intravenous or 
subcutaneous immunoglobulin replacement.  Trimethoprim/sulfamethoxazole, if being given, will 
be stopped 5-10 days prior to bone marrow harvest.     
Central venous access:   If the subject does not already have a central venous access device, 
a central venous access device will be placed for ease of phlebotomy and bone marrow cells and 
drug administration. The decision between percutaneous intravenous central catheter (PICC) line, 
a tunneled central venous catheter, or an implanted sub-cutaneous access device will be made 
by discussions with parents and line placement surgeon. A PICC line may instead be placed as 
a separate procedure with appropriate sedation or a central venous line may be placed during the 
general anesthesia for the bone marrow harvest.  
 
 
 


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7.3 
Treatment Phase: 
Bone marrow harvest: Bone marrow harvest will be under general anesthesia to ≤20 ml/kg.   
For patients older than 2 years of age, where our experience indicates that the yield of bone 
marrow CD34+ cells is lower than in younger children, the patient may be asked to have a “back-
up” bone marrow harvest performed approximately 1-3 months prior to the actual gene transfer 
procedure.  The back-up must contain ≥5x107 nucleated cells/kg from whole bone marrow or 
3x107 mononuclear cells from ficolled marrow. Marrow mononuclear cells will be cryopreserved 
and kept as the back-up.  
Back-up bone marrow: A backup bone marrow inoculum (5x107nucleated cells/kg from whole 
marrow or 3x107 mononuclear cells from ficolled marrow) will be cryopreserved, and CD34+ cells 
will be isolated from the remainder.   
Cell sufficiency criteria and resulting action:  Once the CD34+ cells are isolated and counted, 
the number of CD34+ cells/kg is calculated. 
1)  If a sufficient number of cells (≥1 x 106/kg CD34+ cells) are isolated after the back-up 
was removed, the subject will receive busulfan (See Appendix C), the CD34+ cells will be 
transduced with the EFS-ADA lentiviral vector, and OTL-101 will be re-administered if they meet 
release criteria.  
2)  If less than 5 x 105 CD34+ cells/kg are isolated from the initial harvest, the subject 
would meet subject stopping criteria, would not receive busulfan, would be withdrawn from the 
study, would remain on PEG-ADA and would return to routine medical care.   
3)  If between ≥5 x 105 to <1 x 106/kg CD34+ cells are isolated, the CD34+ cells will be 
cryopreserved as additional back-up and PEG-ADA will be continued.    
The patient will be offered to return later for a second collection to isolate the cells for the 
transduction. If >1.0x106 CD34+ cells/kg are isolated from a second harvest (and 
adequate back-up had been cryopreserved from the first harvest), these cells will be 
transduced with the EFS-ADA lentiviral vector, the subject will receive busulfan, and OTL-
101.  
 
4)  If OTL-101 fails to meet any one of the release criteria and the subject has received 
busulfan, the OTL-101 will not be given, the back-up cells will be administered, the subject would 
meet subject stopping criteria, and s/he would be withdrawn from the study, would remain on 
PEG-ADA and would return to routine medical care.   
 
5) If ≤ 0.5 x 106/kg cells are in OTL-101, which meets release criteria, OTL-101 will be 
administered and then the back-up cells will also be given within 1-2 days after OTL-101 has been 
given.   
7.4 
Post-treatment Phase: 
Post-transplant care:  Subjects will be kept in the hospital for close monitoring for at least 7 days 
post-infusion of the cells. However, they may be kept for a longer period as deemed clinically 
necessary, depending on medical care needed (e.g. CVC care, infections, protective isolation, 


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nutritional support), consequences of toxicity to Busulfan, engraftment of transduced cells and 
immune reconstitution.  Alternatively, for out of town subjects who are clinically stable, post-
transplant care may be transferred to the home physician provided they have agreed to assume 
the post-transplant monitoring.   The monitoring can take place as an in-patient or as an 
outpatient.  In this case, the subject will stay a minimum of 3 days after infusion of the cells at the 
treatment facility (UCLA or NIH) prior to being transferred.  This planned discharge and 
subsequent re-admission to a second facility would not constitute an adverse event. 
   PEG-ADA will be stopped on day +30 post-cell product infusion if the patient received busulfan 
and the final cell product and does not have active infections or other major medical problems. 
   For subjects who have slow recovery of counts after transplant (beyond Day +30), CBC/Diff will 
be performed at least once every 7-10 days, until ANC is >500 cells/mm3.  If after day +42 the 
criteria for non-engraftment are met (persistent ANC < 200/µl or platelets < 20,000/µl) after cell 
infusion on three independent and consecutive determinations over at least ten days, the back-
up cells will be given.  If hematologic reconstitution does not occur by Day +90 from the initial 
infusion of the cell product, prolonged unresponsive pancytopenia will exist.  
Infection prophylaxis:  The subjects will be maintained on prophylactic antibiotics and 
intravenous gamma-globulin replacement and will be closely monitored as an outpatient.  They 
will be readmitted to a hospital when deemed clinically necessary. These reasons include, but are 
not limited to: fever and neutropenia, bacteremia, possible central venous access device infection, 
or other infections that require initial parenteral antibiotics, organ failure, extreme adverse social 
situations, and/or bleeding.   
   Prophylactic antibiotics will be used to provide coverage for Pneumocystis jirovecii pneumonia 
and mucosal yeast:   
 
a) Pneumocystis jirovecii prophylaxis (PJP): Trimethoprim/sulfamethoxazole (TMP/SMX) 
will be stopped prior to marrow harvest and an alternative PJP prophylaxis regimen will be used 
at least until ANC>500 for three consecutive measurements, when TMP/SMX may be resumed.  
Suitable alternative PJP prophylaxis medications include pentamidine (intravenous or 
aerosolized) or atovaquone.  Dapsone should be used with caution, due to its potential 
myelosuppressive effects. PCP prophylaxis may be stopped when CD4>200/ul and PHA 
response is >50% lower limit of lab normal response range (Griffith, 2009).    
 
b) Patients will be given a prophylactic antifungal medication, such as oral nystatin or 
fluconazole in standard doses. This may be stopped when CD3>200/ul.  
   Additional or alternative antibiotics may be administered based on clinical care considerations 
(these will be recorded as Concomitant Medications).   
   Intravenous (or SQ) gamma-globulin will be administered at standard dosage and frequency 
(e.g. 400-600 mg/kg q3-4 weeks). Indications for considering stopping immunoglobulin 
replacement include: absolute CD4>200/L, absolute B cell >100/L, IgA or IgM > LLN for age.  
Immunoglobulin replacement may be continued based on clinical care considerations. 


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7.5 
Extended Follow-up Phase:   
Research Evaluations:   After the infusion of the final cell product, follow-up studies will 
determine whether gene-modified cells have engrafted and are able to produce mature lymphoid 
and hematopoietic cells containing and expressing the ADA gene and the extent and time-course 
of immune reconstitution.  Serial samples of peripheral blood will be analyzed for the frequency 
of cells containing the inserted ADA vector over a two year follow-up phase.   
   Stable persistence of immune function in the absence of PEG-ADA treatment will constitute 
evidence that the gene transfer has led to clinically beneficial effects on immune function.  The 
Phase I/II study will also attempt to determine whether the presence of mature lymphoid cells will 
contain and express the EFS-ADA vector in greater frequency than the subjects in the prior γ-
retroviral vector trials and if those cells will contribute to more rapid or robust immune function.  
   If by 6 months after cell product infusion, there is no evidence of PBMC or erythrocyte ADA 
enzyme activity (over baseline/pre-treatment levels), immune reconstitution (<200/L absolute 
CD3+ T) and no detectable ADA gene-marking in granuloctyes by ddPCR/qPCR (<1/100,000), 
PEG-ADA will be re-started. 
Patients will be monitored for clonal expansion, once gene marking levels exceed 1% (see 
detailed monitoring schedule in Appendix F.       
Long-term follow-up:  After completion of the two years of post-treatment evaluation of this 
study, patients will be offered enrollment in a long-term follow-up study.  We will attempt to 
maintain annual contact with all treated subjects and will obtain samples of peripheral blood cells 
and serum annually for archiving at -80oC, to be available for investigation of potential 
complications related to lentiviral-mediated gene transfer, e.g. to assess the presence of serum 
antibodies to lentiviral vector proteins, the presence of replication-competent lentivirus in 
peripheral blood cells or clonal dominance. 
7.6 
Clinical and Laboratory Evaluations 
7.6.1 Clinical and Research Laboratory Evaluations and Specimen Collection 
Follow-up: 
1. Hematology: hemoglobin, hematocrit, WBC count with differential, platelet count [specimen: 
2mL whole blood anti-coagulated with EDTA; method: automated counter (e.g. Coulter counter, 
or equivalent)] 
2. Biochemistry: albumin, creatinine, total bilirubin, ALT, AST, glucose (non-fasting), total 
protein, calcium, electrolytes, alkaline phosphatase, BUN (specimen: 1mL serum; method: 
automated machine) 
 
Special Assays 
1. Measurements of deoxyadenosine metabolites and ADA activity in RBC (specimen: 2mL 
whole blood anti-coagulated with heparin, method: per protocol in Hershfield lab at Duke 
University). 


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2. Measurement of ADA expression in PBMCs by ADA enzymatic assay (specimen: 4mL whole 
blood anti-coagulated with heparin, method: ADA enzyme research assay kit, Diazyme). 
3. Determination of absolute numbers of CD3+, CD4+ and CD8+ T lymphocytes, CD19+ B 
lymphocytes and CD56/CD16+ NK cells (specimen: 4mL whole blood anti-coagulated with EDTA; 
method: flow cytometry); CD4+/CD45RA+ and CD4+/CD45RO sub-sets, if sufficient cells are 
obtained.  
4. T cell proliferative responses to PHA, tetanus toxoid (specimen: 2-8mL whole blood anti-
coagulated with heparin, method: 3H incorporation or CFSE labeling) 
5. T-cell receptor excision circles (TREC):  As a measure of de novo T lymphopoiesis, T-cell 
receptor excision circles (TREC) will be measured in CD3+ PBMC at serial time-points (and CD4 
and CD8 + cells, if available).  This assay will be performed in the Bone Marrow Lab of Great 
Ormond Street Hospital (GOSH) for Children, London, UK, again to provide consistency between 
the US and UK trials.   
6. TCR Vβ family usage on CD3+ PBMC by flow cytometric analysis will be performed at the 
UCLA and NIH Clinical Immunology Labs. 
7. Quantitative determination of immunoglobulin (IgG, IgA, IgM) levels (specimen: 0.5mL serum, 
method: per clinical laboratory) 
8. Determination of specific antibodies to tetanus toxoid and phosphoribosyl phosphate (PRP) 
(~every 3 months if not receiving IVIg; specimen: 0.1mL serum) 
9. Measurement of the frequency of cells containing the inserted ADA gene in PBMC and 
granulocytes, as well as MACS-sorted T cells and myeloid cells (if quantities permit) (specimen: 
4mL whole blood anti-coagulated with heparin or EDTA, method: quantitative PCR or digital 
droplet PCR) 
10. Determination of replication competent lentivirus (RCL) at baseline, 3, 6, 12, and 24 months 
post-infusion and archived annually thereafter (specimen: 2mL whole blood anti-coagulated with 
heparin; method: DNA PCR at NGVB).  
11. Sample banking at baseline, 3, 6, 12, and 24 months post-infusion and annually thereafter, 
for possible western blot testing (WB) for RCL (specimen: 0.5mL serum, method: WB at NGVB). 
12. Leukemia monitoring at baseline, and at ~6-month intervals for the first five years post-
infusion, then annually for an additional 10 years (15-year total monitoring after infusion).  
Performed only if criteria for triggering leukemia determination are fulfilled (see monitoring plan) 
[specimen: 2mL whole blood anti-coagulated with heparin, method: linear amplification mediated 
(LAM)-PCR] (at NGVB) 
13. Integration Site Analysis:  To monitor vector integration sites, both for safety and scientific 
analyses, genomic DNA from PBMC at specific time-points will be analyzed using non-restrictive 
LAM-PCR and bioinformatic analysis at UCLA and NHGRI. 
A general protocol for investigation of possible clonal proliferations that might develop as 
a result of insertional oncogenesis will be followed.  The purposes of the protocol are to: 


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Provide adequate monitoring so that determination of the need for therapeutic 
intervention is 
made as expeditiously as possible; 
Minimize the risk of either physical or psychological harm of unnecessary interventions,  to 
which subjects are exposed;  
Characterize any clonal proliferation events occurring in gene therapy trials;  
Determine whether any clonal proliferation resulted from insertional oncogenesis;  
Characterize the clonality of the normal, genetically-modified hematopoiesis after lentiviral gene 
therapy of HSC. 
 
(A detailed method and protocol are discussed in Appendix F - Monitoring for Monoclonal 
Expansion or Leukoproliferation.) 
 
Specimen Preparation, Handling and Shipping 
 
Biohazard Containment 
   The vector supernatant will be shipped on dry ice under UN 2814 (infectious substances) and 
UN 1845 (dry ice) from IUPVF to UCLA and NIH.  The bone marrow harvest will be performed in 
an Operating Room at the clinical site. These Operating Rooms meet the national standards for 
the Joint Commission for Accreditation of Healthcare Organizations (JCAHO) and Occupational 
Safety and Health Administration (OSHA).  The cells will then be transported for CD34+ isolation 
in a closed container for processing in the Good Manufacturing Practice (GMP) suite and the 
UCLA Bone Marrow-Stem Cell Laboratory or in the Cell Processing Section of the Department of 
Transfusion Medicine (CPS, DTM) of the NIH Clinical Center.  All viral transduction will be 
performed in the GMP suite, which has its own equipment, including a biosafety cabinet, 
incubator, and centrifuge, as well as negative pressure airflow.  The cells will be washed of all 
viral particles prior to being placed in a syringe for transport and re-infusion.  All needles, blood, 
blood products, viral products and biohazard waste will be handled, shipped, and disposed by 
following the rules and regulations set forth by the UCLA Office of Environmental Health and 
Safety and NIH IBC through recommendations from the Centers for Disease Control and 
Prevention and the National Institutes of Health.  Universal precautions will be used when in 
contact with the patient. All infectious specimens will be transported using packaging mandated 
in the Code of Federal Regulations, 42 CFR Part 72.  
 
Instructions for Specimen Storage 
   The specimens collected for RCL testing at regular time-points will be stored until the 24 month 
time-point results are available.  If there are no detected RCL, then the samples will become the 
property of the principal investigator.  These specimens may be disposed properly, used for 
research purposes, or kept in storage indefinitely.  The long-term study will monitor the status of 
the subject to the 15-year mark. If at this time, the subject has hematopoiesis and immune function 
that does not require PEG-ADA replacement, they have been taken off study, or have passed 
away, then the back-up can be properly disposed, used for research purposes, or kept in storage 
indefinitely. 
 


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Specimen Shipment Preparation, Handling and Storage 
   The substances covered under this section include the blood specimens that will be sent to us 
and other labs for the follow-up laboratory evaluations.  Clinical specimens (blood) will be shipped 
under UN 3373 Category B.  All products and specimens will be packed according to IATA DGRs 
according to the appropriate UN category.  Personnel who will perform these functions will have 
certification from the UCLA or NIH Biosafety Offices for shipping biological materials. 
8 
STUDY SCHEDULE 
8.1 Screening 
Obtaining signed informed consent 
   The principal investigator or co-investigators will invite the subject and subject's family to an 
informal meeting to introduce the research study.  Other investigators, research nurses, the study 
coordinator, etc. on the study may also be present to answer questions.  The subject/subject's 
family will be given a sample copy of the informed consent to take home and read at their leisure.  
The consent will have the investigator's contact information, if they have questions about the 
research study. 
   If the subject/legal representative agrees to participate, the informed consent will take place in 
a 
conference 
room. 
 
Before 
the 
actual 
consent meeting, 
the 
investigator 
and 
subject/representative(s) will have already had informal discussions about the study, and a 
sample copy of the consent will have been given to the subject/representative(s) to read ahead 
of time. 
   At UCLA, consent by teleconference will be obtained in some cases, if the subject and his/her 
legal representative(s) live outside of Los Angeles, for the benefit of the subject.  If this is the 
case, the consent will take place in an office or conference room designated by the subject’s 
home physician.  The home physician will be present with the subject/subject’s legal 
representative(s) during the consent by teleconference between the subject/representative(s) and 
investigator. 
   At the informed consent, the consent is reviewed sentence by sentence, with frequent 
determinations (at least every page) whether there are questions from the potential subject/legal 
representative(s)/family.  Any and all questions will be answered by the investigator(s) to the best 
of his ability.   If the subject is between 7-12 years old, he/she will be asked to read the assent 
form and allowed to ask questions.  A child between 7-12 years old must sign the assent form in 
order to participate in the research study.  Once permission has been given (all applicable 
consents signed), the screening tests to determine eligibility will be scheduled.   
Screening 
   Screening tests will be performed within 60 days prior to enrollment.  We may use test results 
that already exist at the time the informed consent is obtained, provided that the date of the results 
fall within the criteria set above.  


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1. Complete history and physical examination including vital signs (temperature, pulse rate, 
respiratory rate and blood pressure).   
2. Blood tests: 
 
 
a) Comprehensive metabolic panel (albumin, AST/ALT, alkaline phosphatase, total 
protein, total bilirubin, creatinine, BUN, glucose, calcium, Na+, K+, Cl-, CO2) 
     
b) magnesium, phosphate 
 
c) CBC with differential and platelet count 
     
d) INR or PT/PTT 
     
e) HIV-1 and hepatitis B, hepatitis C, CMV, and parvovirus B19 by DNA PCR 
 
f) Peripheral blood for cytogenetic analyses (if cytogenetic testing was not performed 
on cells from amniocentesis) 
 
g) Pregnancy test, if female of child-bearing age 
3. Urinalysis: routine urine chemistry and microscopic examination 
4. Electrocardiogram (ECG) 
5. Echocardiogram (Echo) 
6. Chest X-ray (CXR) 
7. Pulse oximetry 
8.   Biopsy of suspicious skin lesions 
9. Confirmation of ADA deficient SCID* 
* Confirmation of ADA-deficient SCID will be based upon biochemical or genetic demonstration 
of ADA deficiency and T lymphopenia at the time of diagnosis.  The date of these results will likely 
be outside of the parameters set above.  
8.2 Enrollment/Baseline  
   If subjects meet inclusion criteria and have no findings causing exclusion, then subjects will be 
enrolled. 
   Physical exam, specified blood tests, and urinalysis will be performed within 3 days of the bone 
marrow harvest procedure. These studies must meet the inclusion criteria to allow enrolled 
subjects to undergo the bone marrow harvest.  Determinations of ADA enzymatic activity, 
erythrocyte deoxyadenosine nucleotide levels, and immune function will be performed after 
screening and before treatment.  PBMCs and serum will be collected and banked for future RCL 
determination. 
1. Complete physical examination including recording of height (cm), weight (kg), and vital signs 
(temperature, pulse rate, respiratory rate and blood pressure). 
2. Blood tests:  
 
a) Comprehensive metabolic panel (albumin, AST/ALT, alkaline phosphatase, total 
protein, total bilirubin, creatinine, BUN, glucose, calcium, Na+, K+, Cl-, CO2) 
     
b) Magnesium, phosphate  


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c) CBC with differential and platelet count 
     
d) INR or PT/PTT 
 
e) Type and cross for 2 units PRBC 
 
f)  Pregnancy test, if female of child-bearing age 
3. Urinalysis: routine urine chemistry and microscopic examination.  
The following labs must be done prior to infusion of OTL-101: 
4. Measurements of leukocyte or erythrocyte ADA enzymatic activity and erythrocyte 
deoxyadenosine nucleotide levels 
5. Measurement of immune function 
a. Determination of absolute numbers of CD3, CD4 and CD8 T lymphocytes, CD19 B lymphocytes 
and CD56/CD16 NK cells. 
b. Proliferative responses to PHA, Tetanus toxoid and Candida 
c. Measurement of serum immunoglobulin levels (IgG, IgA, IgM) 
d. Measurement of specific antibodies to tetanus, polyribosylphosphate (PRP) 
6.  PBMC and serum banking for RCL determination 
8.3 Treatment 
   If physical exam, blood tests, and urinalysis performed within 3 days of the planned bone 
marrow harvest meet the inclusion criteria, the subjects will undergo the bone marrow harvest 
under general anesthesia.  A total volume of no greater than 20 mL/kg subject body weight will 
be collected in order to isolate CD34+ hematopoietic progenitor cells.  Subjects who meet criteria 
to receive Busulfan (enough cells to freeze back-up and to start culture with ≥ 1 x 106 CD34+ 
cells/kg) will remain hospitalized after the bone marrow harvest, for busulfan administration. 
   Busulfan will be administered as a single IV dose (4 mg/kg) over 3 hours approximately 12-24 
hours after the bone marrow harvest (See Appendix C for Schedule for Busulfan Marrow 
Cytoreduction). Peripheral blood samples will be drawn at the following time-points for busulfan 
area-under-the curve (AUC) pharmacokinetic studies:  immediately following the completion of 
the infusion; at 0, 1, 2, 4, 8 and 13 hours after the end of the infusion. These labs may be drawn 
within 15 minutes of the timepoint, but the actual time in reference to the administration of busulfan 
should be recorded for accurate AUC calculation. 
 


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. 
   If the gene-modified CD34+ cells meet acceptance criteria at the end of the culturing period, 
they will be infused intravenously into the subject (approximately 24-48 hours after busulfan 
administration). 
8.4 Follow-up  
   Following the infusion of the gene-modified cells, the subjects will be evaluated and the following 
exams will be performed (if sufficient samples can be obtained): 
Day +1 
1.  Physical Exam 
2.  Safety/toxicity studies: 
 
a) CBC with differential  
 
b) Serum chemistry panel, electrolytes 
 
Months 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 21, and 24 
1.  Physical Exam 
2.  Safety/toxicity studies: 
 
a) CBC with differential  
 
b) Serum chemistry panel, electrolytes 
 
c) PBMC to be analyzed at 3, 6, 12 and 24 months by PCR for RCL and archived annually, 
thereafter.  
 
d) Serum to be banked at 3, 6, 12 and 24 months and annually thereafter, for possible western 
blot testing for RCR. 
 
e)  Monitoring for leukemia (by LAM-PCR) at approximately 6-month intervals for the first five 
years post-infusion, then annually for an additional 10 years (15-year total monitoring after 
infusion) if criteria for triggering LAM-PCR are fulfilled (see monitoring plan). 
3. Efficacy 
a) Measurements of leukocyte ADA enzymatic activity and deoxyadenosine metabolites in 
RBC 
 
b) Immune function testing 


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i)   Determination of absolute numbers of CD3+, CD4+ and CD8+ T lymphocytes CD19+ B 
lymphocytes and CD56/CD16+ NK cells 
 
ii)  Proliferative responses to PHA, tetanus toxoid. 
 
iii) Measurement of serum immunoglobulin levels (IgG, IgA, IgM) (Q 3 months if not 
receiving IVIg) 
 
iv) Measurement of specific antibodies to tetanus toxoid and phosphoribosyl phosphate  
 
v) TREC  
 
vi) TCR Vβ Usage 
4. Gene transduction/expression: 
a) Measurement of the frequency of cells containing the inserted ADA gene in PBMC and 
granulocytes as well as FACS-sorted T cells and myeloid cells by PCR  
b) Measurement of ADA expression in leukocytes by ADA enzymatic assay and possibly RT 
PCR (if samples are positive by DNA-PCR) 
 
c) Vector integration site analysis 
 
   The allowable window for the follow-up exams/tests is ± 4 weeks. 
   After the 24 month time-point, subjects will be enrolled into a separate long-term follow-up 
protocol, in which they will be followed once every six months for an additional 3 years, and then 
once per year, for an additional 10 years.  The total time that they will be followed after treatment 
is 15 years (see monitoring plan described in Appendix F). 
8.5 Final Study Visit 
   The final visit will occur at year 15 following the infusion of the gene-modified cells.  No special 
procedures or evaluations will be performed at that time.  The participant will be given all current 
contact information of the investigators in case of questions or adverse events possibly related to 
the protocol. 
8.6 Early Termination Visit 
   Participants may be removed from the study early, if they have no detectable gene marked cells 
at two consecutive time-points in the second or later years after treatment.  No special procedures 
or evaluations will be performed at this time.  The participant will be given all current contact 
information of the investigators in case of questions. 
   If voluntary withdrawal occurs post-treatment, the participant will be asked to continue 
scheduled evaluations, if possible, and asked to seek appropriate care under medical supervision 
when necessary.  All efforts will be made to continue contact with participants. 
Where available, limited long-term data (on survival, need for a rescue HSCT or PEG-ADA ERT 
reinstitution) will be collected for withdrawn patients after their exit from the study, until the end of 
the expected study follow-up period. 
 
 


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8.7 Pregnancy Visit 
   It is unlikely that participants will become pregnant while on the study as most of our subjects 
will be pre-pubertal infants and children.  However, follow-up visits will occur as scheduled.  
Additionally, participants are only given the study agent once (during the treatment phase), so 
further administration of the study agent is not an issue. 
8.8 Unscheduled Visits 
   If an unscheduled visit is required, a history and physical exam will be performed and laboratory 
tests will be performed depending on the signs and symptoms related to the unscheduled visit. 
9 
ASSESSMENT OF SAFETY 
9.1 
Specification of Safety Parameters 
   Safety is a primary study outcome measure. 
9.2 
Definition of an Adverse Event (AE) 
   Any unfavorable and unintended sign, symptom, or disease temporally associated with the use 
of the investigational agent, without any judgment about causality.  Grading of AEs will be defined 
by an adapted Pediatric Clinical Toxicity Scale from the NIAID, Division of AIDS (see Appendix 
G). 
9.3 
Definition of a Serious Adverse Event (SAE) 
   Serious Adverse Event (SAE): A Serious Adverse Event is defined as an AE meeting one of the 
following conditions: 
1. Death during the period of protocol defined surveillance 
2. Life Threatening Event (defined as a participant at immediate risk of death at the time of the 
event) 
3. An event requiring inpatient hospitalization or prolongation of existing hospitalization during the 
period of protocol defined surveillance (NOTE:  hospital admission that is due to transfer of 
care for post-transplant monitoring will not be considered an SAE.) 
4. Results in congenital anomaly or birth defect 
5. Results in a persistent or significant disability/incapacity 
   Any other important medical event that may not result in death, be life threatening, or require 
hospitalization, may be considered a serious adverse experience when, based upon appropriate 
medical judgment, the event may jeopardize the participant and may require medical or surgical 
intervention to prevent one of the outcomes listed above.  Examples of such medical events 
include allergic bronchospasm requiring intensive treatment in an emergency room or at home, 
blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development 
of drug dependency or drug abuse. 


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9.4 
Methods and Timing for Assessing, Recording, and Analyzing, 
Managing Safety Parameters 
9.4.1 Methods and Timing for Assessment 
   Following the infusion of the gene-modified cells (study agent), the subject will visit a clinical 
investigator at the following time points: day 1, months 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 21, and 
24.  At each time point, a history (solicited) and physical exam and phlebotomy (unsolicited) for 
lab tests will be performed.  The notes of the history and results of the physical and lab tests will 
be used to determine adverse event.  Notes and results will be recorded on case report forms 
(CRFs).  Any physical and medical condition and lab value outside of normal parameters will be 
noted and compared to the NAIAD Pediatric AIDS Toxicity Table (Appendix G) to determine the 
grade of the abnormal parameter.  In years 3-15, subjects will continue to be followed, albeit less 
often (1-2 times/year), for IO monitoring.  The IO monitoring plan is given in Appendix F. 
9.4.1.1 AE/SAE Grading and Relationship Assignment  
   Intensity of AEs will be assigned using the NIAID Pediatric AIDS Toxicity Table (Appendix G) 
Grade 1 (Mild): events require minimal or no treatment and do not interfere with the patient’s daily 
activities. 
Grade 2 (Moderate): events result in a low level of inconvenience or concern with the therapeutic 
measures.  Moderate events may cause some interference with functioning. 
Grade 3 (Severe): events interrupt a patient’s usual daily activity and may require systemic drug 
therapy or other treatment.  Severe events are usually incapacitating. 
Grade 4 (Life threatening):  Any adverse drug experience that places the patient or participant, 
in the view of the investigator, at immediate risk of death from the reaction as it occurred, i.e., it 
does not include a reaction that had it occurred in a more severe form, might have caused death. 
Grade 5 (Death) 
   Relationship assessment of AEs to the study agent/intervention will be made by the principal 
investigator, as follows: 
Definitely Related  
   There is clear evidence to suggest a causal relationship, and other possible contributing factors 
can be ruled out. The clinical event, including an abnormal laboratory test result, occurs in a 
plausible time relationship to study agent/intervention administration and cannot be explained by 
concurrent disease or other drugs or chemicals. The response to the intervention should be 
clinically plausible. The event must be pharmacologically or phenomenologically definitive. 
Probably Related   
   There is evidence to suggest a causal relationship, and the influence of other factors is unlikely. 
The clinical event, including an abnormal laboratory test result, occurs within a reasonable time 
sequence to administration of the study agent/intervention, is unlikely to be attributed to 


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concurrent disease or other drugs or chemicals, and follows a clinically reasonable response on 
withdrawal or study intervention. 
Possibly Related 
   There is some evidence to suggest a causal relationship (e.g., the event occurred within a 
reasonable time after administration of the trial medication).  However, the influence of other 
factors may have contributed to the event (e.g., the subject’s clinical condition, other concomitant 
events). Although an adverse drug event may rate only as “possible” soon after discovery, it can 
be flagged as requiring more information and later be upgraded to probable or certain as 
appropriate. 
Unlikely  
   A clinical event, including an abnormal laboratory test result, whose temporal relationship to 
study agent/intervention administration makes a causal relationship improbable (e.g., the event 
did not occur within a reasonable time after administration of the trial medication) and in which 
other drugs or chemicals or underlying disease provides plausible explanations (e.g., the subject’s 
clinical condition, other concomitant treatments).  
Not related 
   The AE is completely independent of study agent/intervention administration, and/or evidence 
exists that the event is definitely related to another etiology. There will be an alternative, definitive 
etiology documented by the clinician. 
Expected 
   The clinical event has been described in the informed consent, protocol, or I.B. 
   An expected event related to disease process includes decreased immune function 
(lymphopenia) possibly leading to infection requiring hospitalization.  Hospitalization is considered 
a Grade 3/4 AE, which may last one to several days. 
Unexpected 
   The clinical event has not been described in the informed consent, protocol, or IB. 
9.4.2 Recording/Documentation 
   AEs are documented on a CRF.  The AE CRF will collect the following primary information: 
a.  diagnosis 
 
b.  date of onset 
 
c.  maximum toxicity (using DAIDS toxicity scale) 
 
d.  outcome 
 
e.  date of resolution or death 
 
f.  action taken with trial study agent 
 
g.  withdrawal of subject from study (if applicable) 
 
h.  relatedness to study agent 


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i. seriousness assessment 
   Additional information captured on the CRF will include: 
a.  possible causes of the SAE other than trial medication 
b.  relevant medical conditions 
c.  other relevant risk factors 
d.  trial medication details 
e.  relevant concurrent medications 
   Grade 3 or 4 AEs are reported to the PI or study coordinator, as well as to the Sponsor, within 
48 hours.   
9.4.3 Analysis/Management 
   The subjects are followed by a clinician who has been taught the criteria for SAE.  The CRF 
includes a column for entering the date of resolution of the AE.  The CRF is completed by the 
study coordinator and reviewed by the PI.  The PI decides whether new information on the study 
(or other related studies) should be communicated to the subjects. 
9.5 Reporting Procedures 
9.5.1 General Details for Reporting Procedures 
• 
Adverse Events (AEs) Grades 1 and 2 
o 
Grade 1-2 adverse events will be reported by the clinical investigators to the 
sponsor (by AE log) within 1 week of the office visit.  The sponsor will review and 
confirm the grade of AE. The clinician will follow the AE until resolution or stability. 
• 
Serious Adverse Events (SAEs) Grades 3-4 
All Grade 3 and 4 events will be reported to the Sponsor within 48 hours regardless 
of whether the clinical investigator believes that they are drug related. The clinical 
investigator will be responsible for following the event(s) until resolution or stability. 
The site PI will report the SAE to the IRB and the institutional IBC (if applicable) 
within the guidelines dictated by those entities. 
If the sponsor determines that the event meets reporting requirements to the 
overseeing regulatory committees, the sponsor will notify other investigators and 
the regulatory committees, including the DSMB, FDA, and RAC, within the 
guidelines dictated by each agency (see Section 9.5.2).   
• 
Serious Adverse Events  (SAEs): Grade 5 (death)  
o 
The clinical investigator will notify the site PI immediately.  The PI will notify the 
IRB, IBC, and sponsor within 48 hours.  The sponsor will notify the appropriate 
regulatory agencies, including the DSMB, FDA, OBA RAC and DAIT, NIAID within 
the regulatory limits dictated by those agencies. 


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• 
Events that require reporting in an expedited time frame include leukoproliferative disease 
and death. Leukoproliferation is an SAE of special interest for this product; its development 
in this trial would have significant implications for other studies in the field and therefore it 
will be reported in an expedited time-frame. 
9.5.2 Specific Serious Adverse Event Requirements 
9.5.2.1 PI to Sponsor 
   The site PI will report all SAEs, regardless of causality, to the sponsor within 48 hours of the 
event.  In the investigator’s report to the sponsor, the investigator must include an assessment of 
causality (definitely related, probably related, possibly related, unlikely related, or not related), to 
be considered by the Sponsor when determining whether the event constitutes an expedited 
report to other regulatory agencies.  It is the site PI’s responsibility to report the event to the 
institutional IRB and IBC, according to their reporting guidelines. 
9.5.2.2 Sponsor to FDA 
   We will follow the FDA’s Safety Reporting Requirements for INDs (21 CFR part 312) for safety 
reporting. 
   The sponsor will report, in an IND safety report, only a suspected adverse reaction that is both 
serious and unexpected.  The IND safety report will be reported to the FDA no later than 15 
calendar days after the sponsor’s receipt of the information.  Any unexpected fatal or life-
threatening event, associated with the use of the drug, will be reported to the FDA no later than 7 
calendar days after the sponsor’s receipt of the information.  In all IND safety reports, the sponsor 
will identify all safety reports previously filed with the IND concerning a similar adverse experience 
and will analyze the significance of the SAR in light of the previous reports. 
   Other reports:  According to the current reporting guidelines (Final Rule on FDA Safety 
Reporting, September 2010), the sponsor will also report safety information from other sources, 
including other clinical investigations, animal or in vitro studies, scientific literature, and 
unpublished scientific papers.  Increased occurrences of suspected SARs over that listed in the 
protocol or IB will also be reported. 
9.5.2.3 UCLA to DSMB 
    UCLA will report all SAEs, regardless of causality, to the overseeing DSMB according to their 
reporting procedures.  Serious adverse events will be reported within 10 calendar days after the 
sponsor’s receipt of the information; grade 5 SAEs (death) will be reported within 2 days of the 
sponsor’s receipt of information.  In the sponsor’s report to the DSMB, an assessment of causality 
and an analysis of the significance of the SAE in context of the trial will be included. 
9.5.2.4 UCLA to RAC 
   We will follow the NIH Guidelines (Appendix M-I-C-4. Safety Reporting, April 2016) when 
reporting SUSARs to the RAC. 
   Any SAE that is both unexpected and associated with the use of the gene transfer product will 
be reported to the RAC within 15 calendar days after the sponsor’s initial receipt of the information.  


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Any SAE that is fatal or life-threatening, unexpected and associated with use of the gene transfer 
product will be reported to the RAC within 7 calendar days after the sponsor’s initial receipt of the 
information. 
   All reportable SAEs will be followed until satisfactory resolution or until the clinical investigator 
deems the event to be chronic or the participant to be stable. 
   Anticipated SAEs that are not considered expedited reportable events include: 
• Anemia from the bone marrow harvest 
• Thrombocytopenia, neutropenia, and elevated liver enzymes from busulfan 
All SAEs designated as “not related” to the investigational product under study or to the study 
intervention and all non-serious AEs will be recorded and reported to the regulatory agencies in 
the sponsor’s annual report to these agencies. 
9.6 
Reporting Pregnancy 
   The subjects in this study are infants and children, and thus, pregnancy would not apply to them.  
For adolescents in whom preventive measures fail, we will report pregnancies to the DSMB, FDA, 
IRB, ISPRC, NIAID, and RAC.  The treatment period is a confined period within 3 days and a 
HCG is required within 30 days of initiating the procedure.  Even if there should b the unlikely 
event of pregnancy, there would not be any further administration of study agents.  
9.7 
Type and Duration of the Follow-up of Participants after Adverse 
Events 
   Before the subject leaves the office or hospital, a new appointment will be scheduled to follow 
the AE until the AE resolves or is stable.  AEs that are consistent with the procedure/evaluation 
will be noted on CRFs and re-evaluated at the next follow-up appointment (1-3 months). 
9.8 
Modification of Study Agent(s)/Intervention(s) for a Participant 
   This trial involves a single administration of autologous cells and no modifications are planned.  
9.8.1 Dose / Schedule Modifications for a Participant 
   No dose/schedule modifications will be made.  
9.9 
Halting Rules for the Protocol 
   If any one of the following five instances occurs, new enrollment to the protocol or treatment of 
subjects will be halted, pending investigation of the cause.  Findings will be discussed with the 
FDA, IRB and DSMB and each must agree to conditions by which the study can be resumed 
before enrolling the next patient.  Evaluations of study endpoints of subjects already enrolled and 
who received the final cell product will continue to be performed.   
1.  If there is one death or four grade 4 toxicities (except grade IV busulfan-related hematologic 
serious adverse events {transient leukopenia, anemia, thrombocytopenia resolving within 42 days 
of transplant} which are anticipated and will be reported, but will not be considered stopping 


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criteria).  Additionally, neutropenia (a documented disorder of ADA-deficiency) occurring at any 
time, will be graded for severity but will not count towards the stopping rule. 
2.  If two subjects have prolonged unresponsive pancytopenia. This is defined as initial failure at 
hematologic reconstitution which does not improve following administration of the back-up 
marrow. Failure of hematologic reconstitution is defined as at least any two of the following being: 
ANC <200/mm3, and/or platelets < 20,000/mm3 without transfusions, and/or Hb < 8.0 gm/dl 
without transfusions) on three independent and consecutive determinations over at least ten days 
beyond day +42 from initial infusion of the cell product.  If these conditions are reached, the back-
up marrow will be infused IV.  If failure of hematologic reconstitution persist after the back-up 
marrow has been given through day +90 from the initial infusion of the cell product, prolonged 
unresponsive pancytopenia will exist.   
3. If 0 of 3 successive patients show evidence of engraftment of transduced cells by six months 
of follow-up (all peripheral blood samples negative by ddPCR/qPCR for vector sequences 
(<1/10,000) or by ADA enzyme assay). 
4. If RCL is detected, and confirmed in one subject.   
5. If a subject in this study develops hematological proliferative, monoclonal expansion or 
malignant disease (excluding DFSP).  A thorough investigation of the cause of the proliferation, 
expansion or malignancy, including proviral integration analysis, will be done. 
9.10 Stopping Rules for an Individual Participant/Cohort 
   Participants will be removed from study if an adequate cell product cannot be produced 
(inadequate cell number or failure of cell product to meet release criteria). 
   Participants may be removed from the study, if they have no detectable gene marked cells 
(<1/10,000) at two consecutive time-points six months or later after cell product infusion.   
   No special procedures or evaluations will be performed at the time of withdrawal from the study. 
Patients will resume standard medical care for their underlying disease and/or receive other 
treatments at the decision of the patient and primary physician.  The participant will be given all 
current contact information of the investigators in case of questions. Where available, limited long-
term data (on survival, need for a rescue HSCT or PEG-ADA ERT reinstitution) will be collected 
for withdrawn patients after their exit from the study, until the end of the expected study follow-up 
period. 
9.11 Premature Withdrawal of a Participant 
   If voluntary withdrawal occurs post-treatment, the participant will be asked to continue 
scheduled evaluations, if possible, and asked to seek appropriate care under medical supervision 
when necessary.  All efforts will be made to continue contact with participants.  In addition, efforts 
will be made to obtain consent for use of the data collected to the time of withdrawal, but it will be 
determined by the subject or their representative. Where available, limited long-term data (on 


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survival, need for a rescue HSCT or PEG-ADA ERT reinstitution) will be collected for withdrawn 
patients after their exit from the study, until the end of the expected study follow-up period. 
9.12 Replacement of a Participant Who Discontinues Study Treatment 
   Subjects who discontinue study prior to infusion of gene-modified cells will be replaced. A total 
of 20 subjects will be enrolled. 
10 
CLINICAL MONITORING STRUCTURE 
10.1 Site Monitoring Plan 
Overview 
• 
The monitor will be chosen based on qualifications and experience. 
• 
The monitor will be familiar with the study agent, the protocol, laboratory SOPs, informed 
consent, AEs, and regulatory reporting requirements. 
• 
The duties of the monitor include, but are not limited to ensuring that:  investigators are 
qualified; testing facilities are qualified, study reagents are received, stored, and used 
properly; adherence to protocol; CRFs are completed appropriately, AEs are handled 
properly, protocol amendments are submitted properly and communicated to 
investigators, protocol deviations are documented and corrected; findings are 
communicated to the PI/sponsor. 
• 
Refer to UCLA Clinical Gene Therapy Laboratory SOP 9301 for detailed procedure. 
10.2 Safety Monitoring Plan 
   The NHLBI Gene and Cell Therapy Data Safety Monitoring Board provided oversight for the 
conduct of this trial from July 2013 to September 2016.  Safety monitoring oversight was 
transferred to the UCLA Clinical and Translational Science Institute (CTSI) DSMB in September 
2016. 
10.2.1 Safety Review Plan by the DSMB / SMC 
   Reports will be submitted to the DSMB using their established guidelines, which will include 
latest protocol version, confirmation of RAC, IBC, and IRB approval, enrollment numbers, update 
of subjects, status of the study, and timelines. 
11 
STATISTICAL CONSIDERATIONS 
11.1 Background 
   The objectives of the statistical analyses described in this section are to demonstrate the safety 
and efficacy of the fresh formulation of OTL-101, an autologous, genetically modified CD34+ 
Hematopoietic Stem Cells (HSC) for the treatment of severe combined immunodeficiency due to 


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adenosine deaminase deficiency (ADA-SCID). Full details of the planned statistical analyses for 
this study are found in the statistical analysis plan. 
11.2 Design Considerations and Data 
   This is a subject-level historically controlled, prospective, non-randomized Phase I/II clinical trial 
to assess the safety and efficacy of autologous transplantation of CD34+ cells originated from the 
bone marrow of ADA-SCID infants and children following human ADA cDNA transfer by the EFS-
ADA lentiviral vector. 
   Descriptive results will be presented for the study cohort.  
   Comparisons will be made to three cohorts:  
1) A historical control group consisting of ADA-SCID HSCT-treated subjects lacking a 
medically eligible HLA-matched sibling/family donor and treated at GOSH/UCL (UK) 
or Duke University Children’s Hospital, North Carolina Transplant Center (USA) from 
the year 2000 onward 
2) A historical control group consisting of ADA-SCID HSCT-treated subjects having a 
medically eligible HLA-matched sibling/family donor and treated at GOSH/UCL (UK) 
or Duke University Children’s Hospital, North Carolina Transplant Center (USA) from 
the year 2000 onward   
3) All HSCT-treated subjects from GOSH/UCL or Duke University Children’s Hospital, 
who were treated with HSCT from the year 2000 onward, with any type of donor   
 
Details on the historical HSCT control groups from GOSH/UCL and Duke University Children’s 
Hospital can be found in Appendix H. 
 
   Statistical analyses will be performed, and their outcomes presented, in three stages: 
• 
Interim look – 6 months 
• 
Descriptive statistics for all available subjects 
• 
Primary analysis - Comparison on primary efficacy endpoints to the HSCT control 
cohorts—12 month follow-up analysis 
• 
Descriptive statistics for the complete study cohort, with all patients having reached 
at least 12 month follow-up 
• 
Comparison to HSCT control cohorts on safety, primary efficacy endpoints and 
need of immunoglobulin replacement (IgRT) (main secondary efficacy endpoint).  
• 
End of study analysis: 
• 
Descriptive statistics for the complete study cohort, with all patients having 
completed the study (24-month follow-up) 
• 
Comparison to HSCT control cohorts on safety, primary efficacy endpoints and 
need of immunoglobulin Replacement (IgRT) (main secondary efficacy endpoint).  


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   In each study stage and for all analyses, all relevant data available will be used.  
11.3 Endpoints 
   Study endpoints are provided in Section 4.2. A brief summary of study endpoints is provided 
below.  
11.3.1 Safety 
   Safety endpoints in this study are AEs, including SAEs, with intensity/severity graded by 
Common Terminology Criteria for Adverse Events (CTCAE) criteria, including relation to 
treatment. Special attention will be paid to the following AE types: 
• 
Clinical toxicities 
• 
Exposure to replication-competent lentivirus (RCL)  
• 
Development of monoclonal expansion or leukoproliferative complications from vector 
insertional effects. 
11.3.2 Primary Efficacy 
   The primary efficacy endpoints in this trial are OS and EvFS at 12 months. 
11.3.3 Secondary Efficacy 
   The secondary efficacy endpoints in this trial fall under the following outcome categories 
• 
Overal survival and event free survival at 24 months 
• 
Efficacy of gene transfer/engraftment of HSC  
• 
ADA expression, enzyme activity and detoxification 
• 
Effects of ADA reconstitution on immune phenotype and function – note that need of IgRT 
will be assessed under this category 
• 
Immune reconstitution  
• 
Infection rates  
11.4 Sample Size Considerations 
   Due to the ultra-orphan nature of this indication, the number of subjects for this trial is planned 
based on practical considerations. Specifically, the number of ADA-SCID children meeting 
eligibility criteria in the catchment area is expected to be 4-5 per year. Thus,  
N = 20 subjects is a reasonable enrollment number for a completion of the trial in a reasonable 
timeframe.   
   It should be noted that N = 20 will provide precision of 7% for 100% success (survival) at any 
particular time point and 12% precision if one failure is observed in the 20 subjects. Precision is 
here defined as the half-width of the two-sided 95% CI using the Exact Binomial computations.  
In summary, 20 subjects receiving OTL-101 will participate in this trial. 


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11.5 Analysis Sets 
11.5.1 Safety Analysis Set 
   The safety analysis set will consist of all patients treated with OTL-101 at UCLA/NIH (current 
study cohort) and the complete HSCT historical control cohort, consisting of ADA-SCID subjects 
with any type of donor treated with HSCT at either GOSH/UCL (UK) or Duke University Children’s 
Hospital (USA) from the year 2000 onward. 
11.5.2 Full Analysis Set 
The full analysis set will consist of all patients treated with OTL-101 at UCLA/NIH (current study 
cohort). 
 
The primary analysis set for efficacy from the HSCT historical control cohort will consist of ADA-
SCID patients without a medically eligible HLA-matched sibling/family donor and treated with 
HSCT at either GOSH/UCL or Duke University Children’s Hospital from the year 2000 onwards. 
 
Additional efficacy comparisons will be made with: 
• 
ADA-SCID patients with matched related donors treated with HSCT at either 
GOSH/UCL or Duke University Children’s Hospital from the year 2000 onwards; 
• 
the complete HSCT historical control cohort consisting of ADA-SCID patients with any 
type of donor treated with HSCT at either GOSH/UCL or Duke University Children’s 
Hospital from the year 2000 onwards. 
Note however that for the analysis of primary efficacy, full EFS data for HSCT subjects might only 
be available for the 12-month (primary) and 24-month (end of study) analyses. 
11.6 Statistical Methods 
11.6.1 Overview 
   As noted in Section 11.2, the statistical analysis of this data will be done in three stages, which 
are presented below in Sections 11.6.2, 11.6.3 and 11.6.4.  Since all analyses repeat themselves 
in the different stages, each will be described in detail the first time it appears, which will be 
referred to in subsequent sections where it is repeated. 
   The data will be summarized in tables displaying the mean, standard deviation, median, 
minimum, maximum and number of subjects per group for continuous data (e.g. age, weight) or 
in tables displaying count and percentage for categorical data (e.g. gender, previous treatment). 
Data will be presented by visit, if applicable. Data listing by subject will be provided and, where 
applicable, presented graphically. 
   Statistical analyses will be performed and data appendices created with SAS® V9.4 or higher. 


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11.6.2 Interim Look  
   The interim look will include subjects from UCLA OTL-101 (all subjects followed for at least 6 
months) and aggregate data from HSCT treated subjects treated at GOSH (N ~ 25). The following 
sections describe analyses for both groups, although EvFS and most secondary endpoints may 
not be available for the HSCT historical controls at this stage of the analysis. 
11.6.2.1 
Subject Disposition 
   Subject disposition will be done on the safety analysis set, with disposition provided by UCLA, 
and all HSCT subject groups. Subject disposition will be tabulated; the number of enrolled, 
exposed, prematurely terminated, ongoing and completed subjects will be summarized. A list of 
dropouts will be prepared including treatment received, reason for discontinuation, and time of 
discontinuation.  
11.6.2.2 
Baseline Characteristics 
   Baseline characteristics will be done on the safety analysis set, with characteristics presented 
by UCLA, and all HSCT subject cohorts. The following information collected at baseline will be 
presented: 
• 
Demographics (Age, Gender, etc.) 
• 
Method of diagnosis 
• 
Whether received previous HSCT including: date, transplant cells, donor type and outcome 
• 
Whether received previously PEG-ADA. If yes, for how long 
• 
Whether received previously or is receiving currently immunoglobulin replacement therapy 
• 
Listing of relevant medical history, including infections 
11.6.2.3 
Safety Analyses 
   Safety will be analyzed descriptively by arm over time for the safety population. Coding will be 
done using current MedDRA version or higher. AEs, including SAE’s will be presented by: 
• 
System organ class and preferred term 
   Where appropriate, shift tables will be presented for laboratory parameters, vital signs and 
physical examination.  
11.6.2.4 
Efficacy Analyses 
11.6.2.4.1 
Primary Efficacy Analyses 
   The primary efficacy endpoints, overall survival (OS) and event free survival (EvFS), will be 
analyzed descriptively, including frequencies for each type of event for EvFS. Additionally, the 
following analyses will be done to present and compare the treatment arms, OTL-101 subject and 
HSCT historical control groups, so as to provide as complete a picture as possible of efficacy 
given the small sample size: 
1. OS and EvFS will be described using Kaplan-Meier curves for time to death/event 
bytreatment arm, and a comparison between arms made using the log-rank test. 


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2. OS and EvFS rates at 12 months (for those subjects who have reached the 12-month time 
point) will be described by arm and the difference between arms presented along with 
associated exact binomial CIs.  
11.6.2.4.2 
Secondary Efficacy Analyses 
Main Secondary Endpoint 
• 
Overall survival and event free survival at 24 months 
• 
Use of immunoglobulin replacement therapy. 
Additional Secondary Endpoints 
The following will be presented for the infection endpoint: 
• 
Frequency table for number and percent of patients who experienced 0, 1, 2, or at least 3 
infections, 
• 
Rate of infection will be calculated on the basis of person-years.  
11.6.3 Primary Analysis  
   The primary analysis will include data from the current study where all subjects have reached 
the 12-month time point, HSCT historical controls (with or without HLA-matched related sibling 
donor, evaluated separately and together).  
   The following analyses will be repeated, as described in preceding sections: 
• 
Subject disposition (Section 11.6.2.1)  
• 
Baseline characteristics (Section 11.6.2.2)  
• 
Safety (Section 11.6.2.3)  
• 
Efficacy (Section 11.6.2.4) 
11.6.4 End of study Analysis 
   The end of study analysis will repeat the analyses for the preceding section (Section 11.6.3) for 
the study cohort in which all subjects are expected to have completed 24 month follow-up. 
11.7 Safety Review 
11.7.1 Stopping Rules for the Protocol 
   If any of the following five instances occur, the protocol will be stopped, pending investigation 
of the cause.  Findings will be discussed with the FDA, IRB and DSMB before enrolling the next 
patient.  Study endpoints of subjects already enrolled will continue to be obtained. 
1. If there is one death, or four grade 4 toxicities, not Busulfan-related (transient leukopenia, 
anemia, thrombocytopenia resolving within 42 days of transplant, which are anticipated 
and will be reported, will not be considered stopping criteria).  Additionally, neutropenia (a 


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documented disorder of ADA-deficiency) occurring at any time, will be graded for severity 
but will not count towards the stopping rule. 
2. If two subjects have prolonged pancytopenia, defined as at least any two of the following 
(ANC < 200mm3, and/or platelets < 20,000mm3 without transfusions, and/or Hb < 8.0 g/dL 
without transfusions) on three independent and consecutive determinations over at least 
ten days beyond day +42 after CD34+ cell infusion. 
3. If 0 of 3 successive patients show evidence of engraftment of transduced cells by six 
months of follow-up (all peripheral blood samples negative by PCR for vector sequences 
or by ADA enzyme assay). 
4. If RCL is detected, and confirmed, in one subject.   
5. If a subject in this study develops hematological proliferative, monoclonal expansion or 
malignant disease (excluding DFSP), active enrollment of further subjects will be placed 
on hold.  A thorough investigation of the cause of the proliferation, expansion or 
malignancy, including proviral integration analyses, will be done.  
11.7.2 Stopping Rules for an Individual Participant/Cohort 
   This is a Phase I/II Clinical Trial for which there is a single cohort.  Participants may be removed 
from the study early if they have no detectable gene marked cells at two consecutive time-points 
in the second or later years after treatment.  No special procedures or evaluations will be 
performed at this time.  The participant will be given all current contact information of the 
investigators in case of questions. 
11.7.3 Immunogenicity or Efficacy Review 
1. 
Safety (absence of grade III-IV SAE) 
2. 
Overall/event-free survival (survival without need for HSCT or PEG-ADA ERT) 
3. 
Gene-marking by lineage 
4. 
Erythrocyte ADA enzyme activity 
5. 
Absolute lymphocytes on CBC 
6. 
Absolute number T, B, NK lymphocytes in peripheral blood 
7. 
Lymphocyte proliferation to mitogen (PHA) and antigens (tetanus toxoid) 
8. 
Quantitative immunoglobulins by class (IgG, IgA, IgM) 
9. 
Antibody responses [isohemagglutinins, tetanus toxoid, polyribosylphosphate (PRP)] 
 


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12 QUALITY CONTROL AND QUALITY ASSURANCE 
   We will have SOPs for laboratory procedures, training of staff, equipment preventive 
maintenance, data collection, site monitoring, and audits.  Following written standard operating 
procedures, the monitors will verify that the clinical trial is conducted and data are generated, 
documented (recorded), and reported in compliance with the protocol, GCP, and the applicable 
regulatory requirements. The investigational site will provide direct access to all trial related sites, 
source data/documents, and reports for the purpose of monitoring and auditing by the sponsor, 
and inspection by local and regulatory authorities. 
   The study director will implement quality control procedures beginning with the collection of data 
on CRFs.  CRFs will be checked monthly, and any missing data or data anomalies will be 
communicated to the site(s) for clarification/resolution.  This trial will be performed at both UCLA 
and the NHGRI/NIH, and all QC/QA requirements will apply.  Audits will be conducted at least 
annually. 
 
13 ETHICS/PROTECTION OF HUMAN SUBJECTS 
13.1 Institutional Review Board/Ethics Committee 
   This clinical investigation will not begin until an IND is in effect. 
   The Institutional Review Board (IRB) of the Office for Protection of Research Subjects (OPRS) 
at the University of California, Los Angeles (UCLA), which holds a current FWA, will be 
responsible for the initial and continuing review and approval of this study. Any proposed changes 
will be reported to the IRB. 
   The investigation will be conducted in accordance with all other applicable regulatory 
requirements for GCP. 
13.2 Informed Consent Process 
   Informed consent is a process that is initiated prior to the individual’s agreeing to participate in 
the study and continuing throughout the individual’s study participation.  Extensive discussion of 
risks and possible benefits of this therapy will be provided to the participants and their families.  
Consent forms describing in detail the Study Agent(s)/Intervention(s) study procedures and risks 
are given to the participant and written documentation of informed consent is required prior to 
starting study agent/intervention.  Consent forms will be IRB approved and the participant will be 
asked to read and review the document. Upon reviewing the document, the investigator will 
explain the research study to the participant and answer any questions that may arise.  The 
participants will sign the informed consent document prior to any procedures being done 
specifically for the study.  The participants will have sufficient opportunity to discuss the study and 
process the information in the consent process prior to agreeing to participate.  The participants 
may withdraw consent at any time throughout the course of the trial.  A copy of the informed 
consent document will be given to the participants for their records.  The rights and welfare of the 
participants will be protected by emphasizing to them that the quality of their medical care will not 


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be adversely affected if they decline to participate in this study.  We may also conduct the informed 
consent by teleconference with those individuals who live too far away to conveniently travel to 
UCLA or NIH, where the study will be conducted.  In this case, the informed consent discussion 
will take place in the office of the individual’s personal physician with the physician present. 
13.2.1 Assent or Informed Consent Process (in Case of a Minor) 
   Assent will be obtained from individuals between 7-12 years old. 
 
13.3 Exclusion 
of 
Women, 
Minorities, 
and 
Children 
(Special 
Populations) 
   Women, minorities and children will be included.  
13.4  Participant Confidentiality 
   Participant confidentiality is strictly held in trust by the participating investigators, their staff, and 
the sponsor(s) and their agents.  This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participating subjects. 
The study protocol, documentation, data and all other information generated will be held in strict 
confidence.  No information concerning the study or the data will be released to any unauthorized 
third party, without prior written approval of the sponsor. 
   The study monitor or other authorized representatives of the sponsor may inspect all documents 
and records required to be maintained by the Investigator, including but not limited to, medical 
records (office, clinic or hospital) and pharmacy records for the participants in this study.  The 
clinical study site will permit access to such records. 
   All documents and records with identifying information (e.g. name, DOB, medical record 
number, etc.) will be kept in locked cabinets, and only authorized personnel will have access.  
Records will be kept indefinitely, but for a minimum of 2 years following the discontinuation of the 
study for clinical development.  Data that are presented at meetings or conferences will use 
personal identification numbers (PIN) in lieu of actual names. 
13.5 Study Discontinuation 
   If the study is discontinued, no additional subjects will be recruited or treated. Patients already 
treated will be followed by physicians at the responsible institutions.   There is not a placebo arm 
of the study. 
 
 


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14 
DATA HANDLING AND RECORD KEEPING 
14.1 Data Management Responsibilities 
   Following each scheduled post-infusion visit, the clinician sends the source documents history, 
physical examination, and available laboratory reports to the site coordinator.  The site coordinator 
at each site accumulates all source documents (including other lab reports) and completes the 
CRFs by entering data into the web-based electronic CFRs.  The site PI or designated monitor 
reviews the CRFs against the source documents for accuracy.  The source documents from the 
NIH site (history, physical examination, laboratory reports) are scanned and uploaded into the 
web-based data repository.  At the UCLA site, paper print-outs are made of all transmitted source 
documents and they are filed in a folder for each subject as a secondary back-up; source 
documents from UCLA subjects are similarly stored as paper files.  The NIH site retains all original 
signed documents in its files. AEs will be handled as described in Data Safety Monitoring Plan.  
At the end of the study, CRFs and source documents will be kept indefinitely, but for a minimum 
of 2 years following the discontinuation of the study for clinical development.  All data are collected 
by the sponsor (Orchard Therapeutics, Ltd.), who oversees the compilation of data for archiving 
and interpretation 
   The Protocol Monitor will perform 100% source document verification compared to the data in 
the Electronic data Base at least every 6-8 weeks.  The Program Manager or designated person 
will visit the NIH site prior to initiation of the trial and at least annually, while the site is open for 
enrollment, to oversee operations and data management at the site, as described in SOP 
#9301.03 - CLINICAL TRIAL MONITORING.  If the site is closed to enrollment, or has enrolled 
less than 3 subjects, sponsor monitoring may occur remotely using the electronic database and 
e-mail to resolve discrepancies/deficiencies.  An auditor from the UCLA Clinical and Translational 
Science Institute (CTSI) will perform an audit of the trial documents maintained by UCLA, on a 
yearly basis.  The monitor/auditor will report their findings to UCLA and CTSI, and UCLA will 
submit the findings to the UCLA IRB. 


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14.2 Data Capture Methods 
   Electronic CRFs will be used to collect data.  SIStat will assist the investigators in this study with 
all aspects of data management, quality assurance, security and IT support and, in particular, 
electronic CRF forms/AE tracking.  They will also build a customized web-site with public and 
private portals to facilitate dissemination of data and software, as well as research. 
   The same CRFs will be used at both sites (UCLA and NIH) as web-based electronic data 
capture forms.  The data manager or designated person from each site will deposit the data into 
SIStat using their own log-in ID, within 1 month of the subject’s scheduled follow-up visit, to allow 
time for completion of tests, such as lymphocyte proliferation assays (which may take up to 2-3 
weeks to complete). Copies of source documents from the NIH may be requested to verify 
deposited data. 
   All paper documents and records with patient identifying information (e.g. name, DOB, medical 
record number, etc.) will be kept in locked cabinets, and only authorized personnel will have 
access. Data will be stored electronically via SIStat secure servers at UCLA that feature a 
hierarchical system of password protected logins, 128 bit SSL, the secure socket technology used 
for sensitive transactions on the web and failsafe backup systems.   All computers are password 
protected and only authorized trial personnel have access.  Records will be kept indefinitely 
following the close of the study.  Data that are presented at meetings or conferences will use 


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personal identification numbers (PIN) in lieu of actual names and no other patient specific medical 
information will be released. 
   After each follow-up visit, source documents will be collected and used to complete the CRFs 
for the trial (See Data Capture Methods).  Data will be reviewed quarterly by the Clinical Trial 
Monitoring Group to oversee compliance and safety and to produce scheduled and ad hoc reports 
to DSMB and other oversight entities. 
14.3 Types of Data 
   Types of data collected will include:  
a) Medical records that will be maintained on each subject, including medical history, physical 
examination reports and laboratory results obtained by referring physicians prior to 
enrollment or during follow-up, as well as similar information obtained by the clinical site 
investigators in the course of this study 
b) Laboratory tests for the trial end-points, including those assessing safety (CBC, chemistry 
panel, RCL assays, insertional oncogenesis), and efficacy end-points outcome measure 
(ADA enzyme activity, adenine nucleotide levels, gene marking, clonal diversity immune 
function).  
c) Records of investigational materials (receiving, storage, use, shipping and disposal) will 
also be maintained at each site. 
d) Batch records for each clinical cell processing event.  
14.4 Source documents and Access to Source Data/Documents 
   Source documents for subjects enrolled at a clinical site will be kept at that clinical site. Only 
authorized clinical trial personnel (study coordinators, study director, monitor, PI, etc.) will have 
access to source data/documents.  All documents and records with protected health information 
(e.g. name, DOB, medical record number, SSN, etc.) will be kept in locked cabinets, and only 
authorized personnel will have access.  All computers are password protected and only 
authorized trial personnel have access. 
   Reports to regulatory entities (IRB, FDA, DSMB, NIAID) or public presentations will not contain 
protected health information (names, DOB, addresses, SSN, etc), but will refer to subjects using 
anonymous patient identification numbers (PIN) in lieu of actual names and no other patient-
specific protected health information will be released.  Authorized representative from regulatory 
agencies (FDA, IRB, DSMB and the funding NIAID) will be granted access to records for the 
purposes of QA, reviews, audits, safety evaluations, and study progress. 
14.5 Timing/Reports 
   Data and reports are reviewed at least every 1-3 weeks at meetings with trial personnel at the 
coordinating site.  Joint teleconferences between UCLA and the NIH will be held once every 1 or 
2 months (as schedules allow).  Data from CRFs are compiled quarterly for analysis by the 


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Biostatistics group, and these analyses are reviewed by the Sponsor and trial biostatistician 
quarterly to monitor for stopping rule criteria. 
   Routine reports to regulatory agencies are periodic: biannually to the DSMB (based on their 
schedule) and yearly to the IRB, FDA, OBA RAC and DAIT, NIAID.  Final study reports will be 
generated at the completion of the study. No data is masked and data is coded only for the 
purposes of presenting the study to the public.  A master list, which is only accessible to study 
personnel, will be kept to match the individuals to their coded PIN. 
14.6 Study Records Retention 
   At the end of the study, CRFs, source documents, and investigational product records will be 
kept for a minimum of 2 years.  After two years, records will not be disposed without the Sponsor’s 
authorization.  At the end of the study, a copy of all datasets will be provided to NIAID by the 
protocol Monitor or manager on behalf of the Sponsor.   
14.7 Protocol Deviations 
   A protocol deviation is any non-compliance with the clinical trial protocol, the Manual of 
Operations, or the sponsor’s Investigator’s Brochure.  The noncompliance may be either on the 
part of the participant, the investigator, or the study site staff.  Non-compliance events may be 
noted by any trial personnel, either during the conduct of trial activities or during review of records.  
As a result of deviations, corrective actions are to be developed by the site and implemented 
promptly, including filling out prepared worksheets detailing the violation and the corrective action 
that was taken, and notifying the sponsor within a timely manner.  It will be the responsibility of 
the site to use continuous vigilance to identify and report deviations according to the guidelines 
of the IND sponsor. Protocol deviations will be reported to the local IRB per their guidelines and 
in the annual reports to FDA and NIH RAC.  The site PI/study staff will be responsible for knowing 
and adhering to their IRB requirements. 
15 
PUBLICATION POLICY 
   Publication of the results of this trial will be governed by publication policies of the funding 
institutes. Any presentation, abstract, or manuscript will be made available for review by the NIH 
supporters prior to submission. 


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16 
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Meeting #34 http://www.fda.gov/ohrms/dockets/ac/03/ briefing/ 3924B2_1.pdf 
Vassal G, Deroussent A, Challine D, Hartmann O, Koscielny S, Valteau-Couanet D, Lemerle J, 
and Gouyette A.  Is 600 mg/m2 the appropriate dosage of Busulfan in children undergoing bone 
marrow transplantation?  Blood 79: 2475-2479, 1992. 
Weinthal J, Nolta J, Yu XJ, Lilley J, Uribe L and Kohn DB. Expression of human 
glucocerebrosidase following γ-retroviral vector-mediated transduction of murine hematopoietic 
stem cells. Bone Marrow Transp 8: 393-399, 1991. 
Wang L, Robbins PB, and Kohn DB.  High resolution analysis of cytosine methylation in the 5' 
long terminal repeat of γ-retroviral vectors.  Human Gene Therapy, 9:2321-2330, 1998. 
Williams DA. Expression of introduced genetic sequences in hematopoietic cells following γ-
retroviral-mediated gene transfer. Hum Gene Ther 1:229-239, 1990. 
Zaia JA, Rossi JJ, Krishnan A, Ito JT, Castanotto D, Li S, Yam P, Bauer G, Selander D, Csik S, 
Rossi JM, Li H, Bahn V, Tseng W, Sauter S, Molina A, Parker V, Jolly DJ, Sniecinski I, Forman 
SJ, Kohn DB: Autologous stem cell transplantation using retrovirus transduced peripheral blood 
progenitor cells in HIV-infected persons: Comparison of gene marking post-engraftment with and 
without myeloablative therapy. Blood 94(suppl 1): 642a, 1999. 
Zaia JA, et al, Presented at 14th annual ASGT meeting, June 2009 


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Zollino M, Genuardi M, Bajer J, Tornesello A, Mastrangelo S, Zampino G, Mastrangelo R and 
Neri G.  Constitutional Trisomy 8 and myelodysplasia:  Report of a case and review of the 
literature.  Leuk Res 19: 733-736, 1995. 
Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves 
efficient gene delivery in vivo. Nat Biotechnol 15(9):871-5, 1997.   
Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L and Trono D. Self-inactivating 
lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72(12):9873-80, 1998. 
Zychlinski D, Schambach A, Modlich U, Maetzig T, Meyer J, Grassman E, Mishra A, Baum C.  
Physiological promoters reduce the genotoxic risk of integrating gene vectors. Mol Ther 16:718-
25, 2008.  
 


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17   APPENDICES 
 
 
 
 
 
17.2 APPENDIX B:   SCHEDULE OF PROCEDURES/EVALUATIONS ................................. 117 
17.3 APPENDIX C:  EFS-ADA TRIAL BUSULFAN PHARMACOKINETIC ANALYSIS ........... 127 
17.4 APPENDIX D:  SOP FOR HEMATOPOIETIC STEM CELL PRODUCT AND PATIENT 
SPECIMEN TRANSPORT ..................................................................................... 128 
17.5 APPENDIX E:  SOP FOR TRANSDUCTION ............................................................ 133 
17.6 APPENDIX F:  MONITORING FOR MONOCLONAL EXPANSION OR LEUKOPROLIFERATION 
DUE TO INSERTIONAL ONCOGENESIS .................................................................. 134 
17.7 APPENDIX G:  TOXICITY TABLE FOR GRADING SEVERITY OF ADVERSE EXPERIENCES 
(>3 MONTHS OF AGE) ...................................................................................... 138 
17.8 APPENDIX H:  DETAILS OF HISTORICAL HSCT CONTROL GROUPS ........................ 139 
17.9 APPENDIX I:  LIST OF PROTOCOL CHANGES ....................................................... 142 
 
 
 
 
 
 
 


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17.2 Appendix B: Schedule of Procedures/Evaluations 
Evaluation 
Scr 
B-L 
Pre-
op 
Infu
sion 
BU 
+1d 
Months After Procedure 
1 
2 
3 
4 
5 
6 
8 
10 
12 
15 
18 
21 
24 
P.E. 
X 
X 
- 
 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
History 
X 
X 
- 
 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Comprehensive 
Metabolic Panela 
X 
X 
X 
 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Magnesium, 
phosphate 
X 
- 
X 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
CBC with diff. 
X 
X 
X 
 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Cytogenet. PB 
X 
- 
- 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Pregnancy test* 
X 
- 
- 
 
X 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
INR or PT/PTT 
X 
- 
X 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Type and cross 
- 
- 
X 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
HIV-1, HepB & C, 
CMV, B19 
X 
- 
- 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Urinalysis 
X 
- 
X 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
ECG 
X 
- 
- 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Echo 
X 
- 
- 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
CXR 
X 
- 
- 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Pulse ox. 
X 
- 
- 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
RBC dAXP 
- 
X 
- 
 
- 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
RBC 
ADA 
activity 
- 
X 
- 
 
- 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Immune 
functionb 
- 
X 
- 
 
- 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
TREC, TCRV  
panel 
- 
X 
- 
 
- 
- 
- 
- 
- 
- 
- 
X 
- 
- 
X 
- 
X 
- 
X 
PBMC for RCLc 
- 
X 
- 
 
- 
- 
- 
- 
X 
- 
- 
X 
- 
- 
X 
- 
- 
- 
X 
Serum for WB to 
RCLd 
- 
X 
- 
 
- 
- 
- 
- 
X 
- 
- 
X 
- 
- 
X 
- 
- 
- 
X 
PBMC 
for 
vectorc 
- 
X 
- 
 
- 
- 
X 
X 
X 
X 
X 
Xg 
X 
X 
Xg 
X 
Xg 
X 
Xg 
Busulfan PKe 
- 
- 
- 
 
X 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Myelosupp. Labf 
- 
- 
- 
 
 
X 
X 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Scr = screen 
B-L = Baseline   
+1d = day after infusion   
BU = Busulfan:  See Appendix C for details of administration 
X = to be performed, - = not to be performed 
*For female subject of child-bearing potential 
aincludes albumin, AST/ALT, alkaline phosphatase, total protein, total bilirubin, creatinine, BUN, glucose, 
calcium, Na+, K+, Cl-, total CO2 
bincludes lymphocyte subsets phenotype, quantitative immunoglobulins, antibodies to Tet/PRP (Q 3 months 
if not receiving IVIg), T cell proliferation to PHA/Tet (only at 6, 12, 18, and 24 months and only to Tet if 
immunized against tetanus). 


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cAt each time, any unused PBMC will be cryopreserved in liquid nitrogen, for possible future studies. 
dTo be banked for possible future studies 
eBusulfan PK at 0, 1, 2, 4, 8, 13 hours following IV Busulfan administration, done during hospitalization 
fMyelosuppression Laboratory: Hepatic function tests, CBC/Diff, 2x per week for up to 6 weeks, from day 
+1 until hospital discharge when neutrophil recovery is observed, or longer as indicated.  For subjects who 
have slow recovery of counts after transplant (beyond Day +30), CBC/Diff will be performed at least once 
every 7-10 days until ANC is >500 cells/mm.3 
gAccording to current FDA guidelines, LAM-PCR samples will be obtained ≈  every 6 months, for 5 years, 
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Schedule of Procedures/Evaluations: 
I. Pre-treatment phase: 
A. Screening [performed within 60 days of treatment (except for virologies, which may be 
performed up to 90 days)]: 
The first part of the study will involve performing laboratory tests to determine whether the subject 
meets the inclusion criteria and does not have a contra-indication causing exclusion.  
   1. Complete history and physical examination including recording of height (cm), weight (kg),  
       and vital signs (temperature, pulse rate, respiratory rate and blood pressure) 
   2. Blood tests: 
 
Serum chemistries [albumin, AST/ALT, alkaline phosphatase, total protein, total bilirubin, 
creatinine, BUN, glucose, calcium, electrolytes (Na+, K+, Cl-, CO2)] 
Magnesium, phosphate  
CBC with differential and platelet count 
INR or PT/PTT 
HIV-1, hepatitis B, hepatitis C, CMV, and parvovirus B19 by DNA PCR 
Peripheral blood, or for neonates, cells obtained from amniocentesis, for cytogenetic analyses    
 
 
   3. Urinalysis: routine urine chemistry and microscopic examination 
   4. Electrocardiogram (EKG) 
   5. Echocardiogram (Echo) 
   6. Chest X-ray (CXR) 
   7. Pulse oximetry 
8.  Biopsy of suspicious skin lesions 
9.  Confirmation of ADA-deficient SCID* 
*Confirmation of ADA-deficient SCID will be based upon biochemical demonstration of ADA 
deficiency or documented ADA gene mutation(s) known to cause disease and T and CD3+ cell 
lymphopenia at the time diagnosis was made. 
B.  Baseline measurements (performed prior to the infusion of gene-modified cells): 
   1. 
Leukocyte or erythrocyte ADA enzymatic activity and erythrocyte deoxyadenosine 
nucleotide levels 
2. 
Measurement of immune function 
Determination of absolute numbers of CD3, CD4 and CD8 T lymphocytes; CD19 B lymphocytes; 
and CD56/CD16 NK cells 
Proliferative responses to PHA, tetanus toxoid 
Measurement of serum immunoglobulin levels (IgG, IgA, IgM), specific antibodies to 
isohemagglutinins, tetanus toxoid, polyribosylphosphate {PRP} 
TREC and TCRV panel 
PBMC and serum banking for RCL determination 
II. Treatment Phase:  CD34+ cell isolation, transduction and transplant. 
If patients meet inclusion criteria and have no findings causing exclusion, then treatment will be 
performed.  
A. Bone marrow collection: 
A.1. Pre-operative screening: 


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Pre-operative screening studies will be performed within three days prior to marrow harvest.  
These screening tests are in addition to the standard testing that each clinical site may require for 
their autologous hematopoietic stem cell transplant recipients. Pre-operative studies will include: 
  1. CBC with differential and platelets (can be used as the baseline value) 
  2. INR or PT/PTT 
  3. Magnesium, phosphate 
  4. Comprehensive metabolic (albumin, AST/ALT, alkaline phosphatase, total protein, creatinine, 
total bilirubin, calcium, BUN, glucose, electrolytes) (can be used as the baseline value 
  5. Type and cross for 2 units PRBC (to be done within the duration required by  
      the blood bank at the treatment site) 
  6. Routine urinalysis 
  7. Serum HCG (pregnancy test), if female of child-bearing age 
These studies must meet the inclusion criteria to allow bone marrow harvest to be performed.  
A.2. Bone marrow harvest: 
The patient will be brought to the operating room and be placed under general anesthesia.  The 
region of the posterior superior iliac crests will be sterilized with Betadine™ washes and draped 
in a sterile field. Two physicians will simultaneously aspirate marrow from each crest, taking 
multiple 5-10 ml aspirates to a total volume no greater than 20 ml/kg subject body weight 
(maximum set by the NMDP).  After collection of the marrow, sterile pressure dressings will be 
placed over each aspiration site, the patient will be turned over and a PICC line or central venous 
access device will be inserted (if a central venous access device is not already present), and the 
patient then awakened. Alternatively, a PICC line may be placed with appropriate sedation as a 
separate procedure prior to marrow harvest.  The marrow will be transferred into sterile blood 
bags and transported to: 
a) at UCLA:   UCLA Bone Marrow and Stem Cell Processing Laboratory for initial processing and 
then brought to the GMP facility of the Eli and Edythe Broad Center of Regenerative Medicine 
and Stem Cell Research, Factor Building.  
b) at the NIH:  NIH Clinical Center, Building 10, Cell Processing Section (CPS) of the Department 
of Transfusion Medicine.  
B.  Bone marrow characterization: 
     Up to 2 ml of the bone marrow will be used to characterize the cells: 
Determination of nucleated viable cell count by enumeration in a hemocytometer, coupled with 
trypan blue exclusion 
Complete blood count with differential and platelet count 
Flow cytometry analysis to measure the % CD34+ cells in the clinical lab 
Bacterial culture to determine sterility 
C.  Bone Marrow Backup and Dosage Calculations:  
 
C1.  Bone Marrow Backup: 
The volume of the harvested bone marrow corresponding to 5x107/kg of total nucleated cells 
(TNC) or 3x107/kg of mononuclear cells (MNC) from ficolled marrow will be allocated to the Bone 
Marrow and Stem Cell Processing Laboratory for cryopreservation according to their standard 
operating procedure (SOP).  The TNC = the total white blood cell count (WBC) x volume of the 


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marrow sample; the MNC = the total cell count x volume of the ficolled sample.  Characterization 
and cryopreservation of the back-up will be done according to the SOP of the clinical laboratory.  
The successful isolation of CD34+ cells from treatment phase bone marrow harvest is defined as 
≥ 1 x 106 /kg.  If between 5x105 and 1x106 cells are obtained, they will be cryopreserved as 
additional back-up and the subject offered a second marrow harvest.  If the CD34+ cell yield from 
the second harvest is ≥1x106/kg, the cells will be transduced with the EFS-ADA lentiviral vector.  
If < 1 x 106 /kg cells are obtained from the second harvest, the subject will be withdrawn from 
further participation in the study.  
[NOTE:  If the subject’s umbilical cord blood (UCB) was cryopreserved by a clinical stem cell lab 
at the time of birth, the backup cells may be allocated from the cord blood.  If less than 5x107/kg 
TNC are contained in the cord blood at the time of cryopreservation, the remainder of the backup 
cells will be obtained from the bone marrow at the time of harvest.  If 5x107/kg TNC are obtained 
from the UCB for the backup, the volume obtained from the bone marrow harvest at the time of 
treatment, less 2 ml required for characterization, will be used to isolate CD34+ cells for the 
transduction procedure.] 
C2.  Backup and Dosage Calculations:   
A standard estimate of the minimal numbers of allogeneic bone marrow total nucleated cells used 
for transplant is at least 5 x 107 cells/kg of recipient weight.  This cell number, or 3 x 107 MNC/kg 
from ficolled marrow, will be frozen as a back-up.  The back-up will be used if OTL-101 does not 
meet infusion specifications, or if there is engraftment failure evident after +42 days post cell 
infusion  [ANC < 200 or platelet count < 20,000, repeated and confirmed x3 total determinations).  
With CD34+ cells comprising approximately 1.0% of the mononuclear cells in marrow, this 
translates to an average number of back-up CD34+ cells being approximately 0.5 x 106/kg.  
CD34+ cells will be isolated from TNC in excess of 5 x 107/kg. The cells will then be transduced 
with the vector and, after washing, given back to the subject pending meeting the release criteria.  
If cells do not meet release criteria, they will not be infused.  If the subject has already received 
Busulfan, the subject will be administered only the untransduced back-up cells.           
Therefore, these dosage calculations will be used for assessment of our ability to achieve the 
end-points.  It is difficult to specify the minimal number of transduced CD34+ cells to be infused, 
because it is possible that successful engraftment of transduced stem cells may be achieved with 
lower numbers of engrafting, transduced CD34+ cells than those set by arbitrary criteria.  We 
consider the minimal acceptance criteria for infused, transduced cells to be 0.5 x 106 total cells / 
kg.  
It should be noted that since the subjects will receive prior non-myeloablative conditioning, we will 
cryopreserve untransduced cells.  These will be infused if the final product contains < 0.5 x 106 
cells / kg.  If necessary, these back-up bone marrow cells would be re-infused to overcome more 
severe myelosuppressive effects of the conditioning regimen (persistent ANC < 200/µl or platelets 
< 20,000/µl after day +42  from the day of cell infusion on three independent and consecutive 
determinations over at least ten days).  It is recognized that there is no specific information 
available to extrapolate from the traditional, but empirically-based, numbers of cells needed to 
overcome allogeneic transplant barriers to the number of ex vivo-transduced, autologous cells 
needed for engraftment and therapeutic effects.  Similarly, it should be recognized that 
transduction of clonogenic progenitors poorly predicts transduction of pluripotent stem cells, which 


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can only be assessed by transplantation into recipients and observation of cells produced over 
subsequent months and years.   
Therefore, these observations of the numbers of CD34+ cells obtained and the percentages that 
become transduced will be recorded. However, if < 1 x 106 CD34+ cells/kg are isolated after two 
harvests, subjects will be withdrawn from the study and not receive busulfan.  We will limit total 
infusion dose to 15 x 106 cells per kg, as approved by the FDA/CBER.  If more than 15 X 106 cells 
per kg are isolated after the culturing period, the gene-modified cells that remain after the total 
maximum dose has been given will be disposed or stored for research. 
D.  Administration of Busulfan: 
Subjects who meet criteria to receive Busulfan (enough cells to freeze back-up and to start culture 
with ≥ 1 x 106 CD34+ cells/kg) will remain hospitalized after the bone marrow harvest for Busulfan 
administration, infusion of OTL-101, and post-Busulfan monitoring (minimum of 7 days post-
infusion and may be up to approximately 4 months).  Subjects from out of town may be transferred 
after a minimum of 3 days post-infusion for post-busulfan monitoring by their home physician. 
Subjects will be given a loading dose of the anti-convulsant levetiracetam (10 mg/kg IV slow 
infusion) prior to administration of busulfan, followed by maintenance doses of 5 mg/kg IV every 
day x 2 doses. Antiemetic therapy will also be started prior to busulfan therapy. Busulfan will be 
administered as a single IV dose (4 mg/kg) over 3 hours on day -1. Peripheral blood samples will 
be drawn after the busulfan infusion to measure the busulfan area-under-the-curve 
pharmacokinetics.  At least a 24 hour “wash-out period will be observed from completion of the 
busulfan infusion before OTL-101 is administered on day=0.  The levetiracetam will be 
discontinued 24 hours after the Busulfan infusion on Day= 0.     
BMT day 0 marks the end of the treatment phase of the Busulfan partial marrow cytoreductive 
regimen.  Unless otherwise indicated, anti-emetic medication will also have been discontinued.  If 
OTL-101 meets release specifications, it is administered on this day.  If OTL-101 does not meet 
criteria (e.g. gram or stain positive, endotoxin level too high), OTL-101 will not be given, and the 
back-up cells will be administered. 
E.  Busulfan Pharmacokinetic Determination (Refer to Appendix C for details): 
Blood samples for Busulfan levels would be drawn in dark green-top tubes (containing sodium 
heparin) at the following time-points post-infusion of Busulfan:  immediately following the 
completion of the infusion of intravenous Busulfan, and at 1, 2, 4, 8 and 13 hours after the end of 
the infusion.  Pharmacokinetic studies will be performed in a CLIA-certified laboratory (samples 
may be frozen and shipped for later assay). 
F.  Administration of OTL-101: 
A peripheral intravenous line will be established, if there is no PICC line nor a central venous 
catheter.  Patients will be pre-medicated with an appropriate dosage of acetaminophen (10-15 
mg/kg), PO and Benadryl™ (0.5-1.0 mg/kg) IV/PO, 30-60 minutes prior to infusion.  OTL-101 will 
be infused intravenously through the central or peripheral lines into the subject. The infusion will 
take place in the subject’s hospital room at 3F Mattel Children’s Hospital at UCLA or at the 1NW 
Pediatric Ward of the NIH Clinical Center.  The IV infusion will take less than 15 minutes. 
 
 
 


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G.  Post-infusion testing (Day +1): 
The subjects will remain hospitalized following the infusion and will be monitored for side effects 
during and after the infusion.  The monitoring will include measurements of vital signs every 4 
hours for 24 hours after the infusion.  CBC with differential, platelet count, electrolytes, and 
chemistry panel will be evaluated 24 hours post-infusion. 
H.  Procedure for re-infusion of back-up:  
Should infusion of the back-up cells become necessary, the cells will be thawed and re-infused 
according to the clinical laboratory standard operating procedure. 
I.  PEG-ADA Enzyme Replacement Discontinuation:  
PEG-ADA will be stopped on day +30 post-cell product infusion if the patient received busulfan 
and the final cell product and does not have active infections or other major medical problems.  
Management of PEG-ADA ERT is more fully detailed under Section 6.4.1.    
 
III. Post Treatment Phase 
A.  Sample time-points: 
Follow-up studies will be performed (either as an inpatient or outpatient basis) to assess 
achievement of study end-points.  The outpatient follow-up studies will be performed by the 
subject’s home physician in his/her hometown for the subject’s convenience.  At each of the 
scheduled visits (see “Schedule of Investigation” above), the patients will be seen by one of the 
Clinical Investigators.  At each visit, a complete interval history will be taken, including changes 
or onset of new symptoms, opportunistic infections, changes in medications, and other 
therapeutic modalities.  Physical examinations will also be performed by a Clinical Investigator.  
At months 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 21 and 24 (+/- 4 weeks), patients will have phlebotomy 
performed to obtain peripheral blood for studies.  Studies performed at each of these time-points 
will include: 
Safety/toxicity studies: 
1. CBC with differential, platelet count 
2. Serum metabolic panel, including electrolytes 
3. PBMC to be analyzed at baseline, 3, 6, 12, and 24 months by PCR for RCL, then 
archived annually 
4. Serum to be banked at baseline, 3, 6, 12, and 24 months and annually thereafter, for 
possible western blot testing for RCL 
5. Monitoring for leukemia at baseline, and at approximately 6-month (LAM-PCR) intervals 
for the first five years post-infusion, then annually for an additional 10 years (15-year 
total monitoring after infusion) if criteria for triggering LAM-PCR are fulfilled (see 


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Appendix F for detailed Monitoring Plan for Monoclonal Expansion and 
Insertional Oncogenesis) 
6. Measurements of erythrocyte ADA enzymatic activity and deoxyadenosine metabolites 
in RBC 
Immune function testing: 
7. Determination of absolute numbers of CD3+, CD4+ and CD8+ T lymphocytes, CD19+ 
B lymphocytes and CD56/CD16+ NK cells, and CD4+/CD45RA+ and CD4+/CD45RO+ 
8. Proliferative responses to PHA, tetanus toxoid at baseline, 6, 12, 18, and 24 months 
and to tetanus toxoid only after immunization to tetanus 
9. Measurement of serum immunoglobulin levels (IgG, IgA, IgM)  
10. Specific antibodies to tetanus toxoid, polyribosylphosphate (PRP, Q 3 months if not 
receiving IVIg) 
11. TCR V  panel, TREC 
Gene transduction/expression: 
12. Measurement of the frequency of cells containing the inserted ADA gene in PBMC and 
granulocytes as well as FACS-sorted T cells and myeloid cells by PCR 
13. Measurement of ADA expression in leukocytes and erythrocytes by ADA enzymatic 
assay 
Blood samples to be obtained at each1,2 post-infusion time point. 
 
Test 
 
Tube Type 
 
Minimum Vol. 
 
Destination 
Serum metabolic panel, 
including Electrolytes 
 
Red Top Tube 
 
2.0 ml 
 
Clinical Lab 
 
CBC, differential 
 
Purple Top Tube 
 
1.0 ml 
 
Clinical Lab 
 
Erythrocyte 
ADA 
activity and dAXP 
 
Green Top Tube 
(ice) 
 
2.0 ml 
 
Duke/Hershfield 
 
PBMC for Lymphocyte 
Immunophenotype and 
Proliferation and TCR 
Vβ Usage3 
 
Purple Top Tube 
Green Top Tube 
 
2 X 2.0 ml 
4.0 ml 
Clinical Immunology 
Lab 
and 
Gene 
Therapy Core 
PBMC for ADA enzyme 
assay 
(optional, 
for 
research) 
Green Top Tube 
Purple Top Tube 
 
8.0 ml 
5 ml 
 
Gene Therapy Core 
 


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PBMC 
for 
vector 
(ddPCR/qPCR 
and 
VISA), TREC 
 
PBMC for RCL1 
Serum for WB for RCL2 Tiger Top Tube 
2.0 ml 
Gene Therapy Core 
 
Serum for Ig levels 
 
Tiger Top Tube 
2.0 ml 
Clinical Lab 
1  PBMC for RCL will be done at baseline, 3, 6, 12 & 24 months, then archived annually thereafter 
2 Serum (for Western Blot [WB]) will be archived at baseline, 3, 6, 12 and 24 months, then annually thereafter  
3 Flow cytometry, NIH 
 
Other research tests 
These tests will be performed, only as appropriate, to address outcome(s) and mechanism(s) of 
action of the treatment.  For example, we may look at T cell receptor (TCR) spectratyping from 
PBMCs and/or serum cytokine levels.  No extra blood will be drawn for these tests; tests will be 
performed on existing or left-over samples from the follow-up periods defined above. 
Hierarchy of tests if samples are limited: 
Due to the large number of blood tests planned and the limited volumes of blood that may be 
obtained from young children, it may not be possible to perform every scheduled investigation at 
each time-point.  Therefore, the hierarchy for priority of blood tests will be: 
 
1. Safety studies:  CBC w/diff, platelet count, chemistry panel & electrolytes, LAM-PCR 
(see Schmidt et al 2001, for description of method) 
2. Monitoring ADA levels: leukocyte and erythrocyte ADA enzyme levels and RBC dAXP 
levels 
3. Immunologic function studies 
4. Gene marking/expression 
5. Other research tests 
 
B. Diagnostic bone marrow aspirates: 
Bone marrow aspirates may be requested at 1 or 2 time-points in the first two years after 
treatment.  Bone marrow samples will be used to evaluate the presence of cells containing the 
vector sequences by DNA-PCR of BM cells.  These aspirates will be performed under conscious 
sedation, and parents are free to refuse these post-treatment marrow aspirates, as stated in the 
initial informed consent document. 
 
C. Immunizations: 
When patients have evidence of adequate immune reconstitution (absolute CD4+ 100/mm3)at 
least one year after transplantation, immunization will be given with DT (or DPT, DTaP, or Tdap) 
and IPV every two months, for a total of three dosages.  For children who have IVIg administration 


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stopped, vaccination will follow guidelines for post-allogeneic HSCT vaccinations as in Griffith et 
al, JACI 2009.  If they are below normal, re-immunization with DT will be given.  Annual seasonal 
influenza vaccination (not attenuated nasal) is recommended for subject and family. 
 
D. Extended follow-up:   
The current study plans call for follow-up for a total of two years.  However, the subjects will be 
followed clinically beyond this point, as would any subject with an immunodeficiency disease.  
Standard care would also involve evaluation of immune function at least quarterly by measuring 
lymphocyte numbers and function (PHA, tetanus toxoid blastogenesis). Gene marking will also 
be evaluated as outlined in the monitoring plan, unless no gene-containing cells are seen on 
successive studies over twelve months.   Peripheral blood serum and PBMC will be stored 
annually, in case assays for RCL exposure are needed.  Effort will be made to continue these 
studies as long as possible, although it is not possible to promise life-long follow-up.   The plans 
for life-long follow-up for the detection of RCL and the 15-year follow-up for leukemia monitoring 
are described in Appendix F.  In the case of the death of the subjects, autopsies will be requested 
from appropriate next-of-kin.  Tissues will be analyzed for the presence of gene-containing cells 
and for the presence of RCL. 
 
 


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17.3 Appendix C:  EFS-ADA Trial Busulfan Pharmacokinetic Analysis 
 
Purpose:  Subjects will receive one dose of busulfan, @ 4 mg/kg.  Busulfan blood levels will be 
measured following the busulfan to calculate the area-under the curve (AUC).  Because only a 
single dose of busulfan is given, no dose adjustment will be made.  
  
Method:  Busulfan dose is Busulfex 4 mg/kg IV x 1 dose.  Blood samples are taken at specified 
times after the dose of busulfan is infused.  The busulfan levels in each sample will be measured 
in the laboratory and the projected net AUC calculated. 
 
Sampling:  Busulfan is administered IV over 3 hours.  As soon as busulfan infusion is complete, 
flush CVC with 5cc NS before drawing first level. Draw first level immediately, to measure end-of-
infusion peak (t=0 min).  Then, draw other samples, timed from the end of the busulfan infusion:  
1, 2, 4, 8, and 13 hours after the end of busulfan infusion.   
Sample Times (min.) 
1) 
    0    (immediately at end of 3 hr infusion) 
2) 
  60 
(1 hr) 
3) 
120 
(2 hr) 
4) 
240 
(4 hr) 
5) 
480 
(8 hr) 
6) 
780  
(13 hr) 
The whole blood samples are processed by centrifugation and plasma fractions are frozen at -
20°C until the complete set of samples for the dose are obtained. 
 
Calculation:  Plasma busulfan levels are determined by HPLC.  Busulfan pharmacokinetic 
parameters are calculated by fitting a bi-exponential equation.  Area-under-the curve (AUC) is 
calculated by trapezoidal approximation, while drug clearance (optional) is calculated by using 
the busulfan dose divided by both body weight and the measured AUC. 
 
 


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ANNUAL REVIEW 
PRINCIPAL INVESTIGATOR 
DATE 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Purpose:   
To ensure that hematopoietic progenitor cell products are transported in such a way as 
to protect the integrity of the product and the health and safety of personnel transporting 
the product. 
 
Principles:  Not Applicable. 
 
Definitions:  
 
Excipient: An inert substance used as a diluent or vehicle for a drug. 
 
Materials: 
Room temperature (20 - 25°C) gel ice pack (approx. 900 grams). 
Shipping container (MiniMate Ice Chest  #1 or #2) labeled “Hematopoietic Progenitor 
Cells” and with permanent shipping label inside and outside of container.  See label 
example at end of procedure. 
Wet ice (if applicable). 
Dry ice (if applicable). 
Ziplock®-type plastic bags (9” x 12”), pre-sterilized. 
Liquid Nitrogen and LN2 Shipper. 
 
Specimen Requirements: 
Whole blood, bone marrow, or Hematopoietic Progenitor Cells in excipient. 
 
Safety Precautions: 
Universal precautions are used. All patient specimens are treated as if infected by blood-
borne pathogens, and therefore, gloves must be worn when handling specimens. 
 


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References: 
Foundation for the Accreditation of Hematopoietic Cell Therapy (FACT) Accreditation 
Manual, Section D, 2nd Edition, 2002. 
“Circular of Information for the Use of Cellular Therapy Products”, American Association 
of Blood Banks, America’s Blood Centers, American Red Cross, ASBMT, FACT, NMDP, 
ISCT, 2003. 
 
Procedure: 
Before a cell product is collected and transported, the following orders must be issued: 
An order to collect the cell product. 
An order to receive the cell product in the Bone Marrow (BM)/Hematopoietic Stem Cell 
(HPSC) Lab. 
An order to release the cell product from the BM/HPSC lab to the Clinical Gene Therapy 
Laboratory (CGTL). 
Cell Products: 
Hematopoietic Progenitor Cells, Apheresis (HPC, A) is collected by the University of 
California, Los Angeles Pediatrics/Hematology/Oncology Department and is transported 
in an impenetrable container to the BM/HPSC lab (CHS 46-126) for further processing.   
At the end of the apheresis collection, when prompted by COBE Spectra (or equivalent), 
seal and remove the Hematopoietic Progenitor Cells, Apheresis product, and plasma (if 
collected).  Be sure the product has been labeled before disconnecting and that the label 
is complete. 
Place Hematopoietic Progenitor Cells, Apheresis product in a secondary plastic bag and 
seal to prevent leakage.  Place plasma, if collected, into a separate plastic bag and seal.  
Place any extra samples collected during and/or at the end of the procedure into a 
separate plastic bag and seal. 
Registered Nurse (RN) or Clinical Laboratory Specialist (CLS) brings the product in 
shipping container, as well as accompanying paperwork, from collection site to the 
BM/HPSC lab to complete processing. 
Hematopoietic Progenitor Cells, Marrow (HPC, M) is collected in the OR and transported 
in an impenetrable container (e.g. Room temperature cooler) to the BM/HPSC lab for 
further processing, or it is received in the BM/HPSC lab from the National Marrow Donor 
Program. 
Hematopoietic Progenitor Cells, Umbilical Cord Blood (HPC, UCB) is delivered frozen to 
the BM/HPSC lab. 
Transport of Hematopoietic Progenitor Cell Products, by CGTL personnel, from the 
procurement sites to the BM/HPSC lab (CHS 46-126)  and from the BM/HPSC lab to the 
CGTL (Factor 14-246 or 14-650). 
The product is assigned an identification number. 


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Place a room temperature gel ice pack and thermometer inside a MiniMate Ice Chest (or 
equivalent).  The temperature must be between 20-25C. 
Take the transport container to the product collection site. 
The cell product is released from the BM/HPSC Lab and placed in the plastic bag(s) and 
into the transport container. 
Take the transport container with the product inside, and any relevant paperwork, to the 
final destination. 
When the product arrives at the final destination, it is recorded in the log book, or 
equivalent (e.g. Transduction Worksheet). 
Samples of the product are taken to determine sterility and cell enumeration (e.g. CD34+ 
cells). 
Transport of HPC Product from CGTL to the Pediatric Heme/Onc Unit (3rd floor RRMC) 
for Infusion: 
Infusion of HPC Product (viable, non-frozen): 
After completion of specimen processing, the cells for infusion are put into a sterile 60cc 
syringe for infusion. 
Label the syringe and place in a ziplock-type bag. 
The bag containing the syringe is then placed into an empty ice chest to maintain room 
temperature during transport. 
The syringe is taken to the infusion facility as soon as possible. 
The Disposition of the Final Cellular Product (Form 1000A) will accompany the syringe to 
the infusion center. 
The Disposition of the Final Cellular Product form is completely filled out by all individuals 
indicated. 
When completed, the original document will be retained at the Clinical Gene Therapy 
Laboratory and filed in the study binder of the respective subject.  Additional copies will 
be available upon request to regulatory agencies. 
Transport of HPC Product from CGTL to Control Rate and Liquid Nitrogen Freezers, or 
for Dump Freezing 
The HPC product is transported to the Control Rate Freezer at room temperature. 
The product is frozen following SOP #6000 – Control Rate Freezer. 
The product is removed from the Control Rate Freezer and placed in dry ice. 
The product is taken to the Liquid Nitrogen Freezer in Factor 14-938 and stored. 
The number of samples and their location is recorded in the Freezer Log Book. 
For Dump Freezing, the cells are transported at room temperature to the -20C freezer 
(14-246 Factor) and placed there overnight.  The following day, they are placed in dry ice 
and moved to the Liquid Nitrogen Freezer. 
The number of samples and their location is recorded in the Freezer Log Book. 
Transport for Bedside Thawing: 


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A cart containing a Liquid Nitrogen Shipper and a 37C water bath is prepared. 
The frozen cells are transferred from the Liquid Nitrogen Freezer to the Liquid Nitrogen 
Shipper. 
The cells are taken to the bedside of the patient and thawed in the 37C water bath. 
The cells are aspirated into a sterile syringe. 
The cells are then given to the RN for infusion into the patient. 
 
Calculations:  Not Applicable. 
 
Endpoint:  Not Applicable. 
 
Corrective Action:  Not Applicable. 
 
Attachments: Form 1000A 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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17.5 Appendix E:  SOP for Transduction 
 
 
 


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17.6 Appendix F:  Monitoring for monoclonal expansion or 
leukoproliferation due to insertional oncogenesis 
A general protocol for investigation of possible clonal proliferations that might develop as a result 
of insertional oncogenesis will be followed.  The purposes of the protocol are to: 
Provide adequate monitoring so that determination of the need for therapeutic intervention is 
made as expeditiously as possible; 
Minimize the risk of either physical or psychological harm of unnecessary interventions, to which 
subjects are exposed;  
Characterize any clonal proliferation events occurring in gene therapy trials;  
Determine whether any clonal proliferation resulted from insertional oncogenesis;  
Characterize the clonality of the normal, genetically-modified hematopoiesis after γ-retroviral 
gene therapy of HSC. 
Clonal Monitoring Methods 
     The methods used to characterize clonal expansion in the gene therapy trials for X-SCID 
included LAM-PCR and TCR spectratyping.  LAM-PCR (linear amplification mediated PCR) is 
based upon isolation of genomic DNA from peripheral blood mononuclear cells, primer extension 
with a 5’ biotinylated primer, avidin bead isolation of the products, restriction digest to cut a 3’ site, 
then PCR methods to amplify the antisense strand using a linker-based 3’ primer.  Each unique 
resulting band represents an individual integration site. The sequence can then be identified by 
searches against the human genome to determine the proviral integration site.   
     Information about clonality can also be gained from analyses of immunoreceptor gene 
rearrangements, for example, spectratyping of the TCR for T cells, and by an analogous approach 
for the B cell receptor (BCR) expressed by B lymphocytes.  Since the TCR and Ig immunoreceptor 
genes are the only known genes to normally undergo rearrangement in lymphohematopoietic 
development, analyses of hematopoietic clonality can only be applied to the study of T and B 
lymphocytes, thus precluding monitoring of NK cells or non-lymphoid cells, such as myeloid cells.  
Furthermore, clonal lymphocyte proliferation with certain immunoreceptor rearrangements or with 
multiclonal rearrangements may not be detectable. 
 
Insertional oncogenesis following gene transfer into hematopoietic stem cells could 
theoretically result in either lymphoid or myeloid leukemias.  The techniques used to characterize 
the T cell lymphoproliferation occurring in X-SCID patients after gene therapy may not be 
adequate for other forms of malignant expansion, and it is important that monitoring protocols are 
identified that can be applied to all patients and possible different outcomes. 
 
 


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Protocol for monitoring and evaluation of increasing oligoclonality or 
monoclonality:  
    In order to monitor subjects for evidence of uncontrolled leukocyte proliferation, the following 
protocol will be used:  
1.  Clinical Assessment  
Clinical evaluation for evidence of leukocyte proliferation, or cytopenia - Physical evaluation and 
imaging studies (as indicated) for evidence of lymphadenopathy, hepatomegaly, splenomegaly.   
2.  Screening Laboratory Assessment 
CBC with differential to determine whether there is evidence of leukocytosis, or cytopenia.  
Leukocytosis will be defined as values that are > 15,000 / mm3 on two successive analyses, within 
a clinically-appropriate time-frame and in the absence of signs of infection.  Cytopenia will be 
defined as platelets < 100,000/ul, leukocytes < 1,500/ul, or hemoglobin < 10 g/dl.   
[Routine follow-up will also include serum electrolytes, creatinine, BUN, LDH, uric acid, AST, ALT, 
alkaline phosphatase]. 
 
Quantitative PCR for the frequency of gene-modified peripheral blood mononuclear cells (PBMC).   
 LAM-PCR, if frequency of gene-modified PBMC > 1%.   
If the LAM-PCR results suggest monoclonality, then the following laboratory assessments will be 
performed, as applicable: 
3.   Detailed Laboratory Assessment 
     To determine which cell population may contain an abnormally proliferating clone, 
immunophenotyping1 and FACS sorting2 will be performed on peripheral blood: 
A. Immunophenotyping and determination of absolute numbers of subpopulation: 
     I.  T lymphocytes: Cell surface markers CD3, CD4, CD8, .   If the T lymphocyte number is >2 
S.D. above normal values for age: 
a) If there is evidence of T cell lymphocytosis (CD3), then additional immunophenotypic markers 
may be done to characterize the stage of differentiation, including: CD1, CD2, CD4+/45RA, 
CD4+/45RO, TCR α/β, TCR γ/δ, 
b) If there is evidence of TCR α/β,  T cell lymphocytosis, then staining for TCR Vβ usage will be 
performed, using a broad panel of reagents, e.g., IO Test Beta Mark TM(Beckman Coulter).  
c) If there is evidence of TCR γ/δ T cell lymphocytosis, then TCR γ and TCR δ usage will be 
analyzed with a broad spectrum panel.   
d) If there is an over-represented TCR family, then PCR for respective TCR monoclonality will be 
performed. 
e) TCR spectratyping may also be used to assess/confirm oligo-monoclonality. 
     II. B lymphocytes:  Cell surface markers CD19 and CD20, Igκ and Igλ  If there is evidence of 
abnormal distribution of Igκ or Igλ expression, then IgM, IgD, IgG, IgA staining may also be 
performed 
to 
determine 
if 
a 
clonal 
B 
cell 
population 
is 
present. 
If the B lymphocyte number is >2 S.D. above normal values for age: 
a)  If there is evidence of B cell lymphocytosis (CD19 or CD20), then additional immunophenotypic 
markers may be done to characterize the stage of differentiation, inclujding: CD10, CD27,     
 
1 These assays are expected to require 2-5 ml of blood, depending on the WBC count 
 
2 These assays are expected to require 5-30 ml of blood, depending on the WBC count 
 


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b) Perform PCR or Southern blot for IgH rearrangement to characterize multiple myeloma, 
macroglobulinemia and lymphoma, for example.   
c) Perform PCR for EBV (to evaluate for Epstein-Barr infection). 
d) BCR spectratyping may also be used to assess/confirm oligo-monoclonality. 
     III.  NK cells:  Cell surface markers CD16, CD56, CD57, CD94, CD158, CD3-negative. 
If NK or myeloid cells are sustaining the clonal expansion, however, further immunophenotypic 
assays of the clonality of the hematopoietic lineage are likely to be difficult or impossible, as 
previous studies have depended on X-inactivation in females or the presence of specific 
mutations or cytogenetic abnormalities.  LAM-PCR integration site analysis of total peripheral 
blood leukocytes and sorted myeloid cells will be performed to track clonal changes sustained by 
these cell types.  However, marrow analysis may be useful in determining whether hematopoietic 
differentiation is normal or not.  Marrow examination may also be required if there is significant 
leukocytosis or cytopenia. 
 
B.  
Sorting (if sufficient cells) for LAM-PCR:  
I.  Lymphoid cells: Surface markers CD3 for T-cells and, CD19 for B-lymphocytes. 
II. Myeloid cells: Surface markers CD33, CD13, and/or CD14.  
Genomic material extracted from sorted cells will be used to characterize the site of integration of 
the gene transfer vector: 
Isolation and sequencing of the predominant LAM-PCR fragment. 
Identification of the genomic location of integration. 
Attempt to study the consequences of the integration event in terms of induction or suppression 
of gene expression.    
III. High resolution cytogenetic analyses of blood and/or marrow cells, as indicated. These 
analyses can be performed in the presence of any type of leukocyte clonal proliferation and would 
complement the above studies.        
Monitoring for monoclonal expansion or leukoproliferation due to insertional oncogenesis 
 
 
 
 
 
 
 
 
 
 
 


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17.7 Appendix G:  Toxicity Table For Grading Severity Of Adverse 
Experiences (>3 Months Of Age) 
 
 
 


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17.8 Appendix H:  Details of historical HSCT control groups 
 
1. Objective 
Historical data from ADA-SCID patients who were treated with Hematopoietic Stem Cell 
Transplantation (HSCT) at GOSH/UCL (UK) or Duke University Children’s Hospital (USA) 
during the period of 2000-2016 will be collected. HSCT currently corresponds to the 
standard of care for ADA-SCID patients. These data will be used as historical control 
groups in a current on-going gene therapy study “Phase I/II, controlled, open-label, non-
randomised, single-centre trial to assess the safety and efficacy of EF1αS-ADA lentiviral 
vector mediated gene modification of autologous CD34+ cells from ADA-deficient 
individuals”. The objective is to compare gene therapy versus HSCT to determine the 
optimal treatment. 
2. Study populations 
The control groups will be constituted of patients of 0 to 18 years of age with a confirmed 
diagnosis of ADA deficiency, transplanted at Great Ormond Street Hospital (UK) or Duke 
University Children’s Hospital (USA) between 2000 and 2016. See inclusion criteria page 
64. 
3. Data to be collected – GOSH/UCL 
GOSH hold a registry / clinical database (European Society for Immunodeficiencies – 
ESID and European Bone Marrow Transplant Registry - EBMT) displaying information on 
patients who have received HSCTs. 25 treated patients have been identified between 
2000-2016. The patients have given consent for their data to be entered into this registry 
/ clinical database and to be used for other ethically approved studies. ESID and EBMT 
will allow us to collect the below information: 
• Demographics (gender, date of birth, date of death), 
• Date of treatment with HSCT 
• Method of ADA-SCID diagnosis, 
• Previous HSCT including: date, transplant cells, donor type and outcome and 
whether they needed a second transplant. 
However, to make this historical control group valid for marketing authorisation 
application, our discussions with regulatory agencies suggests that we need to collect 
further information valuable to compare the efficacy and safety of any new treatment in 
research vs. current HSCT standard of care as:  
• 
Previous PEG-ADA ERT (and duration of treatment), 
• 
Previous or current immunoglobulin therapy, 
• 
Listing of relevant medical history, including infections (severe or opportunistic 
infections) 
• 
Vaccination 
• 
Auto-immune disease 
• 
Oncology disease / leukemia 
• 
AEs and SAEs 


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• 
Death and reason for death 
This information is only available from the patient’s records; we therefore plan to re-
consent these patients to access their full medical records, and share the information with 
regulatory agencies and pharmaceutical companies.  
4. Data to be collected – Duke University Children’s Hospital 
Duke University maintain a database of information on patients who have received 
HSCTs. 10 treated patients have been identified between 2000-2016. The patients have 
previously given consent to their data being entered into this database and being used 
for other ethically approved studies. 
The following data will be collected from this control group: 
• 
Demographics (gender, year of birth, age at presentation to site), 
• 
Diagnosis (age at and method of ADA-SCID diagnosis), 
• 
HSCT history, including: age at transplant, type of transplant cells and number, 
donor type and outcome, need for a second transplant, outcome of 
vaccinations post-transplant, 
• 
Use of PEG-ADA ERT and immunoglobulin therapy (before and after 
transplant), including time since transplant if reinitiated post HSCT, 
• 
Graft versus host disease, 
• 
Listing of relevant medical history, including infections (severe, recurrent or 
opportunistic infections) and neurological conditions, 
• 
Previous and Concomitant Treatment, 
• 
Changes in Medical Condition post HSCT, including severe/opportunistic 
infections, emergent neurological events, autoimmune conditions, oncologic 
disease, SAEs, bone/skeletal deformities, physical developmental delays or 
weight loss, 
• 
Lymphocyte subsets (CD3, CD4, CD8, CD19, CD56/CD16 and CD4+/CD45RA 
cell counts) at baseline and at 1-8 months, 9-16 months and 17-30 months post 
HSCT, 
• 
ADA activity & ADA metabolites (plasma deoxyadenosine, ADA activity in 
RBCs, dAXP) at baseline and at 1-8 months, 9-16 months and 17-30 months 
post HSCT, 
• 
Death and reason for death. 
 
5. Data collection process 
This is a retrospective, non-interventional data collection activity; the patients follow 
routine medical practice. We will contact GOSH patients or their authorised representative 
and seek their consent to get access to their medical notes and share the information with 
regulatory agencies and pharmaceutical companies. Teams at GOSH/UCL and Duke 
University Children’s Hospital  will work on the collection of data from databases and 
patients’ records, and the data will be entered into an electronic Case Report Form 
(eCRF). This eCRF will be monitored as per standard process before it can be used for 
statistical analysis and submission to agencies for review and assessment. 


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6. Data Analysis 
The comparison analysis between gene therapy and HSCT will be done based on the 
current gene therapy study objective: 
Survival and event free survival, defined as need to return to PEG-ADA or second 
transplant at 1 and 2 years post treatment. 
Use of immunoglobulin replacement therapy (IgRT) and severe infections post treatment. 
Severe infections, defined as infections requiring hospitalisation or prolonging 
hospitalization and/or documented infections by opportunistic pathogens (interstitial 
pneumonia, intractable diarrhoea) 
 
 


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17.9 
Appendix I:  List of Protocol Changes 
 
Changes made 23 April 2013 (ver. 1.3) from previous version dated 8 October 2012 (ver. 
1.2) 
1. 
Remove measurement of plasma ADA levels as follow-up test 
Reason:  subjects are withdrawn from PEG-ADA ERT 30 days post infusion of 
gene-modified cells, and thus, this test would not be informative 
 
Protocol pages affected:  51, 66, 71, 72, 110, 111, 117 
 
2. 
Remove ophthalmologic exam from baseline screen 
 
Reason:  not needed 
 
Protocol pages affected:  70, 110, 111 
 
3. 
Remove CBC test from Day 0 (Day of Infusion) 
 
Reason:  not needed, subjects already have a CBC drawn on Day +1 
 
Protocol pages affected:  72 
 
4. 
Subjects who discontinue study will be replaced (previously were not replaced) 
 
Reason:  would allow treatment of 10 subjects 
 
Protocol pages affected:  80 
 
5. 
Add identity test to characterization of final product 
 
Reason:  per FDA request 
 
Protocol pages affected:  57 
 
6. 
Change criteria for reinstituting PEG-ADA ERT 
Reason:  clarification, to prevent prematurely re-starting PEG-ADA based on 
previous study data 
 
Protocol pages affected:  62 
 
7. 
Add TCR V panel assay to schedule of procedures 
 
Reason:  correction 
 
Protocol pages affected:  110 
 
8. 
Remove vector marking test from Day +1 (Schedule of Procedures) 
 
Reason:  not needed 
 
Protocol pages affected:  110 
 
 


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9. 
Change description of allocating the back-up bone marrow 
 
Reason:  clarification based on technical procedures at UCLA 
 
Protocol pages affected:  14, 56, 58, 64, 113, 114 
 
10. 
Correct time-points for blood collection of samples for busulfan pharmacokinetics 
 
Reason:  consistency 
 
Protocol pages affected:  71, 115 
 
11. 
Add a scientific reference 
 
Reason:  update 
 
Protocol pages affected:  30, 103 
 
12. 
Update Appendices C, E, and G 
 
Reason:  update 
 
Protocol pages affected:  120, 126, 131 
 
Changes made 13 May 2013 (ver. 1.4) from previous version dated 23 April 2013 
1. 
Clarified the objectives 
Reason:  to emphasize that safety is the primary objective 
Protocol pages affected:  11, 42, 47, 54, 62, 71, 80 
 
2. 
Added IND #15440 to sample label 
 
Reason:  update 
 
Protocol page affected:  59 
 
3. 
Correct grammar (quantitation) 
 
Reason:  correction 
 
Protocol page affected:  81 
 
Changes made 03 July 2013 (ver. 1.4a) from previous version dated 13 May 2013 
1. 
Added Hepatitis C as screening virology test 
 
Reason:  to be consistent with informed consent 
 
Protocol pages affected:  52, 70, 111, 112 
2. 
Change anti-epileptic drug prior to busulfan administration from dilantin/fenytoin to 
Keppra 
 
Reason: hospital procedure 
 
Protocol pages affected:  62, 72, 116 
 
 
 


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Changes made 01 October 2013 (ver. 2) from previous version dated 03 July 2013 
1. Change Inclusion/Exclusion criteria to allow for different reference ranges 
established by different diagnostic labs and to align SCID definition with PIDTC 
guidelines 
Reason: update 
Protocol pages affected:  53, 114 
2. Change the duration of busulfan administration from 2 to 3 hours 
Reason:  to be consistent with hospital practice 
Protocol pages affected:  72, 118, Appendix C 
3. Change the trade name drug “Keppra” to its generic name, levetiracetam 
Reason:  to allow clinical sites flexibility 
Protocol pages affected:  73, 117 
4. Specify the tests in chemistry and immune function panels and describe other tests 
Reason: clarification 
Protocol pages affected:  71, 112-114, 115, 120 
5. Exclude DFSP as a malignancy that would halt enrollment because some ADA-
deficient SCID patients will develop DFSP, and DFSP is not an exclusion criterion 
Reason:  clarification 
Protocol page affected:  81, 87 
6. Change the window for follow-up visits to be +/- 4 weeks to be consistent 
throughout the protocol 
Reason:  consistency 
Protocol page affected: 119 
 
Changes made 10 February 2014 (ver. 3) from previous version dated 01 October 2013 
1. Allow for subjects to be transferred to the care of their  home  physician for 
monitoring 3 days after infusion of gene-modified cells 
Reason:  to allow for flexibility for out-of-town subjects and their families 
Protocol pages affected:  65, 75, 119 
2. Added additional investigators involved in patient care 
Reason:  add to protocol 
Protocol pages affected:  17, 111 
3. Correct error in time of busulfan administration (from 2h to 3h) 
Reason:  error 
Protocol page affected:  123 
 
 
 
 


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Changes made 4 June 2014 (ver. 3.1) from previous version dated 10 February 2014 
1. Change:  Update “Table of Contents”  
Reason:  For accurate pagination 
Protocol pages affected:  iv-vii 
 
2. Change:  Reformat section titles and/or tables and/or margins  
Reason:  Change format for clarity, logical pagination and “Table of Contents” 
update function to work properly 
Protocol pages affected: 10-12, 15-16, 18-19, 23, 107-108, 111-112, 114-115, 
119-120, 123-125, 130-131, 135-136 
 
3. Change:  Updated investigators involved in patient care and updated 
organizational chart  
Reason:  Change in personnel and added new investigators to protocol 
Protocol pages affected:  16, 17, 109, 113 
 
4. Change:  Add PBMC ADA Activity to the Schedule of Evaluations on page 114 
Reason:  Correction; test was omitted 
Protocol pages affected:  113-114 
 
Changes made 13 January 2015 (ver. 4.0) from previous version dated 4 June 2014 
1. Increase number of subjects from 10 to 15 
Reason:  Enrollment of 10 subjects was fulfilled and more subjects have been 
identified.  Preliminary data indicates that the protocol is safe and efficacious. 
Protocol pages affected:  11, 45, 53, 88 
 
 
3. Add prior allogeneic HSCT as an exclusion criteria 
Reason:  do not want to enroll subjects who have had previous exposure to 
chemotherapy 
Protocol pages affected:  55 


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4. Update prophylactic medications requirements 
Reason:  to allow for reasonable clinical management of subjects 
Protocol pages affected:  64-65, 67-68 
 
5. Revise statistics due to increasing the number of subjects to 15 
Reason:  update 
Protocol pages affected:  88 
 
6. Add additional clinicians involved in subject follow-up 
Reason:  update 
Protocol pages affected:  114 
 
7. Clarify when T cell proliferative studies to PHA and tetanus should be conducted 
Reason:  clarification, test not necessary at every follow-up time point 
Protocol pages affected:  115-116, 123 
 
8. Clarify immunization schedule of treated subjects 
Reason:  to allow for reasonable clinical management of subjects 
Protocol page affected:  124 
 
9. Correct the volume of bone marrow that may be harvested.  There was 
inconsistency in 2 places. 
Reason:  correct inconsistency 
Protocol page affected:  74 
 
Changes made 02 June 2015 (version 5.0) from previous version 4.0, 13 January 2015 
1. Add that international normalized ratio (INR) may be used instead of prothrombin 
time (PT) for screening; INR is standard for coagulation studies 
Reason:  update 
Protocol pages affected: 55, 72, 73, 116, 118 
 
2. Increase screening window to 60 days and make consistent throughout protocol 
Reason:  window was too narrow and caused protocol deviations 
Protocol pages affected:  54-55, 72 
 
3. Increase the number of subjects to 20 and correct statistics 
Reason:  enrollment into the trial has occurred faster than anticipated.  An 
additional 2 subjects (past 15) have been preliminarily identified 


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Protocol pages affected:  11, 45, 53, 85, 88 
 
4. Correct the flask size for cell processing 
Reason:  error 
Protocol page affected:  59 
 
5. Change when TMP/SMX may be resumed after busulfan 
Reason:  clinically OK to start when neutrophils have recovered to 500 
Protocol page affected:  68 
 
6. Update clinicians involved in subject follow-up/care 
Reason:  update 
Protocol pages affected:  114-115 
 
Changes made 06 October 2015 (version 6) from previous version 5.0 02 June 2015 
1. Update organizational chart 
Reason:  update 
Protocol page affected:  18 
 
2. Change the window for follow-up visits to be +/- 4 weeks to be consistent 
throughout the protocol 
Reason:  consistency 
Protocol page affected: 51 
 
3. Change method of ADA enzyme measurement in PBMC from the NIH, CLIA-
quality TLC method to UCLA, research-quality Diazyme colorimetric method.  Also 
add that erythrocyte ADA enzyme levels (from CLIA approved lab, Duke 
University) will also be recorded. 
 
 
Protocol pages affected:  52-53, 64, 69-70, 87-88, 90-91. 
 
4. Add that lymphocyte proliferation assay may be performed using 3H thymidine or 
CFSE 
Reason:  most labs are moving away from the radioactive assay to dye-based 
methods that can be measured on a flow cytometer 
Protocol pages affected:  53, 70. 
 


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5. Add that confirmation of ADA enzyme deficiency can also include documentation 
of gene mutation known to cause disease 
Reason:  clarification 
Protocol pages affected:  54, 73, Appendix B. 
 
6. Clarify the infection status eligibility for enrollment 
From:  “Evidence of active opportunistic infection or infection with HIV-1, hepatitis 
B, Hepatitis C, CMV, or parvovirus B 19 by DNA PCR within 60-90 days prior to 
bone marrow harvest.” 
To:  “Evidence of infection with HIV-1, hepatitis B, Hepatitis C, or parvovirus B 19 
by DNA PCR within 90 days prior to bone marrow harvest.  If other infection is 
present, it must be under control (e.g. stable or decreasing viral load) at the time 
of screening.” 
Reason:  clarification 
Protocol pages affected:  55 
 
7. Remove fungal stain requirement for final product release 
Reason:  fungal stain is not a requirement by the FDA for product release.  It was 
added at the request of the UCLA IRB.  However, including the stain delays the 
release of the final product (which is infused fresh) because the central lab at 
RRMC does not perform the test.  The sample has to be driven to the Brentwood 
Annex for the stain.  Additionally, the gram stain will also stain fungi, if present. 
Protocol pages affected:  59-60, Appendix B. 
 
8. Add that for vector copy number (VCN) determination, digital droplet PCR (ddPCR) 
may be used 
Reason:  update of procedures 
Protocol pages affected:  60, 65, 69-70, 83 
 
9. Change the method of mycoplasma detection from the culture method to PCR 
Reason:  update to more rapid PCR method at Labs, Inc, which is a CLIA-
accredited, FDA-registered, and TGA GMP-compliant laboratory 
Protocol page affected:  60 
 
10. Update PEG-ADA re-starting criteria to include change in measurement of ADA 
enzyme activity 
Reason:  to coordinate protocol procedures to be consistent with new changes 
Protocol page affected:  64 
 


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11. Correct the tests included in a chemistry panel to be consistent throughout the 
protocol 
Reason:  correction/consistency 
Protocol pages affected:  69, 73, Appendix B 
 
12. Correct -retro to lentivirus 
Reason:  correct a mistake 
Protocol page affected:  71 
 
13. Remove requirement for recording height and weight at Day +1 after infusion, since 
it is already recorded at screening and baseline 
Reason:  not needed 
Protocol page affected:  73, Appendix B 
 
14. Correct blood draw times for determination of busulfan levels to be consistent with 
previous change 
Reason:  correct a mistake 
Protocol page affected:  75 
 
15. Coordinate descriptions of follow-up exams to be consistent with Schedule of 
Procedures (Appendix B) 
Reason:  clarification 
Protocol pages affected:  75-76, Appendix B 
 
16. Update DSMB reporting requirements 
Reason:  update based on DSMB schedule 
Protocol page affected:  84 
 
17. Update data handling chart 
 
Reason: update 
 
Protocol page affected: 94 
 
18. Update monitoring plan and responsibilities, delete duplicate paragraphs and 
sections noted 
Reason:  Update and correction.  The trial enrolls a small number of subjects (up 
to 20) and thus monthly statistical analysis is excessive. 
Protocol pages affected:  94-98. 
 
19. Update list of clinicians involved in patient care 


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Reason:  update 
Protocol page affected:  17, 116 
 
20. Update scientific references 
 
Reason:  update 
 
Protocol pages affected:  26, 106. 
 
Changes made 02 November 2015 (version 7) from previous version 6, 06 October 2015 
1. Allow ANC ≥500 cells/mm3 alone to be acceptable inclusion criterion without the 
need for analysis of bone marrow biopsy/aspirate. 
Reason: 
not necessary based on previous cases 
Protocol page affected: 55 
 
2. Clarify schedule for collecting myelosuppression labs; does not need to be for 6 
weeks, if not clinically indicated. 
 
Reason:  consistency, clarification 
 
Protocol pages affected:  51, Appendix B 
 
3. Add monitor 
 
Reason:  update 
 
Protocol page affected:  109 
 
4. Update physicians involved in patient follow-up 
 
Reason:  update 
 
Protocol page affected:  112 
 
Changes made 30 March, 2016 (version 8) from previous version 02 November, 2015 
1. Specify on the cell product label that the number of cells is total nucleated cells 
 
Reason:  clarification 
 
Protocol page affected:  62 
 
2. Add additional lab monitoring (weekly CBC/diff) of subjects who are slow to recover 
their cell counts until ANC is >500 cells/mm3 
 
Reason:  DSMB request 
 
Protocol pages affected:  68, 117 
 
 
 
 


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3. Modify study stopping criteria 
 
 
FROM: 
If there is one death or two grade 4 toxicities (except grade IV busulfan-related 
hematologic 
serious 
adverse 
events 
{transient 
leukopenia, 
anemia, 
thrombocytopenia resolving within 42 days of transplant}, which are anticipated 
and will be reported, but will not be considered stopping criteria). 
  
TO: 
If there is one death or four grade 4 toxicities, (except grade IV busulfan-related 
hematologic 
serious 
adverse 
events 
{transient 
leukopenia, 
anemia, 
thrombocytopenia resolving within 42 days of transplant} which are anticipated and 
will be reported, but will not be considered stopping criteria).  Additionally, 
neutropenia (a documented disorder of ADA-deficiency) occurring at any time, will 
be graded for severity but will not count towards the stopping rule. 
 
Reason:  change 
 
Protocol pages affected:  83, 89-90 
 
 
 
 
 
 
 
Changes made 21 October 2016 (version 9) from previous version 8, 30 March 2016 
(pages referenced correspond to “tracked” protocol version) 
 
1. 
Name investigational product “OTL-101” for easy reference 
 
Reason:  clarification 
 
Pages affected:  throughout document 
 
2. 
Remove PBMC ADA activity from efficacy analysis 
Reason:  the PBMC ADA activity assay is research quality.  We will use erythrocyte 
ADA activity, which is performed in CLIA lab at Duke, instead 
Pages affected:  16, 124, 131, Appendix B 
 
3. 
Update results of the -retroviral trial (MND-ADA) 
 
Reason:  update 
 
Pages affected:  36-37 
 


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4. 
Update results and scientific references of insertional oncogenic events in other 
trials using retrovirus 
 
Reason:  update 
 
Page affected:  39 
 
5. 
Update other clinical trials using lentiviral vectors 
 
Reason:  update 
 
Page affected:  41 
 
6. 
Revised Trial Description and Study Objectives 
 
Reason:  revised to match updated statistical section 
 
Pages affected:  52-57, 71 
 
7. 
Update method of mycoplasma testing used for release of cell product 
 
Reason:  update 
 
Page affected  63 
 
8. 
Update DSMB 
 
Reason:  update 
 
Page affected:  87 
 
9. 
Update/revise statistics section (Section 11) 
 
Reason:  revise and update 
 
Pages affected:  87-100 
 
10. 
Update data monitoring procedures 
 
Reason:  change from 80% to 100% source document verification 
 
Page affected:  102 
 
11. 
Update list of clinicians involved in patient care 
 
Reason:  update 
 
Page affected:  123 
 
12. 
Added Appendix for description of HSCT control arm.  Reordered appendices to 
accept new addition 
Reason:  addition to align with new objectives/statistics sections 
Pages affected:  appendices H and I 
 
 


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Changes made 18 April 2017 (version 10) from previous version 9, 21 October 2016 
(pages referenced correspond to “tracked” protocol version) 
 
 
 
 
 
 
2. 
Addition of level 4 sub-headers to sections 2 and 4. 
Reason: administrative reasons allowing the headings to be part of table of 
contents. 
Pages: Sections 2 and 4. 
 
3. 
Primary objectives reworded and presented as separate bullets for ease of 
reading. 
Reason: administrative change 
Page: 12 (protocol summary) and 53 
 
4. 
Secondary objectives, sub-headings of primary and secondary efficacy ave been 
removed and a list of secondary objectives are presented and aligned with the 
statistics section (Section 11). These revisions have also been aligned with the 
endpoints. 
Reason: to align the text of objectives and endpoints with the statistical section 
Pages: 12 (protocol summary), 53, 55 and 56. 
 
5. 
Effects on ADA reconstitution on immune phenotype and function endpoint, text 
from introduction is bulleted as endpoint 11. 
Secondary Efficacy End-point 11:  Infection rates will be also measured under 
immune reconstitution as part of the efficacy assessment of a competent immune 
system. 
Reason:  administrative change. 
Page: 57 
 
6. 
Text added: Signed written informed consent according to guidelines of the IRB 
(UCLA Office of Human Research Protection Program and National Human 
Genome Research Institute (NHGRI) Institutional Review Board.  
Reason: administrative change. 
Page: 59. 
 


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7. 
Added correct level 2 sub-headings to Section 7 in order for these headers to be 
capured in the table of contents. 
Reason: administrative change. 
Pages: 70 to 73. 
 
8. 
Abbreviation correction for Electrocardiogram (EKG changed to ECG). 
Reason: administrative change. 
Page: 77. 
 
9. 
Updated information for reporting safety events to the DSMB in section 9.5.2.3 and 
14.5, due to study oversight being transferred to the UCLA CTSI DSMB in 
September 2016. 
Reason: to comply with UCLA CTSI DSMB standard procedures. 
Page: 86 and 102. 
 
10. 
The statistical considerations section of the protocol has been updated to be 
consistent with the statistical analysis plan generated for the study.  This includes 
defining what data will be reviewed at the interim analysis at 6 months and the final 
analysis.  The text is also refined to match the study objectives and endpoints, and 
distinguishes the different populations to be used for the safety and efficacy 
analyses. 
Reason: Changes to Section 11 (Statistical considerations) to be inline with the 
statistical analysis plan, the objectives, and endpoints of the study. 
Pages: 89 to 95. 
 
11. 
The auditor has changed from UCLA Office of Research Compliance to Clinical 
and Translational Science Institute, and all audit findings will be submitted to UCLA 
IRB, rather than both NIH and UCLA IRBs. 
Reason: Administrative change. 
Page: 99. 
 
12. 
Added correct level 2 sub-headings to Section 17 (Appendices) in order for these 
headers to be capured in the table of contents. 
Reason: administrative change. 
Pages: 114 to 146. 
 
 
 
 


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Changes made 13 November 2017 (version 11) from previous version 10, 18 April 2017 
(pages referenced correspond to “tracked” protocol version) 
 
1. 
Point viii was updated to note that obligations for the conduct of the study have 
been transferred by the IND holder to the CRO, TCTC. 
Reason: Updated following involvement of the CRO, to be in line with standard 
practice. 
Page: ii 
 
2. 
The header for Section 12 of the protocol was added to the Table of contents. 
Reason: Administrative change. 
Page: vi, 91. 
 
3. 
A provision was added for collecting limited long-term data for withdrawn patients 
Reason: tracking the survival of withdrawn patients, as well as their need for a 
rescue HSCT or reinstitution of PEG-ADA ERT, allows to collect better quality trial 
data.  
Page: 75, 82, 83. 
 
4. 
It was clarified that participants will be asked to continue scheduled evaluations if 
voluntary withdrawal occurs, post-treatment only. 
Reason: Updated for clarity. 
Page: 75 and 83. 
 
5. 
The information to be collected in the CRF for all AEs was clarified, and a 
seriousness assessment was added to the list of fields to be completed for each 
AE. It was also noted that grade 3 and 4 AEs should be reported to the Sponsor, 
as well as to the PI or Study Coordinator, within 48 hours. Leukoproliferation was 
noted to be an “SAE of special interest” rather than an “expected SAE”, as it has 
never been observed in relation to OTL-101 so far. 
Reason: Updated for clarity. 
Page: 78 and 79. 
 
6. 
Causality of Serious Adverse events will now be defined as definitely related, 
probably related, possibly related, unlikely related, or not related to the study 
agent. The text was also updated to more precisely define the nature of certain 
events, as Serious Adverse Reactions rather than SAEs. 
Reason: Updated to be in line with standard practice. 
Page: 80. 


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7. 
The language on the RAC reporting requirements was updated to be in line with 
current regulations. The word “expected” was removed as considered to refer only 
to adverse reactions or serious adverse reactions, and modified to “anticipated”. 
Reason: Updated to be in line with currnt regulations and standard practice.  
Page: 81. 
 
8. 
The Statistical Considerations section (Section 11) was updated to include the 
Duke University HSCT control group, as well as to align the language in the section 
to the updated Statistical Analysis Plan. Use of the Duke University HSCT group 
as a comparator for the trial was agreed with the FDA at the End of Phase II 
meeting held on 29Sep2017.  
Reason: The Duke HSCT control group was added to reflect FDA feedback, and 
the language modified to match the updated Statistical Analysis Plan. 
Page: 84 to 88. 
 
9. 
The text was updated to eliminate the requirement for completed eCRF pages to 
be printed and stored in the patients’ medical records, as this was burdensome for 
the site and the monitors. 
Reason: Updated to reduce the burden on site and monitors. 
Page: 93. 
 
10. 
The name and email address of the Regulatory Coordinator were updated. 
Reason: Administrative change. 
Page:109. 
 
11. 
Name and addresses were added for vendors involved in the study (GeneWERK, 
TechnoSTAT and Diamond Pharmacovigilance). 
Reason: Administrative change. 
Page: 112 and 113. 
 
12. 
Appendix H was updated to include the Duke University HSCT control group, and 
to describe what data will be collected for this patient population. Use of this patient 
group as a comparator for the trial was agreed with the FDA at the End of Phase 
II meeting held on 29Sep2017. The paragraph regarding GOSH HSCT patients 
was updated to reflect the fact that 25 subjects treated between 2000 and 2016 
were identified at GOSH, rather than 29. The Data Analysis section was also 
updated to clarify that severe infections will be considered in the comparison 
between trial data and HSCT data, rather than all infections. 


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Reason: the Duke HSCT control group was added to reflect FDA feedback; the 
number of GOSH HSCT patients identified was corrected; the data analysis 
section was updated for clarity. 
Page: 139 to 141. 
 
Changes made 28 March 2018 (version 12) from previous version 11, 13 November 2017 
(pages referenced correspond to “tracked” protocol version) 
 

SAP_001.pdf: 
COVER PAGE
Official Study Title: Autologous Transplantation 
of Bone Marrow CD34+ Stem/Progenitor Cells 
After Addition of a Normal Human ADA 
Complementary DNA (cDNA) by the EFS-ADA 
Lentiviral Vector for Severe Combined 
Immunodeficiency Due to Adenosine Deaminase 
Deficiency (ADA-SCID)
EFS-ADA Statistical Analysis Plan v4.0. dated 
24-Oct-2017
NCT01852071


Statistical Analysis Plan
Study Name: 
Autologous Transplantation of Bone 
Marrow CD34+ Stem/Progenitor Cells 
after Addition of a Normal Human 
ADA cDNA by the EFS-ADA Lentiviral 
Vector for Adenosine Deaminase 
(ADA)-Deficient Severe Combined 
Immunodeficiency (SCID) 
Protocol Number: 
NCT01852071 
Protocol Version: 
10.0 
Protocol Date: 
18 April 2017 
SAP Version 4.0: 
24 October 2017 


Study: NCT01852071 - UCLA Fresh  
Statistical Analysis Plan 
Final Version 4.0 
Page 2 of 27 


Study: NCT01852071 - UCLA Fresh  
 
 
 
Statistical Analysis Plan 
Final Version 4.0 
Page 3 of 27 
Table of Contents 
1 
Introduction ...................................................................................................................... 7 
2 
Study Design .................................................................................................................... 7 
3 
Study Schedule ................................................................................................................. 8 
4 
Study Objectives ............................................................................................................. 10 
4.1 
Primary Objectives ......................................................................................................... 10 
4.2 
Secondary Objectives ..................................................................................................... 10 
5 
Endpoints ........................................................................................................................ 10 
5.1 
Primary Endpoints .......................................................................................................... 10 
5.2 
Secondary Endpoints ...................................................................................................... 10 
6 
Timing of Analyses ........................................................................................................ 11 
7 
Analysis Populations ...................................................................................................... 11 
7.1 
Efficacy Population ........................................................................................................ 11 
7.2 
Safety Population............................................................................................................ 12 
8 
Statistical Methods ......................................................................................................... 12 
8.1 
Subject Disposition ......................................................................................................... 12 
8.1.1 
Summary of Populations ............................................................................. 12 
8.1.2 
Summary of Withdrawals ............................................................................ 12 
8.2 
Demography and Baseline Characteristics ..................................................................... 13 
8.2.1 
Demography ................................................................................................ 13 
8.2.2 
Medical History ........................................................................................... 13 
8.2.3 
ADA Deficiency Diagnosis ......................................................................... 14 
8.2.4 
Disease History ............................................................................................ 14 
8.2.5 
Cytogenetics ................................................................................................ 14 
8.2.6 
Viral PCR .................................................................................................... 15 
8.2.7 
Urinalysis ..................................................................................................... 15 
8.2.8 
Electrocardiogram (ECG) ............................................................................ 15 
8.2.9 
Echocardiogram ........................................................................................... 15 
8.2.10 
Chest X-ray .................................................................................................. 16 
8.2.11 
Pulse Oximetry ............................................................................................ 16 
8.2.12 
Bone Marrow Harvest ................................................................................. 16 
8.2.13 
OTL-101 Administration ............................................................................. 16 
8.2.14 
Transplant Details for the HSCT Controls .................................................. 16 


Study: NCT01852071 - UCLA Fresh  
 
 
 
Statistical Analysis Plan 
Final Version 4.0 
Page 4 of 27 
8.2.15 
Exposure ...................................................................................................... 17 
8.3 
Efficacy Evaluations ....................................................................................................... 17 
8.3.1 
Primary Efficacy Endpoints ........................................................................ 17 
8.3.1.1 
Overall Survival at 12 Months .................................................................... 17 
8.3.1.2 
Event-free Survival at 12 Months ................................................................ 18 
8.3.2 
Secondary Efficacy Endpoints..................................................................... 19 
8.3.2.1 
Overall and Event-free Survival at 24 Months ............................................ 19 
8.3.2.2 
Use of PEG-ADA Enzyme Replacement Therapy ...................................... 22 
8.3.2.3 
Use of Immunoglobulin Replacement Therapy........................................... 19 
8.3.2.4 
Rate of Severe Infections ............................................................................. 19 
8.3.2.5 
Immune Reconstitution ............................................................................... 20 
8.3.2.6 
T Cell Receptor Excision Circles (TREC) in the PB .................................. 20 
8.3.2.7 
Engraftment ................................................................................................. 20 
8.3.2.8 
ADA Expression, Enzyme Activity and Detoxification .............................. 21 
8.3.2.9 
Lymphocyte Proliferative Responses Stimulation Index (SI) ..................... 21 
8.3.2.10 
Antibody Responses .................................................................................... 21 
8.4 
Safety Evaluations .......................................................................................................... 22 
8.4.1 
Adverse Events ............................................................................................ 22 
8.4.2 
RCL Testing ................................................................................................ 24 
8.4.3 
Laboratory Evaluations................................................................................ 24 
8.4.3.1 
Haematology ................................................................................................ 24 
8.4.3.2 
Coagulation Tests ........................................ Error! Bookmark not defined. 
8.4.3.3 
Biochemistry ................................................................................................ 25 
8.4.4 
Vital Signs, Height and Weight ................................................................... 25 
8.4.5 
Physical Examination .................................................................................. 25 
9 
Analysis Software ........................................................................................................... 26 
10 
Changes in Statistical Analysis Plan .............................................................................. 26 
 
 
 
 


Study: NCT01852071 - UCLA Fresh  
 
 
 
Statistical Analysis Plan 
Final Version 4.0 
Page 5 of 27 
List of Abbreviations 
 
ADA 
Adenosine deaminase 
ADA-SCID 
Adenosine deaminase deficient severe combined immunodeficiency 
AE 
Adverse event 
ALT 
Alanine aminotransferase 
AST 
Aspartate aminotransferase 
AXP 
Total adenine ribonucleotide 
AUC 
Area under the curve 
BM 
Bone marrow 
BMT 
Bone marrow transplant 
cDNA 
Complementary Deoxyribonucleic Acid 
CHLA 
Children’s Hospital Los Angeles 
CI 
Confidence interval 
CMV 
Cytomegalovirus 
CNS 
Clinically not significant 
CS 
Clinically significant 
dAXP 
Adenine deoxyribonucleotide 
DFSP 
Dermatofibrosarcoma protuberans 
DNA 
Deoxyribonucleic acid 
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EFS 
Elongation Factor 1α Short form 
ERT 
Enzyme replacement therapy 
GOSH 
Great Ormond Street Hospital 
HIV 
Human immunodeficiency virus 
HLA 
Human leukocyte antigen 
HSC 
Hematopoietic stem cell 
HSCT 
Hematopoietic stem cell transplant 
IgA 
Immunoglobulin A 
IgG 
Immunoglobulin G 
IgM 
Immunoglobulin M 
IgRT 
Immunoglobulin replacement therapy 
LV 
Lentiviral vector 
MCH 
Mean corpuscular haemoglobin 
MCHC 
Mean corpuscular haemoglobin concentration 
MCV 
Mean corpuscular volume 
MedDRA 
Medical Dictionary for Regulatory Activities 
MMRM 
Mixed model for repeated measures 
MUD 
Matched unrelated donor 
NIH 
National Institutes of Health 
NK 
Natural Killer 


Study: NCT01852071 - UCLA Fresh  
 
 
 
Statistical Analysis Plan 
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PBMC 
Peripheral blood mononuclear cells 
PCR 
Polymerase chain reaction 
PEG 
Polyethylene-glycol 
PHA 
Phytohemagglutinin 
PRP 
Polyribosylphosphate 
PT 
Preferred Term 
RBC 
Red blood cell(s) 
RCL 
Replication competent lentivirus 
SAE 
Serious Adverse Event 
SCID 
Severe combined immunodeficiency 
SI 
Stimulation index 
SOC 
System Organ Class 
TCR 
T-cell receptor 
TD 
Transduction 
TREC 
T-cell receptor excision circle 
UCLA 
University of California, Los Angeles 
WBC 
White blood cell(s) 
 
 


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Statistical Analysis Plan 
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1 Introduction 
The trial assesses a new breakthrough therapy composed of ex vivo genetically modified CD34+ 
hematopoietic stem cells (HSC) for the treatment of severe combined immunodeficiency due 
to adenosine deaminase deficiency (ADA-SCID). 
 
The investigational new drug product, OTL-101, includes autologous bone marrow (BM) 
CD34+ HSC from ADA-SCID infants, children and adolescents, transduced ex vivo with a self-
inactivating HIV-1 based lentiviral vector encoding the human ADA cDNA (EFS-ADA LV).  
OTL-101 is used as fresh cell dispersion for infusion after a single non-myeloablative 
conditioning. 
2 Study Design 
This is a historically controlled, open-label, prospective, non-randomized Phase I/II clinical 
trial to assess the safety and efficacy of OTL-101. 
 
OTL-101 will be compared to an historical control group treated with allogeneic Hematopoietic 
Stem Cell Transplantation (HSCT), representative of standard of care. 
  
Two centres are involved in this study: UCLA and the National Institute of Health (NIH) in 
Bethesda, MD. Nineteen subjects have been treated at UCLA and one at the NIH. 
 
 
 


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Statistical Analysis Plan 
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3 Study Schedule 
Evaluation 
Scr 
B-L 
Pre-
op 
Infus
ion 
BU 
+1d 
Months After Procedure 
1 
2 
3 
4 
5 
6 
8 
10 
12 
15 
18 
21 
24 
P.E. 
X 
X 
- 
 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
History 
X 
X 
- 
 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Comprehensive 
Metabolic Panela 
X 
X 
X 
 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Magnesium, 
phosphate 
X 
- 
X 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
CBC with diff. 
X 
X 
X 
 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Cytogenet. PB 
X 
- 
- 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Pregnancy test* 
X 
- 
- 
 
X 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
INR or PT/PTT 
X 
- 
X 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Type and cross 
- 
- 
X 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
HIV-1, HepB & C, 
CMV, B19 
X 
- 
- 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Urinalysis 
X 
- 
X 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
ECG 
X 
- 
- 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Echo 
X 
- 
- 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
CXR 
X 
- 
- 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Pulse ox. 
X 
- 
- 
 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
RBC dAXP 
- 
X 
- 
 
- 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
RBC ADA activity - 
X 
- 
 
- 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Immune functionb 
- 
X 
- 
 
- 
- 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
TREC, TCRV  
panel 
- 
X 
- 
 
- 
- 
- 
- 
- 
- 
- 
X 
- 
- 
X 
- 
X 
- 
X 
PBMC for RCLc 
- 
X 
- 
 
- 
- 
- 
- 
X 
- 
- 
X 
- 
- 
X 
- 
- 
- 
X 


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Statistical Analysis Plan 
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Serum for WB to 
RCLd 
- 
X 
- 
 
- 
- 
- 
- 
X 
- 
- 
X 
- 
- 
X 
- 
- 
- 
X 
PBMC for vectorc 
- 
X 
- 
 
- 
- 
X 
X 
X 
X 
X 
Xg X 
X 
Xg X 
Xg X 
Xg 
Busulfan PKe 
- 
- 
- 
 
X 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Myelosupp. Labf 
- 
- 
- 
 
 
X 
X 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
Scr = screen 
B-L = Baseline 
  
+1d = day after infusion 
  
BU = Busulfan:  See Appendix C for details of administration 
X = to be performed, - = not to be performed 
*For female subject of child-bearing potential 
aincludes albumin, AST/ALT, alkaline phosphatase, total protein, total bilirubin, creatinine, BUN, glucose, calcium, Na+, K+, Cl-, total CO2 
bincludes lymphocyte subsets phenotype, quantitative immunoglobulins, antibodies to Tet/PRP (Q 3 months if not receiving IVIg), T cell proliferation to PHA/Tet (only at 6, 
12, 18, and 24 months and only to Tet if immunized against tetanus). 
cAt each time, any unused PBMC will be cryopreserved in liquid nitrogen, for possible future studies. 
dTo be banked for possible future studies 
eBusulfan PK at 0, 1, 2, 4, 8, 13 hours following IV Busulfan administration, done during hospitalization 
fMyelosuppression Laboratory: Hepatic function tests, CBC/Diff, 2x per week for up to 6 weeks, from day +1 until hospital discharge when neutrophil recovery is observed, 
or longer as indicated.  For subjects who have slow recovery of counts after transplant (beyond Day +30), CBC/Diff will be performed at least once every 7-10 days until ANC 
is >500 cells/mm.3 
gAccording to current FDA guidelines, LAM-PCR samples will be obtained ≈  every 6 months, for 5 years, then annually for an  additional 10 years 


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Statistical Analysis Plan 
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4 Study Objectives 
4.1 Primary Objectives 
The primary objectives in this study are:  
• to examine the safety of autologous transplantation of bone marrow CD34+ cells 
transduced with the EFS-ADA lentiviral vector (OTL-101).  The safety review will 
include the identification of grade III/IV serious adverse events (SAE).  Subjects will 
also be monitored for clinical toxicities, replication competent lentivirus, and 
monoclonal expansion at regular intervals by a clinician, as well as standard blood 
counts and chemistries. 
• to estimate overall and event-free survival by 12 months where failure is defined by 
one of the following endpoints:  death; reinstitution of PEG-ADA; or performance of 
an allogeneic BMT.    
4.2 Secondary Objectives 
The secondary objectives will assess the efficacy of OTL-101, engraftment of transduced cells 
and the extent of ADA expression and immune reconstitution. The secondary outcomes will:  
1. Assess overall and event free survival at 24 months, 
2. Compare overall survival and event free survival at 24 months between patients treated 
with OTL-101 and patients treated with allogeneic HSCT, 
3. Assess the extent of gene transfer in peripheral blood cells,  
4. Assess ADA gene expression by measuring ADA enzymatic activity and adenine 
nucleotides,  
5. Examine the effects of reconstituting ADA gene expression on immune function 
through serial examination of peripheral blood leukocytes and myeloid cells,  
6. Assess immune reconstitution and infection rates, and 
7. Assess use of immunoglobulin replacement therapy.  
5 Endpoints 
5.1 Primary Endpoints 
The primary endpoints are: 
• Safety: the incidence and severity of serious adverse events (SAEs) 
• Efficacy: overall and event-free survival at 12 months, where events are death, 
reinstitution of PEG-ADA therapy and performance of an allogeneic BMT. 
5.2 Secondary Endpoints 
Secondary endpoints for this study are: 
• 
Overall survival and event-free survival at 24 months 
• 
Efficacy of gene transfer/engraftment of HSC  
• 
ADA expression: enzyme activity and detoxification  


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Statistical Analysis Plan 
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• 
Effects on ADA reconstitution on immune phenotype and function (the need for 
IgRT will be assessed under this category) 
• 
Immune reconstitution  
• 
Infection rates 
6 Timing of Analyses 
The statistical analyses of data from this study will be performed in three stages: 
 
Interim look: 
• Performed after all subjects have completed 6 months of follow-up post infusion 
• Descriptive statistics for all available data 
• Comparisons between OTL-101 treated subjects and HSCT control cohorts of 
demographics and overall survival 
 
Primary analysis: 
• Performed after all subjects have completed 12 months of follow-up post infusion  
• Descriptive statistics for all available data 
• Comparison to HSCT control cohorts on overall and event-free survival (primary 
efficacy endpoints), use of immunoglobulin replacement therapy (IgRT) and infection 
rate (key secondary endpoints).  
 
End of study analysis: 
• Performed after all subjects have completed the study (24 months of follow-up post 
infusion)  
• Descriptive statistics for the complete study cohort 
• Comparison to HSCT control cohorts on overall and event-free survival (primary 
efficacy endpoints), use of immunoglobulin replacement therapy (IgRT) and infection 
rate (key secondary endpoints).   
 
7 Analysis Populations 
7.1 Efficacy Population 
The primary efficacy population will consist of all patients treated with OTL-101 at 
UCLA/NIH (current study cohort). 
 
The primary efficacy population from the HSCT historical control cohort will consist of ADA-
SCID patients without a medically eligible HLA-matched sibling/family donor and treated with 
HSCT at either Great Ormond Street Hospital (GOSH) or Duke University from the year 2000 
onwards. 
 
 


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Statistical Analysis Plan 
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Additional efficacy comparisons will be made with: 
• ADA-SCID patients with matched related donors treated with HSCT at either 
GOSH or Duke University from the year 2000 onwards; 
• the complete HSCT historical control cohort consisting of ADA-SCID patients with 
any type of donor treated with HSCT at either GOSH or Duke University from the 
year 2000 onwards. 
7.2 Safety Population 
The safety population will consist of all subjects treated with OTL-101 at UCLA/NIH (current 
study cohort) and the complete HSCT historical control cohort consisting of ADA-SCID 
patients with any type of donor treated with HSCT at either GOSH or Duke University from 
the year 2000 onwards. 
8 Statistical Methods 
In general, continuous variables will be summarised using the mean, standard deviation, 
median, minimum, maximum and number of subjects.  Categorical variables will be 
summarised using number of subjects and percentages. 
 
For all summaries, the HSCT controls will be summarised in the groups of those without a 
matched related donor, those with a matched related donor and all subjects. 
8.1 Subject Disposition 
8.1.1 Summary of Populations 
Number (%) of subjects enrolled (signed informed consent), treated, and in efficacy and safety 
populations will be summarised for the OTL-101 treated and HSCT control groups.  The 
denominator for the percentages will be the number of subjects enrolled.  
 
Should any subjects be excluded from any of the analysis populations, a listing will be 
produced for these subjects noting the reason for exclusion. 
8.1.2 Summary of Withdrawals 
Number (%) of subjects who completed the study period will be summarised along with the 
reason for study withdrawal.  The denominator for the percentages will be the number of 
subjects treated. 
 
At the interim look and the primary analysis, the number (%) completing the designated follow-
up period and ongoing in the study will also be summarized. 
 
Should any subjects have withdrawn from the study, a listing will be produced for these 
subjects noting the reason for study withdrawal, and the time since infusion at withdrawal. 
 


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Statistical Analysis Plan 
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8.2 Demography and Baseline Characteristics 
Demography and baseline characteristics will be summarised for the safety population.   
8.2.1 Demography 
Age in months for the OTL-101 treated subjects will be calculated as:  
12*(date informed consent signed – date of birth)/365.25. 
 
Age in months for the HSCT controls will be captured on the eCRF. 
 
Age in months will be summarized for the OTL-101 treated and HSCT control groups.  Sex, 
race, weight and height at screening will be summarised for the OTL-101 treated group. 
8.2.2 Medical History 
Medical history was collected on two forms in this study.  Each will be summarised separately. 
 
The number (%) of subjects reporting medical history categorized as normal / abnormal 
clinically non-significant (CNS) / abnormal clinically significant (CS) will be summarised by 
body system for the OTL-101 treated subjects.  Should any subject have an abnormal 
evaluation with the clinical significance assessment missing, this will be categorized as 
“abnormal unknown clinical significance” and this category will be added to the summary. 
 
 
For the skin examination, the number (%) of OTL-101 treated subjects with each of the 
following will be summarized: 
• evidence of active malignant disease other than dermatofibrosarcoma protuberans 
(DFSP); 
• evidence of DFSP expected to require anti-neoplastic therapy within the 5 years 
following the infusion of genetically corrected cells; 
• evidence of DFSP expected to be life limiting within the 5 years following the infusion 
of genetically corrected cells. 
 
In addition, the number (%) of OTL-101 treated subjects with each of the following will be 
summarized:  
• evidence of active opportunistic infection; 
• known sensitivity to busulfan; 
• expected survival < 6 months; 
• pregnant; 
• major congenital anomaly. 
 
In addition, the number (%) of subjects reporting medical history at screening in each category 
will be summarised for the OTL-101 treated and the HSCT control groups. 


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Statistical Analysis Plan 
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8.2.3 ADA Deficiency Diagnosis 
Age at diagnosis in months will be calculated for the OTL-101 treated subjects as:  
12*(date of diagnosis – date of birth)/365.25. 
 
For the HSCT control groups, the age at diagnosis will be recorded on the eCRF. 
 
Time since diagnosis in months will be calculated as:  
12*(date of transplant - date of diagnosis)/365.25,  
or, for HSCT control subjects for whom the dates were not recorded, as: 
age in months at transplant - age in months at diagnosis. 
 
Age at diagnosis, time since diagnosis and the value used to diagnose ADA deficiency will be 
summarized using descriptive statistics for the OTL-101 treated subjects.  In addition, the 
number (%) of subjects with each of the following will be summarized: 
• ADA deficient as determined by reference laboratory; 
• with family history of 1st order relative with ADA deficiency and clinical and 
laboratory evidence of severe immunologic deficiency; 
• with evidence of severe immunologic deficiency prior to institution of immune 
restorative therapy; 
• with no healthy, medically eligible HLA-identical sibling with normal immune function 
who may serve as an allogeneic bone marrow donor. 
 
Age at diagnosis and time since diagnosis will be summarized using descriptive statistics for 
the HSCT control groups.  In addition, the number (%) of subjects with each method of 
diagnosis (DNA sequencing/ confirmed absence of <3% enzymatic activity) will be 
summarized. 
8.2.4 Disease History 
Number (%) of OTL-101 treated subjects with each of the following will be summarized: 
• Peri-natal complications (Prematurity / Infection / Other); 
• Method of diagnosis (Newborn screen / Family History / Infection / Other); 
• Siblings (Yes [HLA-Matched] / Yes [HLA-Not Matched] / No); 
• Matched unrelated donor (MUD)/cord search performed; 
• Pulmonary Complications; 
• Feeding History; 
• Prior Surgery. 
8.2.5 Cytogenetics 
Cytogenetics will be assessed at screening and categorised as normal or abnormal, with normal 
results subcategorised as 46XX/46XY/Other. 
 


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Statistical Analysis Plan 
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The number (%) of OTL-101 treated subjects falling into each category for source (PBMCs, 
bone marrow, amniotic fluid), result (normal/abnormal), category of normal result 
(46XX/46XY/Other) will be summarised. 
8.2.6 Viral PCR 
The number (%) of OTL-101 treated subjects with positive/not detected HIV, hepatitis B, 
hepatitis C, CMV and parvovirus B19 will be summarised. 
8.2.7 Coagulation Tests 
Coagulation tests (prothrombin time, partial thromboplastin time and internal normalised ratio) 
will be evaluated at screening and pre-harvest for the OTL-101 treated subjects. 
 
For each parameter, the actual value will be summarised at each visit using descriptive 
statistics. 
 
Coagulation tests will be flagged against the normal range as low/normal/high.  For each 
parameter, the number (%) of subjects with evaluations that were low/normal/high relative to 
the normal range will be summarised by visit. 
8.2.8 Urinalysis 
Urine protein results will be summarized by visit using the number (%) of OTL-101 treated 
subjects with results in each category (0, 1+, 2+, 3+, 4+) of urinalysis result.  In addition, a 
shift table from Screening to Pre-Harvest will be produced. 
8.2.9 Electrocardiogram (ECG) 
An ECG will be taken at screening which will be categorized as normal/ abnormal, not 
clinically significant/ abnormal, clinically significant.  The number (%) of subjects with 
evaluations falling into each category will be summarised.  Should any subject have an 
abnormal evaluation with the clinical significance assessment missing, this will be categorized 
as “abnormal unknown clinical significance” and this category will be added to the summary. 
8.2.10 
Echocardiogram 
An echocardiogram will be taken at screening which will be categorized as normal/ abnormal, 
not clinically significant/ abnormal, clinically significant.  The number (%) of OTL-101 treated 
subjects with evaluations falling into each category will be summarised along with the number 
(%) of subjects with uncorrected congenital cardiac malformation with clinical 
symptomatology.  Should any subject have an abnormal evaluation with the clinical 
significance assessment missing, this will be categorized as “abnormal unknown clinical 
significance” and this category will be added to the summary.  Left ventricular ejection fraction 
will be summarised using descriptive statistics. 
 
In addition, details of any clinically significant findings will be listed.  


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Statistical Analysis Plan 
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8.2.11 
Chest X-ray 
A chest x-ray will be taken at screening which will be categorised as normal/ abnormal, not 
clinically significant/ abnormal, clinically significant.  The number (%) of OTL-101 treated 
subjects with evaluations falling into each category will be summarised.  Should any subject 
have an abnormal evaluation with the clinical significance assessment missing, this will be 
categorized as “abnormal unknown clinical significance” and this category will be added to the 
summary.  In addition, the number (%) of OTL-101 treated subjects with active cardiac disease, 
including clinical evidence of congestive heart failure, cyanosis, hypotension will be 
summarized. 
8.2.12 
Pulse Oximetry 
Oxygen saturation at screening will be summarized for the OTL-101 treated subjects using 
descriptive statistics. 
8.2.13 
Bone Marrow Harvest 
The number (%) of OTL-101 treated subjects with each blood type, for whom harvest can be 
performed, for whom harvest was performed and with central venous access will be 
summarized. 
 
Total number of CD34+ cells harvested and back-up cells collected will be summarised using 
descriptive statistics along with the number (%) of OTL-101 treated subjects with each cell 
type (total nucleated cells / mononuclear cells). 
8.2.14 
Busulfan Pharmacokinetics 
The area under the curve (AUC) for busulfan will be summarised using descriptive statistics 
for the OTL-101 treated subjects. 
8.2.15 
OTL-101 Administration 
The number (%) of subjects who received OTL-101 will be summarised along with the age in 
months at transplant and the number of infusions received (1 or 2). 
 
For each infusion, CD34+ pre-TD vector copy number (copy/cell),  CD34+ post-TD vector 
copy number (copy/cell), CD34+ pre-TD ADA enzyme activity (nmol/min/108 cells), CD34+ 
post-TD ADA enzyme activity (nmol/min/108 cells), subject weight (kg), total CD34+ cells 
infused (x 106 cells), total CD34+ cells infused / kg, infusion volume (mL), duration of infusion 
(min) and infusion rate (mL/min) will be summarized using descriptive statistics. 
8.2.16 
Transplant Details for the HSCT Controls 
Age (months) at transplant, number of CD34+ cells/kg transplanted and busulfan dose, if 
appropriate, will be summarized for each HSCT control group using descriptive statistics. 
 


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Statistical Analysis Plan 
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Number (%) of subjects with each type of transplant cells, transplant donor type, conditioning 
type, receiving enzyme replacement therapy (ERT) prior to the transplant, receiving ERT after 
the transplant, receiving IgRT prior to the transplant, receiving IgRT after the transplant and 
receiving gene therapy after the transplant will be summarized for each HSCT control group. 
8.2.17 
Exposure 
Duration of follow-up in months will be calculated for the OTL-101 treated subjects as:  
12*(date of final evaluation – date of infusion + 1)/365.25. 
 
For the HSCT controls, the duration of follow-up will be recorded on the eCRF. 
 
Duration of follow-up will be summarized for the OTL-101 treated and the HSCT control 
groups using descriptive statistics.  In addition, the total subject-years of follow-up in each 
treatment group will be presented. 
8.3 Efficacy Evaluations 
The primary comparison for all efficacy analyses will be between the OTL-101 treated subjects 
and the allogeneic HSCT controls treated at either GOSH or Duke University without a 
matched related donor.   
 
Secondary comparisons will be between: 
• the OTL-101 treated subjects and the HSCT controls treated at either GOSH or Duke 
University with a matched related donor; 
• the OTL-101 treated subjects and the total group of HSCT controls. 
 
8.3.1 Primary Efficacy Endpoints 
8.3.1.1 
Overall Survival at 12 Months 
Overall survival will be evaluated using the time in months from transplant to either the 
subject’s death or their last evaluation calculated as: 
12*(date of death/last evaluation – date of transplant)/365.25, 
or, for HSCT control subjects for whom the dates were not recorded, as: 
age in months at death/end of follow-up - age in months at transplant.  
 
The number (%) of subjects who were still alive at 12 months post-transplant will be 
summarised.  For the purpose of this analysis, any subject who withdrew from the study prior 
to 12 months, who was known to be alive at the last evaluation, will have a status of 
“unknown”.  The proportion and exact 95% confidence interval (CI) for survivors at 12 months 
post-transplant will be calculated for the OTL-101 treated and each of the HSCT control groups 
along with the difference in proportions (with 95% CI) between the OTL-101 treated and each 
of the control groups, excluding those with unknown status. 
 


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Statistical Analysis Plan 
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Overall survival in each treatment group will also be presented using a Kaplan-Meier curve for 
time to death.  For the purpose of this analysis, any subject who withdrew from the study prior 
to 12 months and was known to be alive at the last evaluation will be censored at the date of 
their last evaluation.  If appropriate, the proportion alive at 12 months and the associated 95% 
confidence interval will be estimated for each treatment group from the Kaplan-Meier curve 
along with the median survival time (and interquartile range), if they can be estimated from the 
data. 
 
If there is at least one event in each group, the log-rank test will be used to compare the 
difference in survival curves between the OTL-101 treated subjects and each of the HSCT 
control groups. 
 
 
In addition, a Bayesian analysis will be carried out.  The prior distribution for the rate of 
survivors in each treatment group will be set to Beta(α=1, β=1) (a conservative non-informative 
prior).  The number of survivors in each group (X) will be estimated and the posterior 
distribution for the rate of survivors in each group will be updated to Beta(α=1 + X, β=1 +N - 
X), where N is the total number of subjects in the group. Histograms will be presented for the 
posterior distributions for each treatment group.   
 
A posterior distribution for the difference between the survival rate in the OTL-101 treated 
group and each of the HSCT control groups will be estimated empirically via simulation with 
10,000 replications.  Based on the distribution above, posterior probabilities for the following 
will be calculated: 
• rate on OTL-101 > rate on control (superiority like test); 
• rate on OTL-101 > rate on control – 10% (non-inferiority like test). 
 
8.3.1.2 
Event-free Survival at 12 Months 
Event-free survival will be evaluated using the time in months from transplant to either the first 
event or their last evaluation calculated as: 
12*(date of event/last evaluation – date of transplant)/365.25,  
or, for HSCT control subjects for whom the dates were not recorded, as: 
age in months at death/end of follow-up - age in months at transplant.   
 
For the purpose of this analysis, the events of interest are:  
• death; 
• reinstitution of PEG-ADA ERT; 
• requirement for a second transplant. 
 
Event-free survival will be summarized in the same way as overall survival described in 
Section 8.3.1.1. 
 


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Statistical Analysis Plan 
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In addition, if there are any events (other than death), the time to each event will be summarized 
in a similar manner to the overall event-free survival. 
8.3.2 Secondary Efficacy Endpoints 
8.3.2.1 
Overall and Event-free Survival at 24 Months 
Overall and event-free survival at 24 months will be summarized in the same way as for the 
analyses at 12 months described in Sections 8.3.1.1 and 8.3.1.2. 
8.3.2.2 
Use of Immunoglobulin Replacement Therapy 
The number (%) of subjects receiving immunoglobulin replacement therapy (IgRT) will be 
summarised by visit for the OTL-101 treated subjects. 
 
The time to cessation of IgRT will be calculated (in months) as: 
 
12*(stopping date of IgRT – date of transplant) /365.25, 
or, for HSCT control subjects for whom the dates were not recorded, as: 
age in months when IgRT stopped – age in months at transplant. 
 
Time to cessation of IgRT will be summarised for the OTL-101 treated and HSCT control 
groups using descriptive statistics and, if appropriate, estimated from a Kaplan-Meier curve in 
which subjects who are still receiving IgRT will be censored at the time of their last visit. 
 
The number (%) of subjects who have stopped treatment with IgRT at 12, 18 and 24 months 
without restarting at any subsequent time point will be summarized for the OTL-101 treated 
and HSCT control groups (data permitting). 
 
In addition, serum immunoglobulin levels (IgG, IgA and IgM) will be measured at each visit 
for the OTL-101 treated subjects.  Levels of IgG, IgA and IgM and changes from baseline will 
be summarised by visit using descriptive statistics.  In addition, plots of the median value and 
range over time on a logarithmic scale will be produced for each parameter along with 
individual plots for each subject which will include age-dependent normal ranges provided by 
the study sites. 
 
8.3.2.3 
Rate of Severe Infections 
Infections will be recorded as adverse events.  The infections of interest in this study are severe 
infections or opportunistic infectious episodes, defined as infections requiring hospitalization 
or prolonging hospitalization and/or documented infections by opportunistic pathogens (e.g. 
interstitial pneumonia, intractable diarrhoea). 
 
The number (%) of subjects reporting severe infections or opportunistic infectious episodes 
along with the number of events reported will be summarized for the OTL-101 treated and 


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HSCT control groups (data permitting).  In addition, the infection rate will be calculated as the 
number of infections/the total follow-up time.  The rate will be calculated both within each 
treatment group and for each individual subject.  The subject infection rate will be summarized 
by treatment group using descriptive statistics.  In addition to the rate for the entire follow-up 
period, the rate will be calculated for both the first 12 months following transplant and for the 
remaining study period. 
8.3.2.4 
Immune Reconstitution  
Immune reconstitution will be assessed using lymphocyte subsets.  Absolute numbers and 
percentages of CD3+, CD4+, CD8+ CD4+CD45RA+, CD4+CD45RO+ T-lymphocytes, 
CD19+ B-lymphocytes and CD16+CD56+ NK cells will be measured. 
 
For each of the lymphocyte subsets, the actual value and change from baseline will be 
summarized by visit for the OTL-101 treated subjects.  The median value and range will be 
plotted over time on a logarithmic scale and individual counts will be plotted along with the 
age-dependent normal ranges reported in Table 2. 
 
In addition, statistical testing of the change from baseline to 6 and 12 months will be performed.  
If the difference is normally distributed, a paired t-test will be used. Otherwise, the Wilcoxon 
signed rank test will be used. 
 
The trend over time will be examined for each count separately, using mixed models for 
repeated measures (MMRM) with visit and baseline count as fixed effects, and subject as a 
random effect, utilizing the compound-symmetry covariance structure. If counts are 
approximately normally distributed they will be analysed on the original scale. If the 
distribution is found to be skewed to the right and a log transformation can normalize the data, 
statistical analysis will be performed on log-transformed data.  If the data are still not normal, 
the data will be ranked in ascending order of magnitude and the analysis will be performed on 
the ranks. 
 
8.3.2.5 
T Cell Receptor Excision Circles (TREC) in the PB 
TREC levels will be summarised for OTL-101 treated subjects at each visit using descriptive 
statistics.  Plots of the median and range over time on a logarithmic scale will be produced.  In 
addition, individual subject plots of TREC levels over time will be produced. 
8.3.2.6 
Engraftment 
Engraftment of transduced cells will be assessed using vector gene marking in granulocytes 
and PBMC. 
 
The vector copy numbers in each type of cell will be summarised for OTL-101 treated subjects 
by visit using descriptive statistics.  In addition, for each cell type, plots of the median and 


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range for copy numbers over time on a logarithmic scale will be produced along with individual 
subject plots. 
8.3.2.7 
ADA Expression, Enzyme Activity and Detoxification  
RBC ADA activity, AXP, dAXP and %dAXP will be summarized by visit for the OTL-101 
treated subjects.  The median value and range will be plotted over time on a logarithmic scale 
as will the values for individual subjects. 
 
In addition, statistical testing of the change in %dAXP from baseline to 6 and 12 months will 
be performed.  If the difference is normally distributed, a paired t-test will be used. Otherwise, 
the Wilcoxon signed rank test will be used. 
 
If the %dAXP values are approximately normally distributed, the trend over time will be 
examined using MMRM with visit and baseline count as fixed effects, and subject as a random 
effect, utilizing the compound-symmetry covariance structure (if it produces an acceptable fit). 
8.3.2.8 
T Cell Receptor Vβ Panel Results 
For each cell type (CD4 and CD8), each of the Vβ results will be summarised for OTL-101 
treated subjects at each visit using descriptive statistics.  A panel of plots of the median and 
range over time on a logarithmic scale will also be produced. 
8.3.2.9 
Lymphocyte Proliferative Responses Stimulation Index 
(SI) 
Lymphocyte proliferative response to PHA, Tetanus, and Candida were collected.  Early in the 
study, these responses were provided by Quest laboratories.  At a later date, these analyses 
were moved to the in-house immunogenetics laboratories at each study site.  Since the results 
from the in-house laboratories are not comparable with those from Quest, they will be 
summarized separately. 
 
The stimulation index will be calculated for each parameter as the sample response/control 
response.  Stimulation indices will be summarized for the OTL-101 treated subjects by visit 
for each parameter, separately for the data from the Quest and in-house laboratories.  In 
addition, for each parameter, the median and range will be plotted on a logarithmic scale over 
time.  These stimulation indices will be compared with the normal reference ranges provided 
by the study sites and the number (%) of subjects with responses above and below the normal 
range will be summarized by visit as well as at any time post infusion. 
 
8.3.2.10 
Antibody Responses 
Antibody responses to Tetanus and Polyribosylphosphate (PRP) were evaluated. 
 


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The response to each of the antibodies will be summarized for the OTL-101 treated subjects 
using descriptive statistics. 
8.3.3 Additional Efficacy Endpoints 
8.3.3.1 
Use of PEG-ADA Enzyme Replacement Therapy 
The time to withdrawal of PEG-ADA ERT will be calculated (in months) as: 
 
12*(stop date of PEG-ADA ERT – date of transplant) /365.25, 
or, for HSCT control subjects for whom the dates were not recorded, as: 
age in months when PEG-ADA stopped – age in months at transplant. 
 
Time to withdrawal of PEG-ADA ERT will be summarised for the OTL-101 treated and HSCT 
control groups using descriptive statistics and, if appropriate, estimated from a Kaplan-Meier 
curve in which subjects who are still receiving PEG-ADA ERT will be censored at the time of 
their last visit. 
8.3.4 Sensitivity Analyses 
Protocol violations in this study will be reviewed prior to database lock in order to determine 
whether any major violations have occurred that could have influenced the outcome of the 
study.  In the event that such violations have occurred, sensitivity analyses will be carried out 
in order to assess the impact of these violations on the efficacy results. 
 
Sensitivity analyses will be carried out for: 
• 
Overall and event free survival (descriptive statistics and Kaplan-Meier analyses) 
• 
Lymphocyte subsets (descriptive statistics and plots) 
• 
Measures of engraftment (descriptive statistics and plots) 
8.4 Safety Evaluations 
8.4.1 Adverse Events 
All adverse events will be coded using MedDRA version 20.0 
8.4.1.1 
Definition of Treatment-emergent Adverse Events 
Adverse events (AEs) will be summarised on the basis of treatment emergence.  An adverse 
event is considered to be treatment emergent if it occurred on or after the date and time of the 
start of the OTL-101 infusion.  If the start time of the event is not present, any event starting 
on the same day as the infusion will be considered to be treatment emergent.  If the start date 
of the adverse event is partial or missing, it will be considered to be treatment emergent if: 
• the day is missing, the month and year are present and are the same as or after the month 
and year of the infusion and the end date is missing or is not before the date of infusion, 
• the day and month are missing, the year is present and is the same as or after the year 
of the infusion and the end date is missing or is not before the date of infusion, 


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• the entire date is missing and the end date is missing or is not before the date of infusion. 
 
8.4.1.2 
Serious Adverse Events 
The primary safety parameter in this study is the incidence and severity of serious adverse 
events (SAEs). 
 
Incidence and frequency of treatment emergent SAEs will be summarised by system organ 
class (SOC) and preferred term (PT): 
• overall, 
• by maximum severity, 
• by maximum relationship to treatment 
• treatment related SAEs.  
 
In each table, if the same SOC or PT is reported on multiple occasions for a single subject, it 
will be included only once in the summary.  In the table by maximum severity, only the most 
severe occurrence for each SOC and PT for each subject will be included in the summary.  In 
the table by relationship, only the most related occurrence for each SOC and PT for each subject 
will be included in the summary. 
 
Any SAEs that are not treatment emergent (occurred prior to the infusion) will be listed. 
 
If there are any deaths, the details will be listed. 
8.4.1.3 
Adverse Events 
Treatment emergent adverse events will be summarised in an overall summary presenting the 
number (%) of subjects with: 
• any AE,  
• AE grade 2 or higher (at least moderate in severity),  
• AE grade 3 or higher (at least severe in severity),  
• treatment related AE,  
• AE leading to study withdrawal, 
• any serious adverse event (SAE),  
• treatment related SAE, 
• SAE leading to death. 
 
Incidence and frequency of treatment emergent AEs will be summarised by system organ class 
(SOC) and preferred term (PT): 
• overall, 
• by maximum severity, 
• by maximum relationship to treatment, 
• treatment related AEs, 


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• AEs leading to study withdrawal (if sufficient events). 
 
In addition, a summary table for the most frequently reported treatment emergent adverse 
events (those reported in 2 or more subjects) will be presented by PT in descending order of 
incidence and frequency. 
 
In each table, if the same SOC or PT is reported on multiple occasions for a single subject, it 
will only be included once in the summary.  In the table by maximum severity, only the most 
severe occurrence for each SOC and PT for each subject will be included in the summary.  In 
the table by maximum relationship, only the most related occurrence for each SOC and PT for 
each subject will be included in the summary. 
 
Any adverse events that are not treatment emergent (occurred prior to the infusion) will be 
listed. 
8.4.2 Concomitant Medications 
Concomitant medications taken during the study will be listed. 
8.4.3 RCL Testing 
Number (%) of OTL-101 treated subjects testing positive for RCL will be summarised by visit 
and at any time. 
8.4.4 Laboratory Evaluations 
8.4.4.1 
Haematology 
Haematology parameters (red blood cell (RBC) count, haemoglobin, haematocrit, mean 
corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular 
haemoglobin concentration (MCHC), white blood cell (WBC), neutrophils, lymphocytes, 
monocytes, eosinophils, basophils and platelet count) will be evaluated over time for the OTL-
101 treated subjects.  The baseline evaluation for each parameter will be the final evaluation 
prior to the OTL-101 infusion. 
 
 For each parameter, the actual value and change from baseline will be summarised at each 
visit using descriptive statistics. 
 
Haematology evaluations will be flagged against the normal range as low/normal/high.  For 
each parameter, the number (%) of subjects with evaluations that were low/normal/high relative 
to the normal range will be summarised by visit.  In addition, shift tables will be constructed 
comparing the flags at the baseline evaluation with those at each post-infusion evaluation and 
the minimum and maximum evaluation post-baseline.  Out of range values will be assessed for 
their clinical significance.  The number (%) of subjects with any clinically significant abnormal 
values will be summarized by visit and at any time post-infusion. 


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8.4.4.2 
Biochemistry 
Biochemistry parameters (Urea Nitrogen, creatinine, total bilirubin, sodium, potassium, 
chloride, calcium, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline 
phosphatase, albumin, total protein, glucose, magnesium, phosphorus, total CO2) will be 
evaluated over time for the OTL-101 treated subjects.  The baseline evaluation for each 
parameter will be the final evaluation prior to the OTL-101 infusion. 
 
For each parameter, the actual value and change from baseline will be summarised at each visit 
using descriptive statistics. 
 
Biochemistry evaluations will be flagged against the normal range as low/normal/high.  For 
each parameter, the number (%) of subjects with evaluations that were low/normal/high relative 
to the normal range will be summarised by visit.  In addition, shift tables will be constructed 
comparing the flags at the baseline evaluation with those at each post-infusion evaluation and 
with the minimum and maximum evaluation post-infusion.  Out of range values will be 
assessed for their clinical significance.  The number (%) of subjects with clinically significant 
abnormal values will be summarized by visit and at any time post-infusion. 
8.4.5 Vital Signs, Height and Weight 
Vital signs (temperature, heart rate, respiratory rate, systolic and diastolic blood pressure), 
height and weight are measured over time for the OTL-101 treated subjects. 
 
The baseline evaluation for each of the vital signs, height and weight will be the final evaluation 
prior to the OTL-101 infusion. 
 
For each parameter, the actual value and change from baseline will be summarised at each visit 
using descriptive statistics. 
 
Each subject’s vital signs be categorized as normal, abnormal not clinically significant or 
abnormal clinically significant at each visit.  The number (%) of subjects with clinically 
significant evaluations will be summarised by visit and at any time post-infusion. 
8.4.6 Physical Examination 
Physical examinations will be performed over time for the OTL-101 treated subjects.  The 
number (%) of subjects with evaluations categorized as normal / abnormal clinically non-
significant / abnormal clinically significant will be summarized by body system at each visit.  
Should any subject have an abnormal evaluation with the clinical significance assessment 
missing, this will be categorized as “abnormal unknown clinical significance” and this category 
will be added to the summary.  An overall summary will also be produced for each body system 
showing the number (%) of subjects with normal evaluations at all pre-treatment evaluations 
who had one or more abnormal evaluations post-treatment, these will be categorized as 
abnormal clinically non-significant / abnormal clinically significant with the worst case being 
used for the summary. 


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9 Analysis Software 
All summaries and analyses will be carried out using SAS version 9.4. 
 
10 Changes in Statistical Analysis Plan 
10.1 Changes in Version 3.0 
 
Since Statistical Analysis Plan version 2.0 was signed off on 14th March, 2017, Version 10.0 
of the protocol has been signed off, so a number of modifications were needed to this SAP in 
order to account for the changes in the protocol.   
 
In addition to these changes, the document has been restructured in order to improve the flow 
and the understandability.  In particular, the description of any derivations of the endpoints has 
been added to the statistical methods section instead of being with the list of endpoints.  Also, 
some of the details relating to programming specifications have been removed and will be 
added to the programming documentation. 
 
Changes of note are: 
 
• Study objectives and endpoints have been reworded to match protocol version 10.0. 
• The age at entry and transplant will be expressed in months rather than years as this is 
more appropriate for the subjects enrolled. 
• The calculation of durations in months has been changed from days/30 to 
12*days/365.25 to be consistent with calculations of age. 
• Inclusion/exclusion criteria will no longer be summarized, but will be listed. 
• A summary of the busulfan AUC has been added. 
• The plots of parameters over time will no longer present the mean +/- 2SE, but the 
median and range as this was more appropriate for the parameters being presented. 
• The infection summaries have been reworded to match the definition of severe 
infections from the protocol.  
• Additional adverse event summaries have been added and it has been explicitly stated 
that all summaries will only include treatment emergent adverse events. 
• Summaries of laboratory evaluations using descriptive statistics have been added. 
• Plots of vital signs over time have been removed as they were not considered to be 
useful. 
• Plots of antibody responses over time and summaries by visit have been removed as the 
data will not be collected over time for individual subjects. 
 


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10.2   Changes in Version 4.0 
Since version 3.0 of the Statistical Analysis Plan was signed off, the sponsor has received 
regulatory approval to include further HSCT control data from Duke University, so this SAP 
has been updated to include these data. 
 
The following additional changes have been made: 
• Details added to clarify the calculation of time to event for subjects in the HSCT control 
group who do not have the dates of events recorded. 
• Tables of reference ranges have been deleted to allow the most up to date ranges 
received from the study sites to be used in the analyses.  
• The summary of coagulation data has been moved from the safety section to the 
baseline section as these data are only collected prior to the transplant. 
• The MedDRA version to be used has been updated to 20.0.